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The Large Range of Inorganic Elements Which Could Normally be Associated with Heparin/HeparanSulphate in vivo Constitute a Haraguchi-Metallomic Matrix which is Suggested to Facilitate theSystemic Management & Protection of Animal Organisms via Signalling by these PolysaccharidesDavid Granta, *Turriff AB53, 6SX UKOriginally drafted in 2007/updated 2009It is proposed that sulphated polysaccharides will become multi-element reservoirs following their equilibration

with the multi-element containing biological fluids in vivo.

SummaryThe uptake of inorganic ions and particles by biological polysaccharides may be a part of a general

poly/oligosaccharide signalling system occurring throughout biota which depends on a coordinated interactionbetween inorganic biochemistry and non-nucleic acid based systems which can influence the proteome. Suchbiological information processing systems have been suggested to exist in order to account (as has been found to berequired in the post-genomic era) for the insufficiency of known control mechanisms (e.g. research into howoligosaccharides generated from pectins, originally thought to influence cellular activities by hormone-like actionsact via influencing Ca ion second messenger activities could suggest the existence of similar polysaccharide-basedsystems in animals). All of the above systems could conceivably by perturbed by the presence of anthropogenicallyintroduced inorganic ions and particles which could perhaps especially target the nitrosative cleavage signalling

pathways which may further depend upon the correct levels of supply of Cu and Zn ion cofactors .

Apart from Ca2+ and other major inorganic ions, trace and ultratrace amounts of

inorganic elements, which seem to have no apparent physiological function, occur in

human blood serum in amounts which are approximately exponentially correlated with

the amounts of such elements in seawater [1]. The existence of a metallomic seawater

range of inorganic elements in blood serum is consistent with the idea that animal life

first started in the sea which obviously is the source of the wide range of inorganic

elements which occur in commercial kelp [2] in association with the cell wall anionic

polysaccharides. A similar polysaccharide-based metallomic matrix can now be

predicted to occur at animal cell surfaces associated with the ultra anionic polysaccharide

structures present in heparin (H) like segments of heparan sulphateb (HS) [3] which seem

to be uniquely constituted to simultaneously sequester a metallomic matrix.

Footnote a

Former industrial and academic research chemist with experience in silicic acid production during silica sol manufacture as describedin British Patent 1,143,019 (to Monsanto Chemical Ltd)Footnote b

E.g.highly anionic side heparan sulphate chains occur in e.g. the sydecans, glypicans, perlecan, agrin and collagen XVIII HSPGswhich abundantly occur at the surface of blood vessels and in glycocalyxes.

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The sequestration and exchange of inorganic elements from biological fluids

by natural polyanionic ion-exchange systems could be pertinent to a fuller understanding

of how polysaccharides contribute to signalling systems throughout biota, a phenomenon

which conceivably contributes to the mechanism by which the nucleus interacts with

the environment so as to influence the evolution of species. In plants, pectin

oligosaccharides were originally supposed to engage in direct hormone-like activities, but

more recent work has identified their prime function to be the modulation of Ca 2+ second

messenger actions[4]; HS in animals is believed to act analogously [5].

Direct evidence for an association of Haraguchi-metallomic matrices with

polysaccharides includes the mass spectrometric analyses of commercial kelp

and various commercial H preparations (cf. data listed in Table I which lists data from

researches aimed at establishing the purity of commercial H and how this is altered

following a standard ion-exchange purification process [6][cf. 7]). More recent ICP-MS

data is for multi-elements leached from heparinized blood collection vessels. Results

are compared in log-log plots in Figs 1.

These data support the notion that animal cell surface anionic polysaccharides willalways

conform to a Haraguchi-type metallomic matrix pattern.

Although the results shown in Table I indicate natural anionic polysaccharide matrices

commonly contain the same range of inorganic elements as those which occur inseawater

and blood serum [1], the anionic polysaccharides apparently become selectively enriched

in the least abundant inorganic elements present in their bathing fluids. Such behaviour

leads to an enrichment of (apparently) non-physiological elements attached to the

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polysaccharides. (Apparently) toxic elements might therefore normally occur in larger

than expected amounts in H and by inference in HS as a result of their in vivo extraction

from biological fluids where they normally occur only in very small quantities.

The presence of inappropriately excessive amount of such elements in H/HS may

therefore contribute to the aetiologies of neurological diseases (such as Alzheimers

disease (AD)) which are believed to involve the dyshomostasis of both metal ions andHS

related signalling systems [7a].

The determination of the inorganic element profile of human and animal anionic

polysaccharides can be suggested to be of potential value in probing the aetiology and

role of such toxic elements in diseases and be diagnostically useful indicators in a similar

but perhaps more exact manner to the determination of the inorganic elements in human

hair [8], (cf., the data in Table I indicate that the majority of the inorganic elements in

porcine H are also approximately correlated with those in standard human hair samples,

cf. Fig 2).

It had been well known for many years [9,10] that a wide range of inorganic elements

can occur in (unpurified, unfractionated) H, and this makes this traditional form of H

unsuitable for use as a blood anticoagulant for some at-risk patients [10] c and for the

determination of the inorganic element contents of any blood fractions which have come

into contact with such H).

footnote cSome previously unaccounted for differences in the chemical reactivity of H reported in the literature [11] could also have arisen fromthe presence of different amounts of inorganic elements in traditional H preparation. While the efficiency of multi-element removal isless for the more highly charged counterions most inorganic elements can be easily removed from anticoagulant H (e.g. Ca2+ or Li+ -substituted H [7]), low molecular weight H and will initially be absent from the antithrombin binding pentasaccharide epitope of Hobtained by direct chemical synthesis [12]]. The author found that Al3+ is bound more strongly to H than a range of othercountercations studied [13] and also that H-Al3+ created a pH 4.6 buffer in solutions containing atmosphere induced HCO3

-

concentrations while a similar saturation of H by physiological counter-cations creates a pH 7.3 buffer system. [The studies reportedin Table I used an Al sample preparation procedure disallowing this SSMS assay method for this element].

Nb. It should be stressed that there is currently no evidence of any toxic actions of those potentially highly toxic elements which occurin small amounts in H , kelp or the apparently analogous multi-element containing traditional medications such as (Ayurvedic)Shilgajit [14] or their USA equivalents [15]. H and HS and other polysaccharides and highly anionic polymers in general may

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actually function to protect the organism against such toxic elements, however, toxic elements may become desorbed during H/HSsystem degradation by excessive NO production or inappropriate heparanase activity (e.g. that associated with neoplasia).

It was noted that the same series of curves (Fig. 2) which describe the alkaline contentsof

the seven mammalian H samples also approximately described the alkaline earth contents

of other (invertebrate) seawater-bathed polysaccharide containing multi-element matrices

[18a]; a more exact mathematical correlation also exists between the total

required amounts of such polysaccharides (which include HS) and habitat salinities of

fifteen species of aquatic invertebrate organisms [18b] which suggests the existence of

some ion-exchange inorganic element sorting processes which could be dependent on the

amounts of anionic polysaccharides present in these species.The occurrence of alkaline earths in five commercial H samples [16], when combined with the similardata for two further H samples listed in Table I, suggests that various multi-inorganic element contents ofH samples arise, in part at least, during the final industrial ion-exchange resin multi-element removal

processes where slight differences in ion exchange procedures are applied to a high-multi-element-content starting H; alternatively some equivalent in vivo partial ion exchange process, e.g., with otherglycosaminoglycans and polyphosphates [17a] may occur. Further work is required to confirm thevalidity of such hypotheses.

Polyphosphate-metal ion-poly--hydroxy butryate [17b] apparently exist at cell surfaces throughout biota; poly--hydroxy butyrate calcium polyphosphate complexes show voltage activated channelbehaviour, be impermeant to Ca2+ Sr2+ and Ba2+ and blocked in a concentration dependent manner byLa3+, Co2+,Cd2+ and Mg2+ ions, in that order ; the polyphosphate-calcium-b-hydroxyl butyrate were suggested tohave been the evolutionary precursors of proteinaceous Ca2+ channels [cf. 17b] (analogous

polyphosphate-calcium-anionic polysaccharide complexes possessing similar activity might, it can nowbe suggested, also have performed a key evolutionary role).

The binding of inorganic elements to these polysaccharides is possibly linked to a servo

control system interlinked to the Golgi biosynthetic apparatus which changes theamounts

as well as the microstructures of the smart polysaccharide-based signaling systems which

are required to optimize the successful acquisition and control by the organism of

inorganic elements and organic nutrients and change these in accordance with

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evolutionary adaptation to environmental niches.

Numerous academic biochemical researchers have made use of H, not to study its metal

ion binding or related inorganic ion exchange properties, but in the perhaps much more

challenging intellectual context of the use of the library of different encoded sequencesof

sulphated sugars present in mast cell H which can be used as models of the complex

HS signalling systems which depend, in a manner somewhat analogous to DNA and

RNA, upon H/HS sequences. However, after such H/HS fragments have been subjected

to similar methods to those used to purify proteins, the necessary inorganic co-factors

may be missed out; and by this inadvertent alteration of the inorganic co-sphere

thereby may downgrade the specificity of the HS signalling, especially forin vitrostudies.

Such attempts have nevertheless been partly successful in unravelling the

biochemistry of the heparan sulphate (HS) side chains of the proteoglycansb [HSPG] of

the heparanome [19] the hypothetical high rank managerial system which is thought

to determine a wide range of biochemical activities [20, 21].

Further indications of the putative roles of inorganic ions in HS biochemistry comesform

studies of the modus operandi of 69Ga scintigraphy [22] which seemed to depend on the

formation of Ga-HS adducts (supporting experiments to these studies showed that HS

binds a range of other multivalent inorganic cations in vivo perhaps analogously to how

such elements bind to H).

Such, relatively specific inorganic interactions, may also be involved in e.g.

the anti-viral binding of HS to virus cell surface binding sites [23], the inhibition of

pathological crystallisation by the binding of H/HS to colloidal sized inorganic particle

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seeds (e.g. [24]), the possible provision of organo-metallic catalyst sites with anti-

oxidant and anti-nitrant activities [25] as well as for the modulation of lipoproteinlipase

and more general lipoprotein glycosaminoglycan interactions (cf. the original Mn2+ - H

method of fractionating lipoproteins) [26] and for the non-enzymic de-N-sulphonation of

HS in vivo (a necessary step in nitrosative deaminative cleavage [27]).

It is now suggested that the topical interest in theoretical modelling of systemicbiological

control systems based on the complex interactions between the proteome and polyanions

[27] must now logically also require the inclusion of the heparanome as well as the

metallome e (such interactive systems might be involved, it can also be suggested in the

evolution of animal species could ultimately be based on mutual interactions between of

the geneome, the proteome and the heparanome-metallome.

Footnote eA term originated by RJP Williams; the scope of this concept has been extended by H Haraguchi.

The heparanome-metallome interfaces the environment directly and also is deeply

involved in possible feedback systems to the geneome [27a]; this is obviously the most

direct way by which animal form can be altered to optimise the species in its habitat

environmental niche. It might be suggested that experimental evidence for this

hypothesis might be obtained by extending the types of studies which were reported in

ref. [18b] where the HS and related polysaccharide systems change to optimise the

aquatic inorganic salt environments. [It should be noted that the above interpretation of

these results was not that made by the authors of ref. [18b] who favoured the alternative

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idea that the different amounts of HS and other polysaccharides were required to counter

the tissue disintegrating effects of an altered osmolality].

For the evolution of other multicellular systems in plants the equivalent polysaccharide-

based-metallome system can be invoked.

Some Roles of Metal Ions in the Heparanome.

Evidence for inorganic signalling via the heparnome includes:

{1} the requirement ofCu and Zn ions for the generation of signalling oligosaccharides

via the nitrosative cleavage of H/HS [28] and

{2} specific inorganic ions are required to allow correct HS epitope protein recognition

and binding [29] i.e. the formation of ternary complexes between specific inorganicions,

H/HS epitopes and growth factor receptors during wound healing and embryogenesis,cf.,

Kan et al., ref. [29h] and

{3} the apparent normal in vivo use of inorganic ions and molecules (e.g. NO) within

servo feedback loops to the HSPG biosynthesis process in the Golgi apparatus inresponse

to stress, e.g., following perturbation of major extracellular physiological inorganic ion

concentrations [30],

to act as sensors of the presence of (insufficiency of) ascorbate [31], glucose [32],oxygen

[33], blood flow rate [34]; the presence of endotoxins [35], the distribution of specific

lipoproteins and other dietary factors, e.g., retinoic acid [36], xylosides [26], and

polyamines [37];

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{4} the possible role of inorganic elements in H/HS for the facilitation

of the further formation of complexes between HS-bound metal ions and nucleic acids,

amino acids, nitric oxide, its metabolites, oxygen, carbon monoxide, carbon dioxide,

sulphur dioxide and thiocyanate.

[This provisional list can perhaps be considerably extended to include the role of metalions in prion biochemistry, neurotransmitters and amyloid proteins, as indicated by thecurrently available published researches which seem to suggest a possible involvement ofHS in neurological dysfunction and related diseases such as chronic fatigue syndrome[38], multiple sclerosis (MS) [39] and Alzheimers disease (AD) [39a]].

A possible example of toxic metal ion perturbation of HSPG signalling has been the

suggested major role of Ba2+intoxication upon the HS-dependent myelin sheath renewal

in the aetiology of MS [39] and dyshomeostasis of Zn2+ in HS-related systems in AD

(reviewed by the author [in internet document 5a]).

Other putative roles for multi-elements in HSPG may be to augment ferroxidase activity [40] and provide for the a fine-tuning ofassociated hydration structures at HSPGs surfaces which may enable proton conduction ; presumably these functions will require

correct inorganic element arrays to be present.The similarity between the hydration behaviour of H metal ion complexes studied as thin films by infrared spectroscopy and man-

made ionomer membranes [41], suggests that H and HS are proton conductors and furthermore this function may be at least partiallydependent on the occurrence of SiO2 in association with these anionic polysaccharides [42].Iinduced metal ion alteration of associated biopolymer including H/HS water activity alteration could be the ultimate reason for anassociated pathological alteration of protein folding (cf. this is also known to be why the Hofmeister effect depends on wateraggregate formation in the presence of inorganic ions [43]).

Appendix

Log-log Correlations Between the Multielements in Natural Polyanionic Materials and Seawater.Using the log-log plot method employed by Haraguchi [3] to demonstrate the approximate correlation

between seawater and blood serum multi-elements with the Microsoft Excel leastsquares fitting programme [from which a comparison of the degree of correlations

between multi-element contents of different media can be made by determining thelinear least squares fitted curve scatter factor r2 (where the correlation is exact forvalues of r2 = 1.0 and absent for r2 = 0)].

Mammalian Multi-Element Sampling (Human Hair & Porcine Heparin)Multielements in heparin were correlated with most results from human hair [8] especially with lesscontaminated 15 element data from schoolboys [8a] which showed r2=0.9. (cf. Figs. 1).

The Alkaline Earth Contents of a Series of Seven Hs [18] (Figs. 2)These were found to be correlated with each other and also with seawater as well as a range of otherseawater-bathed matrices (coral, mollusc shells, a related bone-like material, red and brown algae [18a]).The most easily ion-exchanged element in multi-element H was Ca, which was reduced by a factor of1000 after a single passage through an ion exchange resin column. This allows a numerical estimate to

be made from the residual Ca content of H of the degree of ion exchange of individual matrices.

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This value ranged from 1.0 for mollusc shells etc.(seawater-bathed matrices containing sulphatedpolysaccharides [40]) to 0.0 for the most highly purified H (a Tl H, the multi-element content of which islisted in Table I). Using these values a semi-log plot of the alkaline earth contents of the seven H andfour other putatively related matrices gave a linear correlation with with r2 values for Ca, Sr and Ba beingrespectively 1.00, 0.87 and 0.50. By inference the other natural inorganic element contents of H arehypothesised to be similarly correlated.Multielements in Seawater & Heparin (Figs. 3)

Na and a Tl heparin heparin [Table 1] derived from porcine mast cells showed a positive correlation withr2=0.8 (cf. Figs. 3).The data for seawater are correlated with those for blood serum as discussed in ref [3]. The data for theformer medium are of a higher quality than those currently available for the latter.While the Haraguch log log plot of the multi-elements of blood and seawater showed a correlation valueof r2 = 0.45 a similar treatment of the 38 most abundant elements in heparin showed a higher correlationvalue of = 0.84.Multielements in Marine Algal Biomass (Figs. 4)Alginate-based seaweed biomasses [31] showed typical r2= 0.8 correlations with seawater but were lesswell correlated for Baltic (polluted) seaweed[4a], with r2 = 0.65)).

Multielements in (Geological) Fulvates (Figs. 5)There is a wealth of data available from soil analyses. However, perhaps of especial interest in thecontext of complementary and traditional medical use of dietary supplements is the use of a multi-element containing geological soil humus derived material in Asia and a similar geological depositapparently also employed by indigenous peoples in North America, and now mined for human dietarysupplement use. In Europe, a similar traditional medicine therapeutic use seems to have been put ofmineral wells containing humic matter for skin applications etc..A correlation with biological seawater-like fingerprint is apparent for the multielements in geologicalfulvates [15] r2=0.6 (but other commercially available 70+ multi-element dietary supplements preparedfrom aqueous extracts of humic shale [15a] showed r2=0.05).

Table 1Inorganic Elements in Native (Na) and Countercation-Exchange-Enriched (Tl) Heparin This (toxic? form of

heparin was prepared to investigate heparin by 205Tl NMR (which showed a coalesced pattern of rapid site interchange sites).

Kelp and the ratio of Elements in NaHeparin/human hair (compiled from internet tabulations) Na Heparin Tl+ HeparinA Kelp

B Ratio of Inorganic Na Heparin/ Na Heparin/ CElements Human Hair Earths Crust

Cs 9 0.1 trace 4500 43-------------------------------------------------------------------------------------------------------------------

Tl 8 principal 2.93 640 2000 Na 90000 90 588 3

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

Ti ca.390 4 0.12 ca. 130 (RA)D 0.3 ca. 500 (SVC)D

-------------------------------------------------------------------------------------------------------------------La 7 1 0.19 156 11W 5

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Nd 5 1 128 4Ce 7 3.5 trace 75 4

K 2000 40 12800 100 0.8

Co ca.80 2 12.2 ca.100 0.8Ni ca.170 10 35 ca.100 0.1

Mo 7

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

Cl 5600 1000 36800 ca.10 329Br 130 0.9 trace 4.5 2600I 10

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river and sea water so as to give rise to this approximate but recognisably exponentially correlatedinorganic element distribution pattern.

For H the uptake of multi inorganic ions probably involves the equilibration of animal mast cells

with blood serum with some additional input from impurities present in aqueous solutions and extracted

by H from container surfaces during manufacture, storage etc.

Plant pectins, marine algal polysaccharides, glycosaminoglycans and perhaps also poly-hydroxylbutyrates [17a] can now be predicted, on the basis of preliminary observations of the multi-elementnature of H and reported data for other natural polyanionic matrices, to naturally exist as MFs (as aconsequence of theirin vivo equilibration with multi-element containing biological fluids or naturalwaters). [A different, more specific type of countercation uptake is predicted to occur with

polyphosphates [17b] and phosphate esters]. Some trace and ultratrace elements (such as rare earths andhydrated Fe, Al and Ti oxides which, in possible association with silicic acid, phosphate and Cu asoligomeric inorganic moieties) and proposed that the non-physiological elements in these solutionsshould now be added to the 20 physiological metallomica metal ions; more accurate analytical studieswill be required to identify further such inorganic elements in animal tissues to fulfill the Haraguchihypothesis [3] .

The ability of polyanions to inhibit the crystallization of sparingly soluble salts such as CaCO 3 and BaSO4could create a homoestasis system of supersaturated solutions. Soil water, natural waters like seawater(from the action of humic acid salts) and biological fluids (from the action, at least in the early stages oftheir evolution, by anionic polysaccharides) could be examples of such systems.

It is now also suggested that over-purification of H-like HS epitopes may also

deprive them of their full biological activity. This could explain why highly purified HS

epitopes failed to demonstrate an expected high selectivity between FGFs [49].

It is also suggested that the presence of such multi-elements in anionic polysaccharides facilitates many

more biochemical interactions (including those of H/HS) than have been identified to date, and may

provide a major inorganic ion metallomic-library required by H/HS for wound healing, the

assembly of the embryo as well as various immune, anticoagulant, antioxidant and antinitrant and control

of normal and pathological calcification function.

It is now desirable that the multi-element nature of H/HS be confirmed by others. It is suggested that thisa more exact analysis of this polysaccharide could establish the Haraguchi hypothesis of the existence ofan extended metallomic system in animal tissues.

*Compiled post a fixed-term research study with W.F. Long and F.B. Williamson at Marischal College, University of Aberdeen, U.K.who are thanked for their support. The above hypothesis was also suggested of the authors previous researches at Monsanto Co.,Monsanto Chemicals Ltd., ICI Ltd., International Synthetic Rubber Co. Ltd., and the University of Aberdeen (Departments of SoilScience and Department of Biochemistry/Molecular and Cell Biology) and by discussions with RJP Williams (Oxford University)e

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Note added after the drafting of this report.The internet document entitled Contamination of whole blood/serum from sampling equipment which posted by

ALS tabulates the full ICP-MS determined content of more than 70 elements leached from heparinized bloodcollection tubes by dilute nitric acid; the co-extraction of inorganic elements with H (the amount of inorganic

sulphur present allows the amount of H extracted to be estimated it is evident that per anonic site H is much moreextensively associated with multi-elements than is EDTA.These data fully confirm the hypothesis outlined in the

present article, that H always exists as a seawater-likeHaraguchi-metallomic-matrix;

References

[1]Haraguchi H. Metallomics as integrated biometal science. J Anal. At. Spectrom., 2004; 19: 5-14

[2] Cf., multi-element analysis ofAscophylum nodosum (quoted by several internet sites relating to kelp elements, e.g.http://www/alginure.co.uk/ascophylum-nodosum.html); Kelp Research; Multi-elements of Ecklonia maximahttp://www.gairesearch.co.za/kelp.html; cf., Davis T.A., et al.,App. Biochem. Biotechnol., 2003; 110: 75-909 ).Cf. Truus K. et al.,Proc. Eston. Acad. Sci. Chem., 2001; 50: 95-103.Davis T.A., Llanes F., Volesky B., Diaz-Pulido G., McCook L., Mucci A. 1-H NMR spectroscopic characterization of sodiumalginates extracted from Sargassum spp. and its relevance to heavy metal biosorption. Appl. Biochem. Biotechnol., 2002; 110: 75-90;Davis T.A. Llanes F ., Mucci A., Volesky B. Metal selectivity for for Sargasum spp.in relation to their guluronic acid content and conformation. Environ. Sci. Technol., 2003; 37: 261267. Cf. A review of the

biochemistry of heavy metal biosorption by brown algae. Wat Res 2003: 37; 4311-4333.The existence of a metallomic matrix in alginates has been known for many years cf., Wassermann A. Cation adsorption by brownalgae: the mode of occurrence of alginic acid. Ann. Bot., 1949 NS; 13 (49): 79-88

[3] A typical carefully selected porcine mucosal H (provided by Dr W.E. Lewis, (Runcorn, U.K.) on behalf of a manufacturer, which

formerly produced heparin in the UK ) was studied by SSMS; the full results were given and discussed for this heparin by Moffat C.F.in Synthesis, Characterisation and Application of Chemically Modified Heparins Ph.D. Thesis University of Aberdeen p 187-8. Afuller report of these results, including the SSMS results for the partially purified product of ion exchange resin treatment, appeared inGrant D. Chemistry Preprint Archive, 2000 Oct., 94-104; available at http://preprint.chemweb.com/biochem/0010002 ;The mass spectroscopic multi-element data for mammalian heparins occurred with a similar distribution but in considerably greateramounts than in agarophytes.

(Literature survey/hypothesis files relating to metal ions and HSPG biochemistry and related topics posted on the internet 2000 ondgrant @ ukonline.com; these can not longer be accessed directly via Google but may be obtained from bt.yahoo.com via

bt.internet.comusing the search term ascorbate and nitric oxide in redox control of heparan sulphateorvia. http://www.health-webdirectory.com/directory/heparan

[4] Balluska F., Samaj J., Wojtaszek P., Volkmann D., Menzel D. Cytoskeleton-plasma membrane-cell wall continuum in plants.Emerging links revistited.Plant Physiol., 2003; 133: 482-491; Melotto E., Labovitch J.M. Biologically active cell wall materials.R

Bra., Fisiol. Veg., 1994; 6: 75-82; cf., McNeil M., Darvill A.G., Fry S.C., Albersheim P. Structure and function of the primary cellwall of plants.Annu. Rev. Biochem., 1984; 53: 625-663

[5] Long W.F., Williamson F.B. Glycosaminoglycans, calcium ions and the control of cell proliferation. IRCS J. Med. Sci., 1979; 7:429-434

[6] Grant D., Long W.F., Williamson F.B. Infrared spectroscopy of heparin-cation complexes.Biochem. J., 1987; 244: 143-149

There have been sporadic literature reports of the existence of potentially toxic levels of metalions including V, Cr, Sr, Ba and As in heparin.Alcock N.W., Serum versus plasma for trace metal analysis, Elem. Metab. Man, Anim. Proc. Int. Symp. 4th 1981 (Pub 1982), Eds.J.M. Gawthorme J.M., Howell J.M.M.M.C., White C.L., p. 6578-680; Springer, Berlin;Chem. Abs., 96, 213646Other indications of the occurrence of a range of inorganic elements in heparin wereHeinemannn G.W., Vogt W. Vanadium-contaminated periopertively administered infusion solutions and drugs. Bio.l Trace Elem.

Res. ,2000; 75: 227-234Katz S.A.Amer. Biotechnol. Lab., 1984, 2, 24-30Cf. Jaques L.B. Heparin: and old drug with a new paradigm. Science 1978; 206: 528-533; Amer. Chem. Soc. Adv. Chem. Ser., 187Sect. 23, p349-3and Muzzarelli R.A.A,Polymer Sci. Technol., 1983, 23, 359-374

An inverse exponential relationship could be demonstrated between the seawater/blood serum concentrationsand the degree of (hypothetical) enrichment of heparin or algal polysaccharides in individual elementsfrom such seawater-like media.The least abundant elements seawater of seawater-like bathing fluids seem become selectively enriched

by anionic polysaccharide ion exchange systems. Pitted against a man-made sulphonate-basedcation exchanger by percolation through an ion exchange resin column the Ca content of multi-elementcontaining heparin was reduced by a factor of 1000 but the other elements present were reduced by lesseramounts in the order of ion exchange efficiencyCa>(Cu,Ba,Ti,Mg,Fe,Cs)>Sr> (Co,Cr, Ni, Ga,Zr)>( K?)>Zn>Ag> (La,Nd,Pb,Ce)

[6a]In vitro studies of the binding of metal ions by heparin

http://www.gairesearch.co.za/kelp.html;http://www.gairesearch.co.za/kelp.html;http://preprint.chemweb.com/biochem/0010002http://www.gairesearch.co.za/kelp.html;http://preprint.chemweb.com/biochem/0010002

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Cf., also JR DunstoneBiochem J. 1962; 85: 336-3514and Grant D., Williamson F.B., Long W.F. et al.,Biochem. J., 1991; 275:193-197; ibid., 1992; 287: 849-853; ibid., 1992; 282:601-604; ibid., 1992; 283: 243-246; ibid., 1992; 285: 477-480; 286: 717-720; Biochem. Soc. Trans., 1991; 18:1281-1282; ibid.,18:1282-1283; ibid., 1992; 20: 361S; ibid., 1991; 19: 390S; ibid, 1996; 24: 203S; ibid., 1996; 24:204S; Rabenstein D.L., et al.,Carbohydr Res 1995; 278: 239-256

[7] Celsus Heparin salts (Internet document) http://ww.heparin.com.heparin_salts.html

[7a]The aetiology of Alzheimers and other neurological diseases could be pertinent to the subject matter of the present hypothesis viz.,involving dyshomeosasis of the interdependent HS - metal ions systems including a possible perturbation by Al3+ of Zn2+ -HSinteractions as discussed in an unpublished University of Aberdeen document prepared by Long W.F. and FB Williamson F.B. towhich the author contributed. The literature survey on which this is based was updated in 2000 and posted on the internet (the file can

be accessed at web.ukonline.co.uk/dgrant/dg8/ Cf. also Bush A.I., Tanzi R.E. The galvanization of-amyloid in Alzheimers disease. PNAS, USA 2002; 99: 7317-7319 Cf. also Mani K., et al.,J. Biol. Chem., 2003; 278: 38956-38965 and the quotation from it given in ref. 28

and Sparks D.L., Schreus B.G., PNAS, USA, 2003; 100: 11065-11069

Cf., e.g., Williamson T.G., Mok S.S., Henry A., Cappai R., Lander A., Nurcombe V., Beyreuther K., Masters C.L., Small D.H.,Secreted glypican binds to the amyloid precursor protein of Alzheimers disease (APP) and inhibits APP-induced neurite outgrowth.

J. Biol. Chem., 1996; 49: 31215-31221. Cf., Verbeek M.M., Otte-Holler I., van den Born J., van den Heuvel L.P.W.J., et al. (n.b.Agrin is a major heparan sulfate proteoglycan accumulating in Alzheimers disease brain.Amer. J. Pathol., 1999; 155: 2115-2125)

[8] Rodushkin I., Axelsson M.D. Application of double focusing sector field ICP-MS for multielemental characterization of human

hair and nails Sci. Total Environ., 2000; 250:83-100; ibid., 2000; 262: 21-36[cf., also http://www.analytica.se/hem2001/human /research.asp; cf. Bass D.A., Hickok D., Quig D., Urek K. Trace element analysisin hair: factors determining accuracy, precision, and reliability-statistical data included.Altern Med Rev 2001; 6: 472-481 andBleise A.R., et al., Analytical Qualitey Control Services (Hair Elements Reference Sheet) International Atomic Agency, A-1400,Vienna, Austria]

[8a] Senofonte, Volante N., Caroli S. Assessment of reference values for elements in human hair of urban schoolboys.J. Trace Elem.Med. Bio.,l2000; 14 (1): 6-13

[9] Bowen H.J.M., Trace Elements in Biochemistry Academic, London, 1966 on p. 63 it is noted thatcontaminationcomes form the use of the preservative heparin to prevent the clotting of mammalian blood the majority of thedeterminations of manganese in blood which have been published in the literature appear to be determinations of manganese in adilute solution of heparin

[10] Bohrer D., de Oliveira S.M R., Bertagnolli D., do Nascimento P. C.,et al., Aspectos importantes na determinacao do nivel dealumino no sangue do pacientes em tratamento regular de hemodialise.Revista Brasileira de Analises Clinicas, Rio de Janeiro(RBAC) 2004; 36: 99-103Cf., Bohrer D., Cicerodo do Nascimento P., Becker E., Machado do Carvalho L., Dessuy M. Arsenic species in solutions for

parenteral nutrition.J. Parenteral Enteral. Nutrition 2005; 29: 1-7

[11] Folkman J. et al.,Science 1983; 221: 719

[12] Rosenberg R.D. Redesigning heparin.New Engl. J. Med., 2001; 344: 673-675

[13] Cf. Grant D., Long W.F., Williamson F.B. Zn2+-heparin interaction studies by potentiometric titration.Biochem J1992; 287:849-853 which gave a brief mention of the binding of Al3+ to H; a similar paper describing a full investigation of this binding using

potentiometric titration and freezing point depression methods is awaiting publication

[14]http://www.dhanvantri.com/Minerals_Shilagit.htmCf. Saleem A.M., Gopal V., Rafiullah M.R.M. Bharathidasan P.African J. Trad. Comp. Alt. Med., 2006; 3: 27-36 available athttp://www.bioline.org.br/request?tc06027

[15] US Shilagit Cf., http://www.trccorp.com/faq_lab_analysis.phbCf.,http://www. Healthylivingmtl.com/minerals/ingrediants.htmwww.Coral-Cure.com

http://www.eagle-min.com/mass/htmCf. also http://www.quackwatch.org/01QuackeryRelatedTopics/DSH/colloidal minerals.html(this internet document by J. Pontolillo draws attention to the possible hazard of using geological fulvates as dietary supplements;the heparin/heparan sulphate-like multielement nature of such supplements however tend somewhat to counter the arguments made inthis document)

[15a] http://www.mikeschoice.com/faq.htm

[15b] Tabulation of elements in earths crust (data from McDonough W.F. cited in Table Iofhttp://quake.mit.edu/hilstgroup/CoreMantle/Earthcomo.pdf)

[16] Harrison G.E., Sutton A. Alkaline earths in heparin.Nature, 1963, 197, 809

http://www.dhanvantri.com/Minerals_Shilagit.htmhttp://www.dhanvantri.com/Minerals_Shilagit.htmhttp://ww/http://ww/http://ww/http://www.coral-cure.com/http://quake.mit/http://quake.mit/http://www.dhanvantri.com/Minerals_Shilagit.htmhttp://ww/http://www.coral-cure.com/http://quake.mit/

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[17a] Reusch R.N. Huang R., Bramble L.L. Poly-3-hydroxybutyrate/polyphosphate complexes form voltage activated Ca2+ channelsin the plasma membrane of Escherichia coli. Biophys. J., 1995; 69(3) 754-766 Cf., Reusch R.N. Poly--hydroxybutyrate/calcium

polyphosphate complexes in eukaryotic membranes.Proc. Soc. Expt. Biol. Med., 1989, 191, 377-381

[17b] Van Wazer J.R., Campanella D.A. Structure and properties of the condensed phosphates. IV. Complex ion formation inpolyphosphate solution.J. Amer. Chem. Soc., 1950; 72:655-663 and Van Wazer J.R., Callis C.F. Metal complexing byphosphates Chem. Rev., 1958, 50, 1011-1046

Cf.[18a] Bowen H.J.M. Strontium and barium in sea water and marine organisms. J. Mar. Biol. Assoc. U.K., 1956, 35, 451-458

[18b] Nader H.B., Medeiros M.G.L., Paiva J.F., Jeronimo S.M.B., Ferreira T.M.P.C., Dietrich C.P. A correlation between the sulfatedglycosaminoglycan concentration and degree of salinity of the habitat in fifteen species of the classes Crustracea, Pelecypoda andGastropoda. Comp. Biochem. Physiol., 1983, 76, 433-436Cf. Nader H.B., Ferreira T.M.P.C., Chavante S.F., Toma L., Dietrich C.P., Casu B., Torri G. Maintenenace of heparan sulphatestructure through evolution. Carbohydr. Res., 1988, 184, 292-300

[19] Turnbull J., Powell A., Guimond S. Heparan sulfate: decoding a dynamic multifunctional cell regulator.Trends Cell Biol., 2001, 11, 75-82(The heparaome constitution must, it can now be suggested now be provisionally amended to include specifically augmentedamounts of the rarer inorganic constituents of blood serum}.

[20] Cf., Bernfield M., Gotte M., Park P.W., Reizes O., et al., Function of cell surface heparan sulphate proteoglycans.Ann. Rev.Biochem., 1999, 68, 729-777; Park P.W., Reizes O., Bernfield M. Cell surface heparan sulphate proteoglycans: selectiveregulation of ligand-receptor encounters. J. Biol. Chem., 2000, 275, 29923-29926; Lyon M., Gallagher J.T. Bio-specificsequences and domains in heparan sulphate and regulation of cell growth and adhesion. Matrix Biol., 1998, 17, 485-493;

[21] Cf. Esko J.D., Lindahl U. Molecular diversity of heparan sulphate.J. Chem. Invest., 2001, 108, 169-173 cf. Suppl.,http//www.jci/cgi/content/full/108/2/169/DCI

[22] Results from in vitro and in vivo studies(by Kojima S., Hama Y., Sasaki T., Kuberoda A. Elevated uptake of 67Ga and increasedheparan sulphate content in liver-damaged rats.Eur. J. Nucl. Med., 1983, 8, 52-59 and Y. Hama Y., Sasaki T., Kojima S et al., 67Gaaccumulation and heparan sulphate metabolism in lysosomes. ibid., 1984, 9,51-56) suggest that heparan sulphate proteoglycans areheparin-like multi-element matrices.

[22a] Anti-tumour activites of Ga3+ (Cf., Bernstein L.R. Mechanism of therapeutic activity of gallium.Pharmacol. Rev., 1998; 50:665-682cf. also literature survey given in the internet document http:/ www.titanpharm.com/sci-art-gallium.html)Cf. also the anti-HIV activities of Ga3+ reported by Stapleton J.T. et al.at the39th ICAAC Meeting San Francisco Sept 26-29, 1999)

[H and HS also possess potent anti-HIV activites [23] : could this also be in part related to the natural presence of Ga3+ in thesepolysaccharides?]

[23] De Clercq E. Targets and strategies for the antiviral chemotherapy of AIDS TiPs 1990, 11 (5) 198-205; Chem. Abs.,125,316373qCf.Virology. 1992; 189: 48-52 and BC Herold et al. ibid., 1995; 206: 1108; S Laquere et al.,J Virol. 1998; 72: 979; J Neyts

et al.,Cf., Rusnati M. et al., Biotechnological heparin/heparan sulphate: a novel area of multitarget drug discovery. Curr. Pharmacol.

Design, 2005, 11, 2489-2499; cf. J. Biol. Chem., 273, 16027-16037; ibid., 276, 3254-3256; ibid., 276: 22420-22425 Cf., Bugatti A., et al. with Rusnati M. Heparin-mimicking sulfonic acid polymers as multitarget inhibitors of HIV-1 Tat and

gp120 proteins. Antimicrob. Agents Chemother. Doi ; 10.1128/AAC.01362-06

[24] Grant D., Long W.F., Williamson F.B. Inhibition of glycosaminoglycans of CaCO3 (calcite) crystallization.Biochem. J., 1989; 259: 41-45

Cf.,Med Hypoth. 1992; 38: 49-55; and H Iwada et al.,Proc 7th Urolithiasis Symp., 1994, 185: Chem. Abs. 123, 6960b;Yamaguchi S., Yoshioka T., Utsunomiya M., Koide T., Osafune M., Okuyama A., Sonoda T. Heparan sulfate in the stonematrix and its inhibitory effect on calcium oxalate crystallization. Urol. Res., 1993, 21, 187-192; Chem Abs. 119, 243946t

[25]E.g. Adachi T., Yamazaki N., Tasaki H., Toyokawa T., Yamashita K., Hirano K. Changes in the heparin affinity of extracellular-superoxide dismutase in patients with coronary artery atherosclerosis. Biol. Pharm. Bull. 1998, 21, 1090-1093Grant D., Long W.F., Williamson F.B. A comparison of the antioxidant requirements of proteins with those of synthetic

polymers suggests an antioxidant function for clusters of aromatic and bivalent sulphur-containing amino acid residues.Med. Hypoth., 1989, 28, 245-253

Cf., Albertini R., Passi A., Abuja P.M., De Luca G. The effect of glycosaminoglycans and proteoglycans on lipid peroxidation.Int.J.Mol. Med., 2000, 6, 129-136Albertini R., Rindi S., Passi A., Pallavicini G., De Luca G. Heparin protection against Fe2+ - and Cu2+-mediated oxidationof liposomes,FEBS Lett., 1995, 377, 240-242Ross M.A., Long W.F., Williamson F.B. Inhibition by heparin of Fe(II)-catalysed free-radical peroxidation of linoleic acid.

Biochem. J., 1992, 286, 717-720Grant D., Long W.F., Williamson F.B. Pericellular heparans may contribute to the protection of cells from free radicals. Med.

Hypoth., 1987, 23, 67-71.

Heparin binds Fe(III) oxyhydroxides (including akagaenite) generated by a multi-elementassisted (perhaps Co-dependent) ferroxidase activity [13]; the existence of iron-rich nodules on chitin and

perhaps also potentially with hyaluronan Merce A.L., Marques Carrera L.C., Kelly L., Santos Romanholi L.K., Lobo Recio M.A.

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Aqueous and solid complexes of iron(III) with hyaluronic acid: Potentiometric titration and infrared spectroscopy.J. Inorg. Biochem.,2002, 89, 212-218 [suggest a common occurrence of such a pro-antioxidant functions for polysaccharides.

NMR studies show that while Ca and Na counterions undergo fast intersite exchange on heparin [11],native amounts of Fe and Cu in heparin apparently do not do so].

Cf. also Whitfield D.M., Choay J., Sarkar B. Heavy metal binding to heparin disaccharides. I. Iduronic acid is the main binding site.Biopolymers. 1992, 32, 585-596; cf., ibid., 1992, 32, 597-619

[26] Lindahl U., Hook M, Glycosaminoglycans and their binding to biological macromolecules.Ann. Rev. Biochem., 1978, 47, 385-

417; Kjellen L., Lindahl U. Proteoglycans structures and interactions.Ibid., 1991, 60, 443-465

[27a] [A manuscript outlining this hypothesis has been under consideration by Williamson F.B., but has been delayed because of thisgroup leaders health problems]

[27] Jones L.S., Yazzie B., Middaugh C.R. Polyanions and the proteome. Molecular and Cellular Proteomics 2004, 3, 646-769

Cf., Comper, Genet Phys Processes Dev Biol Form Proc Workshop (1994, pub. 1995) 121; Ed. D. Beysens et al., World ScientificSingapore; Chem. Abs., 124, 79513e

[28] Cappai R., Cheng F., Ciccotosto G.D., Needham B.E., Masters C.L., Multhaup G., Fransson L.A. , Mani K. The amyloidprecursor protein (APP) of Alzheimer disease and its paralog APLP2, modulates the Cu/Zn-nitric oxide-catalysed degradation ofglypican-1 heparan sulphate in vivo. J. Biol. Chem., 2005, 280, 13913-13920Mani K., Cheng F., Havsmark B., David S., Fransson L.A. Involvement of glycosylphosphatidylinositol-linked ceruloplasmin in thecopper/zinc-oxide-dependent degradation of glypican-1 heparan sulphate in rat C6 glioma cells.J. Biol. Chem., 2004, 279, 12918-12923Ding K., Mani K., Cheng F., Belting M., Fransson L.A. Copper-dependent autocleavage of glypican-1 heparan sulphate by nitricoxide derived from intrinsic nitrosothiols.J. Biol. Chem., 2002, 277, 33353-33360Mani K., Jonsson M., Edgren G., Belting M., Fransson L.-A. A novel role for nitric oxide in the endogenous degradation of heparan

sulphate during recycling of glypican-1 in vascular endothelial cells. Glycobiology , 2000, 10, 577-586; Cf. Mani K.,Cheng F., Sandgren S., van den Born J., Havsmark B., Ding K., Fransson L.A. The heparan sulphate-specific spitope10E4 is NO-sensitive and partly inaccessible in glypican-1. ibid., 2004, 14, 599-607

(where the suggestion is made that studies of the extent of S-nitrosylation of [HSPG] glypican-1 and formation of[its nitrosativedegradation products] in healthy and diseased brain tissue may provide for a better understanding of the pathogenesis ofneurodegenerative diseases)Vilar R.E., Ghael D., Li M., Bhagat D.D., Arrigo L.M., Cowman M.K., Dweck H.S., Rosenfeld L. Nitric oxide degradation of heparinand heparan sulphate.Biochem.J., 1997, 324, 473-479

Cf. Pentikanien M.O., Oorni K., Kovanen P.T. Myeloperoxidase and hypochlorite, but not copper ions, oxidize heparin-bound LDLparticles and release them from heparin.Arterioscler. Thomb. Vasc. Biol., 2001, 21, 19 W.D.02-1908

[29] Ca2+ potentiates the aggregation of syndecan-1 transfected cells [a], Ca2+ is required for binding of heparan sulphate to annexin Vand the related potentiation of annexin V assembly on cell surfaces [b]; Ca2+ also is required for HSPG promotion of fibrillin-1microfibrillar assembly [c]; extracellular Ca2+ concentration regulates the distribution and transport of HSPG in rat parathyroid cells

[d]; Ca2+

regulates the binding of H and HS to serpins [e], fibronectin [f] as well as serum amyloid P[g] and is required for thebinding of HS to 2 and 3 integrins, platelet/endothelial cell adhsion molecule-1 (PECAM-1) and L-selectin [h], [j] Ca2+, Mg2+ orMn2+ can differently facilitate basic fibroblast growth factor receptor dimerization [h]; Zn2+ and Ca2+ affect the neutralising ability ofhistidine-rich glycoprotein, [i]; the presence of Zn2+ ions is required to allow HSPG to bind tocollagen XVIII-endostatin [j] and Cu2+

bridges enable the formation of HSPG - prion oligomers [k].(a) Stanley M.J., Liebersbach B.F., Liu W., Anhalt D.J., Sanderson R.D. Heparan sulphate-mediated cell aggregation.J. Biol. Chem.1995, 270, 5077-5083(b) Capila I., Hernaiz M., Mo Y.D., Mealy T.R., et al.,Structure (Cambridge UK) 200l; 9: 57-64; A Lewit-Bentley et al.,Eur. J.

Biochem., 1992, 210, 73-77(c) Tiedemann K., Batge B., Muller P.K., Reinhardt D.P. Interaction of fibrillin-1 with heparin/heparan sulphate, implication formicrofibrillary assembly.J. Biol. Chem., 2001, 276, 36035-36042 (Cf. Calcium dependence)(d) Takeuchi Y., Sakaguchi K., Yanagishita M., Aurbach G.D., Hascall V.C. Extracellular calcium regulates distribution andtransport of heparan sulfate proteoglycans in a rat parathyroid cell line. J. Biol. Chem., 1990, 265, 13661-13668(e) Pike R.N., Buckle A.M., le Bonniek B.F., Church F.C. Control of the coagulation system by serpins. Getting by with a little help

from glycosaminoglycans.FEBS J., 2005, 272, 4842-4851(f) Hayashi K., Madri J.A., Yurchenko P.D. Endothelial cells interact with the core protein of basement membrane perlecan through1 and 3 integrins: an adhesion modulated by glycosaminoglycan. J. Cell Biol., 1992, 119, 945-959

(g) Hamazaki H. Ca2+-mediated association of human serum amyloid P component with heparan sulfate and dermatan sulfate.J BiolChem. 1987; 262: 1456-1460; cf., EH Nielson et al.,APIMS. 1994; 102: 420(h)Kan M., Wang F., Kan M., To B., Gabriel J.L., McKeehan W.L. Divalent cations and heparin/heparan sulfate cooperate to controlassembly and activity of the fibroblast growth factor receptor complex J. Biol. Chem., 1996, 271, 26143-26148;cf., Siegel G., Abletshauser C., Malmsten M., Schmidt A., Winkler K. Reduction of arteriosclerotic nanoplaque formation and size byfluvastin in a receptor-based biosensor model. Cardiovasc. Res., 2003, 58, 696-705; cf. alsoOlgemoller B., Schleicher E.D., Schwaabe S., Guretzki H.-J., Gerbitz K.D. High concentrations of low density lipoprotein decrease

basement membrane-associated heparan sulfate proteoglycan in cultured endothelial cells.FEBS Lett., 1990, 264, 37-39(i) Kazama Y., Koide T. Modulation of protein C inhibitor activity by histidine-rich glycoprotein and platelet factor 4: role of zincand calcium ions in the heparin-neutralizing ability of histidine rich glycoprotein. Thrombosis Haemostasis, 1992, 67, 50(j) Norgard-Sumnicht K.E., Varki N.M., Varki A. Calcium-dependent heparin-like ligands for L-selectin in nonlymphoid endothelialcells Science, 1993; 261: 480-483; cf., Stevenson J.L. Choi S.H., Varki A. Differential metastasis inhibition by clinically relevant

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levels of heparins-correlation with selectin inhibition, not antithrombin action.Clin. Cancer Res., 2005, 11, 7003-7011(j) Richard-Blum S., Feraud O., Lortat-Jacob H., Rencurosi A., Fukai N., Dkhissi F., Vittet D., Imberty A., Olsen B.R., van der Rest M.Characterization of endostatin binding to heparin and heparan sulfate by surface plamon resonance and molecular modeling. Role ofdivalent cations. J. Biol. Chem., 2004, 279, 2927-2936; Lewit-Bentley A., Morera S., Huber R., Bodo G. The effect of metal

binding on the structure of annexin Vand implications for membrane binding.Eur. J. Biochem./FEBS, 1992, 210, 73-77(k) Gonzalez-Iglesias R., Pajares M.A., Ocal C., Espinosa J.C., Oesch B., Gasset M. Prion protein interaction withglycosaminoglycan occurs with the formation of oligomeric complexes stabilized by Cu(II) bridges.J. Mol. Biol., 2002, 319, 527-540Simeon A., Wegrowski Y., Bontemps Y., Maquant F.-X. Expression of glycosaminoglycans and small proteoglycans in wounds:

modulation by the tripeptide-copper complex glycyl-histidyl-L-lysine.J. Invest. Dermato., 2000, 115, 962-968

Other reports of requirements of apparent inorganic cofactors for HSPG interactions are:Zho S., Magura C.E., Hurley W.L. Heparin binding properties of lactoferrin and lysozyme Comp. Biochem. Physiol., 1992, 103B:889-895 (biotinylated heparin binding to lactoferrin was (Na, Ca, Cu, Zn and Fe cation dependent);Hsueh Y.-P., Yang F.-C., Kharazia V., Naisbitt S., Cohen A.R., Weinberg R.J., Sheng M., Direct interaction of CASK/LIN-2 and

syndecan heparan sulphate proteoglycan and their overlapping distribution in neuronal synapses. J. Cell Biol., 1998, 142, 139-151 (a Ca2+ related neuronal CASK/LIN-2 interaction with syndecan HSPG)

Hu W.-L., Regoeczi E., Hepatic heparin sulphate proteoglycan and the recycling of transferrin.Biochem. Cell Biol., 1992, 70, 535-538. (Liver HSPG mimics transferrin receptor reactivity indicating a link between HSPG and Fe biochemistry)Kalea A.Z., Lamari F., Karamanos N.K., Klimis-Zacas D. Dietary manganese affects heparan sulphate concentration and sulfation

pattern in Sprague-Dawley rat aorta.Federation of the European Connective Tissue Societies (FECTs) 2004, XIX, PB (26): 79,Kalea A.Z., Lamari F.N., Theocharis A.D., Schuschke D.A., Karamanos N.K., Klimis-Zacas D.J.,BioMetals, 2006, 19, 535-546(Mn2+ affects HSPG signalling in rat aorta)

Liebel M.A., White A.A.Biochem. Biophys. Res. Commun., Inhibition of the soluble guanylate cyclase from rat lung by sulfatedpolyanions. 1982, 104, 957-1003 (Inhibition of soluble guanylate cyclase by heparin, heparinoids and Mn2+)

[29a]Si (e.g. as silicic acid and its oligomers) could perhaps also be an essential nutrient element for animals because of an, as yetunidentified, but hypothesised essential role in the biosynthesis of HS.Cf. McCarty M.F., Med. Hypoth.,1997, 49, 177-179

Heparin bound to SiO2 also allows the separation of both cations and anions on a single chromatographic column (Takeuchi T. et al.,Ion chromatography using anion exchangers modified with heparin.Analusis 1998, 26, 61-64)[Perhaps this could also depend upon sufficient Ca2+, Mg2+, Mn2+, supply and perhaps also be dependent on trace and ultratraceelement physiological elements which also are present in heparin].

Heparin and other GAGs and polyuronides always occur in association with inorganic Si(Schwarz K. A bound form of silicon in glycosaminoglycans and polyuronides.PNAS, USA 1973, 70, 1608-1612. Silicate clusters atH/HS surface formed by and rare earths may de-toxify e.g., Al3+, Tl+, Pb2+ and Cd2+ .The man-made sulphonate-based ionomer (fuel cell) membrane system which is chemically similar to H/HS [59, 60] in the possessionof arrays of SO3

- groups and in the similar dependence of the degree and strength of hydration pattern of the SO3- groups upon

counterions (maximal for Na+ and minimal for quaternary N+ cations [61,62]) in both types of polymer system function and stabilityimprovement it can be suggested may also be similarly be achieved by the incorporation of colloidal SiO2 particles which for the man-made polymers is known to greatly increase membrane stability.

Amorphous SiO2 particlesa demonstrate a range of quasi-biological activities including an ability, like

non-amorphous crystalline solids, to engage in seeded growth of specific structural modifications which, since this seems also to beaccompanied by an ability of the particles to disintegrate, suggests that they can engage in a cellular activity similar to reproductionwhich can further suggest a modification of the Cairns-Smith hypothesis of how life first started from a SiO2-based template system(such ideas were briefly discussed by Grant D., et al. in Med. Hypoth., 1992, 38, 36-48).An extension of this hypothesis to include the corollary to the spectroscopic evidence that SiO2 particles (which seem to resemble theman-made particles which have biological-like properties) occur in interstellar space in association with polyformaldehyde (a possible

precursor of polysaccharides) could further suggest role for polysaccharide- like molecules (perhaps similar to the polysaccharide-likecomponents of present-day humic materials) in this process.

[30] The Effect of Inorganic Ions on HSPG Biosynthesis:Potentially purely anthrophogenic inorganic ions (Pb2+, Cd2+, Hg(II), and excess F- have been reported to cause a diminution in

HSPG biosynthesis in cell culture experiments; on the other hand the physiological cations Ca2+, Mg2+ and Mn2+ cause anincrease in the biosynthetic rate of HSPG:

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

(Pb, Ca) Fujiwara Y., Kaji T. Suppression of proteoglycan synthesis by calcium ionophore A23187 in cultured vascular endothelialcells: implication of intracellular calcium accumulation in lead inhibition of endothelial proteoglycan synthesis J.HealthSci., 2002; 48: 460-466; cf. also Toxicology, 1999, 133, 159-169

(Cd) Cardenas A., Bernard A., Lauwerys R. Incorporation of [35S] sulfate into glomerular membranes of rats chronicallyexposed to cadmium and its correlation with urinary glycosaminoglycans and proteinuria. Toxicology, 1992, 76, 219-231

(Hg, Mn etc.) Templeton D.M.,Proc Trace Elem Health Disease IUPAC Int Symp 1990, p. 209

(Ca,Mn) Fritz T.A., Gabb M.M., Wei G., Esko J.D. Two N-acetylglucosaminyltransferases catalyse the biosynthsis of heparansulfate.J. Biol. Chem., 1994, 269, 28809-28814 (both enzymes require a divalent cation for activityGlcNAc-TI can use

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both Mn2+ and Ca2+ while GlcNaT-II will use only Mn2+)

(F) Pawalowska-Goral K., Wardas M., Wardas W., Majnusz U. The role of fluoride ions in glycosaminoglycans sulphation incultured fibroblasts.Fluoride, 1998, 31, 193-202

(SO42- transporter) Dawson P.A., Markovich D. Regulation of the mouse Nas1 gene by Vitamin D and thyroid hormone.Pflugers

Arch-Eur. J. Physiol., 2002, 444, 353-359

cf. also (Sr) Cf., Y Henrotin Y., et al., J. Bone Mineral Res., 2001, 16, 299-308the anti-osteoarthritic effect of Sr2+ ions has been attributed to the ability of this hitherto supposed non-physiological ion to increasesulphated polysaccharide biosynthesis.[Cf. also the suggested pathological influence of Ba2+ions upon HS synthesis and function [39] ].(Si) McCarty M.F., Reported anti-atherosclerotic activity of silicon may reflect increased endothelial synthesis of heparan sulfate

proteoglycans Med. Hypoth., 1997, 49, 177-179; Grant D., Long W.F., Williamson F.B. A putative role for colloidal silicates inprimitive evolution deduced in part from their relevance to modern pathological afflictions. Ibid., 1992, 38, 46-48Cf., R.K. Iler, p.762 in The Chemistry of Silica, Wiley, New York, 1979[31] Edward M., Oliver R.F., Biochem. Soc. Trans., 1984, 12, 304; Ibid., 1983, 11, 304; J. Cell Sci., 1986, 85, 217-219

Kao J., Huey G., Kao R., Stern R. Ascorbic acid stimulates production of glycosaminoglycans in cultured fibroblasts. Exp.Mol. Pathol., 1990, 53, 1-10

Cf. Heller R., Munscher-Paulig F., Grabner R., Till U. L-ascorbic acid potentiates nitric oxide synthesis in endothelial cells. J.Biol. Chem., 1999, 274, 8254-8260

[32] Moroano S., Guidobaldi L., Ciprian R., Gabriele A., et al., High glucose modifies heparin sulphate synthesis by mouseglomerular epithelial cellsDiabetes/Metab. Res. Re., 1999, 15, 13-20Cf. M Nishinaage et al.,J. Clin. Invest., 1993, 192, 1381; Chem. Abs., 119, 24338uPaka L., Kako Y., Obunike J.C., Pillarisetti S. Apolipoprotein E containing high density lipoprotein stimulates endothelial productionof heparin sulfate rich in biologically active heparin-like domains. A potential mechanism for the anti-atherogenic actions of vascularapolipoprotein E. J. Biol. Chem., 1999, 274, 4816-4823Cf., Stephen C.C. et al. Role for heparin-binding growth factors in glucose-induced vascular dysfunction.Diabetes, 1998, 47, 1771-1785

[33] Robert G.P., Hardin K.L. Br. J. Dermatol., 1994, 131, 630-633Karlinsky J.B., Rounds S., Farber H.W. Effect of hypoxia on heparan sulfate in bovine aorta and pulmonary artery endothelial cells.Circ. Res., 1992, 71, 782-789Cf. Korenaga R., Ando J., Tsuboi H., Yan W., et al. Laminar flow stimulates ATP- and shear stress-dependent nitric oxide productionin cultured bovine endothelial cells. Biochem. Biophys. Res. Commun., 1994, 198, 213-219and Kouretas P.C., Hannan R.L., Kapur N.S., Hendrickson R., et al., Non-anticoagulant heparin increases endothelial nitric oxidesynthase activity: role of inhibitory guanine nucleotide protein.J. Mol. Cell. Cardiol., 1998, 30, 2669-2682

[34] Grimm J., Keller R., de Groot P.G. Laminar flow induces cell polarity and leads to rearrangement of proteoglycan metabolism in

endothelial cells. Thrombosis Haemostasis, 1988, 60, 437-441Cf. Siegel S., Malmsten G., Lindman B., Flow sensing at the endothelial-blood interface Colloids and Surfaces A: Physiolgical and

Engineering Aspects, 1998, 138, 345- 351

[35] Colburn P., Dietrich C.P., Buonasisi V.Arch. Biochem. Biophys., 1996, 325, 129

[36] (retinoic acid) Zhang L., Schwartz J.J., Miller J., Liu J., Fritze L.M.S. Schworak N.W. Rosenberg R.D.The retinoic acid and cAMP-dependent up-regulation of 3-O-sulfotransferase-1 leads to a dramatic augmentation of anticoagulantlyactive heparan sulfate biosynthesis in F9 embryonal carcinoma cells.J. Biol. Chem. 1998, 273, 27998-28003;cf., Shworak N.W., Shirakawa M., Colliec-Jouault S., Liu J, Mulligan R.C, Birinyi L.K., Rosenberg R.D.Pathway-specific regulation of the synthesis of anticoagulant active heparin sulfate.J. Biol. Chem., 1994, 269, 24941-24952and Zhang L., Yoshida K., Liu J., Rosenberg R.D. Anticoagulant heparan sulfate precursor structures in F9 embryonal carcinomacells.J. Biol. Chem., 1999, 274, 5681-5691

(monensin) Sampaio L.O., Dietrich C.P., Colburn P., Buonassisi V., Nader H.B. Effect of monensin on the sulfation of heparansulfate proteoglycan from endothelial cells. J. Cellular Biochemistry, 1992, 50, 103-110

(+chatechin) Sinn W., Sudhakaran P.R., Von Figura K. Stimulation of heparan sulphate synthesis in cultured rat hepatocytes by (+)catechin. Biochem. J., 1981, 200, 51-57

[37] Belting M., Persson S., Fransson L-A. Proteoglycan involvement in polyamine uptake.Biochem. J., 1999, 338, 317-323Ding K., Sandgren S., Mani K., Belting M., Fransson L-A. Modulation of glypican-1 heparan sulfate structure by inhibition ofendogenous polyamine synthesis. Mapping of spermine-binding sites and heparanase, heparin lyase and nitric oxide/nitrite cleavagesites. J. Biol. Chem., 2001, 276, 46779-46791

Cf., Nishinage M., Ozawa T., Shimada K. Homocysteine, a thrombogenic agent, suppresses anticoagulant heparin sulfate expressionin cultured porcine aortic endothelial cells.J. Clin. Invest., 1993, 192, 1381

[38] Grant D., Williamson F.B. Draft of paper suggesting a nitric oxide/redox dysfunction aetiolgoy of ME/CFS.

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Awaiting comments from acknowledged research leaders involved in this field[This paper arose from a request by FBW that a literature survey into this illness which has no accepted aetiology be conducted; thiswas carried out as private research conducted in part in conjunction with discussions with Dr Vance Spence of the Univeristy ofDundee, Department of Medicine]

[39] Purdey M., Chronic barium intoxication disrupts sulphated proteoglycan synthesis: a hypothesis for the origin of multiplesclerosis. Med. Hypoth., 2004, 62, 746-754[This was a hypothesis based on a global soil and plant inorganic element analysis survey.

The author contributed to this research by communicating the results of research literature surveys to M. Purdey and also byidentifying and arranging visits to MS cluster sites in NE Scotland selected on the basis of a medical thesis]

[40] Williamson F.B., personal communication

[41] The authors studies at Aberdeen University had confirmed the results of previously academic researches (and data fromindustrial uses of sulphonated organic polymer membranes) that heparin (and putatively heparan sulphate) contains a similar systemof interacting SO3

- -associated water cluster stuctures (the size of the cluster vary between individual metal counterions; the greatestdegree and strength of hydration being 6(H2O)-SO3

- for Na+ counterions and the least hydrated for quaternary N+ counterions (cf.Grant D., et al.Biochem. Soc. Trans., 1990, 18, 1293-1294; Ibid., 1983, 11, 96;Ibid., 1984, 12, 302. [These heparin -associatedwater clusters also showed a systematic change in their near infrared water absorptions upon poly-L-lysine and poly-L-arginine

binding (Grant D., et al.Biochem. J., 1991, 277, 569-572.]. It is suggested that further research into the interdependence of GAGhydration, ion binding and migration (i.e. proton conduction and ionomer activity) might confirm that these ubiquitous cell membranecomponents behave analogously to man-made, industrially important, sulphonated organic polymer membrane which seem to employ(heparin/heparn sulphate- like) (H2O)n/SO3

- - counterion modulated water cluster dependent activities for their proton conductive andother related functional activities . (cf. James P.L., et al.J. Materials Sci., 2000, 35, 5111-5119.

[42] The reported effect of the incorporation of colloidal SiO2particles which makes it easier to use such membranes in commerialfuel cells at realistic working temperatures (Adjemian K.J., et al. Silicon oxide Nafion composite membranes for proton-exchangemembrane fuel cell operation at 80-140 degrees C.J. Electrochem. Soc., 2002; 149: A256-261) suggests that the presence of smallSiO2 particles increases the stability of such membranes and this may also be why inorganic Si is also ubiquitously associated withGAGs such as heparin and heparan sulphate in vivo.

[43] E.g., Luck W.A.P. Topics in Current Chemistr,y 1975, 5, 115-180

[44]Grant D., Long W.F., Williamson F.B., N.m.r. spectroscopy of Ca2+-heparin suggests delocalized binding of the cation.Biochem.Soc. Trans., 1991, 19, 390S

[45] Grant D., Long W.F., Williamson F.B., Inhibition by glycosaminoglycans of CaCO3 (calcite) crystallization.Biochem. J. 1989,259, 41-45; Degenerative and inflammatroy diseases may result from defects in antimineralization mechanisms afforded byglycosaminoglycans. Med. Hypoth., 1992, 38, 49-55

[46] Hoch A.R., Reddy M.M., Aiken G.R. Geochimica Cosmochimia Act, 2000, 64, 61-72;Inskeep W.P., Bloom P.R., Soil Sci. Soc. Am. J., 1986, 50, 1431-1437;

cf., Morse J.W.Reviews in Mineralogy Vol 11, Carbonates: Mineralogy and Chemistry p. 227 et seq. (R.J. Reeder and P.H.Ribbe, Eds.) Mineralogcial Society of America;and Suess E., Geochim. Cosmochim. Acta, 1970, 34, 157-168

In a paper which has remained unpublished (to do with issues unrelated to possible public scientific interest) Grant D., Long W.F.,Williamson F.B., et al. compared a range of humic and fulvic acids with glycosaminoglycans as modulators of the seededcrystallization of CaCO3 (using the methods which were described by these authors inBiochem. J., 1989, 259, 41-45). TheAberdeenshire soil-derived polymers (obtained from the Macaulay Inst, Aberdeen) were found to be very potent inhibitors of this

process giving results which, when taken together with those which have now been confirmed in other laboratories, suggest thathumic and fulvic acids act, similarly on a common global basis, as inhibitors of this and other crystallization processes which can besuggested give rise to the homeostasis system of the inorganic element concentrations in seawater. This also suggests that a similar

process involving glycosaminoglycans may have been used (at least in the early stages of their evolution) by multi-cellular animals togenerate and act as a homeostasis buffer for the seawater-like multi-element matrices of animal biological fluids.

[47] Reusch R.N., Sadoff H.L., Putative structure and functions of a poly-beta-hydroxybutyrate/calcium polyphosphate channel inbacterial plasma membranesPNASUSA 1988; 85 (12): 4176-4180 cf., Soc. Exp. Biol. Med., 1989, 191, 377-381Cf., Brown M.R.W., Kornberg A. Inorganic polyphosphate in the origin and survival of speciesPNAS, USA, 2004, 101, 16085-16087;

Kornberg A., Rao N.N., Ault-Riche D. Inorganic polyphosphate: a molecule of many functions.Ann. Rev. Biochem., 1999, 68,89-125.

Cf., Wang L., Fraley C.D., Faridi J., Kornberg A., Roth R.A. Inorganic polyphosphate stimulates mammalian TOR, a kinase involvedin the proliferation of mammary cancer cells.PNAS, USA, 2003, 100, 11249-11254; Tammenkoski M., et al., Human metastasisregulator protein H-prune is a short-chain exopolyphosphatase.Biochemistry, 2008, 47, 9707-9713; Hernandez-Ruiz L., et al.,Inorganic polyphosphate and specific induction of apoptosis in human plasma cells.Haematologica, 2006, 91, 1180-6.(N.b., inorganic polyphosphate has potential to be a highly effective anticancer drug, e.g. against malignant melanoma).

[48] Somers J.M., Tait M.I., Long W.F., Williamson F.B., Activities ofCorallina (Corallinales) and theRhodophyta polymers in themodulation of calcification.Hydrobiologia, 1990, 204/205, 491-497

[49] Kreuger P., Jemeth P., Sanders-Lindberg E., Eliahu L., Ron D., Basilico C., Salmivirta M., Lindahl U. Fibroblast growth factorsshare binding sites in heparan sulphate.Biochem. J., 2005, 389, 145-150

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[50] Henrotin Y., Labasse A., Zheng S.X., Galais P., et al., Strontium ranelate increases cartilage matrix formation.J. Bone MineralRes., 2001, 16, 299-308

[51] Grant D. et al.,Eur. Polym. J., 1974, 10: 481-488;Ibid., 10, 77-83; Ibid.,15, 625-626; J.Polym. Sci. Polymer Lett., 1974, 13, 1-9

[52] M Watanabe M Okada Y Kudo et al.,J. Toxicol. Environ. Health, 2002, 65, 1047-1060;Bermudez E., Mangum J.B. Wong B.A., et al.,Toxicol. Sci.,i 2004, 77, 347-357;Emesowum B., Wojeck B., Koeneman B. et al., An investigation to study the toxicity of titanium dioxide (TiO2)

on human intestinal cells (Caco-2) http://sols. asu. edu/ubep/2007/authors/emesowum_3.phb

[53] Feyzi E., Saldeen T., Larsson E., Lindahl U., Salmivirta M., Age dependent modulation of heparan sulphate structure andfunction.J. Biol. Chem. 1998, 273, 13395-13398

[54] Grant D., Long W.F., Williamson, F.B., Zn2+ - heparin interaction studies by potentiometric titration.Biochem. J., 1992, 287,849-853[This was part of a series of papers dealing with the release of H+ by metal ion binding to H/HS; other metal ions which were studies

by the same method were Mg2+, Mn2+, Fe2+, Fe3+ and Al3+; the articles describing these results remain unpublished for reasonsunconnected to possible scientific merit or public interest, and it is hoped to publish them in due course].

Appendix 2It can be provisionally suggest that a normal fingerprint of multielements in heparin may be a hallmark of

biological purity against which the presence of anthropological inputs may be assessed.Anionic polysaccharides tend to have multi-element profiles which are natural, not artificial [e.g. arisingfrom dust particles [6, Katz; Muzzarelli ], tap water [2] or from leaching from container walls etc. anexception may be Al and Ti both of which can occur in pigments in paint and plastic as well as formZiegler Natta catalyst residues in polyethyelene and polypropylene [47] widely used as food containersetc.

Ti May be a Key Anthropogenic ElementLittle is currently known about the possible effects of intoxication by Ti, an elementwhich is abundant on the earths crust, as well as being widely employed in the form of TiO 2 particles in

paint, ceramics and plastics and perhaps because of this can be especially augmented in H d and

could, together with other potentially other anthropogenically augmented redox active metal ions beespecially disruptive of nitric oxide-HSPG interactions. While such catalyst residues will now normallyoccur in natural waters in such ultra-trace element amounts to seem to pose little direct danger to humanor animal health their gradual enrichment by a continued in vivo sequestration onto H/ HS chains isworthy of further research evaluation.Such an unexpectedly high Ti content in H [cf., ref. 6, Grant et al.) should be further investigatedtogether with other the putative polyolefin catalyst residues [e.g., Cr and V: these were reported to be ofconcern in H cf., ref. 6, Alcock; Heinemann et al.; modern polyolefin catalyst Zr residues also occur inH cf., ref. 6, Grant et al.]Since Ti has no known biological function its presence in association with a pure biopolymer can beassumed to be entirely anthropogenic but the unusually high presence of Ti in H is in contrast to that inthe human hair database [cf. ref. 8 and Table 1].The Ti in H was present as an amorphous-to-X-ray-diffraction chemical form [6], which suggests that it

probably occurs in the nanoparticulate forms which are under current investigation for potential harmfuleffects [48] and arise by newer technologies for TiO 2 particle production; such particles have recently

been suggested to be more toxic than the traditional larger particle forms. Environmental TiO2 couldalso of course arise from rock or soil derived dust (since Ti is an abundant geological element) or fromthe widespread use of Ti metal in engineering (including in prosthetics, where it is however believed to

be well tolerated), from TiO2 the employed in ceramincs, paint, cosmetics and as a pigment in PVC andPE (where it is also believed to be inert). Small TiO 2 particles will also occur following slow adsorptionof ultra-trace amounts of soluble Ti in municipal and natural waters (this may arise both form the largeamount in soils, residues from of coal combustion but perhaps also from the catalyst residues in

polyolefins [traditionally produced using a relatively inefficient first types of TiCl3 or TiCl4-

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MgCl2/Ziegler Natta catalysts which can produce polyolefins containing large amounts of catalystresidues ; Cr is also used in this way in the Philips process and other metals such as V and Zr are usedfor polyolefin production. Polyolefins used for containers as packaging (e.g. present in landfill sites) aswell as used for pipes etc. used in municipal water systems will cause a slow release of Ti, Cr, V and Zrin to the environment. (Polyolefin transition metal catalysts also require Al-alkyl co-catalysts, which

leads to Al-containing residues (this being in addition to the residual Al3+ present from use of aluminiumsalts as flocculants of humic matter; cf. the concern regarding the co-occurrence of Al in H used forkidney dialysis [10]).

The presence of Si in animal tissuesThe high Si content in both human hair, and the association of Si with H/HS [29a,Si] is in accord withthis element being an essential animal nutrient which may be involved in the biochemistry ofglycosaminoglycans, but the details of its physiological actions are, at present, unknown.Si could be a key element linking geological and biological metallomics, suggesting the need fora wider geoglyco-metallome conceptualization by which the inorganic metallome influences theheparanome. The 5900 ppm Si in heparin suggests that in vivo HS side chains may be associated with a

similar or even somewhat more Si than this with, e.g. on average, a tetrameric silicic acid cluster perpolymer chain. Since H is known to form adducts with SiO2 which enable a unique single columnseparation of both cations and anions [[29a], Si, Takeuchi et al.,], some similar function might logically

be proposed for the Si in H/HS.

The interaction of H/HS with Inorganic SurfacesH/HS can also interact in highly selective ways with inorganic surfaces of seed particles used incrysallization kinetic studies, where H/HS, alginates and carrageenans demonstrate morphogenicinhibitory and modulatory actions by selective capping off of active sites at the crystal faces of , e.g.,

barium sulphate, calcium carbonate, phosphate and urate seed crystals (a similar action of H/HS andderived oligosaccharides at urinary and blood vessels walls is thought to protect against pathologicalcalcification [45].

This process seems implicated in the tendency for arteries in aged individuals to calcify more readily

[49].

Ascorbate & Cancer[This was the subject of an internet document Heparan Redox Hypothesis posted by D Grant in 2000cf. ref. [5]The Linus Pauling hypothesis had suggested that ascorbate dietary supplementation provides a practical protectionaganst cancer and virus infections. There however was a lack of a credible mechanism by which this effect ofascorbate could be explained. The known biochemistry of H/HS (anti-cancer, anti-viral) and the ability of ascorbateto increase HS biosynthesis is suggested to provide the required credible mechanism.Ascorbate also induces primary biosynthesis of HS chains with a higher degree of sulphation [31] and therefore

putatively increases the content of H-like epitopes in the HS chains. This will lead to increased multi-inorgani-element binding).Some tumour suppressors turned out to be enzymes required for the biosynthesis of HS (reviewed by Grant, 2000,

ref [5].

The known anti-tumour effect of inorganic Ga3+ ions [22a] present in H and e.g. kelp and fulvates used as dietary supplements, andputatively also present in HS may also play a part in the anti-tumour activities of both H and HS. (In vivo binding of67Ga3+ and othermultivalent cations has been experimentally demonstrated to displace 45Ca from HSPG from damaged in vivo [22] and such HSPGdamage is a hallmark of tumour proliferation and progression (cf., Internet discussion Heparan Redox Hypothesis (Grant, 2000, cfref. [5])

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Ding et al [28] have also established that ascorbate will participate in vivo release of nitric oxide from internal HSPG stores [theseauthors found that ascorbate might be the natural reductant for Cu2+ to give Cu+ needed to catalyse the release in internally stored NOmolecules] in the generation of oligosaccharides from HS during NO-dependent processing of glypican-1 HS which also requires Zn2+

ions [28] (but the reason for this is unknown, but it might be suggested that the release of H+ by this ion [54] may contribute to themechanism of thedeaminative cleavage of HS to give GlcNH2 groups, a process which could be dependent on the release of H

+ closeto the N-SO3

- groups to form NH2 groups by desulphonating them; the H+ seems to arise from the water layer around H/HS; a similar

process may induced by Cu2+ and Fe2+ ions and may also be modulated by polyamines [38]; these H+-dependent processes could bepromoted by along-chain transport of H+ or related ions, as the water layers associated with H/HS are known, in the similar man-madeproton conductor systems to engage in such activities. A direct desulphonation by unliganded redox metal ions of GlcNSO3

- groupsc

of H/HS could enable a rapid response nitrosative oligosaccharide generation in vivo.

*Former industrial and academic research chemist now conducting literature surveys.

Appendix 1[Cf. mathematically exact relationships seem also to exist between seawater quality e.g. as measured by the degree of salinity and theamounts (and type?) of HS and related polysaccharides synthesised by aquatic invertebrates [18a];

FIGURESThe following Figures illustrate the inter-relationships of metallomic matrices. These diagrams wereobtained from other documents which included data from Harrison G.E. and Sutton A., Nature, 1963 (4869) 809.

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