D. Grant (Turriff) draft articles : Page 1. Document 1.Notes on the Inorganic Biochemistry of Heparin/Heparan SulphatePage 115 Document 2

Recent Heparan Sulphate Research May Explain Why a Vegan Diet is Good For You

Page 127 Doc. 3 The emerging paradigm of heparan sulphate related therapy(Doc 3a is an earlier edition of Doc.1);Page 137 Doc. 4 (This article argues for a role of heparan sulphate biochemistry in animal evolution); Page 142 Doc. 5

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DOC. 1. Notes on the Inorganic Biochemistry of Heparin/Heparan SulphateAn Incompletely Edited Scrapbook5/2/09-still needs more editing) Preliminary Draft of a Discussion Document (Drafted for Internet Posting, 2008;

by David Grant, B.Sc., M.Sc., Ph.D. (Turriff, U.K.)*

Research Continuation of Ideas Generated From University of Aberdeen Marishal College Research*

Contents:(page 8)

Summary Introduction Heparin (H) can apparently sequester the full range of inorganic ions which occur in seawater/biological fluids by a mechanism which tends to favour the uptake of the least abundant elements present A suggested inorganic ion nutrient gathering and other functions of extracellular polysaccharides

2(page9)

2.1 (page 10) 2.2

Available information on the association of multi-inorganic-elements with natural polyanionic substances Possible quality control problems with use of H for academic researches and for future therapeutic applications Countercation-specific (polarization-power-related) interation of metal ions with C-(O)O- groups in H/Heparan Sulphate (HS) The Haraguchi Hypothesis Extended to Polysaccharides Metallomics & polysaccharides The Heparanome MetallomeA putative H/HS inorganic environment interface system

2.2-1 2.3 2.4(page 12)

2.4-1(page13)

Summary of the origin of the heparanome-metallome hypothesis

2.4-1-1(page 14)Examples of literature reports of metal ion driven H/HS signaling 2.4-1-2 2.4-2 2.4-3 Comparison of metallomicmulti-inorganic-element arrays in anionic polysaccharides with those in other matrices Further evidence that multivalent inorganic element metal ions binding to H and HS proteoglycans in vivoH/HS is an evolutionary designed flexible metal ion binding system

Polysaccharides, especially the polyuronates and highly anionic glycosaminoglycans, seem to be especially designed to act, in conjunction with other anionic systems, as multi-element metallomic ligands. 2.5(page 16)

Evidence for all-element- decorated H

2.5-1

SSMS-determined inorganic elements associated with an experimental Na H (more accurately Na/Ca = ca.6, H) (believed to represent the inorganic components present in mast cell derived pharmaceutical H before final heavy metal clean-up) SSMS determined inorganic elements associated with an experimental Tl H Summary of apparent effect on metallomic profiles of Heparin leached from blood

2.5-2 2.6

collection containers ICP-MS studies of H 2.6.1(page 18) 2.7(page 18)

Re-evaluation of SSMS Data for NaH and comparison with ICP-MS data Toxic Inorganic Elements in the Natural Anionic Polysaccharides

2.7-1 2.7-2 2.8

Toxic inorganic elements in H Multi-inorganic-elements in common laboratory reagents Other suggested metallomic-related activities of animal polysaccharides: modulation of water supramolecular structure, calcification and deactiviation of Fenton reaction catalysts

2.8-1(page 19) Other cell surface polyanion (putative metallomic) systems: polyphosphate, poly- hydroxybutyrate & teichoic acids 2.8-2 2.9 2.9-1 Possible inorganic phosphate-containing high afffinity metal ion binding sites in H Metal ion assisted polysaccharide-protein binding could be relevant to the current discussions of how different HS polymers selectively binding by variants of FGF in vivo Servo-feedback signaling via the heparanome-metallome could have facitlitated animal evolution Inorganic cofactors for nitrosative structural alteration of H/HS The selective degradation of Heparin/HS by action of redox metal ions How different HS polymers could selectively bind variant of FGF in vivo: hints from lipoprotein binding studies Comparison of Different Multi-Inorganic-Element Matrices

2.9-2(page20) 2.9-3 2.9-4 2.10(page 21)

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3

(page21)

Ca2+ signaling via the heparanome metallome

The suggested primary functions of the heparanome-metallome; The hypothesis that Ca2+ and other inorganic ions modulate HS activitesprimary affect [The Long-Williamson1 hypothesis : HS controls Ca2+activity The control of activities of Ca2+ (e.g. at pericellular environments) were suggested to be the function of HS. This general idea can now, however, be modified to suggest that Ca2+ and other inorganic ions might modulate HS activities by modulating biopoymer water activities which H/HS-protein interactions]

3.1(page 22) In plants Ca2+ and other inorganic ions are believed to modulate polyanonic polysaccharide activities 3.2 Evidence from knockout mice that HS N-SO3- groups potentiate HS-determined Ca2+ activities required for skeletal muscle function (Ndst-1 -/- mice are reported to show reduced Ca2+ kinetics in myotubes)

3.3(page 22) Heparin/HS & control of calcification

3.3-1(page23)N-SO3- is involved in Ca2+ binding by Heparin/HS 3.4 3.4-1 Proposed roles of inorganic borate, silicate, arsenic (oxides) and phosphate attached to H/HS Specific nucleation activities (e.g. afforded by natural SiO2 nanoparticles) may be required for supramolecular structure formation in polysaccharides Polyoxymetalates and biologically relevent Heparin/HS catalytic activity Possible role of anionic polysaccharides in the assembly of polyoxymetalatespolysaccharides with primitive enzymic activity

3.4.-2

3.4-3(page 24) Heparin/HS & Metalloproteinases (MMPs): a putative further example of metal ion (and water structure?) determined activity related effects 3.4-3-1 Modulation of metalloproteinase activity by arsenic suggests a possible therapeutic application of As2O3- H/HS as an anti-cancer agent

3.4-4(page 24) Heparin/HS & Sulfatase activites; postsynthetic editing of HS sequences; role of metallomics? 6-O-Endosulphatase (Sulf) action in smart HS microstructure modulation 3.4-5 Heparin/HS & kallikrein: could exemplify how HS determines water structure/activity HS signaling (thyroid hormone dependent) in skeletal growth & mineralization [inorganic biochemistry related systems] The use use of barium acetate buffer for the electrophoretic separation via selective binding of Ba2+ to Heparin/HS Mineralization (inorganic biochemistry related systems) Thyroid hormone dependent skeletal growth etc. Plaque formation & Heparin/HSThe inhibition of various forms of proteinaceous and inorganic plaques by the most highly sulphated fractions of Heparin/HS and related substances (such as PPS) could be why such preparations show great promise for the therapeutic intervention in those diseases which are believed to be promoted by the formation of such plaques ** [Cf. also Section 6.3].

3.4-6 3.5 3.5-1

3.5-2-1(page26) Phospholipid Heparin/HS interaction with metal ions 3.5-2-2 Polysaccharide metal ion dependent aggregation

3.5-2-3 (page26) Nucleation events (subject to potential inhibition by Heparin/Heparan S) may determine the progression of amyloidoses 3.5-2-4 3.5-3(page 27) 3.5-3-1 Polysaccharide inorganic ion dependent gelation and melting Binding of anionic polysaccharides to crystal surfaces Relevance of control of inorganic crystal morphology to evolution of precursors of life

3.5.4(page29) How information in anionic polysaccharides can be read-off by crystal structures ---------------------------------------------------------------------------------------------------------------------------------4 WATER & LIFE ROLE OF POLYSACCHARIDES

4.1

Water chemistry & compensations between allowable entropy and energy changes Phenomena Related to Water Structure How life depends on the chemistry of water

4.2(page 29) 4.2-1

Possible key role of Wiggins-Water -Structure Possible key role of H/HS surface Wiggins-water-structurePossible role of nucleation of associated water & hydrate phase/conformation change in the metallome-heparanome

4.3(page30) Role of water structure in polysaccharide biochemisty

4.4

General water structuring effects of polysaccharides

4.5(page 31) Bacterial mucilages Mucilage structure adhesion forces 4.6 Evidence for a role of water adheison forces in the anticoagulation mechanism of H and in the binding of poly-L-lysisne and poly-L-arginine to H Evidence for biologicaly relevant polysaccharide-determined water structure adhesion forcesThe binding of poly-L-arginine and poly-l-lysine to an optimally hydrated (Na-counterion containing) Heparin occurs initially via a Heparin-bound water structure adhesion mechanism. This could be observed when a hydrophobic environment was used for near infrared spectroscopic detection of the changes in the overtone water bands asociated with Heparin surface bound water present in mixtures of Heparin and polyamine

Adhesiveness of metal ion dependent water associated with H/HS elicits protein binding similar to how metal ion dependent hydration affects polysaccharide lectin binding etc Evidence for water adhesion forces in H-protein interactions Adhesiveness of metal ion dependent water associated with H/HS may control glycocalyx assembly and calcification 4.74.8

Other evidence for biologically relevant polysaccharide determined water structure adhesion forcesManning (electrostatic) binding of counterions to polyanions

4.9(page 33) Water activity model of metal ion dependent H//HS signaling (Metal ions may participate in H/HS signaling in part via modulation of water activities at H/HS surfaces). Do HS actions ultimately depend on the modulation of water structure to achieve selective protein binding and directed refolding? 4.10(page 34) HS as a proton pump Proton Conduction H/HS probably occur naturally as mixed salt forms in which complex hydration patterns are associated with the sulphate half-ester and Nsulphonate groups which are putativley involved in H+ and Ca2+ transport and energy transduction

Water Channel (HS)Related Proton ConductionArrays of hydrated sulphate half ester groups attached to polysaccharides could be involved in energy (e.g. proton) transduction by a similar mechanism to that by which sulphonated man-made ionomers (in which water clusters are attached to sulphur oxy-anions) are believed to confer conductivity (James et al. 1) and which further seem to be subject to stabilization by the incorporation of similar amounts of inorganic silicate nanoparticles to those which occur naturally with polyuronides (Schwarz1). There may also be roles for in such systems for semiconductor modulator functions of various rare earth ions

4.11(page 35) HS as a mimetic of humic polymer buffers in natural waters

High molecular weight polyanionic (including organic polymer) multi-inorganic-ion sequestering and buffering systems putatively enable bio-friendly water structures to be formed

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Nitric Oxide Biochemistry & H /HS Signaling The selective degradation of H/HS by actions of redox metal ions Metal ions & water structure dependent nitrite cleavage of H/HS Possible key roles of metal ions in the physiological nitrite cleavage of H/HS

5.1 5.2

Early studies of metal ion effects in nitrosative processes 5.3-1 Identifying HS by deaminative cleavage with nitrous acid 5.3-2(page 36) Tyrosine nitration in degenerative diseases 5.3-3 Inorganic ion catalysis of nitrosative reactions associated with stomach cancer 5.4 5.5(page 38)

Internet postings: Ascorbate & Nitric Oxide in Redox Control of Heparan Sulphate Internet posting: Ascorbate & Cancer Additional Hypothesis : Hydrogen Sulphide & Nitric Oxide are Invovled in Redox Control of HS

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6

(page 38)

Relevance of H/HS biochemistry to pathology Putative pathological inorganic element perturbation of H/HS signaling

6.1

6.1-1(page 39) H/HS biochemistry & degenerative diseases 6.1-2 Possible perturbation of H/HS signaling by Ba2+ in multiple sclerosis

6.1-3 Possible perturbation of H/HS signaling by Al3+, Zn2+ (and Cu2+/+) in Alzheimers disease 6.1.4(page 41) H-mimetic pentosan polysufate: anti-misfolded prion & anti- HIV activities 6.1-5(page 42) 6.2 General anti-degenerative disease potency of H/HS

The defective nitrosative cleavage which has been indicated to occur in Niemann-Pick Disease could also be relevant to the etiologies of cancer and other degenerative diseasesPossible role of dyshomeostasis of nitric oxide and cholesterol in Alzheimers disease

6.3(page42)

Cancer & HS Biochemistry. Inhibition of Carcinogenesis by H & H-Like MoleculesOverall HS biochemistry, including the of specific signaling sequences of sulphate half ester anionic groups seems to be influenced by redox status (which include ascorbate, oxygen pressure, homocysteine) and the correct inorganic ions perhaps involved in generation of water structure (but a range of toxic inorganic ions seem to negatively perturb HS biosynthesis).

6.3-1(page 45) 6.4(page 47) 6.5 6.6

Inorganic tumour suppressors and tumour promoters in H

Does HS provide for a servo feedback system which directly interfaces the environment and which allows dietary manipulation to produce beneficially altered HS microstructure? Pacification of redox active iron ions by their incorporation into polysaccharide aggregates Alternative and ethnic medical use of metallomic matrices. Could some of the non-anticoagulant suggested uses of H and claimed benefits of Ayurvedic Shilagit etc. be dependent on the co-occurrence of similar ultratrace elements in these preparations?

---------------------------------------------------------------------------------------------7(page 49) Interference from inorganic constituents in H during blood analysisInterference from multiple inorganic constituents associated with H used as an anticoagulant during determination of inorganic elements in blood ---------------------------------------------------------------------------------------------------------------8 Variation between manufacturers of the degree of final purification of H

9

Could HS signaling generate a systemic biological clock which is speeded up during degenerative disease processes ------------------------------------------------------------------------------------------------------------------10 (page 51) Examples of Literature Reports of Metal Ion Driven H/HS Signaling 10.1 Table I Involvement of metal ions in H/HS interactions thought to be involved in blood anticoagulation, arterial calcification, breast cancer, basic fibroblast factor dimerization, annexin-V binding, endostatin (collagen XVIII), collagen -V and prion interactions, as well as the activities of a range of other proteins and in the nitrosative scission of H/HS 10.2(page 54) Table II Reports of (apparently direct) modulation of HS microstructure and/or HS PG synthesis in response to the presence of inorganic ions etc. 10.3(page 56) Table III Some examples of reported effects of organic molecules on modulation of HS microstructure and/or alteration of HS PG synthesis 10.4 (page60) Table IV Inorganic elemental contents (mg/kg) of kelp, H & human scalp hair Comparison of metallomic multi-inorganic-element arrays in anionic polysaccharides with those in other matrices Examples of metallomic arrays associated with H, kelp & human scalp hair

10.5

Further background to the studies of metal ion binding and the multi-element content of H Comparison of inorganic elements in H and chitosan

10.6(page62) Table IVa10.7

Further Notes

10.8(page 64) Log-Log plots Figs. 1-5 Some interrelationships between multi-inorganic element contents of H-related matrices, seawater, human hair Inorganic element contents compared with H

11

(page 68)

References General reviews of H/HS Reference 1References arranged alphabetically according to first-named author

11.1 11.1- 2 11.2(page 93) 11.2-111.2-2

Further references (and notes) referred to by numberand also arranged according to subject matter

The metallome-heparanome(References to metal ions in H/HS signaling)

(page 93)

Ref. 1A-1 Published peer-reviewed articles which include mention that inorganic ions might participate in H/HS signaling Ref. 1A-2 How metal ions are currently known to assist in H/HS nitrosative signaling Ref. 1A-3 Degenerative diseases: putative roles of H/HS signaling References 1A3-1 H/HS & Cancer (Gene expression seems to be linked to H/HS)

1A3-1-1 1A3-1-1-1 1A3-1-1-2 1A3-1-1-3 1A3-1-1-4 1A3 1-1-4-1 1A3-1-1-5 1A3-1-1-5-1 1A3-1-1-6

Histones Inhibition of Histone Acetyltransfersases (HAT) by GAGs Tumour cell surface HS is a cryptic system which can act both as a promoter or inhibitor of tumor growth System of trans repression by H The Sulf system & cancer Sulf loss influences N-, 2-O, and 6-O sulphation of multiple HS proteoglycans and modulates FGF signaling Glypican & Cancer Glypican 1 HS PG & Cancer Pro-tumor effect of glypican-1 HS PG asociated with human gliomas Glypican 3 HS PG & Cancer Antibody against the 30kD fragment of (Glypican-3 HS) PG with anti-tumour therapeutic potential Glypican-3 HS PG is a potential lung tumor suppressor Glypican 4 Akt

1A3-1-1-6-1 1A3-1-1-6-2 1A3-1-1-6-3 1A3-1-1-7 1A3-1-1-7-1 1A3-1-1-7-2 1A3-1-1-8 1A-1-1-1-8-1 1A-3-1-1-8-2

Glypican 5 HSPG & Cancer Glypican 6 HSPG & Cancer Heparanase & Cancer Selectin, H & Cancer metastasis Heparanase-Akt activity and tumorigenesis Angiogenesis, HS & Cancer Angiopoietin HS & Cancer Endostatin HS PG Zn2+ & Angiogenesis Induced Nitric Oxide Synthase Expression & Cancer Nitric oxide can apparently directly activate MMP-9 Putative modulation of angiogenesis by H/HS Classical research by J Folkman et al Further studies related to VEGF affecting angiogenesis Pericytes and H VEGF antibodies are showing promise as anti-cancer therapeutic agents Dietary polyunsaturated acids & Cancer Glucosamine therapy for degenerative diseases? Earlier reports of relevance to H/HS & Cancer Engelberg (historical) review Other (historical) reviews Tumour suppressors & H/HS Exotosin I (EXT1) and exotosin 2 (EXT2) (are glycotransferases required for biosynthesis of HS) BRCA-1

1A3-1-1-8-3 1A3-1-1-8-4

1A3-1-1-9 1A3-1-1-10 1A3-1-2 1A3-1-2-1 1A3-1-3

1A3-1-3-1

HS can be both pro- and anti-cancer, the difference may be related to HS microstructure

1A3-1-3-2 1A3-1-3-3 1A3-1-3-3-1

Recent reports of potential H-related anti-cancer therapeutics Low molecular weight H and oligosaccharides: anti-cancer effects H derivative for oral administration with claimed potent anticancer activity and low toxicity

(page 102)

1A3-1-3-4 Pentosan polysulfate (PPS) anti-cancer effect 1A3-1-4 Anti-cancer metal and metallioid ions (which could be sequestered from biological fluids by H 1A3-1-5 1A3-1-5-1 1A3-1-6 1A-4 1A-5 Altered HS in transformed cells and cancer tissues Putative differences in H iduronate conformation in transformed cells Water structure & cancer

H and Atherosclerosis H and Dementia H-derived oligosaccharides for inhibiton of senile dementia H/HS claimed to normalize age-related fear response Further Notes on H and Antioxidant Protection (Smoke inhalation injury) Antioxidant & antinitrant activity (related to medulation of superoxide dismutase by H/HS) and sequestration and deactivation of redox active iron, copper & silver ions, etc. by H/HS Further references Refs. 1a1-14; 1b-1h Refs. 6a, 6a-1, 6b-,. 6b-2, 6b-2-1, 6b2-2, 6b2-3; 7-9, 9b-3, 9b-3-1, 9c; 1013, 13a; 14-17 Concluding Remarks Suggestions for Further Research

(page 104)

1A-6

11.3 (page105) 11.3-1

12 12-1 (page 111) APPENDIX Authors Curriciulum Vitae (page 142) Acknowlegements (page 144)

--------------------------------------------------------------------------------------------------------------------------------------------------------------------1. Summary Heparin (H) is believed to be most anionic biopolymer, and has traditionally been used by biochemists to provide a readily available laboratory model of heparan sulphate (HS). Evidence is now discussed which suggests that the biochemical reactivities of H and the H-like segments of HS may be influenced by the presence in the hydrated supramolecular structure of these polysaccharides of small amount of the wide range of inorganic ions which occur in biological fluids and natural waters. Low abundance inorganic solutes are known to become selectively sequestered by the hydrated anionic polysaccharides which occur in the cell walls marine algae and by the polysaccharide-like soil and natural water humic/fulvic polymers. A similar multi-inorganic element sequestration process is now

suggested also to occur with the H-like animal anionic polysaccharides abundantly present in HS at adherent animal cells and extracellular matrices. H/HS seem to preferentially sequester the less abundant ions present in biological fluids. The HS system of polysaccharides which as is now becoming apparent, may serve as a superhub control system which influences a wide range of biological acitivies. HS may, however, have been selected during early stages of animal evolution to serve as a multi-inorganic element nutrient gatherer but later evolved into a general system of inorganic ion regulation of cellular activites by HS proteoglycans. Glycocalyx polysaccharide-reservoir-based inorganic ion (including oligomeric inorganic ion) provision is suggested to allow the heparanome-metallome system to achieve and control a system of delicately balanced HS-(metal ion-[H2O]n) in H-like supramolecular structure which is now proposed to influence HS reactivity as regards protein binding and nitrosative generation of oligosaccharides. An anthopogenic or ortherwise induce inorganic ion dyshomeostasis perturbation of HS biochemical signaling could be of relevance to several pathologies.The control of activities of Ca2+ (e.g. at pericellular enviornments) had formerly been suggested (the Long-Williamson hypothesis) to be a primary function of HS. This general idea can, it is suggested, be usefully modified to suggest that Ca2+ and other inorganic ions can modulate HS activities by affecting biopoymer water activities which affect H/HS-protein interactions

Future biological metallomics research might usefully consider how polysaccharides collect and deploy counterions and other inorganic moieties present in their natural bathing solutions and seek to establish if the ternary association of specific metal ions with HS + proteins is a critical part of the HS signaling mechanism.Keywords: Heparin, heparan sulphate, the heparanome, metallomics, degenerative diseases, cancer, multiple sclerosis, Alzheimers disease, prion misfolding diseases, water structure, hydration, biological clock, inorganic ion contamination of heparin, inorganic elements in blood serum, nitric oxide, nitrite, glycocalyx. Abbreviations: ACE: agiotensin converting enzyme; ChS: chondroitin sulphate; CSE: cystathionine gamma-lyase; DeS: dermatan sulphate; EHDP: ethane hydroxyl, 1,1-diphosphonate; ECM: extracellular matrix; FGF: fibroblast growth factor; H: heparin, HS: heparan sulphate; GAG: glycosaminoglycan; HDL: high density lipoprotein; HIF: hypoxia inducible factor; i.v.: intravenous; iNOS: induced nitric oxide synthase; ICP-MS: inductively coupled plasma mass spectroscopy; I: ionic strength; LDL: low density lipoprotein; LPL: lipoprotein lipase; LPS: lipopolysaccharide; MMP: metalloproteinase; Ndst: N-deacetylase/N-sulfotransferase; PAPS: 3/phosphoadenosine 5/phosphosulfate; PET: polyethylene terephthalate; SLE: systemic lupus erythromatosis; SRCD synchrotron radiation circular dichroism, SSMS: spark source mass spectrometry; TIMP: tissue inhibitor of metalloproteinase; uPA: urokinase plasminogen activator; VEGF: vascular endothelial growth factor.

-----------------------------------------------------------------------------------------------------------------------------Foreword : This document is an attempt to produce a general overview of heparin (H)/heparan sulphate (HS) biochemistry from the perspective of novel academic polysaccharide laboratory researches formerly conducted by the author as a member of a team led by W.F. Long & F.B. Williamson which had led to numerous publications in peer-reviewed journals [cf. Long1, 2003 ]); each of these papers dealt with sub-facets of the results obtained, but no overview of these findings has been attempted before now. Viewed from the perspective of an industrial quality control standard chemist (e.g. where rigorous purification and the reagents employed as monomers, catalysts and solvents etc. are well known to be essential prerequisites for the preparation of polyolefins) it seem now to be apparent that medically employed reagents and polymers (such as glycosaminoglycans) should be subjected to a more thorough evaluation of standard quality control methods in regard to the possible variation in co-purification with these polysaccharides of a wide range of inorganic ions. This phenomenon was formerly regarded as of little fundamental scientific interest being regarded as a relatively trivial random contamination phenomenon which fails to merit the elvel of relevance and scientific value which is required to allow any discussion in mainstream peerreviewed journals. Some recent reports (cf. Boher et al.1) have, however, discussed (albeit in a non-English language peer reviewed journal) the possible risks to patients undergoing kidney dialysis of the presence of potentially toxic elements present in the presently considered type of blood anticoagulant H, which as the most anionic of biologically encountered polymer systems is therefore also the most likely of commonly employed pharmaceutical agents to show an especially enhanced ability for becoming highly enriched in those ultratrace (e.g. counter-cation) metal elements which are now known to exist in biological fluids. This ability may, however be a normal designated property of H and H-like polysacchrides (including HS) and may be of fundamental importance to the in vivo modus operandi of HS PG for the regulation of protein binding by these polysaccharides and also be of relevance following, e.g., anthropogenic, pathological or normal age associated perturbation of blood inorganic ions contents, to a range of pathologies. Inorganic input to the heparanome is suggested to pertain inter alia to the thyroid hormone dependence of inorganic sulphate transport, the biosynthetic thyroid hormone inhibitory roles in HS structure modification and the requirements for inorganic cofactors for the nitrosative scission of HS chains (which generate HS oligosaccharides used as signal messengers) and for metalloproteinase (MMP) control of HS shedding. HS and other polysaccharides putatively also contribute to animal plasma membrane hydration and water activity regulation.

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2. IntroductionHeparin (H) (n.b. a protein and nucleic acid free pharmaceutical industrially prepared polysaccharide mixture) is obtained from its mast cell proteglycans containing highly H-substituted serine glycine rich core protein); H contains abundant salt-forming anionic (sulphate half ester, sugar uronic (carboxylate) side groups which confer upon this polysaccharide system an almost inorganic outer surface.It should be noted that H is a highly heterogeneous system (cf. Nader et al.1, 1981) which does not correspond to what is classically definable as a chemical substance, namely a specific, single formula/structure chemical molecule. H should perhaps more accurately be described as a library of many different chemical molecules, which are structurally related only in the restricted sense of the non-mathematical fuzzy logic used by chemists to label related systems of families of chemical compounds.

The administion of H to humans (e.g. as an aerosol) has been reported to show many health benefits, including the inhibition of asthma, lung damage in hypoxia, bleomycin induced lung fibrosis and the inhibition of lung cancer (cf., Gabr et al. 1 a study of rat lung proteomics following H instillation). For these uses, as well as the traditional use of H as a blood anticoagulant, a quality control problem might be anticipated from a consideration of the available data on the variations in associated inorganic elements arising from different procedures used to remove heavy metals and other unwanted inorganic elements from H. While an approximate similarity is apparent between different Hs (e.g. all seem to approximate to the seawater-metallomic-fingerprint) differences are also evident between the residual multiinorganic element contents of some commercial pharmaceutical H preparations for which analytical data have been made public.A recent study (Rudd et al.,1) which included use of highly sensitive method of synchrotron radiation circular dichroism, has confirmed previous putatively made suggestions (e.g. by the former Aberdeen polysaccharide group, cf., Long, 1,2003 ) which had proposed that HS controls Ca2+activity. It is now evident that the association of Na+, K+, Mg2+, Ca2+, Mn2+, Cu2+ and Fe3+ counterions with H and modified Hs could create structurally distinct uronic acid conformations. This means that the metallomic profile of counterions must be included in considerations of any proposed H/HS signaling system and different counterion forms of H/HS are predicted to have quite different biological activites {e.g. Al3+, Be2+ or Ba2+ intoxication might pathologically perturb growth factor signaling by H/HS , (cf., Purdey, 1, 2004)}.The range of inorganic ions (in addition to Na+ , K+, Mg2+ and Ca2+ ) present biological fluids (and also present in H Tables I and IV) will putatively participate in H/HS ([H2O]n)-inorganic ion-protein adduct formation as well as perhaps participating in self-assembly of formally highly charged GAG chains.

The binding of a range of metal counterions to pharmaceutical H (cf., e.g. Grant, 1997, Long 2003) showed that this polysaccharide system behaved similarly to phosphorus oxyanion systems which had been previously investigated by the author* (both inorganic oxyacid systems show similar systematic shifts in observed infrared absoptions which correlated with chemical reactivity for different salt forms). Studies of H using polarography, equilibrium dialysis, potentiometric titration, osmolytic titration, NMR spectroscopy and infrared spectroscopy 6b-2, 6b2-1 indicated that the counterion binding processes failed to obey the law of mass action as required for ideal solution state dissolved ion interactions; instead, the effective concentration or activity of H remained essentially constant over a wide range of apparent dissolved H concentrations; counterion binding isotherms further demonstrated characteristic discontinuities in the linear isothermal binding curve at characteristic metal ion/polysaccharide ratios.

2.1 A suggested inorganic ion nutrient gathering and other functions of extracellular polysaccharides [N.b. blood serum and other biological fluids contain some 60+ inorganic elements (cf. Haraguchi1)]. A similar range and distribution of inorganic elements pertains both to marine alginates and the animal polysaccharide H, a

circumstance which suggests that the highly anionic polysaccharides in animals behave similarly to those in marine bacteria and algae which act as smart ligands for the simultaneous sequestration of the full range of cations and anions which occur in the natural bathing fluids (cf. Figs.1-6, p. 83-87). Inorganic ion nutrient gathering, it is suggested, could have been the original primary functions of extracellular anionic polysaccharide-rich mucilages. It is further suggested that during the early evolution of animals in the sea, the glycosaminoglycans (including the Hlike polysaccharide system of HS (analogously to carrageenans and alginates in marine algae) provided osmolyte buffer and crystallization control agent functions. The latter encouraged the formation of defined (non-thermodynamic) inorganic salt solution compositions which can be argued to have been critically needed for the maintenance of biological functions. This can explain why the amount of HS and other GAGs present in the tissues of aquatic invertebrates seems to be strictly related mathematically to the salinity of their habitats (Nader et al., 1 (1983) ; 5). Extracellular polyanionic polysaccharides can also provide protection against oxidising radiation (cf., the general ability of H-like molecules to act as anti-free radical agents cf., Grant et al.1, 1987, 1994, 1996. Ross et al.1, Mackintosh et al.1, Long et al.1, 1994. The extracellular polyanionic polysaccharides also can inhibit predation and serve as an effective sink for toxic organic molecules and deactify potentially damaging inorganic particles which can create promote free-radical induced oxidative and nitrative damage as well as unconventional damage via the inappropriate nucleation of phase changes (such as may be required for the formation of toxic proteinaceous fibrils and both calcified and non-calcified plaque formation). A critical influence of polyanionic substances, which is often overlooked by biochemists, is that these substances (exemplified by humic polymers and anionic polysaccharides) can strongly inhibit biologically relevant nucleation dependent phase changes including the preciptiation of insoluble phases from aqueous solutions which also allows such polyanionic substances to regulate water activity. It seems likely that primitive osmolyte balance activities and water structuring activites ultimately can be held responsible for the numerous properties of H-like polysaccharides (e.g. those present in the glycocalyx (cf., RubioGayosso et al.1) which originally was neded to establishe the existence of the first animal organisms in the sea but remain of continued relevance to the ion balance physiological control mechanisms in use in modern highly evolved animal organisms.

2.2 Available information on the association of multi-inorganic-elements with H. Possible quality control problem with use of H for academic researches and proposed therapeutic applicationsThe data currently available suggest that H metallomic profiles approximately conform to the common biological serum/seawater type of distribution and hence are unlikely to have mainly arisen from in vitro sources of post extraction contamination. A preliminary test of the relevance of metallomic thinking to GAG biochemistry is to ask whether the wide range of inorganic elements found to be present in a range of pharmaceutical H samples studied by mass spectroscopic techniques (e.g. Moffat1 and an internet report3b-10 as well as sporadic reports in older literature) conforms to the pattern of blood serum like multi-element distribution in H, which might indicate that the multi-inorganic elements, which seem always to be present in many commercial polysaccharides, will also occur in vivo, or, alternatively they simply represent an artifactual contamination, e.g. arising from industrial processing, or from dust particles. It was originally uncertain whether the characteristic metallomic inorganic element profiles which had been detected in early mass spectrometric evaluations of heparin (H) (cf., Grant1, 2000) applied to to all H samples or simply represented some accidental multi-inorganic-element contamination. Results 3b-10 which have recently been made public by commercial laboratories engaged in blood inorganic element analysis (cf. Table IV and Figs. 6) now tend to confirm the older indications (cf. Bowen3b-1 (cf. also 3b2-8-1) ) that similar multi-inorganic element arrays to those previously found by the Aberdeen polysaccharide group always seem to decorate H. [Multi-inorganic-element analytical data are currently available for alginates e.g., Wassermann1 (and later studies, cf. Table I) which have some resemblances to the analytical data available for H: e.g., leached from blood collection vessels3b-10, obtained from an industrial Na H sample and a derived single counterion enriched salt form (Moffat1 cf., Grant et al., 1, 1987, Grant 1, internet 2000; and data from numerous reports (refs. 3b-1, 3b-2, 3b-3, 3b-4, 3b-5, 3b-6, 3b-7, 3b-8, and 3b-8-1) which suggest a variable presence of individual elements in H].

Irrespective of how the multi-inorganic elements had been accumulated by H, the possible occurrence of variable amounts of inorganic elements in different batches and brands of H poses a serious quality control problem for the use of H as a convenient laboratory model for HS as well as the proposed used of H for the treatment of human diseases for which preliminary reports (reviewed below) suggest is an achievable goal. In the event that the multielement contents of H and HS are confirmed by further studies to be a general phenomenon, a careful reappraisal may be required of some previous H/HS biochemical researches. It will be necessary to draw up appropriate quality control protocols for future researches in this field. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - --- - - - - -- - - - - - - - - - - - - - - - - -- - - - --It be noted that H, perhaps because this is the most anionic biopolymer, H seems have an unusual ability to simultanesously sequester (apart from the wide range of dissolved inorganic ions and molecules) various colloidal sized particles which can occur in biological fluids; apparently H acts thereby in such a manner so as to inhibit the potential seeding by such surfaces of the formation of pathological plaques (vide infra, cf. also Grant et al. 1,1992). H shares this property of the prevention of thermodynamically required precipitations (e.g. of sparingly soluble Ca salts) with other polyanionic substances (e.g. polyphosphate and fulvic acids) by the ability to stabilize aqueous solution supersaturation. ============================================================================ 2.2.-1 Countercation-Specfic [Polarization-Power-Related] Interaction of Metal Ions with C(O)O- groups in H/HS

The carboxylate groups the principal disaccharide repeats in H showed infrared absoptions which altered according to the counterion polarization power. Similar results were reported by Panov & Ovsepyan1, Grant et al. 1 1987b) and Rudd et al.1, confirming that counter cations can directly modify the electronic environments (and putatively the electrostatic activities) of HeparinC(O)-O- groups and thereby contribute to the overall mechanism of how inorganic cations modify the attractive potencies of H/HS. The principal efffect of counterions on HSO3- groups appeared to be the alteration of the water clusters associated with these groups.

2.3 The Haraguchi Hypothesis Extended to Polysaccharides Haraguchi1 noted that most of the elements in the periodic table normally occur in animal cells and discussed the existence of a similar range and distribution of multi inorganic elements in both blood serum and seawater. It is now pointed out that this seawater-like multi-inorganic-element phenomenon is most strikingly illustrated by the highly sulphated anionic animal polysaccharides.Haraguchi seemed to imply that the scientific field (to be termed metallomics) should be concerned inter alia with the roles of the entire seawater range of inorganic ions. He proposed that all elements occur in biological fluids and therefore might be relevant to biochemistry, but he did not specifically discuss any role for polysaccharides in the preferential uptake of such ultra trace elements which could perhaps be required to modulation of water structures present at the surfaces of key information-encoded H and HS anionic polysaccharides which seem to naturally become enriched in such elements. This idea is my extrapolation of his ideas. The only biopolymers for which metallomics were relevant, according to Haraguchi, were proteins. However, it is now suggested that the inorganic element reservoirs seemingly present in anionic polysaccharides may participate in evolved signaling mechanisms and therefore be invovled directly in the transmission of information encoded within the anionic patterns of the ultra anionic animal polysaccharides H and HS so as to enable these polysaccharides to control protein activities.

The Work of Rudd et al. Confirms the Major Relevance of the (Haraguchi) Inorganic Element Phenomenon to How Heparin & Heparan Sulphate Function In Vivo

Rudd et al.1, in a recent groundbreaking study, reported the effect of a range of metal ions on the CD and NMR behaviour of H and modified Hs and also the drastic effect of K+ and Cu2+ substitution of H upon FGF2/FGFR1c action; the presence of different metal counterions creates a distinct chemcial signal which indicates that it is necessary to include such metal ions as part of the information encoding mechanism provided by the H/HS protein control system. An important additional part of this control, it is now suggested, could be provided by the wide range of ultratrace element which occur in biological fluids but which previously were not thought to be of relevance to biochemistry, but which ,nevertheless, seem to become (sometimes) greatly enriched at H/HS surfaces where the could provide an inorganic ion encoded signaling system addtional to that offered by Fe, Cu, Ca, Mg, Zn, K and Na ions implied by Rudd et al., to enable the formation of specific H/HS sugar conformations and electronic phase coupling systems which could behave as discrete biological signals.

2.4 The Heparanome-MetallomeIt is suggested that by combining together two recently proposed hypotheses (Haraguch1 and Turnbull et al.a which discussed the heparanome a and the metallome [putatively analogues of the proteome and the geneome]) gives a useful, but at present tentative, intellectual framework with which to address the roles of inorganic cofactors (and also to address the role of water structures) in H/HS signaling. According to the general principles of physical chemistry, it would be expected that the same range of inorganic ions to those which occur in H, should also occur in HS especially in the H-like segments of HS. {This notion is also in apparent agreement with a number of research publications6}. [for ref. 6 go to page 126]

The possession by anionic polysaccharides of an intricate information endoced bar-code or fuzzy logic microstructure which selectively determines protein folding and signaling is the basis of the heparomea, a mechanism which is thought to enable the HS polysaccharide-based to engage in systemic control of multicellular discriminated behaviour in animals (this is a super-hub, around which numerous biochemical activities can rationaly be proposed to be controlled [perhaps even the most important hub and information system which determines the whole of animal biochemistry] [it might alterntive by viewed as an auxiliary system which enable the genome to perform many more functions than would have otherwise been possible {cf., the much lower than expected number of genes which have been found to occur in humans}. The presence of a correct HS reservoir of inorganic ions may then be supposed to be required for optimum human health. The actions of the heparanome could also be relevant to a fuller understanding of the roles of bacterial, algal and plant anionic polysaccharides. The reactivities of all such biological anionic information systems could however, actually be potentiated via inorganic cofactor effects which in turn could ultimately be potentiated by water structuring effects (although the actual nature of liquid water has not been established perhaps the hypothesis proposed by Wiggins1 provides, at the present time, the most suitable working model). [Viz. the surface multi-ion compositions create exact water activites and ionic strengths which thereby allow their associated polysaccharides to determine the specific folding patterns of associated proteins which rather than possessing intrinsic abilities to fold correctly (as was the traditional view) are now believed to be critically dependent for such folding on the protein surface water activities (cf. WilseRobinson et al.1, Wiggins1)].

J. Turnbull et al. in TRENDS in Cell Biol. 2001, 11 (2) 75-82 suggested the use of the term the heparanome for the new concept of the dynamic expression of HSs possessing differing sugar sequences produced by particular cells or tissues from a precursor in which glucuronic acid-N-acetylglucosamine units are subjected to structural alteration in the Golgi apparatus by sulphation (by 2-O, 6-O and 3-O- sulfotransferases) and epimerization (C5 epimerase) and postsynthetic de-N sulphonation and scission by nitric oxide metabolites and also by the action of 6-O-endosulphatases. These processes also allow for environmental - HS microstructure crosstalk for modulation of HS functions, which are according to L. Kjellin & U. Lindahl (Annu. Rev. Biochem., 1991, 60, 443-465) in a reveiew of the function of proteoglycans of which HS are prominent members: as being present in multicellular animals for the provision of mechanical support, negative charge, regulation of cell migration and aggregation, the development and stabilization of synaptic structures, endothelial regeneration, the stabilization of basement membranes, the modulation of collagen fibrilogenesis including the transparency of the cornea, the regulation of cell growth, urinary trypsin inhibition, provision of a filtration barrier, various roles in morphogeneis, the provision of links to cytoskeleton and ECM, the mediation of adhesion and morphogensis, the assembly of the matrix phosphatidylinositol linkage, the provision of reservoirs for fibroblast growth factors, allow for the regulation of blood coagulation, the mediation of transferrin functions, and the uptake of antigen presentation. The initial step in biosynthesis of H/HS is known to be the formation of the protein linkage region GlcA-Gal-Gal-Xyl-Ser by transfer of xylose from the UDP-xylose to specific Ser residues in the core protein. In their extensive 1999 review of HS systems (the most common members being now known to be glycoylphosphoinositide-linked glypicans and transmembrane syndecans) Bernfield et al. ibid., 68, 729-77, noted that the linkage region, which is the same in HS and chondroitin sulphate ChS, evidently has been highly conserved from flies to humans and that HS provides the most abundant receptor system at adherent animal cell surfaces for tissue morphogenesis and wound repair as well as for host defense and energy metabolism processes which are achieved via interactions including those with the HS molecules present in the extracellular matrix.a

Perhaps the most fully understood epitope in H/HS is the pentsaccharide antithrombin-binding site which occurs most characteristically in mast cell H and in some HSs. Pharmaceutical H, which has been used for many years as a blood anticoagulant, is now available in a variety of salt and molecular weight forms which includes the single epitope, an artificially produced pentasaccharide molecule as well as unfractionated H (Na heparin) from porcine mucosa (but also from bovine lung) which derives a major part of its anticoagulant activity from the antithrombin epitope content.

A putative H/HS inorganic ion environment interface systemIt is suggested in vivo discrimination process by which information held within H/HS sequences is transmitted into biochemical activities requires inorganic cofactors (putatively multi-inorganic elements) suggesting the use of a fuzzy logic, analogue recognition system of a polymer rather than oligomer-associated metallomic array rather than an exact digital type of specific microstructure electrostatic recognition similar to the hydrogen-bonding etc. used by the DNA double helix.

It is proposed that the the multiple inorganic elements which occur in H (and putatively also in HS) and which are also part of an essential glycocalyx reservoir system permits the release of inorganic cofactors in amounts needed to achieve correct biological signaling by the heparanome; this may includes a system of fast track provision to achieve a required inorganic cofactor cocktail to permit a selective H/HS protein binding. Such inorganic cofactors seem to be needed to assist in muscle action in the lung (cf. Jenniskens et al. 1) (and likely also elsewhere including for HS functions performed in the glycocalyx of the heart) and (possibly a range of ) redox metal ions which is needed together with Zn2+ are required for nitrosative scission of HS chains (cf., e.g. Ding et al. 1) and for the modulation of the actions of enzymes invovled in the assembly of H/HS and HS PG disassembly activities of MMPs (e.g. for shedding of HS PGs from plasma membranes).

2.4-1 Summary of the Origin of the Heparanome-Metallome Hypothesis Virtually all elements in the periodic table can be detected in biological samples by commonly used mass spectroscopic multi-inorganic-element analysis techniques. This circumstance prompted Haraguchi1 to update the earlier concept of biochemical metallomics which was restricted to the 20 or so highly essential inorganic elements utilized by biochemical processes previously proposed by R.J.P. Williams. The Haraguchi new biochemical metallomics is now, it seems, to be concerned with all stable elements in the periodic table.

Individually additive contributions of each of these ions present in biological fluids will enable the fine tuning of the structure of liquid water (cf. Luck1). Since Life Depends on Two Kinds of Water (Wiggins1,2008) it follows this chemical system is the most important determinant of all biochemical processes and the most central way metallomics could be of relevance to biology is by the ability of metallomic matrixes to fine tune such water structures. The other primcipal biochemical system for which the new metallomics also seems to be of especial relevance is the anionic polysaccharides which apparently also naturally exist as multi-inorganic-element metallomic matices. This phenomenon is found to the greatest extent with pharmaceutical H, a traditional industrial product which is derived from bovine and porcine mast cell H protoeglycans which has found wide use in medicine as a blood anticoagulant. H is thought to be the most anionic of any biopolymer which confers on this polysaccharide system an ultra-high electrostatic charge which should promote the (chemical nature independent) electrostatic counterion collection process (a mechanism which is at least partly responsible for the uptake of counterions) to enable the simultaneous binding to H of the multitude of types small amounts of metal ions which exist in multi-element containing natural waters including biological fluids. Other H-like medications include marine algal ** and plant-derived xylan fractions (e.g. pentosan polysulfate (PPS)) which are produced by an additional sulphation carried out post-extraction from tissue. These preparations are also suggested to function as metallomic matrices and to require use of multi-inorganic-element mass spectrometric techniques for quality control purposes. All H samples examined to date (and discussed in more detail below) seem to have approximately (log-log) inter-related multi-inorganic element compositions which apparently define them as blood serum/seawater type Haraguchi-newmetallomic matrice systems (cf. Section 10.8 (page 83 et seq.). These metallomic profiles inevitably include small amount of a range of toxic elements the removal of which has been suggested Bohrer et al., 1 to be a critical clinical requirement for certain uses of H. [Different degrees of purification of H achieved by different manufacturers can lead to different degrees of enrichments by the principal conterion (e.g. Ca2+, Na+, Li+ or NH4+) containing different amounts of residual toxic element contents. Such differences in H activites should be addressed by researchers before attempting to use H as a model of HS. This urgently requires further research].2.4-1-1 Examples of Literature Reports of Metal Ion Driven H/HS SignalingCf. Tables I-II (page 66 lists selection of available information on the requirement of inorganic cofactors for H/HS signaling.

The modus operandi of the heparanome is believed to depend on selective interactions between proteins and HS proteoglycans which seem to be less determined by the HS proteglycan core-proteins [cf., Iozzo1, Powell et al.] than the microstructure of the (H)-like segments of the HS polysaccharide side chains, but which also involves the inorganic ions putatively associated with such H-like segments. (HS core proteins will, however, augment the ionic strength (I) at sites of interaction, and thereby also influence the rate of formation of ternary complexes between metal ions, glycosaminoglycans (GAG) and target proteins and may also serve to promote the formation of oligomeric metal ions with enhanced abilities to bind HS side chains). Some examples of the growing literature database on the interdependence of H/HS biochemistry and inorganic factors are collected in Table I which includes a list of reports which have noted that metal ions are required to facilitate H/HS protein binding; in Table II lists reports which have suggested that inorganic ions or particles can also directly modify HS microstructures (evidently by influencing the primary assembly or by postsynthetic structural modification). An essential requirement for inorganic cofactors has been reported to allow the binding of H/HS to endostatin (Ricard-Blum et al.1), of H/HS to annexin-V (Capila et al.,1) and for the H/HS promoted basic fibroblast growth factor receptor assembly process (Kan et al. 1). The Aberdeen polysaccharide group redox hypothesis of H/HS signaling (cf. Grant 1, 2000) putatively also implicates the activities of redox metal ions in the etiologies of degenerative diseases. Such putative roles of the inorganic-element matrix associated with H/HS include the generation of oligomers by the action of nitric oxide metabolites are now known (cf. Ding et al. 1) to be dependent on the presence of Cu and Zn cofactors.

General chemical considerations also suggests that oxygen-metal ion containing (polyoxometallates) and related sulphur bridged metal ions might also be considered as possible cofactors for protein HS interation and regulators of H/HS actitities since a ruthenium red polycation [(NH3)5Ru-O-Ru(NH3)4-O-Ru(NH3)5] 6+ has ben reported (Utsumi & Ota1) to bind to cell surface HS and cause cellular agglutination via a HS intercellular crosslinking mechanism. [N.b. small amounts of Ru were detected by ICP-MS in H]. Further research is warranted into the mechanism by which ruthenium red inhibits immunological responses (Dwyer et al., 1) since HS activities and their modulation by Ru (which can also bind to nitric oxide) could conceivably be implicated in such activities. Ba2+ is known to selectively bind (and precipitate) H (cf., Nader et al. 1, 1981). A perturbation of HS signalling following intoxication by Ba2+ might perturbs FGF-II (b-FGF) assisted myelin sheath renewal and thereby contribute to nervous systems pathologies (this was suggested by Purdey1 to explain an apparent correlation between the presence excess Ba2+ in soil and plant samples and the geographical prevalence of multiple sclerosis (e.g. in North East Scotland)). Other possible candidate metal ion cofactors for aberrant HS signalling include the the rare earth metals (which have found general use as animal growth promoters**) as well as Zr4+ , Ga3+ and Tl+ which seem to be prominent components of industrial H before its final clean up. Be2+ and Al3+ (e.g. the polycations formed of Al3+ which binds more strongly to H when in an aggregated form**, might also promote of HS cell surface polysaccharide activation, deactivation or crosslinking in vivo). (Table III (page 72) lists some analogous effects of organic molecules etc., which influence HS synthesis).2.4-1-2 Comparison of Metallomic Multi-Inorganic-Element Arrays in Anionic Polysaccharides with Those in Other Matrices (Cf., Table IV) Spark source mass spectrometry (SSMS) and inductively coupled plasma mass spectrometry (ICP-MS) multi-element analytical data for several H samples from different manufacturers suggest that H always contains an enhanced seaweed-like inorganic element content (recent ICP-MS reports of blood sampling tube H-associated contaminants suggested3b-10 the co-occurrence with H of the full seawater range of some 80 elements 4). Such single ion enriched Na and Li H (e.g. obtained by use of ion -exchange resin column technology for the preparation of medical-grade H) as well as native H, appears to be greatly enriched in those elements which occur in the least amounts in natural waters, a situation which is reminiscent of the cell wall polysaccharides of plants1a-10 where a similar range of inorganic ions as are believed to contribute to cell wall control over Ca2+ second messenger actions e.g., via the borate crosslink polysaccharide oligosaccharide generator1a-10. The occurrence in marine algal tissues of major cell wall polysaccharides in multi-counterion salt forms rather than, as was originally thought, in a mainly free acid form, was established by A. Wasserman3 and confirmed by W.A.P. Black & R.L. Mitchell3a and later workers. It is now thought that marine algal polysaccharides contain most or all of the inorganic elements (60+ in number) which occur in seawater3b from which solution the anionic polysaccharide ligand is apparently able to simultaneously sequester the full range of inorganic counterions and particles there present, by a mechanism which apparently enables these polymeric ligand systems to become selectively enriched in those elements which are the least abundant in this bathing solution.

Haraguchi4 suggests that an updating of the metallomic concept required that many elements which had hitherto been classified as being non-physiological must now be included as being potentially physiologically relevant since the amounts of these elements in human blood serum are approximately correlated with the amounts present in seawater (as suggested by a log-log correlation between these metallomic matrices4). Possible evidence for the relevance of the former non-physiological elements in blood serum to animal biochemistry is that these elements can become selectively enriched in H (cf. Table IV) suggesting a possible modulatory role of these elements for for H/HS** activities incuding for the formation of ternary polysaccharide-metal ion-protein complexes (e.g. similar to those identified by Cornwell & Kruger1). Table IV (page 77) also compares the metallomic profiles of alginate, H and human hair, a tissue commonly employed for the presence of toxic metals in humans (cf., Fig. 2 (page 83)) Table IVa lists results reported for metal ion impurities present in chitosan (their presence in this case was attributed to their uptake from a final washing with tap water13a but further studies to more fully establish the nature and origian of the multi-inorganic elements in chitosan seems to be warranted).

Polysaccharide-rich biomasses of land plants, brown and red marine algae, as well as mollusc shells, bone, mast cell H and H-like segments of HS may constitute a related system of natural (salinity induced) metallomic matrices (cf., Fig. 3 (page 83) which seems also to be related to the inorganic element contents of human hair15 (cf., Fig.2) (which is currently the most well-researched multi-inorganic element containing matrix) and chitosan13a cf. Table IVa). Human scalp hair (e.g., 15 ; except for Zn, which is augmented in hair) is comparable (cf. ratio A/C Table IV) with the data shown for Na and Tl H. This correlation is best for the least perturbed samples, from schoolboys 15 (Fig.2) The binding of a range of metal counterions to H seemed more akin to a (first stages of a) counterion induced phase-separation process (but where final aggregation into solid particles was strongly inhibited) which nevertheless yielded nanoscale quasi liquid phases of similar structures to the non-crystalline polysaccharide glasses obtainable by casting H and similar polysaccharides onto plane surfaces (a method which is commonly employed in industrial laboratories to study thin films of polymers which was adapted by the author for the evaluation of glycosaminoglycans by attenuated total reflectance and related infrared spectroscopic methods (cf. Grant et al.1, 1987). H when added an anticoagulant for the analysis of metal ions in whole blood has been traditionally associated with interference by a range of metal ions (cf. Bowen 3b-1 and other 3b refs.) which seems to accord with the present hypothesis of a ubiquitous H- associated metallomics scenario. H in mast cells was shown, by histological methods, also to bind {Ru2+ 3}6+ [present as ammonia linked metals in ruthernium red]. A range of similar publications also suggest that multivalent metal ions bind to sulphated polysaccharides when used in histological stains or for the spectroscopic evaluation of H 6. Ruthenium red has also been reported to bind and crosslink HS-like molecules at cell surfaces during agglutination of ascites hepatoma cells by this substance (Utsumo & Oda1). The use of Ga3+ in medical scintigraphic visulization of tissues has similarly been associated with the binding of this metal ion to HS. Related in vitro and in vivo binding studies showed that Ga3+, and a wide range of other multivalent metal ions, bonded strongly to H and HS proteoglycans (Kojima et al., 1, 6). The reported anti-tumour actions of Ga3+ might conceivably arise by a related mechanism.

H and putatively also H-like segments of HS have intrinsically or specifically been designed by evolution to incorporation an additional design features relative to their evolutionary precursors which allows for a more flexible system of metal ion binding (cf. Whitfield et al. 1) [whereby the intrinsically low inherent chelationbinding power for metal ions of older polysaccharides is boosted by use of an iduronate plus some glucuronate rather than exclusively glucuronate (as in more primitive extracellular polysacchrides) and also by using both Osulphate and glucosamine N-sulphonate anionic patterns which seems to enablethe selective but flexible easily alterable mode of binding of the ultratrace elements which occur in both blood serum etc. in order to corrrectly facilitate water structure and the resultant binding of specific HS segments within HS chains to their

designated protein binding sites].Biologically-centred metallomic studies should now perhaps be re-focussed on how the inorganic biochemistry animal anionic polysaccharides function in vivo, including e.g. how they confer tissue protecton. It is indicated that there could be a major role for multi-inorganic elements in the promotion or the inhibition of the formation of plaques in arterial and other tissues including those of the central and peripherial nervous systems.

[A long list can be drawn up of academic laboratory reports (cf. Tables I-III (pages 66-79) which seem to confirm that inorganic cofactors are of potential major importance to H/HS signaling; these findings can be tentatively extrapolated to suggest that hitherto not-hitherto-thought-biologically-relevant ultratrace elements become enriched in H/HS where they serve essential functions such as those responsible for increase animal growth rates and affect thyroid functions etc.** when such elements are added to animal feeds].Perhaps a major system of heparanome-metallome-crosstalk underpins much of animal physiology. 2.4-3 H/HS is an evolutionary designed flexible metal ion binding systemNatural polyanionic systems (e.g. the complex anionic polysaccharides systems of heparin, pectin, alginates as well as the anionic humic materials in soils and natural waters) can apparently simultaneously sequester the full range of inorganic ions which occur in the natural bathing solutions by mechanisms which tend to favour the preferential uptake of the least abundant elements present

2.5 Evidence for all-element decorated H Rabenstein1 has noted that sodium H when injected into the blood stream will bind the Ca2+ ions there present, to convert into the calcium salt form. It is now suggested that while this is probably a good initial approximation of the in vivo behaviour of H, a more accurate description seems to be the formation of a Haraguchi metallomic all-element decorated H similar to that which was first identified by Moffat1 and later discussed on the internet by Grant1 (2000) and confirmed by later reports of ICP-MS analysis to multiple inorganic elements present in of several H samples used in blood collection tubes (cf. Table IV). 2.5-1 SSMS results for Na H [this H was donated by a (former) major manufacturer at Runcorn, U.K] (more accurately this is a Na/Ca = ca. 6 H) Ca (30,000), Si (5900), Cl (5600), K (2000), Mg (1300), Fe (1100), F (890), Cu (730), P (440), Ti (390), Ta (280), Ni (170), Ba (140), Br(130), Co(80), Zn(80), Sr(65), Cr(30), B(25), Ga(20), Pb(16), As(15), I(10), Cs(9), Tl(8), La(7), Ce(7), Mo(7), W(5), Sn(5), Nd(5), Zr(5), Ag(4), Rb(3), V(3), Y(3) , Sb(2) and Cd(1).

This Na H can now be seen to be an especially well-defined example of the type of biological metallomic matrix first identified by Wassermann1 and later (less specifically) discussed by Haraguchi1 where the inorganic elements present in biological matrices approximately correlate with those in seawater and human blood serum.These results were obtained in 1983 by C. M. Moffat1 a graduate student working with W.F. Long and F.B. Williamson (the then directors of a Marischal College (Aberdeen University, Scotland, U.K.) polysaccharide research laboratory) who together with J. R. Bacon, a specialist in multi-inorganic element analysis at the Macaulay Institute (also in Aberdeen) conducted a spark source mass spectrometric (SSMS) multi inorganic element analysis (by a method described e.g. in Filip et al.,1 ) of a carefully selected sample of a typical porcine sodium H (which seems also have been distributed to several academic research laboratories) Moffat extensively dialysed this H against deionised water and following lyophilisation of the non-diffusible material submitted it to SSMS analysis without being pre-ashed. [This H had, however, apparently not been cleaned-up by the manufacturer, e.g. by using the standard final ion exchange single counterion enrichment process; this was possibly a deliberate omission since this H had been specifically allocated for fundamental academic inorganic biochemical studies (and had obviously not been intended for medical use, e.g., as a blood anticoagulant, since it greatly exceeded the Pharmacopeial recommendations for heavy metal contents, but otherwise showed the normal 13C and 1H NMR spectra of porcine H and behaved in the expected manner as a blood coagulant)].

Although the inorganic elements present in this H probably altered only slightly its anticoagulant activities it was suggested by comparison with other H samples containing less associated inorganic elements studies of the binding Zn2+ to this and other H samples (cf. Woodhead et al., 1 and results obtained by other postgraduate student working with F.B. Williamson & W.F. Long) that the presence of different amounts of multielements attached to different H samples might have affected its ability to bind further metal ions and also could have augmented their antioxidant and ferrioxidease activites**. The presence of a number of redox metal ions in H and related substances also suggested augmented redox-metal assisted nitrosative reaction processing by such H-like moleucles (cf. Grant, 1, 2000 internet). This idea has not been properly tested by specifically designed experiments since it only emerged as a possible origin of discrepancies of apparent differences in H samples some years after the original laboratory studies had been completed.In his Ph.D. thesis, Moffat1 had partly reported the SSMS results given in Section 2.5-1 of this document where it is indicated that the major counterion is Na (at ca. 96,000ppm). [Although Moffat1 listed SSMS data for for Ti, Zn, B, V, Co, Ni, Y, Zr and Sb in Table 17 (data for Tl H) in his thesis, he did not list the full results for NaH (which were partly reported in Table 15 of his thesis); this was explained as being due to the possible interferences possibly assoicted with the missed out data which also affected, to a lesser extent those SSMS results which he had chosen to report viz. those for F, As, Br Mo, and Ta. These SSMS data are, however, now shown to be essentially correct since they are comparable to values reported by others using ICP-MS instrumentation (cf., Table IV, vide infra) [results reported later on the Internet for other pharmaceutical, medical grade H samples]. All of commercial H samples examined thusfar seem to contain similar amounts of the same multiple inorganic elements to that derived from the Na H reported in Section 2.5-1 following a single percolation through a cation exchange resin (to give the (Tl+) salt form.

2.5-2 SSMS Determined Inorganic Elements Associated with an Experimental Tl Heparin When the above multi-inorganic Na H (2-1-1) was treated with a standard cation exchange resin procedure the resultant single counter-ion enriched H showed an SSMS spectrum indicating that an effective replacement by the selected single counter-ion had been achieved (cf. Grant 1 (1987)); this procedure produced a form of H similar to the types of (single counterion) H commonly used in medicine as also used as laboratory model for HSc. The results are listed in Table IV (page 69). The inorganic elements present in Na(Ca) nad Tl H are compared in Fig. 1(page76).

This single counterion thallium Hd was prepared by a standard cation exchange process achieved with a sulphonated polystyrene resin (Rohm & Hass Amberlite IR 120) column substituted with Tl+ (Moffat1). The SSMS analyses of the starting Na-H and the resultant Tl-H samples indicated that a single passage through this type standard cation ion exchange column achieved replacement by Tl+ of more then 99% of the principal contaminant inorganic (mainly cat)ions which had been present in this (evidently highly contaminated) starting commercial sodium H (cf. Grant 1(1987)). (A similar effectiveness of use of ion exchange resin columns for the removal of the Al which is present in some commercial H samples has recently been confirmed by Bohrer et al., 1). 2.6 ICP-MS Studies of H3b-10 Dilute Nitric Acid Leachates from Evacuated Plastic Blood Sample Tubes.

Summary of apparent effect on metallomic profiles of H leached from blood collection containers compared to whole H H being a mixture of many individual molecular structures of a related nature which confer different microstructure held information related to the ability to selectively bind counterions and other inorganic ions (as well as proteins). This means that leaching of the H mixture of molecules from polyethylene terephthalate (PET) blood vessel containers (a procedure used to obtain ICP-MS results which have been posted on the internet3b-10) would be expected to fractionate the H in terms of the mass spectroscopic results when compared with mass spectroscopic results obtained by taking, without fractionation, an entire H mixture as received from the manufacturer. A preliminary evaluation of the currently available information on the metallomic profiles of H samples collected in Table IV suggests some fractionation by leaching of H from blood collection tubes by dilute nitric acid applies to the rare earths which seem more abundant in entire H compared to leached H. While the data corresponding to whole H samples used for the preparation of whole blood containers used for clinical analysis is not available to the author an indication of the possible differences between leached H and intact H can be made from a comparison of the available older whole SSMS H and more recent ICP-MS H studies3b-10. [Evidently this information was published to provide backup information on how contaminating inorganic ions are leached from containers and the anticoagulants (including H) used in the makeup of such containers which are believed to be fabricated from PET {which is expected to adsorb H}] The report gives detailed tabulations of ICP-MS results for multiple inorganic elements, which allows the multi-element contents of several commercial samples to be extracted from the reported data; the tabulated data also provided system blanks from non-heparinized tubes. The data confirm earlier indications (Moffat, loc. cit.) that commercial H is a highly substituted multi-inorganic matrix. ICP-MS data for H can be extracted from data which appeared on the Internet in 2005 to indicate that a selective removal of more soluble H fractions occurred during the leaching procedures used to obtain multi-inorganic-element (H-related) for study (cf. discussion of results in footnotes of Table IV). {It is known that H adsorbs at blood collection vessel surfaces, this effect being greater with glass than plastic cf., Tunbridge et al. 1 but this difference may be less for the most up-to-date PET plastic used for blood sampling. Further studies are obviously required to check out this.2.6.1 Re-Evaluation of the Moffat et al. SSMS Data for Na H and Comparison with ICP-MS Data

A re-evaluation of the old Aberdeen (Moffat et al.) SSMS data (by Grant 1 (2000)) indicted that the ion exchange ion replacement procedure which had been used to prepare the Tl H (and conducted in association with the author) had been more complex than required for a simple counterion replacement process. Some of the ultratrace elements present (e.g. cerium) is now suggested to be bound much more strongly to the H than would have been anticipated by a simple electrostatic counterion attachment process (cf. Grant internet, 2000a). The ion exchange process was re-assessed creating skewed but essentially retained the distinct original multiinorganic element matrix related to that of the starting Na-H, albeit this was greatly diluted with a greatly enhanced presence of Tl+ couterions. These data, when compared with those from non-mass-spectroscopic inorganic analytical reports 3b-1 to 3b-8 and (somewhat more accurate ICP-MS) mass spectrometric multi-element analyses data 3b-10 later reported for further H samples confirmed the notion that H and H-like polysaccharides always occur in vivo as multi-element organometallic composite mixtures, which, even after extensive purification, retain a skewed remnant polyinorganic profile related to the original native H metallomic profile (which approximately correlates with the metallomic profiles of seawater, human blood serum and many other biological metallomic matrixese and hence suggests the possibly co-existence of an abundant metallomic array with the (e.g. juxtaposed) polysaccharide chains of H/HS and other proteoglycans in vivo. 2.7 Toxic Inorganic Elements in Natural Anionic Polysaccharides A range of toxic elements occurs in algal polysaccharides, chitin and chitosan as well as in fulvic acid soil and fossilized soil derived humic polymer. The relative amounts of such elements seem to be similar to those present in H which suggests that they arise from a similar process of preferential sequestration by naturally occurring polyanionic polysacchrarides and analogous polycarboxylic acid substituted organic humic polymers**.

2.7-1 Toxic elements in H Currently available evidence suggests that H is an optimum metallomic matrix (hence H is expected also naturally to contain {unless subjected to ion exchange purification} exceptionally large amounts of toxic inorganic elements, (e.g. many commercial H samples were found by Bohrer et al.1,2004 to contain unacceptable amounts of Al (which was efficiently removed by percolation through an ion exchange column); As was also found by Bohrer et al., 1,2005 to occur in commononly used clinical chemicals including H) [N.b., when bound to H (e.g. when used medically as a blood anticoagulant), these elements may, howver, normally be inert in vivo. A similar inertness of toxic metals in the H-like segments of HS proteoglycans can be predicted. Inappropriate nitrosative scission of HS, however, may occur under pathological conditions (cf., e.g., Grant 1, 2000, internet dg2)] which could over-ride the toxic metal pacification mechanism and result in tissue damage arising from release of such toxic heavy metals within the patient. 2.7-2 Multi-inorganic-elements in common laboratory reagents It should be noted that multi-inorganic-elements are also found by modern instrumentation to occur ubiquiteously in various common laboratory reagents (e.g. in simple salts and man-made compounds such as ethylendiaminetetraacetic acid (EDTA) which is used (analogously to H) as an anticoagulant in blood collection tubes sometimes used together with inorganic gels (e.g. those which are silicate-based blood clot stabilizing agents) (cf., 3b-10). The metallomic profiles for EDTA and the clot stabilization gels seemed at first glance to have a H-like multi-inorganic-element nature, but a closer examination suggests that the inorganic profiles of the man-made chemicals are distinctively different and less contaminated that are those of biological ligands; the multielements in the man-made chemicals could also perhaps ultimately be derived from those present in rock salt NaCl (derived ultimately from seawater) but is generally found to be less by one to two orders of magnitude on a molar basis than those which are associated with H. (3b-10). 2.8 Other suggested metallomic-related activities of animal polysaccharides: modulation of water supramolecular structure, calcification and deactiviation of Fenton reaction catalysts Animal polysaccharides are known to modulate normal and pathological calcification and provide antioxidant and antinitrant protection by binding and deactifying toxic metals including inappropriately unliganded redox metals such as Fe3+, Fe2+ and Cu2+. In animals an altered HS biosynthesis seems to occur in direct response to changes in Ca2+, Mg2+ and Mn2+ as well as NO and H2S and putatively other inorganic factors such as silicate nanoparticles and rare earths elements (the presence of optimal amounts of which may be required for optimal vascular health and to achieve promotion of growth and wound healing). This can putatively be achieved by increasing the sequestration power (e.g. for an insufficient presence of essential inorganic elements or for a need to pacify toxic inorganic elements) by an alteration of the Golgi apparatus biosynthesis of HS chains (cf. the reported alteration of HS biosynthesis in reponse to the presence of enhanced amounts of those inorganic elements (such as Pb, Cd and Hg and perhaps also Ba and Al and Be) which are detrimental to health, as noted in Table II. 2.8-1 Other cell surface polyanionic (potential metallomic) systems: polyphosphate, poly- hydroxybutyrate & teichoic acid Various metallomes H/HS, polyphosphate16and poly hydroxybutyrate 16a may determine, perhaps in a cooperative manner, the metal ion binding and associated water activities at cell such surfaces and intracellular organelles.Putative metallomic matrices are constituted by anionic phosphate groups which include apatite minerals and hydroxyapatite composites of bones and teeth and the high molecular weight polyphosphates which have been reported Kornberg 16 to occur at cell surfaces throughtout biota; the association of P with heparin (cf. Table IV) may indicate the occurrence of phosphate-bound metals here also; poly hydroxybutrate which is also apparently present (in a similar to anionic polysaccharides) at the cell surfaces of a wide range of species.

Cf. also associated with Gram positive bacterial peptidoglycans are highly anionic teichoic acids and teichuronic acids (e.g., Bhavsar et al.1 ) which constitute metal ion binding (and water activity regulating) activities. Related phenomenon will also, it is suggested, pertain to the geneome**. 2.8-2 Possible inorganic phosphate containing high affinity metal io binding sites in HThere is a possible role of inorganic phosphate/polyphosphate associated with H for the creation of sparse high affinity metal ion binding sites (these show up in the electrometric titration of H e.g. with Cu2+ (cf. Grushka & Cohen 1). Studies of the binding of Cu2+ to H reported in the literature (Grushka & Cohen1) suggested that H might possess small amounts of high

affinity metal ion binding sites. Later studies suggested that especially in multi-inorganic H suggested the presence of such high affinity Cu2+ binding sites (these might e.g. have been at inorganic P associated with H) were further implicated in the ability of H to act as an antioxidant and perhaps also as a ferrioxidase. Further work is required to more fully establish this possibility.** Use of putatively ion exchange cleaned up Celsus H for modern spectroscopic evaluation of the major mode of Cu2+ binding clearly identified a tetracoordinated Cu2+ ion bound to carboxylic acid group, the ring oxygen of iduronate-2-O-sulphate, the glycosidic oxygen between this reside and the adjacent (towards the reducing end) glucosamine and the 6-O-sulphate group (Rudd et al., 1).

2.9 Metal ion assisted H/HS protein binding A re-evaluation of the authors published (mostly peer-reviewed) studies of how metal ions bind to H (cf. Grant et al., 1, 1984-96 ; cf. also the comprehensive list given by Long1, 2003) is now attempted in the context of an evaluation of the above heparanome-metallome hypothesis. A selection of relevant literature reports are collected in Tables I-III (pages 62-73). A key additional paper which further lends support to the notion that inorganic cofactors are essential for H/HS signaling, is the careful investigation of metal ion binding to modified Hs using modern CD and NMR methods reported by Rudd et al., 1.

2.9-1 Servo Feedback Signaling via the Heparanome-Metallome could have Facilitated Animal Evolution (Cf. also Section 6.3) A system of post-synthetic editing and servo feedback control modulation is suggested by the data collected in Tables I-III (vide infra pages 62-73) to be afforded by the metallome. This is suggested to be achieved by the requirements for metal ions for HS-synthetic and HS-degradating enzymes and the facilitation by metal ions of HS binding to target protein and also the catalysis of nonenzymic pathways of degradation of HS (e.g. by nitric oxide metabolites). These pathways seems to point to a system of similar or greater complexity to that achieved by the previously suggested servo feedback signaling processes which are believed to be afforded by H-degrading (Zn-dependent) endosufatases [cf., Morimoto-Tomita et al.1]. The ability of the heparanome-metallome to interact with the geneome could suggest a mechanism by which alteration of genes might be facilitated via the servo controlled system facilitated by HS in order to counterbalance sustained environmental stresses. This provides a new working hypothesis for how animal evolution could occur**. A system of biofeedback involving altered biosynthetic and postsynthetic alteration of HS microstructure, which evidently can occur in response to various environmental inputs, is suggested by the data currently available Such data includes the effects of inorganic cofactors have been reported to engage in direct metal ion dependent HS segment-protein interactions cf., e.g., Kan et al. 1 cf., also Rudd et al.1, and similar reports listed in Table I and Table II is a collection of reports which suggest that the presence of Na+, Ca2+, Mn2+, Zn2+ and Cu2+ ions can influence HS synthesis and alter HS activity and Table III lists reports which suggests that various small organic molecules, which might influence redox status, e.g., ascorbate, can signal for the upreguation of altered, e.g., more highly sulphated, HS molecules. 2.9-2 Inorganic Cofactors for Nitrosative Structural Alteration of H/HS Inorganic cofactors which can contribute to the nitrosative structural alterations of H/HS (e.g., as described by Cappai et al.1, cf. Ding et al.1) include Cu1+/2+ and Zn2+ ions. The range of inorganic cofactors which occur in pharmaceutical H and which could also occur in HS PG in vivo include ions viz., Ag, Au, Ce and other rare earths, Fe, Hg, Mn, Mo, Ni, Os, Pd Pt, Rh, Ru, Sn,Tl, Ti, U, and Zn (cf. Table IV and also Grant 1, 2000 internet dg2) which should be tested as possible catalysts for augmenting nitric oxide metabolite reactivities. 2.9-3 The Selective Degradation of H/HS by Actions of Redox Metal Ions This includes the older findings of Z. Liu and A.S. Perlin who showed 1c that Cu2+ caused selective free-radical degradation of H and a related study by R.N. Rej et al 1d, which found that trace amounts (ca. 1/185 mol ratio with respect to H) together reactive oxygen containing free-radicals generated from added H2O2, plus ascorbate, caused a marked reduction in the anti-FactorXa activity of H without causing any detectable alteration in the NMR spectrum of the polysaccharide (i.e. the proportion of sites affected was too small to show up in NMR spectra).

The recent study by complementary SRCD, NMR, FTIR and EPR of the site-specific complexation of small amounts of Cu and larger amounts by H reported by Rudd et al.1 extends the understanding of the possible role of Cu as a modulator of H activity. The initial binding phase (15-20 Cu(II)/H molecule) involved specific coordination to four binding sites: the carboxylic acid groups and the ring oxygen of Id2OS, the glycosidic O behind this resiude and the adjacent glucosamine and the 6O-S group. A later binding phase showed little structural specificity. The porcine mucosal H used for this study was obtained from Celsus, USA [who in an internet document cite the Grant et al.1, 1987a work where a somewhat overoptimistic assessment had unwittingly been given of the single counterion nature of standard medical grade H) Nagasawa et al.1 reported that oxidative damage to H caused by actions of redox-active (e.g. iron) ions were of a highly selective nature (e.g. causing preferential destruction of D-glucuoronate rather than L-iduronate anionic groups).

2.9-4 How different HS polymers could selectively bind variants of FGF in vivo: hints from lipoprotein binding studies The divalent cations which facilitate FGF receptor assembly (Kan et al., 1 ,cf . Rudd et al.1)) by HS seems to be a related phenomenon to how multivalent metal ions form ternary complexes with polysaccharides and lipoproteins (the basis of laboratory blood lipoprotein evaluation methods, e.g., using Mn2+ and Ca2+ ion plus sulphated polysaccharides (preferably H) a process which was discovered during early researches into lipoprotein fractionation to allow the efficient precipitation of lipoproteins containing phospholipids and thereby to further provide a quick assay for blood serum LDL/HDL (Cham 1, cf. also e.g. Seidel et al. 1, Cornwell & Kruger1). It is now tentatively proposed that similar metal ion modulated polysaccharide protein interaction could also be relevant to the current discussions of the mechanisms by which different HS polymers selectively bind variants of FGF in vivo (Kreuger et al. 1 cf., Powell et al.1). Early researches had established that exogenous H released lipoprotein lipase (LPL) from (putatively HS at) vascular surfaces (for which action the N-SO3- groups in H/HS were of relevance to the interaction between these polysaccharide and the enzyme). {Later workers (Sivaram et al.1) established that endothelial cells synthesize a Hreleasable high affinity LPL-binding protein hrp(H-releasable protein}. Sulphated polysaccharides including H were found to be u