The following paper was submitted to a vegan publication in 2001 but may not have been considered suitable

for inclusion because at that time the suggested roles of heparan sulfate made in this article were less well known outside polysaccharide scientific circles than is now the case.
Recent Heparan Sulfate Research Results May Help Explain Why a Vegan Diet is Good for You

David Grant Ashbank, AB53 6SX, U.K. Summary. Dietary factors can affect health by influencing heparan sulfate polysaccharide systems on which important tissue protection activities are centered. A vegan diet, which tends to be close to that under which our species evolved may promote health principally by avoiding iron-overload which is associated with inappropriate heparan sulfate degradation by nitric oxide metabolites and the formation of lipid oxidation products which negatively perturb heparan sulfate biosynthesis. Introduction. Of the three major classes of polymer systems used by our cells, the nucleic acids and proteins are better understood than are the polysaccharides. But a picture is gradually emerging in which key sulfated glucosamine-containing polysaccharides especially the heparan sulfate proteoglycans (HSPGs) - are found to be linked to many aspects of health and disease. These polymers are members of the glycosaminoglycan (GAG) proteoglycan families which include chondroitin and keratan sulfates as well as hyaluronan. A critical dependence of human health on heparan sulfate biosynthesis supports the notion that evolutionary pressure, at the very early stages of the evolution of multicellular organisms in our ancestral lineage, led to the selection of these polymers to be the basis of an auxiliary information storage system. If we are allowed to make a computer hardware analogy in which the memory chips are the DNAcontaining genes, then the keyboard and processor are HSPGbased systems providing signaling channels, ion stores and water based osmolality buffers between extracellular and intracellular space. The computer analogy is not an exact one since biological systems seem to use precisely microsequenced polymers for both software and hardware functions. (Although there is poorly understood non-genetically inheritance with which GAGs may even be involved, GAGs are highly dependent on gene products for their biosynthesis). Protein primary sequence structures are

determined by genetic information, but their correct folding (e.g. as required for growth factor modulation, antioxidant anchoring and hemostasis activities) is often determined by binding to HSPGs which may, however, be susceptible to inappropriate oxidative damage under some pathological and adverse dietary situations. Some HSPG Biochemical Systems. Haemostasis was the first tissue-protective HSPG role to be discovered. The inhibition of blood coagulation by heparin, the most sulfated of the heparan sulfate-like polysaccharides (discovered ca. 1915 by McLean & Howell) involves conformational alteration of antithrombin(lll) (AT(lll)) by a highly specific heparin pentasaccharide sequence (as intensively researched by Rosenberg, Lindahl, Choay and others). Dietrich and coworkers predicted a cellular recognition role for HSPGs which seems to be inherent in the complexity of their shape and anionic charge array patterns enhanced by the highly flexible iduronic acid sugar ring (formed by epimerization of the more primitive glucuronic acid CS present in ancestral bacterial polysaccharides) and they recently demonstrated that administration of anticoagulant heparin (including the now popular low molecular weight forms) upregulated AT(lll)-binding sequences in blood vessel cell wall HSPGs and increased their antithrombotic properties. Nitric oxide under some circumstances does likewise, in keeping with recent indications of biofeedback between nitric oxide and heparan sulfate tissue protective systems. HSPG-controlled systems are also employed by organisms for the regulation of cell-cell interactions including cells of the immune system and in regulation of growth factor signaling, most researched for basic fibroblast growth factor (b-FGF). EXT1 and EXTZ anti-tumor factors were recently found to probably be heparan sulfate biosynthesis enzymes (this came as a surprise to many biochemists). HSPGs are centrally involved in lipid processing and can limit transfer of DNA cationic lipid complexes into and within cells. Although the involvement of heparin/HSPG with lipid biochemistry was discovered many decades ago from the routine pharmaceutical use of heparin for inhibition of blood coagulation, a mechanism is currently emerging that suggests that the induction of atherosclerosis may be countered by good lipids typical of vegetable-rich diets (and some fish oils) by promotion of the biosynthesis of beneficially microstructured HSPG for the inhibition of pathological calcification. HSPG involvement in tissue

regeneration in wound healing is part of their global cellular control function which is also highly relevant to embryogenesis (recently reviewed by Perrimon & Bernfield) especially for nervous system development. So it comes less of a surprise to learn that dysfunction of HSPG biochemistry may be implicated in neural degenerative diseases such as BSE and CJD. Heparin/heparan sulfate and analogous substances seem to directly inhibit prion diseases as well as stopping injurious crystal (stone) formation in blood and urinary vessels (including inhibition of calcium oxalate crystallization, a possible metabolite problem potentially associated with long-term high dose Vitamin C (ascorbate) dietary supplementation). Many viruses (including type-O bovine foot and mouth virus) bind to cell wall HSPGs which, together with analogous substances, are broad spectrum antivirals (inhibiting enveloped viruses such as HIV-1, HIV-2, cytomegalovirus, herpes simplex virus (including mutants resistant to acyclovir) togaviruses and rhabdoviruses). HSPG biochemistry involvement together with bacterial adhesion has been proposed for Helicobacter pylon induction of stomach ulceration and cancer. HSPGs are also apparently involved in the biochemistry of endotoxins, polyamines, homocysteine, prostaglandins and nonsteroidal antinflammatory agents and HSPG biosynthesis is adversely affected by toxic heavy metals, excess serum glucose and oxidative stress including that associated with iron overload. Heparin anticoagulant therapy apparently demonstrates anticancer side effects (reviewed by Engelberg in 1998) often associated with restriction of tumor blood vessel growth (angiogenesis). HSPGs and Ageing. Binding to, and supramolecular structure induction of collagens by GAGs, especially by HSPGs, provides very important structural integrity and water-holding properties. A natural depletion, linear with age, in the content of HSPGs (combined with the effect of an apparent natural increase in serum ferritin, the iron storage protein, with age) may even be a major biochemical clock responsible for the ageing process. (Research into atherosclerosis showed a linear depletion of HSPGs with increasing age to occur in total GAGs at human artery surfaces; perhaps this indicates the ultimate secret of beauty and what makes skin look young, possibly allowing future dramatic improvement in the cosmetics trade when such HSPG influenced mechanisms of ageing are more fully understood).

An inverse correlation with age, reported by Aoyagi et al in 1994, shows up in the blood coagulation and fibrinolysis activities of heparin/HSPG-dependent urokinase, plasmin and thrombin. Similar effects observed in Alzheimers disease patients suggested that a derangement of such HSPG-linked processes was accelerated in such degenerative diseases possibly by the effects of trace redox metal ion overload and pathogen perturbations (e.g. situations leading to chronic inflammation) capable of tipping the balances involving HSPG signaling mechanisms. Our general health, as well as the way we look, can be seen then to depend on the structural integrity of the GAG-rich rubbery polymers which hold our cells together in tissue, cartilage and bone structures, and provide binding sites for protective enzymes and growth factors employed in wound healing and hematopoiesis. HSPGs in Antioxidant Defense. The above reasoning is the basis of antioxidant theories of ageing and of ill-health. Degradative damage to the extracellular matrices which, like corrosion of metals or similar degradation of plastics, drastically impairs their performance. An HSPG-based antioxidant theory of health can be made on this basis. Nutrient elements used by antioxidant enzyme defense systems include selenium required for glutathione peroxidase, manganese and iron for intracellular-, and copper and zinc for extracellular-superoxide dismutases (SO Ds). A newly discovered selenium-containing antioxidant is probably another endothelial cell surface, HSPG-bound enzyme. Since heparan sulfate is a linearly codified information polymer (in this way analogous to DNA or RNA) even slight damage at key sites (especially N-sulfonates) may significantly diminish their physiological activities and impair health. The rate at which such defective HSPG is removed and replaced by de novo HSPG biosynthesis obviously require to equate in order to maintain HSPG tissue protective potency. Such processes can, however, be affected by dietary factors. Bench Observations Modeling Oxidant & Nitrant Tissue Damage. Antioxidant protection can be illustrated by a comparison of the effect of antioxidants on test moldings containing trace metal (especially iron-binding) sulfur compounds plus phenolic

antioxidant mixtures developed by industrial chemists in ignorance of the surprisingly similar natural biological antioxidant systems which have only more recently been discovered by biochemists. After storage for two years in a light-free drawer an antioxidant containing test piece retained physical and mechanical properties, but an unprotected test piece had, dramatically, totally disintegrated into dust. Similar damage to HSPG polymer systems can easily be observed in vitro (which is perhaps why, in vivo, these key molecules provide the main blood vessel wall binding sites for extracellular SOD and other antioxidant enzymes). HSPGs Need ANTI-NITRANT PROTECTIONS Nitrous acid is a highly specific and efficient heparin/heparan sulfate nitrosylating-depolymerization agent. Originally the antioxidant concept of avoiding tissue damage in ageing and disease was thought to be centered exclusively on the importance of pro-oxidant (e.g. hydroxyl radical) damage, but the unusually corrosive nature of nitric oxide metabolites needs an extension of this notion to include anti-nitrant protection. Nitric oxide (and therefore its metabolites including nitrous acid) can be critically increased by being induced by cellular (including auto) immune responses elicited against pathological stimulation. Whilst aimed at killing pathogenic organisms such activity may seriously diminish host HSPG if the rate of replacement of HSPG is less than the rate of its destruction, as may easily be the case under vitamin and lipid dietary insufficiency conditions. Despite the fact that nitrous acid in vitro is well known to rapidly cleave heparan sulfate polymers into two-sugar unit fragments (c) and this historically well known reaction continues to be find routine employment in biochemical laboratories for sequencing of HSPG/GAG chain sugar microstructures and also provides a faithful test for the presence of heparan sulfate polymers in GAG mixtures, no chemist in his wildest dreams in olden times would ever have predicted that such vigorous nitrous acid deaminative cleavage could be of major in vivo relevance. How wrong we were. Originally it was thought that nitrogen oxides were only implicated in health as pollutants from car exhausts and the like, but it has now become firmly established that nitric oxide is used very extensively throughout mammalian physiology. Physiological Use of pH for Stabilization of Heparan Sulfate. Amongst the important reasons for the requirement of

maintenance of strict pH control in human physiology is evidently the prevention of nitric oxide metabolite damage to HSPGs; naked (redox active) metal ions (not easily formed in healthy subjects but may occur in chronic sepsis or under adherent macrophages) may override this. Nevertheless the pH 7.4 hydrogen ion buffer stasis system is seen to be an efficient antinitrant defense strategy. Activated Iron and Copper Ion Catalysis of Deaminative cleavage of HSPGs. Vilar et al and Ghael et al, reported in 1997 that deaminative depolymerization of heparan sulfate could occur at physiological pH when the pH buffer used was phosphate based. Such a buffer is often (perhaps even always) contaminated by trace redox metals. The deaminative reaction does not occur in an imidazole buffer. Special nakedness induced in metal ion phosphate adducts then seems a likely culprit for causing serious tissue damage but further research is needed to clarify this. Iron and copper ions were found by in vitro infrared studies (not yet published) to remove sulfonate from glucosamine N-sulphonate. This is thought to be the first stage of deaminative cleavage. It remains to be established if other known redox active ions (especially nickel, manganese, vanadium and titanium) can behave similarly. Challis and colleagues in the1970s had found that iron and copper salts were potent catalysts of nitrosytation of primary amines in studies of carcinogenic nitrosamine formed by reaction of nitrogen oxides with dietary amines; these results provide published confirmation of the likely importance of redox metals in deaminative damage to HSPG systems. Such metal ion and nitric oxide biochemical effects could also be the basis of the pro-cancer threats posed by copper-rich betel nuts, and even explain the carcinogenic effects of asbestos fibers (which evidently involves nitric oxide and iron-activated-bysilicate-surface effects) as well as the effects of silicate plant hairs present in the contaminated bread which had apparently been associated with a previous incidence of increased of oesophageal cancer in Iran. Redox Status, Nitric Oxide Metabolites & HSPGs. Nitrous acid can be directly reductively countered in tissue by

ascorbate and reduced glutathione. Ascorbate will be likely an important factor for control of redox potential but this substance has a kinetically favorable special efficiency for reduction of nitrous acid to nitric oxide (of especial importance to HSPG integrity). Alpha tocopherol (vitamin E), glutathione peroxidase, catalase, vitamins A and K, beta carotene and urate are probably further cofactors for such protection. The effect of naked iron ions and other redox metal ions, may, however, more specifically negate the effects of ascorbate protection and even lead to induction of oxidant damage via ascorbate redox cycling aided by complex formation between ascorbate and redox metal ions. It had long been suspected that nitrite in meat, especially in hams containing nitrite-iron adducts formed during the curing process, was a causative factor for the promotion of stomach cancer, against which a protective role of dietary ascorbate had been evident (ascorbate is abundantly secreted in saliva if dietary intake is sufficient) by protecting against the damaging effects of nitrous acid against HSPGs. Cigarette smoking is also strangely associated with a pro-cancer nitrous acid dependent nitrosamine formation catalyzed by salival thiocyanate secretion (smokers show serum thiocyanate levels directly proportional to the number of cigarettes smoked) and thiocyanate iron adducts may particpate in such nitrosylation reactions (isothiocyanates which show antitumor activity may also participate in tumor redox enzyme iron centre complexing in this case leading to an inhibition of tumor growth). Experiments are required to test if thiocyanate catalyses nitrous acid cleavage of HSPGs in the presence of redox metal ions. A greater incidence in men than in pre-menopausal but not postmenopausal women of upper stomach cancer in which nitrous acid and HSPG biochemistry is implicated is indicative of a pro-cancer role of iron overload.
Relevance of Nitric Oxide Metabolite Damage to HSPG in the Etiology of Degenerative Diseases. Much research effort has been devoted to establishing the totality of the biological roles of nitric oxide which, on its own is an efficient antioxidant and a tissue

protective agent. The current interest in nitric oxide stemmed from the realization that nitric oxide is responsible for blood vessel dilation (a previously identified endothelial-derived relaxing factor turned out to be this simple two atom molecule; such an

unexpected finding was initially greeted with disbelief); nitric oxide has subsequently been found to have a host of other physiological applications (including its use as a second messenger in brain biochemistry). It is believed to be held under homeostasis by binding to haem iron. There are inducible and constitutive forms of nitric oxide synthsase (NOS), L-arginine being the starting point for its biosynthesis. Nitric oxide, calcium and soluble guanylate cyclase biochemistries are interlinked. Nitric oxide was dubbed the molecule of the year by the journal Science in 1992.
Relatively few workers have yet, however, fully considered the implications of potentially very damaging HSPG depolyrnerization processes caused by nitric oxide metabolites inevitably associated with the wide physiological utilization in an oxygen environment of the various nitric oxide mechanisms1. It is likely that in the course of time the significance of the importance of this reaction in the etiology of degenerative diseases will be more generally recognized. It is currently acknowledged however

(e.g. by Salvemini et al 1998) that nitric oxide biochemistry is strongly implicated in the etiology of stroke, multiple sclerosis, Alzheimers disease, amyotrophic lateral sclerosis, Huntingtons and Parkinsons diseases; it is, however, generally believed that redox metal ion dysfunction is involved in the etiology of at least some of these diseases.

The presence of nitric oxide metabolites have been found to be effective markers for the occurrence of arthritis in patients in which nitrate in urine greatly exceed ingested nitrate. by amounts which correlate with the severity of the arthritic diseases (reviewed in 1998 by Stichentoch and Frolich who advocated the therapeutic use of nitric oxide synthase inhibitors; initial trials of these agents produced encouraging results suggesting that further investigations into their effects in other degenerative diseases might be warranted). Successful intervention in osteoarthritis is apparently achieved by glucosamine therapy which may owe its effectiveness to an increased produced in the rate of heparan sulfate biosynthesis as postulated by McCarty in 1997. Dietary glucosamine supplementation coupled with iron chelation therapy as a counter-measure to degenerative diseases, may also warrant investigation.
Another Damaging Nitric Oxide Metabolite is Peroxynitrous Acid.

Tissue damage arising from peroxnitrous acid formed from superoxide anions and nitric oxide is prevented by the elimination of superoxide radical anions by superoxide dismutase. Clusters of tyrosine and cysteine in a number of at-risk proteins previously discussed by the present author in 1992, may perform a similar anti-peroxynitrous acid function since the formation of tyrosine nitrate is an accepted indicator of the presence of peroxynitrite in

tissue. It used to be thought that hydroxyl radicals induced by trace rogue iron ions from superoxide radical anions were the principal perpetrators of ill-health. Peroxynitrite formed from the addition of superoxide to nitric oxide is now believed (following the work of Beckman et al, 1990) to be responsible for much of the DNA and lipid damage formerly attributable to hydroxyl radicals. The possibility of such peroxynitrous acid damage to DNA has received greater literature attention for consideration of possible mechanisms of cancer induction than have the effects of nitrous acid
damage to HSPG which could, however, have greater potential therapeutic relevance. Effect of Lead Ions on HSPG Biosynthesis.

Toxic, or less physiologically utilized, metal ions such as lead and cadmium have also been found to alter heparan sulfate proteoglycan biosynthesis. Lead intoxication from air and water sources by negatively affecting heparan sulfate biochemistry may even account for hypertension (Fujiwara et al 1999). The effect of perturbation of lead, cadmium and perhaps mercury may be contributory factors acting together with dietary insufficiency of ascorbate in causing HSPG system dysfunction.
Toxic Metals Released from Chitosan by Nitrous add Cleavage.

Rapid deaminative nitrous acid cleavage can occur with other glucosamine based polysaccharide systems including chitosan (obtained from deacetylation of exoskeletal chitin) used as a food additive, from which deaminative cleavage will promote the release of bound trace metals including cadmium and mercury originating from marine pollution sources. Although such toxic metals are less dangerous to health when bound to the highly polymeric chitosan, there is a worry about employment of such materials as food additives under conditions where nitrite and nitrous acid might be formed (e.g. in the stomach). Cadmium released from chitosan can diminish glomerular HSPG sulfation and associated anionic density related filtration efficiency.
Disadvanges of Non-Vegan Diets by Impairment of HSPG Protective Mechanisms.

Non-vegan diets may on occasion be associated with various kinds of pathological organisms (and residual chemicals from veterinary interventions) and even prions, which are, of course, often potentially dangerous to human health because of the relatedness of our species to the animal species being used as food. Immune responses elicited against pathological organisms potentially dramatically increase nitrous acid formation with resultant heparan sulfate depolymerization and thereby accelerate tissue damage and ageing.

An especially pertinent danger of pro-oxidant stress arises from chronic iron overload associated with excess consumption of red meat. This is believed to permit de-N-sulfonation of glucosamineN-S03- groups priming the heparan polymer chain for subsequent deaminative cleavage. The vegan diet, of course, goes beyond the vegetarian diet by avoiding the additional hazard posed by inappropriate fats and growth factors present in milk and dairy products of possible involvement in breast and prostate cancer promotion (as discussed by Professor Jane Plant in her recent book).

The effects of cholesterol LDL/HDL (high density lipoprotein/low density lipoprotein) on health are now found to be explicable by the effects of such factors on heparan sulfate biochemsitry e.g. the anti-atherosclerotic effects of HDL- (containing apolipoprotein E) probably stems from up-regulation of AT(lll)-binding heparan sulphate biosynthesis at arterial walls (Paka et al 1999) and saturated lipids present in meat, if consumed to excess, tend to inappropriately diminish biosynthesis of heparan sulfate relative to other GAGs. A further negative lipid-related factor has been identified to be an effect of oxidized lipids which can similarly potentially negate antitissue protection via heparan sulfate mechanisms. Non-vegan Western diet by deviating markedly (including from the required balance of omega three lipids) from that in which our ancestors polysaccharide biochemistry evolved to deal with, potentially impairs heparan sulfate biochemistry for optimum tissue protective efficiency. Pauling: Vitamin c and cancer.

Linus Pauling, a brilliant scientist, incredibly gained two Nobel prizes. Future generations may, however, come to more greatly praise his dietary supplementation discoveries than his chemical or his political accomplishments. His studies on cancer prevention seem to have been poorly accepted in his lifetime and continue to be disbelieved since a plausible biochemical mechanism for them was lacking at that time (a suitably believable mechanism could involve HSPG and nitric oxide biochemistry). Paulings epidemiological evidence of benefit achieved by ascorbate

supplementation against the common cold and cancer appears to be sound. A conference arranged in 1991 in response to the controversy surrounding Paulings claims somewhat restored public credibility in the main thust of his contentions. The cancer studies had been

conducted in Scotland using previous clinical records in place of the paralleled double blind placebo experimental protocol which would perhaps have been more convincing. All patients receiving the vitamin C supplements apparently benefited. Attempts to repeat these studies in the USA were unsuccessful but Pauling believed he had an explanation for this - in the repeat experiments, the US patients died following a too-sudden withdrawal of their ascorbate supplementation as discussed by Pauling in his letters to Chemistry in Britain in 1985. A previous explanation for the anti-cancer benefit of ascorbate had centred on its known effect in hydoxylation of collagen which tends to inhibit cancer cells metastasizing. The additional likely effect on heparan sulphate microstructure and total environment is now seen to be a more significantly wide benefit-providing mechanism. Paulings results suggest that a vegan diet will tend to counter the common cold and inhibit cancer.
Ascorbate-induced increase in heparan sulphate activity and protection

heparan activity by

of

neutralizing the anti-heparan sulfate activity of nitric oxide metabolites may well be the main reason for Paulings findings.

Increased heparan sulfate sulfation and total biosynthetic formation in fibroblast cell culture experiments resulted from ascorbate additions to the culture medium (Edward & Oliver 1983, Kao et al 1992). The significance of this finding is greater than it might at first seem as it is likely to occur with various other cell types as it has been well known for some time that ascorbate was involved in GAG biosynthesis. Further research into this important research finding and its relevance to human health is warranted. Pro-cancer effects of ascorbate have, however, also been reported. Ascorbate can be shown to increase free-radical production in vitro in presence of redox metals like iron. Iron toxicity effects might be capable of reducing or reversing the tissue protective effects of ascorbate. Such iron toxicity has been associated with cardiovascular dysfunction but there may be a dietary link with overtly non-vegan diets.
Salonen et al : Iron Overload and Cardiovascular Disease.

A major beakthrough linking iron overload with heart attack, the major killer in Western society was communicated in 1992 by Salonen and his team from Finland. The origin of this iron toxicity effect is now postulated to include HSPG damage at blood vessel walls caused by the effect of naked iron ions which can prime

heparan sulfate N-sulfonate groups for subsequent deaminative cleavage as well as promote peroxynitrite and hydroxy radical generation via Fenton reactions catalyzed by such iron ions. Iron overload, then, which can arise from a red-meat rich diet can, together with the effects of nitric oxide metabolites (which can arise from dietary arginine via nitric oxide synthase (induced by the immune response to pathogens or normally cellularly produced) as well as from nitrite in meat or from that produced by molybdenumcontaining enzymes of mouth bacteria, is capable of depleting these critical substances by snipping off key N-sulfonate sites or even totally unzipping the polymers which are involved in blood vessel wall sensing and antioxidative protection by providing binding sites for superoxide dismutase and the seleniumcontaining antioxidant molecules. Such effects of iron overload are probably countered by a vegan diet.
The Suggested Biochemical Basis of the Benefit of a Vegan Diet.

The results of chemical and biochemical research summarized here may provide part of the key to understanding why wideranging health benfits ensue from certain ascorbate-rich and lipidfriendly vegetarian and vegan diets (especially if free of natural and man-made toxins). Ascorbate directly reduces nitric oxide metabolites back to tissueprotective nitric oxide, but perhaps of equal or greater importance is that this vitamin is capable of augmenting appropriate heparan sulfate biosynthesis and may be a cofactor in promoting its tissue protective role.
A similar effect has been found with certain vegetable fats) (polyunsaturates especially). A boost of heparan sulfate via ascorbate or lipids is the likely outcome of a vegan diet which has an important additional factor of limiting the effects of haemoglobin iron.

It is now dramatically apparent that many hitherto poorlyunderstood degenerative diseases in which the extracellular matrix is degraded such as bone disease, apoptosis, tissue damage from chronic inflammation involving release of growth factors and cytokines could be potentially linked to inappropriate redox-catalyzed degradation of heparan sulfate by nitric oxide metabolites; these situations are also highly relevant to the etiology of cancer as pointed out by Vilar et al in 1997.
Future 1-leparan Sulfate linked Therapy?

HSPG replacement therapy (e.g. by sulfated polysaccharides derived from artificially sulfated xylans from birch trees or seaweed or pharmaceutical compounds like suramin and analogues) may, in principle, be possible. The current use of dietary glucosamine supplements, popular in alternative medicine, may be to do with boosting HSPG protection. Heparin/heparan sulfate therapy seems to show benefit to Alzheimers and other degenerative disease patients. But a more satisfactory procedure would clearly be to seek non-invasive protection and correction of aberrant heparan sulfate systems through a precise monitoring of dietary control of heparan sulfate status and cofactor disfunction via urinary or tissue sampling and microstructure analysis. Future medical procedures might even allow routine monitoring and correction by such methods. This would need to include a correction of iron (and perhaps other redox metals such as manganese which is required for heparan sulfate biosynthesis enzymes).
Acknowlegements

I am indebted to help received from former colleagues at the University of Aberdeen (especially to Frank Williamson and Bill Long) and I am grateful to Prof KEL McColl of the Dept of Medicine and Therapeutics at the University of Glasgow for discussions of his work.

Selected References & Further Reading

Adachi T Marklund SL (1989) Interaction between human extracellular superoxide dismutase C and sulphated polysaccharides J Biol Chem 264 (15) 8537

Adachi T Yamada H Futenma A Kato K Hirano K (1995) Heparin-induced release of extracellular dismutase form V to plasma J Biochem (Tokyo) 117 (3) 586-90; cf also Adachi et al (1998) Biol Pharm Bull 21 (10) 1090 CA 130 64588h which reported decreased extracellular-superoxide dismutase in patients with coronary artery atherosclerosis
This list of references is truncated.

Appended collected documents with the following titles (obtained from an historical academic research database)
It is thought to be in the public interest to post on the internet the following collection of documents relating to how heparan sulphate can impact on health and potentially be beneficially modulated by a vegan diet.

Combined Contents
1 A vegan diet is a scientifically rational pro-health strategy

1a The study of Si in multielement matrices (e.g. seaweed etc.) 2 Recent heparan sulphate research results may help explain why a vegan diet is good for you Are there easily identified key health master molecules? The occurrence of pro-oxidant situations during chronic inflammation may promote atherosclerosis and

3 4

myalgic encephalomyelitis-chronic fatigue syndrome (ME-CFS) 5 Degradation of heparan sulphate by nitrous acid may promote ME-CFS 6 7 REFERENCES Relevance of heparan sulphate signalling to pathologies Selected shorter list of references

8 9

The major causative factor of CFS may include heparan sulphate biochemical activities which are both nitric oxide and trace metal dependent 10 The possible key role of heparan sulphate deficiency in ME-CFS and autism The non-biological hypothesis of the origin of Me-CFS Anti-viral anionic polysaccharide research may offer hope for an effective ME-CFS therapy
Low level radiation effects on ME-CFS?

11 12

13

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A Vegan Diet is a Scientifically Rational ProHealth Strategy

by David Grant, New Deer, Turriff AB53, May 2013

(These notes are about biochemistry [and which are not intended to be taken as medical advice] were written from the perspective of the current public interest in diet).

A new paradigm: the vegan diet tends to promote heparan sulphate optimization.
Optimization of heparan sulphate (sulfate) [HS] * structure (e.g. by prevention of inappropriate HS de-N sulphonation via boosting the HS Si crosslink content) is a possible mechanism by which a vegan diet achieves major pro-health benefits.
[Nb HS-based Si-containing redox-sensitive sulphated polysaccharide extracellular matrix information sorting systems are subject to exogenous regulation (including by ascorbate and fatty acids and multiple, putatively a metallomic (mineral ion) array) so that a rational regulation of diet may improve the effectiveness of the wound healing and tissue protection mechanisms and perhaps also the cognitive processes which are afforded by HS metallome crosstalk].

The idea that a vegan diet improves health by processes which require HS integrity arose informally from discussions of lab. work done at a UK University. [This idea has, however, not been properly reported in public]. These studies of HS suggested that when

undamaged, HS is pro-health but when damaged, HS a driver of disease processes. The crux of the benefit of a vegan diet seems to be that it could accentuate the former and minimize the latter.
It seems that any dietary factor which can boost correctly-structured HS will tend to be pro-health.

Ascorbate and omega 3 fatty acids, retinoic acid and magnesium (also correct other minerals) in vegan diets boost such good-HS. A similar effect may also be achieved by inorganic Si present in unrefined plant food. Dietary supplementation with glucosamine can potentially also augment HS. (This may be why this supplement provides a wide-ranging pro-heath benefit cf. Bell et al. (2012) PMID 22828954. On the other hand (red) meat eating can create too much active iron (in iron overload) seriously damages HS via de-N-sulphonation (this equates to bad-HS). (This is likely to be why serum ferritin, a marker of iron overload associated with eating red meat was found by JT Salonen et al in 1992 [cf. Circulation 86 803-11; PMID 1516192] to be highly correlated with cardiovascular mortality in Eastern Finnish men). While the currently-published literature tends to confirm the idea that a vegan diet can be very beneficial to heath, it seems that it will be necessary to produce an entirely new write-up linking together the available published information on vegan diets so as directly to focus directly on how the unique health promoting effects of these diets might be mediated by HS.

Although the conventionally published literature seems not yet to have fully made the critical link between veganism, health and HS integrity, it is nevertheless a useful starting off point to review the peer-reviewed literature.

Numerous separate scientific papers (which do not cite each other) discuss the benefits of veganism how in vivo HS biosynthesis may be aided by dietary supplementations. These contributions when considered together have, I believe, come close to revealing how the pro-health effects of HS integrity and the benefits of a good (especially a vegan) are closely interlinked phenomena.
In 1999 (PMID 10687887) MF McCarty proposed that the health benefit of a vegan diet may be achieved by keeping blood sugar in check. [In 1999 it was less understood than is the case today that a too-high blood sugar level can seriously damage HS. If blood sugar gets out of control, HS is now known to get degraded and such degradation is now thought to be is a major cause of tissue damage in diabetes]. Writing in 1997 the same author had also postulated [PMID 9140889] that in vivo HS might be augmented via a dietary by supplementation with glucosamine. (N.b. HS is an alternating polymer of glucosamine and uronic acid {i.e. glucosamine is a biosynthetic precursor of HS}. Such dietary supplementation has become very popular in alternative medicine (and is now also acknowledged by conventional medicine) for use in therapeutic intervention in osteoarthritis, and for apparently providing a range of other major pro-health benefits cf PMID 22828954 and 23139249]). HS, also it seems, might also potentially be capable of being boosted by dietary silicon [PMID 9278931]. A possible mechanism by which such a boost arises is the ability of inorganic silicic acid to crosslink HS polysaccharide chains {a mechanism suggested by Klaus Schwarz in 1973 cf Ralph K Iler} and thereby to diminish the probability of an otherwise too- fast degradation rate of HS which might occur e.g. under pro-oxidant and pro-nitrant situations. HS is also potentially similarly boosted by ascorbate (Vit C) supplementation which

increases the anionic (sulphate half ester and N-sulphonate) density and thereby increases barrier function (this may be why ascorbate-derivative supplementation has been shown, at least under certain conditions, to combat the progression of cancer, cf. e.g. PMID 16075276].

Work at Aberdeen U. had indicated that HS-like polymers may naturally contain very large amounts of Si (confirming a similar prior report by K Schwarz [PMID 4268099]) and it was further suggested that such bound Si might boost the antioxidant (and anti-toxic metal including aluminium) capacity of the HS or provide inorganic crosslinkages which can potentially reduce any inappropriate degradation of HS. Dietary supplements such ascorbate, glucosamine and silicic acid (or silica sols) may therefore combat cardiovascular risk, osteoarthritis and other diseases via an HSsupramolecular structure upgrade mechanism.
A paper entitled Vegan proteins may reduce risk of cancer, obesity, and cardiovascular disease by promoting increased glucagon activity by MF McCarty in Medical Hypotheses 1999 53 (6) 459-85 [PMID 10687887] (for which the full text is available free on the internet at

http://img2.timg.co.il/forum/1_155647027.pdf. is a good starting-off point which can be followed by 78 other papers which cite it) which discusses how a vegan diet might improve the actions of enzyme systems which control the level of blood sugar. This article draws attention to how veganism can increase
glucagon activity (which raises serum glucose levels, an effect which is opposite to that of insulin, these acting as a servo control of blood glucose level) which leads to improved weight control, and how low fat vegan diets may be especially protective as regards breast prostate and colon cancer.

*HS is a hallmark of all adherent cells of multicellular animals where it acts as a high-level quasicomputational system (using sorting of information held in sugar sequences and anionic patterns). It is biosynthesized in animals by a rout that recreates the evolutionary process starting off from a bacterialstyle polysaccharide. Plants separated during evolution early on and use different sugar systems for intracellular signaling and structure than do animals so that glucosamine is not directly available from plant-based diet. But it is in principle available from fungi (mushrooms are essential pro-heath components of a good vegan diet as they provide B vitamins [cf. B12 ex bacteria on surfaces of unwashed mushrooms] and also structural fibre- chitin-chitosan ----glucosamine). Cell culture experiments (in petrie dishes) have shown that a range of nutrients (mostly which are enhanced in vegan diets) can enhance the production of optimally structured HS so as to improve the pro-health microstructure of HS in a manner which also boosts lifespan. Ascorbate boosts HS of higher degree of sulphation (and combats nitrite damage to HS). {It is believed that freshly biosynthesized HS microstructure varies systematically with age so as to, in the absence of disease, directly determines lifespan [cf. E Feyzi et al PMID 9593669]; a corollary is that diminution of HS integrity at the human aorta surface could impair health by speeding up the ageing process}. Healthdepleting alteration in HS can be demonstrated to result from e.g. glucose dyshomeostasis, from adverse cholesterol HDL/LDL, from heavy metal intoxication or from chronic pathological bacterial infection (producing nitrite and free radicals) including from bacterial toxins or from excessive cellular immune system stress.
The idea that HS is the prime dietary-accessible key pro-health control system is clearly indicated by the wealth of published papers on HS and also to some extent confirmed by the available scientific reviews of HS. None of these however fully deal with the entire extent to which HS can influence human and animal physiology. This extreme extent became apparent slowly during an in-depth reevaluation of published and new information collected during a 10+ year programme conducted during tenure of a research fellowship a.

The idea that a vegan diet protects HS from inappropriate degradation arose during HS scientific research conducted in a major UK University (where bench and literature research data seemed to potentially link the optimization of HS-afforded tissue protection to a low active iron or vegan diet but this idea did not achieve the formally scientific status of a published article because of a disruption of the research programme by non-academic- related circumstances). ---------------------------------------------------------------------------------------HS (and heparin a mast cell highly sulphated specialized anticoagulant HS) is especially essential for blood vessel health. HS also provides multi- anti-pathogen activities including antiviral actions. Cancers seem to be HS defect diseases. Cf. e.g. the tumour metastasis process proceeds via HS degradation. Work done in numerous labs internationally confirm that HS provides a multi-pathway activity system of health promotion and wound healing which suggests that the principal cause of death in Western societies is perhaps due to deficiencies in HS protection promoted by toxins and poor diet. HS is probably a prime metallome i.e. it selectively accumulates all of the inorganic elements required for animal nutrition.
a

The preliminary write up of these ideas has been posted on the internet in a series on notes starting from 2000 which further discussed how HS activity can be modified by a range of inorganic minerals and other common dietary factors (cf. e.g. preliminary drafts published as (please excuse spelling errors remaining) Publication 2 Web Scribd }. {Cf. Table II ; Humans have been indicated to have the smallest extracellular inorganic pool of all animals} Feb 17 2010 and ~WRL005 Scribd; (2012) Draft Silica-Scribd; www.scribd.com/doc/104804240/Draft-Silica

Optimum tissue protective HS sulphation and Si content conferring optimum supramolecular hydration and molecular weight structure and optimum pro-health effectiveness is potentially provided by an optimum vegan diet.

A recent hypothesis (Marx and Gilon) has suggested that a multi-trace metal ion array (presumably similar to the heparin metallome array) whch is associated with the extracellualar matrix surrounding neurons is the ultimate basis of cognition and memory. The likely influence of diet in maintaining the proposed multi-inorganic array points to a possible benefit of a vegan diet having multi-elements associated with plant polysaccharides for the promotion of cognitive efficiency and mental health.

1a Summary from AU-research related doc entitled

The study of Si in multielement matrices (e.g. seaweed etc.)
The study of Si in multielement matricesa (such as occur in association with seaweed alginates and human hair) might contribute to an increased understanding of interconnections betwen geology, inorganic chemistry and biochemistry (and be relevant to the elucidation of the early stages of the evolution of biota) Si (after Na and Ca) was the third most abundant of the 37 elements asociated with a (non NIH) referece standard sodium heparin b, which suggests a similar situation could also occur with HS proteoglycans c. It is suggested that such matrices consist of a SiO2sol/gel system stabilised by an association with a range of inorganic oxyanions (suphate, phosphate, borate, arsenate, vanadate, tungstate and perhpas aluminate) onto which is adsorbed a range of inorganaic cations. Evidence was presented for the possible presence of this type of matrix in vivo in amounts ranging up to several percent of dry weight, but most of this seems to be lost during the standard extraction and puification procedures which has led to the existence of perhaps an almost stoichiometric amout of Si in polyurionides in plants and glycosaminoglycans in animal tissues to have been largely overloded by biochemists. Hints fom industrial reserach on the behaviour of related SiO2 compounds suggest that these kinds of bio-SiO2 materials (i.e. polyuronide and glycosaminoglycan water glass composites) could perform hghly evolved functions such as ion sorting, proton gradient formation and the modulation of normal and pathological crystallizaton and serve (e.g. in animal glycocalyxes) as slowrelease agents for growth factors (e.g. for basic fibroblast growth factor) and metal ion catalysts (e.g. those such as Cu and Zn which initiate the nitric oxide metbolite scission fo HS). a Cf. the suggestion by H Haraguchi (2004) for the need for a new branch of science (metallomics) for such studies. b This typical industrial preparation was kindy donated by a major pharmaceuticl company to serve as a secondary referece standard for sodium heparin, as a substitute for the Mathews and Cifonelli NIH reference standard heparin. c Further work is warrented to more fully investigate the Si contents of polysaccharides in vivo especially those form vascular glycocalyxes and urinary deposits. d Si was not removed from the (non-NIH) Na heparin by an extensive dialysis against water; a similar resistnce to the removal toSi from hyaluronan and pectin by autoclaving aqueous solutions or treating with a 8M urea solution had led to the proposal that the Si in these poyuronides occurs in C-O-Si linked structures ( cf K Schwarz 1973). As most of the bound Si could be remoed form the Na heparin by a mld process of cation exchange resin percolation in which Na+ was replaced by Tl+ (a process whch is accompanied by a diminution of the countercation-dependent hydration of the polysacccharide

DOCUMENT 1a

molecules) the Si associated wuith the polyurionides of plants and glycosaminoglycans of animal tissues is now proposed to mostly occur as colloidal SiO2 derivatives. Cf also Grant D (Sept 23,2010) ~WRL005-Scribd [sect 5-5-5 Silicon] and Grant D et al Med Hypoth 1992 38 46-48 (A putative role for colloidal silicates in primitive evolution deduced in part form fheir relevance to modern pathological afflications).
] [Cf also Exley et al have discussed how chronic fatigue syndrome (a putative HS-defect illness, vide infra) might arise from an iatrogenic aluminium intoxication; Al3+ binding to HS causes severe alteration in properties (as indicated by Al3+ -heparin binding studies conducted at Aberdeen U.); the idea that Si, e.g. dissolved silicic acid in blood, might protect against aluminium intoxication was originally suggested by Birchall and Chapell; this idea could be further extended to include possible vascular Si-O-HS protection against toxic effects of aluminium].

The following paper was submitted to a vegan publication in 2001 butit may not have been considered suitable for inclusion because at that time the suggested roles of heparan sulphate made in this article were less well known outside polysaccharide scientific circles than is now the case.

Recent Heparan Sulphate Research Results May Help Explain Why a Vegan Diet is Good for You

David Grant Ashbank, AB53 6SX, U.K. Summary. Dietary factors can affect health by influencing heparan sulphate polysaccharide systems on which important tissue protection activities are centred. A vegan diet, which tends to be close to that under which our species evolved may promote health principally by avoiding iron-overload which is associated with inappropriate heparan sulphate degradation by nitric oxide metabolites and the formation of lipid oxidation products which negatively perturb heparan sulphate biosynthesis. Introduction.

Of the three major classes of polymer systems used by our cells, the nucleic acids and proteins are better understood than are the polysaccharides. But a picture is gradually emerging in which key sulphated glucosamine-containing polysaccharides especially the heparan sulphate proteoglycans (HSPGs) - are found to be linked to many aspects of health and disease. These polymers are members of the glycosaminoglycan (GAG) proteoglycan families which include chondroitin and keratan sulphates as well as hyaluronan. A critical dependence of human health on heparan sulphate biosynthesis supports the notion that evolutionary pressure, at the very early stages of the evolution of multicellular organisms in our ancestral lineage, led to the selection of these polymers to be the basis of an auxiliary information storage system. If we are allowed to make a computer hardware analogy in which the memory chips are the DNAcontaining genes, then the keyboard and processor are HSPGbased systems providing signaling channels, ion stores and water based osmolality buffers between extracellular and intracellular space. The computer analogy is not an exact one since biological systems seem to use precisely microsequenced polymers for both software and hardware functions. (Although there is poorly understood non-genetically inheritance with which GAGs may even be involved, GAGs are highly dependent on gene products for their biosynthesis). Protein primary sequence structures are determined by genetic information, but their correct folding (e.g. as required for growth factor modulation, antioxidant anchoring and haemostasis activities) is often determined by binding to HSPGs which may, however, be susceptible to inappropriate oxidative damage under some pathological and adverse dietary situations. Some HSPG Biochemical Systems. Haemostasis was the first tissue-protective HSPG role to be discovered. The inhibition of blood coagulation by heparin, the most sulphated of the heparan sulphate-like polysaccharides (discovered ca. 1916 by Jay McLean & William H Howell) involves conformational alteration of antithrombin(lll) (AT(lll)) by a highly specific heparin pentasaccharide sequence (as intensively researched by Robert D Rosenberg, Ulf Lindahl, Jean Choay and others). CP Dietrich and coworkers predicted a cellular recognition role for HSPGs which seems to be inherent in the complexity of their shape and anionic charge array patterns enhanced by the highly flexible iduoronic acid sugar ring (formed by epimerization of the more primitive glucuronic acid CS present in ancestral

bacterial polysaccharides) and they recently demonstrated that administration of anticoagulant heparin (including the now popular low molecular weight forms) upregulated AT(lll)-binding sequences in blood vessel cell wall HSPGs and increased their antithrombotic properties. Nitric oxide under some circumstances does likewise, in keeping with recent indications of biofeedback between nitric oxide and heparan sulphate tissue protective systems. HSPG-controlled systems are also employed by organisms for the regulation of cell-cell interactions including cells of the immune system and in regulation of growth factor signalling, most researched for basic fibroblast growth factor (b-FGF). EXT1 and EXT2 anti-tumour factors were recently found to probably be heparan sulphate biosynthesis enzymes (this came as a surprise to many biochemists). HSPGs are centrally involved in lipid processing and can limit transfer of DNA cationic lipid complexes into and within cells. Although the involvement of heparin/HSPG with lipid biochemistry was discovered many decades ago from the routine pharmaceutical use of heparin for inhibition of blood coagulation, a mechanism is currently emerging that suggests that the induction of atherosclerosis may be countered by good lipids typical of vegetable-rich diets (and some fish oils) by promotion of the biosynthesis of beneficially microstructured HSPG for the inhibition of pathological calcification. HSPG involvement in tissue regeneration in wound healing is part of their global cellular control function which is also highly relevant to embryogenesis (recently reviewed by N Perrimon & M Bernfield) especially for nervous system development. So it comes less of a surprise to learn that dysfunction of HSPG biochemistry may be implicated in neural degenerative diseases such as BSE and CJD. Heparin/heparan sulphate and analogous substances seem to directly inhibit prion diseases as well as stopping injurious crystal (stone) formation in blood and urinary vessels (including inhibition of calcium oxalate crystallization, a possible metabolite problem potentially associated with long-term high dose Vitamin C (ascorbate) dietary supplementation). Many viruses (including type-O bovine foot and mouth virus) bind to cell wall HSPGs which, together with analogous substances, are broad spectrum antivirals (inhibiting enveloped viruses such as HIV-1, HIV-2, cytomegalovirus, herpes simplex virus (including mutants resistant to acyclovir) togaviruses and rhabdoviruses). HSPG biochemistry involvement together with bacterial adhesion has been proposed for Helicobacter pylon induction of stomach

ulceration and cancer. HSPGs are also apparently involved in the biochemistry of endotoxins, polyamines, homocysteine, prostaglandins and nonsteroidal antinflammatory agents and HSPG biosynthesis is adversely affected by toxic heavy metals, excess serum glucose and oxidative stress including that associated with iron overload. Heparin anticoagulant therapy apparently demonstrates anticancer side effects (reviewed by H Engelberg in 1999) often associated with restriction of tumour blood vessel growth (angiogenesis). HSPGs and Ageing. Binding to, and supramolecular structure induction of collagens by GAGs, especially by HSPGs, provides very important structural integrity and water-holding properties. A natural depletion, linear with age, in the content of HSPGs (combined with the effect of an apparent natural increase in serum ferritin, the iron storage protein, with age) may even be a major biochemical clock responsible for the ageing process. (Research into atherosclerosis showed a linear depeletion of HSPGs with increasing age to occur in total GAGs at human artery surfaces; perhaps this indicates the ultimate secret of beauty and what makes skin look young, possibly allowing future dramatic improvement in the cosmetics trade when such HSPG influenced mechanisms of ageing are more fully understood). An inverse correlation with age, reported by T Aoyagi et al in 1994, shows up in the blood coagulation and fibrinolysis activities of heparin/HSPG-dependent urokinase, plasmin and thrombin. Similar effects observed in Alzheimers disease patients suggested that a derangement of such HSPG-linked processes was accelerated in such degenerative diseases possibly by the effects of trace redox metal ion overload and pathogen perturbations (e.g. situations leading to chronic inflammation) capable of tipping the balances involving HSPG signalling mechanisms. Our general health, as well as the way we look, can be seen then to depend on the structural integrity of the GAG-rich rubbery polymers which hold our cells together in tissue, cartilage and bone structures, and provide binding sites for protective enzymes and growth factors employed in wound healing and haematopoiesis.

HSPGs in Antioxidant Defense. The above reasoning is the basis of antioxidant theories of ageing and of ill-health. Degradative damage to the extracellular matrices which, like corrosion of metals or similar degradation of plastics, drastically impairs their performance. An HSPG-based antioxidant theory of health can be made on this basis. Nutrient elements used by antioxidant enzyme defense systems include selenium required for glutathione peroxidase, manganese and iron for intracellular-, and copper and zinc for extracellular-superoxide dismutases (SODs). A newly discovered selenium-containing antioxidant is probably another endothelial cell surface, HSPG-bound enzyme. Since heparan sulphate is a linearly codified information polymer (in this way analogous to DNA or RNA) even slight damage at key sites (especially N-sulphonates) may significantly diminish their physiological activities and impair health. The rate at which such defective HSPG is removed and replaced by de novo HSPG biosynthesis obviously require to equate in order to maintain HSPG tissue protective potency. Such processes can, however, be affected by dietary factors. Bench Observations Modeling Oxidant & Nitrant Tissue Damage. Antioxidant protection can be illustrated by a comparison of the effect of antioxidants on industrial plastic test mouldings containing trace metal (especially iron-binding) sulphur compounds plus phenolic antioxidant mixtures developed by industrial chemists in ignorance of the surprisingly similar natural biological antioxidant systems which have only more recently been discovered by biochemists. (Cf Grant et al 1989a). After storage for two years in a light-free drawer an antioxidant containing test piece retained physical and mechanical properties, but an unprotected test piece had, dramatically, totally disintegrated into dust. Similar damage to HSPG polymer systems can easily be observed in vitro (which is perhaps why, in vivo, these key molecules provide the main blood vessel wall binding sites for extracellular SOD and other antioxidant enzymes). HSPGs Need ANTI-NITRANT PROTECTIONS Nitrous acid is a highly specific and efficient heparin/heparan sulphate nitrosylating-depolymerization agent. Originally the antioxidant concept of avoiding tissue damage in ageing and disease was thought to be centred exclusively on the importance of pro-oxidant (e.g. hydroxyl radical) damage, but

the unusually corrosive nature of nitric oxide metabolites needs an extension of this notion to include anti-nitrant protection. Nitric oxide (and therefore its metabolites including nitrous acid) can be critically increased by being induced by cellular (including auto) immune responses elicited against pathological stimuli. Whilst aimed at killing pathogenic organisms such activity may seriously diminish host HSPG if the rate of replacement of HSPG is less than the rate of its destruction, as may easily be the case under vitamin and lipid dietary insufficiency conditions. Despite the fact that nitrous acid in vitro is well known to rapidly cleave heparan sulphate polymers into two-sugar unit fragments and this historically well known reaction continues to be find routine employment in biochemical laboratories for sequencing of HSPG/GAG chain sugar microstructures and also provides a faithful test for the presence of heparan sulphate polymers in GAG mixtures, no chemist in his wildest dreams in olden times would ever have predicted that such vigorous nitrous acid deaminative cleavage could be of major in vivo relevance. How wrong we were. Originally it was thought that nitrogen oxides were only implicated in health as pollutants from car exhausts and the like, but it has now become firmly established that nitric oxide is used very extensively throughout mammalian physiology. Physiological Use of pH for Stabilization of Heparan Sulphate. Amongst the important reasons for the requirement of maintenance of strict pH control in human physiology is evidently the prevention of nitric oxide metabolite damage to HSPGs. Naked (redox active) metal ions (not easily formed in healthy subjects but may occur in chronic sepsis or under adherent macrophages) may override this. Nevertheless the pH 7.4 hydrogen ion buffer stasis system is seen to be an efficient antinitrant defense strategy. Activated Iron and Copper Ion Catalysis of Deaminative cleavage of HSPGs. RE Vilar et al and D Ghael et al, reported in 1997 that deaminative depolymerization of heparan sulphate could occur at physiological pH when the pH buffer used was phosphate based. Such a buffer is often (perhaps even always) contaminated by trace redox metals. The deaminative reaction does not occur in an imidazole buffer. Special nakedness induced in metal ion phosphate adducts then seems a likely culprit for causing serious tissue damage but further

research is needed to clarify this. Iron and copper ions were found by in vitro infrared studies (not yet published) to remove sulphonate from glucosamine N-sulphonate. This is thought to be the first stage of deaminative cleavage. It remains to be established if other known redox active ions (especially nickel, manganese, vanadium and titanium) can behave similarly. BC Challis and colleagues in the1970s had found that iron and copper salts were potent catalysts of nitrosylation of primary amines in studies of carcinogenic nitrosamine formed by reaction of nitrogen oxides with dietary amines; these results provide published confirmation of the likely importance of redox metals in deaminative damage to HSPG systems. Such metal ion and nitric oxide biochemical effects could also be the basis of the pro-cancer threats posed by copper-rich betel nuts, and even explain the carcinogenic effects of asbestos fibres (which evidently involves nitric oxide and iron-activated-by-silicatesurface effects) as well as the effects of silicate plant hairs present in the contaminated bread which had apparently been associated with a previous incidence of increased of oesophageal cancer in Iran. Redox Status, Nitric Oxide Metabolites & HSPGs. Nitrous acid can be directly reductively countered in tissue by ascorbate and reduced glutathione. Ascorbate will be likely an important factor for control of redox potential but this substance has a kinetically favourable special efficiency for reduction of nitrous acid to nitric oxide (of especial importance to HSPG integrity). Alpha tocopherol (vitamin E), glutathione peroxidase, catalase, vitamins A and K, beta carotene and urate are probably further cofactors for such protection. The effect of naked iron ions and other redox metal ions, may, however, more specifically negate the effects of ascorbate protection and even lead to induction of oxidant damage via ascorbate redox cycling aided by complex formation between ascorbate and redox metal ions. It had long been suspected that nitrite in meat, especially in hams containing nitrite-iron adducts formed during the curing process, was a causative factor for the promotion of stomach cancer, against which a protective role of dietary ascorbate had been

evident (ascorbate is abundantly secreted in saliva if dietary intake is sufficient) by protecting against the damaging effects of nitrous acid against HSPGs. Cigarette smoking is also strangely associated with a pro-cancer nitrous acid dependent nitrosamine formation catalysed by salival thiocyanate secretion (smokers show serum thiocyanate levels directly proportional to the number of cigarettes smoked) and thiocyanate iron adducts may particpate in such nitrosylation reactions (isothiocyanates which show antitumour activity may also participate in tumour redox enzyme iron centre complexing in this case leading to an inhibition of tumour growth). Experiments are required to test if thiocyanate catalyses nitrous acid cleavage of HSPGs in the presence of redox metal ions. A greater incidence in men than in pre-menopausal but not postmenopausal women of upper stomach cancer in which nitrous acid and HSPG biochemistry is implicated is indicative of a pro-cancer role of iron overload.
Relevance of Nitric Oxide Metabolite Damage to HSPG in the Aetiology of Degenerative Diseases. Much research effort has been devoted to establishing the totality of the biological roles of nitric oxide which, on its own is an efficient antioxidant and a tissue

protective agent. The current interest in nitric oxide stemmed from the realization that nitric oxide is responsible for blood vessel dilation (a previously identified endothelial-derived relaxing factor turned out to be this simple two atom molecule; such an unexpected finding was initially greeted with disbelief); nitric oxide has subsequently been found to have a host of other physiological applications (including its use as a second messenger in brain biochemistry). It is believed to be held under homostasis by binding to haem iron. There are inducible and constitutive forms of nitric oxide synthase (NOS), L-arginine being the starting point for its biosynthesis. Nitric oxide, calcium and soluble guanylate cyclase biochemistries are interlinked. Nitric oxide was dubbed the molecule of the year by the journal Science in 1992.

Relatively few workers have yet, however, fully considered the implications of potentially very damaging HSPG depolyrnerization processes caused by nitric oxide metabolites inevitably associated with the wide physiological utilization in an oxygen environment of the various nitric oxide mechanisms1. It is likely that in the course of time the significance of the importance of this reaction in the aetiology of degenerative diseases will be more generally recognized. It is currently acknowledged however (e.g. by D Salvemini et al 1998) that nitric oxide

biochemistry is strongly implicated in the aetiology of stroke, multiple sclerosis, Alzheimers disease, amylotrophic lateral sclerosis, Huntingtons and Parkinsons diseases; it is, however, generally believed that redox metal ion dysfunction is involved in the aetiology of at least some of these diseases.

The presence of nitric oxide metabolites have been found to be effective markers for the occurrence of arthritis in patients in which nitrate in urine greatly exceed ingested nitrate. by amounts which correlate with the severity of the arthritic diseases (reviewed in 1998 by DO Stichentoch and JC Frlich who advocated the therapeutic use of nitric oxide synthase inhibitors; initial trials of these agents produced encouraging results suggesting that further investigations into their effects in other degenerative diseases might be warranted). Successful intervention in osteoarthritis is apparently achieved by glucosamine therapy which may owe its effectiveness to an increased produced in the rate of heparan sulphate biosynthesis as postulated by MF McCarty in 1997. Dietary glucosamine supplementation coupled with iron chelation therapy as a counter-measure to degenerative diseases, may also warrant investigation.
Another Damaging Nitric Oxide Metabolite is Peroxynitrous Acid.

Tissue damage arising from peroxnitrous acid formed from superoxide anions and nitric oxide is prevented by the elimination of superoxide radical anions by superoxide dismutase. Clusters of tyrosine and cysteine in a number of at-risk proteins previously discussed by the present author in 1989, may perform a similar anti-peroxynitrous acid function since the formation of tyrosine nitrate is an accepted indicator of the presence of peroxynitrite in tissue. It used to be thought that hydroxyl radicals induced by trace rogue iron ions from superoxide radical anions were the principal perpetrators of ill-health. Peroxynitrite formed from the addition of superoxide to nitric oxide is now believed (following the work of JS Beckman et al, 1990) to be responsible for much of the DNA and lipid damage formerly attributable to hydroxyl radicals. The possibility of such peroxynitrous acid damage to DNA has received greater literature attention for consideration of possible mechanisms of cancer induction than have the effects of nitrous acid
damage to HSPG which could, however, have greater potential therapeutic relevance. Effect of Lead Ions on HSPG Biosynthesis.

Toxic, or less physiologically utilized, metal ions such as lead and cadmium have also been found to alter heparan sulphate proteoglycan biosynthesis. Lead intoxication from air and water sources by negatively affecting heparan sulphate biochemistry may even account for hypertension (K Fujiwara and T Kaji 1999). The effect of perturbation of lead, cadmium and perhaps mercury may be contributory factors acting together with dietary insufficiency of ascorbate in causing HSPG system dysfunction.

Toxic Metals Released from Chitosan by Nitrous add Cleavage.

Rapid deaminative nitrous acid cleavage can occur with other glucosamine based polysaccharide systems including chitosan (obtained from deacetylation of exoskeletal chitin) used as a food additive, from which deaminative cleavage will promote the release of bound trace metals including cadmium and mercury originating from marine pollution sources. Although such toxic metals are less dangerous to health when bound to the highly polymeric chitosan, there is a worry about employment of such materials as food additives under conditions where nitrite and nitrous acid might be formed (e.g. in the stomach). Cadmium released from chitosan can diminish glomerular HSPG sulphation and associated anionic density related filtration efficiency.
Disadvanges of Non-Vegan Diets by Impairment of HSPG Protective Mechanisms.

Non-vegan diets may on occasion be associated with various kinds of pathological organisms (and residual chemicals from veterinary interventions) and even prions, which are, of course, often potentially dangerous to human health because of the relatedness of our species to the animal species being used as food. Immune responses elicited against pathological organisms potentially dramatically increase nitrous acid formation with resultant heparan sulphate depolymerization and thereby accelerate tissue damage and ageing. An especially pertinent danger of pro-oxidant stress arises from chronic iron overload associated with excess consumption of red meat. This is believed to permit de-N-sulphonation of glucosamineN-SO3- groups priming the heparan polymer chain for subsequent deaminative cleavage. The vegan diet, of course, goes beyond the vegetarian diet by avoiding the additional hazard posed by inappropriate fats and growth factors present in milk and dairy products of possible involvement in breast and prostate cancer promotion (as discussed by Professor Jane Plant in her recent book).

The effects of cholesterol LDL/HDL (high density lipoprotein/low density lipoprotein) on health are now found to be explicable by the effects of such factors on heparan sulphate biochemistry e.g. the antiatherosclerotic effects of HDL- (containing apolipoprotein

E) probably stems from up-regulation of AT(lll)-binding heparan sulphate biosynthesis at arterial walls (L Paka et al 1999) and saturated lipids present in meat, if consumed to excess, tend to inappropriately diminish biosynthesis of heparan sulphate relative to other GAGs. A further negative lipid-related factor has been identified to be an effect of oxidised lipids which can similarly potentially negate anti-tissue protection via heparan sulphate mechanisms. Non-vegan Western diet by deviating markedly (including from the required balance of omega three lipids) from that in which our ancestors polysaccharide biochemistry evolved to deal with, potentially impairs heparan sulphate biochemistry for optimum tissue protective efficiency. Linus C Pauling: Vitamin c and cancer.

Linus Pauling, a brilliant scientist, incredibly gained two Nobel prizes. Future generations may, however, come to more greatly praise his dietary supplementation discoveries than his chemical or his political accomplishments. His studies on cancer prevention seem to have been poorly accepted in his lifetime and continue to be disbelieved since a plausible biochemical mechanism for them was lacking at that time (a suitably believable mechanism could involve HSPG and nitric oxide biochemistry). Paulings epidemiological evidence of benefit achieved by ascorbate

supplementation against the common cold and cancer appears to be sound. A conference arranged in 1991 in response to the controversy surrounding Paulings claims somewhat restored public credibility in the main thrust of his contentions. The cancer studies had been conducted in Scotland using previous clinical records in place of the paralleled double blind placebo experimental protocol which would perhaps have been more convincing. All patients receiving the vitamin C supplements apparently benefited. Attempts to repeat these studies in the USA were unsuccessful but Pauling believed he had an explanation for this - in the repeat experiments, the US patients died following a too-sudden withdrawal of their ascorbate supplementation as discussed by Pauling in his letters to Chemistry in Britain in 1985. A previous explanation for the anti-cancer benefit of ascorbate had centred on its known effect in hydroxylation of collagen which tends to inhibit cancer cells metastasizing. The additional likely effect on heparan sulphate microstructure and total environment is now seen to be a more significantly wide benefit-providing mechanism. Paulings results suggest that a vegan diet will tend to counter the

common cold and inhibit cancer.

Ascorbate-induced increase in heparan sulphate activity and protection of heparan activity

by neutralizing the anti-heparan sulphate activity of nitric oxide metabolites may well be the main reason for Paulings findings. Increased heparan sulphate sulphation and total biosynthetic formation in fibroblast cell culture experiments resulted from ascorbate additions to the culture medium (M Edward & RF Oliver 1983, cf J Kao et al 1992). The significance of this finding is greater than it might at first seem as it is likely to occur with various other cell types as it has been well known for some time that ascorbate was involved in GAG biosynthesis. Further research into this important research finding and its relevance to human health is warranted.

Pro-cancer effects of ascorbate have, however, also been reported. Ascorbate can be shown to increase free-radical production in vitro in presence of redox metals like iron. Iron toxicity effects might be capable of reducing or reversing the tissue protective effects of ascorbate. Such iron toxicity has been associated with cardiovascular dysfunction but there may be a dietary link with overtly non-vegan diets.
Salonen et al : Iron Overload and Cardiovascualr Disease.

A major breakthrough linking iron overload with heart attack, the major killer in Western society was communicated in 1992 by Salonen and his team from Finland. The origin of this iron toxicity effect is now postulated to include HSPG damage at blood vessel walls caused by the effect of naked iron ions which can prime heparan sulphate N-sulphonate groups for subsequent deaminative cleavage as well as promote peroxynitrite and hydroxy radical generation via Fenton reactions catalysed by such iron ions. Iron overload, then, which can arise from a red-meat rich diet can, together with the effects of nitric oxide metabolites (which can arise from dietary arginine via nitric oxide synthase (induced by the immune response to pathogens or normally cellularly produced) as well as from nitrite in meat or from that produced by molybdenumcontaining enzymes of mouth bacteria, is capable of depleting these critical substances by snipping off key N-sulphonate sites or even totally unzipping the polymers which are involved in blood vessel wall sensing and antioxidative protection by providing binding sites for superoxide dismutase and the seleniumcontaining antioxidant molecules. Such effects of iron overload are probably countered by a vegan diet.
The Suggested Biochemical Basis of the Benefit of a Vegan Diet.

The results of chemical and biochemical research summarized

here may provide part of the key to understanding why wideranging health benefits ensue from certain ascorbate-rich and lipidfriendly vegetarian and vegan diets (especially if free of natural and man-made toxins). Ascorbate directly reduces nitric oxide metabolites back to tissueprotective nitric oxide, but perhaps of equal or greater importance is that this vitamin is capable of augmenting appropriate heparan sulphate biosynthesis and may be a cofactor in promoting its tissue protective role.
A similar effect has been found with certain vegetable fats) (polyunsaturates especially). A boost of heparan sulphate via ascorbate or lipids is the likely outcome of a vegan diet which has an important additional factor of limiting the effects of ~haemoglobin iron.

It is now dramatically apparent that many hitherto poorlyunderstood degenerative diseases in which the extracellular matrix is degraded such as bone disease, apoptosis, tissue damage from chronic inflammation involving release of growth factors and cytokines could be potentially linked to inappropriate redox-catalysed degradation of heparan sulphate by nitric oxide metabolites; these situations are also highly relevant to the aetiology of cancer as pointed out by Vilar et al in 1997.
Future: heparan Sulphate linked therapy?

HSPG replacement therapy (e.g. by sulphated polysaccharides derived from artificially sulphated xylans from birch trees or seaweed or pharmaceutical compounds like suramin and analogues) may, in principle, be possible. The current use of dietary glucosamine supplements, popular in alternative medicine, may be to do with boosting HSPG protection. Heparin/HS therapy seems to show benefit to Alzheimers and other degenerative disease patients. But a more satisfactory procedure would clearly be to seek non-invasive protection and correction of aberrant HS systems through a precise monitoring of dietary control of heparan sulphate status and cofactor dysfunction via urinary or tissue sampling and microstructure analysis. Future medical procedures might even allow routine monitoring and correction by such methods. This would need to include a correction of iron (and perhaps other redox metals such as manganese which is required for

heparan sulphate biosynthesis enzymes).

Acknowlegements

I am indebted to help received from former colleagues at the University of Aberdeen (especially to Frank Williamson and Bill Long) and I am grateful to Prof KEL McColl of the Dept of Medicine and Therapeutics at the University of Glasgow for discussions of his work. References : vide infra in section

ADDN. INSERT. DOCs

1.

Contents 1 Key health master molecules 1a The heparanome is centrally implicated in health 1b General well-being provision
1c Heparin/heparin & lifespan

Inserted doc 1 The occurrence of pro-oxidant situations during chronic inflammation Inserted doc 2 Alteration of heparin promotes ME-CFS, heart disease and atherosclerosis by a Ca related mechanism
Degradation of HS by Nitrous Acid
REFERENCES Relevance of HS signalling to pathology

Selected short ref list Inserted document 3 Possible key role of HS deficiency in both ME-CFS and autism Low level electromagnetic radiation effect on water structure and ME-CFS Quantized electromagnetic field effects

Are There Easily Identifiable Key Health Master Molecules?

It should be noted that nitric oxide plays multiple physiological roles in animals (being involved e.g. in signalling and neurological activities); these are additional to its intrinsic antioxidant protective effects. Nitric oxide signalling had been widely employed by animals - so much so that nitric oxide might even be regarded as a prime pro-health small molecule. It is appropriate therefore to confer upon nitric oxide master molecule status.

[Cf. C Wheatley (Med Hypoth 2006 67 124-42, cf J Nutr Environ Med 2007 16(3-4) 181-211) has also applied the master molecule concept to a nitric oxide regulator [Vitamin B12 (cobalamin)]; this vitamin has been suggested to regulate nitric oxide synthases, a circumstance which allows for the dietary supplementation by B12 to achieve facile renormalization of nitric oxide tissue contents and thereby to combat a wide range of pro-inflammatory situations. The common use of aspirin as an anti-inflammatory agent may, at least in part, also depend on the ability of this substance to improve nitric oxide status. This may also be at least part of the reason why aspirin may reduce the risk of cancer metastasis (cf Rothwell PM et al Lancet 2012 379 (9826) 1591-1601). [Cf. a perceived insufficiency or defect in the homeostasis of nitric oxide (especially in the situation of ascorbate deficiency which leads to an alteration in nitric oxide metabolism in favour of the production of excessive peroxynitrite and nitrite) is thought to accelerate the formation of tissue- and biomolecule-damaging reactive oxygen species (ROS), as well as the stated reactive nitrogen species. {Peroxynitrite and nitrous acid. its salts molecules which are putatively critically implicated in the etiology of several types of inflammatory conditions which could include (some and perhaps most types of) cancer and also perhaps the mystery illness, chronic fatigue (Chronic Fatigue Syndrome, CFS), had been suggested by PM Pall (cf inserted document vide infra) to arise because of the dysregulation of nitric oxide, a circumstance which further suggests a possible beneficial therapeutic use of Vitamin B12 and/or aspirin to alleviate the symptoms of CFS}. Enhanced nitric oxide production is apparently associated with endometriosis, a disease which seems further to be associated with xenobiotic intoxication (putatively with dioxins and related halogenated aromatic substances formed accidentally during pyrolysis conditions commonly encountered in numerous industrial and municipal waste disposal processes).

Another major molecular, putatively key, master health system, however, which also interacts significantly with nitric oxide, is the animal glycosaminoglycan system. This contains as a major component the nitric-oxide-sensitive, highly anionic polysaccharide (coreprotein- held) side chain system consisting of heparan sulphate, a copolymer of uronic acids (mainly iduronic acid but with some glucuronic acid) alternating with glucosamine, GlcNH 2. Most of the NH2 groups in heparan sulphate are sulphonated. This confers upon them a considerably enhanced chemical stability including against reaction with nitric oxide or nitric oxide metabolites. If, however, following the establishment of chronic cellular immunological responses against recalcitrant infecting organisms, or accumulation (especially in fatty tissues) of anthropogenic xenobiotic toxins (especially dioxins and related molecules), there can become established a chronic pro-oxidant stress which puts at high risk the tissue regulatory managerial processing functions centred on GlcNSO3- -dependent heparan sulphate-held codes. These can become (partly) deleted as a consequence of oxidative/nitrosative stress. Specifically, key single diseases such as interstitial cystitis and endometriosis (putatively a nitric oxide dyshomeostasis illness which can be countered by exogenous heparin) and a wide range of more complex degenerative diseases, putatively arise by this mechanism. It is now suggested that a wide cause of disease is the relative ease of alteration of those (heparinsensitive) nitrosative heparan sulphate deaminative reaction processes which can be observed under in vitro conditions (and which can, e.g. be followed by infrared spectroscopy in the lab).
The above ideas were engendered following a comprehensive review (conducted as part of a funded academic research programme hosted by the former polysaccharides lab at Marischal College, Aberdeen University and

continued informally thereafter) of the now extensive literature survey on the heparanome-held information processing system. This system should perhaps now be included (additional e.g. to DNA, nitric oxide and the drug aspirin and perhaps also calcium and bisphosphonates) in the list of health-confering-master-substances which are closely associated with the nitric oxide signalling systems. Just as damage to DNA-encoded genetic information (mutation) can lead to cancer and a range other diseases, so also can damage to the information-encoded heparnome sugar sequences potentially widely impact on all aspects of human and animal health. Heparanome deficiencies putatively (and perhaps surprisingly) must, it is suggested, play key etiological roles in (perhaps) most diseases. The Si-containing endothelial carbohydrate-rich glycocalyx contains abundant HS (>CS>hyaluronan) which provides a barrier function for transvascular exchange of water and solutes(including plasma borne macromolecules, e.g. superoxide dismutase etc., etc.); it is shed in response to inflammation and is centrally implicated in vascular homeostasis (cf. Gao DE and Lipowsky HH Microvasc Res 2010 80 394-401). Hypoxia may increase macrophage motility by decreasing HS PG biosynthesis (Asplund A et al J Leukoc Biol 2009 1901393). Defective N-sulphation of HS limit PDGF-BB binding and pericyte recruitment in vascular development (Abrahamson A et al Genes Dev 2007 21 316-331). In addition to HS there are possible key role of the glycosaminoglycans versican and hyaluronan in atherosclerosis (cf Karangelis DE et al Curr Med Chem 2010 17 (33) 4018-2). Galactoseaminogalactans such as CS and DS bind LDL. Lower HS may be accompanied by inappropriately high CS + DS + hyaluonan. Exposure to non-esterified fatty acids (NEFA) increased the ChS:HS ratio and encourged LDL binding and thereby promoted atherogenesis (Rodriguiez-Lee M et al Arterioscl Thromb Vasc Biol 2006 26 130-135; a similar action may give rise to calcific aortic valve disease (cf Grande-Allen KJ et al Cardioivasc Res 2007 76 19-28 Sulphation levels per se of CS and HS PGs seem to control cellular and in particular neural maturation. This, if defective, seems to lead to pathologies, including neurological ones (cf Karus M et al Neural Dev 2012 7 20 PMCID 3423038).

[Some Abbreviations Used HS: heparan sulphate (sulfate); CS: chondroitin sulphate; DS: dermatan sulphate: PG: proteoglycan; LDL: low density lipoprotein [(bad) cholesterol]. CFS: Chronic Fatigue Syndrome; ME: myalgic encepalomyelitis; FM: fibromyalgia; ALS amyotrophic lateral sclerosis; MS multiple sclerosis; AD: Alzheimers disease; CF: cystic fibrosis; RA: rheumatoid arthritis, SLE systemic lupus erythematosus. FGF fibroblast growth factor; FGFR fibroblast growth factor receptor SOD superoxide dismutase RNOS reactive nitrogen oxygen species; ROS reactive oxygen species]

1a The heparanome is centrally implicated in health

Hypothesis: The Heparanome Centrally Controls Health
The simple idea, that the heparanome centrally controls health via a catholic tissue regulation ability, implies that the protective maintenance of the integrity of the heparanome may combat many or all disease processes. This idea also requires a re-assessment of much semi-empirical data which have suggested that a useful therapeutic intervention may be achieved in a wide range of degenerative diseases via dietary regulation. An extensive literature seems to suggest that the chemical structure and integrity of the heparanome, and how this can be repaired, may allow for a fuller understanding of the mechanism by which non-invasive therapeutic benefit might be achieved through dietary alteration. Re-stating: the amount and microstructure of heparan can be beneficially altered via an improvement of

microstructure which is under the control of redox status etc. and which in turn can be changed for the worse or for the better by diet, being positively affected e.g. by the dietary presence of the correct vitamins, fatty acids, inorganic silicon (e.g. as silica sol particles or silicic acid) mineral ions, etc., and negatively affected by excess glucose, toxic metal ions, environmental toxins etc. Heparan can most easily be upgraded by nitric oxide, copper, magnesium and zinc. Heparan can also be improved by the exogenous administration of heparin or heparin-like drugs which enter an apparent servo control system which can putatively apparently upgrade the heparan system microstructure at the primary biosynthesis stage. This process evidently combats perceived extracellular threats. Paulings high-ascorbate-anti-cancer strategy may not, it is now suggested, principally have been achieved by the commonly-believed collagen crosslinking mechanism, but instead by an ascorbatedetermined beneficial alteration of the tissue redox status which signals for an upgrading of the heparan microstructure (e.g. by the ability of ascorbate to increase the degree of sulphation of heparan and to diminish the amount of inappropriate nitrosative scission which can be demonstrated to arise via nitrite attack which can be neutralised by ascorbate, as easily shown in vitro in the lab). The wider glycome literature confirms the above hypothesis. A related hypothesis suggests that the extracellular matrix secreted by vascular cells modulates proteoglycan synthesis. Adverse alterations to this process lead e.g. to an increased LDL uptake and resultant vascular dysfunction; (cf. e.g. Vijayaogopal P and Menon PV Atherosclerosis 2005 178 (1) 75-82). Heparan sulphate proteoglycans seem to act in so many pathways that they must be assumed to be true top-rank master-molecule systems which works in tandem with but only in partial control by the genome. The heparin/heparin PG system is a major or indeed the principal epigenetic control system. The heparin/heparin information sorting systems are control agents for multiple classes, perhaps eventually all of the physiological and pathological functions; e.g. they influence ageing, cell-cell adhesion, cell-matrix adhesion, cellular proliferation, differentiation and tissue development, wound healing and oncogeneis (transformation, tumour angiogenesis and metastatasis), circadian rhythm, as well as in neurological signalling which putatively includes the mechanism of memory as well as the immune system and also act directly and indirectly to increased anti-pathogen protection. I.e. multiple heparan dependent tissue protection systems exist which are of very much greater scope than the more commonly known, historically first found heparanome function: the blood anticoagulation tissue protective functions of the heparin type of heparan sulphates. In agreement with the heparan master-system-for-health-concept, it has been reported that the detailed microstructure of heparan, uniquely amongst biomolecular systems, varies systematically with animal age (and also changes dramatically between normal and transformed cells). 1b General Well-Being Provision Heparan repair by dietary factors (e.g. those which have indicated independently but empirically to be generally beneficial to health) is a general and very wide-ranging mechanism of health promotion.
While the heparan information-code-system is known to be much more complex than that of DNA (it apparently, however, lacks any the easily discernible nucleic acid-like double helix replicating activity but some hints of a self association may be suggest future developments in this field), its widest ranging central control functions seem (as indicated from a study of the current literature) most obviously to be used for the management of the modification of (water-structure/activity determined) protein conformation (including for the growth factors involved in development and wound healing); this behaviour is sufficiently widely observed for the system of heparan sulphates that they can now justifiably be accorded the rank of an ome system terminology, namely the heparanome which may have an analogous reach for a fuller understanding of fuzzy logic system mathematical computational mechanisms used by muticellular animals, additional to that the genome in biota. It should be noted that while the enzymes which are currently known to be required for the primary biosynthesis of primary sugar units which can be assembled into heparan sulphate and also their supporting protein backbone cores are of course entirely genome-derived, some heparan information codes are also potentially altered by a non-genetic, nitric-oxide-dependent postsynthetic modification processes. This genome-independent process may indicate that for animals and other multicellualar classes of organisms the heparan and related types of polysaccharides provide an overhead encoded control systems which can act independently of the nucleic acids). The heparnome may, even, it might be suggested, have started off at the start of multicellular animal evolution as a prime buffer against the total extracellular environmental insult, e.g. it offering major buffer action for the creation of the correct

multi-inorganic-element-ion-achieved water activity (osmolality) [cf Grant 2000] and also for the combating of a wide range of pathogenic organisms both but restricting their entry into the organisms but also by inhibiting the biochemical activity of invading organisms. Additional primitive tissue protective activities of heparan include the provision of primary and secondary (protein dependent) antioxidant activity and the prevention of the growth and the limitation of the toxic activity of overtly pathological crystal formations (which is evidently a part of the wider osmoliaty control which is apparently essential to the integrity of cells and their assemblages {cf Grant et al 1992a}). 1c Heparin/Heparin & Lifespan Heparan held information also apparently establishes the intrinsic lifespan of individuals. I t apparently determines the average longevity of individual animal species as well as affords a mechanism by which any adverse change of environmental conditions can cause heparan sulphate management to evolve an appropriate future restructuring of animals tissues and organism appendages etc. in order to combat new environmental challenges.

A key part of the above putative heparan-dependent mechanisms is considered to be achievable by a post-biosynthetic modification of heparan by the nitric oxide metabolites which create short chain heparan sulphate fragments which exhibit hormone-like activities and which can enter the cell and signal to the nucleus. The primary biosynthesis of heparan seems also to become altered in response to such heparin-like-hormone signals. (This is a potential servo-feedback system). Exogenously applied heparin or semisynthetic heparinoids, putatively exert direct therapeutic, hormone like, actions which combat chronic inflammatory situations, therefore potentially allowing heparin, it is proposed, to combat degenerative diseases by this mechanism.
The heparin-afforded tissue protection facility may also be achievable by use of dietary glucosamine. It should be noted that in 1953 JH Quastel and A Cantero reported on the inhibition of tumour growth by D-glucosamine (cf. Nature. 1953; 171: 252-4). [This paper noted that in a mouse model of cancer, a significant inhibition of tumour growth was apparently achieved by the dietary supplementation by glucosamine]. It should be noted that if some of this glucosmine which is a primarly heparan building block substance survives the digestive system, it could inhibit tumour heparanase or directly allow for a fast boost of anti-tumour heparan sulphate biosynthesis (as apparently happens in the laboratory in studies of some transformed animal cell cultures in vitro).

This above ideas about the possible therapeutic use of nitric oxide and heparan should, it is now suggested, be followed up by further studies. Such studies should start off from a consideration of the circumstance that tumour malignancy and therefore the mortality of cancer is known to correlate negatively with aspirin usage [cf Rothwell PM et al Lancet 2011 9826] (which may perhaps arise because of the amelioration of nitric oxide benefit by this drug) and also with the presence of tumour secreted heparanase (an enzyme which cleaves heparan sulphate by a mechanism which is entirely enzymatic). The nitric oxide dependent process of nonenzymatic cleavage of heparan sulphate which arises as a consequence of defective nitric oxide metabolism, however, also creates a similar effect, but in this case the heparin-like molecules which are formed can also inhibit heparanase can therefore also can diminish heparanase promoted metastasis. This activity, it is now suggested, may also be augmentable by aspirin. A series of currently unpublished rough draft documents which are of possible wider public have been inserted at the end of this note. (These reviews were written following literature searches undertaken by the author some ten years ago at the request of Dr FB Williamson [then an academic researcher at Aberdeen University specialising (in conjunction with Dr (later Prof) WF Long) in polysaccharide, including heparan sulphate, biochemistry] who had been diagnosed with and who wished to find any published information which perhaps in conjunction with both published an unpublished results from this own lab, might give hints on how to design possible therapeutic interventive strategies for CFS]). The literature reviews (which were derived largely from a Chemical Abstracts search with the assistance of University and other libraries as well as with discussion with numerous persons who are too numerous to be listed here, who are gratefully thanked for their assistance) focused on the possible manner in which heparin/heparan sulphate (sulfate) biochemistry and especially the ideas relating to iron catalysed nitrosative scission of heparan sulphate suggested by Aberdeen University polysaccharide research might be relevant to the etiology of CFS viz a dyshomeostasis of nitric oxide HS signalling arising as a consequence of chronic inflammation [which also suggests that CFS and fibromyalgia and other chornic inflammatory conditions might also have a related etiology, as also

Chronic Fatigue Syndrome (CFS)

could atherosclerosis (an idea also arrived at, albeit from a different perspective, by Elder & berall [Schweiz, Z Ganzheitsmed 2011, 23 109-120] which tends to support the hypothesis that CFS and atherosclerosis may share common molecular risk factors). The earlier laboratory studies (conducted by F.B. Williamson , it should be noted, had suggested that a defective heparan-sulphate-associated dysregulation of some heparan iron sequestration processes might conceivably directly contribute to the etiology of ME/CFS. This research appears to support the idea that iron balance dysregulation may be centrally associated with atherosclerosis (cf. Sullivan JI Exp Biol Med 2007 232 (8) 1014-1020) and the hypothesis that that CFS and atherosclerosis have overlapping etiologies. The minutes of the discussions held between D Grant (Turriff AB53), F B Williamson (Aberdeen AB15) and which also sometimes included Vance Spence (University of Dundee) from 2003, suggested the above ideas which lead to new general hypothesis of ME-CFS. This is outlined in the first page of document 1. Document 2, dated 11 November 2005, which is reproduced here intact, is a corrected version of a paper written in 2003 (which had been prepared by the author for possible use during a (3rd October 2003) Royal Society of Edinburgh Wellcome Trust Workshop entitled: New developments in the biology of Chronic Fatigue Syndrome with emphasis on the anti-viral pathway and oxidative stress and how these impact on the vascular endothelium which had been held at West Park Conference Centre Dundee and had been attended by the author. [A later extension of the Williamson et al chronic inflammatory anti-heparan hypothesis of CFS further suggested that a major mechanism which stimulates the acceleration of heparan sulphate ageing (a process which diminishes the ability of heparan sulphate to perform its multiple tissueprotective roles) might arise from intoxication by those, now globally distributed (anthropogenic) xenobiotics, which are commonly present in the Western diet (following enrichment up the food chain which ending up in fatty tissues). The pro-inflammatory endocrine disruption process leading to CFS and other mystery illnesses seemed to be exemplified most dramatically by the halogenated aromatic substances e.g. dioxin-like substances, including perhaps most especially hexachorobenzene (which although banned under international protocol is enabled by its unique chemistry to be formed spontaneously in relatively large amounts and becomes transported intercontinentally from its probable source, the incineration of a wide variety of organic starting molecule waste products when heating occurs in chloride ion or organochlorine molecule copper and iron or antimony containing environments). It turned out that the affected academic who had commissioned the ME-CFS literature survey had also apparently been subjected to topical therapeutic hexachlorobenzene insecticide application. (Hexachlorobenzene or a related substance had apparently been applied to his arms therapeutically as a treatment for scabies). There are confirmatory reports in that literature that human intoxication by residual insecticide hexachlorobenzene (as well as benzene hexachloride) insecticide intoxication correlates with the presence of ME- CFS in affected individuals who also demonstrate a chronic and systemic oxidative stress. This, according to the heparan sulphate literature potentially could adversely alter heparan sulphatedetermined nervous system repair mechanisms in individuals who have become intoxicated by these insecticides. {Cf. internet posting accessible via search term Amey Bischop hexachlorobenzene}. It should be noted that an ATSDR public health statement available on the internet indicates that low levels of hexachlorobenzene have been found in the fatty tissues of almost all people tested. These amounts are most likely the result of low levels in food].

Prostacyclin, Heparin/HS and CFS

Prostacycin is known to inhibit platelet aggregation and be a vaso dilator. The HS physiological control system is apparently involved in the in vivo mediation of prostacylin activitiy. The perturbation of nitric oxide biochemistry in CFS may (e.g. in combination with mild iron dyshomeostasis) create abnormal nitrosative oligosaccharides which increase prostacyclin sensitivity. This may be a major cause of CFS.
The vascular endothelium is a potential source of immune system signalling oligos derived from the vascular heparin/HS system [which is subject to nitric oxide depolymerization as well as boost in primary biosynthesis via exogenous heparin or heparin-like drug administration].

It should be noted that VA Spence F Khan and JGF Belch found evidence for an enhanced sensitivity of prostacylcin-dependent vascular responses in patients with CFS. These authors studied cutaneous vascular responses to acetylcholine, a procedure which could also be used to classify different CFS-like illnesses. The crux of the CFS etiology mystery may be the answer to why, when most diseases are accompanied by a blunted response to acetylcholine, the opposite is true for CFS. Perhaps this is because the blood in CFS patients contains specific hormones which stimulate prostcyclin production. Candidate possible hormone-like oligosaccharides which may be responsible for this could be similar to those which exist in low molecular weight heparin [LMWH] but not in unfractionated heparin {cf Kaji et al found that LMWH (but not unfractionated heparin) enhances prostacyclin production in cultured human endothelial cells. (Such enhancement was induced at 0.1U/mland above after 6h of treatment).
{Heparin degradation products (oligosaccharides) present in LMWH (but not the original form of heparin) therefore seem to stimulate endothelial production of prostacyclin. It should be noted that changes in endothelial function which are also apparent in preeclampsia can also be combatted by LMWH (cf Mello et al)}.

Berg reported that pharmaceutical heparin (and related drugs) can alleviate the symptoms of some types of CFS (considerd to be in such cases produced by an irregularity in the blood coagulation system, resembling the antiphospholipid antibody [Hughes] syndrome). Self-administered heparin anti-CFS therapy (as indicated in internet blogs) seem to offer major relief from the symptoms of CFS, but in a manner unrelated to the anticoagulation action of heparin. While the administration of heparin is reported to often successfully alleviate the symptioms of CFS this apparently can occur in a highly irregular manner. For their possible use in the controlled therapeutic intervention in CFS further research seems to be required in order to define under what conditions heparin-like drugs may undergo in vivo decomposition to stimulate prostacyclin production. For the normalization of prostacylin other specific hormone-like HS-derived oligosaccharides may be requried. Alternatively the neutralization of proprostacycin oligos (e.g. by polyamines) may be required to combat CFS.

The possible beneficial effect of a vegan diet against mystery inflammatory disorders. was confirmed by a Finnish epidemiological study Ref [3] [K Kaartinen et al PMID 11093597; cf. also 11 other studies in PubMed] for fibromyalgia. That a vegan diet may also benefit chronic fatigue syndrome (which is of related etiology to fibromyalgia) is an idea which also worthy of consideration. A range of internet blogs etc. [accessed on May 25 2013 via the search terms vegan diet fibromyalgia chronic fatigue syndrome] support the notion that chronic fatigue syndrome is also improved by a vegan or vegetable rich diet.

INSERTED DOCUMENT 1 (first page only shown of document 1)

The Occurrence of Pro-Oxidant Situations during Chronic Inflammation may Promote Atherosclerosis and Myalgic Encephalomyelitis-Chronic Fatigue Syndrome (ME-CFS) by Depletion of Heparan Sulphate Dependent Antioxidant & Antinitrant Activities & Suggests a Parallel Pathological Effect of Nitration and Heparan Sulphate Deaminative Cleavage David Grant a Frank B Williamsonb [footnotes to include addresses and (most recent) affiliation]

Heparan sulphate (HS) occurring as proteoglycan side chains at adherent animal cells as well as in the form of soluble proteoglyans and oligosaccharides are utilized by conserved cellular control systems in all multicellular animal organisms. HS provides ligand binding sites by which it modulates the activity of numerous signalling proteins. The system which have been most fully investigated are in haemostasis and fibroblast growth factor (FGF) signalling (Bernfield et al 1999); recent interest has extended to embryogenesis (Perriman & Bernfield 2000). [Cf. also Casu et al 2010]. Each type of cell seems to have a characteristically coded HS microstructure (cf. Lyon & Gallagher 1999). Since postsythetic enzymatic processing of HS (for the patterning of cells in the avian and likely more general embryogenesis) is performed by an extracellular sulphatase (Dhoot et al 2001), it is clear that even minor postsynthetic alterations to HS, which are possible consequences of oxidant and nitrant damage, may potentially influence how HS is able to effectively modulate cellular differentiation, immune responses and wound healing. Atherosclerosis is associated with a depletion of HS-dependent endothelial SOD activity (Adachi et al 1995) and an apparent, linear, also age-dependent depletion in the proportion of HS in total glycosaminoglycans (GAGS) in atherosclerosis-affected cerebral artery tissues (Murata & Yokayama 1989). The detailed microstructure of HS extracted from related endothelial surface tissues also shows a direct age-dependent alteration (Feyzi, 1998). The above reports suggest the hypothesis that an artificially enhanced ageing of HS may occur under pathological conditions. Such HS damage (including defective signalling for biosynthesis) may, e.g., be centrally involved in etiologies of atherosclerosis and related illnesses (which likely include MECFS). This change in HS could further be associated with free iron effects which are the evident basis of the relationship between serum ferritin and cardiovascular dysfunction (Salonen et al 1990) and atherosclerosis (cf. Sullivan 1992). The unusual aetiology of ME-CFS seems also to be explicable as arising form a dysfunction of nitric oxide biochemistry perhaps centred on the toxic effects of peroxynitrite (Pall 2000), nitrous acid or other nitric oxide metabolites. Literature discussions of the origin of ME-CFS have also included observations of Herpes Simplex 6 viral infection which according to other literature reports can alter (endothelial) HS biosynthetic processes.
Cf Bernfield M et al(1999) Functions of cell surface heparan sulfate proteoglycans Annu Rev Biochem 68 729-777 Dhoot GK et al (2001) Regulation of Wnt signalling and embryo patterning by an extracellular sulfatase Science 293 1663-1666 Feyzi E et al (1998) Age-dependent modulation of heparan sulfate structure and function J Biol Chem 273 (220 13305-13398 Lyon M Gallagher JT (1998) Biospecific sequences and domains of heparan sulphate and the regulation of cell growth and adhesion Matrix Biol 17 (7) 485-493 Pall ML (2000) Elevated, sustained peroxynitrite levels as the cause of chronic fatigue syndrome Med Hypotheses 54 115-125; Cf ibid 57 139-145 and J Chronic Fatigue Syndrome 7 45-58; Murata K Yokoyama Y (1989) Acidic glycosaminoglycans in human atherosclerotic cerebral arterial tissues Atherosclerosis (Shannon) 78 (1) 69-79 Chem Abs 111 151343a Perrimon N Bernfield M (2000) Specificities of heparan sulphate proteoglycans in developmental processes Nature 404 725-728 Salonen JK et al (1992)High stored iron levels are associated with excess risk of myocardial infarction in eastern Finnish men Circulation 86 (3) 803-811 Sullivan JL (1992) ibid 86 (3) 1036-1037.

INSERTED DOCUMENT 2 Degradation of Heparan Sulphate* (HS) by Nitrous Acid may Promote Myalgic Encephalomyelitis-Chronic Fatigue Syndrome (ME-CFS)a and Related Afflictions
D Grantb & FB Williamsonc [footnotes including addresses and (most recent) affiliations] Draft for e.g. Medical Hypotheses or Internet
Contents 1. Introduction 2. Hypothesis 3. Alteration of HS may Contribute to ME-CFS, Heart Disease & Atherosclerosis by a CaRelated Mechanism 4. Heparin/HGS Therapy for ME/CFS? 5. Interactive nitric oxide and HS biochemistries. Dietary and other Factors which Influence HS Biochemistry 7. Potential Anti-RNOS Protection Mechanisms 8. Suggested further experiments References X. Relevance of Heparan Sulphate Signalling, a Likely Important Control System in Animal Biochemistry, to Pathology
Y. 1 The major causative factors of CFS may include heparan sulphate biochemical activities which are both nitric oxide and trace metal ion dependent Y2 Effects of Cadmium, Lead, Mercury & Aluminium Y.3 RNOS include Peroynitrous and Nitrous Acid Y.4 Possible Relevance of Copper Intoxication, Niitric Oxide, Heparin/Heparan Sulpahte on Platelet Activity, in the Etiology of CFS Y-5. Heparin/Heparan Sulphate Biochemistry & Copper Status Y.6 Ageing & Iron Overload may Augment Niitrant Damage in Arthritic Disease and Neurological Dysfunction Y.7 Model of Heparan Sulpahte Recycling Y.8 The ability to chelate trace redox metals by cysteine tyrosine antioxidants Y.9 Necessity for the Existence of a Physiological Anti-Nitrant Protection System
6. Tyrosine-3-O-NO2

1. Introduction Reactive nitrogen oxygen species (RNOS), which include peroxynitrite, are known to promote numerous illnesses associated with the formation of nitrotyrosine in proteins (cf Ischiropoulos (1998)) [1]. Peroxynitrite, the specific product of the reaction of nitric oxide with the superoxide anion was suggested by Pall et al (2000) [1] to be centrally implicated in the aetiology of ME-CFS. A major sub-set of CFS patients were reported (Berg) to apparently benefit from heparin therapy [1]. Since the formation of superoxide is attenuated in part by extracellular superoxide dismutase (ECSOD) bound at endothelial surface heparan sulphate (HS) (e.g., cf. Adachi & Marklund 1989) any depletion of these polysaccharides will increase the likelihood of the formation of peroxynitrite at blood vessel walls. Peroxynitrite can further transform into nitrous acid, a more damaging nitric oxide metabolite which efficiently cleaves HS chains and also damages various other key biochemical molecules It is worth exploring the circumstances where this cleavage might occur. Under physiological conditions peroxynitrite equilibrates with nitrite (Pfeiffer et al 1997) which is also readily formed by the direct oxidation of nitric oxide, and although reductants like ascorbate and glutathione can reverse this process, these are subject to depletion by dietary insufficiency or under pathological stress situations.

Local acidic conditions necessary for nitrous acid formation from nitrite may follow from acidosis produced under chronic inflammation conditions. Prolonged immunological secretion of hypochlorous acid (HOCl) could generate from peroxynitrite or nitrite damaging amounts
of nitrous acid. This is a well-established reagent in organic chemistry, it (or a reactive intermediate such as N2O3 or as yet undetermined active nitrogen species, derived from it) specifically transforms numerous organic amines by their initial nitrosation, diazonium ion formation and further reaction, such as the transformation of primary amines, into alcohols: [C-NH2 C-OH], HNO2 a reaction which is responsible for an efficient mutation of RNA. Nitrous acid as well as cleaving HS also cross-links DNA.

Known redox metal catalysis of related nitrosative reactions (e g Challis et al 1978) suggests an additional catalytic role for trace iron and copper. Such ions are believed to become activated in low molecular weight form by acidosis (cf Lipscomb et al 1998). Thiocyanate (Boyland & Walker 1974; Fan & Tannenboum 1973) also promotes nitrosative reactions perhaps also influenced by the effects of trace redox ions. 2. Hypothesis We hypothesise that ME-CFS and related afflictions arise as a consequence of the disturbance of the wide-range tissue-protection and immunological signalling activities afforded by HS.
Nb HS is now acknowledged to act as a central system for control of numerous protein activities (Bernfield et al 1999) and may also be involved in Ca2+ - dependent signalling (Long & Williamson 1979; Grant et al 1992b). These HS functions may be disturbed, it is proposed, by an aberrant nitric oxide metabolite activation caused by chronic inflammation, acidosis and redox ion dyshomeostasis which leads to an altered biosynthesis as well as the pathological degradation of HS proteoglycans.

ME-CFS-type illnesses can apparently (also?) arise as a consequence of their triggering by an intoxication by acetylcholinesterase inhibitors (e g by insecticides or chemical warfare agents) and/or chronic viral or bacterial infections [2] which are believed to lead to a chronic immune system dysfunction (causing an induced lipid peroxidation which leads to HS biosynthesis and metabolism perturbations, (cf., e.g. Pillarisetti) and increases in nitric oxide synthase (affecting both the induced Ca-independent {in macrophages}as well as the Ca-dependent route in endoethelial cells) leading to an overproduction of HOCl and nitric oxide and its metabolites, which, by inappropriately degrading HS, it is now suggested, can induce CFS. This HS degradation is also suggested to be augmented by the effects of dyshomestasis of redox metal ions [especially copper and iron e.g. by a diminution of transferrin such as that which is known to be associated with Alzheimers and Parkinsons diseases (cf. Lipscombe et al 1998 ) as well as by the effects of environmentally augmented toxic metals such as lead, mercury (cf Templeton 1991) and aluminium [a neurotoxin which inhibits superoxide dismutase activity (Shainkin-Kestenbaum 1989) and which is a likely promoter of dialysis dementia and (putatively also) Alzheimers disease)] which can alter HS microstructure both during and after biosynthesis. The formation of excessive amounts of nitric oxide metabolites will lead to further glutathione depletion and the generation of a pro-oxidant state in which together with the effect of an associated elevation of serum homocysteine (a characteristic indicator of ME-CFS). Homocysteine is also known (Nishinga et al 1993, Shimada) to inhibit endothelial HS biosynthesis. Inappropriate deaminative cleavage of HS catalysed by redox ions, is likely to postsynthetically further deplete endothelial HS. Such circumstances may adversely alter HS-dependent biochemical signalling and set up a chronic procoagulant state (cf. Berg, 1999) which further affects neurological function in which HS signalling is implicated. The aetiology of ME-may be further influenced by the depletion of HS-protected blood vessel calcification (cf Grant et al 1992a) and alteration in poorly understood processes by which intracellular Ca2+ is believed to be involved in signalling for HS biosynthesis (Fujiwara & Kaji 2002) and also for wound repair (e.g. via fibroblast growth factor signalling cf. Kan et al 1996).

The above scenarios are also relevant to any discussion of the apparent pathological relationships between the aetiologies of ME-CFS and arteriosclerosis.

3. Alteration of HS may Contribute to ME-CFS, Heart Disease & Atherosclerosis by a Calcium Related Mechanism. An alteration to HS microstructure and associated biochemistry which is associated with depletion of HS-dependent superoxide dismutase activity (Adachi 1995) which could encourage the process of blood vessel calcification, which has also been associated with the arteriosclerotic processes. Cf, an age-related diminution of HS has been found to be present in atherosclerosis affected cerebral arteries (Murata & Yokayama 1989). This idea of an involvement of depletion of HS anti-calcification activity is also in accord with the circumstance that differences in pro- and anti-atherogenic lipoproteins can putatively be directly attributed to their effects in increasing or decreasing the amounts of correctly-structured subendothelial HS (this perhaps especially pertaining to heparan chains of perlecan proteoglycan (cf. Pillarisetti 2000). A possible additional HS-dependent mechanism associated with atherosclerosis is the putative servo feedback system which affects endothelium integrity via the actions of hormone-like HS oligosaccharides (also called endogenous heparin {cf Engelberg who correlated the variation of this factor in blood with the present of LDL cholesterol as an antiatherosclerotic determinant which inhibits atherosclerosis and other degenerative diseases; this activity may partly explain why heparin seems able to dramatically counter the symptoms of CFS in a subset of CFS-affected subjects (Berg et al 1998)}). The age-dependent alteration in the sulphation pattern of HS was considered (Feyzi et al 1998) to enhance pathological platelet adherence to endothelial surfaces and thereby to promote atherosclerosis. The observed alteration in HS biosynthesis and consequent alteration of HS patterning may, however be an attempt by the animal heparanome protection system to optimise the HS-afforded anticalcification tissue protection.
It should be noted that evidence that HS microstructure alterations may lead to atherosclerosis both by lipid and non-lipid mechanisms as suggested by primate animal model studies (Kramsch et al 1981). Insulin-deficient diabetes mellitus (Type I DM) is associated in increase in atherosclerosis which might be explicable by the perturbation of the liver HS-dependent lipid metabolism caused directly by a high blood glucose which has been found to impair HS biosynthesis (cf Ebara et al 2000) or by the effect of altered HS hormonal actions involving an additional input by Ca 2+. Evidence for a non-lipid atherogenic process in humans was suggested by the observation that the formation of arterial atheroma plaques (studied in a primate model) could be reversed by the administration of several types of anti-calcification diphosphonates (bisphosphonates) (Kramsch et al 1981). These substances are amongst the most efficient inhibitors of calcification known. They, however, actually possess a very similar anti-calcification potency to that demonstrated by intact HS (Grant et al 1989a). Such diphosphonates are reported also to prevent artificial heart valve calcification when incorporated into these constructs to allow for slow diphosphonate release (Levy et al 1985) confirming that, in humans, diphosphonates can be effective surrogates for HS for the inhibition of the calcification of heart valves . (Such diphosphonates might, therefore, according to our general hypothesis, also be potential therapeutic agents for ME-CFS if the arteriosclerosis-like aetiology of ME-CFS is correct. Such a hypothesis would concur with the apparent benefit of heparin therapy to both arteriosclerosis (Engleberg, 1984) and ME-CFS (Berg et al 1999). It should also be noted in this context that unfractionated heparin shows a similar anticalcification activity to HS (Grant et al 1989a)).

Heparin and HS fragment signalling which include effect of normal or pathological RNOS cleavage may also be involved in the modulation of vascular smooth muscle cell activity, including that

determined by Ca2+ - and nitric oxide dependent systems (e.g. Au et al 1992). The damaging effects on the cardiovascular system of iron dyshomeostasis may include a promotion of pathological RNOS disturbance affecting heparin/HS biochemistry which might contribute to the reason why there is an epidemiological association between moderately elevated serum ferritin levels and ischemic heart disease (Salonen et al 1990). Cf. Sullivan (1990) highlighted the relevance of these findings to the establishment of the mechanism of the arteriosclerotic process. Although serum ferritin apparently can increase with age in the absence of any obvious disease (Jarrett et al 1988), it is tempting to speculate that undetected tissue damage which results from such and apparent age-only ferritin elevation will inevitably be a contributor to the general diminution of body function which is associated with the normal ageing process.

4. Heparin/HS Therapy for ME-CFS? That the aetiology of ME-CFS could be dependent on the status of endothelial HS and the amount of circulating endogenous heparin in the bloodstream is confirmed by the reported benefit of a low dose heparin therapy for CFS (Berg et al 1999). The apparent success of such a treatment could be due to the hormonal actions of such HS oligosaccharides as well as, e g, to the ability of heparin to release superoxide dismutase from endothelial HS binding sites as well as the ability of exogenous heparin to augment the primary biosynthesis of HS. [Exogenous heparin has been reported to increases the biosynthesis rate of anticoagulant HS by endothelial cells (Pinhal et al 1998)]. The ability of anti-atherogenic lipoprotein-rich diets to improve HS tissue protection (cf. Pillarisetti 2000) might also suggest that such diets might also improve ME-CFS symptoms]. Other treatments which have been suggested to alleviate the symptoms of CFS such as ascorbate supplementation (internet) could similarly act by the effect of the action of ascorbate as a booster of HS, especially more highly sulphated HS biosynthesis (cf. Edward and Oliver 1984). It should be noted that the reported inhibition by treatment with the heparin-mimetic, pentosan sulphate of allergic encephalomyelities in rats (Willenborg & Parish 1988) may be a phenomenon related to the alleviation of the symptoms of ME-CFS by heparin. 5. Interactive Nitric Oxide and HS Biochemistries. Dietary and other Factors which Influence HS Biochemistry HS (occurring as proteolgycans) has been highly conserved throughout animal evolution (Nader et al 1988) and provides for the regulation of a wide range of physiological functions, including those of the immune system (cf. Bernfield et al 1999; cf. also Norgard-Sumnicht 1993) and the less well understood roles of HS and NO in neurological functions. Defects in both of these biochemical systems are probably also implicated, but by a currently unknown mechanisms, in the aetiology of Alzheimers disease (cf. Fukuchi et al 1998). HS proteoglycans, involved in both antioxidant (Adachi et al 1995) and also potentially as direct antinitrants, are potentially sensitive to damage inflicted on the specific glucosamine-N-SO 3- groups which are known to be required for specific of protein site interlinking [e.g. in antithrombotic, immune function signalling, growth factor activity, antioxidant activity dependent on protection against iron initiated damage (Ross et al 1992) and he inhibition of vascular calcification (Grant et al 1989, 1992a]. Pathological alteration of the biosynthesis of endothelial HS may be promoted by atherogenic lipoproteins; [cf. lysolecithin which has been reported to reduce endothelial HS, perhaps via a stimulation of heparanase secretion from endothelial cells, a process which can reduces monocyte adhesion (Sivaram et al 1995), other effects on HSPGs of oxidised lipoproteins and lipids (cf. Pillarisetti 2000, Paka 1999; cf also Chang et al 2000), pathogenic organisms which attach to cell surface HSPGs (cf. a Herpes virus was found to diminish HS biosynthesis (Kaner et al 1990) and a

similar bacterial digestive tract surface HS biochemical perturbation may also arise from Helicobacter pylori infection (cf. Ascenciio et al 1995, Chmiela et al 1995)]. The type of endotoxin perturbation of HS biosynthesis demonstrated (by Colburn 1996) could further suggest that a variety of specific bacterial endotoxins might perhaps differentially alter HS biosynthesis. The inhibition of biosynthesis of HS has also been reported to arise from the effects of elevated homocysteine (Nishinga et al 1993), toxic metals (e.g. lead (Fujiwara & Kaju 1999), cadmium (Crdenas et al 1992) mercury, manganese and nickel (Templeton 1991) [more controversially also by magnesium manganese and fluoride ions (this latter circumstance might require that a specific pro-CFS effect of excess fluoride intoxication might be anticipated)]. HS biosynthesis seems highly sensitive to a negative perturbation by elevated glucose (which has been reported to inhibit glomerular HS production (Morano et al 1999) and putatively is a major mechanism of tissue damage in diabetes. Trace lead and mercury which could also be a consequence of the (inappropriate?) use of dental amalgams; this circumstance might significantly adversely affect endothelial HS integrity in genetically susceptible individuals. It is suggested that it may be worthwhile to study the effect of mixtures of environmental toxins including the above with the aim of establishing if synergistic effects arise between the above toxins and other agents such as plasticizers, organohalogen flame retardants and organophosphate insecticides, titanium, aluminium, silicates and silicone implant matrices etc. The net effect upon endothelial HS biosynthesis (as well eg as a function of genetic variability) is suggested to be a useful indicator of the existence of an aetiology based on HS dysfunction. Glucosamine dietary supplementation, it may also be putatively hypothesised, could additionally counter atherosclerosis and other related illnesses by a straightforward mechanism based on the enhancement of the biosynthesis of HS (McCarty 1997). Ascorbate may act similarly by selectively boosting highly sulphated HS biosynthesis (Edward & Oliver 1983) and exogenous heparin administration has been indicated to signal (as it were as a hormone) for an augmentation of anticoagulant (antithrombin (III) site rich) HS pentasaccharide segment biosynthesis within endothelial cells (Pinhal et al 1998). The hormone-like fragments of HS (i.e. HS oligosaccharides) may act as cell-cell signalling molecules analogously to the signalling in plants which have been ascribed to the hormone effects of oligosaccharides (McNeil et al, 1984); this may allow for the possible intervention in pathologies by specific HS oligosaccharides which hitherto have demonstrated a variety of non-anticoagulant benefits by unknown mechanisms, such as the heparin-like oligosaccharides which been reported to produce an improvement in cognitive function in humans or in a rat model of Alzheimers disease. The administration of such heparin/HS-like hormone-like neurological dysfunction-correcting glycosaminoglycans (Ferrero et al 1989) may perhaps be effective when orally administered, however, only when the oligosaccharides are administered in the form of their conjugates with suitable chemical carriers. Prior to the discovery that nitric oxide is a major animal biological signalling messenger, a wellestablished and highly reliable test of the occurrence of HS in glycosaminglycan mixtures was the observation of the deaminative cleavage of HS by nitrous acid (e.g. Shively & Conrad 1970, Delaney & Conrad 1983, Bienkowski & Conrad 1985). [The fractionation of the oligosaccharides formed by this nitrosative scission permits approximate sugar sequencing to be determined in these linearly sugar microstructure sequence-encoded information carriers]. While this reaction is routinely accomplished in the lab in vitro at pH 1.5, a more selective deamination seems to occur at pH 5.6 and perhaps also at higher pH values (cf Vilar et al 1997). The latter deaminative cleavages were also apparently found to be highly sensitive to the chemical nature of the buffer solutions used, suggesting that small amounts of iron and other redox metals which co-occur with those (phosphate-based) buffers which promote nitrosative cleavage at physiological pH, achieve this cleavage critically because of contamination of the buffer by trace iron and other redox metal ions. This phenomenon is obviously conceivably highly relevant to pro-atherosclerosis and pro-CFS situations as it could mimic the perhaps common modestly iron overload conditions which are associated with Western diets and lifestyles.

There is currently a growing awareness of the possibility that deaminative cleavage of HS is part of a intra and extracellular signalling system involving ascorbate, copper and zinc ions as well as polyamines which apparently direct the biosynthesis of HS chains containing designed amounts of free GlcNH2 groups pre-primed for nitrous acid deaminative cleavage (Mani et al 2000; Ding et al 2001. {N.b. these authors discovered that ascorbate can boost of HS recycling by the promotion of Cu(I) redox cycling in which Cu(II) reduction by ascorbate stimulates a nitric oxide release from thiol stores (including those present in the HSPG core proteins). Belting et al 2001 discussed how HS regulates polyamine cellular uptake which therefore may also impinge on nitrosative signalling via HS alteration effects. Aberrant control of nitrous acid generating systems could lead to the dysfunction of this system and such effects may be relevant to various degenerative and chronic disease processes. 6. Tyrosine-3-O-NO2 Previously assumed to be a specific indicator for the occurrence of peroxynitrite, the formation of tyrosine-3-O-NO2 in proteins also indicates the potential in vivo occurrence of excess nitrous acid [which is reported to nitrate tyrosine as well as the phenolic groups present in dietary flavenoid and polyphenol antioxidants (Oldreive et al 1998); (such substances could serve as auxiliary anti nitrous acid protectives)]. Tyrosine nitration is also observed to be produced by nitrite plus HOCl (cf Whiteman et al 1999) which confirms he notion that HOCl might contribute to an in situ nitrous acid formation mechanism which arises as a consequence of cellular immunological HOCl secretions which, under chronic immunological activation conditions may overwhelm the various anti-nitrous acid protective systems on which the integrity of the multi-functional tissue protection afforded by HS ultimately depends. [It should be noted that NO2 is also a potential in vivo reagent for the formation of tyrosine-3-ONO2 from tyrosyl radicals generated by immunological myeloperoxidase activity also involved in HOCl production (Van Dalen et al 2000)]. 3-nitrotyrosine in the urine of rats is reported to arise from an exposure to sarin and pyridostigmine (Abu-Quare et al 2001). Intoxication by these agents has been associated with a CFS-like condition (Gulf war syndrome). We now suggest that a chronic pro-oxidant state exists in these afflictions which might perhaps better named as a pro-nitrant states coupled with a dyshomeostasis (or intoxication by) redox metal ions (copper, iron and perhaps should also tentatively include manganese as well as nickel, vanadium, titanium and silver) which are potential catalysts for various nitrosation and mechanistically related HS deamination processes which initiate a cascade of depletion of HS tissue protection mechanisms. A related HS-depeletion process may also arise from the effect of thiocyanate present in body fluids at concentration levels which can be correlated with the intensity of tobacco smoking. (It should be noted that thiocyanate catalyses the chemical reaction process of organic molecule nitrosylation (Fan & Tannenbaum (1973); McColl et al 2000); this reaction may perhaps also be promoted the presence of trace amounts of free iron ions. These two mechanisms provide a link between nitrosative damage to HS and the promotion of degenerative diseases as a result of tobacco smoking. 7. Potential Anti-RNOS Protection Mechanisms The potentially severe tissue-damaging activities of the various reactive nitrogen oxygen species (RNOS) which include peroxynitrite and nitrous acid (and may also be subject to thiocyanate and redox metal catalysis) suggests that, in animal tissues, there must normally exist a range of physiological antinitrant protection systems. Failure of such systems (including dietary insufficiencies) could be part of the cause of the promotion of a number of diseases, especially those in which HS biochemistry is implicated, but the aetiology of which have hitherto been a puzzle, and which are now suggested to include the immunological dysfunctional phenomena which apparently give rise to ME-CFS.

[Major anti nitric oxide metabolite protection will be provided inter alia by haemoglobin (forming stable nitric oxide adducts) and the thiol adduct and reductant activity of glutathione as well as by the unique reducing power of ascorbate for nitrite reconversion to nitric oxide]. Anti-nitrant protection may also be provided in vivo by those proteins which containing conjugated cysteine-tyrosines which allow stable radical formation. Such systems may also possess redox metal electron transfer potential similar to that employed in the reactive centre of ribonucleotide reductase which has been suggested (Grant et al 1989a) to serve as in vivo antioxidants. An example is the multi-kringle apo lipoprotein a (apo-Lp(a)) ) (such kringle structures can also bind to the glycosaminoglycans heparin and heparan sulphate (Stephens et al 1992)). apo-Lp(a) is proposed to initially attach RNOS at cysteine for subsequent promote adjacent tyrosine nitration . Although Lp(a) is a strong marker of oxidative damage, or even a pathogen (but by an unknown mechanism (Romero et al 2000)), the associated apoLp(a) protein has a likely antioxidant/antinitrant function. It could even be a surrogate for ascorbate (Rath & Pauling, 1990) since it has evolved in species which have recently lost the ability to synthesise this important chemical reductant, antinitrant and antioxidant. Under glutathione deficiency conditions, associated with chronic viral infections, recycling of oxidised ascorbate will, it should be noted, also be compromised. An established benefit of ascorbate is its strengthening effect on the extracellular matrix (ECM) by the promotion of collagen hydroxylation; an additional known property of ascorbate, however of potential relevance to extracellular matrix supramolecular structure and the etiology of ME-CFS, is its effect on augmentation and modulation of HS biosynthesis (Edward & Oliver1984, Kao et al 1990); ascorbate may also be involved in intracellular HS biochemistry as a reductant for conversion of Cu2+ to Cu+ which has been indicated to increase the catalytic activity of Cu ion for release of nitric oxide from intracellular thiol stores. Normal nitric oxide metabolite processing of HS at unsubstituted GlcNH2 groups, is apparently influenced by polyamine signalling (Mani et al 1989, cf. Ding et al 2000). Dysfunction of anti-nitrant protection, it is now suggested, will lead to damaging protein tyrosine nitration such as that most enhanced in ALS (Ferrante et al 1997) where nitrite/nitrous effects could produce nerve demyelination (cf Zielasek et al 1992) a process which may be relevant to tissue damage associated with ME-CFS. 8. Suggested Further Experiments New techniques are required to monitor the possible formation of nitrous acid and other RNOS species in vivo. Further experimental work is needed to advance knowledge of the relevance of nitrous acid as a pathological agent and if apo-Lp(a) and similar activated tyrosines are endogenous anti-nitrous acid protection agents and of relevance for design of improved therapeutic interventions. A candidate for such further research might seek to answer the question: Does nitrosation of the (HS and divalent cation dependent) endothelial adhesion molecule PECAM-1 (DeLisser et al 1993) damage its function and promote hypercoagulable states? Nitrous acid has long been known to mutate nucleic acids and cleave HS by attacking glucosamine C2 residues. The reaction mechanisms of these reactions are not fully understood but likely involve RNOS derivatives other than nitrite/nitrous acid per se. Further studies are also required to advance knowledge in this field. Since peroxynitrite/peroxynitrous is believed to equilibrate with nitrite/nitrous acid under physiological conditions (Pfeifer et al 1997) any initially formed peroxynitrite, generated from nitric oxide plus superoxide anion, may generate active RNOS intermediates in common with nitrous acid. Further research is urgently required to establish the mechanism of such nitrous acid reactions in vivo. [A clue about potential reactive intermediates involved in such nitrous acid damage, however, seems to be offered by the observation that a similar deaminative cleavage of intact heparin to that expected for nitrous acid is readily produced by nitric oxide (not evidently requiring the pre-formation of nitrite or further metabolites) in phosphate (but not when an imidazole buffer was employed) occurs at physiological pH in vitro (Vilar et al 1997). Further experimental work is also required to probe the relevance of these observations to pathology. We suggest that the trace redox metals inevitably present

in phosphate buffers could have contributed to these unexpected results. A possible explanation might be that a formation of local acidosis creates the acidic conditions previously assumed to be necessary to generate nitrous acid from nitrite, but the formation of an active redox metal RNOS adduct should not be discounted as a substituted glucosamine activator since the in vivo intracellular nitrite scission of heparan sulphate, evidently used for signalling, in and between endothelial cells studied in culture, can be inhibited by copper chelators (Mani et al 2000); furthermore, copper binding to heparin seems to be a highly specific process as is the catalysis of oxidative cleavage of heparin (Liu & Perlin 1994). Copper and iron catalysis of de-N sulphonation of heparin which primes for subsequent deaminative cleavage has also been indicated to occur as suggested by preliminary investigations (Grant et al 1995) but this idea requires further study. The importance of redox metal ions and other catalysts for nitrite damage to amines is supported by older findings that the nitrosation of amines, which occur by mechanisms which are probably related to nitrous acid reactions, are strongly catalysed by various redox ions including of copper, iron and silver (Challis 1978). Inappropriate deamination of HS proteoglycans, as well as the deamination of key amines including pterin derivatives and serotonin, might occur in the aetiology of ME/CFS by attack by a nitrous acid route (or the derived RNOS) process augmented under pro-oxidant, pro-acidosis conditions augmented by the catalytic effects of redox metal (especially copper) ions as affecting blood vessel heparan sulphate acid biochemistry which may further perturb platelet activation known to be dependent on heparin, nitric oxide and subject to redox dependent copper ion catalysis of nitrosothiol activity (Gordge et al 1995). Heparin/heparan may furthermore exist naturally in a multi-counterion form containing many trace redox metal ions (Grant 2000) and part of its function may be to act for a sink to prevent redox disturbance, a function at risk from aberrant scission of these polysaccharides by RNOS which may allow release of catalysts which promote further nitrant damage. As noted above, phosphonate therapy by replacing some of the activitiesof HS, might potentially alleviate the symptoms of ME-CFS. Such phosphonates are well-established pharmacological agents. 9. NOTES Other diseases than CFS which might be promoted by nitrous acid, in addition to atherosclerosis include multiple sclerosis (MS), Alzheimers disease (AD), cystic fibrosis (CF), amylotrophic lateral sclerosis (ALS), rheumatoid arthritis (RA), fibromyalgia (FM) and systemic lupus erythematosus SLM). HS proteoglycans, are potentially involved in both antioxidant (Adachi et al 1995) and anti-nitrant damage limitation, are themselves sensitive to damage of specific structures such as glucosamine-N-SO3- required for specific protein interaction with HS chains. It is now proposed such damage can be caused by redox-metal-catalysed acidosis as well as RNOS disruption of key signalling by poly and oligosaccharides derived from HS. a- SUMMARY Chronic Fatigue Syndrome(CFS)-Myalgic Encephalomyelitits(ME) is a RNOS Disease The etiology of CFS (termed myalgic encephalomyelitis (ME) in the UK) and related afflictions, the incidence of which is reported to be higher in women than in men (which is consistent with the sex ratios of nitric oxide generation), was formerly a mystery, but is currently believed to be due to a systemic chronic dysfunction initiated by pathogen lipopolysaccharide etc. cytokine stimulated nitric oxide secretion (with an associated increased pterin derivative markers effect). A herpes viral infection mechanism could cause a diminution of key (antioxidant, antithrombotic and antinitrant) heparan sulphate tissue protective activity at endothelial surfaces. Excess nitrogen oxides of redox states increased by the induced pro-oxidant milieu are believed to promote a disturbance of disulphide balance (including lipoic acid and glutathione), ion channels (with a possible alteration in calcium - magnesium systems), neuroendocrine, mitochondrial DNA, and the haemostatic balance (a hypercoagulability state seems to be induced which may include abnormal platelet activation), perturbation of the Krebs cycle [alteration in urinary succinate (Clifton et al 2001)

being diagnostic] and other glycolytic pathways, as well as cytochrome p450 liver dysfunction and impairment of the hypothalamic pituitary adrenal axis. Mycoplasmal infections inside blood leukocytes have been reported for CFS and patients of other chronic illnesses (Nicholson et al 2001) which are consistent with the existence of a chronic stimulation of immunolgical nitric oxide metabolite formation.

REFERENCES
[including selected additional refs added when this document was revised May 2013]

[1]
Berg D Berg LH Couvaras J Harrison H (1999) Chronic fatigue syndrome (CFS) &/or fibromyalgia (FM) as a variation of antiphospholipid antibody syndrome (APS): an explanatory model and approach to laboratory diagnosis Blood Coagulation Fibrinolysis 10 435-438 Heparin therapy for CFS Cf Dr David Berg Recent findings in the hypercoagulable state found in CFS Success in treating this condition with heparin. Internet reports

# For other ailments [cf thus annotated refs]

The possible future use of heparin therapy extends to:

[cf thus annotated refs]

Heparin/HS is implicated in wide ranging physiological activity control systems it providing a major mechanism for the regulation of protein function as well as for binding of key proteins at blood vessel walls (antithrombin(III), heparin cofactor II, antioxidant enzymes (superoxide dismutase, and other antioxidant molecules) and the provision of anti pathogen activity (antibacterial antiviral and perhaps antiprion) and inhibition of pathological calcification (Grant et al 1989). List of other proposed functions of include Provision of negative charge Provision of mechanical support Regulation of cell migration adhesion and aggregation Regulation of blood coagulation and platelet activity Regulation of cell growth e.g. provision of reservoir and activity regulation of growth factors e.g. fibroblast growth factors Morphogen in embryogenesis and wound healing Role in development and stabilization of synaptic structure Endothelial regeneration Stabilisation of basement membranes Modulation of fibrillogenesis including in the cornea Interlink between cytoskeleton and ECM Assembly of matrix phosphatidyl-inositol linkage Mediation of transferrin activity Uptake role in antigen presentation Modulation of zinc finger DNA gene activation Pathogen binding and potential antagonism Inhibition of urinary trypsin

Inhibition of urinary bacterial adhesion Inhibition of urinary calcification Inhibition of tumour growth and metastasis Provision of filtration barrier Flow sensing at endothelium

Adachi T & Marklund SL (1989)

Interaction between extracellular superoxide dismutase and sulphated polysaccharides J Biol Chem 264 (15) 8537-8541 Adachi T et al (1995) Heparin -induced release of extracellular-superoxide dismutase form V to plasma J Biochem (Tokyo) 117 (3) 586-590 Cf also (1998) Changes in heparin affinity of extracellular -superoxide dismutase in patients with coronary artery atherosclerosis Biol Pharm Bull 21 (10) 1090-1093 Chem Abs 130 64588h (form V EC-SOD, determined by heparin injection, is depleted in atherosclerosis patients suggesting that depletion of heparan-SOD contributes to this disease) Cf Sandstroem J et al (1993) Biochem J 294 (3) 853-857 Chem Abs 119 200199j

Ischiropoulos H (1998) Arch Biochem Biophys 356 1-11 [2]

There are many reports that viral (e.g. herpes types) and bacterial infections are associated with CFS. Cf Nijs J Nicolson GL De Becker P Coomans D De Meirleir K (2002) High prevalence of Mycoplasma infection among European chronic fatigue syndrome patients. Examination of four Mycoplasma species in blood of chronic fatigue syndrome patients FEMS Immunity & Matrix Microbiol 34 209-214 It should be noted that commonly used antibiotics are ineffective against viruses but that heparan sulphate (heparin-like)-related agents show promise as broad spectrum anti-virals and may be a major part of the endogenous mucus layer which acts as an anti-viral and anti-pathogen system, but dysfunction of which may promote CFS and other illnesses.

Other References Listed Alphabetically According to First-named Author with numbered reference referral to text position

[6-4]

Abu-Qare AW Abou-Donia MB (2001) Combined exposure to sarin and pyridostigmine bromide increased levels of rat urinary 3-nitrotyrosine and 8-hydroxy-2'-deoxyguanosine, biomarkes of oxidative stress Toxicol Lett 123 (1) 51-58 Cf Abu-Qare AW Suliman HB Abou-Donia MB (2001) Induction of urinary excretion of 3-nitrotyrosine , a marker of oxidative stress, following administration of pyridostigmine bromide, DEET (N,N-diethyl-m-toluamide) and permethrin, alone and in combination in rats Ibid 121 (2) 127-134
*[1-3][3-1][9-1]

Adachi T & Marklund SL (1989)

[ 5-5]

Cf. Adachi T (1995)

Adachi T Marklund SL (1989) Interaction between human extracellular superoxide dismutase C and sulphated polysaccharides J Biol Chem 264 (15) 8537-8541 Cf Adachi T et al (1995) Heparin-induced release of extracellular dismutase form V to plasma J Biochem (Tokyo) 117 (3) 586-590 {1-1-2}

Ahn B Han BS Kim DJ Ohshima H (1999) Immunohistochemical localization of inducible nitric oxide synthase and 3-nitrotyrosine in rat liver tumors induced by N-nitrosodiethylamine Carcinogenesis 20 1337-1344
Alcock NM (1982) Proc Int Symp Trace Elem Metab Man Anim 4th 1981 (Publ 1982) p 678 Springer Berlin, Ed: Howell J McG White CL Chem Abs 96 213646j [Pharmaceutical heparin can be a source of elevated plasma Mn2+] [cf Kan et al; Mn3+ in CFS?;] [This reference is one of many which show HS may sequester a wide range of redox metals etc., cf Grant 2000]

Aoyagi T et al (1994) Age-dependent decreases in fibrinolytic enzyme activities in serum of healthy subjects Biol Pharm Biol 17 (2) 348-351 PMID 8205137
*[1-3-1]

Arteel GE Franken S Kapper J Sies H (2000) Binding of selenoprotein P to heparin: characterization with surface plasmon resonance Biol Chem 381 (3) 265-268 Chem Abs 133 85803p (Selenoprotein P antioxidant probably [like SOD] binds to endothelial wall heparan sulphate)

*[5-9]

Ascencio F Hansson H-A Larm O Wadstrm T (1995) Helicobacter pylori interacts with heparin and heparin-dependent growth factors FEMS Immunol Med Microbiol 12 265-272 Cf also J Med Microbiol 38 240
*[3-9]

Au YP Kenagy RD Clowes AW (1992) Heparin selectively inhibits the transcription of tissue-type plasminogen activator in primate arterial smooth muscle cells during mitogenesis J Biol Chem 267 (5) 3438-3444

[x-10]

Baker LP Chen Q Peng HB (1992) Induction of acetylcholine receptor clustering by native polystyrene beads. Implication of an endogenous muscle-derived signalling system J Cell Sci 102 543-555
[y-20]

Bando Y Aki K (1990) Superoxide-mediated release of iron from ferritin by some flavoenzymes Biochem Biophys Res Commun 168 (2) 389-395
Bandel C et al (2002) Abundance of interstitial heparan sulfate in granuloma annulare but not in other mucinous skin diseases J Cutan Pathol 29 (9) 524-528 studied granuloma annulare (GA). These authors commented that shedding seems less evident in those skin diseases where mucin deposition occurs but in which inflammation is absent)]. Comment and follow on internet search. [From 10-14-12 re-reading of notes made for 2003 Chronic Fatigue Syndrome Workshop] (Possible relevance to the fuller understanding etiology of CFS ) INSERTED NOTE Idea: Oligosaccharide hormone dysfunction may promote a range of diseases but the administration of suitable replacement oligosaccharides might combat these disease processes by countering chronic inflammation. GA, IC, FM and ME-CFS vide infra A perturbation of the regulated shedding processes by which heparan sulphate (fragments) modulate the biologic actions of growth factors and cytokines [especially as inflammatory process modulators] may be pertinent to a fuller understanding of (all) inflammatory skin diseases and epithelial surface defect diseases. Putatively these include fibromyalgia and CFS. It should be noted that thyroid dysfunction is a related process which impacts on mitochondrial calcium [energy] dysfunction- putatively this is also implicated in CFS]; It should also be noted that Mycobacterial infection as in CFS can induce GA. {Cf co-occurrence of GA with autoimmune thyroditis (cf Vasquez-Lopez et al (2003) J Am Acad Dermatol 48 (4) 517-520)} Search term autoimmune thyroiditis CFS -> [Cf Mary Shomon & Donna Yeaw September 1997 Chronic Fatigue Syndrome, Fibromyalgia, and Autoimmune Thyroid Disease cited Hilgers A Frank J (1994) Wien Med Wochenschr PMID 7856214 (microsomal thyroid antibodies occur in CFS patients). Hyaluronan and fibromyalgia (FM). Gamace et al Med Hypoth 2000 thyroid dysfunction in FM -> GAG shedding especially serum hyaluronan. [This is likely to co-occur with heparan-like fragments] A suggested diagnostic for fibromyalgia (a CFS-like illness) is alteration of serum GAGs especially increased hyaluronan indicative of chronic inflammation. Another chronic inflammatory disease (the bladder epithelium disease) interstitial cystitis (IC) is reported to be inhibited by the administration of a heparan-fragment-mimetic pentosan polysulfate (an artificially sulphated plant xylan) which may combat HS fragment hormone release dysfunction associated with bladder smooth muscle cell parasympathetic nervous system impulse transmission dysfunction}].

{1-1-1} Beckman JS Ischiropoulos H Zhu L van der Woerld M Smith C Chen J Harrison J Martin JC Tsai M (1992) Kinetics of superoxide dismutase- and iron-catalysed nitration of phenolics by peroxynitrite Arch Biochem Biophys 298 (2) 438-445 Cf Beckman JS et al (1990) Apparent hydroxyl radical production by peroxynitrite: Implications for endothelial injury from nitric oxide and superoxide PNAS USA 87 1620-1624 Bell GA et al (2012) Use of glucosamine and chondroitin in relation to mortality Eur J Epidemiol 27 (8) 593-603 PMID 22828954 This n=77510 study found that current use of glucosamine was associated with a significant decreased risk of death from cancer and a large risk reduction for death from respiratory diseases. {cf also Kantor ED et al PMID 23139249 who in a n=9947 study found that high sensitivity serum C-reactive protein hsCRP concentration, a marker of inflammation, is reduced in glucosamine, chondroitin and fish oil supplement users}

[These studies lend support the hypothesis that dietary glucosamine confers improved health via an augmentation of HS-afforded general tissue protective actions] {Cf also Herrero-Beumont G et al (2007) Glucosamine sulfate in the treatment of knee osteoarthritis symptoms Arthritis Rheum 56 (2) 555-567 confirmed the efficiecy of the use of this supplement for the alleviation of the symptoms of knee osteoarthritis} [5-30] Belting M Persson S Fransson L-A (1999) Proteoglycan involvement in polyamine uptake Biochem J 338 317-323
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Berg D Berg LH Couvaras J Harrison H (1999)

Chronic fatigue syndrome (CFS) &/or fibromyalgia (FM) as a variation of antiphospholipid antibody syndrome (APS): an explanatory model and approach to laboratory diagnosis Blood Coagulation Fibrinolysis 10 1-4 (pre-publicn. internet file)
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Bernfield M Gtte M Park PW Reizes O Fitzgerald ML Lincecum J Zako M (1999)

Functions of cell surface heparan sulfate proteoglycans Annu Rev Biochem 68 729-777 [5-26] Bienkowski MJ Conrad HE (1985) Structural characterization of the oligosaccharides formed by depolymerization of heparin with nitrous acid J Biol Chem 260 (1) 356-365 Chem Abs 102 132387r

Birchall JD Chappell JC (1988) The chemistry of aluminium and silicon in relation to Alzheimers disease Clin Chem 34 265-267
Bishop JR Schuksz M Esko JD (2007) Heparan sulphate proteoglycans fine-tune mammalian physiology Nature 446 1030-1037 [1-7] Boyland E Walker SA (1974) Effect of thiocyanate on nitrosation of amines Nature 248 601-602 [x-9] Brandan E Maldonado M Garrido J Inestrosa NC (1985) Anchorage of collagen-tailed acetylcholinesterase to the extracellular matrix is mediated by heparan sulfate proteoglycans J Cell Biol 101 985-992 Burg A Cohen H Meyerstein D (2000) The reaction mechanism of nitrosothiols with Cu(I) J Biol Inorg Chem 5 (2) 213-217 Chem Abs 133 116355p (Cu(I) and other metal ions and their complexes catalyse the decomposition of nitrosothiols) *Campbell JH Renncik RE Kalevitch SG Campbell GR (1992) Heparan sulfate-degrading enzymes induce modulation of smooth muscle phenotype Exp Cell Res 200 156-167 [5-14] Crdensas A Bernard A Lauwerys R (1992) Incorporation of [35]sulfate into glomerular membranes of rats chroniclly exposed to cadmium and its relation with urinary glycosaminoglycans and proteinuria

Toxicology 76 219-231 Casu B et al (2010) Heparin-derived heparan sulfate mimics that modulate inflammation and cancer Matrix Biol 29 (6) 442-52 [PMCID 3057403] {x-4-n} Cathcart RF (1991) A unique function for ascorbate Medical Hypotheses 35 32-37 (Cf The Lancet (1990) 335 235 glutathione deficiency associated with chronic HIV infection countered by massive ascorbate administration; chronic infection by other viruses may similarly cause glutathione depletion) [1-5] [8-7] Challis BC Edwards A Hunma RR Kyrtopoulos SA Outram JR (1978) Rapid formation of N-nitrosamines from nitrogen oxides under neutral and alkaline conditions IARC Sci Publ 19 (Environ Aspects N-Nitroso Compounds) 127-142 Chem Abs 89 1296583c Cf also Challis BC Outram JR (1978) Rapid formation of N-nitrosamines from nitric oxide in the presence of silver(I) salts JCS Chem Commun 16 707-8 [5-16]{x-4-n} Chang MY Potter-Perigo S Tsoi C Chait A Wight TN (2000) Oxidized low density lipoproteins regulate synthesis of monkey aortic smooth muscle cell proteoglycans that have enhanced native low density lipoprotein binding properties J Biol Chem 275 (7) 4766-4773 [5-10] Chmiela M Paziak-Domanska B Rudnicka W Wadstrm, T (1995) The role of heparan sulphate-binding activity of Helicobacter bacteria in their adhesion to murine macrophages APMIS 103 469-474

[9-2] Clifton BP McGregor NR Fulcher G Dunstan RH King K Niblett S Hoskin L Roberts TK Butt HL Dunsmore J Klineberg J (2000/1) Deregulation of succinate metabolism in a cohort of patients with defined chronic fatigue syndrome [Alison Hunter Memorial Foundation Conference, December 2000, Sydney, Australia http://www.ahmf.org/0.1cliftonbligh.html Internet report of conference see: http://www.estead.com/files/Third] [5-12] Colburn P Dietrich CP Buonassisi V (1996) Alterations of heparan sulfate moieties in cultured endothelial cells exposed to endotoxin Arch Biochem Biophys 325 (1) 129-138 David G Bai XM Van der Schueren B Cassiman J-J Van Den Berge H (1992) Developmental changes in heparan sulfate expression: in situ detection with monoclonal antibodies (Differential binding and regulation requires -NSO3- groups of heparan sulphate) J Cell Biol 119 (4) 961-975 *David G Bernfield M (1998) The emerging roles of cell surface heparan sulfate proteoglycans Matrix Biol 17 (7) 461-463 Chem Abs 130 149916p (Includes a review of pathological organisms which bind to cell surface heparan sulphate proteoglycans) *de Beer F Hendriks WL van Vark LC Kamerling SWA van Dijk KW Hofker MH Smelt AH Havekes LM (1999) Binding of VLDL to heparan sulphate proteoglycans requires lipoprotein lipase, whereas apoE only modulates binding affinity Arterioscler Thromb Vasc Biol 19 (3) 633-637 Chem Abs 131 1911h

*De Clercq E (1990)

Trends Pharmacol Sci [TiPS] 11 198-205 (Antiviral effect of heparin and fragments including those produced by nitrous acid may exhibit broad antiviral activities inhibiting HIV-1 and HIV-2, cytomegalovirus, herpes simples including mutants resistant to acycolvir, togaviruses and rhabdoviruses). [5-25] Delaney SR Conrad HE (1983) Changes in disaccharide composition of heparan sulphate fractions with increasing degrees of sulphation Biochem J 209 315-322 (One of the many classical studies using nitrous acid degradation of heparan sulphate) [8-1] DeLisser HM Yan HC Newman PJ Muller WA Buck CA Albelda SM (1993) Platelet/endothelial cell adhesion molecule-1 (CD31)-mediated cellular aggregation involves cell surface glycosaminoglycans J Biol Chem 268 (21) 16037-16046 de Saram K McNeil KL Khokher S Ritter JM Chowienczyk (2002) Divergent effect of vitamin C on relaxation of rabbit aortic ring to acetylcholine and nitric oxide donors Brit J Pharmacol 135 1044-1050 *Dhoot GK Gustafsson MK Ai X Sun W Standiford DM Emerson CP Jr (2001) Regulation of Wnt signaling and embryo patterning by an extracellular sulfatase Science 293 1663-1666 (Heparan sulphate regulates avian growth factor signalling via post synthetic processing; this potentially opens a window for extracellular environmental factor alteration of this process) Dietrich CP Cf Nader HB et al (1987) Heparin sequences in the heparan sulfate chains of an endotheial cell proteoglycan PNAS USA 84 (11) 3565-3569 Cf also Nader HB et al (1999) Brazilian Journal of Medical and Biological Research 32 (5) 529-538

Ding K et al (2001) N-unsubstituted glucosamine in heparan sulfate of recycling glypican-1 from suramin-treated and -deprived endothelial cells J Biol Chem 276 (6) 3885-3894 Ding K Sandgren S Mani K Belting M Fransson L-A (2001) Modulation of glypican-1 heparan sulfate structure by inhibition of endogenous polyamine synthesis. Mapping of spermine-binding sites and heparanase, heparin lyase and nitric oxide/nitrite cleavage sites ibid, 276 (50) 46779-46791; cf Mani K et al (2006) Glycobiol 16 (8) 711-718
[5-29][7-8]

Di Stasi AM Mallozzi C Macchia G Petrucci TC Minetti M (1999) Peroxynitrite induces tyrosine nitration and modulates tyrosine phosphorylation of synaptic proteins J Neurochem 73 727-735
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*Drzeniek Z Stcker G Siebertz B Just U Schroeder T Ostertag W Haubeck H-D (1999) Heparan sulfate proteoglycan expression is induced during early erythroid differentiation of multipotent hematopoietic stem cells Blood 93 (9) 2884-2897 [3-3] Ebara T Conde K Kako Y Liu Y Xu Y Ramakrishnan R Goldberg IJ Schachter NS (2000) Delayed catabolism of apoB-48 lipoproteins due to decreased heparan sulfate proteoglycan production in diabetic mice J Clin Invest 105 (12) 1807-1818

*Edelberg MJ Conrad HE Pizzo SV (1991) Heparin oligosacchrides enhance tissue type plasminogen activator: a relation between oligosaccharide length and stimulation of plasminogen activation Biochemisty 30 (4) 10999-11003 Chem Abs 115 229145s (Smaller oligosaccharides bind exclusively to tissue type plasminogen activator, the larger interact with both this and plasminogen) [4-5][7-5]{x-4-1} Edward M Oliver RF (1984) Ascorbate increases the sulphation of glycosaminoglycans synthesised by human skin fibroblasts Biochem Soc Trans 12 304; cf. J Cell Sci 1983 64 245-254 [5-20] Cf ibid (1983) 11 383 and [x-4] Kao J Huey G Kao R Stern R (1990) Ascorbic acid stimulates production of glycosaminoglycans in cultured fibroblasts Exp Mol Pathol 53 1-10 PMID 2209807 (Heparan sulphate is boosted by ascorbate) cf also Hornig B et al (1998) Vitamin C improves endothelial function of conduit arteries in patients with chronic heart failure Circulation 97 (4) 363-368 [5-20] Edward M Oliver RF (1983) Changes in synthesis and distribution of sulphated glycosaminoglycans in human skin fibroblasts upon ascorbate feeding J Cell Sci 64 245-254 Biochem Soc Trans 11, 386

[3-2-a][3-7]

Engleberg H (1984)

Heparin and the atherosclerotic process Pharmacol Rev 36 (2) 91-102 [3-7-1] *Engleberg H (1999) Actions of heparin that may affect the malignant process Cancer 85 (2) 257-272 (Review of anti-metastasis, apoptosis, reverse transcriptase, telomerase and topoisomerase (modulations by heparin) [1-1-1-n] Espey MG Miranda KM Thomas DD Xavier S Citrin D Vitek MP Wink DA (2002) A chemical perspective on the interplay between NO, reactive oxygen species and reactive nitrogen oxide species Ann New York Acad Sci 962 195-206

Exley C et al (2009) A role for body burden of aluminium in vaccineassociated macrophage myofascitis and chronic fatigue syndrome Med Hypotheses 72 (2) 135-139 [A single case was reported where the potential dangers associated with aluminium adjuvants were

identified. This provides a posssible mechansim by which the cascade of immunogloical events which are associated with autoimmune conditions including chronic fatigue syndrome, may arise]
[1-1-1][1-8][6-5] Fan T-Y Tannenbaum SR (1973) Factors influencing the rate of formation of nitrosomorpholine from morpholine and nitrite: Acceleration by thiocyanate and other anions J Agric Food Chem 21 (2) 237-240 [7-9][1-1-1] Ferrante RJ Browne SE Shinobu LA Bowling AC Baik MJ MacGarvey U Kowall NW Brown RH Jr Beal MF (1997) Evidence of increased oxidative damage in both sporadic and familial amyotrophic lateral sclerosis J Neurochem 69 2064-2074 [1-1-1] Ferrante RJ Shinobu LA Schulz JB Mathews RT Thomas CE Kowall NW Gurney ME Beal MF (1997) Increased 3-nitrotyrosine and oxidative damage in mice with a human copper/zinc superoxide dismutase mutation Ann Neurol 42 326-334 #[5-23] Ferrero ME Santini V Mantovanti M Prino G (1989) Does successful treatment with glycosaminoglycan polysulfate in senile dementia indicate the pathogenic mechanism of the disease? Biochem Soc Trans 17 360 (Ateroid, a HS containing glycosaminoglycan mixture, used therapeutically for AD) (cf Lorens SA Guschwan M Hata N van de Kar LD Walenga JM Fareed (1991) Behavioural, endocrinal, and neurochemical effects of sulfomucopolysaccharide treatment on the aged Fischer 344 rat Semi-Throm Hemost 17 supplem 2 164-173 [3-2] Feyzi E Saldeen T Larsson E Lindahl U Salmivirta M (1998) Age-dependent modulation of heparan sulfate structure and function J Biol Chem 273 (22) 13395-13398 #Fillit H Lahita R (1991) Antibodies to vascular heparan sulfate proteoglycan in patients with systemic lupus erythematosus Autoimmunity 9 159-164 [1-1-1m] Francoeur C Denis M (1995) Nitric oxide and IL8 as inflammatory components of cystic firosis (CF) Inflammation (New York) 19 (5) 587-598 Chem Abs 124 6745x
[2-8][5-13]

Fujiwara Y Kaji T (2002)

Suppression of proteoglycan synthesis by calcium ionophore A23187 in cultured vascular endothelial cells: implication of intracellular calcium accumulation in lead inhibition of endothelial proteoglycan synthesis J Health Sci 48 (5) 460-466 Cf also Toxicology (1999)133 (2,3) 159-169 (Lead inhibits endothelial heparan sulphate biosynthesis probably via a calcium signalling effect) Fujiwara K Kaji T (1999) Lead inhibits the core protein synthesis of a large heparan sulfate proteoglycan perlecan by proliferating vascular endothelial cells in culture

Toxicology 133 (2.3) 159-169 Chem Abs 131 154569c Fujiwara Y Tsumura N Yamamoto C Kaji T Differential effects of cadmium on proteoglycan synthesis of arterial smooth muscle cells: increase in small dermatan sulfate proteoglycans , biclycan and decorin, in the extracellular matrix at low cell density Toxicology 2002 170 (1-2) 89-101 (Cadmium causes atherosclerosis in experimental animals. This could be arise because of the ability of cadmium at non cytotoxic levels to alter the composition of glycosaminoglycans in favour of those which accumulate at vascular walls in atherosclerosis (where an increase in small chrondoritin/dermatan proteoglycans especially biglycan and decorin and a decrease in other proteoglycans including heparan sulphate which was confirmed by vascular smooth muscular cell culture experiments. [5-4] Fukuchi K-I Hart M Li L (1998) Alzheimers disease and heparan sulphate proteoglycan Froniers in Bioscience 3 d327-337 *# Gabizon R Meiner Z Halimi M Ben-Sasson SA (1993) Heparin-like molecules bind differentially to prion-proteins and change their intracellular metabolic fate J Cell Physiol 157 (2) 319-325 *Gilat D Hershkoviz R Goldkorn I Cahalon L Korner G Vlodavsky I Lider O (1995) Molecular behaviour adapts to context: heparanase functions as an extracellular matrix-degrading enzyme or as a T cell adhesive molecule, depending on local pH J Exp Med 181 (5) 1929-1934 Chem Abs 122 288881w [x-2] Goldstein JA (1993) Nitroglycerin: A potent mediator for hyperfusion in CFS Cf Chronic Fatigue Syndrome: The limbic hypothesis The Haworth Medical Press CA USA (Internet) The Haworth Medical Press Vol 1 in the Haworth Library of the medical /neurobiology of somatic disorders http/www.me-cvs.nl/index.php?pageid=30228printlink=true8highlight=cfs [8-8][y-18] Gordge MP Meyer DJ Hothersall J Neild GH Payne NN Noronha-Dutra A (1995) Copper chelation induced reduction of the biological activity of S-nitrosothiols Br J Pharmacol 114 (5) 1083-1089 Chem Abs 122 204815s Cf Pietraforte D Mallozzi C Scorza G Minetti M (1995) Role of thiols in the targeting of S-nitrosothiols to red blood cells Biochemistry 34 (21) 7177-7185 Chem Abs 122 289483f (Platelet activation studies)
[y-29-1]

Grant D Long WF Williamson FB (1987) Pericellular heparans may contribute to the protection of cells from free radicals Med Hypoth 23 67-71
[3-5][5-7] [7-1][x-6][y-42]

Grant D Long WF Williamson FB (1989a) A comparison of antioxidant requirements of proteins with those of synthetic polymers suggests an antioxidant function for clusters of aromatic and bivalent sulphur-containing amino acid residues Med Hypoth 28 245-253
[During a meeting which had been set up in 2003 to extend the scope of this 1989 hypothesis (that cysteine-tyrosine (C..Y) couplings are broad spectrum antioxidants) to include anti- nitric oxide metabolite actions of CY, a coauthor of the above paper who had been diagnosed with CFS requested that further literature researches [on the C..Y antioxidant hypothesis (now being carried on informally)) should then become re-focussed on the etiology of CFSs. Of possible relevance to this task it was considered that the iron ion binding

ability of C..Y s and their reaction products seemed likely to create a system of antioxidant and antinitrant defence via a pacification of iron and other redox metal ions combined with the ability of S=O containing molecules (e.g. sulphated polysaccharides and the oxidation products of cysteine (acting similarly to the S=O group in taurine which is known to possess antioxidant protection activity). The facility for tyrosine enzymatic phosphorylation or sulphation blockage by tyrosine nitration (which is a well-known major marker of oxidative stress) was also thought to be of possible relevance. The systems of cysteine-tyrosine juxtapositons in proteins might therefore serve as a front line anti-active nitrogen as well as an anti-active oxygen defence system. Could heparin + CY (synergistic/) antioxidant/antintrant agents as well as offering novel etiological insights into CFS also offer new therapies for these mystery afflictions?]

*[y-28]

Grant D Long WF Williamson FB (1989b) Inhibition of glycosaminoglycans of CaCO3 (calcite) crystallization Biochem J 259 41-45
[y-28-1]

Grant D Long WF Williamson FB (1992a) Degenerative and inflammatory diseases may result from defects in antimineralization mechanism afforded by glycosaminoglycans Med Hypoth 38 49-55
[Free-radical generating sites (e.g. adsorbed redox metal ions including iron at calcium salt crystal surfaces) were suggested to arise pathologically and promote tissue damage as an ultimate result of defects in heparan sulphate afforded anti-crystallization activities. The accumulation of iron ions in joints and other local cartilage areas apparently predisposes such tissues to form apatite-like structures on calcium carbonate surfaces if the process is not inhibited. Such pathologically formed hydroxyapatite putatively further promotes degenerative diseases including cancer]

Grant D Long WF Williamson FB (1992) A putative tole for colloidal silicates in primitive evolution deduced in part from their relevance to modern pathological afflictions Med Hypotheses 38 46-48 [The ability of SiO2 sols to self seed similar daughter particles suggests a similar role in early evolution of life; the modern association of SiO 2 with polysaccharides may be a relict phenomenon from early events in prebiology]
{Cf Draft Silica-Scribd www.scribd.com/doc/104804240/Draft-Silica Sept 32012} [2-3] Grant D Long WF Williamson FB (1992b) A study of Ca2+-heparin complex-formation by polarimetry Biochem J 282 601-604 Grant D Long WF Moffat CF Williamson FB (1992c) Cu2+-heparin interaction studied by polarimetry Biochem J 283 243-246 [8-9] Grant D (2000) Multi-ion content of heparin (David Grant, 10 October 2000/10:47 biochem/0010002) available online at http://preprint.chemweb.com cf. internet search term Alcock NM Proc Int Symp Trace Elem Metab Man Anim 4th 1981 (pub 1982) document, Grant D (2012) INSERTED DOCUMENT 2 for Draft Silica-Scribd www.scribd.com/doc/104804322/2/INSERTED-DOCUMENT-2 [x-18] Grant D Long WF Williamson FB (2002) A study of Mn2+ heparin and Al3+ heparin complex formation by potentiometric titration To be submitted to Biochem J [y-29] Grant D Long WF Mackintosh G Williamson FB (1996) Heparins as essential antioxidants Biochem Soc Trans 24 (2) 1943 Chem Abs 125 75677j

Cf. (1994) Agents Actions 41 Special Conf Issue C214-215 Grant D (2100) ~WRL005-Scribd http://www.scribd.com/doc/37999700 [Polysaccharides whcih act inter alia as tissue protctive systems, which if defective, can promote major illnesses] (2012) A Hypothesis1a37-Scribd, and Doctype HTML Public IaA-Scribd www.scribd.com/doc/85182650/Doctype-HTML-Public-1a-A *Halper J Carter BJ (1989) Modification of human carcinoma S-13 cells by heparin and growth factors J Cell Physiol 141 (1) 16-23 Chem Abs 111188341a (EGF and insulin like growth factor 1 (IGF 1) were not able to overcome heparin induced inhibition of cell growth (due to strong complexing by heparin) but such inhibition was partly overcome by TGF (DATA) and FGF -1 (weaker heparin complexes formed). Heparin-like substances present in the extracellular matrix play an important role in the control of epithelial growth) [1-1-b] Han SW Kim H (1996) Ginsenosides stimulate endogenous production of NO in rat kidney Int J Biochem Cell Biol 28(5) 573-580 Chem Abs 125 75776r [1-1-1] Hanazawa T Kharitonov SA Barnes PJ (2000) Increased nitrotyrosine in exhaled breath condensate of patients with asthma Am J Respir Crit Care Med 162 1273-1276 *Harenby J et al (1994) Haemostaseologia (Stuttgart) 14 (1) 16 Chem Abs 122 311523s (Heparin binding to leucocytes may contribute to antifibrinolytic function of granulocytes, monocytes and lymphocytes)

Haraguchi H (2004) Metallomics as integrated biometal science J Anal At Spectrom 19 5-14

[y-22] Hedlund BE Hallaway PE (1993) High-dose systemic iron chelation attenuates reperfusion injury Biochem Soc Trans 21 340-343 [1-1-1] Heller R Mnsche-Paulig F Grbner R Till U (1999) L-ascorbate potentiates nitric oxide synthesis in endothelial cells J Biol Chem 274 (12) 8254-8260 Chem Abs 131 13677z * Hiebert LM McDuffie NM (1989) The intracellular uptake and protracted release of exogenous heparin by cultured endothelial cells Artery (Fulton, Mich) 16 (4) 208-222 Chem Abs 111 49858k

[1-1-b] Hawkins RD Son H Arancio O (1998) Nitric oxide as a retrograde messenger during long-term potentiation in hippocampus Prog Brain Res 118 155-172 Chem Abs 130 165908t

(Review of NO in brain development, plasticity and disease) #*[1-1-b] Heigold S Sers C Bechtel W Ivanovas B Schfer R Bauer G (2002) Nitric oxide mediates apoptosis induction selectively in transformed fibroblasts compared to nontransformed fibroblasts Carcinogenesis 23 924-941 #* [1-1-b] Hibbs JBJ Westenfelder C Taintor R Vavrin Z Kablitz C Baranowski RL Ward JH Menlove RL McMurray MP Kushner JP Samlowski WE (1992) Evidence for cytokine-inducible nitric oxide synthesis for L-arginine in patients receiving interleukin-2 therapy J Clin Invest 89 867-877
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Himmelreich U Kuchel PW (1997) 13-C NMR studies of transmembrane electron transfer to extracellular ferricyanide in human erythrocytes Eur J Biochem 246 (3) 638-645 (Ascorbate reduction linked to glycolysis. Large differences between apparently healthy subjects) [Cf Erythrocyte sedimentation rates are known to be markedly altered in ME-CFS. Optical microscopy can also show erythrocyte alteration in CFS {Oxidative stress induced haemolysis of erythrocytes e.g. induced by some types of silica sols may be a useful marker of erythrocyte damage}] [y-32][1-1-n] Hoffmann J Haendeler J Aicher A Rssig L Vasa M Zeiher AM Dimmeler S (2001) Aging enhances the sensitivity of endothelial cells toward apoptotic stimuli. Important role of nitric oxide Circ Res 89 709-715 [x-5] {x-4-n} Hornig B Arakawa N Kohler C Drexler H (1998) Vitamin C improves endothelial function of conduit arteries in patients with chronic heart failure Circulation 97 (4) 363-368 *Hornebeck W Lafuma C Robert L Mczr M Mczr E (1994) Heparin and its derivatives modulate serine proteinases (SERPS), serine proteinase inhibitors (SERPINS) balance: Physiopathological relevance Pathol Res Pract 190 (9-10) 895-902 Chem Abs 122 77358t

Iler RK (1979) The chemistry of silica: solubility, polymerization, colloid and surface properties and biochemsitry Wiley
[1-1-1] Ignarro LJ (1992) Haem-dependent activation of cytosolic guanylate cyclase by nitric oxide: a widespread signal transduction mechanism Biochem Soc Trans 20 455-469; cf. Blood Vessels 1991 28 67-73 [x-8] Inestrosa NC Matthew WD Reiness CG Hall ZW Reichart LF (1985) Atypical distribution of asymmetric acetylcholinesterase in mutant PC12 pheochromocytoma cells lacking a cell surface heparan sulfate proteoglycan J Neurochem 45(1) 86-94
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Ischiropoulos H (1998)
Biological tyrosine nitration: a pathophysiological function of nitric oxide and reactive oxygen species Arch Biochem Biophys 356 1-11 [1-1-d] Ishihara M (1998) A new degradative pathway for heparan sulphate proteoglycan Trends Glycosci Glycotechnol 10 (54) 331-332 Chem Abs 130 63940e (The balance between NO and superoxide may determine which glycosaminoglycan component of the ECM is destroyed and explain how NO and reactive oxygen species may participate in the regulation of the ECM metabolism) *Itoh K Nakao A Kishimoto W Takagi H (1995) Heparin effects on superoxide production by neutrophils Eur Surg Res 27 (3) 184-188 Chem Abs 122 306167z (Unfractionated heparin at low doses increases but at high doses suppresses neutrophil superoxide production; low molecular weight heparin has less effect) [3-12] Jarrett DRJ Grosset AB Rowland Payne CME (1989) Ageing as a cause of raised serum ferritin in the absence of disease J Clin Exp Gerontol 11 (3-4) 145-154 *Ji Z-S Sanad DA Mahley RW (1995) Intravenous heparinase inhibits remnant lipoprotein clearance from the plasma and uptake by the liver: in vivo role of heparan sulphate proteoglycans J Lipids Res 36 (3) 583-592 Chem Abs 122 236012u (Heparan sulphate proteoglycans are involved in the uptake of apoE lipoprotein by the liver) *Kaji T Yamamoto C Sakamoto M (1991) Low molecular weight heparin [LMWH] enhances prostacyclin production by cultured human endothelial cells Chem Pharm Bull 39 3368-3369 Chem Abs 116 165989f (Prostacylin potentiates vascular dilation and inhibits platelet aggregation; LMWH but not unfractionated heparin [UFH] induces an increase in proacyclin production. This indicates that the oligosaccharide-type rather than the un-degraded heparin is bio-active under the studied conditions. This points to a similar possible role of oligosaccharides from HS (nitrosative and heparanase) degradation) [2-8-1][y-7] Kaji T Yamamoto C Sakamoto M (1991) Effect of lead on the glycosaminoglycans metabolism of bovine endothelial cells in culture Toxicology 68 249-257 (cf Fujiwara Y Kaji T (1999) ibid 133 (2,3) 15-1699 Chem Abs 131 154569) (Lead decreases heparan sulphate proteoglycan biosynthesis) Kaji T Ohkawara S Nakajimoa M Yamamoto C Fujiwara Y Miyajima S Kiuzynu F (1997) ibid 118 (1) 1-10; Cf Fujiwara Y et al (2000) ibid 154 (1-3) 9-19
[2-8-2]

Kaji T Miyajima S Yamamoto C Fujiwara Y Sakurai S Yamagishi S Sueishi K Yamamoto H (2000) Selective increase in decorin core mRNA level in cultured vascular smooth muscle cells after exposure to advanced glycation end products J Health Sci 46 (3) 223-227 Chem Abs 133 41544p [AGE products was reported to leave the heparan sulphate bearing proteoglycan core mRNAs unchanged while causing augmentation of the putatively pro-atherosclerotic (LDL-binding which tends to be augmented in diabetes) non-heparan sulphate glyosaminoglycans, chondroitin and dermatan sulphate; such proteoglycans can act to cancel the anti-atheroma effects of the heparan sulphate protection system] [7-6][x-4] Kao J Huey G Kao R Stern R (1990)

Ascorbic acid stimulates production of glycosaminglycans in cultured fibroblasts Exp Mol Pathol 53 1-10 PMID 2209807 (Heparan sulphate is boosted by ascorbate) *[4-7] Kan M Wang F To B Gabriel JL McKeehan WL (1996) Divalent cations and heparin/heparan sulfate cooperate to control assembly and activity of the fibroblast growth factor receptor complex J Biol Chem 271 (42) 26143-26148 (related to phosphorylation control) *Kan M Wu X Wang F McKeegan WL (1999) Specificity of factors determined by heparan sulfate in a binary complex with receptor kinase Ibid 274 15946-15952
[LATER COMMENT impairment of nervous system function and wound healing by perturbation, e.g. by Pb2+ Cd2+ Hg2+ Mn3+ or Sb(III, V) of the Ca2+ ( Mg2+ or Mn2+) essential cofactors for FGFR HSPG- assisted processing could promote CFS and other degenerative diseases]

[5-8] Kaner R-J Iozzo RV Ziaie Z Kefalides NA (1990) Inhibition of proteoglycan synthesis in human endothelial cells after infection with herpes simplex virus type I in vitro Am J Respir Cell Mol Biol 2 423-431 [1-1-1] Kanner J Harel S Granit R (1991) Nitric oxide as an antioxidant Arch Biochem Biophys 289 (1) 130-136 (cf also Hinshelwood CN (1958) The structure of physical chemistry, Oxford, the Clarendon Press p 395) *Karlsson K Marklund SL (1988) Extracellular superoxide dismutase association with cell surface bound sulphated glycosaminoglycans Basic Life Sci 49 (Oxygen Radicals in Biol Med) 647-50 Chem Abs 111 131597b Cf Biochem J 242 55 {x-4-n} Karlinsky JB Rounds S Farber HW (1992) Effects of hypoxia on heparan sulphate in bovine aortic and pulmonary artery endothelial cells Circ Res 71 (4) 782-792 Chem Abs 118 78363v (Hypoxia induced decreased sulphation of heparan sulphate but increased the AT(III) binding site content)

Kaartinen K et al (2000) Vegan diet alleviates fibromyalgia symptoms Scand J Rheumatol 29 (5) 308-313

Kerr JR et al (2008) Gene expression subtypes in patients with chronic fatigue syndrome/myalgic encephalomyelitis J Infect Dis 197 1171-1184
[Highly represented functions were haematological disease and function,
immunological disease and function, cancer, cell death, immune response and virus infection; infection clustering of gene expression revealed seven CFS/ME subtypes. Several genes of the IL6 signalling pathways were identified (but not IL6 per se). The theme of neurological disease was indicated including organophosphate effects similar to GWS intoxication in a subset of patients] cf. Zhang L et al. J Clin Pathol 2010 63 153-164 [There was evidence for subtypespecific relationships for Epstein-Barr and enterovirus in ME/CFS]

[x-7][3-10-n] Kry V Orlovsk M Stanckov M Risko M Zlnaj D (1992)

Urinary glycosaminoglycan excretion in rheumatic diseases Clin Chem 38 (6) 841-846 (Diagnostic alteration of urinary GAGs det. by dimethlymethylene blue (but not by alcian blue) in the relative order of the effect: rheumatoid arthritis>osteoarthritis>ankylosing spondylitis>normal subjects) [1-1-d] Kirchner JJ Hopkins PB (1991) Nitrous acid cross-links duplex DNA fragments through deoxyguanosine residues at the sequence 5' -CG J Amer Chem Soc 113 4681-4682 *Kjelln L Lindahl U (1991) Proteoglycans: structures and interactions Annu Rev Biochem 60 443-475 [1-1-c] Kouretas PC et al (1998) Non-anticoagulant heparin increases endothelial nitric oxide synthase activity: role of inhibitory guanine nucleotide proteins J Mol Cell Cardiol 30 (12) 2669-2682 Chem Abs 130 246661j [1-1-b] Korenaga E et al (1994) Laminar flow stimulates TP- and shear stress-dependent NO production. Nitric oxide production by bovine endothelial cells is related to shear stress Biochem Biophys Res Commun 198 (1) 213-219 Chem Abs 120 103328p [3-4] Kramsch DM Aspen AJ Rozler LJ Lynn J(1981) Atherosclerosis: prevention by agents not affecting abnormal levels of blood lipids Science 213 1511-1512 [The apparent prevention of atherosclerosis in primates by the administration of bisphosphonates might also indicate a a potential therapeutic intervention strategy for ME-CFS] [1-1-b] Kroncke K-D Klotz L-O Suscheck CV Sies H (2002) Comparing nitrosative versus oxidative stress toward zinc finger -dependent transcription. Unique role for NO J Biol Chem 277 13294-13301 [3-10-n] Lahiri B Lau PS Pousada M Stanton D Danishefsky I (1992) Depolymerization of heparin by complexed ferrous ions Arch Biochem Biophys 293 54-60 (Ascorbate promotes limited degradation of heparin) (cf Metcalf DD et al (1990) who also found that Fentons reagent and activated phagocytes degraded heparin and also Horner AA (1971) had reported that rat skin heparin was degraded by ascorbate due to the presence of trace [redox] metals) (cf also Nagasawa K Uchiyama H Sato N Hatano A (1992) Chemical change involved in the oxidative-reduction depolymerization of heparin Carbohydr Res 236 165-180 Chem Abs 118 97491b) [3-10-n] Laihev JP (1996) Bio Metals 9 (1) 10 Chem Abs 124 109969t (Fe(II) selectively degrades heparin) *Larnkjaer A Nykjaer A Olivecrona G Thgersen H Ostergaard PB (1995) Structure of heparin fragments with high affinity for lipoprotein lipase binding to 2-macroglobuliin receptor /low-density-lipoprotein-receptor-related protein by heparin fragments

Biochem J 307 (1) 205-214 Chem Abs 122 285358v [x-11] Lauri SE Kaukinen S Kinnunen T Ylinen A Imai S Kaila K Taira T Rauvala H (1999) Regulatory role and molecular interactions of a cell-surface heparan sulfate proteoglycan (Nsyndecan) in hippocampal long-term potentiation J Neurosci 19(4) 1226-1235 [1-1-n] LaVoie MJ Hastings TG (1999) Peroxynitrite- and nitrite-induced oxidation of dopamine: implications for nitric oxide in dopaminergic cell loss J Neurochem 73 2546-2554
Leong Jm et al (1998) Different classes of proteoglycans contribute to the attachment of Borrelia burgdorferi to cultured endothelial and brain cells. Infection Immunity Mar 1998 994-999 (Vance Spence e-mail 22/8/04) (The Lyme disease spirochete recognizes and binds heparan and dermatan sulphate; binding was inhibited by heparinase or heparitinase but not by chondroitinaseABC; binding could be inhibited by heparin)

[3-6] Levy RJ Wolfrum J Schoen FJ Hawley MA Lund SA Langer R (1985) Inhibition of calcification of bioprosthetic heart valves by local controlled-release diphosphonate Science 228 190-192 [The diphosphonate used, ethane hydroxydiphosphonate, exhibits similar anti calcification activity to HS (Grant et al 1989b) which tends to confirm a hypotheis that HS could provide this function in blood and heart vessels] Cf Golomb Gee A SAIO Trans 1986 32 91) 587-590 [1-1-a] Leteux C Chai W Nagai K Herbert CG Lawson AM Feizi T (2001) 10E4 antigen of scrapie lesions contains an unusual nonsulfated heparan motif J Biol Chem 276 (16) 12539-12545 (Such GlcNH2 groups would be expected to rapidly react with nitrous acid) [1-1-b] Li L Chaikof EL (2002) Mechanical stress regulates syndecan-4 expression and redistribution in vascular smooth muscle cells Arterioscler Thromb Vasc Biol 22 (1) 61-68 [3-5] Liang JN Chakrabarti B Ayotte L Perlin AS (1982) An essential role for the 2-sulfamino group in the interaction of calcium ion with heparin Carbohydr Res 106 101-109 *Liang OD Ascencio F Fransson LA Wadstroem T (1992) Binding of heparan sulphate to Staphylococcus aureus Infect Immunol 60(3) 899-906 Chem Abs 116 169797g *Libeu CP Lund-Katz S Phillips MC Wehrli S Herniz MJ Capila I Linhardt RJ Raffa RL Newhouse YM Zhou F Weisgraber KH (2001) New insights into the heparan sulfate proteoglycan-binding activity of apolipoprotein E J Biol Chem 276 (42) 38-44 (Defective binding of ApoE to HSPG is associated with atherosclerosis via a defective liver clearance, a phenomenon which is also relevant to Alzheimers disease neural plaque formation) [1-1-c] Liebel MA White AA (1982) Inhibition of the soluble guanylate cyclase from rat lung by sulfated polyanions Biochem Biophys Res Commun 104 (3) 957-964

*Lind T Tufaro F McCormick C Lindahl U Lidholt K (1998) The putative tumor suppressors EXT1 and EXT2 are glycosyltransferases required for the biosynthesis of heparan sulfate J Biol Chem 273 (41) 26265-26268 Chem Abs 130 64007t [1-6][2-4][3-10] Lipscomb DC Gorman LG Traystman RJ Hurn PD (1998) Low molecular weight iron in cerebral ischemic acidosis in vivo Stroke 29 487-493 [8-5] Liu Z Perlin AS (1994) Evidence of a selective free radical degradation of heparin mediated by cupric ion Carbohydr Res 255 183-191 Cf Rej RN Holme KR Perlin AS (1990) Marked stereoselectivity in the binding of copper ions by heparin. Contrasts with the binding of gadolimium and calcium ions Carbohydr Res 207 143-152 Liu J et al (2006) Antimetastatic effect of a lipophilic ascorbic acid derivative with antioxidation through inhibition of tumor invasion Cancer Chemother Pharmacol 57 584-590 [PMID 16075276] [2-2] Long WF Williamson FB (1979) Glycosaminoglycans, calcium ions and the control of cell proliferation IRCS Journal of Medical Science 7 4239-423 Cf Long WF Research and Scholarship-University of Aberdeen www.abdn.ac.uk/~bch118/publications2003march.doc [1-1-1] Love S (1999) Oxidative stress in brain ischemia Brain Pathol 9(1) 119-131 Chem Abs 98 132290c (Peroxynitrite involved in neurodegeneration via NAD+ depletion, excessive DNA damage and polyadenylation) *Luster AD Greenberg SM Leder P (1995) The IP-10 chemokine binds to a specific cell surface heparan sulfate shared with platelet factor 4 and inhibits endothelial cell proliferation J Exp Med 182 (1) 219-231 Chem Abs 123 53934x [Angiostasis. IP-10 is induced in response to IFN ] *Lyon M Gallagher JT (1998) Biospecific sequences and domains of heparan sulphate and the regulation of cell growth and adhesion Matrix Biol 17 (7) 485-493 *Lyon M Gallagher JT (1994) Heparan sulfate oligosaccharides having hepatocyte growth factor (HGF) binding affinity PCT Int Appl WO 94 21,689 Chem Abs 122 47510u (HGF binds to oligosaccharides with a high proportion of 6-O sulphate hexosamines, claimed as therapeutic agents) Marx G Gilon C The molecular basis of memory. Pt 2: Chemistry of the tripartite mechanism ACS Chem Neurosci (Publicn. date Feb 18 2013) DOI: 10.1021/cn300237r [This paper apparently proposes that (human and other animal) brain function and especially memory, depends on the differentiated binding of inorganic counterion arrays to the extracellular matrix (presumably heparan sulphate {metallome} dominated) which surrounds the neuron.

The univalent and divalent or higher valent metal ion interactions are suggested to respectively create short and long term memory. The authors write we propose a tripartite mechanism to describe the processing of cognitive information (cog-info) comprising the (1) neuron (2) surrounding neural extracellular matrix (nECM) and (3) numerous trace metals distributed therein.... The neuron is encased in a polyanionic nECM lattice doped with metals (n>10) wherein it processes (computes) and stores cog-info. Each [nECM: metal] complex is the molecular correlate of a cognitive unit of information (cuinfo), similar to a computer bit. These are induced / sensed by the neuron via surface ion iontophoretic and electroelastic (piezoelectric) sensors. The generic cuinfo are used by neurons to biochemically encode and store cog-info in a rapid, energy efficient, but computational expansive manner]. This hypothesis could further suggest that the integrity of the HSmetallome (including the completeness of multiple trace inorganic element loading, which is likely to be diet-dependent and vegan-diet promoted) can optimize cognition and memory function; a corollary to this idea is that toxic metal ions e.g. Al 3+ which perturb HS biochemistry may also promote memory-loss (e.g. in neurodegenerative diseases) by a related nECM:Al3+ mechanism. [1-1-1] Mahler HR Cordes EH (1966) Biological Chemistry Harper & Row, New York, cf p 163 (Nitrous acid causes DNA mutagenesis, adenine is transformed to hypoxanthine, guanine to xanthine and cytosine to uracil; related hydrazine-induced processes, with diazonium ion formation and alkylation, stimulate DNA cross-linking; hydroylamine is also a mutagen especially active at converting cytosine to uracil) *Majack RA Bornstein P (1984) Heparin and related glycosaminoglycans modulate the secretory phenotype of vascular smooth muscle cells J Cell Biol 99 1688-1695 * Manaster J Chezar J Shurtz-Swirski R Shapiro G Tendler Y Kristal B Shasha SM Sela S (1996) Heparin induces apoptosis in human peripheral blood neutrophils Br J Haematol 94 (1) 48-52 Chem Abs 125 158180t *Mandil AK et al (1995) Kidney Int 48 (5) 1506 (Heparin induced endothelial cell cytoskeletal reorganization is a possible mechanism for vascular relaxation) [5-28] [7-7][8-4] Mani K Jnsson M Edgren G Belting M Fransson L- (2000) A novel role for nitric oxide in the endogenous degradation of heparan sulfate during recycling of glypican-1 in vascular endothelial cells Glycobiology 10 (6) 577-586 (Cu and NO dependent internal degradation of heparan sulphate during recycling of glypican-1 in vascular endothelial cells) {3-10-n} Metcalfe DD Thompson HL Klebanoff SJ Henderson WR (1990) Oxidative degradation of rat mast-cell heparin Biochem J 272 51-57 (Normal neutrophils, but not those from subjects with chronic granuloma disease, oxidatively degrade rat mast cell heparin (protoeglycan). At low Fe concentrations the Haber Weiss reaction involving superoxide (O2- .) predominates but at high Fe concentrations the Fenton reaction (involving H2O2 + Fe2+ reactions) was indicated to be responsible for the reactive oxygen species which degraded both solubilized and granule-associated heparin protoglycan)

[5-19][y-35]{x-4-n} McCarty MF (1997) Glucosamine may retard atherosclerosis by promoting endothelial production of heparan sulphate PG Medical Hypotheses 48 (3) 245-251 Chem Abs 127 12883u; PMID 10687887 Reported antiatherosclerotic activity of silicon may reflect increased endothelial synthesis of heparan sulfate ibid. 49(2) 175-176 (1999) Vegan proteins may reduce risk of cancer, obesity, and cardiovascular disease by promoting increased glucagon activity ibid 53 (6) 459-485 (2009) with Barroso-Aranda J and Contieras F The low-methionine content of vegan diets may make methionine restriction feasible as a life extension strategy ibid 72 (2)125--128
McCarty also noted in the above paper that Current epidemiology appears to be reasonably consistent with the thesis that animal product ingestion is primarily responsible for the high incidence of Western cancers in urbanized societies MF McCarty in an e-book (2008) entitled Staying Lean and Healthy into Ripe Old Age and in Med Hypoth 2009 72 125-8 [PMID 18789600] notes that the pro-health effects of a vegan diet may also arise by the encouragement of methionine restriction (which is thought to extend lifespan) {DG added comment. The methionine connection to lifespan can also be linked to blood homocysteine via HS}. McCarty had previously noted in 1999 that amino acids from vegan diets have a powerful beneficial pro-health effects compared with amino acids from meat diets. This difference shows up in differences produced in the modulation the secretion of both insulin and glucagon the system responsible for sugar storage and utilization (and thereby allowing correct weight control and blood vessel integrity via the promotion of good HDL cholesterol). McCarty writes: The concept that the high intake of essential amino acids characteristic of Western diets, by colluding with high fat intake to induce excessive insulin growth factor-1 (IGF-1) activity, constitutes a grave risk factor for many deadly diseases Many of the major cancer killers can be characterized as Western cancers based on the fact that their age-adjusted incidences are astoundingly higher in modern urbanized nations than in Asia and other cultures whose traditional diets are low in fats and animal products. Two exceptions should be noted, however, in Western cohort or case-controlled studies, dairy products sometimes appear to be protective with respect to several cancers, quite possibly because these foods are often major dietary sources of calcium and vitamin D. Similarly fish ingestion is sometimes reported as protective, marine fish can be rich in vitamin D as well as in long chain omega-3 polyunsaturated fats that inhibit cancer promotion and growth in numerous animal cancer models. There may, according to McCarty (1999) also be additional benefits from low fat vegan diets. A vegan diet, aside from its deficit of vitamin B12 activity (readily compensated by supplementation)is typically more micronutrient-dense (per calories) than the diets favored by omnivores, high in protective phytochemicals and fiber and usually somewhat lower in fat- especially saturated fat. Fears that a vegan diet may be inadequate in protein quality or quantity were unfounded. Vegans tend to have low serum lipids, lean physiques, later puberty and decreased risk of certain Western cancers and rheumatoid arthritis. A vegan diet, preceded by a one-week juice fast, is a traditional treatment for rheumatoid arthritis and other inflammatory disorders at Nordic health spas. Vegan diets inhibit cardiovascular diseases. Vegan diets may also inhibit the progression of multiple sclerosis].

[3-10-n] McColl KEL Grant J et al Seminar papers on the role of iron, nitric oxide and heparan sulphate dysfunction in degenerative diseases presented at the University of Glasgow (1998,1999) by D Grant (cf. Grant 2000) [5-22] McNeil M Darvill AG Fry SC Albersheim P (1984) Structure and function of the primary cell walls of plants Ann Rev Biochem 53 625-663 *Majack RA Cook SC Bornstein P (1985) Platelet-derived growth factor and heparin-like glycosaminoglycans regulate thombospondin synthesis and deposition in the matrix by smooth muscle cells J Cell Biol 101(3) 1059-1070 {3-10-n} Minetti M Forte T Soriani M Quaresima V Menditto A Ferrari M (1992)

Iron-induced ascorbate oxidation in plasma as monitored by ascorbate free-radical formation. No spin-trapping evidence for the hydroxyl radical in iron-overloaded plasma Biochem J 282 459-465 Mello G et al (2005) Low molecular weight heparin [LMWH] lowers the recurrence rate of preeclampsia and restores the physiological vascular changes in angiotensin converting enzyme DD in women Hypertension 45 (1) 86-91 [1-1-c] Miriami M et al (1995) Promotion of nitric oxide formation by heparin in cultured aortic endothelial cells from spontaneously hypertensive rats Clin Exp Pharmacol Physiol 22 (suppl 1 S146-147) Mishra-Gorur K Castellot JJ Jr (1999) [1-1-p] Heparin rapidly and selectively regulates protein tyrosine phosphorylation in vascular smooth muscle cells J Cell Physiol 178 (2) 205-215 Cf also Trends Glycosci Glycotechnol 10 (52) 193 [1-1-v] Misra A et al (1996) Clin Exp Immunol 104 (3) 406-411 Chem Abs 125 84448n (Dengue virus induced nitrite kills spleen cells by induction of a toxic factor) Cf. Int J Expt Pathol 1996 75 (5) 237-242 [1-1-d] Moeslinger T Friedl R Volf I Brunner M Koller E Spiekermann PG (2000) Inhibition of inducible nitric oxide synthase by oxidised lipoprotein(a) in a murine macrophage cell line FEBS Lett 478 (1,2) 95-99 {1-1-5} {1-1-4} Mohr S Stamler JS Brne B (1996) Posttranslational modification of glyceraldehyde-3-phosphate dehydrogenase by Snitrosylation and subsequent NADH attachment J Biol Chem 271 (8) 4209-4214 (Glycolytic enzymes are modified by nitric oxide) [5-17]{x-4-n} Morano S Guidobaldi L Cipriani R Gabriele A Pantellini F Medici F DErme M Di Maraio U (1999) High glucose modifies heparan sulphate synthesis by mouse glomerular epithelial cells Diabetes Metab Res Rev 15 (1) 13-20 (30mmol glucose (6 x physiol level) produced 50% decrease in proteoglycan biosynthesis mostly centred on the cell layer heparan sulphate proteoglycans) [cf Raats CJI et al (2000) Kidney Int 57 385-400 who found that glomerular heparan sulphate apparently was both diminished in amount and was less sulphated under the conditions of excess blood glucose which occur in diabetes] [3-1][y-30] Murata K Yokoyama Y (1989) Acidic glycosaminoglycans in human atherosclerotic cerebral arterial tissue Atherosclerosis 78 (1) 69-79 Chem Abs 111 151343q (Data were reported but not discussed suggesting a linear depletion of heparan sulphate as a function of age) [1-1-1] Myers PR Minor RL Jr Guerra R Jr Bates JN Harrision DG (1990) Vasorelaxant properties of the endothelium-derived relaxing factor more closely resemble S-nitrosocysteine than nitric oxide Nature 345 161-163 (Nitric oxide in physiological solutions spontaneously forms nitrite but adducts formed between

nitric oxide and cysteine thiols may prevent this. Proteins rich in cysteines may provide antinitrite/nitrous acid protection) [5-1] Nader HB Ferreira TMPC Toma L Chavante SF Toma L Dietrich CP Casu B Torri G (1988) Maintenance of heparan sulphate through evolution. Chemical and enzymic degradation , and 13Cn.m.r. -spectral evidence Carbohydr Res 184 292-300 (cf Dietrich CP et al (1996) Arch Biochem Biophys 325 (1) 129-138 Cf. Heparan sulfate and heparins : similar compounds performing the same functions in vertebrates and invertebrates) Cf also Nader HB et al Brazilian Journal of Medical and Biological Research 1999 32 529-538 [3-10-n] Nagasawa K Uchiyama H Sato N Hatano A (1992) Chemical change involved in the oxidative-reductive depolymerization of heparin Carbohydr Res 236 165-180 Chem Abs 118 97491b *Neyts J Snoeck R Schols D Balzarini J Esko JD Van Schapdael A De Clercq E (1992) Virology 189 (1) 48 Chem Abs 117 62372r (Cytomegolavirus attachment to heparan sulphate) [2] Nijs J et al (vide supra) [2-6][5-11] Nishinga M Ozawa T Shimada K (1993) Inhibition of the expression of anticoagulant heparan sulphate by H2O2. Suppression of heparan sulphate expression by homocysteine in porcine endothelial cultures J Clin Invest 192 (3) 1381-1386 Chem Abs 119 24338u [5-3][y-25] Norgard-Sumnicht K Varki A (1995) Endothelial heparan sulphate proteolgycans that bind L selectin have glucosamine residues with unsubstituted amino groups J Biol Chem 270 (20) 12012-12024 Chem Abs 123 28146j (Free GlcNH2 groups in endothelial but not on CHO heparan sulphates) cf Norgard-Sumnicht K et al (1993) Science 261 480-483 Chem Abs 119 115311r (L-selectin recognises heparin-like chains which can be the physiological ligand mediating leucocyte trafficking) [6-1] Oldreive C Rice-Evans C Paganga G (1998) The effect of dietary flavanoids and phenolics on the formation of nitrotyrosine by acidic nitrite http://www.oxyclubcalifornia.org/OCC1998/1998 abstracts/poster0.7html [Nitrous acid reacts with tyrosine to produce 3-nitrotyrosine in vitro inhibited by dietary antioxidants which may become nitrated. Ascorbate does not inhibit (perhaps since it is not nitrated but phenolic antioxidants (epicatechin>epigallocatechin>ferulic acid >rutin seem to be nitrated more efficiently than tyrosine and are effective anti-nitrants)] [1-1-d] Pacelli R Wink DA Cook JA Krisha MC De Graff W Friedman N Tsokos M Samuni A Mitchell JB (1995) Nitric oxide potentiates hydrogen peroxide killing of E coli J Exp Med 182 (5) 1469-1479 Chem Abs 123 283591n (Nitrous acid may be responsible for the anti-bacterial action of nitric oxide)
[5-8-1][x-6]

Paka L Kako Y Obunike JC Pillarisetti S (1999) Apolipoprotein E containing high density lipoprotein stimulates endothelial production of heparan sulfate rich in biologically active heparin-like domains. A potent mechanism for the antiatherosclerotic action of vascular apolipoprotein E J Biol Chem 274 (8) 4816-4823 (cf Chang MY Potter-Perigo S Tsoi C Chait A Wight TN Oxidised low density lipoproteins regulate synthesis of monkey aortic smooth muscle cell proteoglycans that have enhanced native low density lipoprotein binding properties ibid. 275 (7) 4766-4773)

[1-2]

Pall ML (2000) Elevated, sustained peroxynitrite levels as the cause of chronic fatigue syndrome Med Hypotheses 54 115-125; cf ibid 57 139-145 and J Chronic Fatigue Syndrome 7 45-58 and Ann NY Acad Sci 933 323-329 [1-2] Pall ML (2001) Cobalamin [Vitamin B12] used in chronic fatigue syndrome therapy is a nitric oxide scavenger ibid, 2001 8 39-44 cf http://molecular.biosciences .wsu.edu/Faculty/pall.html Pall ML Satterlee JD (2001) Elevated nitric oxide/peroxynitrite mechanism for the common etiolgy of multiple chemical sensitivity, chronic fatigue syndrome and post-traumatic stress disorder Ann NY Acad Sci 933 323-329 [cf also Internet discussion by J Kamsteeg New theory (Hypothesis of Pall) for induction of chronic fatigue (CFS)? of the Pall hypothesis of the aetiology of CFS [avail confirmed 10/12;
Heparan sulphate binds extracellular superoxide dismutase (SOD) which is released by heparin (cf Ascensio loc cit) this allowing heparin to inhibit the formation of peroxynitrite (which formed from nitric oxide and peroxide anion) and this mechanism may in part explain why heparin seems to dramatically counter CFS in a subset of affected individuals (cf Berg loc cit); also Wright [a UK medic] pers commun ]

*Park PW Reizes O Bernfield M (2000) Cell surface heparan sulfate proteoglycans: selective regulators of ligand-receptor encounters J Biol Chem 275 (39) 29923-29926 [y-37] Pauling L (1991) In: Block G Henson DE Levine M (Eds) Ascorbic acid: biologic functions and relation to cancer Proc Conf Natl Inst Health Bethesda MD Am J Clin Nutr 54 1252S-1298S; cf. J Natl Cancer Inst 83 (8) 547-550, (Whilst this conference supported previous claims of an anti-cancer activity of ascorbate, a possible heparan sulphate mechanism for this was not discussed. Ascorbate boosts heparan sulphate biosynthesis in cell culture (Edward and Oliver (1984); Kao et al (1990)); tumour suppressors EXI and EXTII are heparan sulphate biosynthesis enzymes and heparin shows anti- tumour activity (Englebert 1999)) *#Prez M Wandosell F Colao C Avila J (1998) Sulphated glycosaminoglycans prevent the neurotoxicity of a human prion protein fragment Biochem J 335 369-374 *Perrimon N Bernfield M (2000) Specificities of heparan proteoglycans in developmental processes Nature 404 725-728 (Wnt and Hedgehog signalling is heparan sulphate dependent) [1-1-1a] Peterkofsky B (1991) Ascorbate requirement for hydroxylation and secretion of procollagen: relationship to inhibition of collagen synthesis in scurvy Am J Clin Nutr 54 1135-1140S [1-4][8-2] Pfeiffer S Gorren ACF Schmidt K Werner ER Hansert B Bohle DS Mayer B (1997) Metabolic fate of peroxynitrite in aqueous solution. Reaction with nitric oxide and pH-dependent decomposition to nitrite and oxygen in a 2:1 stoichiometry J Biol Chem 272 3465-3470 Cf. also Pfeiffer S Lass A Schmidt K Mayer B (2001) Protein tyrosine nitration in cytokine-activated murine macrophages

Involvement of a peroxidase/nitrite pathway rather than peroxynitrite Ibid 273 (42) 27280-27282 Cf Pfeiffer S Mayer B (1998) Lack of tyrosine nitration by peroxynitrite generated at physiological pH Ibid 273 (42) 27280-27285
{1-1-1}

Pignatelli B Li C-Q Bofretta P Chen Q Ahrens W Nyberg F Mukeria A Bruske-Hohlfeld I Fortes C Constantinescu V Ischiropoulos H Ohshima H (2001) Nitrated and oxidized plasma proteins in smokers and lung cancer patients Cancer Res 61 778-784
[4-2-1][5-8-1]

Pillarisetti S (2000) Lipoprotein modulation of subendothelial heparan sulfate proteoglycans (perlecan) and atherogenicity Trends Cardiovasc Med 10 60-65 A review of vascular HS (especially perlecan HSPG); HS is well known for it lipid processing roles. A negative regulation of HS by atherogenic molecules is balanced by a positive regulation by antiatherogenic molecules. The pro- and anti- lipoproteins which cause changes in HS core protein biosynthesis and HS metabolism rate can be identified as being the outcome of pro- and antiatherogenic dietary inputs {Apo E and apoE HDL improve HS tissue protection by the modulation of lipoprotein metabolism, monocyte attachment, smooth muscle cell proliferation as well as protection against oxidative and nitrosative damage to tissue, calcification, pathogen binding, thromobosis and nitric oxide regulation. Perhaps anti-atherogenic-lipoprotein-rich diets might also improve ME-CFS symptoms}. [4-2] [5-21] Pinhal MA Walenga JM Jeske W Hoppensteadt D Dietrich CP Fareed J Nader HB (1994) Antithrombotic agents stimulate the synthesis and modify the sulphation pattern of a heparan sulfate proteoglycan from endothelial cells Thromb Res 74 (2) 143-153 Plant, Jane (Prof) Your Life in Your Hands Virgin Books 2002 Purdey, Mark (contination of site by Purdey Nigel) The www.purdeyenvironment.com/CFS%20Page-htm discusses how susceptible indviduals may become affected with CFS as a consequence of toxic exposure from a variery of sources which have increassed in recent decades; these include organophosphate, organochlorine and volatile organic compounds. Mark Purdey proposed (in consultation with the author) a Ba-HS growth factor perturbation mechansim of myelin sheath renewal [this was published in a peer reviewed paper, Medical Hypotheses 2004 62(5) 746754 (an epidemiolgical investigation which suggested that barium intoxication may contribute to the prevalence of multiple sclerosis, including in NE Scotland)] cf www.purdeyenvironment.com [1-1-1] Quijano C Hernandez-Saavedra Castro L McCord JM Freeman BA Radi R (2001) Reaction of peroxynitrite with Mn-superoxide dismutase. Role of the metal center in decomposition kinetics and nitration J Biol Chem 276 11631-11638 [3-7] Ragazzi E Chinellato A (1995) Heparin: pharmacological potentials from atherosclerosis to asthma

Gen Pharmacol 26 (4) 691-701 Chem Abs 122 305698m [7-4] Rath M Pauling L (1990) Hypothesis: lipoprotein(a) is a surrogate for ascorbate Proc Natl Acad Sci USA 87 6204-6207 [1-1-n] Recchia FA Vogel TR Hintze TH (2000) Nitric oxide metabolites accumulates in erythrocytes in proportion to CO2 and HCO3concentrations Am J Physiol 279 (2 Part 2) H852 (Great variability in plasma nitric oxide concentration is dependent on CO2/HCO3-) [1-1] Reiter CD Teng R-J Beckman JS (2000) Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite J Biol Chem 275 (42) 32460-32466 [Mentions prior knowledge of the protective effect of ascorbate and thiols for tyrosine nitration by nitrogen dioxide, decreased phenoxy pKa in certain protein environments and and the catalytic effect of redox metals such as Mn within proteins affecting nitration of adjacent tyrosines]
[Pfeiffer & Mayer (1998) loc cit, had failed to consider the inhibitory effect of urate in an in vitro superoxide anion generation procedure experiment which had failed to demonstrate the nitration of tyrosine at physiological pH but a later study by Pfeiffer et al ofRAW macrophages activated with lipopolysaccharide or zymosan A causing inducible NO synthase expression and observed tyrosine nitration were found not to depend on superoxide production but seemed rather to depend on peroxidase reaction products]

[y-16] Rej RN Holme KR Perlin AS (1990) Marked stereoselectivity in the binding of copper ions by heparin. Contrasts with the binding of gadolinium and calcium ions Carbohydr Res 207 143-152 [7-3] Romero FI Khamashta MA Hughes GR (2000) Lipoprotein(a) oxidation and autoantibodies: a new path in atherothrombosis Lupus 9 (3) 206-209 The augmentation of Lp(a) in response to nitrosative stress evidently (Moeslinger et al 2000) induces a down regulation of induced nitric oxide synthase in murine macrophages by oxidsed Lp(a)). (A review confirming a conventional view that lipoprotein(a) presents an outstanding pathogen, acting by a unknown mechanism (or is an outstanding marker of other agent such as nitric oxide metabolite tissue injury?) High plasma levels are associated with (arterioslclerotic) chronic inflammatory attack on the arterial wall (oxidised low density lipoprotein) also relevant to the aetiology of anti-phospholipid (Hughes) syndrome (APS) and systemic lupus erythematosis (SLE)) [1-1-n] Rosen DR Siddique T Patterson D Figlewicz DA Sapp P Hentati A Donaldson D Goto J ORegan JP Deng HX et al (1993) Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis Nature 362 59-62 [5-6] Ross MA Long WF Williamson FB (1992) Inhibition by heparin of iron (II)-catalysed free-radical peroxidation of linolenic acid Biochem J 286 (3) 717-720 Chem Abs 117 187524y (Heparin N-SO3- groups are important for the observed antioxidant activity) cf Ross MA Long WF Williamson FB Moffat CF (1992) Effect of chemically modified heparins, and of heparin fragments, on Fe(II)-catalysed peroxidation of linolenic acid

Biochem Soc Trans 20 216S (It was suggested that heparin acts as an antioxidant by binding and sequestering Fe(II) ions) [3-10]

Salonen JT Nyyssnen K Korpela H Tuomelehto J Seppnen R Salonen R (1992)

High stored iron levels are associated with excess risk of myocardial infarction in eastern Finnish men Circulation 86 (3) 803-811

[1-1-n] Salvemini D Jensen MP Riley DP Misko TP (1998) Therapeutic manipulations of peroxynitrite Drug News Perspect 11 (4) 204-214 Chem Abs 129 254229u (Nitric oxide metabolites are strongly implicated in the etiology of stroke, multiple sclerosis, Alzheimers disease, amylotrophic lateral sclerosis, Huntingtons and Parkinsons diseases) [x-17] Schlemmer E Scharnagel H Hofffmann MM Gschwendrer C Wieland H Maerz W The cellular uptake of lipoprotein A (Lp(a)) is mediated by apolipoprotein a and the LDL receptor related protein http://www.kenes.com/73eas/program/abstracts/240.doc Complexing with apolipoprotein a increases the binding of low density and receptor associated protein (RAP). Inhibition of heparan sulphate proteoglycan biosynthesis diminished the binding of Lp(a) to RPA suggesting that cellular uptake of Lp(a) may involve cell surface heparan sulphate.

Schwarz K (1973) A bound form of silicon in glycosaminogycans and polyuronides PNAS USA 70 (5) 1608-1612
[y-7a]

Shainkin-Kestenbaum R Adler AJ Berlyne GM Caruso C (1989) Effect of aluminium on superoxide dismutase Clinical Science 77 (5) 463-466 [Aluminium at concentrations similar to those found in the serum of uraemic patients inhibits superoxide dismutase; this inhibition may promote aluminium dementia and carcinogenic processes which are known to be more common in uraemic subjects; this research group also reported that nickel and chromium also inhibit SOD in a similar manner to aluminium but these authors also reported that SOD was also found by them to be inhibited by silicon; {others e.g. J.D. Birchall cf Lancet 1988 332 (8616) 1008-1010 have however suggested that silicic acid and its polymers may confer biological protection against aluminium intoxication}]
{AU research suggested that Al3+ binds to heparin and HS suggesting that cell surface HS may act as a protection against (small amounts of) Al3+ intoxication} Although current literature on veganism scarcely mentions HS, this polysaccharide system putatively contains polyanionic S(O) and Si(O)-[Schwarz crosslinks]- which can behave as an redox (ascorbate and other dietary component) sensitive information system which apparently is able to play numerous roles in animal physiology so as also to make it likely that the influence of diet on HS biochemsitry must have a major influence on health, HS has also become of especial interest in the post-humangenome-project era (having a too-low complexity of the transcribed DNA) which now seeks to identify some (perhaps far-back-) epigenetic system which is apparently needed to account for the complexity of animal biochemistry. HS fits this bill as HS is known to enable embryogenesis and numerous other complex signalling processes so that HS is a prime candidate for a sought-after overlord -informationcontrol system; this HS system may additionally require Si. The current growing awareness of the power of HS + Si as a pro-health control system suggests that in the future a major revolution in healthcare could arise based on the paradigm that the best way of improving health, of promoting longevity and of preventing and treating numerous diseases may be the achievement of an optimally-structured extracellular matrix heparin/HS polysaccharide system {with optimally positioned O- and

N-sulphated alternate polymer of glucosamine and uronic acid microstructures and associated Si linked metallome}. [5-11]

Shimada K (1994) Rinsho Byori 42 (4) 746 Chem Abs 121 6206k (Homocysteine suppresses heparan sulphate anticoagulant activity in vasculature endothelial cells; blood contains homocysteine in both oxidised and reduced forms; higher homocysteine is associated with Alzheimer's disease; increasing intake of folic acid, vitamin B12 and vitamin B6 reduce blood homocysteine concentrations). {B12 is believed to be a major nitric oxide control agent} [1-1-1] Shively JE Conrad HE (1970) Stoicheometry of the nitrous acid deaminative cleavage of model amino sugar glycosaides and glycoasminoglycuronans Biochemistry 9 (1) 33-43
[x-7]

Shimpo M Ikeda U Maeda Y Ohya K Murakami Y Shimada K (2000) Effect of aspirin-like-drugs on nitric oxide synthesis in rat vascular smooth muscle cells Hypertension 35 (5) 1085-1091 *Siegel G Malmsten M Lindman B (1998) Flow sensing at the endothelium-blood interface Colloids and Surfaces A: Physiochemical and Engineering Aspects 138 345-351 [y-8] Singh RJ Hogg N Joseph J Kalyanaraman (1996) Mechanism of nitric oxide release from S-nitrosothiols J Biol Chem 271 (31) 18596-18603 [Cu+ is a more effective catlayst than Cu2+ for release of NO from thiol stores] (Effects of redox metals as catalysts) [5-8-1] Sivaran P Obunike JC Goldberg IJ (1995) Lysolecithin-induced alteration of subendothelial heparan sulfate proteoglycans increases monocyte binding to matrix J Biol Chem 270 (50) 29760-29765 [An etiological role of alteration of HS in atherosclerosis was confirmed to arise by the effect of modified lipoproteins upon endothelial HS etc (it causing an apparent major decreased sulphate incorporation in the subendothelium matrix by a process equivalent to heparanase degradation which leads to the diminution of the HS content of this tissue) with a consequent increase of monocyte binding] [y-14] Sorenson RJR (1984) Copper complexes in biochemistry and pharmacology Chemistry in Britain 1110-1113

Spence VA Khan F Belch JGF (2000) Enhanced sensitivity of the peripheral cholinergic vascular response in patients with chronic fatigue syndrome Am J Med 108 (9) 736-739 PMID 10924652; [Cf also Spence VA et al (2004) Prostacylins Leuko Essent Fatty Acids 70 (4) 403-407]
[7-2] Stephens RW Bokman AM Myhnen HT Reisberg T Tapiovaara H Pedersen N GroendahlHansen J Llinas M Vaheri A (1992)

Heparin binding to the urokinase kringle domain Biochemistry 31 7572-7579 Cf Schlemmer E Scharnagel H Hoffmann MM Gschwendner C Wieland H Maerz W The cellular uptake of lipoprotein A (Lp(a) is mediated by apolipoprotein a and the LDL receptor related protein (RAP) http://www.kenes.com/73eas/program/abstracts/240.doc Inhibition of heparan sulphate biosynthesis diminished the binding of Lp(a) to RPA. The cellular uptake of Lp(a) may involve cell surface heparan sulphate *Stringer SE Mayer-Proschel M Kalyani A Rao M Gallagher JT (1999) Heparin is a unique marker of progenitors in the glial cell lineage J Biol Chem 274 (36) 25455-25460 (Heparin previously thought to be restricted to mast cells was found, in a totally Nsulphonated form) to be an important determinant of the developing nervous system in concert with specific microstructured heparan sulphates which characteristics of differentiated glial subpopulations) [x-7][y-36] Stichtenoch DO Frlich JC (1998) Nitric oxide and inflammatory joint diseases Brit J Rheumatol 37 246-257
[3-11]

Sullivan JL (1992) Stored iron and ischemic heart disease. Empirical support for a new paradigm Circulation 86 (3) 1036-1037 (Editorial comment on the findings of Salonen et al (1992) loc cit that iron status measured by serum ferritin has a major role in the etiology of acute mycoardial infarction and enhances the notion that dyshomeostasis of iron or its dietary enhancement may be involved in the aetiology of atherosclerosis and related illnesses) Sun H Berquin IM Edwards IJ (2005) Omega-3 polyunsaturated fatty acids regulate syndecan-1 expression in human breast cancer cells Cancer Res 2005 65 4442 cf Hu Y et al (2013) 15-lipoxygenase-1-mediated metabolism of docosahexaenoic acid is requried for syndecan-1 signaling and apoptosis in prostate cancer cells Cardinogenesis 34 (1) 176-182 [n-3PUFA metabolite is required for heparan sulphate mediated anti-tumour effect] *Takeuchi Y Sakaguchi K Yanagishita M Aurbach GD Hascall VC (1990) Extracellular calcium regulates distribution and transport of heparan sulfate proteoglycans in a rat parathyroid cell line J Biol Chem 265 (23) 13661-13668

Tanaka Y et al (1998) Heparan sulfate on endothelium efficiently induces integrin-mediated T cell adhesion by immobilizing chemokines in patients with rheumatoid synovitis Arthritis Rheum 41 (8) 1365-1377

[2-5][5-15] Templeton DM (1991) Metal-proteoglycan interactions in the regulation of renal mesangial cells, implications for metalinduced nephropathy Proc Trace Element Disease 1991

Ed: A Aito (Proc JO Nord Trace Elem Soc/UPAC Int Symp 1990) Roy Soc Chem Cambridge UK Chem Abs 111 1294071z [This paper suggests that Hg intoxication inter alia diminishes HS] *Tiedemann K Btge B Mller PK Reinhardt DP (2001) Interactions of fibrillin-1 with heparin/heparan sulfate, implications for microfibrillar assembly J Biol Chem 276 (38) 36035-42 (Ca2+ dependence of extracellular micorfibrils (fibrillin-1) binding to HSPG [x-8-1] Torres JC Behrens MI Inestrosa NC (1983) Neuronal 16S acetylcholineesterase is solublized by heparin Biochem J 215 (1) 201-204 Chem Abs 99 154263u [6-3] Van Dalen CJ Winterbourn CC Senthilmohan R Kettle AJ (2000) Nitrite as a substrate and inhibitor of myeloperoxidase Implications for nitration and hypochlorous acid production at sites in inflammation J Biol Chem 275 (16) 11638-11644 [1-1-3] Van der Loo B Labugger R Skepper JN Bachschmid M Kilo J Powell JM PalaciosCallender M Erusalimsky JD Quaschning T Malinski T Gygi D Ullrich V Lscher TF (2000) Enhanced peroxynitrite formation is associated with vascular aging J Exp Med 192 (12) 1731-1744 [5-27][8-3] Vilar RE Ghael D Li M Bhagat DD Arrigo LM Cowman MK Dweck HS Rosenfeld L (1997) Nitric oxide degradation of heparin and heparan sulphate Biochem J 324 (2) 473-479 Cf also Ghael D et al (1997) Ibid 324 473-497 and Biochem Mol Biol Int 43 (1) 183-188 Chem Abs 128 39743j Virtanen JK et al (2012) Serum long chain n-3 polyunsaturated fatty acids, mercury, and risk of sudden cardiac death in men: a prospective population-based study PLos One 7 (7) e4106 PMID 22815906 [The toxic effect of Hg offsets the benefit of n-3 polyunsaturated fatty acids. Both occur in fish. The negative effect of Hg on HS [cf Templeton . loc cit] putatively offsets the beneficial effect of n-3PUFA on HS This source of Hg would not be present in vegan diet] *Webb LMC et al (1993) Binding of heparan sulphate enhances neutrophil responses to IL8 Proc Natl Acad Sci USA 909 (15) 7158-7162 Chem Abs 119 201478h (HS promotes IL8 dependent transmigration of neutrophils and protects tissue from lytic enzymes released by them) [6-2] Whiteman M Spencer JPE Jenner A Halliwell B (1999) Hypochlorous acid-induced DNA base modification: potentiation by nitrite. Biomarkers of DNA damage by reactive oxygen species Biochem Biophys Res Commun 257 572-576 Cf also Whitewell M Halliwell B (1996) Quoted by Oldreive et al (1998) DATA (formation of tyrosine nitrate by the action of HOCl and nitrite) and http://www/med.nus.edu.sg/bioweb/faculty/metthew/matt_web%20.htm [4-6] Willenborg DO Parish CR (1988) Inhibition of allergic encephalomyelitis in rats by treatment with sulfated polysaccharides

J Immunology 140 3401-3405 *Yang L Yang Y-C (1995) Heparin inhibits the expression of IL-11 and granulocyte -macrophage colony stimulating factor GMC-SF in primate bone marrow stromal fibroblasts through mRNA destabilisation Blood 86 (7) 2526-2533 (Novel regulation mechanism of cytokines potentiated by heparin, via post-transcription mRNA degradation) *Yoshizumi M Kourembanas S Temizer DH Cambria RP Quertermous T Lee M-E (1992) Tumor necrosis factor increases transcription of the heparin binding epidermal growth factorlike growth factor gene in vascular endothelial cells J Biol Chem 267 (14) 9467-9469 (Heparin binding epidermal growth factor is a more potent mitogen than epidermal growth factor and platelet derived growth factor and is implicated in the etiology of atheroscerosis) *YuraY Iga H Kondo Y Harada K Tsujimoto A Yanagawa T Yoshida A Sato M (1992) Heparan sulphate as a mediator of herpes simplex virus binding to basement membrane J Invest Dermatol 98 (4) 494-489 Chem Abs 116 212108e (GlcNSO3- is essential for herpes simplex binding to HS) Zhang X-P Tada H Wang Z Hintze TH (2002) cAMP signal transduction, a potential compensatory pathway for coronary endothelial NO production after heart failure Arterioscler Thromb Vasc Biol 22 1273-1278 [-Adrenergic receptor agonists are believed to cause vasodilation by increasing intracellular cAMP in smooth muscle also increase nitric oxide production impairment of which is associated with heart failure in endothelial cells. There is evidence that many classical vasodilators actually function, at least in part, via nitric oxide-mediated vasodilation.] [7-10] Zielasek J Tausch M Toyka KV Hartung H-P (1992) Production of nitrite by neonatal microglical brain macrophages Cellular Immunol 141 111-120 (Upon incubation with bacterial lipopolysaccharide or rat interferon - produced nitrite (from nitric oxide) may promote demyelenating diseases).

Relevance of Heparan Sulphate Signalling (a Likely Important Control System in Animal Biochemistry), to Pathology X. Oligosaccharide signalling has been established (stemming from the work of Albersheim) to provide an intracellular hormone-like activity in plants. An analogous system, based on heparan sulphate proteoglycan signalling, may also be present in animals involving a complex signalling mechanism between heparan sulphate fragment

oligosaccharide which can produce feedback to initiate altered distant heparan sulphate biosynthesis (in the Golgi apparatus involving various isoforms of Ndeacetylase/sulfotransferase which produce different N-unsubstituted glucosamines microstructures which are also potentially affected by N desulphation by sulphamidases as well as by non-enzymic processes). A post-synthetic processing is also achieved by the action of enzymic (heparanases) as well as by non-enzymic (nitric oxide/ nitrite). X-1. Does a Resetting of the Proposed Heparan - RNOS Signalling System via Administration of Heparin or NO Donors Account Explain the Reported Therapeutic Benefit of Such Agents in CFS? This model used for discussion of the aetiology of CFS agrees with reported alleviation of CFS symptoms upon administration of either heparin or nitric oxide donors such as the successful intervention in CFS recently reported following low dose heparin administration (Berg et al 1999) (which is equivalent to an appropriate heparan sulphate fragment) or by the of the successful, at least temporary, alleviation of CFS symptoms by the administration of the nitric oxide booster, nitroglycerin (Goldstein 1993). X-2.CFS and Ageing The CFS condition, which is usually reversible, and is not restricted to aged individuals, is unlikely to simply be an ageing defect, and although ageing effects may contribute to a diminution of vascular heparan sulphate and associated chronic nitric oxide metabolite imbalance, further promoted by additional cellular immune responses following organophosphate intoxication, viral or bacterial infections or inappropriate immunological treatments may induce an inadequacy of superoxide dismutatse activity (e.g. consequent on deactivation of mitochondrial MnSOD by nitric oxide) producing a chronic pro-oxidant scenario which induces altered heparan sulphate biosynthesis (which is known to be affected by the presence of H2O2 and elevation in homocysteine levels (Nishinaga et al 1993)). X-3. CFS & Superoxide Dismutase Activity Defective superoxide dysmutase activity is implicated in excessive reactive nitric oxide metabolite formation. A genetic dysfunction of SOD is a likely prime cause of ALS, a disease which is also associated with RNOS damage. Extracellular (Cu/Zn) superoxide dismutase is bound to blood vessel wall heparan sulphate chains and diminution of these will disrupt the normal mechanism of SOD release. Whereas nitric oxide is a potent antioxidant, and as it possesses the facility as a small gaseous molecule for rapidly penetrating membranes and organelles, its rapid conversion to nitrite in the presence of oxygen abolishes this important tissue protective function, and, under prooxidant conditions, or direct reaction with the superoxide anion, the formation of peroxynitrite arises from addition of nitrite to the superoxide anion. The spatial juxtaposition of superoxide dismutase normally is required to allow nitric oxide synthases to function as a nitric oxide generator otherwise oxidised metabolites such as peroxynitite are produced. Aluminium has been reported to inhibit the activity of superoxide dismutase and intoxication by this metal should therefore be included as a potential risk factor for arthritic and neurodegenerative diseases and perhaps also for CFS. X-4. Ascorbate, Glucose, Glucosamine, Lipids & Homocysteine The above molecules apparently affect heparan sulphate proteoglycan assembly. Low ascorbate, excess glucose or homocysteine, inappropriate lipids or toxic metal ions evidently perturb the biosynthesis of heparan sulphate by directly affecting the polysaccharide chain assembly in the Golgi apparatus, but the details of how this is achieved are yet to be established. Apparent beneficial effects of glucosamine or ascorbate dietary supplementation are readily explicable as being due to a boosting effect of these substances on heparan sulphate biosynthesis. Ascorbate was reported to greatly augment the biosynthesis of heparan sulphate in bovine fibroblasts (Kao et al 1990) confirming prior reports by Edward & Oliver (1984) who studied human fibroblasts.

That ascorbate also functions in a similar manner in vivo by boosting endothelial heparan sulphate is suggested by an observed improvement of endothelial function of conduit arteries in patients with chronic heart failure (Hornig et al 1998). Ascorbate can augment heparan sulphate activity by at least two mechanisms. Both nitric oxide and heparan sulphate production by endothelial cells are stimulated by ascorbate. Nitric oxide biochemistry is affected by ascorbate which highly efficiently reduces nitrous acid back to nitric oxide thereby sparing heparan sulphate from direct nitrous acid nitrite uncatalysed deaminative cleavage at unsubstituted glucosamine sites. Dietary supplementation by ascorbate is suggested to alleviate the symptoms of CFS by this mechanism. Related cell culture experimental data suggests similar effects are achieved by a favourable dietary lipid composition (oxidised lipoprotein components have demonstrated an ability to diminish heparan sulphate biosynthesis but apolipoprotein E containing high density lipoprotein stimulated endothelial production of heparan sulphate rich in heparin-like domains (Paka et al 1999) suggesting that an appropriate dietary intervention for the alleviation of the symptoms of CFS may be a rational therapeutic strategy. A high serum glucose could, however, be detrimental as this diminishes related liver heparan sulphate biosynthesis for lipid processing. Altered lipids putatively signal for altered heparan sulphate biosynthesis in endothelial cells. Enhanced urinary nitrate excretion is a feature of elevated nitric oxide activity and this, together with altered urinary glycosaminoglycan secretion, characterises arthritic diseases (Stichenoch & Frolich (1987). Further research is required to establish if similar correlations apply to CFS. X-5. Acetylcholine Processing by Heparan Sulphate. Targets for RNOS Heparan sulphate proteoglycans have been implicated in the assembly of acetylcholinesterase within neuronal cells (Inestrosa et al 1985) and collagen-tailed acetylcholinesterase is anchored to the extracellular matrix by heparan sulphate proteoglycans (Brandon et al 1985). Further heparan sulphate activities of possible relevance to the etiology of in CFS include acetylchloine receptor processing; heparan sulphate appears to modulate receptor clustering detectable in model systems (Baker et al 1992), and such effects perturbed by altered heparan sulphate could affect neurological functions both in the central and peripheral nervous systems. Related heparan sulphate effects have been implicated in the biochemistry of learning (Lauri 1999). There could also be a heparan sulphate dependent effect in the etiology of the familiar form of amyotrophic lateral sclerosis triggered by insufficiency of SOD genetic defect (Rosen et al 1993, cf. Calder 1995) in which current thinking implicates active nitrogen oxygen substances generated from nitrous acid plus superoxide as the major damaging agent (Cookson & Shaw 1999, cf Love 1999). Ferrero et al (1989) reported that an improved cognitive function in Alzheimers disease patients following administration of a heparan sulphate containing pharmaceutical preparation which may implicate improved neurotransmitter function. X-6. Cysteine-Tyrosine Containing Antinitrant Proteins as Antinitrant Drugs It is suggested that a therapeutic intervention in nitrous acid driven pathologies might be achieved by the administration of suitable cysteine tyrosine anti-nitrants. These substances are analogues of industrially-used plastic antioxidants which are believed to function by stopping reactive radical degradative chain reactions and also by providing liganding sites for the sequestration of trace redox active metals which are capable of initiating such chain reaction processes (Grant et al 1989). The binding and stabilisation of nitric oxide linked cysteine is a further major mechanism of preventing its further conversion to nitrite. Such structures are now proposed to have this function in fibrin, immunoglobins and especially in such multi-kringle structures as apolipoprotein a (which has been suggested (Rath & Pauling 1990) to be a surrogate for ascorbate in animals lacking the ability to biosynthesise this antioxidant). Apolipoprotein a has also been reported to access cells via attachment to heparan sulphate proteoglycans at the cell surface (Schlemmer et al 2002). Depletion of this point of

entry may impair intracellular anti-nitrant protection. We now further suggest that these molecules provide a key anti reactive nitrogen oxygen species control system applicable to lipid rich as well as aqueous phases which functions in addition to the proposed haemoglobin and glutathione nitric oxide control systems. The cysteine tyrosine system is suggested to back up the evident highly kinetically efficient aqueous-phase-only ascorbate-based redox control of nitric oxide. (Ascorbate rapidly reverses the nitrite formation reaction but only in water-rich milieu and the activity of lipoprotein-a should provide protection for lipid rich environments). Naturally occurring phenolics and flavenoid antioxidants, may serving additionally as antinitrants present in foods such as chocolate may have a similar benefit. X.7 Aspirin Aspirin has been associated with increased nitric oxide synthase activity in vascular smooth muscle cells (Shimpo et al 2000) and may benefit CFS sufferers by this effect. Further research is required to test this idea. X.8 Heparin with Physiological Counter Ions Contributes to Physiological pH Buffering Small amounts of CO2/HCO3- generate pro-nitrous acid pH values are corrected by the presence of heparin as studied by in vitro pH titrations suggestive of a similar function for correctly formulated heparan sulphate at blood vessel walls. Aberrantly damaged heparan sulphate may further promote its own destruction by encouraging adjacent acidosis and nitrous acid deaminative cleavage. Titration of aqueous solutions exposed to atmospheric CO2 with heparins with physiological counter ions quickly creates a physiological pH buffer. Non-physiological Al3+ heparinate however formed a pH 4.3 buffer (Grant et al 2002).

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Selected Shorter List of References (Sub-selection List)

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Belting M Persson S Fransson L-A (1999) Proteoglycan involvement in polyamine uptake Biochem J 338 317-323

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AHMF Conference 2001 Internet report of conference *David G Bernfield M (1998) The emerging roles of cell surface heparan sulfate proteoglycans Matrix Biol 17 (7) 461-463 Chem Abs 130 14991p

(Review includes listing of viruses , bacterial and protozoa which attach to cell surface heparan sulphates and may be inhibited by heparan sulphate oligosaccharides)

Ding K et al (2001) N-unsubstituted glucosamine in heparan sulfate of recycling glypican-1 from suramin-treated and-deprived endothelial cells J Biol Chem 276 (6) 3885-34894 Cf Ding K Sandgren S Mani K Belting M Fransson L-A (2001) Modulations of glypican-1 heparan sulfate structure by inhibition of endogenous polyamine synthesis. Mapping of spermine-binding sites and heparanase, heparin lyase and nitric oxide/nitrite cleavage sites. ibid., 276 (50) 46779467912 [5-20] (1984) Ascorbate increases the sulphation of glycosaminoglycans synthesised by human skin fibroblasts Biochem Soc Trans., 12 304 [1-1-1]Fan T-Y Tannenbaum SR (1973) Factors influencing the rate of formation of nitrosomorpholine from morpholine and nitrite: acceleration by thiocyanate and other anions J Agric Food Chem 21 (2) 237Ferrero ME Santini V Mantovani M Prino G (1989) Biochem Soc Trans 360 (Ateroid (a heparan sulphate containing preparation) therapy for Alzheimers disease (cf Lorens SA Guschwanm M Hake N van den Kan LD Waldnyo JM (1991) Behavioural, endocrinal, and neurochemical effects of sulfomucopolysaccharide treatment on the aged Fischer 344 male rat Semin-Thromb Hemost 17 supplem 2 164) Feyzi E Saldeen T Larsson E Lindahl U Salmivirta M (1998) Age-dependent modulation of heparan sulphate structure and function J Biol Chem 273 (22) 13395-13398 Fujiwara Y Kaji T (2002) Suppression of proteoglycan synthesis by calcium ionophore A23187 in cultured vascular endothelial cells: implication of intracellular calcium accumulation in lead inhibition of endothelial proteoglycan synthesis J Health Sci 48 460-466 Fukuchi K I et al (1998) Alzheimers disease and heparan sulphate proteoglycans Front Biosci 3 327-337 Chem Abs 29 3327e Goldstein JA (1993) Nitroglycerin: A potent mediator for hyperfusion in CFS Cf Chronic Fatigue Syndrome: The limbic hypothesis Gordge MP Meyer DJ Hothersall J Neild GH Payne NN Noronha-Dutra A (1995) Copper chelation induces reduction of the biological activity of S-nitrosothiols Br J Pharmacol 114 (5) 1083-1089 Grant D Long WF Williamson FB (1987) Pericellular heparans may contribute to the protection of cells from free radicals Med Hypoth 23 67-72 Grant D Long WF Williamson FB (1989a) A comparison of antioxidant requirements of proteins with those of synthetic polymers suggests an antioxidant function for clusters of aromatic and bivalent sulphur-containing amino acid residues Med Hypoth 29 245-253 Grant D Long WF Williamson FB (1989b) Inhibition by glycosaminoglycans of CaCO3 (calcite) crystallization Biochem J 259 41-55 Chem Abs 110 171240x [2-7] Grant D Long WF Williamson FB (1992a)

Degenerative and inflammatory diseases may result from defects in antimineralization mechanism afforded by glycosaminoglycans ibid 38 49-55

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[2-2] Long WF Williamson FB (1979) Glycosaminoglycans, calcium ions and the control of cell proliferation IRCS J Med Sci 7 429-434 Luster AD Greenberg SM Leder P(1995) The IP-10 chemokine binds to a specific cell surface heparan sulfate shared with platelet factor 4 and inhibits endothelial cell proliferation J Exp Med 182 (1) 219-231 Chem Abs 123 53934x

McCarty MF (1997)

Glucosamine may retard atherosclerosis by promoting endothelial production of heparan sulphate proteoglycans Med Hypoth 48 (3) 245 Chem Abs 127 12883u
Manaster J Chezar J Schurz-Swirski R Shapiro G Tendler Y Kristal B Shasha SM Sela S (1996)

Heparin induces apoptosis in human peripheral blood neutrophils

Br J Haematol 94 (1) 48 Chem Abs 125 158180t Mani K Jonsson M Edgren G Belting M Fransson L-A (2000) A novel role for nitric oxide in the endogenous degradation of heparan sulfate during recycling of glypican-1 in vascular endothelial cells Glycobiology 10 (6) 577-586 Mishra-Gorur Castellot JJ Jr (1999) Heparin rapidly and selectively regulates protein tyrosine phosphorylation in vascular smooth muscle cells J Cell Physiol 1788 (2) 205 Misra A et al (1996) Clin Exp Immun 104 (3) 406 Chem Abs 125 84448n {1-1-5} Moeslinger T et al Inhibition of inducible nitric oxide synthase by oxidized lipoprotein(a) in a murine macrophage cell line FEBS Lett 478 (1,2) 95-99 Morano S Guidobaldi L Cipriani R Gabriele A Pantellini F Medici F DErme M DiMario U (1999) High glucose modifies heparan sulphate biosynthesis by mouse glomerular epithelial cells Diabetes/Metab Res Rev 15(1)13-20 Chem Abs 131 57278z Myers PR et al (1999) Vasorelaxant properties of the endothelium-derived relaxing factor more closely resemble S-nitrosocysteine than nitric oxide Nature 345 161-163 Nagasawa K Uchiyama H Sato N Hatano A (1992) Chemical change involved in the oxidative-reductive depolymerisation of heparin Carbohydr Res 236 165-180 Chem Abs 118 97491b

[x-3][x-4-n] Nishinga M et al (1993) Inhibition of the expression of anticoagulant heparan sulphate by H2O2. Suppression of heparan sulphate expression by homocysteine in porcine endothelial cell cultures J Clin Invest 192 (3) 1381 Chem Abs 119 24338u [1-1-1] Nitric oxide is an efficient antioxidant, but its metabolite nitrite especially in the form of nitrous acid is also able highly efficiently, to inappropriately degrade heparan sulphate, mutate RNA and cross link DNA as well as deactivating biological amines such as serotonin. (A routine laboratory employment of the sensitive and specific nitrous acid deaminative cleavage for the detection and sequencing of HS far predated the discovery that nitric oxide was a major physiological agent.)
[y-25]Norgard-Sumnichi K Varhi A (1995) Endothelial heparan sulphate proteoglycans that bind L-selectin have glucosamine residues with unsubstituted amino groups J Biol Chem 279 (20) 12012 Chem Abs 123, 28146j Oldreive C Rice-Evans C Paganga G (1998) The effect of dietary flavenoids and phenolics on the formation of nitrotyrosine by acidic nitrite http://www.oxyclubcalifornia.org/OCC1998/1998abstracts/poster0.7html

Pacelli R Wink DA Cok JA Krisha MC DE Graff W Friedman N Tsokos M Samuni A Mitchell JB (1995) NO potentiates hydrogen peroxide killing of E coli J Exp Med 182 (5) 1469-79 CA 123 283591n

(Nitrous acid: likely in vivo utilisation as anti-pathogen. Acidosis induced by pathogens can be utilised by this mechanism)
[x-6] Paka L Kako Y Obunike JC Pillarisetti S (1999)

Apoliprotein E containing high density lipoprotein stimulates endothelial production of heparan sulphate rich in biologically active heparin-like domains. A potent mechanism for the anti-atherosclerotic action of vascular apolipoprotein E J Bio Chem 274 (8) 4816-4823 Perez M Wandosell F Colaco C Avila J (1998) Sulphated glycosaminoglycans prevent neurotoxicity of human prion protein fragments Biochem J 335 (2) 369-374 Chem Abs 130 76099n Pfeiffer S Gorren ACF Schmidt K Werner ER Hansert B Scott Bohle D Mayer B (1997) Metabolic fate of peroxynitrite in aqueous solution Reaction with nitric oxide and pH-dependent decomposition to nitrite and oxygen in a 2:1 stoichiometry J Biol Chem 272 2465-3470 CHECK Pfeiffer S Lass A Schmidt K Mayer B (2001) Protein tyrosine nitration in cytokine-activated murine macrophages Involvement of a peroxidase/nitrite pathway rather than peroxynitrite ibid 276 36 34051-34058 cf Pfeiffer S Mayer B (1998) Lack of tyrosine nitration by peroxynitrite generated at physiological pH ibid 273 (42) 27280-27285 Pinhal MAS Walenga JM Jeske W Hoppenstaede D Dietrich CP Fareed J Nader HB (1994) Antithrombotic agents stimulate the synthesis and modify the sulphation pattern of a heparan sulphate proteoglycan from endothelial cells Thromb Res 74 (2) 143 Chem Abs DATA 289743j

Rath M Pauling L (1990) Hypothesis: liporotein(a) is a surrogate for ascorbate Proc Natl Acad Sci USA 87 6204-6207 Reiter et al J Biol Chem 275 (42) 32460 Romero FI Khamashta MA Hughes GRV (2000) Lipoprotein (a) oxidation and autoantibodies. New path in atherothrombosis Lupus 9(3) 206-209

Siegel G Malmsten M Lindman B (1998) Colloids and Surface A Physiochemical and Engineering Aspects 138 345-351 (Elsevier) (Evidence for heparan sulphate involvement in flow sensing at the endothelium-blood interface) Singh RJ Hogg N Joseph J Kalyanaraman (1996) Mechanism of nitric oxide release from S-nitrosothiols J Biol Chem 271 (31) 18596-18603 Chem Abs 125 135916w [5-18] Sivaram P Obunike JC Goldberg IJ (1995) Lysolecithin-induced alteration of subendothelial heparan sulfate proteoglycans increases monocyte binding to matrix J Biol Chem 270 (59) 29760-29765 Stephens RW Bokman AM Myohanen HT Reisberg T Tapiovaara H Pedersen N Grondahl-Hansen J Llinas M Vaheri A (1992) Heparin binding to the urokinase kringle domain Biochemistry 31 7572-7579

Van der Loo Labugger R Skepper JN Bachschmid M Kilo J Powell JM Palacios-Callender M Erusalimsky JD Quaschning T Malinski T Gygi D Ullrich V Luscher TF (2000)

Enhanced peroxynitrite formation is associated with vascular aging

J Exp Med 192 1731-1744 Vilar RE Ghael D Li M Bhagat DD Arrigo LM Cowman MK Dweck HS Rosenfeld L (1997)

Nitric oxide degradation of heparin and heparan sulfate Biochem J 324 473-479
Cf also Ghael D et al (1997)

Ibid 324 473-497 also Biochem Mol Biol Int 183 Chem Abs 128 39743j Willenborg DO Parish CR (1988) Inhibition of allergic encephalomyelitis in rats by treatment with sulfated polysaccharides J Immunol 140 3401-3405 [1-1-n]Zielasek J Tausch M Toyka KV Hartung H-P (1992) Production of nitrite by neonatal rat microglial brain macrophages Cellular Immunol 141 111-120

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The major causative factors of CFS may include heparan sulphate biochemical activities which are both nitric oxide and trace redox metal ion dependent.

Y.1
Nitric oxide/nitrite intracellular and extracellular scission of heparan sulphate is, however, believed to be a normal cellular process involved in signalling both within and between cells. The release of nitric oxide from intracellular stores is catalysed by Cu ions, more effectively by Cu(I) than by Cu(II) (Singh et al 1996). Ascorbate may enhance nitric oxide release by reducing Cu(II) ions. Under physiological conditions peroxynitrite forms an equilibrium with nitrite which renders possible the formation of highly toxic nitrous acid, it can rapidly deaminatively cleave heparan sulphate at unprotected de-N sulphonated glucosamines. Furthermore a deaminative cleavage of N-sulphated (normally protecting against this process by N-sulphation) heparin at physiological pH has been reported (Vilar et al 1997) to occur in phosphate buffer but not in imidazole buffer. This is suggestive of effects of trace redox impurities acting as catalysts for this reaction since these are ubiquitous contaminants of such phosphate buffers. Prior studies of related nitrosylation processes had also identified redox metals as having such potent catalytic activity fort the formation of pro-cancer nitroamines (Challis 1978). Iron and copper ions also appear to catalyse the de-N-sulphonation of heparin rendering it immediately active for nitrous acid deaminatve cleavage (Grant 1996). Heparin, including low molecular weight heparin is known to promote biosynthesis of anticoagulant heparan sulphate proteoglycan for endothelial cells surfaces, restoring that which has been subject to inappropriate post synthetic alteration by active nitric oxide metabolites. Nitric oxide also affects blood vessel biochemistry it having a major blood vessel tone regulatory function locally modulated by cytokine activity. Local reduction in blood vessel wall heparan sulphate by enzymic and non-enzymic degradation be a component of normal physiological pH control. That CFS signalling dysfunction correction may be achieved by the administration of pharmaceutically produced heparan sulphate fragments or their equivalents such as heparin or heparinoids, is in agreement with recent reports.

Y.2 Effects of Cadmium, Lead, Mercury & Aluminium Trace toxic metal ions such cadmium, lead and mercury have been found in cell culture experiments to be potent disruptors of heparan sulphate biosynthesis perhaps ultimately by perturbing calcium dependent signalling involved as a regulator of this process (cf Templeton; Cardenas et al., Kaji et al).

The effects of dental amalgam leakage is a possible risk factor for promotion of CFS by this mechanism. Shainkin-Kestenbaum et al reported that aluminium ions at amounts similar to what is found in the serum of uremic patients, inhibits superoxide dismutase activity. This pro-oxidant (and putatively also pro-nitrant) effect of aluminium ions will also tend to diminish heparan sulphate integrity. It is believed that haemoglobin provides the principal mechanisms for physiological nitric oxide homeostasis in which nitric oxide forms derivatives with both the iron centres and active sites on the surrounding protein sites. More specific anti-nitrant protection, however, is also likely to be provided the specially favourable chemical reductant activity of ascorbate, which is uniquely efficient at reducing nitrite to nitric oxide, as well as by the antioxidant (reductant) and other activity of glutathione (and related thiols) which can additionally prevent the formation of nitrite by the formation of stable nitrosothiols which release nitric oxide when required e.g. under the influence of Cu(I) catalysis. Various redox active transition metals, if present in a unprotected from, are likely to catalyse nitric oxide release from nitrosothiols (cf Singh et al 1996). We now further suggest that multiple clusters of cysteines and tyrosines present in serum proteins of which lipoprotein a, previously suggested to be an antioxidant (Rath & Pauling 1991)(having the dual ability to chelate trace redox metals and break reactive oxygen chain reactions (cf Grant et al 1989)), is conceivably specialised for this newly proposed additional function by the extra provision of an anti-nitrant protection facility. Y.3 RNOS include Peroxynitrous and Nitrous Acid The pro-oxidant state which induces peroxynitrite formation has been suggested to promote CSF) and also promotes tissue damage including at blood vessel walls especially affecting endothelial heparan sulphate dependent processes (such as anticoagulation) (Pall 2000). There is some disagreement as to the exact nature of the molecular species responsible for such damage. Since peroxynitrite exists in an equilibrium at physiological pH with nitrite which further equilibrates to form nitrous acid under acidic but physiologically relevant conditions, it is possible that nitrous acid is the actual molecular species which is responsible for the tissue damaging activity which previously has been ascribed to peroxynitrite. Nitrous acid will be obviously enhanced under acidosis conditions. Nitrous acid has been known for over a century to efficiently react with amines which include neurotransmitters and nucleic acids. Such nitrite and nitrous acid redox metal catalysis may be part of the supposed activity of peroxynitrite metabolite effect which has previously been suggested to promote degenerative disease processes and which has been characterised by the detection of the nitration of tyrosine phenolic groups in proteins e.g. in body fluids. Such groups have been identified (Ischiropoulos 1998; Reiter et al 1999) as convenient markers of arthritis, atherosclerosis, motor neurone disease, Alzheimers disease, Parkinsons disease and asthma; and CFS? Further studies are required to fully establish if this is the case). Dysfunction of antioxidantrelated protection is proposed to promote tissue damage associated with the extensive formation of 3-nitrotyrosine residues in proteins which is a characteristic feature of atherosclerosis and arthritis and it is now proposed the disease processes of chronic fatigue syndrome.

Y.4 Possible Relevance of Copper Intoxication, Nitric Oxide, Heparin/Heparan Sulphate on Platelet Activity, in the Etiology of CFS Copper is an acute phase agent. It has been suggested that a common health risk arises from common deficiency of copper in Western diets (Sorenson). But the possibility of copper overload and copper intoxication (e.g. arising as a consequence of use of copper water pipes in soft water areas) as a factor in the etiology of CFS is worth examining, perhaps the presence of inappropriate amounts of copper

contributing to an inappropriate activation of platelets as well as the catalysis of an inappropriate deamination of heparan sulphate by nitric oxide metabolites which may potentially influence endothelial function. It should also be noted, however that both heparin and nitric oxide potentiate platelet activity. Caeruloplasmin is an abundant serum protein which functions for the binding and transport of copper. It is also amongst the group of proteins identified as containing cysteine-tyrosines which may function as anti-nitrant protectants as well as possessing superoxide dismutase activity. A potential dysfunction in copper homoeostasis may arise due to augmented RNOS disruption of Cu transport. It is suggested that experimental evidence of such an effect might be sought by the analysis of blood copper and caeruloplasmin in CFS patients.

Y-5. Heparin/Heparan Sulphate Biochemistry & Copper Status Heparin provides a useful model for physiochemical studies of metal ion binding of relevance to heparan sulphate biochemistry (Although a form of heparan sulphate from glial cells more strongly resembles heparin than does average heparan sulphate, and antithrombin binding heparin -like heparan sulphate can be generated from endothelial cells. It should also be noted, however, that the more usual, less sulphated form of heparan sulphate also sufficiently resembles heparin in its general sugar chemical structure to permit it act as a heparin surrogate. Divalent ions including copper are known to modulate growth factor activity, for basic fibroblast growth factor, of importance for the repair of damaged endothelium and angiogenesis. Differences in copper ion binding to heparins had been proposed to be useful to determine their potential anticoagulant activity. Copper ions bind to heparin in a characteristic manner different from other counterions (Grant et al 1992c; Rej et al). A pharmaceutically-prepared heparin contained appreciable copper, but not all of this copper was easily removable by percolation though a sulphonate ion exchange resin. This phenomenon was thought to be relevant to the understanding of how trace copper ions show unusually specific paramagnetic broadening of heparin resonances under physiologically relevant slightly acidic conditions but not at normal physiological pH (Liu et al), suggesting that such copper and may participate in specific cleavage reactions of heparin by RNOS and reactive oxygen species such as peroxide (DATA) by a range of mechanisms dependent on the various copper pool which are attached to heparan sulphate at different binding strengths. Copper is also implicated in nitric oxide biochemical activities. The intracellular release of nitrite for processing of heparan sulphate may be achieved in cell cultures by use of copper chelators which allow diminution of release of nitric oxide from thiol stores via a catalytic effect of Cu(I) and to a lesser extent by Cu(II). Platelet activity was altered by copper chelators which induced a reduction of the biological activity of S-nitrocysteine and S-nitroglutathione for thrombin induced platelet aggregation. Here also the biological activity of Cu(I) is greater than Cu(II) (Gordge et al 1995). Y.6 Ageing & Iron Overload may Augment Nitrant Damage in Arthritic Diseases and Neurological Dysfunction An increase in serum ferritin has been evidenced to occur in absence of disease (Jarrett et al 1989). Superoxide mediates the release of iron from ferritin by some flavoenzymes such as NADHlipoamide dehydrogenase (Bando & Aki 1990). Augmented serum ferritin (iron overload) has also been associated with cardiovascular dysfunction and mortality (Salonen et al 1990) [this dysfunction was putatively associated with an impairment of blood vessel wall function caused by the induction of radical chain processes promoted by reactive oxygen or reactive oxygen nitrogen species. This hypothesis is consistent with reports that attenuation of reperfusion injury is produced by high dose systemic iron chelation therapy (e.g. Hedlund & Hallaway 1993)]. Iron catalysis of amine nitrosylation by nitrous acid or related intermediates has also been documented (Challis 1979) and this possibility suggests that trace iron may sensitize ironassociated heparin and heparan sulphate chains for subsequent deaminative cleavage by nitric

oxide or nitrous acid. Direct damage to heparan sulphate could also result from an iron ion catalysis of reactive oxygen attack as well as de-N-sulphonation of heparan sulphate enabling facile subsequent nitrous acid cleavage (Grant et al 1995). De-N-sulphonated glucosamines, pre-primed for nitrous acid cleavage, have been observed in endothelial heparan sulphate chains (Norgaard-Sumnichi & Varki 1995). Heparan-sulphate-dependent nitrite effects, whilst normally involved in intracellular signalling processes (Ding et al 2000), are also subject regulation by polyamine signalling, but there are further hints that aberrant trace redox metal dyshomeostasis could perturb this process also. Furthermore, trace active iron may also promote the nucleation of pathological calcification. It should be noted that inappropriately altered (morphology) of iron rich particles ingested by macrophages have been suggested to contribute to an inappropriate immunological nitric oxide secretion (Williamson et al 1994). It is likely that heparan sulphate provides a major anti-calcification mechanism for blood as well as urinary vessel walls and defects in such activity will contribute to various diseases (Grant et al 1992). The presence of N-sulphonate groups is required for this function , de-N-sulphonated heparin being inactive. The removal of sulphonate groups is possibly catalysed by redox metals as well as by protons formed under local acidic pH conditions generated near coordinating iron ions (Grant et al 1996). The observed age-dependent decrease in heparan sulphate content of cerebral arteriosclerotic vessels reported by Murata and Yokoyama (1989), was apparently accompanied by an ageincrease in other glycosaminoglycans co-existing with heparan sulphate, including those which lack anti-calcification activity or promote calcification (demonstrated in vitro to be the situation with dermatan sulphate). Advanced glycation end products (AGE) are also augmented in ageing and are also associated with vascular complications such as atherosclerosis . AGE were reported to selectively increase decorin (dermatan sulphate bearing) core protein mRNA (Kaji et al 2000) and therefore likely will promote calcificationassociated blood vessel wall dysfunction. Ageing also impairs endothelial cell nitric oxide synthase activity and, apparently, (cf. Hoffmann et al 2001), also enhances the sensitivity of endothelial cells towards apoptotic stimuli as well as systematically alters the biosynthesis of specific microstructured heparan sulphate chains in endothelial cells (Feyzi et al 1998). The above scenario may even be an attempt by the organ to correct for a defective anti-calcification protection provided by postsynthetically degraded heparan sulphate, via a feedback signalling mechanism involving calcium. [Calcium signalling has been implicated as a control factor in endothelial heparan sulphate biosynthesis, increased intracellular calcium being associated with diminished heparan sulphate proteoglycan biosynthesis and this effect, consequent on lead intoxication may account for the detrimental effect of lead (Fujiwara & Kaji 2002) and other toxic metal ions (cadmium mercury and aluminium) on endothelial function]

Y.7 Model of Heparan Sulphate Recycling Environmental stresses may induce rapid alteration in the systems which are sensitive to oligosaccharide turnover rates by actions of environmental stress on nitrous acid or equivalent reactive nitrogen chemical agents. The rate of oligosaccharide generation and turnover may be influenced by redox status and serve to meet various physiological requirements. Malfunction of such feedback systems may, however, trigger a formation of adverse but stable interactive states following degenerate loop signalling errors which manifest in chronic illhealth states such as CFS. Such chronic states might potentially be corrected, however, according to this hypothetical model, by the re-stabilisation of any incorrect reactive nitrogen balance by an increased input of appropriate signalling oligosaccharides, appropriate heparanases, or by the provision of additional nitric oxide e.g. by administered as nitroglycerin, arginine or by nitric oxide synthase inhibitors. This situation is suggested to be analogous to the use of glucosamine sulphate which has been found useful in the treatment of arthritic illnesses by likely boosting heparan sulphate biosynthesis (McCarty 1997) which also benefit from treatment with nitric oxide synthase inhibitors (Stichtenoch & Frlich 1998), and

perhaps also explains the efficiacy of the use of ascorbate supplementation, which similarly could augment heparan sulphate biosynthesis, for the treatment of cancer (Pauling et al 1991). Ascorbate, of course rapidly reacts with nitrous acid to produce nitric oxide i.e. it reverses the effect of oxygen on nitric oxide which, in the absence of a correct amount of ascorbate may lead to the formation of inappropriate amounts of damaging nitrous acid the putative key agent responsible for the etiology of various degenerative diseases because of its ability to inappropriately deaminatively cleave heparan sulphate (the highest ranking system manager which confers multi-facet tissue protection).

Y.8 The ability to chelate trace redox metals by cysteine tyrosine (CY) antioxidants (cf Grant et al 1989) is now proposed to enable these CY proteins also to act as efficient anti-nitrants. Experimental evidence for the relevance of such a mechanism to CFS should be sought. It should be noted that Nitrous Acid, has been long known to efficiently react with amines including those present in nucleic acids and cause mutation thereof.

Y.9 Necessity for the Existence of a Physiological Anti-Nitrant Protection System Nitric oxide widely used as a signalling molecule and as an anti-pathogen inflammatory agent will require the back-up of specialised anti-nitrant mechanisms to shield tissues against various active nitrogen-based oxidants; this protection may be of greater importance to tissue integrity than the more fully understood antioxidant protection. Defects in such anti-nitrant protection may therefore be the root causes of many pathological actions of RNOS including those ascribed to peroxynitrite/nitrite which are known to promote various diseases associated with the nitration of tyrosine residues in proteins which is now known to characterise more than 80 human illnesses (Ischiropoulos, 1998). Unprotected nitric oxide is rapidly converted in the presence of oxygen in amounts commonly present in tissue to nitrite, which can rapidly add H+ to produce nitrous acid under physiologically relevant pH conditions as well as under pathologically induced acidosis conditions. The precise mechanism of nitrous acid deamination is not known, but it may involve, on occasion, a chemical entity distinct from HNO2 or from N2O3 which may actually be the most damaging agent since separate mechanisms of a nitrous acid like metabolite formation seems to arise including a peroxynitrite (nitrite plus superoxide anion) route, and another active nitrogen moiety formed by a direct route from nitric oxide at physiological pH under conditions where highly active adducts with redox metal ions such as copper and iron, may be formed. The assumption that peroxynitrite per se is the active factor in the promotion of CFS is questionable and the role of nitrous acid or other nitrous acid derived molecules might be considered for this role. Although haemoglobin is believed to be a major sink for the complexing of nitric oxide, facilitating its physiological control, of especial additional importance for reversing the oxidation of nitric oxide are the chemical reductants, ascorbate, glutathione and more generally cysteine residues in proteins. These substances and sequences are therefore likely to be utilised biologically as anti-nitrant agents. A further anti-nitrant system is, however, suggested to be provided by cysteine tryrosine cluster groupings as exemplified by the kringle structures present in lipoprotein a and related structures in fibrinogen. It should be noted that kringle and related structures also bind to heparin/heparan sulphate polysaccharides which include amongst their functions wide-ranging tissue protection enabled by glucosamine-based polysaccharides, especially heparin/heparan sulphates. These polysaccharides may also provide a buffer against RNA mutation (including the inhibition of pathological RNA virus mutation) and DNA cross linking. While nitrite appears to be a normal method of in vivo intracellular and probably extracellular processing of heparan sulphate chains (cf Mani et al 2000; Ding et al 2001), nitrous acid, if allowed to accumulate in excessive amounts, will potentially cause inappropriate deaminative cleavage of extracellular heparan sulphate chains and disrupt their extensive signalling mechanisms (cf Bernfield et al 1999) especially those

involving modulation of immune responses, nervous impulse transmissions and wound healing. Grant et al (1989) indicated that conserved cysteines and tyrosine in numerous proteins could provide antioxidant protection by a mechanism similar to that used commercially in polyolefin antioxidants in which bivalent sulphur by acting partly as redox metal sequesters further act synergistically with hindered phenols to provide a long-lived, oxidation reaction chain reaction breaking protection system.
This hypothesis was ultimately derived from an update of prior research projects on heparin/heparan sulphate summarised in www.abdn.ac.uk/~bch118/publications2003march.doc which progresed ideas which had been origianlly aired in IRCS J Med Sci (1979), 7 429-434, a predictive article by WF Long & FB Williamson who led the former Polysaccharide Research Group at Marischal College, University of Aberdeen, UK, AB9 1AS)

b Ashbank,Turriff AB53 and Cults, Aberdeen AB15, UK [Footnotes]

a ME-CFS is putatively a related cohort of illnesses by inappropriate overactivation of induced nitric oxide synthases coupled with a depletion of anti - RNOS activities causing an elevation of peroxynitrite, nitrite and nitrous acid metabolites which damage HS and key amines under conditions of acidosis and redox metal ion dyshomeostasis.

INSERTED DOCUMENT 3

The Possible Key Role of Heparan Sulphate Deficiency in Autism and ME-CFS (Myalgic Encephalomyelitis - Chronic Fatigue Syndrome)
Autism and ME-CFs may both be heparan-sulphate-defect-dependent diseases. Since heparan sulphate is susceptible to dietary (e.g. ascorbate and good lipids) biosynthetic upgrade and nitrosative stress depletion, this hypothesis also suggests that suitable beneficial dietary interventions related to redox system rebalancing might be designed for these diseases. Research conducted at the polysaccharide group lab at Aberdeen University and associated discussions between ex-members of this group had postulated that diverse diseases including chronic fatigue syndrome and autism could have a common etiological origin, namely a dysfunction of the heparan sulphate tissue protection and management system. This idea is indirectly supported by the published

and unpublished results of this research group and also by a wide range of published articles from other groups. A recent report that a heparan sulfate (HS) deficiency found in a mouse model of autism also shows up in a preliminary human study of 4 postmortem autistic compared with age-matched controls (BL Pearson Behav Brain Res 2013 243 138-45) now tends to confirm the validity of the idea that autism is a HS-defect-driven diseasea. The idea that defects in HS biochemistry cause autism seems to offer a novel conceptualization which might lead to improved therapeutic intervention strategies. A further concept worthy of consideration is that ME-CFS and autism may both share common etiological features and this is due to underlying HS defects (differing between the diseases). This possibility is supported by internet-available information b. The focus of future research should, it is therefore suggested, especially consider the possible dependence of autism and ME-CFS on heparan sulphate deficiency. Footnotes a BL Pearson MJ Corley A Vasconcellos DC Blanchard RJ Blanchard Heparan sulfate deficiency in autistic postmortem brain tissue from the subventricular zone of the lateral ventricles Behav Brain Res 2013 Apr 15 243 138-45 [The cellular organization, cell proliferation , and constituents of the extracellular matrix such as Nsulfated heparan sulfate (HS) and laminin in postmortem brain tissue of n=4 young to elderly individuals with autism and age matched controls (studied by immunofluorescence techniques), showed a strong and systematic reduction in HS with increasing age as a possible useful marker of autism. These preliminary human subject findings tended to confirm earlier indications from a mouse model of autism which showed that autism was associated with genetic modifications which reduced HS and genetic variants in HS-related genes]. b Cf. www.psychologytoday.com/blog/complementary-medicine/201106/is-autism-related-cfs-andfibromyalgia where J Teitelbaum noted that many specialists who work with autism as well as CFS and fibromyalgia find that there is a major overlap between the three conditions. He suspected that given a specific genetic makeup, the very same processes that trigger CFS and fibromyalgia in adults can trigger autism in children. {The blog also reported benefits from L-carnitine and acupuncture techniques}. Cf. www.prohealth.com/library/showarticle.cfm?libid=14407 GL Nicholson et al compared blood infections in cohorts of CFS and autistic subjects and noted that similar to CFS patients a large subset of neurobehavioural disease patients (ASD) show evidence of chronic infections. Although there were significant differences in median age and diagnoses between the two groups of patients, they tended to have similar incidence of three types of chronic infections.

A (5/5/13) Sunday Times (UK) Magazine article reported interviews with scientists currently engaged in finding causes (and possible cures) for the debilitating condition Myalgic Encephalomyelitis (ME) (which reported on a sustained terror campaign requiring some of the interviewed scientists to be under police protection). The article especially discussed the outcome of a recent controversial research finding: the supposed role of retrovirus XMRV in CFS (which was later withdrawn). I t seemed that a specific mouse retrovirus was a possible or even a likely cause of the disease and might have suggested that antiretroviral (e.g. anti-HIV) therapy might alleviate ME-CFS. The article also noted that in the UK the current psychological mental illness related talking therapy intervention therapy was considered to be the most appropriate method of alleviating the symptoms of ME-CFS and funding for other ideas was sparse. This seems to be based on the idea that ME-CFS is simply a psychosomatic illness.

The Non-Biological Hypothesis of the Origin of ME-CFS.

This idea was in part supported by a Gaelic language TV channel in Scotland which interviewed with UK researches in the ME-CFS field in the UK. The international research database does not, however, support a non-biological origin of ME-CFS. It seems much more likely that the cause of ME-CFS is multi-factorial and triggered by some general immune dysfunction e.g. following a bacterial and/or viral infection. The following research articles support a biological basis of ME-CFS. JR Kerr et al J Clin Pathol 2008 61, 1-2, ibid., 730-9; cf JR Kerr and DK Mattey Clin Infect Dis 2008 46 e83-7{cf also JKS Chia and AY Chia ibid 2008 63 156-64) Cf Review Chronic fatigue syndrome LD Devanur and JR Kerr J Clin Virol 2006 doi:10.1016/j.jcv.2006.08.013; cf also L Lorusso et al Autoimmun Rev doi:10.1016/j.autrev.2008.08.003. S Galbraith et al J Infect Dis 2011 204 10 1632-40 (N.b this study of post-infective fatigue syndrome found no reliable peripheral blood gene expression correlation); H Naess et al In Vivo 2010 24 185-8 (This 873 patient [75.3%F] study showed that those with initial infection [77%] as a precipitating factor more often acute onset of fatigue, tender lymph nodes and myalgia as well as improvement of fatigue on referral); J Zhang et al J Clin Pathol 2010 63 156-64 studied differential gene expression of 88 genes with ME/CFS; the two most common infectious triggers were reported to be Epstein-Barr virus and enterovirus. B Grinde suggested (Med Hypoth 2008 doi:10.1016/j.mehy.2008.03.014) that a normally benign virus, e.g. one of the circoviruses which are chronically present in the majority of people but rarely tested for diagnostically, following other virus infection which compromises the blood brain barrier (n.b. this has a major heparan sulphate component which is subject to disruption by nitrosative stress), allows the circovirus or other normally benign viruses (including very unconventional ones which are not yet accepted to exist by medial sciences) to enter the brain. The HS system may have evolved to protect against such currently not-accepted types of pathogens. This idea at least is worth thinking about. Could stealth/virtual particles have pathological input by somehow being able under some additional stimulus be able to activate the immune response? If so such an activation of the immune response may be blocked by natural mammalian anti-viral pathways. These include those dependent on heparan sulphate.

Anti-Viral Anionic Polysaccharide Researches May Offer Hope for an Effective ME-CFS Therapy

An Aberdeen University research program had found that heparin-like drugs and semisynthetic heparinoids (e.g. xylan sulphate) were potent anti-retroviral agents, These drugs were putatively highly effective against HIV-1 etc. A xylan sulphate fraction prepared from a local seaweed was found by the Aberdeen group to have an especially high activity. This Marischal College polysaccharide work, which gradually slowed and stopped during the nineties, but had confirmed work conducted in several other laboratories had been funded by the Scottish Home and Health Dept who seemed to wish to continue their funding but this was disrupted when a lead researcher in this program was required to cease work because of the debilitating effect of ME-CFS. A continuation of thinking was attempted on

a post-academic-home based research and discussion group basis, to which I contributed. A focus of this discussion was a (Royal Society of Edinburgh) University of Dundee workshop in 2003. I was asked to prepare a literature survey for attendance at this meeting. This led to two suggestions based on the idea that the underlying disease process was dependent on heparan sulphate dysfunction. (Draft documents about this were eventually posted on the internet). One of these especially drew attention to the possible effect of hexachlorobenzene (HCB) as a pro-inflammatory stimulus which negatively affects heparan sulphate functions..
A clue about how ME-CFS might have be triggered by HCB came from the circumstance that the above academic who had become afflicted with ME-CFS intimated that previously when he had suffered from scabies and been treated topically for this infestation with the aromatic insecticide HCB. This and related substances arise globally during pyrolysis of industrial waste products. Although their deliberate manufacture and use is prohibited by the Stockholm Protocol the accidental manufacture of HCB still continues. Such environmental contamination may be at least part of the cause of ME-CFS and othe mystery illnesses.

A second suggestion for a hypothesis of the origin of ME-CFS was that: dysfunction of the extracellular matrix control system of sulphated polysaccharides under nitric oxide (copper) nitrous acid attack promotes ME-CFS. An update internet search conducted on 9 May 2013 showed up numerous general reviews of current ME-CFS researches. Anyone can do their own internet literature search on ME-CFS and come up with a similar list of reports. I used a search term copper radiation field effect in ME-CFS which had been selected on the basis of an Aberdeen University -linked ME-CFS 2003 and after academic scientific discussion group topics of interest, which revealed the following site: Chronic Fatigue Syndrome (ME,CFS or CFIDS)charles_w.tripod.com/cfs.html I have jotted down from this site refs my selection topics (as influenced by the historical Aberdeen group discussions): Thyroid (receptors)[depression of ACTH and cortisol secretion; Cheney [TH1 and TH2 balance] ; DHEA; orthostatic intolerance; brain scans; NFkappa B; incr. IGA, IgM; consistent low erythrocyte sedimentation rate abnormality; ECG abnormalities; citrate in urine : possible Mg loss; different RNA; Canadian Consensus Panel; ME-CFS victims never get the common cold because of chronic activation of the immune system; Fibromyalgia different pain mechanisms (dopamine) F>M possibly related to hippocampus; 80% of fibromyalgia victims have CFIDS; similarity of side effects of statin therapy with fibromyalgia; low molecular weight R Nase L increased activity correlated with severity of CFIDS (but this is normal in fibromyalgia) ; 2-5 Synthetase /RNase abnormalities (also found with [other] viral illnesses) {environmental toxins in the presence of heat shock proteins may also be able to activate this pathway}; Epstein Barr virus, other herpes type viruses; Retrovirus work E De Freitas U Pennsylvania ultimately found HTLV-II-like genes associated with CFIDS (1991) seems to have been the stimulus for the XMRV virus work (Judy Mikovits). XMRV seems to have been a laboratory contamination false result; stealth virus; Mycoplasma; (cf Gulf War Syndrome Mycoplasma fermentans; autism, boron supplementation therapy (for mercury intoxication?) ; circoviruses; magnesium and potassium calcium activated BKCa pump (antibacterial); Defect in platelet immune function; Cysteine; glutathinone; Vitamin B6; antioxidant; cytochrome p450 ; EPA Coagulation system dysfunction Hughes syndrome. {DG {Aberdeen} insert; cf HEPARIN therapy for ME-CFS} Fluoride (connective tissue disorders; almininium Alzheimers disease) Lyme disease; Aspartame; Pollution (exhaust gas, smoke, cigarette smoke, solvent fumes etc may exacerbate virus-related immune damage); Endometriosis; Nitric oxide [Pall] S-adenosylmethionine; Ciguatera toxin; Mercury;

Strong electromagnetic fields {Also? Weak and very weak electromagnetic fields}
Major physiological elements Vitamin D Vitamin C

Acetylcarnitine NADH Molybdenum Caffeine Zinc Sitosterol Irritable bowel syndrome Opioid antagonist drug Naltrexone It is of interest that links can be made between the heparan sulphate (sulfate) (HS) tissue protection system and all of the above topics. This may simply be because the HS system is a high level system manager which eventually can be linked to all of animal physiology. It however may also mean that therapeutic alteration of HS (which can be accomplished by dietary intervention) might offer a rational therapeutic approach to the alleviation of the symptoms of ME-CFS and other (mystery) illnesses. This idea derives from researches conducted at the Marischal College University of Aberdeen polysaccharide laboratory. This work which stopped apparently because of the effect of ME-CFS on the lead researcher.
Some eleven years ago, an Aberdeen University library literature research initiative to derive hypotheses on the etiolgy of ME-CFS was launched at the behest of Dr FB Williamson (a former polysaccharide research group leader at Marischal College, University of Aberdeen); this was extended over the intervening years. The preliminary results had been discussed with a University of Dundee research group under Vance Spence for attendance at a 2003 Royal Society of Edinburgh workshop (set up in part to draw up grant proposals for promotion of research into ME-CFS). In the event Dr Williamson had been too ill to attend the workshop so that his paper on heparan-nitrosative stress as an etiological feature of ME-CFS paper had not presented at that meeting. The Dundee researches had shown that ME-CFS and related illnesses damage the blood flow (capillary) system as shown up by use a novel diagnostic method employing acetylcholine to sense nitric oxide-dependent signalling defects. (This protocol seemed to distinguish between subtypes of ME-CFS and organophosphate poisoning). Other principal speakers at the 2003 Dundee workshop had discussed the role of chronic viral infections and anti-viral therapeutic approaches to blood pressure defects [orthostatic intolerance] in ME-CFS).

My original 2003 and sporadically ongoing literature search and related discussions (with the above scientists and Dr Gwen Kennedy, also of Dundee U.) and my attendance at the above 2003 Dundee ME-CFS workshop led to two draft documents outlining quite different ideas which, however, seem to merit further research. Both of these derived hypotheses advanced for discussion as being of possible value for understanding the etiology of ME-CFS were derived from the FBW research interests in polysaccharides heparin/heparan sulphate and especially how these physiological system mangers are under the control of redox balance (as a part of a nitric oxide metal ion signaling process) which putatively had become disturbed in ME-CFS patients). The heparin-like molecules and extracellular polysaccharides in general also possess water structure modifying abilities which may also be relevant to the etiologies of ME-CFS diseases.

OTHER LATER IDEAS

Low Level Radiation Effect on Water Structure in Tissue- Could This Cause ME-CFS? E.g via a Possible Mechanism of Alteration Of Heparin/Heparan Sulphate (Hydration-Dependent) Signalling. An apparent demonstration of how very weak electromagnetic fields could apparently alter a putatively biological relevant signalling ability of anionic polysaccharides was suggested by the observation that the ability of heparin-like molecules to inhibit calcification can apparently be subject to significant interference by the juxtaposition of the reaction vessel to an adjacent electronic circuit; this

conclusions was reached during attempts to set up an automatic crystal growth crystal inhibition set-up apparatus for the study of the inhibition of this process by modified heparin etc. molecules. (There seem to be are other experimental studies reported in the literature which supports the concept of the existence of such effects). The heparin/heparan sulphates, together with glycosaminoglycans in general are hydrated and the activities of these molecules depends, at least in part, upon the activities of the associated water molecules and the modulation of associated water structure. This idea had been a general focus of the Marischal College polysaccharide research group (Cf the internet-document-listed publications of a comanager of the is group with FB Williamson, WF Long, 2003, [cf. www.abdn.ac.uk/~bch118/publications2003march.doc]. The Earths magnetic field is thought to provide a map (which may conceivably be located in the extracellular matrix encoded in anionic patterns of hydrated glycosaminoglycans) but which can also be inherited and so allow the migration of animals (cf. e.g. KJ Lohmann et al Curr Opin Neurobiol 2012 22 336-42) Cf also: DC magnetic fields affect light-elicited ionic current fluxes in turtle retinas (MS Raybourn Science 1983 220 715). The activity of enzymes can be altered by external low intensity electromagnetic fields (cf. V Vojisavljevic et al doi:10.1109/IEMBS.2010.5626786). There are numerous reported effects of electromagnetic field which potentially effect human health both positively and negatively (RN Kostoff and CGY Lau Technol Forecast Soc Change 2013) Could the effect of abnormal electromagnetic fields also alter the hydration dependent biochemical responses of heparan sulphate so as to promote illness like ME-CFS? A range of effects are known to arise in biological systems from the effects of electromagnetic fields (cf RH Funk et al Prog Histochem Cytol 2009). The effect of low level electromagnetic fields seems to alter cognition (cf WR Adey, 1975-1993 papers). Memory and cognition are also thought to be facilitated in part by heparan sulphate-dependent systems. The redox system control of heparin/heparan involves nitric oxide and copper (zinc catalysed effects). Copper dyshomeostasis (or intoxication) may be a key factor in the etiology of ME/CFS. Copper becomes naturally elevated in acute phase inflammatory conditions. The heparin and putatively heparan hydration structure and its dependence on counterion mimics the Nafion system of proton conductors. The above processes might allow an input from the Earths magnetic field or the quantized vacuum radiation field into interactions with the copper (zinc) nitric oxide heparan redox servo-feedback system of animal physiology control (cf publications from the Fransson group in Sweden; similar work had been underway at Aberdeen University) which it is now suggested could also be facilitated in the hydration associated with the heparan field by analogy with a proposed Del Guidice mechanism (vide infra) by which supramolecular ordering and long distance electron transport was suggested to be facilitated in aqueous colloidal dispersions via the interactions between laser like actions of dipoles in water molecules and the quantized vacuum radiation field. This leads to a tentative hypothesis that ME-CFS could arise from a heparan redox disturbance (perhaps centred on copper/zinc ratio alteration or iron overload (the latter from diet) coupled with multi-factor heparan diminution (e.g. vial bacterial endotoxin, altered glucose homeostasis, thyroid factor, sulphate depletion magnesium and manganese dyshomeostasis, high cholesterol LDL, fluoride and aluminium (aluminum) intoxication) critically additionally compromised by the disturbance of background radiation, native vacuum field background, produced by anthropogenic electromagnetic field intoxication superposition.
Quantized electromagnetic field effects: The existence in the vacuum of a system of quantized electromagnetic fields is a central part of physics. This idea replaced the aether of James Clark Maxwell (the vacuum was pure aether). The spontaneous emission of electrons required the presence of a background wave action inherent in the vacuum.

Cf the fluctuation of particle antiparticle pairs advanced by Dirac, a concept which was later extended by Richard Feynman etc. This became the foundation of theory of standard model which rationalized the particle zoo the multitude of sub-atomic particles which had been found from high energy collision experiments. This model was confirmed by the recent announcement of the detection of the Higgs boson which confirms that mass is produced by interaction of those particles which can have mass with the all-pervasive Higgs field. The proposal by Meessen, the quantization of length (and time) extends this idea.

The existence of vacuum energy (and other quantum mechanical-related phenomena such as reverse time cf. the Evans fluctuation theorem) per se may also underpin all chemical reactivity to a much greater extent than has heretofore been recognized and explain the hitherto puzzling extrathermodynamic linear free energy correlations (a major set of these is the widespread enthalpy-entropy compensation in kinetic rate constants [k=AexpEa/RT] which commonly show up for a single class of reaction type, an accurately linear dependence between logA on E a; this phenomenon has most commonly reported for biochemical molecules reacting in water, but is also found to arise widely for temperaturedependent effects throughout chemistry and biochemistry (cf Scribd Apr 3 2012 The Evans Fluctuation Theroem might explain enthalpy entropy compensation). The vacuum field system seems to facilitate the enthalpy-entropy compensation process.
This has repercussions for an altered understanding of biochemistry and medicine. That the quantized electromagnetic field interacts with the system of dipoles in liquid water as part of a mechanism of setting up a free electron laser effect has been shown to be theoretically possible (by Del Giudice et al [1988 Phys Rev Lett 61 1085]) and related quantized electromagnetic field calculations suggested that long-range structuring of water adjacent to surfaces should occur as a consequence of the interaction of the intrinsic wave-like nature of water dipole interactions and the quantized electromagnetic field in the radiation gauge (of the vacuum). These predicted water structures are very long range. Speculation about this possibility (made by Giuliano Preparata and those influenced by this scientist) has suggested a mechanism of how acupuncture is facilitated by meridian channels. These were postulated to depend on the (linked up through vacuum energy effects) of the hydration cuffs on the polysaccharide side chains of extracelluar matrices. This links up heparan sulphate biochemistry to radiation filed effects. Could this interaction be relevant to a fuller understanding of the mechanism of heparan sulphate signaling?

The publications of BL Reid independently suggest that virtual energy of the vacuum interacts significantly with biological systems and could play fundamental roles in metabolism. If these ideas are at least partly correct then defects in the transduction of the vacuum energy with the biological access systems used from transduction of this complex field could impair energy use by organisms. Cf G Preparatas ideas. Anthropogenic perturbation of such interactions might be at least part of the background situation which sets up chronic fatigue. Input from electromagnetic pollution might translate into chronic fatigue. The currently increasingly complex man-made electromagnetic field superposition on the quantum electromagnetic field under which or animal evolved may alter the above type of poorly understood long range hydrogen-bonding systems upon which the Chinese acupuncture and other presently notexplained ethnic medical phenomena may depend. The modus operandi of the polysaccharide codes (which evidently differ markedly from those of the nucleic acids) may require some kind of longdistance fuzzy recognition (not the lock and key type of recognition used by nucleic acids) facilitated by the radiation field. This electronic effect which is currently very poorly understood with respect to human health could have major relevance to the fuller understanding of the etiologies of several mystery illnesses which have apparently arisen in tandem with the increasing complexities of electromagnetic field in urbanized societies. [A further obviously bizarre idea (arising from attempts in industrial lab to find explanations for extrathermodynamic determinants of chemical and biochemical reactivity suggested) that there may exist types of virtual virus which are analogous to the virtual particles of physics; (these virtual particle antiparticle pairs of the vacuum started off as mathematical abstractions but eventually became to be considered as having some actual reality which affect real physical and chemical systems. A topical example is the Higgs boson the detection of which confirmed the existence of the Higgs field of the standard model which is thought to confer mass and hence is ultimately responsible for chemistry and biology. While the Higgs boson has a very short lifetime, the possible effect of the electromagnetic field in the radiation gauge on interactions between dipoles in water molecules has been theoretically predicted (by E Del Giudice et al., Phys Rev Lett 1988 61 1085-8) to affect systems over much longer periods of times e.g. 10-14s, and may therefore conceivably be, e.g., the basis (cf. internet files citing

this Del Giudice paper, commenting on the theories of G Preparata) of homeopathy and Chinese acupuncture, as well as, perhaps, the reported nucleic acid-like segment-like signals which are reported to be induced in virtual space but which also (highly controversially) can also be translated into real space]. This kind of virtual pathogen might, conceivably, at least in part, contribute to the etiology of both autism, ME-CFS as well as a wide range of other illnesses].