MANNITOL
CHEMISTRY, USES AND
POTENTIAL SIDE EFFECTS
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PHARMACOLOGY - RESEARCH, SAFETY
TESTING AND REGULATION
BIOTECHNOLOGY IN AGRICULTURE,
INDUSTRY AND MEDICINE
MANNITOL
CHEMISTRY, USES AND
POTENTIAL SIDE EFFECTS
PAOLO FUBINI
EDITOR
New York
Copyright 2013 by Nova Science Publishers, Inc.
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Preface vii
Chapter 1 Utilization and Production of D-Mannitol by Bacteria 1
Josef Deutscher, Houda Bouraoui, Meriem Derkaoui
and Philippe Joyet
Chapter 2 Concentration of Mannitol and Other Soluble
Carbohydrates in the Crustose Lichen Rhizocarpon
Geographicum 21
Richard A. Armstrong
Chapter 3 Mannitol: Disease-Related Changes and their Use
for Clinical Disorders 41
David Caldern Guzmn, Gerardo Barragn Meja,
Hugo Jurez Olgun and Ernestina Hernndez Garca
Chapter 4 Use of Mannitol in Thermal Energy Storage Applications 63
Luisa F. Cabeza, Camila Barreneche, Antoni Gil
and A. Ins Fernndez
Chapter 5 Chiral Phosphorous Ligands Derived from D-Mannitol:
Synthesis and their Applications in Asymmetric Catalysis 87
Yingwei Zhao, Lei Yang and Hanmin Huang
Index 113
PREFACE
In this book, the authors present topical research in the study of the
chemistry, uses and potential side effects of mannitol. Topics discussed
include the utilization and production of D-mannitol by bacteria; concentration
of mannitol and other soluble carbohydrates in the crustose lichen rhizocarpon
geographicum; disease-related changes and mannitols use for clinical
disorders; use of mannitol in thermal energy storage applications; and chiral
phosphorous ligands derived from D-mannitol.
Chapter 1 - Certain plants, yeasts, algae, lichen and fungi produce large
amounts of D-mannitol and many bacteria developed the capacity to utilize
this naturally occurring carbon and energy source. For that purpose they use
different transport systems and catabolic pathways. Some bacteria transport D-
mannitol via ion-driven co-transport systems or ABC transporters without
modification of their substrate. Intracellular D-mannitol is subsequently
converted into fructose by the D-mannitol 2-dehydrogenase MtlD (l) and
fructose is phosphorylated to the glycolytic intermediate fructose-6-P.
Numerous other bacteria take up D-mannitol via the phosphoenolpyruvate
(PEP): carbohydrate phosphotransferase system (PTS), which catalyzes the
transport and concomitant phosphorylation of its substrates. D-mannitol
transported by the PTS therefore arrives as D-mannitol-1-P in bacterial cells.
The enzyme D-mannitol-1-P 5-dehydrogenase MtlD(s) converts D-mannitol-
1-P into fructose-6-P. The genes encoding the different types of transport and
catabolic enzymes are usually organized within the mtl operon. Transcription
of the mtl operon is also controlled by various mechanisms, which allow the
expression of the mtl genes only when mannitol is present in the growth
medium. The mtl operon is usually also submitted to carbon catabolite
repression, which prevents its expression when an efficiently metabolizable
viii Paolo Fubini
carbon source, such as glucose, is present. Bacteria not only utilize mannitol,
but some are also able to produce D-mannitol from fructose with the aid of the
enzyme Mdh. This is of biotechnological interest and improvement of the
yield of mannitol biosynthesis might allow a cheaper and more efficient
production of D-mannitol compared to the industrial production via catalytic
reduction of fructose.
Chapter 2 - In symbiotic lichens which have Trebouxia as the algal
partner, photosynthesis by the algae results in the production of the soluble
carbohydrate ribitol which is then transported to the fungus where it is
converted to arabitol and mannitol. Within the fungus, arabitol may act as a
short-term carbohydrate reserve while mannitol may have a more protective
function and be important in stress resistance. The concentrations of ribitol,
arabitol, and mannitol were measured, using gas chromatography, in the
central areolae and marginal hypothallus of the crustose lichen Rhizocarpon
geographicum (L.) DC. growing on slate rocks in north Wales, UK. The
concentrations of all three soluble carbohydrates were greater in the central
areolae than in the marginal prothallus. In addition, the ratio of mannitol in the
prothallus to that in the areolae was least in July. The concentration of an
individual carbohydrate in the prothallus was correlated primarily with the
concentrations of the other carbohydrates in the prothallus and not to their
concentrations in the areolae. Low concentration of ribitol, arabitol, and
mannitol in the marginal prothallus compared with the central areolae suggests
either a lower demand for carbohydrate by the prothallus or limited transport
from areolae to prothallus and may explain the low growth rates of this
species. In addition, soluble carbohydrates appear to be partitioned differently
through the year with an increase in mannitol compared with arabitol in more
stressful periods.
Chapter 3 - Mannitol, a white, crystalline alcohol, is derived from sugar
by reduction. The pathway to obtain mannitol from natural products is through
hydrogenation of fructose, which is formed from either starch or sugar. This
substance is present in a wide variety of natural products, and in almost all
plants. It is used as an osmotic diuretic agent and a weak renal vasodilator.
Aqueous solutions of mannitol are mildly acidic and sometimes such solutions
are used to decrease the pH. Mannitol is clinically used in osmotherapy to
temporarily reduce acute intracranial pressure while waiting for the definitive
treatment. It is also used to treat patients with oliguric renal failure.
Consequently, mannitol increases water and Na+ excretion, thereby decreasing
extracellular fluid volume. It can also be used as a facilitating agent for
transporting drug agents directly into the brain. This chapter reviews the
Preface ix
and ). To test the d-mannitol thermal behaviour at pilot plant scale, 150 kg of
d-mannitol were introduced in a storage tank which was designed as shell-and-
tubes heat exchanger. Results show that applying different cooling conditions
produces d-mannitol polymorphic changes. Moreover, it has been shown that
the working range (between 135C and 175C) is adequate for pilot plant
experiments.
Chapter 5 - As a kind of readily available carbohydrate, D-mannitol has
been widely used in the synthesis of chiral phosphorous ligands. This chapter
reviews the design and synthesis of various mono- and diphosphorous ligands
based on D-mannitol backbone. Also, the successful applications of these
ligands in asymmetric catalysis, such as enantioselective hydrogenation and
enantioselective conjugate addition, are summarized.
In: Mannitol ISBN: 978-1-62808-762-8
Editor: Paolo Fubini 2013 Nova Science Publishers, Inc.
Chapter 1
ABSTRACT
Certain plants, yeasts, algae, lichen and fungi produce large amounts of
D-mannitol and many bacteria developed the capacity to utilize this naturally
occurring carbon and energy source. For that purpose they use different
transport systems and catabolic pathways. Some bacteria transport D-
mannitol via ion-driven co-transport systems or ABC transporters without
modification of their substrate. Intracellular D-mannitol is subsequently
converted into fructose by the D-mannitol 2-dehydrogenase MtlD (l) and
fructose is phosphorylated to the glycolytic intermediate fructose-6-P.
Numerous other bacteria take up D-mannitol via the phosphoenolpyruvate
(PEP): carbohydrate phosphotransferase system (PTS), which catalyzes the
transport and concomitant phosphorylation of its substrates. D-mannitol
*
Corresponding author: Josef.Deutscher@grignon.inra.fr.
2 Josef Deutscher, Houda Bouraoui, Meriem Derkaoui et al.
INTRODUCTION
The hexitol D-mannitol is by far the most abundant sugar alcohol in nature.
This sugar alcohol has the same stereochemical configuration as mannose and
therefore possesses a two-fold symmetry axis. Rotation of the hexitol by 180
provides a molecule with identical configuration and the 1- and 6-position are
therefore indistinguishable. Nevertheless, phosphorylated mannitol is usually
referred to as mannitol-1-P and not as mannitol-6-P. D-Mannitol is produced in
relatively large quantities for example by certain marine algae. In the brown
seaweed Laminaria japonica mannitol is the most abundant carbohydrate.
Enterobacter sp. JMP3 was recently shown to efficiently convert D-manitol
produced by the seaweed into bioethanol [1]. In algae the polyol exerts multiple
functions, such as osmoregulation, storage, regeneration of reducing power, and
scavenging of active oxygen species [2, 3]. In these organisms, D-mannitol is
mainly produced from fructose-6-P, which is reduced to D-mannitol-1-P by the
enzyme D-mannitol-1-P 5-dehydrogenase and subsequently dephosphorylated to
D-mannitol [4]. When D-mannitol-producing algae are transferred from a saline
medium (sea water) to fresh water most of the accumulated intracellular D-
mannitol is released into the environment by a yet unknown efflux system [5, 6].
In higher vascular plants D-mannitol is also one of the major photosynthetic
products [7] and also protects against osmotic pressure and stress [2]. In celery
and some related plants biosynthesis does not seem to occur from fructose-6-P but
from mannose-6-P, which is reduced by an NADPH-dependent mannose-6-P 1-
reductase to D-mannitol-1-P [8, 9]. Finally, a D-mannitol-1-P-specific
phosphatase dephosphorylates D-mannitol-1-P to D-mannitol [6, 10]. Some plants
Utilization and Production of D-Mannitol by Bacteria 3
Figure 1. Schematic presentation of the three different known D-mannitol uptake systems,
the catabolic pathways of D-mannitol and a potential D-mannitol efflux system present in
some heterofermentative lactic acid bacteria. D-Mannitol can be transported by bacteria
via ion-driven permeases (MtlT), ABC transport systems (MtlEFGK) or PTS permeases
(MtlA or MtlA and MtlF). D-Mannitol transported by the PTS arrives as D-mannitol-1-P
in the cell and is subsequently converted to the glycolytic intermediate D-fructose-6-P by a
short-chain alcohol dehydrogenase MtlD(s). D-Mannitol taken up by an ion-driven
permease or an ABC transport system is first oxidized to D-fructose by a long-chain
alcohol dehydrogenase MtlD (l) and subsequently phosphorylated to D-fructose-6-P. MtlE
of the ABC transport complex is a D-mannitol binding protein located in the periplasm,
MtlK is an ATP hydrolyzing protein providing the energy for the transport process and
MtlF and MtlG are two membrane-spanning proteins. Some heterofermentative lactic acid
bacteria produce D-mannitol from D-fructose in an NADPH-requiring reaction catalyzed
by the enzyme Mdh, another type of mannitol 2-dehydrogenase. D-Mannitol might be
secreted into the medium via an ABC efflux system, the genes of which are frequently
located in the vicinity of the mdh gene.
Utilization and Production of D-Mannitol by Bacteria 5
encoded by the mtl operon, MtlZ, strongly resembles fructokinase from Vibrio
alginolyticus and has indeed been shown to convert fructose formed from D-
mannitol or D-glucitol by the D-mannitol dehydrogenase MtlD(l) into fructose-6-
P [16]. The other kinase MtlY phosphorylates xylulose formed by MtlD(l) from
arabitol into xylulose-5-P [16].
Gram-positive organisms including firmicutes and actinobacteria as well as
enterobacteriaceae take up D-mannitol via a PTS and phosphorylate it already
during its transport to D-mannitol-1-P, which is subsequently converted to
fructose-6-P by the enzyme D-mannitol-1-P 5-dehydrogenase (MtlD).
Unfortunately, the nomenclature is ambiguous because both, the D-mannitol 2-
dehydrogenase of bacteria transporting D-mannitol via ion-driven transporters or
ABC transport systems as well as D-mannitol-1-P 5-dehydrogenase of bacteria
transporting D-mannitol via a PTS were called MtlD. D-Mannitol-1-P 5-
dehydrogenases and D-mannitol 2-dehydrogenase have a different length (about
370 and 500 amino acids, respectively) and belong to the short-chain and long-
chain alcohol dehydrogenases [15]. In this article they will be distinguished as
MtlD(l) for the long-chain and MtlD(s) for the short-chain alcohol
dehydrogenases. The two types of alcohol dehydrogenases probably have the
same evolutionary origin, because despite their different lengths the C-terminal
part of mannitol-1-P 5-dehydrogenases from firmicutes and enterobacteriaceae
exhibits significant sequence similarity (about 40%) to the central part of mannitol
dehydrogenases from corynebacteria and pseudomonadaceae.
In order to phosphorylate its substrate during the transport step four of the
ususally five PTS components form a phosphorylation cascade (Figure 3).
Enzyme I (EI) autophosphorylates with PEP and transfers the phosphoryl group to
the histidyl residue of the second general PTS protein HPr. P~His-HPr
phosphorylates one of usually several sugar-specific EIIA components and
P~EIIA donates its phosphoryl group to the cognate EIIB. In the last step, P~EIIB
phosphorylates the carbohydrate (sugar, sugar alcohol, amino sugar and many
other sugar derivatives) bound to the corresponding membrane integral EIIC.
Phosphorylation of the carbohydrate lowers the affinity for its EIIC and the
phosphorylated substrate is released into the cytoplasm (Figure 3) [18]. The EII
components are frequently fused together providing one single protein. This is the
case for the D-mannitol-specific MtlA protein of E. coli and other
enterobacteriaceae, which is composed in the following order of the EIIC, EIIB
and EIIA domains. Several actinobacteria, such as Arthrobacter aurescens, also
possess an EIICBAMtl protein composed of the three mannitol-specific domains.
This is also true for Corynebacterium durum, while, as mentioned above, most
other corynebacteria possess an ion-driven transporter MtlT [14]. In contrast, in
Utilization and Production of D-Mannitol by Bacteria 7
mtlADR operon controls expression of the mannitol operon of this organism [41].
It is tempting to assume that MtlR regulates the amount of MtlS in response to the
presence or absence of D-mannitol.
The mtlR gene of the -proteobacterium P. fluorescens strain DSM50106
encodes a regulatory protein of about 34 kDa belonging to the AraC/XylS family
of transcription regulators [42], which contain a C-terminal HTH DNA binding
domain [43]. P. fluorescens MtlR was identified as transcription activator, which
when produced in E. coli allowed the mannitol-induced expression of the galK
reporter gene fused to the promoter region of the P. fluorescens mannitol operon.
In the presence of D-mannitol purified MtlR specifically binds to a DNA fragment
containing the promoter/operator region of the mtlEFGKDZY operon (Figure 2),
which encodes the four proteins of an ABC transporter, a D-mannitol 2-
dehydrogenase and two carbohydrate kinases (Figure 2). P. fluorescens MtlR is
probably activated not only by mannitol, but also by glucitol and arabitol, because
as mentioned before these two polyols are also taken up by the ABC transporter
[16]. In P. fluorescens strain SBW25 a gene encoding a protein nearly identical to
MtlR of strain DSM50106 is located just upstream from the mannitol operon
(Figure 2). In other strains (SS101) the mtlR gene is loctaed far away from the mtl
operon.
In firmicutes the expression of the mannitol operon is also controlled by a
transcription activator called MtlR [21, 27, 44, 45]. However, MtlR of firmicutes
is not at all related to MtlR of -proteobacteria. MtlR of firmicutes is composed of
an N-terminal DNA binding domain and an Mga-like domain followed by four
regulatory domains containing potential PTS phosphorylation sites (Figure 4)
[46]. The penultimate regulatory domain resembles EIIBGat and the last one
EIIAMtl PTS components. They are preceded by two domains which were called
PTS regulation domains (PRD). PRDs are found in antiterminators and
transcription activators of firmicutes, actinobacteria and certain proteobacteria
[18]. Each PRD usually contains two conserved histidyl residues potentially
phosphorylated by PTS components. The MtlR transcription activators of
firmicutes therefore possess up to six presumed PTS phosphorylation sites, five of
them being histidines and one being a cysteine. The activity of PRD-containing
transcription regulators is indeed controlled by PTS-catalyzed phosphorylation at
two or sometimes three of the potential phosphorylation sites. Although the
sequence of PRD-containing MtlR proteins is strongly conserved it seems that
their mode of regulation can largely vary. PRD-containing MtlRs are usually
controlled by two PTS-catalyzed phosphorylations with antagonistic effects on
their activity. One is catalyzed by P~His-HPr, the other by one of the two
mannitol-specific PTS components (Figure 4). In addition, in certain cases
Utilization and Production of D-Mannitol by Bacteria 11
Figure 4. The different regulatory mechanisms controlling the activity of MtlR from B.
subtilis and G. stearothermophilus. Both proteins need to be phosphorylated by PEP, EI
and HPr at the first conserved histidine in PRD2. This phosphorylation is prevented by the
uptake of an efficiently metabolizable carbon source, such as glucose, and it serves as
CCR mechanism. MtlR from G. stearothermophilus becomes also phosphorylated by PEP,
EI, HPr, EIIAMtl and EIIBMtl at His-598 in the EIIAMtl-like domain and this
phosphorylation inhibits the transcription activation function. It is prevented when D-
mannitol is taken up via the PTS, because under these conditions the phosphoryl group of
the P~EIIBMtl domain is mainly transferred to D-mannitol bound to EIICMtl. It therefore
functions as an induction mechanism for the mtl operon. The inhibitory phosphorylation of
MtlR from B. subtilis occurs at Cys-419 in the EIIBGat-like domain and it is catalyzed by
PEP, EI, HPr and EIIAMtl. In addition, a third condition needs to be fulfilled in order to
render MtlR from B. subtilis active: It needs to be sequestered to the membrane by
interacting with the unphosphorylated EIIBMtl domain of the mannitol-specific permease
MtlA.
The first PRD-containing MtlR protein intensively studied was MtlR from
Geobacillus stearothermophilus (previously called Bacillus stearothermophilus).
The PRD-containing MtlR binds to an about 50 bp DNA region located upstream
from the mtlARFD promoter. The affinity of MtlR for its DNA target site was
strongly affected by its phosphorylation state. Phosphorylation of MtlR by PEP,
EI and HPr at the histidines in PRD2 enhanced the binding affinity about a 100-
fold compared to unphosphorylated MtlR. In contrast, when MtlR was
12 Josef Deutscher, Houda Bouraoui, Meriem Derkaoui et al.
phosphorylated in the presence of PEP, EI, HPr, EIIAMtl and the soluble EIIBMtl
domain of MtlA the transcription activator exhibited a 10-fold lower affinity for
its operator site than unphosphorylated MtlR [44]. Under the latter conditions,
MtlR is expected to be phosphorylated at the conserved histidine(s) in PRD2 by
P~His-HPr as well as at His-598 in the EIIAMtl-like domain by P~EIIBMtl.
MtlR from G. stearothermophilus and B. subtilis exhibit 41% amino acid
sequence identity. It was therefore not surprising that both proteins become
activated by phosphorylation in PRD2 catalyzed by P~His-HPr (Figrue 4).
However, significant differences in the regulation of their activities by the
mannitol-specific PTS components were observed. Phosphorylation of the B.
subtilis PRD-containing MtlR at His-599 in the EIIAMtl-like domain by P~EIIBMtl
has only a slight inhibitory effect on its transcription activator function and a third
phosphorylation turned out to be more important. B. subtilis MtlR was the first
protein for which phosphorylation by an EIIA protein at the conserved cysteine
residue (Cys-419) in the EIIBGat-like domain has been demonstrated. This
phosphorylation exerts the main inhibitory effect on transcription activation by B.
subtilis MtlR [27]. Replacement of Cys-419 with an alanine or deletion of the
EIIAMtl-encoding mtlF gene leads to strong constitutive expression of the lacZ
reporter gene fused to the mtlA promoter [27, 48]. Surprisingly, when in addition
to mtlF the DNA fragment encoding the EIIBMtl domain of MtlA was also deleted,
MtlR was only poorly active. The EIIBMtl domain therefore seems to be required
for MtlR activity. Indeed, the unphosphorylated EIIBMtl domain of MtlA was
found to interact with the two C-terminal EIIBGat- and EIIAMtl-like domains of
MtlR and this interaction is necessary to render MtlR active [47]. Phosphorylated
EIIBMtl does not interact with MtlR [46]. However, it does not seem to be the
interaction with EIIBMtl itself that is necessary for MtlR activation, but rather the
contact with the hydrophobic membrane or a specific membrane component,
because production of EIIBMtl as a distinct soluble cytoplasmic protein inhibited
the MtlR transcription activator function.
Interestingly, EIIBMtl of V. cholerae has recently been shown to be involved
in the regulation of biofilm formation [49]. Growth on mannitol induces the
expression of the vps biofilm matrix exopolysaccharide synthesis genes.
Expression of the vps genes and biofim accumulation were also induced by
ectopic expression of the DNA fragment encoding soluble cytoplasmic EIIBMtl. It
was proposed that this mechanism allows the marine bacterium V. cholerae
attachment to mannitol producing algae and therefore would play an important
role in habitat selection.
When an efficiently metabolized PTS substrate, such as glucose, is taken up
by firmicutes, HPr will be mainly present as unphosphorylated and seryl-
Utilization and Production of D-Mannitol by Bacteria 13
phosphorylated protein and the very small amount of P~His-HPr [50] will lead to
only poor phosphorylation of MtlR in PRD2; the transcription activator will
consequently be inactive (Figure 4). This regulatory concept is supported by the
observation that B. subtilis ptsH or ptsI mutants do not express the PmtlA-lacZ
fusion [27]. The almost complete absence of P~His-HPr-mediated MtlR
phosphorylation during the uptake of glucose therefore serves as a CCR
mechanism. In contrast, the mannitol-specific EIIA and EIIB components are only
dephosphorylated when D-mannitol is transported by the PTS. The presence of D-
mannitol therefore prevents the inactivation of MtlR by phosphorylation of its
EIIBGat-like domain by P~EIIAMtl (B. subtilis) or of its EIIAMtl-like domain by the
P~EIIBMtl domain of MtlA (G. stearothermophilus) (Figure 4). The almost
complete absence of these phosphorylations in the presence of D-mannitol is
therefore used as an induction mechanism. In addition, MtlR of B. subtilis needs
to interact with the unphosphorylated EIIBMtl domain of MtlA and to be
sequestered to the membrane in order to be active (Figure 4). As explained above,
unphosphorylated EIIBMtl prevails in firmicutes when they transport D-mannitol
via the PTS. EIIBMtl-mediated MtlR sequestration to the membrane therefore
serves as a second induction mechanism.
L. casei has also been shown to transport mannitol via a PTS [51] and its
MtlR exhibits about 44% sequence similarity when compared to MtlR from G.
stearothermophilus or B. subtilis. However, in the L. casei protein the second
conserved His in PRD2 (His-399) is replaced with a tyrosine. Replacement of
either one of the two conserved histidines in PRD2 with another amino acid was
found to prevent the phosphorylation of MtlR from G. stearothermophilus and B.
subtilis by P~His-HPr [26, 27]. Similarly, preliminary experiments suggest that
owing to the His-Tyr replacement L. casei MtlR is also not phosphorylated by
P~His-HPr. In fact, in contrast to B. subtilis MtlR the L. casei regulator does not
seem to require activation via phosphorylation by PEP, EI and HPr, because
deletion of ptsI (EI) was found to lead to strong constitutive expression of the
mtlARFD operon of the lactic acid bacterium (P. Joyet and J. Deutscher,
unpublished results). In conclusion, these results demonstrate that although the
PRD-containing transcription activators of firmicutes exhibit a high degree of
sequence similarity the detailed mechanisms regulating their activity can largely
vary and need to be studied for each regulator and each species.
Owing to the absence of P~His-HPr-catalyzed phosphorylation of MtlR, CCR
of the L. casei mtlARFD operon is probably only mediated by the main catabolite
repression mechanism operative in firmicutes; it involves the catabolite control
protein A (CcpA) [52] and seryl-phosphorylated HPr (P-Ser-HPr) [53]. The
uptake of glucose leads to an increase of FBP and a decrease of inorganic
14 Josef Deutscher, Houda Bouraoui, Meriem Derkaoui et al.
phosphate. These conditions stimulate the kinase function of the bifunctional HPr
kinase/phosphorylase (HprK/P) [54, 55] and a major part of HPr is therefore
converted into P-Ser-HPr [50]. In the presence of glycolytic intermediates P-Ser-
HPr binds to CcpA [56] and allows the transcription regulator to interact with
catabolite response elements (cre) [57]. These operator sites precede most
catabolic transcription units and binding of CcpA/P-Ser-HPr either inhibits or
stimulates their expression leading to CCR or carbon catabolite activation (CCA),
respectively. The mtl operon of B. subtilis has indeed been shown to be submitted
to CcpA/P-Ser-HPr-mediated CCR. Mutants deleted for hprK, the gene encoding
HprK/P, or producing a mutant HPr in which Ser-46 is replaced with an alanine
exhibit strongly reduced CCR for the mtl operon [50, 58]. In B. subtilis, the
remaining repression is due to the above described MtlR-mediated CCR. CCR of
the mtl operon in enterobacteriaceae is probably mediated by the complex formed
by cyclic-AMP and the cAMP receptor protein (cAM/Crp), which stimulates the
expression of catabolic genes. In enterobacteriaceae, the uptake of an efficiently
utilized carbon source, such as glucose, lowers the cAMP level [59] and the
cAMP/Crp complex is no longer formed. As a consequence catabolic genes are
only poorly expressed. Competition of the glucose- and mannitol-specific PTSs
for the common phosphoryl donor P~His-HPr has also been discussed as a CCR
mechanism for carbohydrates taken up via a PTS. According to this concept,
P~His-HPr would donate its phosphoryl group preferentially to EIIAGlc and less
frequently to other EIIAs, such as EIIAMtl.
Although several actinobacteria transport D-mannitol via a PTS (MtlA) and
many of them possess PRD-containing transcription activators, in none of them is
the expression of the mannitol operon controlled by a PRD-containing MtlR. The
mtlAD PTS operon of several Arthrobacter and Leifsoniae species is preceded by
a gene encoding a repressor of the TetR family, which probably controls the
expression of the mannitol operon. No gene encoding a putative transcription
regulator is found in the vicinity of the D-mannitol PTS operon in several other
actinobacteria and the regulation of their expression remains obscure. As already
mentioned before, the mtlTD operon present in corynebacteria is usually
controlled by a DeoR-like repressor.
mtlF (EIIAMtl) were constructed and were indeed found to produce intracellular
D-mannitol from glucose at a concentration of about 70 mM and a yield of 30% to
40% [71, 72]. In another attempt, D-mannitol-1-P dehydrogenase and D-mannitol
1-phosphatase were produced in an ldh deletion strain of L. lactis. This allowed
the transformation of D-fructose-6-P into D-mannitol-1-P followed by its
dephosphorylation to D-mannitol with an about 50% yield [73]. Other organisms,
such as E. coli, were also genetically modified for improved D-mannitol
production. Usually the mannitol-2 dehydrogenase, Mdh, from different microbes
was synthesized in the genetically modifed strains [3, 17].
Finally, it should be mentioned that bacterial D-mannitol-1-P 5-
dehydrogenases have been introduced into transgenic plants, such as tobacco [74],
in order to transform D-fructose-6-P into D-mannitol-1-P, which is subsequently
dephosphorylated by the plant D-mannitol 1-phosphatase to mannitol. The
increased production of D-mannitol by the plants provided a protection against
osmolytic stress and droughts.
CONCLUSION
D-Mannitol, the most abundant sugar alcohol in nature, is an important
carbon and energy source for bacteria. This is underlined by the fact that during
evolution bacteria developped at least three different types of D-mannitol-specific
transport systems, two different types of catabolic routes and four different modes
of regulation of mtl operon expression. Especially the uptake and phosphorylation
of the hexitol by the PTS allows its efficient utilization. Nevertheless, glucose and
probably a few other carbohydrates are preferentially utilized and repress the
synthesis of the enzymes necessary for D-mannitol metabolism.
A few bacteria, mainly heterofermentative lactic acid bacteria, are able to
produce themselves D-mannitol from D-fructose. Seen the increasing use of D-
mannitol in the food industry and in medicine, the biotechnological production of
the hexitol by genetically modified bacteria growing on cheap carbon sources,
such as sugar cane melasses, is expected to significantly lower the costs of its
production. However, the organisms genetically modified so far still need to be
further optimized in order to make the biotechnological synthesis competitive
with the present chemical production of D-mannitol by reduction of fructose. The
synthesis of D-mannitol in plants normally not producing this hexitol and its use
as osmoprotectant and as protectant during droughts is very promising as well, but
also needs further optimization.
Utilization and Production of D-Mannitol by Bacteria 17
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Editor: Paolo Fubini 2013 Nova Science Publishers, Inc.
Chapter 2
CONCENTRATION OF MANNITOL
AND OTHER SOLUBLE CARBOHYDRATES
IN THE CRUSTOSE LICHEN RHIZOCARPON
GEOGRAPHICUM
Richard A. Armstrong
Department of Vision Sciences, Aston University, Birmingham, UK
ABSTRACT
In symbiotic lichens which have Trebouxia as the algal partner,
photosynthesis by the algae results in the production of the soluble
carbohydrate ribitol which is then transported to the fungus where it is
converted to arabitol and mannitol. Within the fungus, arabitol may act as a
short-term carbohydrate reserve while mannitol may have a more protective
function and be important in stress resistance. The concentrations of ribitol,
arabitol, and mannitol were measured, using gas chromatography, in the
central areolae and marginal hypothallus of the crustose lichen Rhizocarpon
geographicum (L.) DC. growing on slate rocks in north Wales, UK. The
concentrations of all three soluble carbohydrates were greater in the central
areolae than in the marginal prothallus. In addition, the ratio of mannitol in
the prothallus to that in the areolae was least in July. The concentration of an
individual carbohydrate in the prothallus was correlated primarily with the
INTRODUCTION
A lichen is an intimate association between an alga and a fungus and regarded
as one of the best examples of mutualism or symbiosis involving
microorganisms (Armstrong, 2011). The lichen thallus is highly structured but
in different species shows varying degrees of integration of the two symbionts. A
typical lichen is composed mainly of fungal hyphae with eucaryotic algal cells
embedded in an upper cortical layer. The algal partner carries out photosynthesis
and supplies the fungus with carbohydrate but there is little experimental evidence
to suggest that the fungus supplies nutrients directly to the alga (Smith and
Douglas, 1987). If there is a benefit to the alga, it may lie in the protection offered
by the thallus, thus extending the range of habitats that can be potentially
occupied by the alga.
There are three common lichen growth forms. In fruticose lichens, the thallus
is attached to the substratum at a single point and forms a complex branched
structure. By contrast, in foliose lichens, the thallus comprises a series of radially
arranged leaf-like marginal lobes, while crustose-type lichens comprise a thin
crust tightly attached to the surface of rock or tree bark. Endolithic lichens, in
which the lichen lives within the surface layers of the rock, are the most extreme
example of the crustose lifestyle (Armstrong and Bradwell, 2010). In endolithic
species, the upper cortex is absent while the algae and fungal hyphae are scattered
within the surface layers of the substratum. Most crustose species, however, have
a distinct upper cortex, an algal layer, and fungal medulla. In some species, the
margin of the thallus is diffuse and not sharply demarcated, but in others is
delimited by a non-lichenized fungal prothallus. One of the most widely
distributed species of the latter type is Rhizocarpon geographicum (L.) DC.
Mannitol in Rhizocarpon geographicum 23
STRUCTURE OF RHIZOCARPON
R. geographicum consists of a flat basal plate of black fungal tissue termed
the prothallus. Discreet areolae (Figure 2), containing the algal cells, develop in
association with this fungal prothallus but the prothallus extends beyond the outer
edge of the areolae to form a marginal ring normally 1 - 3 mm in width
(Armstrong and Bradwell, 2001).
Figure 1. Thalli of Rhizocarpon geographicum (L.) DC. growing on a slate rock surface in
north Wales, UK. The non-lichenized black prothallus is clearly visible especially at the
margin of the thalli (Bar = 1 cm).
24 Richard A. Armstrong
Figure 2. Structure of a typical areole of the lichen Rhizocarpon geographicum (L.) DC.
Note the colourless cuticle covering the cortex the algae occur in clusters in a distinct layer
below the cortex. Throughout the areolae, fungal hyphae grow vertically upwards from the
basal region.
Development of Rhizocarpon
prothallus. Free-living Trebouxia cells are often the first to colonize a bare
substratum and such cells can be detected on the surface before any lichen thallus
has become established (Mukhtar et al., 1994).
Growth of Rhizocarpon
areolae grew at similar rates as adjacent, intact thalli for a period of two months,
but growth then declined and the hypothalli fragmented and disappeared from the
surface within six months. Hence, if the prothallus is using exogenous supplies of
carbohydrate, concentrations on the surface appear to be insufficient to maintain
growth other than for short periods of time.
An alternative explanation for the results, however, is that the prothallus is
using carbohydrate reserves in the periphery for growth and once these were
exhausted, the fungal hyphae die. In a further experiment (Armstrong and Smith,
1987), individual thalli of R. geographicum were removed from rock surfaces,
each on a small piece of smooth slate. Complete removal of the central areolae
resulted in no measurable RaGR of the prothallus over a period of 18 months.
Removal of the areolae to within 1 and 2 mm of the prothallus, however,
significantly reduced growth in proportion to the width of the areolae present.
These results suggest that carbohydrate is supplied to the marginal prothallus by
the central areolae and that pioneer algal cells trapped in the prothallus do not
produce sufficient carbohydrate for growth processes.
Hence, the slow growth of R. geographicum could be a consequence of its
primitive growth form and a direct result of the problem of transferring
carbohydrate from the central areolae to the marginal prothallus. Nutrient transfer
may occur only within the immediate vicinity of each individual areola (Innes,
1985) and therefore mature areolae located at the margin may be the most
important contributor to the growth of the prothallus.
Carbohydrates in Rhizocarpon
Objectives
The study was carried out at a site in South Gwynedd, north Wales (Grid Ref.
SN 6196) in an area of Ordovician slate rock (Armstrong, 1974). Slate outcrops
varying in surface area from 2 30 m2 are a common feature of the hillsides in
this region. These surfaces possess a rich lichen flora characteristic of siliceous
rock in the north and west of the UK (James et al., 1977) and include communities
with a high proportion of crustose species (Armstrong, 1974). R. geographicum is
a member of several different communities at the site, especially on south-facing
rock surfaces (Armstrong, 1974; 2002).
Taxonomy of Rhizocarpon
Sampling of R. Geographicum
No deleterious effects of the transplant procedure were observed and the thalli
grew on the boards at similar rates to those in situ on south-facing rock surfaces
(Armstrong, 2002). Samples of areolae and prothallus were collected from
randomly chosen thalli on 1 November 1997, 1 February 1998, 1 April 1998, and
1 July 1998. Four replicate samples of the marginal prothallus and areolae (10
50 mg) were then obtained on each occasion by gently scraping the thalli with a
scalpel under a dissecting microscope. Samples of prothallus were taken as close
to the growing edge as possible and of areolae within 1 cm of the marginal
prothallus. Care was taken to avoid contaminating the prothallus samples with
developing areolae. Samples were stored in 80% ethanol in a refrigerator and
were analysed within one week of collection.
Measurement of Carbohydrates
CONCENTRATION OF CARBOHYDRATES
IN RHIZOCARPON
Figure 3. A typical gas chromatogram trace obtained from a sample of areolae collected on
1 July. Peak 4 is arabitol, peak 5 ribitol, and peak 9 mannitol.
VARIATION IN CARBOHYDRATES
BETWEEN SAMPLE TIMES
The levels of ribitol arabitol, and mannitol in areolae and prothallus at each
sample time is shown in Figure 4. Apart from some overall differences in
carbohydrate levels (F = 3.34, P < 0.05), the analysis of variance suggested little
variation in individual carbohydrates between sample days. The ratio of each
carbohydrate in the prothallus to that in the areolae at each sample time is shown
in Figure 5.
The ratios are considerably less than unity for each carbohydrate but vary
through the year. The ratio for mannitol is least in July suggesting particularly low
levels in the prothallus relative to the areolae at this sample time.
Figure 4. Levels of the carbohydrates ribitol, arabitol and mannitol (m per mg extracted
tissue) within the areolae and prothallus of Rhizocarpon geographicum. Analysis of
variance (ANOVA): Sample period F = 3.34 (P < 0.05); Soluble carbohydrate F = 15.72 (P
< 0.001); Sample period x Carbohydrate F = 2.25 (P > 0.05), Prothallus/areolae F = 116.67
(P< 0.001); Prothallus/Areolae x Sample period F = 2.84 (P > 0.05); Prothallus/areolae x
Carbohydrate F = 7.76 (P < 0.01).
34 Richard A. Armstrong
Figure 5. The ratios of ribitol, arabitol, and mannitol in the prothallus to areolae in
Rhizocarpon geographicum.
RH RA AH AA MH MA
RH - - - - - -
RA 0.02 - - - - -
AH 0.24 -0.15 - - - -
AA -0.13 0.87** 0.05 - - -
MH 0.72** 0.05 0.77** 0.01 - -
MA 0.61** 0.31 -0.04 0.28 0.28 -
the central areolae through hyphal connections (Armstrong and Smith, 1987;
1994), then carbohydrates are partitioned differently in the prothallus and largely
independently from the areolae.
Figure 6. The possible interrelationships between ribitol, arabitol, and mannitol in the
marginal prothallus and central areolae in Rhizocarpon geographicum (R = Ribitol, A =
arabitol, M = Mannitol).
Mannitol in Rhizocarpon geographicum 37
CONCLUSION
The crustose lichen R. geographicum has an unusual thallus structure
consisting of discrete granules (areolae) containing the algal component growing
in association with a non-lichenised fungal prothallus that extends beyond the
areolae to form a marginal ring. R. geographicum grows at substantially lower
rates than foliose species especially in arctic and alpine environments (Armstrong
and Bradwell, 2010). The supply of carbohydrate to the marginal prothallus may
be a limiting factor and although there is some evidence that the prothallus can
use exogenous sources of carbohydrate (Armstrong and Smith, 1996),
concentrations are unlikely to be sufficient to support growth. Poor rates of
translocation from the areolae to the prothallus may explain the slow growth of
this species.
There has been speculation that R. geographicum may represent one of the
most primitive types of lichen. The existence of a marginal, non-lichenised
prothallus appears to be important to the survival of this species. If the existing
prothallus is removed, a new prothallus is developed within a year, regenerating
first by retreat of the marginal areolae and then by new prothallus growth
(Armstrong and Smith, 1987). It is possible that this growth form is actually an
adaptation to ensure slow growth and consequently, a lower demand for nutrients
from the environment. As a result, a greater concentration of the products of
photosynthesis, such as mannitol, can be allocated to stress resistance rather than
growth thus enabling Rhizocarpon to colonise more extreme environments than
most foliose species.
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38 Richard A. Armstrong
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III. The importance of the rewetting phase. New Phytologist 1976; 77: 115-
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Editor: Paolo Fubini 2013 Nova Science Publishers, Inc.
Chapter 3
ABSTRACT
Mannitol, a white, crystalline alcohol, is derived from sugar by
reduction. The pathway to obtain mannitol from natural products is through
hydrogenation of fructose, which is formed from either starch or sugar. This
substance is present in a wide variety of natural products, and in almost all
plants. It is used as an osmotic diuretic agent and a weak renal vasodilator.
Aqueous solutions of mannitol are mildly acidic and sometimes such
solutions are used to decrease the pH. Mannitol is clinically used in
osmotherapy to temporarily reduce acute intracranial pressure while waiting
for the definitive treatment. It is also used to treat patients with oliguric renal
failure. Consequently, mannitol increases water and Na+ excretion, thereby
decreasing extracellular fluid volume. It can also be used as a facilitating
agent for transporting drug agents directly into the brain. This paper reviews
the possible mechanisms of mannitol and its metabolite that are involved in
common clinical disorders. Moreover, the analytical techniques and
42 D. Caldern Guzmn, G. Barragn Meja, H. Jurez Olgun et al.
INTRODUCTION
Mannitol is a white, crystalline sugar alcohol. The chemical formula is
C6H8(OH)6 (Figure 1) (table 1). It is used as an osmotic diuretic agent and a weak
renal vasodilator. It was originally isolated from the secretions of flowering ash
and was called manna for its resemblance to the Biblical food [1].
Figure 1. Mannitol.
FRUCTOSE MANNITOL
FRUCTOSE-6-PHOSPHATE
GLUCOSE GLUCOSE-6-PHOSPHATE
Figure 3. Sucrose.
Figure 4. Fructose.
Mannitol is one of the most abundant energy and carbon storage molecules in
nature, produced by a plethora of organisms, including bacteria, yeasts, fungi,
algae and many plants [3]. D-Mannitol is the predominant carbon compound in
conidiospores of the filamentous fungus, Aspergillus niger, and constitutes 0 to
15% of the dry weight. Mannitol 1-phosphate dehydrogenase and mannitol 1-
phosphate phosphatase form the major metabolic pathway for mannitol
biosynthesis in A. niger. Under stress conditions, mannitol appears to be essential
for the protection of A. niger spores against cell damage [4].
Fermentation by microorganisms is a possible alternative to traditional
industrial synthesis, producing much higher yields of mannitol, with minimal to
no side products. Fructose has been discovered in mannitol metabolic pathway
(the mannitol cycle in fungi) in a type of red algae (Caloglossa leprieurii), and it
is highly possible that other microorganisms employ such pathways [5]. A class of
lactic acid bacteria, labeled heterofermentative because of their multiple
fermentation pathways, converts either three fructose molecules, or two fructose
44 D. Caldern Guzmn, G. Barragn Meja, H. Jurez Olgun et al.
plus a glucose (figure 5) molecule to two mannitol molecules and one molecule of
lactic acid (figure 6), acetic acid, and carbon dioxide each.
Figure 5. Glucose.
GLYCOGEN
NADH
MANNITOL - 1 - P
NADH+
FRUCTOSE MANNITOL
Figure 8. Ethanol.
Figure 9. Polyvinylpyrrolidone.
For the past 90 years, mannitol has come to be the most widely used
hyperosmolar solution to treat elevated intracranial pressure. Mannitol is
clinically used in osmotherapy in head traumas as a temporary management to
reduce acutely raised intracranial pressure until more definitive treatment can be
applied [8]. It is also used to treat patients with oliguric renal failure. The
administration is intravenous and the filtration is by the glomeruli of the kidney.
46 D. Caldern Guzmn, G. Barragn Meja, H. Jurez Olgun et al.
However, its reabsorption in the renal tubules is not possible. This results in
decreased water and Na+ reabsorption due to the osmotic effect it exerts.
Consequently, mannitol increases water and Na+ excretion, thereby decreasing
extracellular fluid volume.
In a recent study of 140 patients hospitalized in a Mexican General Hospital
due to different clinical disorders, nearly 8% of these patients were treated with
mannitol (bottle 250ml in solution 20%), however, ambulatory emergency,
pregnant and newborn patients were not included in the study (table 2),
Mannitol can also be used as a facilitating agent for the transportation of
pharmaceuticals [9], and did not appear to alter the effectiveness of the drugs [10].
The arteries of the bloodbrain barrier (BBB) are much more selective than
normal arteries. Normally, molecules can diffuse into tissues through gaps
between the endothelial cells of the blood vessels. However, what enters the brain
must be much more rigorously controlled. The endothelial cells of the bloodbrain
barrier are connected by tight junctions, and simple diffusion through them is
impossible. For this, the need of active transport that requires energy and that can
only transport molecules whose receptor signals exist in the arterial endothelial
cells.
Mannitol is capable of opening this barrier by temporarily shrinking the
endothelial cells and simultaneously stretches the tight junctions between them
[11]. The combination of sodium and mannitol contributes to establish a higher
osmotic gradient across BBB and, furthermore, the progressive accumulation of
mannitol in the ischemic brain tissue counteracts its therapeutic efficacy on
cerebral edema [12].
Mannitol is commonly used in the circuit prime of a heart lung machine
during cardiopulmonary bypass. The presence of mannitol preserves renal
function during the times of low blood flow and pressure, while the patient is on
bypass. The solution prevents the swelling of endothelial cells in the kidney,
which may have otherwise reduced blood flow to this area and provoke cell
damage. Mannitol is contraindicated in patients with anuria and congestive heart
failure [13] (Table 3).
To clarify the mechanism underlying hyperosmotic-induced renal fibrosis in
renal distal tubule cells, Chiang and cols. [27] showed that hyperosmolarity
significantly enhances the susceptibility to exogenous transforming growth factor
(TGF)-beta1, as mannitol (27.5 mM) significantly enhanced the TGF-beta1-
induced increase in fibronectin levels.
Table 2. Clinical disorders in hospitalized Mexican patients (Hospital of 300 beds)
Men Women
Clinical Diseases Age Clinical Diseases age
Maxillary abscess 66 Anemia 66
Perianal abscess 35 Cerebral aneurism 79
Right suprarenal adenoma 44 Bilateral arthralgia 51
Cervical adenopathy 38 Right leg arthrodesis 73
Appendicitis 39 Right hand arthroplasty 61
Appendicitis 33 Total arthroplasty of right hip 79
Acute appendicitis 48 Total arthroplasty of left shoulder 75
Dehydration of tissue in surgical cleaning 25 Arthroscopy of left knee + medial disk meniscus 17
Renal cancer 55 Asthma 50
Hypovolemic shock 38 Bilateral breast biopsy 60
Septic shock 85 Bronchiolitis 1
Percutaneous surgery of left kidney 51 Colon cancer 69
Radical cystectomy + neobladder 46 Breast cancer and bone metastasis 63
Acute cholecistitis 55 Cancer of the bladder 46
Renauteral cholic 49 Bilateral cerebral carotid 51
Hypertensive crisis 56 Lynphangitis in left leg 56
Ventriculoperitoneal shunt 43 Cerebritis 40
Lumbosacral decompression 73 Cholecystectomy 36
Rotators and tendon clamp tear manguito 46 Nissen cholecystectomy 26
Respiratory distress 1 Cholecystitis 83
Valvular double lesion 52 Alitiasic cholecystitis by steroids 31
Abdominal pain 31 Colon-lumbar in study 19
Abdominal pain in study 58 Coxartrosis 62
Lumbar pain 47 Convulsive crisis 65
Table 2. (Continued)
Men Women
Clinical Diseases Age Clinical Diseases age
Chest pain 54 Epileptic crisis of origin to determine 81
Chest pain in study 44 Septal deformity of hypertrophy 41
Encephalitis mastitis 65 Dermo lipopexia 55
Encephalitis mastitis 65 Abdominal pain 59
Encephalopathy 74 Abdominal pain 38
Metabolic encephalopathy 61 Abdominal pain 15
Diverticular sickness 79 Abdominal pain 39
Vascular cerebral diseases 60 Abdominal pain 19
Ischemic CVA 82 Abdominal pain 72
Fever 20 Abdominal wall seroma drainage 47
Fever 52 Epilepsy 26
Lumbar FX, Left knee FX 29 Pyloric stenosis 51
Decompensated diabetic gastroenteritis 22 Ischemic CVA 80
Infectious gastroenteritis 1 Cerebral-vascular accident (CVA) 74
Gepi 55 Fever + diarrhea 21
Laparoscopic heminefrectomy 56 Right hinge fracture 61
Hemorrhoids and hemicolectomy 61 Anterior cervical fusion 48
Acute myocardial infarction 57 Anterior cervical fusion 60
Airway infections 67 Gastroenteritis 17
Influenza H1N1 1 Glioma 31
Congestive cardiac insufficiency 66 Hemorrhoids 24
Respiratory insufficiency 85 Abdominal wall embedded hernia 62
Melan vs. Angioma lesion of small intestine 65 Total hysterectomy 49
Lymphoma 26 Regional ileitis 61
Men Women
Clinical Diseases Age Clinical Diseases age
Left renoureteral lithiasis 45 Respiratory insufficiency 53
Ureteral lithiasis 26 Medullar liberation of C4C5 and C5C6 31
Community acquired pneumonia 3 Ganglio-centinela mastectomy 63
Right pneumonia 53 Ganglio-centinela mastectomy 44
Nasal obstruction 41 Pneumonia 67
Intestinal 75 Pneumonia 84
Intestinal occlusion 75 Basal pneumonia 78
Peritonitis 80 Pneumonitis 32
Plastia of right ankle Achilles tendon 72 Intestinal occlusion 59
Thoracic aorta postoperative dissection 63 Inguinal plasty 62
Open radical prostate 68 Grade 11 polycontuntion strain 25
Molar fracture reduction 53 Open radical prostate 60
Resection of right renal cyst and laparoscopy 73 Sarcoma of the radius 13
Resection of mandibular tumor 24 Leukemoide reaction 39
Prostate RTU 74 Reconstruction of bile ducts 28
Vesicle tumor RTU 67 Gastroesophagic reflux 27
Digestive tube bleeding 63 Ritirectomy in study 59
Lower digestive tube bleeding 48 Left knee meniscal rupture 35
Kaposi sarcoma. Neurosiphilis 47 Lower digestive tube bleeding 72
Gaseous gangrene sepsis. Diabetic foot 42 Secon reconstruction TX 42
Syncope in study 19 Painful abdominal syndrome 63
Abdominal tumor 32 Fever syndrome 33
Mediastinal tumors in study 56 Thyroidectomy 54
Varicocele 66 Thyroidectomy 39
Table 3. Recent studies of mannitol used on clinical disorders
These cells were also resistant to other apoptotic stimuli that were
activated via the intrinsic cell death pathway, while remaining sensitive to
extrinsic apoptotic stimuli. Interestingly, these osmotic stress resistant cells
showed no increase in anti-apoptotic proteins, and released cytochrome C
from its mitochondria following exposure to intrinsic apoptotic stimuli [45].
Mannitol is also the basis of Bronchitol which was developed by the
Australian pharmaceutical company (Pharmaxis) as a treatment for cystic
fibrosis and bronchiectasis [46]. Mannitol is orally inhaled as a dry powder
through what is known as an osmohaler and osmotically draws water into the
lungs to thin the thick, sticky mucus characteristic of cystic fibrosis. This is
intended to make it easier for the sufferer to cough the mucus out during
physiotherapy. The critical characteristic of mannitol is its particle size
distribution. It can also be used to temporarily encapsulate a sharp object while
passing through the venous system [47]. Because mannitol dissolves readily in
blood, it can be used in combination with other drugs (table 5).
An intracarotid injection of high molarity mannitol (1.41.6M) causes the
contents of the artery to be hyperosmotic to the cell. Water leaves the cell and
enters the artery in order to recreate an osmotic equilibrium. This loss of water
causes the cells to shrivel and shrink, stretching the tight junctions between the
cells [61]. The newly formed gap reaches its peak within five minutes after
mannitol injection, and stays widely open for thirty minutes. During this time
span, drugs injected into the artery can easily diffuse through the gaps between
cells directly into the brain [62].
Table 5. (Continued)
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In: Mannitol ISBN: 978-1-62808-762-8
Editor: Paolo Fubini 2013 Nova Science Publishers, Inc.
Chapter 4
ABSTRACT
Nowadays thermal energy storage (TES) systems are proposed as
one of the most powerful technologies to be charged with heat (or cold)
and hold energy over time by shifting demand over time to reduce peak
loads and facilitating the greater use of renewable energy by storing the
energy produced so it can coincide with demand. TES systems are able to
store energy as sensible heat leading with temperature increment of the
storage medium, as latent heat storing energy using the latent heat
produced when a phase change state occur using phase change materials
PCM, and as chemical reaction energy storing energy using a
exothermic/endothermic reversible reaction by thermochemical materials
(TCM). Solar cooling and air-conditioning is a technology that allows
coincidence of solar gains with cooling loads reducing peak loads created
by air-conditioning. TES systems can be coupled between absorption
chillers and solar collectors in order to use the energy stored when the
there is a peak load or the system is practically discharged. In addition,
phase change materials PCM candidates must fulfill several conditions
to be used as storage materials: melting point of PCM must be closed to
selected work temperature range, high latent heat and high specific heat,
elevate thermal conductivity (solid and liquid state) to support charging
and discharging processes inside the storage system. Additionally, the
change volume during phase change transformation must be minimum, as
well as the pressure vapor, allowing the use of conventional containers.
Moreover, it must melt congruently with minimum subcooling and it
must be chemically stable. D-mannitol has a phase change temperature at
167 C and a phase change enthalpy is around 316 kJkg-1. These
thermophysical properties turn d-mannitol as a perfect candidate to be
used as PCM and it was studied with this purpose. D-mannitol was
characterized performing differential scanning calorimetry under dynamic
mode using a 0.5 Kmin-1 heating rate between 25 C and 200 C. This
substance was cycled several times and results shows 3 different thermal
behavior: The first one was a single peak at 167 C, the second has
double peak at 156 C and 167 C, and the third thermal behavior is a
single peak at 157 C. Accordingly, there is a polymorphic transformation
which was studied with FT-IR and two different phases were identified:
-phase and -phase. Then, a temperature range was established to work
with this substance as PCM between 135 C and 175 C. This working
range includes the phase change transformation of the two phases under
analysis ( and ). To test the d-mannitol thermal behaviour at pilot plant
scale, 150 kg of d-mannitol were introduced in a storage tank which was
designed as shell-and-tubes heat exchanger. Results show that applying
different cooling conditions produces d-mannitol polymorphic changes.
Moreover, it has been shown that the working range (between 135C and
175C) is adequate for pilot plant experiments.
1. INTRODUCTION
Energy efficiency and generation are key points in the development of
countries and these factors have direct impact with their own inner production
which is priority important as well considering both emerging economies and
the countries of the OECD (Organization for Economic and Co-operation and
Development). Currently, energy demand has increased dramatically; making
OECD countries to invest a part of their budgets for research and development
to create new alternatives to the adjustment of energy demand and generation.
Use of Mannitol in Thermal Energy Storage Applications 65
Due to the current high price of oil and the increase of CO2 emissions,
unrivalled alternative is renewable energy, which do not involve any
environmental impact when they are implement to generate electricity or
energy.
Leading renewable energy is consolidated solar energy to heat domestic
hot water (DHW) via thermal panels or allows the generation of electricity by
installing photovoltaic panels locally (in a house/building) or in large solar
plants generating large amount of energy (see Figure 1). The foremost
advantage of solar energy is its low cost (it is the cheapest renewable energy)
and widespread availability.
However, solar energy presents a drawback which is not easily solvable:
this renewable energy is time depending and in some applications this fact
produces a mismatch between energy demand and energy supply.
A possible solution to this drawback raised above is the installation of
thermal energy storage (TES) systems. These systems are able to store the
excess energy produced by charging different materials and discharging them
during peaks of energy demand or when it is necessary.
Thermal energy storage (TES) will play a key role in the successful
application of thermal heating and cooling technologies. Meanwhile, the
International Energy Agency (IEA) suggests energy storage as a possible
66 Luisa F. Cabeza, Camila Barreneche, Antoni Gil et al.
solution for the energy problems described, often focusing on materials and
systems for building and industrial applications.
Thermal energy can be stored following three processes: as sensible heat,
as latent heat or as thermochemical energy. Furthermore, TES systems can be
charged with heat (or cold) and hold energy over time by shifting demand and
reducing the peak loads. These facts will facilitate the greater use of renewable
energy.
Abhat et al. [1] proposed in 1983 a classification of materials to be used as
thermal energy storage materials which is shown in Figure 2.
Figure 2. Classification of substances used as PCM for thermal energy storage [1].
4.18 kJkg-1K-1. Sensible heat storage has two main advantages: it is cheap
and without the risks derived from the use of toxic materials.
Heat capacity is given from content of material used, volume or mass.
Sensible heat is often used with solids like stone or brick, or liquids as water
(high specific heat as mentioned).
Temperature (C)
Stored heat
Figure 3. Thermal energy storage profile vs. temperature when heat is stored as
sensible heat.
Temperature (C)
Stored heat
Figure 4. Thermal energy storage profile vs. temperature when heat is stored as latent.
CHARGE
STORE
DISCHARGE
TES systems for solar cooling work between 140 C and 200 C and
materials to be used as PCM for this application must own a phase change
temperature close to this temperature range. Furthermore, another important
requirement is the storage capacity: the PCM selected has to perform the
highest heat storage capacity through the highest latent heat of fusion.
Materials with this requirement found in the literature are listed in Table 1
[5-7].
Table 1 shows that the material with highest phase change enthalpy and
with a suitable melting temperature for this application is d-mannitol. Thereby,
it is a potential candidate to be used as PCM for thermal energy storage in
solar cooling applications.
This material present several polymorphic phases and thermophysical
properties of these phases are slightly different. This information is listed in
Use of Mannitol in Thermal Energy Storage Applications 71
Table 2. All phases have the temperature of phase change within the
application temperature range.
2. POLYMORPHISM AT LABORATORY
Thermophysical properties behavior of PCM must be studied previously
of the implementation of one material to be used in one application. In fact,
this is a key point and has to be accomplished before the putting this material
into practice.
One of the most powerful technics to characterize the thermophysical
response of materials/substances is differential scanning calorimetry (DSC)
[2], and it is widely used to perform this kind of characterization of PCM at
laboratory scale. This technique consists on heating up one sample and one
blank (which is a completely empty crucible) in order to remove the signal
from the crucible by itself. The obtained signal will be the thermophysical
behavior of the sample under study. Both crucibles must be heated under an
inert atmosphere (normally 50-80 ml/min N2 flow) by using a constant and
controlled heating rate. For PCM analysis slow heating rate is recommended
(~0.5 C/min) [15].
72 Luisa F. Cabeza, Camila Barreneche, Antoni Gil et al.
a
Use of Mannitol in Thermal Energy Storage Applications 73
Figure 7. Three different thermal behaviors obtained with DSC analysis of d-mannitol
during melting process.
74 Luisa F. Cabeza, Camila Barreneche, Antoni Gil et al.
Figure 8. On the left FTIR bands of d-mannitol with no cycles of DSC; on the right
FTIR bands of d-mannitol cycled 5 times by DSC.
Use of Mannitol in Thermal Energy Storage Applications 75
double peak during the fourth cycle. Finally, the -phase is formed again
33.3% of the time because this phase is more stable than the other one and its
formation is favored.
1 Peak 2 Peaks
Tm, (C) Tm, (C) Tm, (C) Tm, (C)
0% 100% 0%
1st Cycle
--- 168.5 1 --- ---
18% 18% 64%
2nd Cycle
157 0 166 0 152 3 167 2
50% 0% 50%
3rd Cycle
156 0 --- 153 2 167 1
0% 0% 100%
4th Cycle
--- --- 152 2 164 0
0% 33.33% 66.66%
5th Cycle
--- 164 0 149 1 164 0
during the charging process and an air heat exchanger of 20 kWe to cool down
the HTF and simulate the real energy consumption.
Figure 9. High temperature pilot plant to test PCM for solar cooling applications.
Figure 10. TES unit constructed: a) U shape tubes bundle; b) position of the
temperature sensors.
210
200
190
Temperature [C]
180
170
160
150
140
PCM temperature
130
HTF inlet temperature
120
0:00 0:28 0:57 1:26 1:55
Time [h:min]
Figure 11. D-mannitol melting process in the pilot plant during Experiment A.
210
200
190
Temperature [C]
180
170
160
150
140
PCM temperature
130
HTF inlet temperature
120
0:00 0:28 0:57 1:26 1:55
Time [h:min]
Figure 12. D-mannitol melting process in the pilot plant during Experiment B.
80 Luisa F. Cabeza, Camila Barreneche, Antoni Gil et al.
Figure 13. XRD analysis of d-mannitol cycled in the TES unit (-phase - sample from
Experiment A).
Figure 14. XRD analysis of d-mannitol cycled with TES unit (-phase - sample from
Experiment B.
Use of Mannitol in Thermal Energy Storage Applications 81
Moreover, results obtained for experiment B are plotted in Figure 12. The
temperature range of phase change obtained was between 150 C and 162 C.
This temperature range corresponds to a-phase or d-phase as Table 2 listed.
In order to discern which phase is obtained after each experiment
performed, two samples were analyzed X-ray diffraction (XRD). Each sample
was gathered after experiment A and experiment B, respectively.
Diffractograms of these two samples analyzed are presented in Figure 13 and
Figure 14.
These results were compared with patterns of XPERT HIGHSCORE
PLUS (Panalyatical) database. The sample gathered after experiment A
corresponds to -phase (Figure 13) and the sample gathered after experiment
B is linked to -phase (Figure 14).
Samples gathered after each experiment (A and B) were also analyzed
with DSC in order to compare the results obtained at pilot plant scale with
those obtained at laboratory scale considering exactly the same crystalline
phase.
The DSC results and pilot plant results are overlapped in Figure 15 where
curves of melting enthalpy vs. temperature are plotted. The melting range
obtained by DSC is shorter than the high temperature pilot plant. This fact is
due to the amount of material and the dimension of the equipment used.
Figure 15. Comparison of results obtained in the TES unit and obtained by DSC.
82 Luisa F. Cabeza, Camila Barreneche, Antoni Gil et al.
CONCLUSION
Thermal energy storage (TES) systems are proposed as one of the most
powerful technologies to facilitate a greater use of renewable energy by
storing the energy produced so it can coincide with demand. TES systems are
able to store energy as latent heat storing energy using the latent heat produced
when a phase change state occur using phase change materials (PCM). PCM
Use of Mannitol in Thermal Energy Storage Applications 83
REFERENCES
[1] Abhat. Low temperature latent heat thermal energy storage: heat storage
materials. Solar Energy 30,313332: 1983.
[2] Mehling H, Cabeza LF. Heat and cold storage with PCM. Springer-
Verlag; 2008. ISBN-13: 9783540685562.
[3] L.F. Cabeza, A. Castell, C. Barreneche, A. de Gracia, A.I. Fernndez.
Materials used as PCM in thermal energy storage in buildings: A review.
Renewable and Sustainable Energy Reviews 15, 1675-1695: 2011.
84 Luisa F. Cabeza, Camila Barreneche, Antoni Gil et al.
Chapter 5
ABSTRACT
As a kind of readily available carbohydrate, D-mannitol has been
widely used in the synthesis of chiral phosphorous ligands. This chapter
reviews the design and synthesis of various mono- and diphosphorous
ligands based on D-mannitol backbone. Also, the successful applications
of these ligands in asymmetric catalysis, such as enantioselective
hydrogenation and enantioselective conjugate addition, are summarized.
1. INTRODUCTION
In the past twenty years, the design and synthesis of highly efficient chiral
ligands for asymmetric catalysis had made considerable achievements. [1]
Among the development of suitable ligands for specific transition metal
catalyzed enantioselective reactions such as asymmetric hydrogenations, [2, 3]
much effort has been made to construct chiral backbones by utilizing the
resources existing in natural chiral pools. Carbohydrates have been widely
used for enlarging the amounts of potential superior ligands because they are
easily accessible, commercially available, and often of high enantiomeric
purity. [4] Most importantly, their highly functionalized property makes it
straightforward to design novel ligands bearing specific structures through
modifications.
D-Mannitol is commonly obtained via the hydrogenation of fructose,
which is formed from either starch or sucrose. As an important carbohydrate, it
has been extensively used in organic synthesis with several applications, such
as ligands, [5] polymers and the preparation of small chiral building blocks,
[6] and key intermediates in total synthesis. [7] Versatile ligands using D-
mannitol as a backbone have been synthesized, and successfully applied in
asymmetric catalytic reactions. The role of functional group derived from
mannitol in the ligands structure could be divided into two types: (1) as the
main chiral backbone; and (2) as an additional group. Genarally, the mannitol
fragment could provide fair degree of rigidity and flexibility to enhance the
enantioselectivity of the whole ligand. In some case, the mannitol moiety may
also act as hemilabile coordination group. [8, 9]
groups at C1 and C3 positions and other two hydroxyl groups remained with
furnishingthe chiral 1,4-diol.
Using the tetrol 9 as starting material, the chiral 1,4-diols like 10a with
specific spatial configuration could be obtained (Scheme 6). [19, 20]
Depending on the specific reaction conditions, two diastereomeric
bis(epoxides) (11a and 11b) were selectively prepared, respectively. The
intramolecular SN2 reaction of the tosylate reversed the absolute configuration
of C2 chiral center from R to S. The bis(epoxides) were then reduced with
LiAlH4 in THF to the corresponding diols (10a and 10b). The epoxides could
also serve as electrophiles for copper-mediated ring opening to afford 12a and
12b. This synthetic strategy allows flexibility in steric tuning of the final
phospholane ligands.
The following work carried out by Wangs group [30] refer to the
application of these phosphite ligands in the asymmetric Cu-catalyzed
enantioselective conjugate addition of diethylzinc to cyclic enones.
The value of the enantiomeric excess is controlled by the cooperative
effect between the mannitol backbone (C-3 and C-4) and the binaphthol
phosphite moiety.
When ligand 22a was used, up to 71% ee was obtained for addition
reaction of cyclopentenone under the optimized conditions.
Chiral Phosphorous Ligands Derived from D-Mannitol 97
Table 3. (Continued)
comparable to the results achieved with Rh-DuPhos catalysts and higher than
that of with an Rh-RoPhos catalyst.
Zhangs group also synthesized a ferrocene-bridged polysubstituted
phospholane ligand 39 with the same phosphorous cycle as ligands 35a and
36a. The Rh-complex with this ligand showed high enantioselectivity and
reactivity in the asymmetric hydrogenation of -dehydroamino acid. Up to
over 99% ee and 10 000 TON were achieved with this catalytic system.
Riegers group developed a flexible approach to generate monodentate
phosphoramidites differing in the absolute configuration of carbon atoms and
the steric demand of the substituents. Refluxing the isopropylidene ketal
protected diols with hexamethyltriaminophosphane (HMTAP) in toluene could
afford phosphoramidites 43-47 (Scheme 21). [19, 20] The synthesized Rh
complexes bearing two monodentate phosphoramidite ligands (43-47) were
tested in the asymmetric hydrogenation of -(acetamido)cinnamic acid. The
ligands 43a-47a and 43b only produced a small excess of N-acetyl-(R)-
phenylalanine with lower ee values ranging from 3 to 36% Ligand 47b
remarkably gave an 89% ee for (S)-enantiomer, which may be due to front
orientation effect of the phenyl fragments.
Almost at the same time of Brner and Zhangs work, [36] RajanBabus
group [37, 38] also discovered a versatile route for the synthesis of various
functionalized mono and bisphospholane ligands. The RoPhos-like ligands
35b was also efficiently prepared with different absolute configuration. The
method also allowed the approach toward biphosphinite 48b and biphosphine
49b from the diol 10b. Ligand 50b bearing a potentially hemilabile tert-
butylthioether functionality at the -position was also successfully prepared
with the same synthetic strategy (Scheme 22).
These ligands were evaluated by palladium(0)-catalyzed addition of
dimethyl malonate to (E)-1,3-diphenylprop-2-enyl acetate (Table 6).
It is illustrated that the sense of induction is controlled by the chirality of
the C2 and C5 atoms in all cases. Excellent results could be obtained by using
phospholanes 33b and 35b. The appropriate phosphine 49b gave higher ee
than its diastereotopic structure 49a. The ligand 50 containing chelating
phosphorus and sulfur atoms did not lead to decent enantioselectivity.
Brners group introduced a couple of hydroxylmethyl groups into the
phospholane cycles for enhancing the solubility of the corresponding Rh
complex 52 (Rh(BASPHOS)) in water [15]. Starting from the diol 6, the chiral
Chiral Phosphorous Ligands Derived from D-Mannitol 107
ligand 51 was successfully prepared in good yield (Scheme 23). The Rh(I)
complex 52 was applied in the asymmetric hydrogenation of the water-soluble
2-acetamido acrylic acid and its ester with water as solvent. Up to 99.6% ee
was obtained for the hydrogenation of the acid [15].
RajanBabus group found that the hydroxyl RoPhoses 37a, 38a and 37b
could be successful used in the asymmetric hydrogenation of methyl
acetamidoacrylate in aqueous media. They could be recycled with no loss of
activity or selectivity in 1:1 methanol/water.
The authors also synthesized the free hydroxyl ligand 53, in which the
method for protection and de-protection of hydroxyls was different from that
described by Brners group (Scheme 24) [39]. Ligand 53 was found giving
the best results in hydrogenation in neat water. The enantioselectivity and
recyclability is outstanding with the catalyst derived from this ligand. In four
sequential runs, ~99% ee and >90% isolated yield (100% conversion) were
obtained for the hydrogenation of methyl acetamidoacrylate in neat water.
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INDEX
antisense, 9
# anuria, 46
aorta, 49
21st century, 59
apoptosis, 51, 54, 60
apoptotic mechanisms, 59
A appendicitis, 47
areolae, viii, 21, 23, 24, 25, 26, 27, 28, 29,
ABC transporters, vii, 1, 3 30, 31, 32, 33, 34, 35, 36, 37, 38
acetic acid, 44 arteries, 46
acid, 3, 4, 15, 25, 44, 55, 62, 71, 90, 92, 94, artery, 53
95, 98, 104, 105, 107 arthralgia, 47
acidic, viii, 41, 42, 90 arthrodesis, 47
acrylic acid, 107 arthroplasty, 47
active oxygen, 2 assessment, 50
active transport, 46 asthma, 50, 54
acute lung injury, 52, 59 atmosphere, 71
adaptation, 37 atoms, 106
additives, 45 ATP, 3, 4, 5
adenoma, 47 attachment, 12
adenopathy, 47
adjustment, 64
B
adults, 50, 57
age, 23, 47, 48, 49, 51
Bacillus subtilis, 8
alanine, 12, 14, 53
bacteria, vii, 1, 3, 4, 5, 6, 7, 14, 15, 16, 43
Alaska, 26, 39
bacterial cells, vii, 2, 3
algae, vii, viii, 1, 2, 12, 21, 22, 24, 28, 43
bacterium, 12, 13
alimentation, 1
BBB, 46
amino, 4, 6, 12, 13, 30, 60
behaviors, 72, 73
amino acid(s), 4, 6, 12, 13, 60
Bilateral, 47
ANOVA, 33
bile, 49, 55
antioxidant, 51, 52, 54, 59
114 Index
F
H
fermentation, 43
fibroblasts, 52, 58 habitat, 12, 22, 26
fibrosis, 46, 50, 51, 53, 57 half-life, 51
filtration, 45 harvesting, 65
flexibility, 88, 92, 94, 109 head injury, 58
flora, 29 head trauma, 45, 52
fluid, viii, 41, 46, 69, 76, 82 heat capacity, 66
food, 14, 16, 42 heat transfer, 66, 69, 76, 82, 84
food industry, 16 heating rate, ix, 64, 71, 83
food products, 14 hemodialysis, 56
formation, 12, 25, 52, 75 hemoglobin, 52, 58
formula, 42 hepatic failure, 57
fragments, 105 hernia, 48
France, 1 hexane, 97
freedom, 100 histidine, 11, 12
fructose, vii, viii, 1, 2, 3, 4, 5, 6, 14, 15, 16, homogeneity, 8
31, 41, 42, 43, 88 human, 51, 52, 58, 59, 60, 62
FTIR, 74, 75 human brain, 60
functionalization, 109 human skin, 58
fungi, vii, 1, 3, 32, 39, 43, 44 hydrogen, 15, 52
fungus, viii, 21, 22, 27, 28, 43 hydrogen gas, 15
fusion, 13, 25, 48, 70, 76 hydrogenation, viii, x, 14, 15, 41, 42, 87, 88,
90, 93, 94, 95, 98, 99, 100, 102, 103,
104, 105, 107, 108, 109
G hydrolysis, 90
hydroquinone, 82
gangrene, 49
hydroxyl, 52, 88, 92, 98, 103, 104, 108
gastroenteritis, 48
hydroxyl groups, 89, 92, 98, 104
genes, vii, 2, 3, 4, 5, 8, 9, 12, 14, 15
hypertonic saline, 56, 57, 59
genus, 25, 28 hypertrophy, 48
geometry, 98 hysterectomy, 48
glucose, viii, 2, 8, 11, 12, 13, 15, 16, 31, 44,
52, 58
glutathione, 52, 56 I
glycerol, 3, 32
glycine, 53 ID, 17
Index 117
plants, vii, viii, 1, 2, 16, 39, 40, 41, 43, 44, reconstruction, 49
65 recovery, 51, 60
platelets, 59 regenerate, 15
platform, 102 regeneration, 2, 15, 25
platinum, 60 renal dysfunction, 52
pneumonia, 49 renal failure, viii, 41, 45
polar, 34, 40 renewable energy, ix, 63, 65, 66, 82
pollution, 58 repression, vii, 2, 3, 13
polymer(s), 56, 88 repressor, 8, 9, 14
polymerase, 9 requirements, 69, 104
polymorphism, 85 researchers, 67
pools, 88 reserves, 27
population, 29, 51 residues, 10, 51
positive correlation, 35 resins, 30
power generation, 84 resistance, viii, 21, 34, 37, 59
preparation, iv, 88, 95 resources, 88
preservation, 60 response, 9, 10, 14, 54, 59, 71
prevention, 60 RH, 35
promoter, 9, 10, 11, 12 rhodium, 95, 100
proteasome, 51 rights, iv
protection, 16, 22, 27, 43, 54, 108 rings, 92, 101
proteins, 4, 5, 7, 8, 9, 10, 11, 12, 27, 53 risks, 67
prothallus, viii, 21, 22, 23, 24, 25, 26, 27, RNA, 9
28, 29, 30, 31, 32, 33, 34, 35, 36, 37 roots, 3, 61
purity, 88 routes, 3, 16
PVP, 45 rowing, 23
runoff, 26
Q
S
quantification, 30
savings, 69
secretion, 55
R selectivity, 108
sensitivity, 54, 60
radicals, 52
sensors, 77, 78
radiotherapy, 50, 57
sepsis, 49
radius, 49
serum, 61, 62
rainfall, 34
services, iv
RE, 38 shape, 24, 77, 78
reaction time, 93 shock, 47
reactions, 88, 98, 108
side effects, vii
reactive oxygen, 58, 59
signals, 46
reactivity, 105
signs, 51
receptors, 51, 57
skeletal muscle, 59
recommendations, iv
skeleton, 98
120 Index
skin, 52 surplus, 69
small intestine, 48 survival, 37
sodium, 46, 54, 60 susceptibility, 46, 51, 57
software, 30 swelling, 46, 50, 51, 58
solar collectors, ix, 64, 69, 70 Switzerland, 26, 40
solid state, 69 symbiosis, 22, 39
solidification, 74, 77, 79 symmetry, 2
solubility, 106 syndrome, 49, 54, 61
solution, 29, 45, 46, 65, 66, 69, 103 synthesis, x, 3, 12, 16, 28, 42, 43, 87, 88,
sorption, 84 90, 95, 98, 102, 106, 108
Spain, 63, 76
species, viii, 2, 13, 14, 22, 25, 28, 29, 32,
34, 37, 39, 58, 59 T
specific heat, ix, 64, 66, 67, 83
target, 11
spectroscopy, 8, 58
techniques, ix, 39, 41
speculation, 37
technologies, ix, 63, 82
sputum, 50
technology, ix, 63
stability, 51, 56
temperature, ix, 30, 34, 63, 66, 67, 68, 69,
starch, viii, 41, 42, 43, 53, 60, 88
70, 71, 74, 76, 77, 78, 79, 81, 82, 83, 84
state(s), ix, 11, 63, 66, 82, 84
tendon, 47, 49
stenosis, 48
testing, 62, 85
steroids, 47
tetrahydrofuran, 92
stimulation, 54
TGF, 46, 51
storage, vii, ix, 2, 43, 63, 65, 66, 67, 68, 69,
therapy, 50, 56, 58, 59
70, 77, 82, 83, 84, 85
thermal energy, vii, ix, 63, 65, 66, 70, 82,
stress, viii, 2, 16, 21, 27, 28, 34, 37, 43, 51,
83, 84, 85
55, 59
thermal energy storage (TES), ix, 63, 65
stretching, 53
thermal properties, 77, 83
structural changes, 8, 9
thermochemical materials (TCM), ix, 63
structure, 22, 37, 75, 88, 106
tissue, 23, 24, 25, 32, 33, 39, 46, 47
styrene, 95, 96
tobacco, 16
substitution, 101
toluene, 97, 105, 107
substrate(s), vii, 1, 3, 6, 9, 12, 93, 100, 104
transcription, 5, 8, 9, 10, 11, 12, 13, 14
success rate, 54
transformation, ix, 16, 43, 64, 83, 88
sucrose, 31, 43, 55, 88
transformations, 74
Sugar alcohols, 39
transforming growth factor, 46, 57
sugarcane, 15
transition metal, 88, 109
sulfate, 102
translocation, 26, 37
sulfur, 106
transplant, 30
sulfuric acid, 88
transport, vii, viii, 1, 3, 4, 5, 6, 8, 13, 14, 16,
Sun, 58
22, 28, 32, 46, 53
suppression, 60
transportation, 46
surface area, 29
traumatic brain injury, 50, 59
surface layer, 22
treatment, viii, 41, 45, 50, 51, 53, 54, 56, 57,
surgical resection, 50
58, 61
Index 121
V
Y
vapor, ix, 64
yield, viii, 2, 15, 43, 45, 90, 92, 107, 108
variables, 35, 36
vasodilator, viii, 41, 42
vision, 51 Z
zoospore, 40