The Possible Relevance of Metallomics to Heparan Sulphate Biochemistry

Heparin and putatively also heparan sulphate may provide a high capacity multi-inorganic-element reservoir
system.

The heparanome and metallome are suggested to interact with each other to enable the modulation of a
wide range of biological processes in animals.

David Grant
(A hypothesis compiled from research carried out at Marischal College, University of Aberdeen*
and follow-on internet research to 20/4/05. Document Modified 18/5/08. Reformatted and updated
11/8//09 and 25/9/10)

Summary

The occurrence of a wide range of inorganic elements in seawater and biological fluids may

be of relevance to the fuller understanding of the biological activities of anionic polysaccharides

which tend to acquire an H. Haraguchi ‘all-element’ metallomic stoicheometry under in vivo conditions.

It is suggested that such metallomic matrices are associated with all animal cell surface and extracellular

matrix anionic polysaccharides which could provide a system of osmolyte balance as well as the essential

inorganic cofactors (e.g., including Ca2+, Mg2+ and the Mn2+ and Cu2+ ions which are known to be required

for the linkage of target proteins to specific anionic patterns in heparan sulphate to as well for the
provision

of Cu2+ (Cu+) and Zn2+ ions which are believed to be essential requirements for the nitrosative scission of

heparan sulphate).

Introduction

The concept of the metallome was originally suggested by R.J.P. Williams (1)

but was later re-defined by H. Haraguchi (2) to emphasize the existence of

inter-relationships between the multielement profiles of biological and

geological matrices, e.g., between human blood serum and seawater.

(It should be noted that such inter-relationships became more apparent following

the use of mass spectrometric methods for the assay of inorganic elements in

biological materials (e.g., nails and hair (3)).

The anionic polysaccharides (e.g. of marine algae (4)) are also known
to form metallomic matrices.

A re-evaluation of the relevance of the occurrence of seawater-like

multi-inorganic-matrices throughout biota now suggests that the

‘metallome’ could also be of relevance as the ‘heparanome’**.

Metallomic Aspects of the Chemical and Biochemical Activities of Polyanionic Biopolymers

A recent review (3) of biological metallomics was restricted to the proteome (cf. about a third of proteins

show a high affinity for Cu, Fe, Zn and Mo metal ions ions which are well known to serve as reactive

centres in metalloproteins).

The extension of the concept of metallomics to anionic polysaccharides also seems warranted

since these complex biopolymers occur widely throughout biota in extracellular locations

allowing them to to serve as inorganic ion buffers. Their inorganic element composition

seems also to define them (at least in the case of the anionic cell surface polysaccharide sof marine algae)
to

as Haraguchi-type metallomic matrices (2)).

For multicellular animals similarly constituted anionic polysaccharides may also occur at cell surfaces
and

extracelular matrices where they may create the original (ancient) seawater-like water activity in which

the ancestors of the present terrestrial animals originally evolved. Evidence in favour of such a cell
surface

water-activity regulating function (that cell surface polysaccharides can create a favourable osmolyte

balance systems at cell surfaces) is that the cell surface anionic polysaccharides (especially the heparan

sulphates) extracted from of a range of fifteen species of aquatic animal organisms varied considerably in

amounts but with a mathematical exactness in which the amounts required to be present depended

exponentailly on the salinity of the the habitats (2-1).

A further characteristic behaviour of such cell surface seawater or seawater-like solution bathed anionic

polysaccharides is their ability to preferentially sequester those inorganic ions which occur

in the least amounts in their natural bathing solutions. This suggests some osmolyte function exists for

those many inorganic elements which can become enriched at anionic polysaccharides, including those in

animals but have not previously been indicated to have any physiological function.

It is further pointed out, however, that while the cell surface glycocalyxes and extracellular matrices
may exist in the ‘all-element’ forms in vivo they may not end up as this type of matrix in the laboratory.

It is likely that such matrices are subject to inappropriate over-purification during standard laboratory

work-up procedures. It has been formerly been assumed that the natural multi-inorganic metallomic

matrix nature of anionic polysacharide systems is of trivial scientific importance.

This assumption needs now to be questioned.

It seems that heparin and the related heparin-like segments of heparan sulphate proteoglycans

could be the most uniquely relevant types of biological ligands with which to conduct biological

metallomics research since this type of highly sulphated polysaccharide structure is the most

anionic type of any known biopolmer.

Experimental evidence that anionic polysaccharides bathed with multi-inorganc containing solutions
become anionic polysaccharides.

While a range of multivalent metal ions have been previously reported to bind to heparan sulphate

proteoglycans (HSPGs) under both in vitro and in vivo conditions studies in the context of elucidation of

the mechanism of scintigraphic imaging (5-5), the principal evidence that heparin/heparan sulphate

occur naturally as metallomic matrices is provided by the kind of chemical analysis data which is

summarised below (cf. also Figs. 1) which perhaps indicates that that an approachingly

similar-to-seawater ‘full’ range of inorganic elements co-occurs with pharmaceutical heparin.

This circumstance seems most likely to arise from the occurrence of this type of heparin

in the originating tissue (mast cells) and not from any post-extraction contamination

of the heparin (e.g. from dust or inorganic elements present in chemical reagents or

vessels employed in the industrial processes employed for removing heparin from

its tissue of origin).

The idea that heparin might be a metallomic matrix was first suggested from the

consideration of the results obtained by use of a standard soil science laboratory

spark source mass spectroscopic analysis (5) of a specially provided sample of

pharmaceutical heparin (provided by a major manufacturer). This idea was later

confirmed by multi-element analytical investigation of heparin using inductively coupled

plasma arc mass spectroscopy (ALS, 5-1-1) which allows for the determination of

the full elemental composition of a series of different heparinized blood collection

tube extracts.

The total analytical data which are now currently available in the public domain seems

to confirm the original hypothesis that heparin-like polysaccharides are always chemically

consituted as full Haraguchi-type metallomic matrices.

Summary of the inorganic element content as µg/g of heparin

This was restricted for Aberdeen U. results (5) to the amount of elements present in amounts greater than
1µg/g which are listed thus: ( ) and [ ] respectively for the elemental composition of Na porcine mucosal
heparin and a modified heparin obtained following single counterion [Tl] ion-exchange column
enrichment of this Na heparin; raw data listing the total inorganic element ICP-MS results for extracts of
heparinized blood collection tubes given in an ALS internet document (ref. 5-1-1) confirm the general
metallomic nature of heparin for which the inorganic element contents, indicated by { }, were calculated
on the assumption that the S found by ICP-MS was derived from the sulphate half ester groups of
heparin:

Na ca. 9% of dry weight, Ca (30000) [30]{171}, Si (5900)[100]{116}, Cl (5600)[1000]{n.d.}, K (2000)[40]{23},

Mg(1300)[10]{?}, Fe (1100)[10]{5}, F (890)[4](n.d.}, Cu (730)[5]{16}, P (440)[30]{61}, Ti (<390)[4]{0.2}, Ni
(<170)[10]{8}, Ba (140)[1]{1}, Br (130)[0.9]{?}, Zn (80)[7]{16}, Sr (65)[1.4]{2}, Co (<80)[2]{<0.01},
B (<25)[1]{10}, Ga (20)[1]{0.005}, As (15)[1]{<78}, Pb (16)[4]{0.8}, I (10)[<1]{1.3}, Cs (9)[0.7]{0.01},
Tl(8) [principal counterion]{0.2}, Mo (7)[<1]{0.01}, La (7)[4]{0.005}, Ce (7)[3.5]{0.02}, Ag (4)[0.5]{0.002}, Sn
(5)[0.3]{<0.3}, Nd (5)[1]{0.01},W (5)[<1]{<0.02}, Zr (5)[0.3]{0.05}, Rb (3)[0.3]{0.1},V(3)[<1]{<0.3}, Mn (1)
[<0.1]{0.4}, Cd(1)[1.5]{0.3}); these data confirm previous reports (cf. refs. 5-1 and 5-1-2) that Ca, Si, Sr, Ba, Cu,
Zn, As, Al, Mn, Cr and V normally occur in heparin. (Cf. also the reports (5-2, 5-3) that heparin binds Eu3+ and Cu2+
under physiological conditions and further relevant data reported in refs. 5-3-1, 5-4, 5-4-1 and 5-5).
It is evident that such multi-inorganic element matrices, were they to be replicated in vivo, could act as
reservoirs for the provision of physiologicaly essential metal ions including those required for the
formation of inter- and intra-molecular linkages involving polysaccharides.
It should be noted that the biological metallomic matrices commonly include a range of
elements (e.g. As and Al) in addition to the twenty or so metal ions which are currently
known to be biochemically essential for animals.

While the phenomenon of the presence of small amounts of a large number of inorganic elements is fairly well known to occur with
for numerous laboratory reagents including e.g. ethylendiamine tetraacetic acid (EDTA), the amount of the metallomic range of
inorganic elements which can be sequestered by heparin seems to be uniquely greater than is the case with other ligands, in keeping
with this substance being the most anionic biopolymer.

Putative Mutual Interaction Between the Heparanome and the Metallome

Haraguchi-metallomics concepts are now suggested to be especial relevance to the heparanome** (6), the

putative animal cell surface and extracellular matrix polysaccharide-based signalling system which is

believed to depend on the temporal and spatial regulation processes afforded by modulation of the ultra-

anionic heparin-like segments within the HSPG polysaccharide side-chains which enables the

orchestration of the activity of numerous proteins including via their incalcation interactions with
information encoded heparin-like fragments (7-10).

Possible Relevance of the Co-Occurrence of Metalloid and ‘Non-Physiological’ Inorganic Elements in

Heparin

Anionic polysaccharides seem ubiquitously to contain appreciable amounts of metalloid elements such

as P, B and Si as well as a range of non-physiological metal ions such as the rare earths.

The ability of such metalloid elements in their oxy-anionic forms to act as secondary sites for the binding
of

metal cations is likely to be part of the mechanism by which natural anionic poysaccharides acquire a full

Haraguchi metallomic matrix.

This circumstance suggests that the biochemical efficiency of the heparan sulphate side chains of HSPGs

which are currently believed to play major roles in animal biochemistry e.g., for the modulation of
growth

factor signalling in embryogenesis, wound healing and during the progression of cancer may, at least
partly,

be dependent of the ubiquitous attachment of the full naturakl range of inorganic elements to these

polysaccharides.

Furthermore since such putative biological activity-determining inorganic elements can be inadventantly

depleted or enhanced during common laboratory purification and fractionation procedures this can be

proposed to lead to the formation of artefactal heparan fraction generation, a circumstance which must, in

the past, have led to a mis-assignment, of perhaps numerous, supposed heparan sulphate-determined

effects and their correlations.

It seems worthwhile to emphasize the possibility that laboratory protocols for reseach in this

field may require to be modified to allow for the preparation of appropriate multi-element chemical
forms

of such polysaccharides to correctly reproduce the metallomic forms of these molecules which occur in

vivo.

Log-log correlations between metallomic matrice inorganic element contents

An inverse exponential relationship was indicated to exist

between the enrichment achieved by the uptake to the polysaccharide and the concentration of individual
inorganics present in the bathing solution (i.e. the least abundant elements which occur in seawater-like

bathing solutions also tend to be more avidly sequestered by heparin). .

The type of approximate correlation which shows up between the inorganic element composition of

seawater and human blood serum (cf. Fig. 1 of ref. 2) is also found to exist with heparin.

Thus log-log correlations can be demonstrated between the inorganic element contents of heparin and

human blood serum (cf. Figs.1) as well as human hair where multi-inorganic elements are presumable

linked to the fibrous protein component cf. Rodushkin & Axelson, Supplementary References); such

correlations are demonstrated by heparins from different manufacturers as well as for single counterion

enriched heparin obtained by passage through a sulphonated polystyrene ion exchange column (a process

commonly employed to achieve medical grade heparin).

Figs 1. Examples of Log-Log Plots Between Seawater (& Seawater-Related Blood Serum)
& Heparin Multi-Element Contents
Fig1a

A Heparanome-Metallome Crosstalk Hypothesis

The hypothesis that metal ions determine much of heparan sulphate biochemistry is consistent with a

large number of literature reports exemplified by data collected in Tables I and II which indicate

that inorganic cations can be absolutely required to achieve a range of biochemical processes

mediated by heparan sulphate.

These include blood haemostasis, antioxidant defence and growth factor assembly

processes (cf., Kan et al. 1966 (11) cf., Rudd et al. (12); Guimond et al. (13)); (cf. also Grant (14)).

Possible Pathological Relevance of Anthropogenic Perturbation of Normal Metallomic Matrices

The apparent epidemiological correlation between the prevalence of multiple sclerosis

(e.g. in North East Scotland) and the geological, environmental anthropogenic contamination

of soil and vegetation with Ba2+ (15) could suggeste that a perturbation of

divalent-metal-ion-dependent heparan sulphate growth factor signalling (cf. 11) (required

for wound healing) might contribute to the aetiology of this disease.

The presence of inappropriate amount of the redox metal ions may also promote an inappropriate

augmentation of the nitrosative scission of heparan sulphate which could be of possible relevance to a

fuller understanding of the aetiology of several degenerative diseases

(cf. Grant et al., 2000 Supplementary References).

[Cf. metal and other inorganic element dependence of heparin/heparan sulphate actions (11-18)

include the modulation of enzymic (19) and nitrosative scission (20) processes which yield messenger

oligosaccharides which appear to mediate cellular immune function, angiogenesis and

the inhibiton of inappropriate cellular proliferation (21-24)].

Metal ions, and perhaps inorganic silicate colloidal particles, which seem always to be associated with

anionic polysaccharides throughout biota, may also conceivably contribute to the proposed heparan
sulphate

dependent ‘trans acting, smart glue’ trapping cell-cell communication mechanism proposed by Jakobsson
et

al. (21).

Inorganic cations might be able to directly regulate growth factor activities by mechanisms which include

the alteration of the conformation of the adjacent polysaccharide chains (cf., ref. 25).
Specific binding of highly toxic e.g., nuclides to polysaccharide metallomic matrices and well as

other toxic metals such as Hg, Cd and Pb to polysaccharide metallomic matrices might serve as a

tissue-protective system at cell surfaces and extracellular matrices. (The occurrence of metllomic
matrices

might e.g., enhance the free-radical quenching abilities of polysaccharide-based antioxidant defense

systems (cf., 28-4).

When the above possibilities are brought to the full attention of the polysaccharide research scientific

community, a task which this article seeks to accomplish, further examples of the modulation of heparan

sulphate signalling by inorganic elements, might eventually, it is suggested, be discovered.

Table I

Examples of facilitation by divalent cations of the binding of heparin/heparan

to proteins.

Cation Process Involving Metal Ion Determined

Heparin/Heparan Sulphate Protein Linkage References
or Analogous Crosslinking |

__________________________________________________________________________

M2+ ions Assembly of Annexin-V Capila et al. 2001, ref. (17)

at Phospholipid membranes

-------------------------------------------------------------------------------------------------------------------------------------

Mn2+ Serum lipid (LDL) aggregate formation

Heparin -M2+ -Phospholipid Lindahl & Hook 1978, ref. (8)
-----------------------------------------------------------------------------------------------------------------------------------
M ion
2+
Modulation of FGF-2 activity Kan et al. 1996, ref. (11)
Ca2+, divalent cation required for dimerisation of
Mg2+ FGF receptor for activation
or Mn2+
-------------------------------------------------------------------------------------------------------------------------------------
Ca2+ Heparin-Mn+-LDL linking Keskes et al. 1983, ref. (29-1)
-------------------------------------------------------------------------------------------------------------------------------------
Ca 2+
Factor X - Prothrombin complex Ofosu et al. 1982, ref. (29-2)
-----------------------------------------------------------------------------------------------------------------------------------
Ca2+ Inhibition of heparin binding to Thrombin Spreight & Griffith 1983, ref. (29-3)
------------------------------------------------------------------------------------------------------------------------------------
Ca2+ Fibronectin Hayashi & Yamada 1982, ref. (29-4)
-------------------------------------------------------------------------------------------------------------------------------------
Ca 2+
β2 and β3 Integrins, Discussed by Kan et al. 1996, ref. (11)
-------------------------------------------------------------------------------------------------------------------------------------
Ca2+ Platelet/Endothelial
Cell Adhesion Molecule-1 (PECAN-1) Discussed by Kan et al. 1996, ref. (11)
-------------------------------------------------------------------------------------------------------------------------------------
Ca 2+
Cadhedrin, Discussed by Kan et al. 1996 ref. (11)
-------------------------------------------------------------------------------------------------------------------------------------
Ca2+ L-Selectin, Koenig et al. 1998, ref. (29-5)
Norgard-Sumnich et al. 1993, ref (29-6)
--------------------------------------------------------------------------------------------------------------------------------
Ca2+ Serum Amyloid P (SAP) Nielson et al. 1994, ref. (29-7)
-----------------------------------------------------------------------------------------------------------------------------------
Ca2+ Porcine Brain Synaptosome Shinjo et al. 2004, ref (29-8)
Ca-ATPase inhibition Zhao & Zhang 2003, ref. (29-9)

-----------------------------------------------------------------------------------------------------------------------------------
Ca2+ Recycling of heparan sulphate proteoglycans by parathyroid Takeuchi et al. 1990, ref. (29-10)
cell lines

-----------------------------------------------------------------------------------------------------------------------------------
Ca2+ Dependent -Heparan Sulphate-Neurological Activity

Ca2+ Signaling in neurons induced by S100A4 Kiryushko et al. 2006, ref. (29-11)
and heparan sulphate
may act as co-receptors of S100 proteins in neurons

-------------------------------------------------------------------------------------------------------------------------------------
Ca2+/Na+ Smooth muscle Na+/Ca2+ exchanger inhibition
heparin fragments bearing C4-5 unsaturation Schinjo et al. 2004, ref. (29-8)
at the non-reducing end, produced by
bacterial lyase activity

Heparin binds with high affinity to Knaus et al. 1990, ref. (29-12)
voltage-dependent L-type Ca2+ channels

Vascular flow sensor potentiated

via Ca2+/Na+ induced heparan conformation alteration Siegel et al. 1998, ref (29-13)

Zn2+ (and Ca2+ ) Modulation of heparan sulphate binding

Zn2+ Inhibition of Fibroblast growth factor-2 (FGF-2) activity; Ricard Blum et al. 2004 ref. (16)
Endostatin binding
---------------------------------------------------------------------------------------------------------------------------------
Zn2+ Modulation of MRP-8/14 S100 activity Robinson et al. 2002, ref. (29-14)
-----------------------------------------------------------------------------------------------------------------------------------

Zn2+ Fibrillin-1, Tidemann et al. 2001, ref. (29-15)

-----------------------------------------------------------------------------------------------------------------------------------
Zn2+ H-Kininogen. Stanley et al. 1995, ref. (29-16)
-------------------------------------------------------------------------------------------------------------------------------------
Zn 2+
Histidine Rich Glycoprotein Jones et al. 2006, ref. (29-17)
-------------------------------------------------------------------------------------------------------------------------------------
Zn2+ Histamine Kerp 1963, ref (29-18)
-------------------------------------------------------------------------------------------------------------------------------------
Cu2+ Fibrinogen Lages & Stivala 1973, ref. (29-19)
-------------------------------------------------------------------------------------------------------------------------------------
Cu 2+
Prion Proteins Gonzalez-Inglesias et al. 2002, ref. (29-20)
----------------------------------------------------------------------------------------------------------------------------------
Ca2+ ( or Mg2+) Inhibition of heparin binding to Antithrombin(III) Yamane et al. 1983, ref. (29-21)

-----------------------------------------------------------------------------------------------------------------------------------------------------------------
Deaminative cleavage of heparan sulphate via heparan sulphate (e.g. via core protein storage of nitric oxide)

Metal ion modulation promotes scission of syndecan-1 heparan sulphate

chains by NO metabolites via
Cu+ and Cu2+ redox recycling (putatively involving ascorbate)
which enables heparan sulphate oligosaccharide generation e.g. , Ding et al.., 2002, ref. (20)

A possibly related process is the inhibition by

sulphated polysaccharides of a
Mn2 - dependent soluble guanylate cyclase Liebel et al. 1982, ref. (29-22)

The nitrosative scission of heparin/heparan sulphate at physiological pH was found to occur in the presence of a phosphate buffer but
not an imidazole buffer (Vilar et al., 1997, ref. (16a-1)). This might suggest that trace amounts of redox metals such as Cu and Fe,
known to occur in phosphate buffers, promote this reaction.
-------------------------------------------------------------------------------------------------------------------------------------------------------------------
Amyloid Formation
Zn2+ 50nM Zn2+ promotion of binding of heparin to
Amyloid Precursor Protein (APP) Masters et al. 1993, ref. (29-23)

Inhibition of APP proteolysis by heparin is abolished by Zn2+

---------------------------------------------------------------------------------------------------------------------------------------------

Inorganic Crystal Formation

Calcium Oxalate Crystals (or precursor high oxalate concentration) Borges et al., 2005, ref. (29-24)
Inhibtion of formation by heparan sulphate

Table II
Examples of Reports of The Effects of Inorganic Cations and Anions on Heparan Sulphate Biosynthesis
Mn2+ Promotion of heparan sulphate A.Z. Kalea et al., Biometals 2006, 19,
biosynthesis 535-546 (ref. 29-25)

----------------------------------------- ------------------------------------
α-GlcNAc transferase I can use both Mn2+ T.A. Fritz et al.,J. Biol. Chem., 1994, 269,
and Ca2+ but 28809-28814 (ref. 29-26)
α-GlcAc transferase II can use only Mn2+

Mg2+ Effect of Mg2+ deficiency on the P. Jaya and P.A. Kurup,J. Biosci., 1986,
metabolism of glycosaminoglycans, 10, 487-493 (ref. 29-27)
including heparan sulphate

Ca2+ Suppression of proteoglycan including

heparan sulphate synthesis by calcium
ionophore A23187 in cultured
vascular endothelial cells Y. Fujiwara and T. Kaji , J. Health Sci.,
------------------------------------------ --------------------------------------- 2002, 48, 460-466
Pb2+ Implication of intracellular calcium (ref. 29-28)
accumulation in Pb2+ inhibition of
endothelial proteoglycan including
heparan sulphate biosynthesis

Cd2+ Inhibition of the incorporation of [35-S] A. Cardenas et al.,Toxicology

sulphate into glomerular membranes of 1992, 76, 219-231 (ref.29-29)
rats chronically exposed to Cd2+ and its
relation with urinary glycosaminoglycans
and proteinuria

Hg2+, Ni2+ Inhibition of glomerular heparan sulphate D.M. Templeton, Proc. Trace Element
biosynthesis. Health Disease, IUPAC Int. Symp.,1990,
Metal-proteoglycan interactions in the p. 209-219 (ref. 29-30)
regulation of renal mesangial cells:
implication for metal induced
nephropathy.
SiO2 Putative promotion of heparan sulphate M.F. McCarty
biosynthesis Med Hypoth.,1997, 49, 177-179

Si, putatively as SiO2 nanoparticles, may Cf., R.M. Iler The Chemistry of Silica ,
always occur in association with Wiley, 1979, cf. p. 762 (ref. 29-31)
heparin/heparan sulphate and D. Grant et al. Med Hypoth 1992 38
46-48 cf., Additional References)

F- F- ions inhibit heparan sulphate sulphation M. Pawalowska Goral et al., Fluoride,

1998, 31, 193-201 (ref. 29-32)
SeO4 2-
Inhibits heparan sulphate biosynthesis C.P. Dietrich et al., FASEB J., 1988, 2, 56-
59 (ref. 29-33)
Further Discussion & Suggestions for Future Research

The roles of metal ions in heparin/heparan sulphate biochemistry summarized in Tables I and II indicate

that metal ions could commonly be required for a range of biochemical activities which are facilitated by

heparan sulphate. This includes the binding (of specific segments of) heparin/heparan sulphate to

target phospholipids and proteins as well as for the generation of the oligosacchrides which are believed

to act as messenger molecules invovled in cell-cell communication. The role of metal ions in the latter

process includes the Cu2+ + e  Cu+ redox cycling which is required for the release of nitric oxide from

conserved cysteines present in heparan sulphate proteoglycan core protein stores, a process which is

believed to contribute (20) to the regulation of oligosaccharide generation via the deaminative cleavage

of heparan sulphate chains by nitric oxide metabolites (for which reaction there also seems to be a less
well-

defined catalytic role of Zn2+ ions, which also indirectly affect the alternative mechanism of
oligosaccharide

production via the action of heparanase which has recently been recently indicated (by Zcharia et al.,

(19)) to occur in conjunction with metalloproteinase activity).

Other indications of the importance of metal ions in glycosaminoglycan biochemistry are that the

physico-chemical behaviour of cartilage is believed to be modulated e.g. for calcification and swelling
(30)

by an association of metal ions with anionic polysaccharides and their aggregates.

In related studies, heparin was found to interact more strongly with monovalent and divalent

metal cations than did the other glycosaminoglycans which are the main components of cartilage (31).

The ability of glycosaminoglycans to inhibit calcification (32), a phenomenon which has been suggested

to be of critical importance to the prevenetion of blood vessel wall calcification (33), is (putatively)

also affected by the secondary inorganic elements (which may be augmented by anthropogenic input)

which are likely to become enriched in cell wall heparan sulphates since anionic extracellular
polysaccharides seem generally to have the ability to simultaneously bind and selectively become
enriched

in the least abundant ultratrace elements which occur in natural waters and biological fluids.

It should be noted that this multi-element sequestration property is also the basis of the use of the algal

biomass of kelp for plant and animal nutrition (4) and for its use for the removal toxic heavy metal ions

from polluted waters (4-1).

This suggests that toxic metal ion trapping by animal polysaccharides may serve a similar function

to the above.

A further potential function of metal and other small inorganic ions in the heparanome is an apparent

servo feedback system causing the presence of specific inorganic ions and inorganic surfaces

(summarised in Table II) to signal for the de novo synthesis of heparan sulphates putatively generated in

reponse to the extracellular presence of such inorganic entities via alteration of heparan sulphate in the

Golgi apparatus.

The provision of inorganic cofactors for the above processes may be achieved by an inbuilt reservoir

system consists of a metallomic array of inorganic elements attached to heparin/heparan sulphate

protoeglycans.

Heparin seems to be chemically constituted so as to simultaneously bind the full range of seawater

elements in both cationic, anionic and non-ionic forms.

(Heparin in addition to having the ability to bind a wide range of counter cations can (paradoxically) also
avidly binds anions (including the halides, sulphate, silicate, borate and phosphate) as well as colloidal
particles (e.g. sparingly soluble alkaline earth carbonates as well as iron oxides) apparently so as to
achieve a well-defined seawater-related metallomic matrix status inorganic element reservoir.
Heparin-like segments within heparan sulphate polysaccharide chains are predicted to behave similarly).

While Ca2+ (10) and Zn2+ are the most common currently known inorganic cofactors for

heparanome actions (cf. Table I) it is suggested that an in depth study of the inorganic elements silicon

titanium, the rare earths, manganese, phosphorus, vanadium, arsenic and gallium, which apparently

consistently occur in heparin, could be of possible physiological relevance and should be evaluated as

possible essential cofactor components for specific heparanome activities.

Heparan sulphates were reported by Nader et al., to occur in greater abundance in those marine
invertebrates with habitats having greater salinities; this relationship seems to be a mathematically exact
one for sulphated glycosaminoglycan contents (34). This phenomenon, and it’s apparent continuation in
more complex animals (exemplified by the ionic filtration roles of heparan sulphate in glomerular
basement membranes (34-1)) agrees with the concept that the original biological function of heparan
sulphate polysaccharides may have been for the regulation of the ionic balance at the cell surface, and
perhaps also to sequester essential nutrient elements from the bathing solutions. The need for an
improved, readily reversible, sequestration seems to have arisen at the time of the first evolution of
multicellular animals in the sea which was coincident with the evolution of an isomerase enzyme to
convert the glucuronic acid residues of the precursor bacterial-like polysaccharides in order to yield the
improved metal ion chelation abilities of iduronate groups which can flexibly bind and release metal ions
(36). This process can be suggested to have further evolved into a complex morphogenic mechanisms
involving crosstalk beteen the metallome and the heparnome sulphates with feedback processes allowing
postsynthetic modification involving nitric oxide which is putatively used for control of morphogenesis in
multicellular animals. This complex postsynthetic modification process (which enable a rational
alteration of the signalling code and produces oligosaccharides) by both enzymic and non-enzymic
pathways can achieve direct and indirect sensitivity to the presence of both inorganic ions and organic
molecules (e.g. ascorbate, retinodic acid, fatty acids, etc. [more fully listed and referenced in refs. 14]).

The reports listed in Table I and Table II, taken together, might suggest that heparanome associated metal
ions could normally be important components of the modus operadi of this complex information system
in which inorganic ions and particles apparently can act (via servo feedback modes?) to change the
heparan sulphate microstructure in response to external stresses. This phenomenon is most apparent for
the altered synthesis of heparan sulphates in parathyroid cells in response to changes in extracellular Ca2+
concentrations (29-10) and for the alteration of synthesis of heparan sulphates towards a form more able
to effectively combat calcium oxalate crystal stone formation (29-24)).

Metal Ions & Heparan Sulphate Cooperate for Control of Fibroblast Growth Factor Activity
Attempts to correlate fibroblast growth factor variant activities (a phenomenon which is
likely to be under a strict temporal and positional control) with the binding behaviour of
separated-out and purified specific heparan sulphate-core-anion pattern oligosacchrides,
failed (as reported by Kreuger et al. (35)) to show any of the expected discriminated binding.
This had been assumed by these authors to occur by a mechanism which recognised only
the naked anionic patterns present in the individual oligosaccharides. But, if it is correct
as is now suggested, that some specific counterion(s) may also be needed to complete the signal,
it is conceivable that these were removed during standard column purification of oligosaccharides.
Ricard-Blum et al. (16) reported an example of this effect. Zn2+ ions were found to be
essentially required to allow endostatin to bind to heparan sulphate only after the inadvertantly removal
of
Zn2+ can cause failure of this specific binding to occur.
Rudd et al. (12) and Guimond et al. (13) have more recently reported in depth studies of the specific
binding of a range of metal ions to heparin/heparan sulphate in a manner able to greatly influence basic
fibroblast growth factor receptor signalling activity and which supports the idea that inorganic elements,
including K+ and Cu2+ could be essential components of a heparan sulphate microstructure “signal”.

The pathological alteration of metal ions dependent heparan sulphate growth factor activity regulation
may be of prime relevance to the aetiology of cancer since oncogenic cellular transformation is generally
accompanied by a dramatic alteration in microstructure with a characteristic diminution in amounts of
those high anionic heparan sulphates present at cell surfaces which are able to strongly bind metal ions
(cf. e.g. ref. 37 and refs. 14).
The pro-oxidant situation which is known to be associated with degenerative diseases has been proposed
to accelerate a diminution of tissue protection via the non-enzymic degradation of heparan sulphate by
nitric oxide dependent routes (Grant et al., 1997, Grant, 2000 cf., Supplementary References) and
perhaps also by other redox-active-metal-dependent reactive oxygen free-radical formation (cf. 38).
Exogenously applied heparin metal ion complexes and related polysaccharide preparations can, however,
apparently confer general tissue protection against degenerative disease, putatively including cancer, by a
mechanism which, at least partly, depends on their ability to inhibit free-radical degradation processes
via the deactivation of redox active metal ions (cf. refs 28, 28-1, 28-2, cf. 39). This tissue protection
seems to require the sequestration by heparin via a phase change, high-affinity binding process (cf. 28)
which may further depend on the retention by heparin of silica and other compnents of its ‘native’
seawater metallomic array. When such multi-elements are removed during ‘purification’, the heparin
apparently becomes inactive for such actions.
Further work is however required to confirm these preliminary findings.

The following ‘metallome-heparanome’ sub-hypotheses can be suggested.

1) Heparan sulphate proteoglycans are so structured as to facilitate the initial uptake, rapid transport and
release of nutrient elements and promote heavy metal detoxification processes.

2) Anionic polysaccharides may generate specific conformations as a consequence of the binding of

specific types of metal ions. These conformations may play direct roles in heparan sulphate signalling

3) The heparan sulphate nutrient element sequestration action contains a feedback loop to altered heparan
sulphate synthesis for the generation of altered anionic patterns which potentially can signal for an
altered tissue and organ assembly process (principally via the regulation of the provision and the
activation of growth factors and their receptors). This may have enabled the evolution of animal species
to occur in response to inorganic nutrient stress, oxidative stress or nitrosative stress.

It is likely that individual heparin preparations will show differences in multi-element contents due to
original in vivo multi-element contents and work-up procedure; further work is warranted to more fully
study this.

References
(1)
R.J.P. Williams (Oxford University)
Personal communication.

(2)
H. Haraguchi
Metallomics as integrated biometal science.
J Anal. At. Spectrom., 2004, 19, 5-14;
cf.
http://www.apchem.nagoya-u.ac.jp/06-III-3/index-e.html.

[The use of modern mass spectroscopic techniques suggests the common occurrence of 50+ inorganic
elements in a range of biologically relevant matrices (where these often occur with a similar abundance and type
to those which occur in the sea). This adds support to the notion that life began in the sea. The origin and
comparison of the inorganic multielement contents of different geological and biological matrices seems to be
the
basis of Haraguchi’s suggestion for redefined metallomics.
Since well-defined ‘Haraguchi-type’ multi-inorganic-element profiles are known to be associated with
glycosaminoglycans (especially with the heparin/heparan sulphate family) and with marine algal
polysaccharides and perhaps also with pectin and soil humic (organic) matter (e.g. fulvates), these substances,
rather than proteins (with the possible exception of the fibrous protein presumably repsonsible for the multi-
inorganic element content of hair) are now suggested to be the most highly relevant classes of compounds for
futher metallomic studies].
Haraguchi metallomics is the study of all of the inorganic elements, and not just those which are
currently accepted as being biologically relevant, and critically draws attention to the occurrence of non-
physiological inorganic elements which ubiquitously show up, albeit mostly in very small
concentrations, in a range of biologically relevant matrices).
Haraguchi matrices may eventually even be found actually to include all stable elements.
At least some of these may perform presently unsuspected biological functions and could be critically
relevant to the fuller understanding of animal nutrition.
The human blood serum and seawater (cf. ref. 2, Fig. 1) which supports the idea of the
likely central role of seawater compostion in the early evolution of animals could also suggest that
this inorganic ion driving force may continue to drive animal evolution in a terrestrial environment.
Animal cell surface anionic polysaccharides are positioned as extracellular antennae analogously
to the marine extracellular alginate polysaccharides which interface seawater from which they acquire a
full
Harguchi inorganic element array. Heparin (and therefore also the heparin like segments
of heparan sulphates) probably achieve metallomic matrix chemical constitutions in this manner
It is now proposed that this type of phenomenon which is faily well known to occur with
marine alginates (4) also applies to the analogously sited animal cell surface heparan
sulphates which are mainly located in extracellular locations which facilitates the
sequestration of ions etc. which occur in extracellular fluids.
The algal cell surface anionic polysaccharides which build Haraguchi-metallomic
arrays from seawater seem to share a similar nutrient gathering function with
the animal glycosaminoglycans.

(2-1) cf., H. B Nader et al., ref. (34) vide infra

(3)
S. Mounicou, J. Szpunar and R. Lobinski
Metallomics: the concept and methodology.
Chem. Soc. Rev., 2009, 38, 1119-1138;
doi:10.1039/b713633c.

[This review is largely restricted to the relvance of metallomics as defined by R.J. P. Williams, to proteins].

(3)
Multi-inorganic elements associated with human hair
cf., Rudushkin, I. and M. Axelsson Supplementary References.

(4)
A. Wassermann
Cation adsorption by brown algae. The mode of occurrence of alginic acid.
Ann. Bot. N.S., 1949; 13 (49): 80-88;
cf.,
W.A.P. Black and R.L. Mitchell
Trace elements in the common brown algae and in sea water.
J. Mar. Biol. Assoc. U.K., 1952, 30 (3) 575-584.
[These authors noted that “Seawater probably contains all the chemical elements, although a number of them
have
not yet been detected”].
The above 1949 study by Black and Mitchell clearly suggest that anionic polysaccharides in the cell walls of
marine algae occur in the form of multi-inorganic metal ‘salts’.

[More recent studies also further confirm this idea. The multi-element contents, in e.g., kelp, has been indicated
to derive largely from the in vivo binding by the extracellular anionic polysaccharides present in the algal cell
walls of the ionic and particulate multi-inorganic elements present in seawater,
E.g., K. Truus, M. Vaher and I.Taure,
Proc. Estonian Acad. Sci. Chem., 2001, 50, 95-103,
and
The multi-element analysis of Ascophylum nodosum
reported at http://www/alginure.co.uk/ascophylum-nodosum.html contain data
were apparently provided by the Norwegian Institute of Seaweed Research.
Cf. also T.A. Davis et al. (Supplementary references) ; the multi-elements in Ecklonia maxima given in
http://www.gairesearch.co.za/kelp.html.

(4.1) Polyanionic algal polysaccharides have also been reported to be useful for the removal of heavy metals
from
water supplies cf., T.A. Davis et al. (Supplementary References)].
(5)
The ultra-anonic ubiquitous adherent animal cell heparin (a member of the heparan sulphate
family and also the most anionic biopolymer) apparently occurs, as is demonstrated below, in a
similar multi-inorganic chemically-constituted form to the cell surface anioic polysaccharides
which occur in marine algae (4).
(It should also be noted in this context that heparin is also believed to be the most
anionic known biopolymer known).

A sodium porcine mucosal heparin sample (further details of which are given by C.F. Moffat in:
Synthesis,“Characterisation and applications of chemically modified heparins”, Ph.D. Thesis, University
of Aberdeen, Department of Biochemistry, 1987 and briefly by D. Grant et al., in Biochem. J.,1987, 244,
p. 143, cf., also, D. Grant in Chemistry Preprint Archive, 2000, October, 2000, (10), 94-103) was donated
by a former major UK manufacturer as being suitable for fundamental chemical/biochemical
investigations. (It should also be noted that this heparin appeared not to correspond to the “overpurified”
form of heparin obtained after the ion exchange reduction of multi-elements needed to achieve suitable
low heavy metal ion contents preferred for pharmaceutical use); spark source mass spectrometry showed
the presence in this heparin (following extensive dialysis) of major amounts of a seawater-like
distribution of inorganic elements; a lesser amount (but also a seawater-like distribution) was obtained
after partial removal of the associated inorganic elements detailed by D. Grant (1987) loc.cit. This
standard ion replacement method was found to be highly effective for most elements by less so for Pb2+,
Ce4+ and other rare earth ions.

(5-1)
That a wide range of metal ions occur in commercial heparins in the manner established in the above
paragraph, is also in aggreement with older reports in the scintific literature including the articles by:
H.J.M. Bowen, who in “Trace Elements in Biochemistry” Academic press, London, 1966, p. 63,
noted an ubiquitous presence of barium, calcium, copper, manganese, strontium and zinc ions in
heparin. The presence of such ‘contaminants’ demanded tha careful consideration be made of
appropriate blanks before attempting to use blood collection tubes containing this anticoagulant for
the evaluation of the contents of several the above mentioned and other metal ions in blood
fractions.
The importance of the above advice was confirmed in 2005 by The ALS Laboratory Group (a major
Swedish-headquartered international analytical service provider) who brought to the attention of, inter
alia, medical and other analytical scientists, the wide range of inorganic elements which could be leached
from commercial heparinized blood collection tubes into blood samples, cf.,
(5-1-1)
http://www.alsglobal.se/hem2005/pdf/blood_collection_tubes.eng.pdf
(downloaded on 8 Aug 2008 and 13 Aug 2009).
(This document provides detailed ICP-MS determined multi-inorganic-element analyses information,
including possible blanks, allowing the accurate estimate of metal ion ‘contaminants present in heparin
molecules and other reagent employed in blood collection tubes; these data were apparently published to
allow for a greater awareness by interested parties in possible sources of error in the determination of the
inorganic element contents of blood sample fractions. Since the ICP-MS data include the amounts of
inorganic sulphur present, this enables a back calculation to be made of both heparin and its associated
inorganic elements which can be leached from several brands of blood collection tubes by the dilute
nitric acid extraction methpd used. It is likely, however that the described method of obtaining solutions
of heparin-inorganic element complexes, underestimates the amounts of Co, Mo, La, Ce, Ag, Nd and W
associated with the starting heparin coated commercial PET blood collection tubes).
It should also be noted that a similar range of inorganic elements (but in much lower stoicheometric amounts occur
in ethylendiaminetetraacetic acid (EDTA), which is also used as an anticoagulant in blood collection tubes).
[N.b., this report did not consider any wider relevance of these data].
A full ‘all element’ seawater-like inorganic element distribution is likely for such systems.

Other published studies of inorganic element ‘contamination’ of heparin include:

(5-1-2)
G.F. Harrsion and A Sutton (Nature, 1963 (4869) 809) who reported the presence
 of significant amounts (this being of concern for biomedical applications) of calcium, strontium and
barium in several heparin samples.

N.W. Alcock (Elem. Metab. Man Anim. Proc. Int. Symp., 4th., 1981 (Pub. 1982) Eds. J.M.
Gawthorme, J.M.M.M.C. Howell, C.L. White p. 678-680, Springer, Berlin; Chem. Abs., 96,
213646) noted the presence of potentially toxic amounts of manganese and chromium in some
heparins.

S.A. Katz Amer. Biotechnology (1984) Lab., 2, 24-30 reviewed previous reports of the
presence of calcium, copper, manganese, strontium and zinc in heparin (and suggested that the
source of such elements was laboratory dust).

G. Heinemann and W. Vogt (J. Biol.Trace Elem. Res., 2000, 75, 227-234) noted the
occurrence of variable amounts of vanadium in heparin

D. Bohrer et al. reported the arsenic contents (Parenteral Enteral Nutrition,

2004, 29 (1) 1-7) and the aluminium contents (RBAC (Brasil), 2005, 36 (2) 99-103) of
heparin.
It should be noted that the aluminium ions in heparin were also shown to be easily removed
by cation exchange resin column percolation (this purification procedure was recommended
for heparin which was to be medically employed for anticoagulantion of blood during dialysis).

While brine or untreated municipal water used for work-up of polysaccharide extracts using during
industrial scale manufacture of laboratory reagents and pharmaceutical heparin may, in part, be the
source of the multielements shown to co-occur with heparin (the possibility of which requires further
research) it is suggested that a more likely explanation for the experimentally observed multi-inorganic
element status of heparin is the in vivo equilibration of heparin with the dissolved or dispersed
multielement ionic components present in all or most biological fluids (which have incidentially a multi-
elemental composition which is approximately similar to seawater).

(5-2)
Eu3+ heparin complex formation facilitates heparin assay in blood samples
M. Rizk, Y. El-Shabrawy, N.A. Zakhan, S.S. Toubar and L.A. Carreira
Spectroscopic determination of heparin sodium using Eu(III) as a probe ion.
Spectroscop. Lett., 1995, 28 (8) 1235-1239.
[Eu3+ formed an adduct with heparin with log10K=5.079].

(5-3)
R.N. Rej, K.R. Holme and A.S. Perlin
Marked stereoselectivity in the binding of copper ions by heparin.
Contrasts with the binding of gadolinium and calcium ions.
Carbohydr. Res., 1990, 207, 143-152.
[Trace amounts (ca 1/185 mol ratio with respect to heparin) of Cu2+ together with
a slight molar excess of H2O2 + ascorbate reduced (e.g., by 30% over 30 min at 40oC)
the anti-FactorXa activity of heparin without causing any detectable alteration in the
NMR spectrum of the heparin; Fe2+ caused a less selective alteration in the heparin.
Evidently Cu2+ engages in more site specific binding to heparin than does Fe2+].
Cf.
Z. Liu and A.S. Perlin
Evidence of a selective free radical degradation of heparin mediated by cupric ion.
 Carbohydr. Res., 1994, 255,183-191.

(5-3-1)
Multi-phase nature of Cu2+ heparin interaction
There is evidence for physiologically relevant alteration in heparin activity by the binding of Cu2+
ions (5-3), and 13C-NMR studies suggest the existence of complex multi-phase relationships
in Cu2+ heparin interactions. Individual cations bind to heparin in a markedly not electrostaticlly
defined manner (5-4), cf., a number of studies have confirmed that heparin-like polysaccharides
also avidly bind inorganic anions (5-4-1) which should be ruled out if electrostatics dominated the
binding of inorganic moieties to heparin
Removal of paramagnetic ions from solution by insoluble colloid formation
between such ions and anionic polysacharides. A putative mechanism of antioxidant protection.
Pharmaceutical heparin was suggested, from NMR spectroscopic evidence, to be capable of
binding paramagnetic ions in aggregated insoluble forms
[cf. also Grant D. et al., Additional References, in Supplementary References, vide infra;
Anionic polysaccharides may provide antioxidant protection by this method
by sequestering Cu2+ cf. (28), Fe2+ (28-1, 28-2) and Fe3+ (28-3).
Chitosan and hyaluronan have also been suggested to act as antioxidants by this type of mechanism
(28-4)].

D. Grant et al.,
13C n.m.r. spectroscopy of Cu2+- heparin suggests phase separation of the complex from Cu2+ in
aqueous solution.
Biochem. Soc. Trans. 1992; 20: 214S.
Cf., R.A. Albertini et al., who discussed the protective effects afforded by heparin against Cu2+
induced
oxidation of human LDL in FEBS Lett., 1995, 377, 240-242; ibid., 383 155-158 cf., also 28-3, 28-4
and
http://electra.chemistry.upatras.ge/fects/final/w-shops.htm.

Cf. also the following articles by D. Grant et al.

Cation interaction with heparin at antithrombin-binding sites may differ

from that occurring elsewhere in the polymer.
Cell Biology International Reports, 1987, 11, 220.

Cation interactions with heparins/heparans are not explicable in terms of simple electrostatic effects.
Cell Biology International Reports, 1987,11, 221.

Cation complexation with heparin studied by 13C-NMR spectroscopy.

IRCS J Med. Sci., 1986, 14, 903-904.

Binding of copper to heparin. Physiochemical studies suggest interaction of

pharmacological significance .
Brit. Soc. Cell. Biol., 1985 36, Suppl. 8 (Abstr. 26), 13.

And the following articles by other former memebers of the Aberdeen polysaccharide group:

M.A. Ross, W.F. Long WF , F.B. Williamson and C.F. Moffat

Effect of chemically modified heparins , and of heparin fragments on
Fe(II)-catalysed peroxidation of linoleic acid.
Biochem Soc Trans., 1992, 20, 216s.
[Cf . Abstracts of the 641st Meeting of the Biochemical Society
Royal Holloway and Bedford New College London,
17-20 Dec 1991 p. 56 Abstract No. 159].

M.F. Williamson, W.F. Long and F.B. Williamson

Effect of heparin on the UV absorption properties of Fe(II) and Fe(III).
 Biochem. Soc. Trans. 1992, 20, 360s.

N.E. Woodhead, W.F. Long and F.B. Williamson

Zinc ion binding by heparin.
Biochem. Soc. Trans., 1983, 11, 96-97;
Cf., these authors, Zinc binding by heparin.
Analysis by equilibrium dialysis suggests
the occurrence of two, entropy-driven processes
Biochem. J., 1985, 237, 281-284.

Cf. also
D. Grant et al. Supplementary References

(5-4)
While the results of in vitro studies of ion binding by heparin by
D. Grant W.F. Long and F.B. Williamson
(A study of Ca2+ heparin complex formation by polarimetry
Biochem J., 1992 282, 601-604) suggested that the association between Ca2+ and heparin in
aqueous environments cannot be described adequately in terms of simple electrostatic
interactions and resembles in some respects the formation of mineral-like colloidal states; cf. also
the paper (by these authors, ibid 1991, 277, 569-571) which highlighted the possible critical
role of clusters of water molecules (which are beleived to be associated with heparin sulphate
half-ester anionic groups) in the inorganic-element binding process; other in depth studies of the
binding of a range of types of metal ions to heparin (in Supplementary References by D. Grant et al.
collected in the Appendix, vide infra) suggest that binding affinity of counterions to heparin could
alter in the presence of more than one other competetive counterion to create a system of mutual
adjustment of apparent binding affinities which seems ‘designed’ to discourage the formation of any
single counterion substituted heparin complexes.
This behaviour accords with the putative ability of heparin to acquire a full metallomic array of
inorganic elements via the process of simultaneous affinity variation so that appreciable amounts of
the
wide range of different types of inorganic ions and particles which are present in the natural
biological
bathing solutions become simultaneously bound each at sufficiently great amounts so as to create a
full
Hargauchi matrix.

(5-4-1)
Binding of anions to heparin
Cf., J.R. Helbert and M.A. Marini
Structural studies of heparin II. Exchangeable anions.
Biochim. Biophys. Acta, 1964, 83, 120-122.
[Heparin binds SO42- so that the normal presence of inorganic sulphate in heparin can increases the
total amount of sulphur ‘in heparin’ by a factor of 2+ greater than that due to the presence of sulphate
half-ester and N-sulphonate groups].
Cf.,
K.H. Simon
Naturwiss Rundschau, 1982, 11, 452-455,
and
L.B. Jaques
Heparin: an old drug with a new paradigm.
Science, 1979, 206, 528-533.
Cf also., Fo-We (Forschungs und Verweltungs Anstalt)
Brit. Pat. Appl. 890,622 (1962).
[Formation of complexes between heparin and inorganic salts].

Cf.
T. Takeuchi , Anon. Safni, T. Miwa, Y. Hashimoto and H Moriyama
 Ion chromatography using anion exchangers modified with heparin.
Analusis, 1998, 26, 61-64.
[A form of heparin- based ion exchange chromatography can separate cations and anions on a single
column; the mechanism appears to depend on the ability of heparin-SiO2 to act both as a cation and
anion selective binding system].

(5-5)
Y. Hama, T. Sasaki, S. Kojima and A. Kuberoda
67-Ga accumulation and heparan sulfate metabolism.
Eur. J. Nucl. Med., 1984, 9, 51-56; Chem. Abs., 100, 188079t.

Cf., S. Kojima, Y. Hama, T. Sasaki and A. Kuberoda

Elevated uptake of 67-Ga and increased heparan sulphate content
in liver-damaged rats.
Eur. J. Nucl. Med., 1983, 8, 52-59,
and Y. Hama, S. Kojima, and A. Kuberoda
Relation of heparan sulphate content and
67-Ga uptake in various tissues of rats.
Kagaku Igaku, 1982, 19 (6) 855-861; Chem Abs., 97, 211645e.
[67-Ga forms stronger complexes with heparan sulphate
than other GAGs allowing tissue distribution of heparan sulphate to be visualised]
S. Kojima
Uptake of 67-Ga citrate in tissue and heparan sulphate.
Radioisotopes, 1986, 35 (8) 437-445; Chem. Abs., 191815j.
Cf. also ref. 13
and
e.g. D.S. Millbraith et. al.,
Eur. Pat. Appl., EP 55028; Chem. Abs., 97, 168966;
A. Mostbeck et al.,
Nuklearmedizin Suppt. (Stuttgart), 1981, 18, 343.
[Heparin complexes of 111In and 99Tc may also be used for medical imaging].

(6)
J. Turnbull, A. Powell and S. Guimond
Trends Cell Biol., 2001, 11, 75-82.
[Heparan sulphate protoglycans (HSPGs), glypicans, syndecans, agrin etc.) constitute a major
multicellular animal cellular control system which is believed to be functionally dependent on the
presence of conserved sugar sequences required for specific interactions with proteins].

(6-1)
This could be of especial relevance to the post human geneome era which seeks to understand how the
unexpectedly small size of the human genome can serve such highly complex species as humans which
may be due to the ability of heparan sulphate to hold and process information.

Cf., D. Fernig, The University of Liverpool http://www.newlightsource.org/events/presentations/NLS

%20life%20science%20Fernig. Pdf
Decoding the matrix: dynamics of the key regulatior of cell-cell communication.
[This presentation seems to suggest that the information encoded in heparan sulphate may be more
pertinent to future medical research than is the information stored in the genome; it was noted that while
the original estimate of the number of protein encoding genes in humans was ca. 1,000,000 it is now
belived that this number is similar to that of Caenorhabditis elegans 20,000. {Although Fernig mentions
35,000 for the protein encoding genes present in Homo sapiens more recent work (Clamp et al.
Supplementary References) suggests that this value is ca.20,500].
(7)
H.B. Nader, T.M.P.C. Ferreira, L. Toma,
S.F. Chavante, C.P. Dietrich, B. Casu and G. Torri
Maintenance of heparan sulphate through evolution.
Carbohydr. Res., 1988, 184, 292-300.

(8)
U. Lindahl and M. Höök
Glycosaminoglycans and their binding to biological macromolecules.
Ann. Rev. Biochem., 1978, 47, 385-417.

(9)
L. Kjellén and U. Lindahl
Proteoglycans: structures and interactions.
Annu. Rev. Biochem., 1991, 60, 443-465.

(10)
M. Lyon and J.T. Gallagher
Biospecific sequence and domains of heparan sulphate and the regulation of cell
growth and adhesion.
Matrix Biol., 1998, 17 (7) 485-493.
Cf .
J.T. Gallagher
Heparan sulfate: growth control with a restricted sequence menu.
J. Clin. Invest., 2001, 108 (3) 357-361.

(10-1)
M. Bernfield, M. Götte, P.W. Park, O. Reizes, M.L. Fitzgerald, J. Lincecum, and M Zako
Functions of cell surface heparan sulfate proteoglycans.
Ann. Rev. Biochem., 1999, 68, 729-777.

(10-2)
N. Perrimon and M. Bernfield
Specificities of heparan sulphate proteoglycans in developmental processes.
Nature, 2000, 404, 725-728.
Cf.,
J. Biol. Chem., 2000, 275, 29923-29926

(10-3)
P.W. Park, O. Reizes and M. Bernfield
Specificities of heparan sulphate proteoglycans : selective regulators of
ligand-receptor encounters.
J. Biol. Chem., 2000, 275, 29923-29926.

(11)
M. Kan, F. Wang, M. Kan, B. To, J.L Gabriel and W.L. McKeehan
Divalent cations and heparin/heparan sulfate cooperate to control assembly and
activity of the fibroblast growth factor receptor complex.
J. Biol. Chem., 1996, 271, 26143-26148.
[These authors reviewed the other cell surface recognition molecules whose structures
and activities are modulated by divalent cations and heparan sulphate or other
glycosaminoglycans in the pericellular matrix, including
β 2 and β3 integrins, platelet/endothelial cell adhesion molecule-1 (PECAM-1)
cadhedrin and L-selectin].
Cf.,
M. Kan, X. Wu, F. Wang and W.L. McKeegan
Specificity of fibroblast growth factors determined by heparan sulfate in a binary complex with
 Receptor kinase.
J. Biol. Chem., 1999, 274, 15947-15952.
[Anticoagulant heparan sulphate is required for FGF receptor divalent cation
dependent association between heparan sulphate and the exodomain].

(12)
T.R. Rudd, S.E. Guimond, M.A. Skidmore, L. Duchesne, M. Guerrini, G. Torri, C. Cosentino,
A. Brown, D.T. Clarke, J.E. Turnbull, D.G. Fernig and E.A. Yates
Influence of substitution patterns and cation binding in conformation and activity of heparin
derivatives.
Glycobiology, 2007, 17 (9) 983-993
[The traditional view that signalling by heparin/heparan sulphate depends only on the
polysaccharide
anionic sequence is indicated to be incorrect, since the binding of individual cations including K+
and Cu2+ seems dramatically to alter the activities of such polysaccharide anionic sequences (e.g.
those required for regulation of fibroblast growth factor and receptor activity); this work
strengthens
the notion that the heparanome-metallome interaction concept is a valid hypothesis for how
heparin/heparan sulphate signalling occurs in animal biochemistry].

Cf., T.R. Rudd, M.A. Skidmore, S.E. Guimond, M. Guerrini, C. Cosentino, R. Edge, A. Brown,
D.T. Clarke, G. Torri, J.E. Turnbull, R.J. Nichols, D.G. Fernig and E.A. Yates
Site-specific interactions of copper(II) ions with heparin revealed with complementary (SRCD,
NMR, FTIR and EPR) spectroscopic techniques.
Carbohydr. Res., 2008, 343, 2184-2193.

(13)
S.E. Guimond, T.R. Rudd, M.A. Skidmore, A. Ori, D. Gaudesi, C. Cosentino, M. Guerrini, R. Edge,
D. Collison, E. McInnes, G. Torri, J.E. Turnbull, D.G. Fernig and E.A. Yates.
Cations modulate polysaccharide structure to determine FGF-FGFR signaling. A comparison of
signaling and inhibitory polysaccharide interactions with FGF-1 in solution.
Biochemistry, 2009, 48, 4772-4779.

(14) D. Grant 2009

www.scribd.com/doc/26994439/Publication-2-Web
2010
www.scribd.com/doc/38671527/WRL2521 -Revised
www.scribd.com/doc/34178257/WRL1873-With-Fig

(15)
M. Purdey
Chronic barium intoxication disrupts sulphated proteoglycan synthesis:
A hypothesis for the origin of multiple sclerosis.
Med. Hypotheses., 2004, 62, 746-754.

[Cf., Anthropogenic perturbation of heparin/heparan sulphate signalling.

Some of the reported requirements for the presence of specific divalent metal ions (e.g. Ca2+ or Zn2+) for
heparin/heparan sulphate binding to proteins which is listed in Table I can also be rationally suggested to
be subject to anthropogenic perturbation (e.g. by Ba, Pb, Cd or Hg ).
The X-ray crystal structure of a heparin oligosaccharide annexin-V adduct indicates how interlinking Ca2+ ions
might influence the aggregation of annexin V at plasma membranes but this activity might be perturbed by Ba2+
which is reported to disturb Ca2+ binding to annexin-V this being of relevance for annexin ion channel building,
anticoagulant and cellular apoptosis activity.
While Ba2+ occurs in pharmaceutical porcine heparin and also in human scalp hair, there is a larger variation in the
reported values for Ba2+ in human hair consistent with an environmental intoxication source of this metal].
(16)
S. Ricard-Blum, O. Féraud, H. Lortat-Jacob, A. Rencurosi, N. Fukai, F. Dkhissi, D. Vittet,
A. Imberty, B.R. Olsen and M. van der Rest
Characterization of endostatin binding to heparin and heparan sulfate by
surface plasmon resonance and molecular modelling: role of divalent cations.
J. Biol. Chem., 2004, 279, 2927-2936.

(17)
I. Capila, M.J. Hernáiz, T.R. Mealy, B. Campos, J.R. Dedman, R.J. Linhardt and B.A. Seaton
Annexin V-heparin oligosaccharide complex suggests heparan sulfate-mediated
assembly on cell surfaces.
Structure (Camb.) 2001, 9, 57-64.

(18)
A. Lewit-Bentley, S. Morera, R. Huber and G. Bodo
The effect of metal binding on the structure of annexin V and implications for
membrane binding.
Eur. J. Biochem., 1992, 210, 73-77.
(19)
Heparanase degradation is now known to occur in a Zn-dependent metalloproteinase-dependent
manner
Cf., E. Zcharia, J. Jia, X. Zhang, L. Baraz, U. Lindahl, T. Peretz, I. Vlodavsky and J.-P. Li
Newly generated heparanase knock-out mice unravel co-regulation of heparanase and matrix
metalloproteinases.
PLoS ONE 2009, 4 (4): e5181Epub 2009 Apr.10.
[This suggests that the generation of key oligosaccharides messenger molecules is achieved by a
‘smart’ system in which Zn2+-dependent metalloproteinsases act in concert with heparanase].
Bacterial heparanase activity is also metal ion dependent
Cf., e.g.,
C.F. Moffat, W.F. Long, M.W. McLean and F.B. Williamson
Heparanase-(II)-catalysed degradation of N-propionylated heparin.
Biochem. Soc. Trans., 1997, 25, S654.
Heparanase II from Flavobacterium heparinum. Action on chemically modified heparins.
Eur. J. Biochem., 1991, 197, 449-459.
Heparanase II from Flavobacterium heparinum. HPLC analysis of the saccharides generated from
chemically modified heparin.
Ibid., 1991, 202, 531-541.

(20)
Nitrous acid degradation of heparin/heparan sulphate - metal ion effects
K. Ding, K. Mani, F. Cheng, M. Belting and L.-Å. Fransson
Copper-dependent autocleavage of glypican-1 heparan sulfate by nitric oxide
derived from intrinsic nitrosothiols.
J. Biol. Chem., 2002, 277 (36) 33353-33360;
cf., K. Mani, M. Jonsson, G. Edgren, M. Belting and L.-Å. Fransson
Glycobiology, 2000, 10, 577-586.
[Endogenous internal degradation of heparan sulphate during recycling of
glypican-1 in vascular endothelial cells].

{Cf., M. Belting, S. Persson and L.-A. Fransson

Proteoglycan involvement in polyamine uptake.
Biochem. J.,1999, 338, 317-323}.

Cf., R.E. Vilar, D. Ghael, M. Li, D.D. Bhagat, L.M. Arrigo, M.K. Cowman, H.S. Dweck
 and L. Rosenfeld
Nitric oxide degradation of heparin and heparan sulphate.
Biochem. J., 1997, 324, 473-497.
Cf., D. Ghael, M. Mileva, H.S. Dweck and L. Rosenfeld
Biochem. Mol. Biol. Int., 1997, 43, 183-188.
[The reason why different buffers support different degrees of reactivity of nitrite
for deaminative cleavage of heparin/heparan sulphate can be deduced to arise from
the presence of trace amounts of copper and iron in phosphate buffers; n.b. this
possibility was not discussed by these authors but arises from unpublished work
carried out in the field by the Author at Aberdeen University].
{Rapid movement of bound ions along anionic polysaccharide chains apparently
promotes formation of nanoparticles of Fe3+ oxides and thereby protects against
oxidative damage (a possible normal function of hyaluronan in vivo, cf. ref. (28-4) ) .
It is possible that the formation of such Fe(III) nanoparticles is reversed by the action of nitric oxide.
Toxic metal ions may therefore play an potentially important role in the
perturbation of such antioxidative activities (suggesting a further hypothesies for how excessive
nitric oxide production may promote of degenerative diseases including cancer and multiple sclerosis
(the subject of a communication in preparation)}.

(21) L. Jakobsson
Press release from Uppsala University.
“….an entirely new mechanism for how communication can be regulated between cells”.
[This was apparently enabled by a glue-like heparan sulphate mechnism
which can support angiogenesis via vascular endothelial growth factor [VEGF] growth factor
activation using heparan sulphate provided by smooth muscle cells, a trans activation mechanism].
Cf., e.g., Medical News Today, 11 May 2006,
http://www.medicalnewstoday.com/articles/43115.php.
Cf., L. Jakobsson, J. Kreuger, K. Holmborn, L. Lunden, I. Eriksson, L. Kjellén and
L. Claessen-Welsh
Heparan sulfate in trans potentiates VEGFR-mediated angiogenesis.
Dev. Cell, 2006, 10, 625-634.

(22)
M. Herbert and J.P. Maffrand
J. Cell Physiol., 1989, 138, 424-432.
[Oligosaccharides following internalization show antiproliferative effects on
vascular endothelial and smooth muscle cells].

(23)
R. Hahnenberger, A.M. Jakobson, A. Ansari, T. Wehler, C.M. Svahn and U. Lindahl
Low-sulphated oligosaccharides derived from heparan sulphate inhibit normal angiogenesis.
Glycobiology, 1993, 3 (6), 567-573.
[Inhibition of angiogenesis by low sulphated oligosaccharides from heparan sulphate
for which endothelial cell surface-bound heparan sulphate proteoglycans may
constitute a pool of precursors for anti-angiogenic polysaccharides].

(24)
S. Ihrcho, L.E. Wrenshall, B.J. Lindman and J.L. Platt
Immunol. Today, 1993, 14, 500-501; Chem. Abs., 120, 51877v.
[Review on the release of heparan sulphate from endothelial cells during
inflammation and possible regulation by soluble heparan sulphate fragments
of the functioning of lymphocytes at sites of inflammation].

(25)
Boyd J., F.B. Williamson and J. Gettins
Physico-chemical study of heparin, Evidence for a calcium-induced
co-operative conformational transition.
 J. Mol. Biol., 1980, 137, 175-190.
Cf., W.F. Long and F.B. Williamson
Glycosaminolycans, calcium ions and the control of cell proliferation.
IRCS J. Med. Sci., 1979, 7, 429-434;
[Cf., related studies, of the inorganic biochemistry of heparin/heparan sulphate by the Aberdeen
polysaccharide group listed in the Supplementary References].

(26) Cf., Grant D., W.F. Long and F.B. Williamson:

Infrared spectroscopy of heparin-cation complexes
Biochem. J., 1987, 244, 143-149

Zn2+-heparin interaction studied by potentiometric titration

Ibid., 1992, 287, 849-853;

A potentiometric titration study of the interaction of heparin with metal cations

Ibid., 1992, 285, 477-480;
Similarity and dissimilarity in aspects of the binding to heparin of Ca2+ and Zn2+
as revealed by potentiometric titration
Biochem. Soc. Trans., 1996, 24, 203S;

N.m.r spectroscopy of Ca2+-heparin suggests delocalized binding of the cation

Abstracts of the 641st Meeting of the Biochemical Society, Issued with The Biochemist,
Royal Holloway and Bedford New College 17-20 December 1991,
p.56 Abstract No 157.

(27)
D. Grant (2000) Seminar presentation and discussion University of Glasgow .
(Discussions Relating to Ascorbate/Heparan Sulphate /Upper Stomach Cancer etc.)
http://web.ukonline.co.uk/dgrant/dg2/ also http://web.ukonline.co.uk/dgrant/dg1/
also http://web.ukonline.co.uk/dgrant/dg4/ and http://web/ukonline.co.uk/dgrant/dg5/
cf. http://web.ukonline.co.uk/dgrant/dg8
These notes suggested that the anti-cancer activity of ascorbate is more likely arise from the roles of redox plus non
redox metals in ascorbate/nitric oxide determined heparan sulphate fuzzy logic control systems than via collagen or
DNA-damage dependent mechanisms (cf., Linus Pauling had suggested that the anti-cancer activity of ascorbate was
due to a general promotion of collagen crosslinking and Albert Szent Gyorgi had suggested that anti-cancer actions
of ascorbate were due to a DNA protection antioxidant/ DNA (‘quantum dot’ physics mechanism [cf. also the DNA
semiconductor hypothesis of B. Marczynski, Med. Hypotheses, 1988, 26 (4) 239-249 (Carcinogensis as the result of
the deficiency of some trace elements)].

(28)
D. Grant, W.F. Long, C.F. Moffat and F.B. Williamson
Cu2+-heparin interaction studied by polarimetry.
Biochem. J., 1992, 283, 243-246.
Cf., There is a lack of paramagnetic broadening of NMR
absorbances in heparin containing 1100ppm Fe and 730ppm Cu [these values were obtained from
spark source mass spectroscopic analysis (Moffat, Colin F, Ph.D. Thesis
“Synthesis Characterisation and Applications of Chemically Modified Heparin”
University of Aberdeen 1987].
Cf. also
D. Grant., W.F. Long and F.B. Williamson
Complexation of Fe2+ ions by heparin.
Biochem. Soc. Trans., 1992, 20, 361s.
[This is not a simple thermodynamicically driven process].
(28-1)
M.A. Ross, W.F. Long and F.B. Williamson
Inhibition by heparin of Fe(II)-catalysed free-radical peroxidation of linolenic acid.
Biochem. J., 1992, 286, 717-720.
Cf. M.A. Ross et al.,
Heparin inhibits potentiation of thiobarbituric acid reactive substances in
the presence of linoleic acid and Fe2+ ions.
Biochem. Soc. Trans., 1992, 20(4) 364s.
Cf. D. Grant et al,. ibid., 1996, 24 194S and ref. 28-2
(28-2)
D. Grant, W.F. Long and F.B. Williamson
Pericellular heparans may contribute to the protection of cells from free radicals.
Med. Hypotheses, 1987, 23, 67-71.
Cf. also Ross, Marion A “Heparin as an Antioxidant” M.Sc. Thesis University of Aberdeen,1992.
D. Grant, W.F. Long, G. Mackintosh and F.B. Williamson
The antioxidant activity of heparin.
Biochem. Soc. Trans., 1996, 24, 194S.
[Cf. R. Albertini, A. Passi , P.M. Abjuja and G. De Luca
The effect of glycosaminoglycans on lipid peroxidation.
Int. J. Mol. Med. 2000, 6,129-136.
R. Albertini, S. Rindi, A. Passi, G. Palladini, G., Pallavicini and G. De Luca
The effect of heparin on Cu2+-mediated oxidation of human low-density lipoproteins.
FEBS Lett., 1995, 377, 240-242], [cf. also http://electra.chemistry.upatras.gr/fects/final/w-shops.htm].
And refs 28-3 and 28-4.]
(28-3)
M.F. Williamson, W.F. Long and F.B. Williamson
The effect of heparin on the u.v. absorption properties of Fe(II) and Fe(III).
Biochem. Soc. Trans., 1992, 20, 360S.

(28-4)
A.L. Merce, L.C. Marques Carrera, L.K. Santos Romanholi and M.A. Lobo Recio
Aqueous and solid complexes of iron (III) with hyaluronic acid.
Potentiometric titrations and infrared spectroscopy studies.
J. Inorg. Biochem., 2002, 89, 212-218.
[Hyaluronan promoted Fe3+ nanoparticle formation was described in this paper].
Cf., P. Sipos, O. Berkesi, H, Tombacz, T.G. St Pierre and J. Webb
Formation of spheroidal iron(III) nanoparticles sterically stabilized by chitosan in aqueous solutions.
J. Inorg. Biochem., 2003, 95, 55-63.
[Iron-containing nanoparticles form at chitosan surfaces; a similar process may occur at heparin
surfaces; heparin has also been observed to induce the formation of akagenite (βFe(O)(OH))
fibrils{which were identified by the X-ray powder diffraction pattern of this crystalline form}
starting with aqueous solutions of Fe(II), apparently formed via by a process which implicated the
existence of an intrinsic ferroxidase activity of heparin, a property which is perhaps most especially
demonstrated by the natural multi-inorganic elemental forms of heparin rather than by the artifactal
‘more highly purified’ forms of heparin {F.B. Williamson formerly of Aberdeen U., personal
communication}].

Tables I & II (Specific References)

(29-1)
E. Kecskes, K.G. Bucki, P.I. Bauer., R. Machovich., and I. Horvath
Thromb. Haemost., 1983, 49, 138-141; Chem. Abs., 99, 3504x.
[Heparin forms a complex with LDL in the presence of Ca2+; this complex retains the anticoagulant
activity of the heparin].

(29-2)
F.A. Ofosu, G. Modi, A.L. Cerskus, J. Hirsh and M.A. Blajchman
Ca binding to heparin inhibits phospholipid dependent assembly of Factor X and
prothrombin activated complex.
Thromb Res., 1982, 28 (4) 487-497; Chem. Abs., 98, 69550.

(29-3)
M.O. Speight and M.J. Griffith
Calcium inhibits the heparin-catalysed antithrombin III/thrombin reaction by
decreasing the apparent binding of heparin for thrombin.
Arch. Biochem. Biophys., 1983, 225 (2) 958-963.

(29-4)
M. Hayashi, and K.M. Yamada
Divalent cation modulation of fibronectin binding to heparin and to DNA.
J. Biol. Chem., 1982, 257 (9) 5263-5267.

(29-5)
A. Koenig, K. Norgard-Sumnicht, R. Linhardt and R. Varki
Differential interaction of heparin and heparan sulfate glycosaminoglycans
with the selectins. Implications for the use of unfractionated and low molecular
weight heparin as therapeutic agents.
J. Clin. Invest., 1998, 101 (4) 877-889.

(29-6)
K.E. Norgard-Sumnicht, N.M. Varki, and A.Varki
Calcium-dependent heparin-like ligands for L-selectin in nonlymphoid
endothelial cells.
Science, 1993, 261, 480-483.

(29-7)
E.H. Nielson, I.J. Sørensen, K. Vilsgaard, O. Andersen and S.E. Svehag
Calcium enhanced aggregation of serum amyloid P and its inhibition by the ligands
heparin and heparan sulphate. An electron microscope and immunoelectrophoretic study.
APMIS, 1994, 102, (6) 420-426; Chem. Abs., 122, 181976a.

(29-8)
S.K. Shinjo, I.L. Tersariol, V. Oliveira, C.R. Nakaie, M.E. Oshiro, A.T. Ferreira,
I.A. Santos, C.P. Dietrich and H.B. Nader
Heparin and heparan sulfate disaccharides bind to the exchanger inhibitor
peptide region of the Na+/Ca2+ exchanger and reduce the cytosolic calcium
of smooth muscle cell lines. Requirement of C4-C5 unsatruation at 1->4 glycosidic linkage
for activity
J. Biol. Chem., 2004, 277 (50) 48227-48233.

(29-9)
Y. Zhao and X. Zhang
Heparin inhibits the reconstituted plasma membrane Ca-ATPase from
porcine brain synaptosome.
Glycoconjugate J., 2002, 19, 373-378.

(29-10)
Y. Takeuchi, K. Sakaguchi, M. Yanagishita, G.D. Aurbach and V.C. Hascall
Extracellular calcium regulates distribution and transport of heparan sulfate
proteoglycans in a rat parathyroid cell line.
J. Biol. Chem., 1990, 265 (23) 13661-13668.
Cf. Y. Takeuchi, M. Yanagashita and V.C. Hascall
Recycling of transferrin receptors and heparan sulfate proteoglycans in a
rat parathyroid cell line.
J. Biol. Chem., 1992, 267 (21) 14685-14690.
(29-11)
D. Kiryushko, V. Novitskaya, V. Soroka, J. Klingelhofer, E. Lukanidin, V. Berezin, and E. Bock
Molecular mechanism of Ca2+ signaling in neurons induced by the S100A4 protein.
Mol. Cell. Biol., 2006, 26 (9) 3625-3638.

(29-12)
H.-G. Knaus, F. Scheffauer, C. Romanin, H.-G. Schindler and H. Glossmann
Heparin binds with high affinity to voltage-dependent L-type Ca2+ channels.
Evidence for an agonistic action.
J. Biol. Chem., 1990, 265, 11156-11166.

(29-13)
G. Siegel, M. Malmsten and B. Lindman
Flow sensing at the endothelium-blood interface.
Colloids and Surfaces A: Physiochemical and Engineering Aspects, 1998, 138, 384-351.
[Heparan sulphate may serve as a vascular flow sensor via conformation changes elicited mechanically
and electrostatically by Na + and Ca2+ cation binding].

(29-14)
M.J. Robinson, P. Tessier, R. Poulsom and N. Hogg
The S100 family heterodimer, MRP-8/14, binds with high affinity to heparin and heparan sulfate
glycosaminoglycans on endothelial cells.
J. Biol. Chem., 2002, 277 (5) 3658-3665.

(29-15)
Tiedemann K., B. Batge, P.K. Muller and D.P. Reinhardt
Interactions of fibrillin-1 with heparin/heparan sulfate, implications for microfibrillar assembly.
J. Biol. Chem., 2001, 276 (38) 36035-36042.
[Ca2+ dependence of extracellular microfibrils fibrillin-1 binding to
heparan sulphate proteoglycan].

(29-16)
M.J. Stanley, B.F. Liebersbach, W. Liu, D.J. Anhalt and R.D. Sanderson
Heparan sulfate-mediated cell aggregation.
J. Biol. Chem., 1995, 270 (10) 5077-5083
[Aggregation of syndecan-1-transfected cells mediation by divalent cations].

(29-17)
A.L. Jones, M.D. Hulett and C.R. Parish
Histidine rich glycoprotein HRG- a novel adapter protein in plasma that
modulates the immune vascular and coagulation systems.
Immunol. Cell Biol., 2005, 83 (2) 106-118.

(29-18)
L. Kerp
Importance of zinc for histamine storage in mast cells.
Intern. Arch. Allergy Apppl. Immunol., 1963, 22, 112-123.
Cf. L. Kerp and G. Steinhauser
On a ternary heparin-metal histamine-complex.
Klin. Wochschr., 1961, 39, 762-764.

(29-19)
B. Lages and S.S. Stivala
Copper ion binding and heparin interactions of human fibrinogen..
Biopolymers, 1973, 12, 961-974.

(29-20)
R. Gonzáles-Iglesias, M.A. Pajares, C. Ocal, J.C. Espoinosa, B. Oesch and M. Gasset
Prion protein interaction with glycosaminoglycan occurs with the formation of
oligomeric complexes stabilized by Cu(II) bridges.
J. Mol. Biol., 2002, 319, 527-540.

(29-21)
Y. Yamane, S. Saito and T. Koizumi
Effects of calcium and magnesium on the anticoagulant action of heparin
Chem. Pharm. Bull (Tokyo) 1983, 31, (9) 3214-3221; Chem. Abs., 99, 205875e

(29-22)
M.A. Liebel and A.A. White
Inhibition of the soluble guanylate cyclase from rat lung by sulphated polyanions.
Biochem. Biophys. Res. Commun., 1982, 104 (3) 957-964; Chem. Abs., 96,138749q.

(29-23)
C.L. Masters et al.
PCT Int. Appl., WO 9310459 (1993); Chem. Abs., 119, 136893b.
[Alzheimer’s disease is treated by modulation of metal ion/ heparin/amyloid precursor protein (APP);
Zn2+ at 50nM promoted heparin binding to APP;
Zn2+ abolished a protective effect afforded by heparin of proteolysis of APP].

(29-24)
F.T. Borges, Y.M. Michelacci, J.A. Aguiar, M.A. Dalboni, A.S. Garófalo and N. Schor
Characterization of glycosaminoglycans in tubular epithelial cells: Calcium oxalate and oxalate ions
effects.
Kidney Int., 2005, 68, 1630-1642.
[Kidney tubular cells apparently can upregulate the synthesis of {specifically microstructured?}
glycosaminoglycans when cultured in the presence of calcium oxalate crystals or high concentration of
oxalate which can induce the formation of (harmful) calcium oxalate crystals. A servo feedback system
could be suggested to exist here; specifically microstructured heparan sulphate molecules ‘designed’ to
protect kidney cells from such calcium oxalate crystals or the precursors of such crystals. N.b.,
heparin/heparan sulphate is well known to be an effective inhibitor of calcium oxalate crystallization].

(29-25)
A.Z. Kalea, F.N. Lamari, A.D. Theocharis, D.A. Schuschke, N.K. Karamanos and D.J. Klimis-Zacas
Dietary manganese affects the concentration, composition and sulfation pattern of heparan sulfate
glycosaminoglycans in Sprague-Dawley rat aorta.
Biometals, 2006, 19 (5) 535-546.

(29-26)
T.A. Fritz, M.M. Gabb, G. Wei and J.D. Esko
Two N-acetylgluocosaminyltransferases catalyse the biosynthesis of heparan sulfate.
J. Biol. Chem., 1994, 269 (46) 28809-28814.
[α-GlcNAc transferase I can use both Mn2+ and Ca2+ while
α-GlcNAc transferase II can only use Mn2+. Mn2+ status may therefore
affect heparan sulphate proteoglycan biosynthesis by this mechanism].

(29-27)
P. Jaya and P.A. Kurup
Effect of magnesium deficiency on the metabolism of glycosaminoglycans in rats.
J. Biosci., 1986, 10, 487-493.

(29-28)
Y. Fujiwara and T. Kaji
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., 2002, 48, 460-466.
Cf., Y. Fujiwara, and J. Kaji
Lead inhibits the core protein synthesis of a large heparan sulfate proteoglycan perlecan
by proliferating vascular endothelial cells in culture.
Toxicology, 1999, 133 (2,3) 159-169; Chem. Abs., 131, 154569.
Cf. T. Kaji, C. Yamamoto and M. Sakamoto
Effect of lead on the glycosaminoglycan metabolism of bovine aortic endothelial cells
in culture.
Ibid., 1991, 68, 249-257.

(29-29)
A. Cárdenas, A. Bernard and R. Lauwerys
Incorporation of [35-S] sulfate into glomerular membranes of rats chronically
exposed to cadmium and its relation with urinary glycosaminoglycans and proteinuria.
Toxicology, 1992, 76, 219-231.

(29-30)
D.M. Templeton
Metal-proteoglycan interactions in the regulation of renal mesangial cells:
Implications for metal induced nephropathy.
Proc. Trace Element Health Disease IUPAC Int. Symp.,1990, p. 209-219; Ed., Aito A.

(29-31)
M.F. McCarty
Reported anti atherosclerotic activity of silicon may reflect increased
endothelial synthesis of heparan sulfate proteoglycans.
Med. Hypotheses, 1997, 49, 175-176.
Cf., R.M. Iler, The Chemistry of Silica , Wiley, 1979, cf. p. 762.

(29-32)
K. Pawlowska-Góral, M. Wardas, W. Wardas and U. Majnusz
The role of fluoride ions in glycosaminoglycan sulphation in cultured fibroblasts.
Fluoride, 1998, 31, 193-202.

(29-33)
C.P. Dietrich, H.B. Nader, V. Buonassisi and P. Colburn
Inhibition of synthesis of heparan sulfate by selenate: possible dependence on sulfation for chain
polymerization.
FASEB J., 1988, 2, 56-59.

(30)
R.D. Campo
Effects of cations on cartilage structure: swelling of growth plate and degradation of proteoglycans
induced by chelators of divalent cations.
Calcif. Tissue Int., 1988, 43 (2) 108-121.

(31)
L. Lerner and D.A. Torchia
A multinuclear NMR study of the interactions of cations with proteoglycans
heparin and Ficoll.
J. Biol. Chem., 1986, 261, 12706-12714.
[23-Na, 39-K, 25-Mg and 43-Ca NMR relaxation assessment of cation binding to
heparin].
(32)
D. Grant, W.F. Long and F.B. Williamson
Inhibition by glycosaminoglycans of CaCO3 (calcite) crystallization
Biochem. J., 1989, 259, 41-45.
 Cf.,
Degenerative and inflammatory diseases may result from defects in
antimineralization mechanism afforded by glycosaminglycans (ref. 33).

(33)
D. Grant, W.F.Long and F.B. Williamson
Med. Hypotheses, 1992, 38, 49-55.
[Multi-element containing anionic polysaccharides may be present as inorganic-crystal-like
anionic
structures at solution interfaces.
In a range of related studies (reported as yet only as conference posters and in M.Sc. theses from
Aberdeen University) it was reported that inorganic crystal surfaces can discriminate (or “read
off”)
polysaccharide microstructures.
It is of interest in this context that unliganded free iron ions can catalyse the de-N-sulphonation of
heparin suggesting that iron overload could diminishes heparan sulphate tissue protection].

(34)
H.B. Nader, M.G.L. Medeiros. J.F. Paiva, V.M.P. Paiva, S.M.B. Jerônimo, T.M.P.C. Ferreira
and C.P. Dietrich
A correlation between the sulphated glycosaminoglycan concentration and degree of salinity of the
habitiat in fifteen species of the classes Cructacea, Pelecypoda and Gastropoda.
Comp. Biochem. Physiol., 1983, 76, 433-436.
[The mathematically exact nature of the increasing amounts of sulphated polysaccharides
especially
heparan sulphates present in tissues exposed to habitats with increasing salinity confirms the
existence of osmoeregulatory and/or inorganic ion nutrient element sequestration role for heparan
sulphate in animal biochemistry].

(34-1)
H. Morita, A. Yoshimura, K. Inui, T. Ideura, H. Watanabe, L. Wang, R. Soininen and K.
Tryggvason
Heparan sulfate of perlecan is involved in glomerular filtration.
J. Am.Soc. Nephrol., 2005, 16, 1703-1710.

(35)
J. Kreuger, P. Jemth, E. Sanders-Lindberg, L. Eliahu, D. Ron, C. Basilico, M. Salmivirta and
U. Lindahl
Fibroblast growth factors share binding sites in heparan sulphate.
Biochem. J., 2005, 389, 145-150.

(36)
D.M. Whitfield., J. Choay and B. Sarkar
Heavy metal binding to heparin disaccharides. I.
Iduronic acid is the main binding site.
Biopolymers, 1992, 32, 585-596;
Heavy metal binding to heparin disaccharides. II.
First evidence for zinc chelation.
Ibid., 1992, 32, 597-619.

(37)
E.g. N.E. Woodhead, W.F. Long and F.B. Williamson
The heparan sulphates of normal and virus-transformed hamster fibroblasts
Biochem. Soc. Trans., 1981, 9, 555-556
N.E. Woodhead, W.F. Long WF, F.B. Williamson and W.J. Harris
ibid., 1984, 12, 300-301; and these authors, (1986)
Heparan sulphates from fibroblasts exhibiting a temperature-dependent transformed growth trait.
IRCS J. Med. Sci. (Lib. Comp.) 14, 427-428.
 W.F. Long and F.B. Williamson
Heparan structure and the modulation of angiogenesis..
Med. Hypotheses, 1984, 13, 385-394.
D. Grant, W.F. Long and F.B. Williamson
Differences in the properties of heparans from BHK and PyY cells.
Eur. J. Cell Biol., 1985, 36, 14.
Infrared and proton nuclear magnetic resonance spectroscopy of carbohydrates from BHK and
PyY cells.
ibid., 36,14.
A role for glycosaminoglycans in cellular adhesion of relevance to the cancer state.
Biochem. Soc.Trans., 1985, 13, 388-389.

Numerous other laboratories have studied cancer-related pathological alterations of heparan

sulphate e.g., P. Tapanadechopone et al. (Supplmentary References) but
a full review is outwith the scope of this article.

(38)
B. Lahiri, P.S. Lai, M. Pousada, D. Stanton, and I. Danishefsky
Depolymerization of heparin by complexed ferrous ions.
Arch. Biochem. Biophys., 1992, 293, 54-60.
Cf.,
J.P. Lahiev
Bio Metals, 1996, 9 (1) 10; Chem. Abs., 124, 109969t
[Fe(II) selectively degrades heparin].
Cf.,
Z. Liu and A.S. Perlin
Evidence of a selective free radical degradation of heparin, mediated by cupric ion.
Carbohydr. Res., 1994, 255, 183-191

(39)
D. Grant, W.F. Long, G. Mackintosh and F.B. Williamson
Roles of heparins and heparans in inflammatory aspects of cancer and the potential of
heparinoids as anti-cancer drugs.
Proc. Int. Congr. Inflamm., Vienna, 10-15 Oct. 1993.

APPENDIX

Supplementary References
Listed Alphabetically According to First Named Author

Ayotte L., and A.S. Perlin

Nmr spectrosocpy observations related to the function of sulfate
groups in heparin. Calcium binding vs. biological activity.
Carbohydr. Res., 1986, 145 (2) 267-277.

Bocca B
Cf. Visconti A. et al., Ann 1st Super Sanita 2005, 41 (2) 217-222

Bodini M.E., and D.T. Sawyer

Electrochemical and spectroscopic studies of manganese(II), (III) and (IV) gluconate
complexes 2. Reactivity and equilbriums with molecular oxygen and hydrogen peroxide.
J. Amer. Chem. Soc., 1976, 98 (26) 8366-8371.
[Formation of hydroxyl radicals by Fenton type reactions from Mn(II) also
similar reactions from V(IV), Cr(II), Ti(III) and Fe(II) discussed].

Burger, K., F. Gaizer, M. Pékli, G.Takácsi Nagy and J. Siemroth

The effect of cations on the calcium ion coordination of heparin.
Inorganica Chimica Acta, 1984 92, 173-176.
[Ca and Zn selective electrodes show complex effects of Li, Na, K and Mg on
Ca binding; Zn more strongly bound than Ca and Zn binding strongly
reduces K binding].

Casu B., P. Oreste, A. Naggi, G. Torri, G. Zoppetti, G. Sporteoletti and F. de Santis

EDTA-free heparins…
European Patent Application No. 0245813 (1987).

Clamp M., B. Fry, M. Kamal, X. Xie, J. Cuff, M.F. Lin, M. Kellis, K. Lindblad-Toh and E.S. Lander
Distinguishing protein-coding and noncoding genes in the human genome.
PNAS, USA, 2007, 104, 19428-19433
doi:10.1073/pnas.0709013104.

Cochran D.L.
Glycosaminoglycan stimulation of calcium release from mouse calvariae.
Specificity for hyaluronic acid and dermatan sulfate.
Calcif. Tissue Int., 1994, 41, 79-85.
[Hyaluronic acid was more stimulatory of Ca2+ release from bone cultures than
heparin which although inactive alone caused the release of Ca2+ in the presence
of parathyroid hormone; diminished heparin/heparan sulphate affected by ageing,
is associated with increased proportion of hyaluronate and dermatan sulphate
which would tend to augment Ca2+ mobilisation].

Dais P., Q.J. Peng and A.S. Perlin

A relationship between 13-C-chemical-shift displacements.
and counterion-condensation theory, in the binding of calcium ion by heparin.
Carbohydr. Res., 1987, 168, 163-179.

Davis T.A., B. Volesky and A. Mucci

A review of the biochemistry of heavy metal biosorption by brown algae.
Water Res., 2003, 37 (18) 4311-4330.
T.A. Davis, F. Llanes, G. Volesky, G. Diaz-Pulido, L. McCook and A. Mucci
1H-NMR study of Na alginates extracted from Sargassum spp. in relation
to heavy metal biosorption.
Appl. Biochem. Biotechnol., 2003, 110, 75-90.
[Cell wall constituents such as alginate and fucoidan are chiefly responsible for
the passive removal of toxic heavy metals such as Cd, Cu, Zn, Pb, Cr and Hg].
T.A. Davis, F. Llanes, B.Volesky and A. Mucci
Metal selectivity of Sargassum spp. and their alginates in relation
to their alpha-1 guluronic acid content and conformation.
Environ. Sci. Technol., 2003, 37, 261-267.
Cf. O. Raize, Y. Argaman and S. Yannai
Mechanism of biosorption of different heavy metals by brown marine macroalgae.
Biotechnology and Bioengineering, 2004, 87, 451-458;
B. Larsen, Proc. Int. Seaweed Symp Xth, Gothenburg (1980)
de Gruyter, Berlin, 1981, pp 7-34;
Cf., F. Mo, T.J. Broback and I.R. Siddiqui
Carbohydr. Res., 1985, 145, 13.
[Alginate from brown seaweed occurred as a mixed Na-Mg-Ca-Sr salt]
In agarophytes, the polysaccharide agarose present in the extracellular mucilage is
believed to provide such a function as well as to create a firm hydrated gel].

Dunstone J.R.
Ion-exchange reactions between acid mucopolysaccharides and various cations.
Biochem. J., 1962, 85, 336-351.
Cf.,
J.E. Scott
In The Chemical Physiology of Mucopolysacharides. Binding in solutions containing acid
mucopolysaccharides; Ch.12, p. 171-187;
Ed., Guiliano Quintarelli; Churchill, London (1968).

Fransson L.-Å., I. Carlstedt, L. Coster and A. Malmstrom

Binding of transferrin to the core protein of fibroblast protoheparan sulfate.
Proc. Natl. Acad. Sci., USA, 1984, 81 (18) 5657-5661.
(However, cf., A. Schmidtchen et al.
Biochem. J., 1990, 265 (1) 289-300, did not confirm the original findings).

Grant D. et al., Additional References

Heparin was subjected to numerous in vitro metal ions binding studies; these confirm the existence of
complex polysaccharide binding mechanisms for metal ions.
It was found that while the Manning electrostatic polyelectrolyte counterion binding theory correctly
predicts the tendency for heparin to attract counterions more strongly accordingly to the value of the
counterion charge, to show a characteristic discontinuity in the binding isotherms for individual
counterions and to predict that a wide range of counterions can simutaneously bind to this polyanion, it
was found that not all equally charged counterions bound at similar strength, that the discontinuity for
individual counterion binding occurred at ratios of cation/anion which disagreed (cf. ref. 26) with
Manning theory predictions. Electrostatic binding theories also fail to account for a well-established
paradox that ultra-anionic heparin to binds strongly to SO42- (ref. 5-4-1) and other anions. In depth
studies of the mechanism of binding of counterions to heparin (cf. refs. 26 and 5-3-1) however suggested
an alternative model of counterion binding to heparin invovling a key role for a phase change engulfment
processes triggered by an altered hydration and hydrogen bonding associated with the non-electrostatic
association of inorganic ions with the polyol group of polysaccharides; this process may be the origin of
the ability of polysaccharides to bind a wide range of inorganic ions. This binding process is however
subject to additional modulation of binding by the presence of the additional electrostatic fields present in
heparin (and heparin-like segments of heparan sulphate).
It should be noted that heparin and heparan sulphate are uniquely complex and, amongst the anionic
polysaccharides, show greatest structural diversity (to which the bound inorganic ions, it is now
suggested, must also contribute). This flexible diversity apparently facilitates the performance of
complex signalling and control functions throughout a wide range of activities in animal biochemistry. It
is therefore predicted that the precise physical chemical and ultrastructural details of the counterion
profile (including an ability to engage in variable rapid site exchange (cf. ref. 28)) and the possible
perturbation of this by anthropogenic input) could be highly relevant to the in vivo molecular
(/supramolecular) mechanisms which control the heparanome.

Grant D., W.F. Long and F.B. Williamson

Analysis by infrared spectroscopy of the association of water and metal ions with heparin.
Biochem. Soc. Trans., 1983, 11, 96.

Grant D., C.F. Moffat, W.F. Long and F.B. Williamson

Altered water structure in mixtures of heparin and metal ions.
Biochem. Soc. Trans., 1984, 12, 302.

Grant D., W.F., Long and F.B. Williamson

A role for glycosaminoglycans in cellular adhesion of relevance to the cancer state.
Biochem. Soc. Trans., 1985, 13, 389.

Grant D., W.F., Long and F.B. Williamson

Pericellular heparans may contribute to the protection of cells from free radicals.
Med. Hypotheses, 1987, 23 (1) 67-71.

Grant D., W.F. Long and F.B. Williamson

A model of two conformational forms of heparins/heparans suggested by infrared
spectroscopy.
Med. Hypotheses, 1987, 24 (2) 131-136.
Grant D., W.F. Long and F.B. Williamson
Effect of heparin on dismutation of superoxide anion.
Biochem. Soc. Trans., 1988, 16, 1030-1031.

Grant D., W.F. Long and F.B. Williamson

A model of Ca2+ -heparin interaction.
Biochem Soc. Trans., 1988, 16, 1028-1029.
The presence of more than one counterion (studied with Na+ + Ca2+) caused a blurring of
the chemical distinctions between the counterions which tended to undergo random fast
exchange distribution with a similar binding strength.

This and other NMR studies by D. Grant et al., suggested co-occurrence with heparin and related
polysaccharides of a complex system wherby inorganic ions are held both in rapid site change locations,
at more secure locations and at phase-separated locations. The relative amounts of different types of
inorganic elements which are present, putatively under equilibrium conditions, seems to indicate the
existence of a mechanism which favours the preferential sequestration of the least abundant elements
present in natural multi-element bathing solutions, cf. Fig1a).

Grant D., W.F. Long, C.F. Moffat and F.B. Williamson

Infrared spectroscopy of chemically modified heparins.
Biochem. J., 1989, 261, 1035-1038.

The presence of more than one counterion (studied with Na+ + Ca2+) caused a blurring of the chemical
distinctions between the counterions which tended to undergo random fast exchange distribution with a
similar binding strength.

Grant, D., W.F. Long., C.F. Moffat and F.B. Williamson

Infrared spectroscopy as a method of investigating the conformation
of iduronate saccharide residues in glycosaminoglycans
Biochem. Soc. Trans., 1990, 18, 1277-1279

Grant D., W.F. Long and F.B. Williamson

A possible ring conformational change of iduronate residues detected by ir
spectroscopy of aqueous solutions of lithium heparin
Biochem. Soc. Trans., 1990, 18 (6) 1281-1282;

Grant D., W.F. Long and F.B. Williamson

The dependence on counter-cation of the degree of hydration of heparin
Biochem. Soc. Trans., 1990, 18 (6) 1283-1284

Grant D., W.F. Long and F. B. Williamson

Infrared spectroscopy of heparin suggests that the region 750-950cm -1
is sensitive to changes in iduronate ring conformation;
Biochem. J., 1991, 275 (1) 193-197

Grant D., W.F. Long and F.B. Williamson

Heparin-polypeptide interaction. Near-i.r spectroscopy in an anhydrous
dispersant allows the involvement of polymer-associated water to be assessed.
Biochem. J. 1991, 277 (2) 569-571

Grant D., W. F. Long and F.B. Williamson

Examination of cation-heparin interaction by potentiometric titration
Abstracts of the 641st Meeting of the Biochemical Society Issued with The Biochemist,
Royal Holloway and Bedford New College 17-20 December 1991, p. 56 Abstract No 158;

Grant D., C.F. Moffat., W.F. Long W.F. and F.B. Williamson
Ca2+ -heparin interaction investigated polarimetrically
Biochem. Soc. Trans., 1991, 19 (4) 391S

Grant D., C.F. Moffat., W.F. Long and F.B. Willliamson

A relationship between cation-induced changes in heparin optical rotation and
heparin-cation association constants
Biochem. Soc. Trans., 1991, 19 (4) 392S

Grant D., C.F. Moffat, W.F. Long and F.B. Williamson

Optical rotation changes in chemically modified heparins as a guide to
anionic groups involved in Ca2+ binding
Biochem. Soc. Trans., 1991, 19 (4) 393S

Grant D., C.F. Moffat, W.F. Long and F.B. Williamson

Carboxylate symmetric stretching frequencies and optical rotation shifts of
heparin cation complexes
Biochem. Soc. Trans., 1991, 19 (4) 394S

Grant D., W.F. Long and F.B. Williamson

I.r. spectrosocpic analysis of heparin-polypeptide interaction
Biochem. Soc. Trans., 1991, 19 (4) 395S

Grant D., W.F. Long, C.F. Moffat and F.B. Williamson

Polarimetry of mixtures of Cu(II) ions and chemically modified heparins
Biochem. Soc. Trans., 1991, 20 (1) 2S

Grant D., W.F. Long and F.B. Williamson

IR spectroscopy of heparan sulphates isolated from the surfaces of normal and
virally transformed fibroblasts
NIR News, 1992, 19-21
Cf., Grant D., W.F. Long and F.B. Williamson
NIR spectroscopy shows that animal cell adhesion to non-biological solid surfaces
may require surface water structuring
NIR News, 1992, 22-24
(Proc. Int. Conf., Aberdeen, Near Infrared Spectroscopy, 1991 pub. 1992
Ed. Murray I,, and Cowe I.A.
VCA Weinheim Germany; Chem. Abs., 118 164498z)

Grant D., W.F. Long and F.B. Williamson

Multiple-specular-reflectance i.r. spectrocospy of
glycosaminoglycan-cetylpyridinium complexes
Biochem. Soc. Trans., 1992, 20(1) 4S

Grant D., W.F. Long and F.B. Williamson

A putative role for colloidal silicates in primitive evolution deduced in part form their
relevance to modern pathological afflictions
Med. Hypotheses., 1992, 38, 46-48
(Later discussions took this hypothesis further. The original background to this hypothesis was the
observations partly reported in Brit Pats 1143014 and 1136016 of biological-like self assembly of silica
sols. Different types of silica sols underwent self seeding of new particle growth and therefore a system
of different silica sols (generated by slight difference in initial conditions) seemed to be potentially
capable of competing with each other for the acquisition of low molecular weight (water soluble) silicic
acid and thereby suggested a hypothesis for the generation of high levels of molecular complexity and
cellular structures such as the precursors of modern organisms. The natural ability form structures of
increasing complex structures over geological timescales (akin to a process of Darwinian evolution) can
be suggested to have been driven by the unique abilities of silica sols in this regard; such advantages for
obtaining nutrient for growth (initially silicic acid) might have been achieved by the evolution of motility
this needing energy generation e.g. via mechanisms using sequestered inorganic energy-rich phosphate
structures in the pores of the silica sols; primitive pro-biological polysilicates would have been capable
from the start of multi-ion adsorption from seawater; evolution of polysaccharides proteins and nucleic
acids would have followed from advantages produced by these systems for gaining nutrients. Cf., Grant
D., W.F. Long and F.B. Williamson, Degenerative and inflammatory diseases may result from defects in
antimineralization mechanisms afforded by glycosaminoglycans
Med Hypotheses, 1992, 38, 49-55.
This article argued that polyanions similar to polyphosphates and glycosaminoglycans
may have influenced early biological evolution by modulating the morphology,
surface chemistry and activity of polyoxyanionic minerals such as silica and apatite

Grant D., W.F. Long and F.B. Williamson

The binding of Pt(II) to heparin
Biochem. Soc. Trans., 1996, 24 (2) 204S

Grant D., W.F. Long and F.B. Williamson

Incompletely published (but displayed as conference posters) of studies conducted by procedures similar
to those described in ref. (32) showed that the carrageenans and other anionic polysaccharides as well
natural polyanions such as humic materials, by binding to nascent crystallization nuclei also inhibit
CaCO3 crystallization; the highly efficient action of humic polymers is probably of major relevance to
global CO2 balance in the sea since this seems to potentially be subject to anthropogenic perturbation by
land-derived humic matter e.g. produced following deforestations and intensive agriculture].

Grant D., W.F. Long, F.B. Williamson

(1997) Unpublished
The ability of nitrite (which is believed to be the major nitric oxide metabolite which is active
in the above heparan sulphate signalling pathways) to react with unsubstituted glucosamines now known
to be formed duing the primary biosynthesis as well as by post-synthetic events in heparan sulphate
chains and thereby to create a range of signalling oligosaccharides is dependent on metal ion catalysis
(iron ions can, e.g. catlayse a de-N-sulphonation process which is required to generate active sites for
nitrite cleavage). Inorganic cofactors which can affect nitrosative signalling by the heparome probably
also include, apart from metal ions, a range of gas molecules (which may include hydrogen sulphide,
hydrogen monoxide, hydrogen peroxide) as well as nitric oxide.
Cf., Grant D., W.F. Long, G. Macintosh and F.B. Williamson
Antioxidant activity of heparin
Biochem. Soc. Trans., 1996, 24 (2) 194S

Grant D. (2000)
http://web.ukonline.com.uk/dgrant/dg5

Grant D. (2000)
Ascorbate and nitric oxide in redox control of heparan sulphate
http//www.ukonline.co.uk/dgrant/dg4
[Are the roles of inorganic cofactors intrinsically different between heparan sulphate and DNA?
It may be intrinsically imperative to strongly hinder the access of redox metal ions to DNA. Heparan sulphate occurs abundantly at
uniquely accessible extracellular sites in contrast with the DNA location. The suggested normal physiolgical heparan sulphate
multielement sequestration behaviour seems intrinsically to contrast strongly with the perceived situation with DNA which it might be
anticipated, must be shielded from potential disruption of its encoded information e.g. by Fenton reactions inducible by contact with
damaging metal ions which is prevented in eukaryotes by holding DNA intracellularly, shielded by the cytoskeleton and histones and
subject to the protective actions of antioxidants, high affinity metal ion binding proteins and specific damage correction repair
mechanisms. Hence although both heparin/heparan sulphate and DNA contain conceptually similar linearly encoded information
systems, quite different regulation of metal ion interactions may be required for proper function of these two linearly information
encoded biopolymer systems].

Grant D. (2005)
Metallome-Heparanome Crosstalk Hypothesis
Although there are a large number of possible in vivo relevant multielement biological ligands additional
to specific metal ion ligands such as calmodulin, transferrin and caeruloplasmin (e.g. nucleic acids,
proteins, polysaccharides and thousands of types of lower molecular weight biomolecules) of which the
extracellular polysaccharides seem uniquely placed to associate with metal ions, (at often modest but
physiologically relevant affinities) with metal ions present in multielement solutions such as blood serum
(1) by several mechanisms (e.g., electrostatic, hydrogen-bonding and phase change engulfment).
Multi-metal-ion and multielement binding may determine the behaviour of the unusually ultra-anionic
biological polysaccharide systems including the heparan oligosaccharides are apparently involved in as
yet poorly understood servo-feedback intracelluar signalling involving inorganic ions and particles.
Metal ion binding studies of heparin, and mass spectroscopic multielement analysis (by SSMS and ICP-
MS) show that heparin acts as an efficient multielement matrix, this was formerly thought to be of trivial
significance arising from contamination during extraction and work-up procedures. However, such
polysaccharide inorganic complexes may normally arise from an equilibration with physiological media
which normally contain a similar large range of dissolved ions to those present in seawater (or blood
serum). The multielement character of heparin and heparan sulphate is of obvious relevance to the
mechanism of heparanome protein interactions designed for subsequent easy release of a wide variety of
metal ions required, e.g., for specific nucleic acid, protein or polysaccharide structure building and
related functions. An absolute requirement for specific divalent metal ions can potentiate signalling by
growth factors and could be critically relevant for fundamental studies of the biological roles of
heparin/heparan sulphate--metal ion--protein and heparan sulphate--metal ion--nucleic acid interactions
and wider mechanisms.
{The multielement contents of biological samples, whole cells and complex multi molecular protein,
organic and inorganic component solutions such as blood serum and geological matrices such as sea
water are now thought to be of fundamental interest to the fuller understanding of the roles of metal ions
in biology. Highly anionic extracellular polysaccharides could provide suitable metallomic ligands.
Heparin, it is now suggested is perhaps the single most relevant such ligand since this is the most ultra
anionic polysaccharide in biology. The uniquely high multielement binding capacity heparin can
uniquely provide insight into mammalian metal ion presence. Further study of the mass spectrosocpic
multielement evaluation of polysaccharides derived from human and animal tissues is warranted}.

Grushka E. and A.S. Cohen

The binding of Cu(II) and Zn(II) ions by heparin
Anal. Lett., 1982, 15 (B16), 1277-1288

Hamazaki H.
Ca2+-mediated association of human serum amyloid P component with
heparan sulfate and dermatan sulfate
J. Biol. Chem., 1987, 262, 1456-1460
(No binding was observed in the absence of added Ca2+ but other
M2+ ions studied (M: Ba, Cu, Mg, Mn and Sr) did not promote binding)

Herwats L., P. Laszlo and P. Genard

How heparin binds sodium: a sodium-23 NMR study
Noveau J de Chemie, 1977, 1 (2) 173-176

Hu W.-L., and R. Regoeczi

Hepatic heparan sulphate proteoglycan and the recycling of transferrin
Biochem. Cell Biol., 1992, 70 (7) 535-538

Iler R.K.
The Chemistry of Silica
Wiley, New York, 1979
(Cf., p. 762
[“silica is bound in tissues with glycosaminoglycans and polyuronides.
About 800 ppm SiO2 was bound to purified hyaluronic acid, chondroitin 4-sulfate
and heparan sulfate. Silica is also reported to be bound to pectin and alginic acid…
Some association of polysaccharide with silica has probably existed since life began”]
Jorpes J.E.
Heparin: A mucopolysaccharide and an active antithrombotic drug
Circulation, 1959, 19, 87-91
[A historical review of the discovery and early use of heparin as an anticoagulant]
{The high ash content of heparin had noted from the start of scientific interest in heparin. Previously this
was regarded as ‘impurities’}
N.b., heparin extracted from (mast cells) in mammalan tissue is a pharmaceutical agent and a convenient
model for elucidating the behaviour of the structurally related heparan sulphate proteoglycan system
management system which apparently most often depends for its activity on the information encoded in
heparin-like segments.

Karlinsky J.B. and R.H. Goldstein

Regulation of sulfated glycosaminoglycan production by
prostaglandin E2 in cultured lung fibroblasts
J. Lab. Clin. Med., 1989, 114 (2) 176-184

Kazama Y. and Koide T.

Role of Zn and Ca ions in the heparin neutralizing ability of histidine rich glycoprotein
Thromb Haemostasis, 1992, 67 (1) 50-55; Chem. Abs., 114,187724t

Kjellén L. and U. Lindahl

Proteoglycans: structures and interactions
Annu. Rev. Biochem., 1991, 60, 443-475

Lewit-Bentley A., S. Morera, R. Huber and G. Bodo

The effect of metal binding on the structure of annexin V and implications for
membrane binding
Eur. J. Biochem., 1992, 210, 73-77

Liang J.N., B. Chakrabarti, L. Ayotte and A.S. Perlin

An essential role for the 2-sulfamino group in the interaction of
calcium ion with heparin
Carbohydr. Res., 1982, 106, 101-109

Luck W.
Ber Bunsenges Phys, Chem., 1965, 69(1) 69
(Cf. also ibid., 826 and Kleeberg E. Ed., (Proc. Symp. 2-3 Aprl 1987, Marburg, FRG in Honor of the 65th
Birthway of Werner A.P. Luck) “Interactions of Water in Ionic and Nonionic Hydrates”, Springer Verlag
Berlin, 1987;
Luck W.A.P.
Is life mainly inorganic chemistry?
Topics Curr. Chem., 1976, 5, 113-180
[Salt effects on the association of water: the explains the Hofmeister effect which may in turn explain
the role of water clusters associated with sulphated polyanions as modulators of protein folding]
[If life is mainly inorganic chemistry then the metallome is obviously a key player in he most
fundamental processes determining the basic life processes]

Lyon M.E., D. Bremner, T. Laha, S. Malik, P.J. Henderson and M.A. Kenny
Specific heparin properties interfere with simultaneous measurement of
ionized Mg and ionized Ca
Clin. Biochem., 1995, 28 (1) 79-84
[Time dependent bias was observed in ionized Mg and Ca
concentrations with Zn heparin but not with Li or electrolyte balanced heparin]

Lyon, M., J.A. Deakin and J.T. Gallagher

Liver heparan sulfate structure. A novel molecular design.
J. Biol. Chem., 1994, 269 (15) 11208-112115.

McKeehan W.L., X. Wu and M. Kan

Requirement for anticoagulant heparan sulfate in the fibroblast growth factor
receptor complex
J. Biol Chem., 1999, 274 (31) 21511-21514

Murata K., and Y. Yokoyama

Acidic glycosaminoglycans in human atherosclerotic cerebral arterial tissue
Atherosclerosis (Shannon, Irl.) 1989, 78 (1) 69-79; Chem. Abs., 111, 151343a
[Age-dependent alteration in abundance of heparan sulphate at
arterial walls]
(In a related study by E. Feyzi, T. Saldeen, J. Larssen U. Lindahl and M Salmivirta
Age-dependent modulation of heparan sulfate structure and function
Biochem. J. 1998, 273, 13795-13798
it was proposed that lifespan in humans is determined by
heparan sulphate microstructural alteration during ageing)

Muzzarelli R.A.A
Heparin-like substances and blood compatible polymers obtained from
chitin and chitosan
Polymer Science Technology, 1983, 23, 359-374
[This paper suggests that the multi-inorganic elements which are associated with
these anioic polysaccharides derives from tap water]

Nagasawa K., H. Uchiyama, N. Sato and A. Hatano

Chemical change involved in the oxidative-reductive depolymerization of heparin
Carbohydr. Res., 1992, 236, 165-180

Ohkubo Y., Tsukada F., Kohno H., and Kubodera A.

Relationship between binding activity of 67-Ga and low sulphated acid
glycosaminoglycans
Nuclear Med. Biol.,1989, 16 (4) 343-346; Chem. Abs., 111, 111668d

Panov V.P., and A.M. Ovespan

Study of heparin salts by spectroscopic methods (translated)
Vysokomol. Soedin., 1984, Ser., A 26(9) 1963-1970 ; Chem. Abs., 102, 113831q

Parish R.F., and W.R. Fair

Selective binding of zinc ions to heparin rather than to other glycosaminoglycans
Biochem,.J., 1981, 193, 407-410

Percival E., and R.H. McDowell

Chemistry and Enzymology of Marine Algal Polysaccharides
Academic Press, London and New York (1967) cf., p. 19

Rabenstein D.L., J.M. Robert and J. Peng

Multinuclear magnetic resonance studies of the interaction of inorganic
cations with heparin
Carbohydr. Res., 1995, 278, 239-256

Renne T., J. Dedio, G. David and W. Muller Esterl

J. Biol. Chem., 2000, 275 (43) 33688-33696
[Zn2+ promotes the association of heparan sulphate and (biotinylated heparan sulphate-) kininogen]

Rodushkin I., and M.D. Axelsson

Application of double focussing sector field ICP-MS for multielemental characterization of human hair and
nails.
Part I. Analytical methodology
Sci. Tot. Environ., 2000, 250, 83-100; cf., ibid., 2000, 262, 21-6
Cf., also V.N. Senofonte and S. Caroli, J. Trace Elem. Med. Biol., 2000, 14, 6-13;
D.A. Bass et al., Altern. Med. Rev., 2001, 6, 472-481
A.R. Bleise et al., Analytical Quality Control Services (Hair Elements Reference Sheet) Interntional Atomic
Energy Agency, A1499, Vienna , Austria, and
http://www.analytica.se/hem2001/human/research.asp.)

Senofonte Violante N., and S. Caroli

Assessment of references values for elements in human hair of urban schoolboys
J. Trace Elem. Med. Biol., 2000, 14(1) 6-13

Schlemmer U.
Studies of the binding of copper, zinc and calcium to pectin, alginate, carrageenan
and gum guar in HCO3 -- CO2 buffer.
Food Chem., 1989, 32 (3) 223-234.

Stringer, S.E., M. Mayer-Proschel, A. Kalyani, M. Rao and J.T. Gallagher

Heparin is a unique marker of progenitors of the glial cell lineage.
J. Biol. Chem., 1999, 274 (36) 25455-25460.

Tajmir-Riahi H.-A.
D-Glucose adducts with zinc-group metal ions. Synthesis and spectroscopic and structural
characterization of Zn(II), Cd(II) and Hg(II) complexes with D-glucose, and the effects of metal-ion
binding on the sugar anomeric structures.
Carbohydr. Res., 1989, 190, 29-37.

Tapanadechopone P., S. Tumova, X. Jiang and J.R. Couchman

Epidermal transformation leads to increased perlecan synthesis with heparin-binding growth
factor affinity.
Biochem. J., 2001, 355 (2) 517-527.

Templeton D.M.
Acceleration of the mercury-induced aquation of bromopentammine Co(III)
by naturally occurring glycosaminoglycans.
Can. J. Chem., 1987, 65, 2411-2421.

Timpl R.
Structure and biological activity of basement membrane proteins.
Eur. J. Biochem., 1989, 180, 487-502.

Toida T.E. with R.J. Linhardt, et al.

Detection of GAGs Cu(II) complex in capillary electrophoresis
Electrophoresis, 1996, 17, 341-346; J. Chromatog., 1997, 787 (1-2) 266-270

Vandewalle B., F. Revillon, L. Hornez and J. Lefebvre

Calcium regulation of heparan sulphate proteoglycans in breast cancer cells
J. Cancer Res. Clin. Oncol., 1994, 120 (7) 389-392 ; Chem. Abs., 121,105487j

Wiggins P(M)
Life depends on tow kinds of water
PLoS ONE 2008 Jan 9 3(1)e1406
Cf. also http://www.Isbu.ac.uk/water/monograph200904pw.pdf
and
Physica A2002, 414, 458

Williams R.J.P.
The biochemistry of sodium, potassium, magnesium and calcium
Quart. Rev., 1970, 24 (3) 331-365

Yamaguchi S., T. Yoshioka, M. Utsunomiya, T. Koide, M. Osafune, A. Okuyana and T. Sonada

Heparan sulfate in the stone matrix and its inhibitory effect on calcium oxalate crystallization
Urol. Res., 1993, 21 (3) 187-192; Chem. Abs., 119, 243946t
[Heparan sulphate is a potent inhibitor of calcium oxalate crystallization in vivo]

Zou S., C.E. Magura and W.L. Hurley

Heparin-binding properties of lactoferrin and lysozyme
Comp. Biochem. Physiol., 1992, 103B (4) 889-895
[Biotinylated heparin binding to lactoferrin was dependent on Na, Ca, Cu, Zn and Fe cations]
_____________________________________________________________________________________

A heparan sulphate-environment feedback system which can act to determine alteration in animal
speciation must of course eventually alter DNA. This could be closely related to an interaction of
mutational activity and nitrosative signalling processes. The current list of reported interactions between
the heparanome and the genome seem to suggest this possiblilty but for a more complete understanding
of the phenomenon of evolution of species it must also, it can be suggested, be established why the
presence of liquid water is so essential for life. It can be argued that sugars and polysaccharides
including heparan sulphate must play a key role in the regulation of water activity. (Cf. the interspecies
dependence heparan sulphate contents of tissues of aquatic invertebrates were found to be related in a
mathematically exact manner to the salinity and therefore potential osmolyte stress of their habitats (this
information was reported by H.B. Nader et al. in ref. 34); a related phenomenon is the influence of
inorganic ion concentrations upon Hofmeister effects, which in turn are ascribable to an action of such
ions on water structure e.g. as discussed by W. Luck and P. Wiggins (cf. references cited in the
Appendix). It can be demonstrated that heparin/heparan sulphate metal ion complexation impacts upon
this phenomenon (e.g. as reported by Grant et al, in the references listed in the Appendix and in refs. 14).

Loss of Essential Metal Ion Cofactors During Purification

Although metal ions are known (cf., Table I) to have critical roles in the modulation of heparin/heparan
sulphate - protein interactions, this may only be discovered fortuitously, as exemplified by how the
requirement for the presence of Zn2+ ions to permit endostatin - heparan sulphate binding was discovered
(16). Whilst binding to Zn2+ will normally occur in vivo, this binding was found to be it is abolished in
vitro unless Zn2+ ions, evidently removed during column polysaccharide gel purification processes, were
reintroduced. Required metal ions can evidently also bind strongly to the polysaccharides used for usual
chromatographic separations which may therefore be the unexpected source of experimental errors.
In vivo, sufficient metal ions for facilitation of the correct protein-heparan sulphate interactions will
normally be present in common physiological fluids but may be absent in sufficient amounts after
column gel fractionations or in physiological saline solutions prepared by use of chemically pure sodium
chloride.

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The metal ion dependent postsynthetic alteration of information encoded in coded heparin/heparan is not
restricted, as is DNA, by a requirement to preserve genetic information, so that while the entire signal in
heparan sulphate chains is believed to be of physiological significance (e.g. as a sort of biological
postcode for defining particular cellular types and locations) the details of this code have not yet been
established owing to the lack of sequencing methods as good as those available for nucleic acids) the
heparan sulphate chains are normally utilised after being specifically modified to generate fragments
containing information packets apparently ‘designed’ to be read at distant sites. It is now suggested that
inorganic elements are required to create the encoded information present in heparin/heparan sulphate
system (including the oligosaccharide messengers).

**The heparanome
The heparanome is the name recently suggested for the system of animal polysaccharides which
contain evolutionary conserved (7) domains of mineral-like anionic arrays of sequences of highly
sulphated polyiduronate/glucuronate glucosamine N-sulphonate residues.
Studies of HSPG biochemistry suggest an especially important role for such information
encoded sequences occurring in side chain polysaccharide structures which act like biological postcodes
to facilitate the interaction with conserved HSPG binding sites in proteins.
They have well-established roles in morphogenesis (providing a reservoir and control system for basic
fibroblast growth factor and its receptors, regulation of embryo assembly) mediation of adhesion and
morphogenesis, the provision of links betwen cytoskeleton and extracellular matrix, modulation of
antioxidant activity including the anchoring of endothelial antioxidant enzymes, the assembly of matrix
phosphatidyl-inositol linkages, the regulation of blood coagulation and apoptosis, the modulation of
synaptic and neurological activity, and implication of invovlement in the mechanisms of memory, cognition and
ageing.
Although such activities are more pronounced for HSPGs than for other glycosaminoglycans,
the latter however share various primitive functions with heparan sulphates especially in the provision
of ion and pH balance, water activity, mechanical support, modulation of collagen fibrillogenesis
including the transparency of the cornea.
Glycosaminoglycans generally provide for a regulation of calcification, cell migration, aggregation and
development, a filtration barrier and stabilization of basement membrane, synaptic structures,
endothelial surfaces, mediation of transferrin uptake and antigen presentation.
Heparin, a cocktail of heparan sulphate-like molecules produced by mast cells present in many animals
(but not apparently in rabbits) is believed to be the most highly anionic polysaccharide biological-molecule type.
It is manufactured and purified commercially to provide a largely protein-free pharmaceutical agent which is widely
used for blood anticoagulation, an action which is mainly attributable to its content of an
antithrombin (III) pentasaccharide binding sequence which is also present in the anticoagulant-type-HSPG which
can be induced to form at vascular walls, from which it can be released by proteolytic or nitrosative cleavage (processes
which are also putatively subject to perturbation by inappropriate multielement constituents in blood serum).

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*Home based research continued from former institutional affiliated research at the University of
Aberdeen (Marischal College) and discussions with F.B. Williamson and other former Aberdeen
Polysaccharide Group members and others including R.J.P. Williams (Oxford University) and K.E.L.
McColl (Glasgow University).

The multi-inorganic element nature of heparin (as a state of matter) is indirectly fully confirmed by the
major international analytical laboratory ALS, vide supra (in an internet report which seems to be aimed at
commercial end-users) who evidently conducted mass spectrometric elemental composition determinations of at
 least two different brands of sodium and lithium heparin. These results confirmed that these heparins contained
a
similar multi-element presence to heparins which had been analalysed by Aberdeen University (these were a Na
and a derived Tl heparin.
(N.b. these multi- element contents, although greatly reduced in amounts for most elements are still considered
sufficiently great to define all heparins, even ‘purified’ or ‘single counterion’ heparins as multi-element
metallomic
matrices. These results suggest that heparin is putatively a provider of the range of metal ions which are
utilised
as biochemical cofactors. If this hypothesis is confirmed then the range of inorganic cofactors used by animal
biology is greater than that which is currently believed to be the case.
Problems of reproducibility of inorganic residues in heparin can apparently however also arise from differences
in
work-up procedures between manufacturers. This may become a serious problem when industrially prepared
heparin is used as a laboratory model for heparan sulphate.
A potential metal ion toxicity problem may also arise (e.g. with some industrial heparins identified by D. Bohrer
et
al., RBAc (Brasil) 2004, 36 (2) 99-103) in which the occurrence of sufficiently large amount of Al indicated
their
unsuitability especially for kidney dialysis patient use, without prior passage through an ion exchange column to
reduce the amount of such Al to an acceptably low level).

Appendix

Comparison of log-log plot of apparent enrichment of multielements in algal biomass and heparin
from seawater (like bathing) source of the multielements