Progress Report of a Home-Based Research InitiativeHeparan Sulphate & Health.A new medical paradigmDavid Grant, PhD, New Deer, Turriff AB53 6SX, 30/7/07

Polysaccharides which act inter alia as tissue protection systems, which if defective, can promote major illnesses. A long term literature survey* into the biochemistry of such polysaccharidessuggests that novel therapeutic intervention systems are possible based on the current knowledge of such systems. The potential importance to medicine of such polysaccharides can hardly be overstated since even using the state of current knowledge, a paradigm shift in therapeutics is now currently available for therapeutic exploitation based on the revolutionary notion that animal and human “health” may arise principally or even solely from an optimisation of the activities of such polysaccharides the most active of which are sulphated polysaccharide-rich proteoglycans of the heparin/heparan sulphate class which may allow the administration of designer polysaccharides-based therapeutic agents, or the dietary modifications needed to augment their in vivo synthesis may allow a successful intervention in illnesses for which conventional therapies and concepts are currently relatively unsuccessful (i.e., most degenerative diseases including some intractable diseases, e.g., malaria, viral infections, cancers, prion diseases, Alzheimer’s disease and various arthritic conditions and perhaps also major psychoses and chronic fatigue syndromes). This is a credible scenario if, as seems possible, heparan sulphate is a “master” system which “drives” a wide range of “slave” biochemistry systems which include those which influence human and animal health including proteases (e.g., serpins), lipoprotein lipases, growth factors and probably also prions.Successful therapeutic intervention based on such polysaccharides might eventually be possible for all diseases and related studies may even allow a fuller understanding of memory, cognition and intervention in the ageing process.

Heparin/heparan sulphate resembles DNA in being a somewhat analogous but more complex linear chemical encoded information system enabling the provision of a high ranking management system of protein activity regulation with feedback to the nucleus via enzymic and metal ion dependent nitric oxide generation of oligosaccharide messengers which appear to engage in servo-feedback loops which can be entered by the exogenous administration of correct signalling oligosaccharides or their mimetics which could allow a simply achieved augmentation of tissue protection afforded by the heparan sulphate-based tissue protective systems.

*[This is a personal, now home-based, informal project of searches of scientific literature prompted by previous laboratory work in a heparin/heparan sulphate and related laboratory research multi-disciplinary research group at Marischal College, Aberdeen headed by Dr FB Williamson and Prof WF Long (cf. who has listed on the internet his publications, which include most of the former research group’s output). The pro-health strategy based on heparan sulphate biochemistry is of obvious wide potential interest** , e.g., the human requirement for sufficient dietary ascorbate can be, at least partly, explained by the effects of ascorbate on heparan sulphate biosynthesis and the neutralisation by ascorbate of nitrous acid, a potent heparan sulphate depolymerisation agent. A number of other dietary factors similarly impact positively or negatively on heparan sulphate biosynthesis or postsynthetic modification [e.g. beneficial sodium, magnesium manganese and calcium ions, retinoic acid etc., on the one hand, and the down- regulation of heparan sulphate proteoglycan biosynthesis by toxic inorganic elements, endotoxins, excess glucose, oxidised lipids, a facility which suggests that the heparin/heparan sulphate tissue protection systems might be optimised by diet alone].**About ten years ago the beneficial effects on human health of heparan sulphate had been given wide publicity by being mentioned on BBC2 television Newsnight. A scientist was insisting that heparan sulphate would in the future become as widely used as aspirin as a cure-all.

Relevance of Prions & Inorganic Ions to Heparin/Heparan Sulphate Biochemistry.

Contents 1. Introduction 2. Natural Polyanionic Organic and Inorganic Polymers 2-1 Heparin/Heparan Sulphate & Glycosaminoglycans 2-2 The Heparanome 3. Role of Nitric Oxide in Biochemistry 3-1 Role of tyrosine nitration in nitrosative stress 3-2 Role of nitric oxide in heparan sulphate biochemistry 3-3 Putative Roles of Heparan Sulphate and Nitric Oxide in Prion Diseases 4. The Metallome

4-1 Seawater & “Seawater-Related” Matrices The Complexity of the Inorganic Profiles of Biological Fluids

This could correspond to a requirement of multi-elements to create the correct osmolyte balance(and related water supramolecular structure and influence of protein hydration and folding)

4-2 The Heparanome & the Metallome Must Mutually Interact in Health & Disease 4-3 The Heparin (Metallome) Provides a Model for the Heparanome

4-5 Inorganic Factors Affecting Heparan Sulphate Biosynthesis 4-6 Alteration of Metalloproteinase Shedding of Heparan Sulphate by Exogenous Heparin & Pentosan Polysulphate (PPS) 5. Protein Folding

5.1 The Putative Driving Force of Water Structure Relating to Protein Folding 5-2 Heparin/Heparan Sulphate & PPS Affect Both Protein Folding, Inorganic Supramolecular Structure Formation & Crystallisation

5-3 Possible roles of metal ions in prion misfolding neurodegenerative diseases 5-3-1 Manganese 5-3-2 Iron nickel & chromium 5-3-3 Aluminium & Beryllium The promotion of dementia by aluminium intoxication 5-3-3-1 Recent evidence for the presence of aluminium in heparin and other parenteral nutrition-related substances 5-3-4 Cadmium 5-3-5 Silicon

5-4 Inorganic Sulphate6 Some of Mark Purdey’s Contributions7 Promotion of Nerological Diseases by Pesticide Exposure7-1 Perturbation of Nitrosative Heparan Sulphate Signalling by Metal (e.g. Manganese &

Iron) Ion Dyshomeostasis 8. Similar metal ion-water cluster binding of sulphonated ionomers and heparin 9. Some Related Aberdeen U. Polysaccharide Research Results 9-1 Heparin /Heparan Sulphate 9-2 Pentosan Polysulphate Research 9-3 Spark source mass spectra of heparin 7-3-1 Cation exchange resin replacement of multi-elements from heparin 7-3-2 The Occurrence of Thallium, Strontium & Gallium in Heparin 10. References & Further Notes 11. Addendum

1.

The prion only hypothesis of transmissible spongiform encepalopathies (TSEs) can now be stated to be firmly established as noted by Lee & Coughey (2007) in a comment on the recent report by the Supattopone groupa (there have been doubts since the 1960s regarding the protein-only hypothesis of scrapie fator infectivity since nucleic acids might have co-purified in ultratrace amounts with infective prions (PrPSc) and could have been ultimately responsible for prion infectivity). Polyanions, including synthetic polynuceotides were, however, found by Supattopone et al., to be essential cofactors for the conversion of cellular to aberrant prion suggesting that viral nucleic acids might conceivably similarly promote the pathological conformational transformation. [Synthetic polyanion cofactors studied by Supattopone et al., can be seen as substitutes for the heparan sulphates of the cell surface which are the most the likely cofactors for the in vivo process of scrapie infection being apparently required both to enable prion entry into the cell and also to allow the misfolding process].

If the polyanion present naturally as the cofactor in TSEs is heparan sulphate, the complex nature of this polyanionic system needs to be addressed to fully understand the aetiology of TSEs.It is likely that heparan sulphate like the related system heparin is a metallomic matrix which responds to environmental metal ions and this can explain the mechanisms of toxicity which seem to depend on a dyshomeostasis of metal ions such as copper and manganese (and perhaps also iron, nickel, chromium, silver, cadmium and mercury other redox-active metal ions as well as barium, strontium, aluminium, silicon and perhaps also beryllium) which might directly creating infectious polymers or promote their further aggregations into toxic plaques, processes which also seem to involve heparan sulphate proteoglycans (perhaps normally controlled by the heparan sulphate system which if this becomes defective, could lead to plaque formation but the actual central aetiological defect resides in the heparan sulphate system allowing an eventual promotion of prion diseases). Defects in such heparan sulphate tissue protection could also be the central aetiological feature linking prion TSEs with the other non-prion neurodegenerative processes such as Alzheimer’s disease, Parkinson’s disease etc. Since the heparan sulphate signalling system putatively incorporates roles for the second messenger nitric oxide and its metabolites a useful diagnostic hallmark of such dysfunctions can be suggested to be the existence in affected patients of extensive nitration of tyrosine [this phenomenon has been associated with a wide range of degenerative diseases].The roles in neurodegenerative processes of the dyshomeostasis of metal ions such as manganese, copper and iron (e.g., as discussed by Case, 2005) should be carefully reconsidered in the light of the proposed heparan sulphate - nitric oxide biochemistry linkage which has been unravelled by the Fransson et al., group at Lund U. and the requirement for heparan sulpahte as a cofactor in TSE infectivity.Trevitt & Collinge were as late as 2006 apparently somewhat reluctantly to acknowledge that heparan-sulphate-related-biochemistry might be centrally implicated in prion diseases, and these authors were also unaware of the possibility of successful therapeutic intervention in heparan sulpahte biochemisty using heparinoids, e.g., pentosan polysulfate (PPS). Important studies of PPS in human subjects could have considerable significance for pointing the way forward for the fuller understanding and inhibition of prion diseases, other neurodegenerative diseases, as well as for initiating researches which might lead to a fuller understanding of heparan sulphate biochemistry and its putative employment therapeutically. aSupattopone et al. generated infective PrPSc from pure compounds [PrPC lipids and a synthetic polyanion using prion amplification (PMCA) a procedure due to Saa et al., 2006) where the use of sonication enables the achievement of very high yields of infectious prions starting from ultratrace amounts of infectious particles (which are believed to consist of aggregates of ca. 20 prion monomers)].

2. A Range of Natural Polyanionic Molecules Show Related Metal Ion Binding PropertiesThe ability to bind metal ions can be suggested to potentially have a profound effect on protein activity by influencing water structure and protein folding.The range of natural polyanionic molecules for which such activities might be anticipated includeinorganic polysilicates, inorganic polyphosphate, poly-hydroxy-butryate, nucleic acids, polyadenylated proteins, heparin/heparan sulphate, the other sulphated glycosaminoglycans and hyaluronic acid, the marine algal polysaccharides (e.g. agar, carrageenan, alginic acid), plant polysaccharides (e.g., pectins and xylans), chitin, bacterial polysaccharides and the major environmentally important but less well defined, colloidal organic-inorganic matrices termed humic and fulvic acids.The role of purely inorganic polyanions in biology should be stressed since the purely inorganic polyanionic system, polyphosphate seems to be a ubiquitous component of all cell surfaces throughout biota, (this system of compounds is also widely used for industrial applications including those where their binding to inorganic ions and surfaces is exploited); such long-chain inorganic polyphosphates share with pentosan polysulphate (PPS) the ability to act as effective blood anticoagulant (but via different mechanisms; heparin, the more efficient anticoagulant catalyses antithrombin action whereas PPS acts via Factor VIIIa (Wagenvoord et al., 1988) and ‘heparin cofactor II’ (HCFII) (but also subject to modulation of the effects by Ca2+). Long chain inorganic polyphosphates also potentiate thrombin inhibition by HCFII to a similar extent as PPS. Other biological functions of inorganic polyphosphate which resemble those of heparin/heparan sulphate and PPS include the direct metal ion dependent modulation of inorganic structural formation (as applies to the prevention of unwanted crystal formation in urinary and blood systems, and the modulaton of the mineralization of bone tissue). Exogenous heparin and PPS seem both to effectively inhibit pathological accumulation of extracellular matrix in glomerulosclerosis. The mechanisms of action of heparin and PPS appear to be different. (PPS is believed to act via metalloproteinases and their inhibitors (Elliot et al., 1999)). Glomerulosclerosis may arise as a more general consequence of metal ion dysfunction (including the presence of toxic metal ions) which perturbs the heparan sulphate control process for mesangial cell proliferation, dysfunction of which promotes sclerosis (cf. Templeton, 1991).It should be noted that the entirely inorganic long chain polyphosphates resemble heparin/heparan sulphate for actions such as putative regulators of gene expression, apoptosis and cellular proliferation e.g. by fibroblast growth factor (cf. Shiba et al. 2003; cf., Kan et al. 1996 who drew attention to the role of metal ions in such processes). It might be suggested that the ability of these polyanionic molecules to combine with and regulate the activity of inorganic metal ions in biological fluids is the prime modus operandi of their biological effects.A further corollary to this is that inorganic ions will directly influence the supramolecular structure of water and the water bound to proteins and thereby influence protein folding (cf., Robinson & Cho etc.).

The in vitro binding of inorganic counterions to various polyanionic molecules have been extensively studied. Inorganic polyphosphates seem to form high affinity covalent, metal complexes. This contrasts to the behaviour of heparin, humic acid and DNA which act as less selective ligands for metal ions and for which the most widely believed hypothesis is that due to Manning in which counterion uptake is held to depend only upon the value of the positive charge of the countercation. (Such elements as Fe3+, Ga3+, Ce3+ and La3+, which are present in ultratrace amounts in natural waters, are predicted by the Manning theory to become selectively bound to these natural polyanionic polymers in agreement with experimental observations). Heparin, perhaps surprisingly, apparently also demonstrate the same counterion dependent effect on –OSO3

- associated water clusters as man-made, sulphonated polystyrene ion exchange resins and similar materials (e.g., Nafion®) used industrially as ionomers and proton conductors which are believed to require such metal ions related clusters of water molecules as the functionally active sites.

The (possible inorganic ion determined) interaction between glycosaminoglycans and natural inorganic polymer systems (especially inorganic polyphosphates and the similar but more complex system of inorganic polysilicates) may also be employed by organisms to modify the activities of glycosaminoglycans in vivo by the incorporation of the inorganic polyanonic systems into the sulphated polysaccharide systems.

Such inorganic systems also have the necessary complexity and putative information holding abilities to constitute possible pre-biotic cellular systems (c.f Grant et al. 1992a) and this ability may be retained in modern organisms

2.1 The Unique Roles of Heparin/Heparan Sulphate & Glycosaminoglycans in Animal OrganismsThe highly anionic polysaccharide system of heparan sulphate polysaccharides is believed to constitute a major system of animal tissue protection, knowledge of which has the potential to be further developed for the rational design of drugs to combat a wide range of human and animal diseases. The idea that polysaccharides could provide such benefit is often thought to be highly unconventional and therefore adherents of alternative hypotheses relating to human health tend to vigorously oppose researches aimed at achieving such polysaccharide-based therapeutics.

Heparin (discovered by McLean & Howell in 1913) is a member of the wide all-animal cell surface heparan sulphate system (cf., Kraemer, 1968) and is further part of the wider glycosaminoglycan (GAGs) system (these were originally termed mucopolysaccharides) which include other proteoglycans: chondroitins (researched in the 19th century, e.g., by Morner) dermatan sulphate, keratan sulphate and the free polysaccharide, hyaluronan. It is now believed (from work with C. elegans) that the heparan sulphate system is older than other GAGs (perhaps excepting hyaluronic acid). The biosynthesis of heparan sulphate (elucidated by Lindahl) seems to replicate its evolutionary history starting off as a polysaccharide resembling that of bacterial walls (heparanosan). It should be noted that heparin occurs in vivo principally in mast cells present in many mammals (but not in rabbits) and also in tissues of invertebrates, but heparan sulphate proteoglycans are present at almost all animal cell surfaces as an abundant receptor and these contain heparin-like segments. The review articles on heparin by Jaques (1978), are still worth reading as they reveal the wide range of activities and types of molecule which were known some fifty years ago to be affected by heparin interactions.

2.2 The Heparanome The “heparanome” (a term due to Turnbull, 2001) is a putative high level biological system management system which employs the heparan sulphate coded information held in anionic and other chemical signal patterns. The “heparanome” includes heparan sulphate proteoglycans (e.g., syndecan, glipcan, agrin, collagen XVIII etc.) constitute a codon information system which is analogous to the geneomic DNA , RNA system (but the heparanome apparently lacks the facility for self replication by a system analogous to the DNA template mechanism). Owing to its availability in large amounts, and because it consists of more than a hundred related polysaccharides (Nader et al., 1981), pharmaceutical heparin can provide a library of the differently sulphated anionic sequences which are present in the heparan sulphate side chains of heparan sulphate proteoglycans and thus act as a convenient model system of the entire heparanome.

The heparan sulphate system is now thought to be a key modulator of embryogenesis in all animals. The body plan of flies and man seem to be controlled by a similar heparan sulphate directed processes (which involves post-synthetic modifications). Interest in the heparanome was especially increased following the discovery that the assembly of the embryo is apparently greatly influenced by this. E.g. studies of the fruitfly and humans revealed similar heparan sulphate and lipid (cholesterol) dependent signalling (HhN hedgehog in Drosphila melanogastser) dependent events in flies had human equivalents which interacted with human tumour inhibitors EXT-1 and EXT-2 (heparan sulphate assembly enzymes (cf. Ingham, 1999). The roles of heparin/heparan sulphate in modulating angiogenesis and apoptosis is enabling new anti-cancer stratagems to be developed.Heparan sulphate has now also been directly associated with the biological clock [circadian rhythm –(Kuberan et al., 2004); perhaps determining Ca2+ ion concentration pulsation] and vascular ageing (Feizi et al. 1998) (heparan sulphate is also thought either (via the information encoded in its microstructure) to directly determined or be determined by the ageing process; heparan sulphate is also believed to be involved in cognition and memory (e.g., Ethell et al., 1999).

A problem had been both with the determination of the exact molecular structure and the modus operandi of the heparanome (does it require a highly specific DNA-like structure/sequence integrity or

is it a looser fuzzy logic system ?) One idea is that an exact anionic sequence recognition system may be required to correctly specify gives a postcode-like identification of self and to identify cells within a co-ordinate system within the organism.

Inorganic elements bound to the heparin/heparan sulphate polymer may contribute as functional components within this complex signalling system.There seems however to be a lack of awareness of this possibility.From a practical point of view the ability of heparin-like molecules to pick up inorganic contaminants from storage vessels etc represents a potential major quality control problem. The ability of heparin to bind Al3+ should be highlighted in this context.Recent studies have shown this element to be a common contaminant in commercial heparin.

The heparan sulphate animal protection and protein folding control system may also be at least partly redundant in more evolved higher animals where alternative systems have substituted for roles played by these polysaccharides in more primitive animals but in the most highly evolved species the heparan sulphate system is principally retained for wound healing and tissue remodelling processes occurring during the earlier stages of development. Animal evolution might also occur by a process of crosstalk between the heparanome and the genome.The relatively un-researched role of associated inorganic profiles (the metallome) might improve the understanding of this.

The putative role of heparan sulphate in animals as a system manager or back-up system manager can explain why a wide range of diseases can respond to heparin therapy.

The anticoagulation activity of heparin was the activity was the first to find use (starting around 1937).The molecular basis of such activity (the heparin pentasaccharide signalling sequence) was established by Lindahl et al., Rosenberg et al. (and Casu et al., who contributed to the spectroscopic identification of heparin conformations etc.). A parallel dermatan sulphate, heparin cofactor II mechanism of blood anticoagulation was also identified. Heparinoids like the plant xylan based pentosan polysulfate (SP54) were also found to act as a blood anticoagulant by substituting into the heparin cofactor II system as well as interacting strongly with Factor VIIIa.

That plant-derived polysaccharides have a useful pharmacological role in animals confirms the evolutionary ancient employment of polysaccharides throughout biota.

3. Role of Nitric Oxide in Animal BiochemistryThis had become firmly established by about 1992 (when nitric oxide was “the molecule of the year”).

3-1 Role of tyrosine nitration in nitrosative stressThe occurrence of tyrosine nitration has been associated with a wide range of diseases.(cf., the review by Ischiropoulos); {n.b. tyrosine nitration also occurs with Alzheimer’s but less prominentaly with prion diseases where the infective agent seems to diminish nitric oxide synthase activity so that TSEs might be promoted by dyshomeostasis which diminishes nitric oxide signalling, including that which invovles heparan sulphate).Peroxynitrite, formed from the reaction of superoxide anions with nitric oxide, is believed to be the major reagent which leads to tyrosine nitration. It is also likely that specialised protective anti-nitrosative agents exist (e.g., but controversially, apolipoprotein-a, a protein with an unusual abundance of kringle structures which could act as sinks for reactive nitrogen species; other examples may be those proteins containing clusters of cysteines and tyrosines, cf. Grant, et al.,1989 or quercetin [an apparently effective antidote to chronic fatigue syndrome, a disease which seems likely to be promoted by nitrosative stress, and possibly related neurological damage; heparin seems also to be of benefit to some types of chronic fatigue syndrome patients].

3.2 Role of Nitric Oxide in Heparan Sulphate BiochemistryA sound argument can be made for a central role of nitric oxide metabolite action in heparan sulphate biochemistry in which the nitrosative scission process creates signalling oligosaccharides (the

Fransson group are now leaders in this field; others who have contributed include Vilar et al., 1997. (This was also the theme of an internet discussion by Grant in 2000). A convenient method of establishing the presence and quantifying heparan sulphate in mixtures with other GAGs is the use of nitrous acid which causes a quantitative scission of heparan sulphate generating oligosaccharides. This was not originally thought to be of relevance to any in vivo situations, but recently this assumption has needed to be revised following the discovery during the last 20 years that nitric oxide is a major messenger molecules in animal biochemistry. The important second messenger nitric oxide seems naturally to create oligosaccharides from heparan sulphate as part of a complex intracellular signalling system which also requires copper and perhaps zinc ions.Heparan sulphate is degraded by two distinct routes one involving the enzyme heparanase and the other a redox metal-ion / nitric oxide route by which oligosaccharides having “hormone” like activities are formed via nitrosative scission of heparin/heparan sulphate chains.

Heparan sulphate oligosaccharides apparently act as messengers to induce intracellular trafficking and also engage in extracellular functions.

It is now postulated, on the basis of partly unpublished research carried out at Aberdeen U. that those inorganic factors and lipids/water structuring effects which affect such nitric oxide influence on heparan sulphate signalling are also likely to contribute to the aetiology of the wide range of degenerative diseases which have been associated with aberrant protein nitration (these include cancer, atherosclerosis, arthritis, Alzheimer’s disease, prion diseases and other neurodegenerative disorders including Niemann-Pick disease (where there is a dyshomeostasis of cholesterol which is known from studies of Drosophila, to be jointly involved in control of heparan sulphate growth factor signalling).

3.3 Putative Roles of Heparan Sulphate and Nitric Oxide in Prion Diseases

Diseases where nitrosative stress or lack of nitric oxide exists could also involve dysfunction of heparan sulphate biochemistry. This includes prion diseases, cf.:

“Scrapie infection of neuroblastoma cells precludes nitric oxide production when the cells were challenged with lipoprotein (Lindgren 2003) (suggesting that studies of the deaminative cleavage of glypican-1 heparan sulphate may provide for a better understanding of the pathogensis of neurodegenerative disease…. Copper deficiency has also been associated with neurodegenerative disease” [Mani et al. 2004).

It seems likely that the successful therapeutic intervention by heparan sulphate mimetics such as pentosan polysulphate in pathological conditions depends on the ability of such mimetics to substitute for such endogenous oligosaccharide messengers which if the nitric oxide supply is diminished as a result of infective prions the amounts of heparan sulphate oligomers will also be reduced. A possible feedback system may use heparin-like fragments of heparan sulphate as hormone-like signalling entities to signal for or inhibit gene expression. In this way plant xylan derivatives could fit into the animal cell feedback system and reset the stress response seeming to act as direct anti-pathogens. (Pentosan polysulphate was also reported to be more highly active for the potentation of the mitogenic action via stimulation of DNA synthesis in 3T3 fibroblasts than is low molecular weight heparin (Robinson et al., 1992)).

4. The MetallomeThis was suggested as a system of biochemical relevance by RJP Williams. A recent contributor to this field was H Haraguchi who proposed in 2004 that a much larger number of inorganic elements than the 20 which had been proposed by Williams to naturally occur in animal fluids. Haraguchi also emphasised the relevance of the approximate correlation which exists between the multi-elements present in biological fluids like blood serum and seawater and mentioned heparin in a table of bio topics for which metallomics was suggested to be relevant. The metallome seems to be a key sub-system of the “phenome” (Varki) which also includes the geneome, the proteome and the heparanome.As suggested above if the basis of life hinges on the unique physical chemical properties of liquid water, this explains the importance in biochemistry of molecules (such as polysaccharides, osmolytes

such as urea and inorganic including metal ions which can most efficiently modulate this physical chemistry.

4.1 Seawater & “Seawater-Related” Metallomic Matrices

Polyanionic molecules can be predicted to offer binding sites for a variety of naturally occurring metal ions present in biological fluids and other natural waters. Seawater

The sea is believed to be the location of the origin of life. The idea that blood serum resembles seawater seems to have originally been due to work done in Toronto ca. 1905 by Macallum.

Seawater is now believed to contain all elements.

The dissolved or suspended inorganic elements in the sea seem to exist under a form of homeostasis which has been effectively retained over geological time, but how this is achieved is unclear. A possible mechanism could involve natural polyanionic organic humic colloids (humic matter seems to be present in the sea in amounts of ca.1-2 ppm which from in vitro crystallisation experiments conducted by the author at Aberdeen U, is a sufficient amount to allow natural humic matter to inhibit and regulate the formation of inorganic crystals (especially the ability to create stable supersaturated solutions of e.g. of CaCO3 and BaSO4). [Humic matter is formed by the degradation of living organisms, in a process in which those residual chemical structures which have a high intrinsic chemical stability like polymethyene or polyuronates or their metal complexes resist biodegradation and attain average 14C lifetime of ca.1000 years. A humic polymer could also have formed pre-biotically from the ‘primordial soup’ (e.g. by the polymerisation of formaldehyde and the pyrolysis of the polymer obtained). Anionic polysaccharides such as heparan sulphate seem to share with humic matter the ability to create a seawater-like mixture of inorganic solutes and this may have been their original role during the evolution of early animal ancestors in the sea some 1000 million years ago].

Some confirmation of this idea comes from the apparently exact mathematical relationship which appears to exist between salinity and amounts of these sulphated polysaccharide required to regulate the osmolyte balance in primitive animal organisms (Nader at al., 1983).

The Complexity of the Inorganic Profiles of Biological Fluids.

Biological fluids are complex mixtures of inorganic ions, all of which can apparently can bind simultaneously to long chain biological antennae by relatively poorly understood electrostatic plus other attractive forces allowing them to collect inorganic ions (e.g. 50+ in number (Haraguchi, 2004)) and inorganic motes from blood serum and other biological fluids [a collection of redox + non-redox-active metal and non-metal ions and small inorganic and organometallic particles which exist separately from the more studied and numerous protein components]).

4.2 The Heparanome & the Metallome Must Mutually Interact in Health & DiseaseThe biological heparanome will create a multi-inorganic-element containing glycocalyx. This could serve as an inorganic element reservoir in animals in a similar manner to anionic polysaccharides in marine organisms (e.g., the alginate components of brown seaweed which are known to occur in vivo as multi-element arrays, as originally demonstrated by Wassermann in 1949).

[The efficient uptake of inorganic ions and particles by polysaccharides may likewise be of value for protection against radionuclides; this method was apparently used for human subjects following Chernobyl; such uptake of radionuclides by heparan sulphate in vivo seems also to be an important part of mechanism by which radionuclide imaging (scintigraphy) detects tumours (cf. e.g., Kojima et al., 1983); the provision of a range of essential inorganic nutrients by the heparanome may also be why it is targeted by pathological organisms such as Toxoplasma gondii (cf., Bishop et al., 2005)].

Recent publications by the Fransson group etc., have shed light on how redox metals (Cu has been studied but it can be suggested that this work be extended to the other heparin-associted redox metals which include Fe, Mn, Ni, Cr, Ag and Ce) might contribute to nitric oxide-dependent heparin/heparan sulphate signalling and its disturbance.

The author has suggested that the multi-elements in heparin and by implication heparan sulphate are also correlated with biological fluids and seawater and therefore the unique ability of heparin and related molecules to act as reservoirs of such multi-inorganic elements should, it might be suggested, be a central theme of further metallomic and heparan sulphate research.

The co-existence of a range of inorganic elements with these highly anionic sulphated polymers (of which heparin/heparan sulphate are the most well characterised examples) may per se be part of a wider information holding and processing system used by animals.

(This may function in co-operation with the system of inorganic polyphosphates (Kornberg, 2006, the Nobel laureate, has noted that this important ubiquitous cell surface polyanionic system has largely ignored by almost all biochemists [cf., however, the paper of Shiba et al., loc. cit. who listed the range of biochemical activities which these polyanionic molecules are known to influence, this range being reminiscent of the behaviour of heparin/heparan sulphate]). It is suggested to be of especial significance that these polyanionic molecules share with heparin the ability to bind metal ions and to inhibit seeded crystallisation processes. (e.g. as described by Grant et al. 1987; cf. Grant et al. 1992b).There seems, however, to have been no reported attempts to determine the effects of polyphosphates on protein (e.g. prion) misfolding.

[In a similar manner to how inorganic phosphates in the form of hydoxyapatite columns can fractionate DNA, hydroxyapatite can fractionate heparin (Marion Ross, Aberdeen U. studies). Heparin also removes material from such columns producing stable colloidal suspension of a heparin-Ca-hydoxyapatite (This was studied by infrared spectroscopy the author). It is also known that hydroxapatite minerals are multi-inorganic-element holding matrices (cf., Grant et al., 1992b); natural multi-element containing bone-like materials may therefore be able to crosslink to heparin/heparan sulpahte in a manner which reflects both the inorganic element profile and the microstructure of the heparin/heparan sulphate.This can be suggested to especially apply to the inorganic elements Si, P, B, Fe and lanthanide ion-containing structures which could co-exist with phosphate containing groups associated with heparin/heparan which may thereby putative contribute to the modus operandi of heparan sulphate including its modulation of protein folding.

The puzzling phenomenon of the strong binding to heparin of those inorganic elements which occur as anions (e.g. sulphate) was noted by Jaques (1978). The belief that the binding mechanism of inorganic elements to heparin is entirely electrostatic seems to be contradicted by this behaviour. A similar problem arises in accounting for the association of (anionic) silicates with heparin. That this phenomenon may have major physiological relevance is suggested by the recent report that heparin-silica based chromatographic system can efficiently separate both cations and anions on a single column (Takeuchi et al.,1998). It should be noted that all natural polyuronides including heparin and heparan sulphate occur in association with inorganic silicon in some unknown chemical form; this might improve the biological ion pumping ability of sulphated polysaccharides (the possibility of such a system is suggested by the employment in industry of chemically related polysulponated organic polymers as ion conductors in which the additional presence of inorganic silica particles is known to confer improved performance (vide infra). (The analogous biological cell membrane heparan “mineral” system could, however, now have become redundant for ion pumping activities in modern organisms which functions are usually regarded as being entirely provided by protein-based ion channels and pumps.)

4.6 Metalloproteinase Shedding of Heparan Sulphate is Affected by Exogenous Heparin or PPS

Shedding by metal ion-(Zn2+) dependent metalloproteinases of syndecan ectodomain heparan sulphate chains attached to residual core protein are believed to generate soluble extracellular heparan sulphate proteoglycan effectors of a wide range of biochemical activities (including those of adhesion molecules, cytokines, growth factors and their receptors) (cf., Bernfield et al.).Exogenous PPS or heparin can modify the processes affected (but somewhat differently).[Matrix metalloproteinase MMP-2 was significantly increased after treatment with PPS or heparin but heparin could also decreased MMP-9; heparin or PPS also increased the formation of tissue inhibitor of metalloproteinases, TIMP-1 and TIMP-3 (cf. Elliott et al.)].

5 Protein Folding

5.1 The Putative Driving Force of Water Structure Relating to Protein Folding

Towards the end of the nineteenth century Hofmeister studied the effect of inorganic ions etc. on protein denaturation. Denaturation can be approximately equated with misfolding. The Hofmeister series ranks solutes for this ability. Half a century later WAP Luck found good experimental evidence that the Hofmeister series could be attributed to the effect of these solutes on the supramolecular structure of liquid water.

Protein folding is also commonly attributed to a phase boundary regulatory hydrophobicity/hydrophilicity balance determined by the amino acid sequences of the protein. This could be due ultimately to an influence of pore and surface attractive and repulsive forces with solute, to which glycosylation will contribute (n.b. biological fluids are a multi-inorganic element seawater-like mixture; commonly used buffers may not replicate this exactly and lead to erroneous indications in vitro studies of protein folding; another problem for discussion of model systems for protein folding is possible differences in solute composition of yeast vs. mammalian). In a model of protein folding using the two-state neighbour model, it was proposed that polar groups promote the formation of low density iceIh-type bonding in their neighbourhood, whereas nonpolar groups tend to promote the high density ice II-type structure. Large changes in the neighbouring water structures are thereby induced which induce protein folding which was proposed to depend on a delicate balance between outer hydration effects and inner hydration (cf. Robinson & Cho, 1999).

Another ancient system of molecular chaperones are the heat shock proteins which have water activity regulation roles related to control of protein folding. There are hints that mammalian heparanase may have relict barely detectable amino acid sequence homologywith hydrolases generally and also apparently with some heat shock proteins, suggesting a more ancient common ancestor of both.

5-2 Heparin/Heparan Sulphate & PPS Affect Both Protein Folding & Inorganic Crystallisation

Polysaccharides, especially anionic polysaccharides like alginates and heparin are known to behave as highly active morphogens for all kinds of organic and inorganic phase boundary regulation, allowing the control of both organic and inorganic crystallisation and protein folding including folding of protein-metal ion complexes.

[Protein folding is subject to modulation by glycosylation both O and N linked as well as glycosylationdue to ionic linkage (especially the association with heparin/heparan sulphate)].

Heparin/heparan sulphate is involved in binding to and altering the conformation of proteins (this is most firmly established for antithrombin) as well as regulating the form and occurrence of calcium oxalate as well as for purely inorganic particles thereby functioning as general in vivo chaperones to protect tissues from the damaging effects of all kinds of toxic particles and crystals (cf., Grant et al. 1992a).

A renowned geneticist (Lima-de-Faria) has suggested that the processes which govern the modulation of [all kinds of] crystal formation (especially for the control of the seeding process which often involves a rate controlling influence of change in hydration) is the ultimate basis of biology, and by inference is also highly relevant to pathologies. e.g., of cancer. This includes purely inorganic crystal formation e.g. the inorganic particles which promote urinary cancer induced by saccharin in male rats (Cohen et al. 2000)).

5. 3 Possible Roles of Metal Ions in Prion Misfolding Neurodegenerative Diseases

The role of heparin (and its mimetics, e.g., PPS) for inhibiting the adverse effects of misfolded proteins could include the inhibition of the mechanism of toxicity of iron and manganese ion linked prions and their aggregates. Iron oxidation state three and manganese oxidation state four particles; such particles may putatively promote both protein misfolding and (in an analogous manner to the toxic forms of asbestos which is believed to depend on associated iron) promote the formation of damaging reactive oxygen and nitrogen containing free-radicals.

5-3--1 ManganeseManganese is a redox metal which under oxidative stress could yield higher oxidation states which can be potentially highly damaging. Manganese in oxidation state three would promote oxidative degradation and in oxidation state four could form sparingly soluble seeds which could cause pathological protein misfolding. Manganese is also required for correct heparan sulphate synthesis and altered manganese nutrition could adversely alter heparan sulphate biosynthesis (Kalea et al., 2006); manganese can also induce inducible nitric oxide synthase in neurological situations (cf., Bae et al., 2006).Experimental evidence for possible roles of manganese in prion diseases include the studies of Treiber et al. (2006) who reported that manganese could misfold prions in the yeast cytosol environment (the idea that manganese toxicity might determine prion diseases was originally proposed by Mark Purdey)

and the work of Quaglio et al. (2001) who reported that copper ions could form a protease resistant misfolded prions (but distinct from that of the scrapie isoform).

5-3-2 Iron

Iron has recently been reported (Basu et al., 2007) to modualte the formation of protinase K-resistant prion protein and therefor be a key inorganic putative element in the in vivo promotion of tissue damage by prion aggregate metal ion complexes.

Iron is also an especially pertinent candidate metal which (via an associated proton release), could contribute to the transport of specific ions (e.g. of copper) by prions.

Fe(II) is toxic and strictly controlled but the amount present (in equilibrium with serum ferritin (a major iron (III) storage protein) which increases with age in the absence of disease Jarrett et al ); ferritin is also believed to facilitate PrPsc uptake in the intestine (Mishra et al., 2004).Salonen et al. found a high epidemiological correlation between excess ferritin iron and cardiovascular mortality (this might be linked to heparan sulphate status since heparin/heparan sulphate can apparently convert Fe(II) to non-toxic Fe(III) polymeric oxy-hydroxy forms. But over-purified heparin seems not to do this, a possible reason being a requirement for the presence of trace amounts of cobalt to act as the oxidation catalyst (FB Wiliamson et al., Aberdeen U research results [also G. Mackintosh unpublished Aberdeen U. work which also included studies of PPS in this context). Further experiments to confirm this work are required.Other polysaccharides, including hyaluronic acid may similarly contribute to the pacification of toxic iron.Merce et al. reported the protective action of removal of Fe(III) by hyaluronan and Sipos et al. have reported that spherical colloidal iron rich particles, which tie up Fe in an inaccessible form, attach to chitosan.The high anionic change on heparin/heparan sulphate may endow these polysaccharides with especially highly efficient capacities for detoxification of excess inorganic ions.As with other heparan sulphate proteoglycan functions those for iron ion transport etc.may be partly redundant.Liver heparan sulphate proteoglycan seems to have an independent transferrin-like role (Hu et al.) Transferrin and ferritin and other proteins which have high iron binding power are probably the principle extracellular transport and storage ligands for iron.

5-3-3 Aluminium & BerylliumThe promotion of dementia by aluminium intoxication This is a well established phenomenon.It is also conceivable, from a pure-chemistry perspective, and environmental protection agency internet-available documentation, rather than from any hard medical evidence, that aluminium other inorganic elements could to have entered the food chain in Guam. This could include beryllium which apparently can be detected in ground waters arising from environmental pollution by large amount of beryllium-containing containing bearings from a major military aircraft engine servicing facility could suggest that this element might have been involved in the aetiology of the dementia of Guam.The ubiquitous presence of Al3+ in the environment in man-made chemical forms, however, could suggest that this element could have a wider but largely unacknowledged potential role in promoting neurological dysfunction. Aluminium is commonly present in many chemical reagents used for preparation of solutions and in drugs including heparin which are widely used medically (cf. Bohrer et al., 2004).An unusual mode of binding of Al3+ to heparin (unpublished Aberdeen U. studies) and by inference to heparan sulphate may be relevant to the suggested importance of this element in pathologies which have been associated with biological actions of heparan sulphate proteoglycans. (N.b. Al3+ intoxication is believed to promote dialysis dementia; this could be relevant to the elucidation of the role of environmental factors in the aetiology of sporadic neurodegenerative disorders Cf. “Aluminium in Food and the Environment” Royal Society of Chemistry [Proc Symp. held on 17 May, 1988 in London, Ed. R Massey & D Taylor]Special Publication, No.73 (cf., p.20-36 in which JA Edwardson et al. (of the MRH Neurochemical Pathology Unit Newcastle General Hospital) discussed the possible neurotoxic effect of aluminium)).

There are numerous more recent references to this problem, e.g., Perl, 2006.

The problem of the co-occurrence of numerous inorganic elements in heparin was of practical concern e.g., by HJM Bowen (1966) in a standard classical text on inorganic biochemical analysis, warns against using heparin during preparation of solutions for any inorganic element analysis in blood because of the presence of such inorganic elements in commercial heparin. This had especially prevented measurement of the manganese contents in heparinized blood samples, and had led to a long delay in establishing the normal levels of this element in blood serum..Aluminium, beryllium, thallium and cadmium were, however, not included in the list of heparin-associated elements given by Bowen, nor were aluminium and beryllium assayed for in the later Aberdeen U. studies of CF Moffat (1985) (who had used an aluminium spark source mass spectroscopic method). Moffat, however measured the amounts of 38 inorganic elements which were present at >1ppm in a pharmaceutical industry research grade sodium heparin batch (which, incidentally, contained little (< 1 ppm) manganese); this heparin seems also to have contained some thallium and cadmium (detected at 1.5 ppm in an ion-exchange resin purified sample derived from the same starting pharmaceutical sodium heparin).The lack of thallium poisoning in patients who had been assumed to have been administered heparin containing ca 7ppm thallium was, however, puzzling (Moffat, 1985). It might have pointed to the existence a thallium de-toxification mechanism afforded by heparin/heparan sulphate.It should be noted that a similar amount of thallium seems to be associated with anionic polysaccharides present in kelp, used as a human food. The presence of a full-seawater-like range of multielements (which of course includes aluminium, thallium and cadmium) in pharmaceutical heparin is in agreement with the general results reported in the CF Moffat (1987, Aberdeen U.) Ph.D. thesis.

The classical method of purification of heparin (Howell, 1928) had used Lloyd’s reagent (precipitated aluminium silicate), a method which was continued by later workers (Scott & Charles, 1933) in purification processes which also employed CdCl2 and Ba(OH)2 and NaCl (described as brine in a later Patent, conceivably containing the multi-elements of seawater).The Dietrich group (Straus et al., 1984) described a method of preparing heparin for laboratory studies using “Celite” (SiO2, diatomaceous earth) based on fractional precipiation of heparin from other GAGs as the K salt, avoiding the possibility of Al Cd and Ba contamination inherent in the Scott & Charles method.Choay S.A. (France) described in 1973, in a UK Patent, a method of preparing a Ca-enriched heparin starting from a 10-12% aqueous solution of Na heparinate which had an anticoagulant activity of 166 IU/mg, and respective contents of NH4

+ ,Ca, Na, K and Mg of 0.1, <0.1, 11.5, 0.5 and <0.1, and also was free of heavy metals and proteins; to this solution solid CaCl2 was introduced, the pH adjusted to 7.5 by the addtiion of CaO, followed by dialysis and filtration on an asbestos plate, the pH brought to 6.5 by adding HCl and then EtOH added to give a mixed Na Ca heparinate which was dissolved in distilled water to which solution CaCl2 was added, agitiated for e.g. 12 hours at pH = 7.3 followed by dialysis against deminerlised water to give a dialysate to which was added a bacteriostatic agent (to 0.3%) metacresol; this solution was then filtered through asbestos and after standing 2h, reprecipitated with EtOH to give “Na-free, Ca heparinate” with an anticoagulant activity of 160IU/mg.

A method was described by Kerey G, et al., (1986) of precipitating heparin of pharmacopoeial purity, preferably calcium heparinate using a similar method but apparently avoiding filtration through asbestos employed precipitation by quaternary ammonium salts of cetyl pyridinium chloride or Hyamine in which the formation of an insoluble phase from the ion exchanged multi-element form of heparin present in aqueous solutions which are known to contain heavy metals and other inorganic anions (is evidently the origin of the multi-inorganic element nature of heparin) is displaced towards a sparingly soluble single salt form which can later be transformed into a desired single salt form.

The presence of Si, Al, Cd, Ba etc., in some heparin may, however, be due to the continued use of classical methods for the preparation of commercial heparin, by the use of asbestos or similar filtration aids as well as the uptake of such elements present in trace or ultratrace amounts in biolgical fluids such as blood serum.

5-3-3-1 Recent evidence for the presence of aluminium in heparin and other parenteral nutrition-related substances[a predictable problem for such highly polyanionic drugs but which urgently requires further clarification and possible rectification

in order to promote the future attainment of the full potential of heparin/heparan sulphate and heparinoid therapeutics]

In a recent studies relating to the safety of substances used during hemodialysis treatments the occurrence in heparin of aluminium [Bohrer D et al., in RBAC (Brasil). 2004; 36(2) 99-103] and arsenic [J Parenteral Nutr 2005: 29 (1) 1-7] was quantified. The following is an excerpt from the RBAC article relating to aluminium, translated from Portuguese:- [Summary (in English): “The elevated toxicity of aluminium for renal insufficiency patients is well documented in the literature. The toxic action of this element is so elevated that an annual control of the serum aluminium level of the patients on regular hemodialysis treatment is required by the (Agencia Nacional de Vilancia [Brasil]). Due to the ubiquity of aluminium, the analysis demands special care to avoid contamination. In this work the most important issues related to this analysis from sample collection till the aluminium quantification itself, (were) considered in order to carry the analysis successfully. The measures to avoid contamination are relevant because of the low limit that put the patient at risk. Aluminium levels above 30g/L mean intoxication and determine a treatment with chelating agent to reduce serum aluminium level. As this treatment is not free from side effects, it is very important that reliable results are obtained in the serum aluminium analysis.”]DG Transl. Table III (p.102, of the Bohrer et al. RBAC article) Al (g/L or g/g) (present as an impurity in the reagents studied).Nitric acid Merck superpure 110+1g/L Merck distilled 10+1mg/LEDTA (sodium salt) Merck 3.37+0.7 g/g 3.37ppm[data also given for sulphuric acid, trichloroacetic acid) Sodium tungstate Merck 26.5+1.70mg/g 26.5ppmNa2SO4 Reagen 26.5+3.77g/g 26.5ppm

Heparin bovine (sodium salt) Sigma 0.0 g/g (Odd that the Sigma results were reported less precisely;

could this mean that these values were not independently verified?)

Heparin porcine (sodium salt) Sigma 0.0 g/g Heparin porcine (ammonium salt) Sigma 0.0 g/g Commercial heparins 5000 IU/ml (ca. 30mg/L) Fujisawa 732+23 g/L Roche 783+51 g/L ca. 35ppm Cristalia 72+6 g/L 25000 IU/ml (ca. 150mg/L) Eurofarma 436+40g/L Roche lot1 137+2g/L Roche lot2 168+3g/L Roche lot3 190+5g/L

It could be suggested that aluminium should routinely be removed from all commercial heparins prior to use of this drug in humans, especially for kidney dialysis patients. This may be easily achieved by percolation through a cation exchange resin column; an example of the high efficiency of this procedure being given in Fig. 1 in the Bohrer et al. RBAC paper which shows the amount of aluminium in heparin before and after ion exchange replacement of Al3+ by Na+ on a cation (exchange) column, where before and after the first and subsequent uses of the column the Al3+ contents in the eluted heparin solutions were determined to be 350, 0,0,0, and ca. 15g/L (i.e., the column became saturated with Al3+ after three elutions).

The presence of variable amounts of aluminium and other toxic elements (e.g. lead, arsenic thallium and cadmium) in commercial heparin- a predicatable problem with highly polyanionic drugs which urgently requires further investigation and rectification in order to achieve the future attainment of the full potential of heparin/heparan sulphate and heparinoid therapeutics

5-3-4 CadmiumCadmium has also been reported to occur in many commercial zinc dietary supplements where its occurrence is thought to promote prostate cancer, the etiology of which is almost entirely unknown, but a possible mechanism could involve disruption by Cd2+ of heparan sulphate growth factor signalling or metalloproteinase release of soluble heparan sulphates from cell surfaces).

5-3-5 Silicon

The details are at present far from clear regarding the roles of the essential element silicon seems to be held under strict homeostatic control in human blood (Bisse et al., 2005).Si is probably present as forms of silica especially colloidal silica sol particles rather than silicate esters, which can, however, form with rare sugars.It may co-exist with P in the form of long chain polyphosphate (cf. Grant et al. 1992a; cf. Kornberg, 2006); this suggests such a coexistence may also occur with heparin/heparan sulphate.These molecules could be present in small amounts in the full inorganic metallomic array associated with heparin/heparan sulphate. There have been indications that inorganic silica has been associated with polysaccharides from earliest stages of the evolution of biota (cf., Iler 1978 and Grant et al., 1992a;) and could therefore be especially important as contributors to the biological activity of the biopolymers with which they are associated in modern organisms. In this context silica is known to enhance the stability and functional activity of the industrially important (e.g. for fuel cell use) man-made sulphonated polymers which have almost identical metal ion and water binding properties to those of heparin/heparan sulphate (cf. Adjeman et al., 2002; James et al., 2000 and Grant et al. 1990).

Another key role of inorganic silica particles in polysaccharide chemistry and in biology in general (including the triggering of protein folding) may be to act as a nucleating or seeding agent. FB Williamson, former sen. lecturer and polysaccharide group manager Aberdeen U., (personal communication) in this context further recommended as essential reading the treatise by Lima de Faria (1988) which draws attention to the close similarities and suggested related mechanisms which determine both inorganic and biological morphologies.Differences between results of reported studies may be due to different nucleating agents inadvertently present or absent form the experimental conditions usedHeparan/heparan sulphate and nucleic acids may both require nucleation/seeding for their normal functions. Applied to iron ion binding by heparin/heparan sulphate and other polysaccharides as a mechanism of antioxidant protection this binding may additionally require the presence of some nucleating agent normally present in vivo but not necessarily in vitro. (Some literature reports which had failed to confirm earlier Aberdeen U. reports that heparin could act directly as an antioxidant may have been affected by the absence of such nucleating agents whereas other investigators (Albertini et al., 1996) who unwittingly had included the necessary nucleating agents in their experimental conditions, confirmed that heparin could demonstrate a copper ion binding antioxidant activity which was relevant to in vivo lipid oxidation conditions.

5.4 Inorganic SulphateThe effectivness of the tissue protection afforded by the more highly sulphated heparan sulphates might be subject to its limitation under conditions of insufficiency of inorganic sulphate needed for generation of the biological sulphation agent phosphoadenylylsulphate (PAPS). (During discussions with Mark Purdey, it became apparent that defective heparan sulphate tissue protection functions might arise either from a direct lack of dietary inorganic sulphate or from deficiencies of the function of the inorganic sulphate transporter which apparently requires [a light quality dependent] vitamin D isoform and thyroid factor cofactors (Dawson & Markovich). [A light intensity dependence of blood serum inorganic sulphate concentration is consistent with the observation by Krijgsheld et al. that this could exhibit a circadian rhythm.]A hitherto puzzling, but fairly consistent, global phenomenon of the latitudinal variation in the incidence of multiple sclerosis, which seems to be most exactly observed throughout Australia, might conceivably have arisen, it was proposed, from the effect of the latitudinal variation in light intensity on the supply of viamin D isoform required for the correct function of the inorganic sulphate transporter needed for heparan sulphate sulphation [cf., the heparan sulphate (and divalent cation-) dependent growth factor mediatied mylein sheath repair (Mark Purdey, 2004, loc. cit., hypothesis of the aetiology of this disease]. A relatively recent deficiency of inorganic sulphate in agricultural soils (e.g., in the U.K.) which has evidntly arisen from the changeover from coal burning to low sulphur oil burning for electricity generation, has been suggested to be a major enivonmental promoter of such diseases as autism and chronic fatigue syndrome and suggestions that these may be alleviated by dietary supplements of inorganic sulphate or organic molecules which convert to inorganic sulphate in vivo. On the other hand, a preliminary (unbublished) study by the author (at Aberdeen U.) of the effect of a diminution of the inorganic sulphate concentration in BHK fibroblast cell cultures did not show up any obvious easy-to-achieve reduction in the degree of sulphation of heparan sulphate. Evidently such cells can maintain the necessary sulphation of heparan sulphate, e.g., by an ability to replendishing insufficient inorganic sulphate by producing this from sulphur-containing amino acids. A diminution of the sulphation of proteoglycans in cartilage as a result of diminished presence of inorganic sulphate has, however, been reported by, e.g., Ito et al., for epiphyseal cartilages in culture but these authors found no relationship was observed between the serum sulphate concentration and the extent of sulphation of xenobiotics. Silbert et al., (cf. Ito et al.) reported that human skin fibroblasts cultured under conditions of inorganic sulphate depletion showed a markedly diminished sulphation of chondroitin sulphate but the sulphation of dermatan sulphate was preserved to a greater degree (but little epimerization of glucouronic acid to iduronic acid had occurred in the absence of sulphation; diminished inorganic sulphate also tended to increase the relative proportion of dermatan sulphate). Other, yet to be identified, factors may be required to facilitate the diminution of heparan sulphate sulphation under conditions of inorganic sulphate insufficiency.

6 Some of Mark Purdey’s Contributions to the Understanding of the Aietiology of TSEsThe self-educated scientist, the late Mark Purdey, (working from High Barn Farm, Elmworthy, Taunton, UK) will be remembered as the originator of a number of novel ideas relating to prion and other neurodegenerative and related diseases. These include putative roles of intoxication by insecticides and possible roles of manganese and copper ion dyshomeostasis in prion diseases (cf. the 2005 review by Case and the later findings of Treiber et al., 2006 on manganese induction of prion misfolding in yeast cytosol; barium and other toxic metal intoxication might also perturb of heparan sulphate signalling (of possible relevance to the aetiology of multiple sclerosis) and the effects of

barium and strontium on prion related heparan sulphate biochemistry and the occurrence of a ferritin-related mechanism of pathogenic particle generation of relevance to the aetiology of TSEs. A role of metal ions in heparin/heparan sulphate biochemistry may also pertain to the mechanism by which pentosan polysulphate, a mimetic of heparin/heparan sulphate anionic sulphated polysaccharides, can act as an efficient therapeutic agent for a range of diseases including prion diseases.The possibility that pentosan polysulphate can provide credible therapeutic benefit for variant Creutzfeld-Jacob diesease patients is a major milestone for the encouragement of researches aimed at the more general application of therapeutic agents based on heparan sulphate biochemistry.

Mark Purdey publicised the relevance of inorganic element intoxication in prion and other neurodegenerative diseases. This included Kuru and the dementia of Guam (personal communication).The putative role of manganese intoxication in these diseases was given a wide public coverage in a film shown on TV (BBC 2 Correspondent, on 25/3/01) which presented his manganese-based hypothesis of the origin of prion diseases. [This hypothesis seems to have been submitted to Medical Hypotheses on 1 April 1998, accepted by the referees on 29 October 1998 but delayed for publication until 2000 (Med Hypotheses. 2000; 54 (2) 278-306]. This paper suggested that the substitution of a foreign cation (e.g. of manganese) for copper ions normally bound to prions might initiate TSEs.These ideas have probably influenced the researches of D. Brown and G. Multhaup,

Subsequent papers by Mark on related hypotheses seem to have been more quickly accepted for publication (e.g. that entitled “Does an ultra violet photooxidation of the manganese-loaded/copper-depleted prion protein in the retina initiate the pathogensis of TSE?” was received on 20 December 2000 and accepted on 9 January 2001) and “Metal microcrystal pollutants; the heat resistant, transmissible nucleating agents that initiate the pathogenesis of TSEs?”, was received by Medical Hypotheses on 8 March 2005 and accepted for publication on 9 March, 2005. The occurrence of elevated levels of the following metals in antlers of deer suggested roles for Ag/Ba/Sr piezoelectric crystals in the aetiology of chronic wasting disease (CWD).

A further paper entitled: “Does an infrasonic acoustic shock wave resonance of the manganese 3+ loaded/copper depleted prion protein initiate the pathogenesis of TSE” was received by Med Hypotheses on 22 August 2002 and published later.The idea that acoustic shock could trigger prion diseases seems to have been supported by the use of sonication by Saa et al, 2006, as a key requirement for the efficient seeding of infective prion aggregates in high yield.

7 Promotion of Neurological Diseases by Pesticide Exposure Mark Purdey’s original BSE hypothesis (Med Hypotheses. 1996; 46: 429-54) had suggested that chronic pesticide [e.g. organophosphate pesticide] exposure might have been involved in the augmentation of cellular prion formation and its misfolding. Pesticide exposure has also been associated with common, putatively neurological, conditions including Gulf war syndrome, organophosphate poisoning and chronic fatigue syndrome (myalgic encephalomyelitis) for which literature reports suggest that the accumulation of chlorinated pesticides in tissues of human subjects might be correlated with the incidence of this disease in such subjects. It should be noted that contamination of animal food chains by chlorinated pesticides is augmented by products of similar potential neurological disease promoting activities formed during the incineration of all chlorinated organic substances [which generate hexachlorobenzene and its metabolites (substances with a dioxin-like activity)]. Mark’s brother Nigel Purdey has suggested on his internet site that his own intoxication by chlorinated pesticides might have been the origin of his chronic fatigue syndrome. Dr FB Williamson (retd sen. lecturer, polysaccharide group, Aberdeen U. who has also been chronically affected with such a disease) favoured a hypothesis (arrived at during discussions with the author and Vance Spence, Dundee U.) of the aetiology of this disease involving the dyshomeostasis of redox chemistry of iron (and the possible involvement of nitric oxide and heparan sulphate in this). The recently suggested pro-oxidant role of iron-prion aggregate formation (Basu et al., 2007) and the now established likely role of heparan sulphate in prion diseases, appears to be a related hypothesis.(The author had also previously engaged in industrial research studies of the formation of hexachlorobenzene by the pyrolysis of chlorinated organic substances molecules and the use of chlorinated organic molecules as catalysts of transition metal oxidation to improve polyolefin yields (e.g., Grant, 1974, loc. cit.) in which the pro-oxidant effect of perchlorocarbons is utilised to re-oxidise spent transition metal catalysts. This background now prompts an additional hypothesis of TSEs that such prion diseases and other neurological conditions may be promoted by the ultratrace presence of chlorinated organo-chlorine, dioxin-like, incinerator off-gas product exposure. This could explain a pro-oxidant scenario in which higher oxidation states of manganese could be generated in situ. There is a known to be an accumulation of such substances in the fatty tissue of animals following their accumulation and transmission in the food chain which has led to an attempt by International Treaty (The Stockholm Convention) to limit their manufacture and use).

7-1 Perturbation of Nitrosative Heparan Sulphate Signalling by Metal (e.g., Manganese & Iron) Ion Dyshomesotasis

Inorganic ions could affect prion diseases by their direct influence on the associated heparan sulphate chaperone/hormone system, e.g. via perturbation of those metal ions (especially copper and zinc) required for nitrosative signalling and divalent metal ions (e.g. calcium, magnesium and manganese) needed for fibroblast growth factor receptor assembly [Kan et al., 1996].This scenario is in accord with the discovery of the existence of unsubstituted glucosamine residues (pre-primed for nitrosative cleavage) within those heparan sulphate chains, recognized by the monoclonal antibody 10E4, which co-locate with scrapie lesions (Leteux et al., 2001). A further corollary to this finding is that the prion diseases could arise directly as a consequence of the dyshomeosasis in the nitric oxide - un-substituted glucosamines intracellular signaling system (which is believed to be involved intracellularly in directing the activity and trafficking of intracellular organelles).

It is conceivable that manganese ions could especially perturb nitrosative signaling and this is the reason for their suspected involvement in the aetiology of the pathology of TSEs (cf. Purdey, 2000) as well as in the cholesterol dysfunctional Niemann-Pick disease (Mani et al,. 2007).Manganese ions liberated from glycocalyx stores could produce excess primed sites for nitric oxide scission.

[The endocytosis of heparan sulphate metal ion complexes may allow small amounts of redox metal ions to enter the cell cytosol but remain within the polysaccharide milieu in which copper ions jumpbetween selected carboxylate groups alone the heparan chains (a process discovered to occur with heparin by Rej et al. in 1994)].

7.b Metal Ions Which Are Known To Crosslink Heparan Sulphate to Target Proteins

The metallomic matrix on heparan sulphate seems likely to provide a range of essential inorganic cofactors for the selective crosslinking of heparan sulphate (H) microstructures to target proteins. The following are examples of protein-metal ion-H:annexin five-Ca2+-H (Capila et al, 2001) endostatin-Zn2+-H (Richard-Blum et al., 2005), prion- Cu(II) -H (Gonzalez-Iglesias et al. 2002)fibroblast growth factor receptor dimer-Ca2+-H (Kan et al. 1996)

Heavy Metal Ions in HeparinThe original impure form of heparin, discovered and shown to be a blood anticoagulant by J McLean in 1916 was too toxic for human application perhaps partly because of its high toxic heavy metal contents, but by around 70 years ago, a less toxic form of this drug had become available allowing it to be routinely used for the inhibition of blood coagulation during surgery. To achieve this (and to comply with US and Europeran Pharmacopeia for heavy metal contents of heparin) a large reduction of the original heavy metal contents of native heparin seems to have been required (e.g., achievable by fractional precipitation using barium, calcium and copper salts followed by standard technological ‘clean-up’ using metal ion complexing resins, highly cationic ion exchange resins or soluble complexing agents, e.g., ethylenediamine tetracarboxylic acid (EDTA). (The actual methods used would have varied between manufacturers who commonly keep these details secret). (Infusion of EDTA, it should also be mentioned, is also widely employed in a controversial alternative medical procedure to remove vascular plaque which seems to have key inorganic heavy metal ion glycocalyx components).Further evidence that the efficient heavy metal ion complexing properties of EDTA was used to ‘clean up’ heparin is provided by the report (Casu et al. 1987) that all commercial heparins available to these authors showed the well defined NMR signals of EDTA superposed on those of heparin, the presence of such EDTA was, furthermore, a likely cause of dangerous uncontrolled bleeding in some patients receiving heparin anticoagulation therapy. Gatti, Casu et al. (1979) had previously briefly reported the use of a complexing resin Chelex100 to remove excess paramagnetic (e.g iron, manganese and copper)

metal ions from native heparin in order to minimise the resultant paramagnetic perturbation of the NMR spectra; simialar paramagnetic metal ions were also apparently amongst the “impurities” detectable by NMR in the commercial heparins which had been available to Neville et al. (1988) in Canada.

Phase Change Model of Heavy Metal Binding to Heparin/Heparan SulphateThis is a related phenomenon to the formation of iron-rich particles attached to chitosan discussed above.A Patent (Fo-We, 1962) had described how heparin forms unusual inorganic complexes containing both inorganic cations (e.g. of copper, manganese and cobalt) and anions (e.g. iodide).

This suggests an attachment to heparin by inorganic ions occurs as ion clusters which resemble a crystalline or sub-crystalline structure rather than separately bound ions produced by a normal reversible thermodynamic equilibrium binding to a conventional ligand where bound ions can equilibrate freely between the various molecules dissolved in a homogeneous solution phase. In agreement with this concept the Aberdeen polysaccharide group also found that the uptake of inorganic ions by heparin was physically equivalent to a phase change process (a kinetcally controlled rather than a thermodynamically reversible process) (cf., Prof. WF Long, Aberdeen U., 2003, [internet]). The physics of phase change also imply the requirement for nucleation of phase change and therefore all heparin/heparan sulphate binding phenomena might further require to be nucleated. (The importance of nucleation of phase change is a phenomenon often encountered in polysaccharide research laboratories (e.g. discussed by FB Williamson, Ph.D, Thesis, Edinburgh University, 1968).

Some Aberdeen U. Heparin, Heparan Sulphate & Pentosan Polysulphate ResearchA PPS research programme was funded some 15 years ago by the Scottish Home & Health Department (to WF Long, FB Williamson Department of Molecular & Cell Biology). The aim wasto identify heparin fractions and heparinoid antiviral especially anti-HIV agents in the context of safety of blood transfusion. This especially centred on xylan sulphates having a related structure to SP54 butfrom a marine algal source. The author was the bench chemist most centrally involved in the collection of algae (Palmaria palmata) and the extraction and fractionation of PPS and in writing of reports, literature surveys etc. relating to this research. The most readily sulphated and of highest antiviral activity were polysaccharides which had been partially degraded by endogenous xylanase. A byproduct was a sulphonated pyridine derivative which could assist in monitoring the pH change during work-up. Fractionation of crude sulphated xylans were performed using Sephadex G25 and G75 columns as well as charge density fractionation on a Whatman DE52 diethylaminoethylcellulose columns. Fractions were studied by infrared spectroscopy using a multiple reflectance procedure (adapted from the authors prior industrial lab. experience). This allowed much smaller samples to be studied than would have been possible by other methods.In addition a large number of other classes polyanionic substances were tested for their anti-HIV activity. This included lignin derivatives, soil humus fractions, fulvic and humic acid and uronic–acid rich fungal extracts, The anti-HIV-1 activities were evaluated by AG Dalgelish (St Mary’s Hospital Medical School, London using AZT (azidothymidine “Zidovudine” a 2/,3/-dideoxynucleoside analogue) as the reference standard). The most effective preparation was also independently assayed for anti-HIV activity as arranged by Mr W Forbes (Edinburgh). It was concluded that a fraction of algal PPS could provide an optimised anti-HIV activity.

It could be suggested that this information could possibly be of value for the improvement of anti-prion effectiveness of xylan sulphates.

A number of reports from other groups also confirmed this potent anti-viral effect, including anti-HIV-1 activity to be a property of various kinds of sulphated polyanionic molecules(This list is extensive; such compounds include antimoniotungstate, suramin, polyvinylsulphate,glycyrrhizin sulphate, lignin derivatives and various kind of sulphated polysaccharides especially those derived from marine algae; these compounds are of potential interest as anti-prion agents).

The birch wood derived xylan which had been modified by sulphation pharmaceutical product [SP54] (from Benechemie, Munich) was similar in potency and chemical structure to the sulphated algal xylans prepared from Palmaria. [The algal product might however be somewhat more active].

A possible problem with SP54 could be the presence of residual pyridine groups (from the solvent used) which had become covalently attached to some of the PPS molecules.This could be avoided if confirmed to be a problem (e.g. the toxicity in high dose performance of high dose use of PPS for the treatment of vCJD as reported by Whittle et al 2006).

The Aberdeen group also investigated sulphated xylans for their metal ion binding antioxidants and pathological inorganic crystallisation inhibitor activity in comparison with that of heparin. Also studied in animal models as anti-cancer agents. Related studies used red blood cell evaluations. [Gillian Mackintosh, Ph.D. thesis Aberdeen U.].

A related project with Marathon oil had also sought to use sulphated polysaccharides as borehole anti-scale agents. These were tested by the State University of New York by G.H. Nancollas (who originally developed the methods used when he had been at Glasgow University where the author had for a short time worked in his laboratory).

It seemed desirable to set up a commercial enterprise based on our findings which were for this reason not published, but the group manager, at about this time, became incapacitated by a mysterious illness which eventually caused him to take early retirement. Former polysaccharide group members invited in (e.g. after-work-elsewhere) attempts to unravel the cause of this illness (“yuppie flu”/fibromyalgia/ myalgic encephalomyelitis/chronic fatigue syndrome). Nitrosative stress seemed to be involved. As with many illnesses heparin therapy had been reported to be of major benefit.

Cation Exchange Resin Replacement of Multi-Elements from Heparin

Studies by the Aberdeen group (initiated by C.F. Moffat, [at that time a postgraduate student] and directed as regards the selection of the analytical method, spark source mass spectrometry used, by F.B. Williamson) in co-operation with the Macaulay Institute, Aberdeen, who had expertise in a related measurement of multi-elements in soil samples, had included an investigation into the efficiency of the use of sulphonated polystyrene ion exchange resin (Amberlite IR120) for replacement of counterions and other bound inorganic components in heparin the give the commonly pharmaceutically employed ‘single salt form’ of heparin; a facile reduction by two orders of magnitude could readily be achieved in the calcium content of a standard industrially produced multi-element containing sodium heparin but the efficiency of removal of other 37 inorganic elements simultaneously held by this heparin was progressively less, being least efficient for cerium for which the reduction factor was 2. Comparison of these results with previous reports lends some support for the idea that all heparins could be related multi-element matrix systems. [A comparison of the Aberdeen results with a study reported in 1964 by Sutton & Harrison had shown related alkaline earth contents in five commercial heparin samples could be arranged on a single plot to include the Aberdeen results for two further heparins, one commercial and one derived from the commercial heparin by employment of a standard industrial heparin '‘clean-up'’ ion exchange procedure. All of these commercial heparins or the commercial-heparin derived purified heparin seemed to form a single series consistent with their description as examples of different degrees of ‘cleaned-up’ heparin starting from some approximately similar kind of multi-inorganic-element heparins present in the in vivo starting materials].

Whilst there are indications that the presence of apparently toxic metal ions may not pose any unacceptable risk to the public, certain toxic metal ions in certain heparin preparations have recently been suggested to be potentially harmful cf., Bohrer, 2004; there have been prior reports of a similar nature (by Alcock in 1983) and Heinemann & Vogt in 2000). Mn2+ in heparin can give misleading results for assay this element in blood which led Bowen in 1966 to the suggest that because of the common co-occurrence of a range of inorganic elements with heparin this anticoagulant should never be used for inorganic element analysis in blood.

Different amounts of complexed inorganic elements in heparins from different manufacturers could explain the lack of activity of some heparins for modulation of vascular smooth muscle cell

proliferation (Castellot et al., 1999) and the regression of tumours via inhibition angiogenisis (Folkman, 1983)). This possibility arises since inorganic elements may be an absolute requirement for the enactment of the full biological activity of heparin/heparan sulphates. Probable examples of this are that zinc is required for endostatin to heparin (and by inference in heparan sulphate (collagen XVIII) interaction (Ricard-Blum et al., 2004) and bivalent calcium etc. ions are required for the heparan sulphate assisted fibroblast growth factor receptor dimersiation (Kan et al., 1996) as well as Zn2+ / Ca2+ for a heparan sulphate related therapy for Alzheimer’s disease (cf., Masters, 1993). Also, Ca2+ potentiates the binding of heparan sulphate and dermatan sulphate (Hamaguchi, 1992) to human serum amyloid P; this was suggested to protect against aberrant Alzheimer peptide fibril formation (Janciauskiene et al., 1995); also the processing of amyloid or other misfolded proteins by cells may involve the copper-nitric oxide deaminative cleavage signalling process, which if defective under conditions of insufficiency or excess of nitric oxide, ascorbate or transition metal oxidant could be a major part of the aetiology of TSEs. (cf., Mani et al., 2007). [Scrapie infection of neuroblastoma cells precludes nitric oxide production when the cells are challenged by lipoprotein suggesting that alteration in nitrosative cleavage of heparan sulphate may be an essential part of the aetiology of TSEs (this idea was originally suggested by Mani et al., 2004); it should be noted that a hallmark of degenerative diseases in general which has become apparent over the last ca.15 years is the occurrence of tyrosine nitration, a marker of nitrosative stress under which conditions heparan sulphate biochemical signalling will be affected].

The Occurrence of Thallium, Strontium & Gallium in Heparin

Moffat (1987) had found Tl to occur in pharmaceutical heparin and assumed that this situation applied to all heparins, he also noted that there were no literature reports of thallium poisoning associated with heparin anticoagululation, therefore it seemed that that this highly toxic metal might become biologically inert when attached to heparin. Tl also probably occurs in ultratrace amounts in blood serum and in seawater (cf. Haraguchi, 2004) from which it is sequestered by kelp (in a similar manner to its uptake by heparin from animal biological fluids) and likewise there are no reports of any thallium intoxication from this source from the use of kelp as a major food ingredients in Japan and China. Gallium is also present in kelp and in heparin – this is perhaps a contributory factor in the anti-tumour action attributable to these agents as Ga3+ alone is well known to have this effect; a similar situation exists for Sr2+ , which is present in both heparin and in kelp and again Ga3+ alone or in conjunction with such polysaccharide matrices is believed to be able to assist in bone formation. Traditional medicine in India, China and America use multi-element-containing geological organic polyanionic fulvates which are apparently effective therapeutic agents for numerous ailments. Since the multi-inorganic-element profiles of these materials seem to also be correlated with those of heparin and kelp it might be suggested that their apparent effectiveness could be at least partly due to their multi- inorganic element nature.

The notion that alginate in marine algae was a multi-counterion salt form rather than being present as the free alginic acid seems to have been established by Wassermann in 1949 (confirmed by studies conducted by WAP Black (Institute of Seaweed Research Musselburgh) and RL Mitchell (The Macaulay Institute, Aberdeen) at about that time.The multi-element compositions in these matrices are now reported also to be correlated with those of the Aberdeen group heparin samples as well as with the inorganic element content of seawater.[Harguchi (2004) discussed the relatedness between the inorganic profiles of seawater and blood serum in his discourse promoting the idea that non-physiological elements should be included within the definition of biological metallomics].

The ultimate reason for the requirement for animals for a seawater-like multielement extracellular bathing solution could be that the exact arrangement of the hydrogen bonded water molecules is dependent on the sum of the Hofmeister structuring effect of dissolved ions. [There is much experimental evidence which tends to support this idea]. This could also be the ultimate driving force for protein folding and explain why non physiological elements (defined as those which are found in X-ray structures of proteins) occur in blood serum].

That sulphated polysaccharides in animal tissues (including heparan sulphate) have a primitive function for sequestration and buffering of inorganic ions from seawater is to some extent supported by the

findings that for aquatic animal species (e.g., molluscs etc. which exist in intimate tissue contact with seawater) the heparan sulphate (and related polysaccharide) contents of the entire organism increases in an exact mathematical relatedness with the degree of salinity of the habitat (Nader et al., 1983 collected such data for fifteen species). This scenario seems to ultimately derive from the ability of heparan sulphate biosynthesis to respond to the status of inorganic element concentrations in extracellular environments. The kidney, which is the main organ in higher animals believed to be responsible for inorganic ion homeostasis and which contains functionally active heparan sulphate but this is not believed to be directly responsible for this homeostasis but to be required only as a structural component; nevertheless kidney glomerular heparan sulphate biosynthesis changes in direct response to extracellular [Na+] (Jyothirnmayi et al., 1995).

Dietary factors and heparan sulphate biosynthesis

The effect of seawater and aquatic organism brackish water composition on the amount of heparan sulphate required by invertebrates (Nader et al., 1983), discussed above, will principally arise from the effect of sodium concentration [Na+] on the biosynthesis of heparan sulphate (and includes signalling for alteration in the degree of sulphation). A wide-ranging literature survey suggests that numerous reports document similar interactions between a wide range of other dietary factors (both organic and inorganic) and heparan sulphate biochemistry which suggests that this is how this high level management system for animal biology is a transducer between the environment and the genome allowing the direction of animal evolution in response to restriction of food supply.

A general principle seems to be that all dietary factors can affect heparan sulphate biosynthesis.This includes manganese which was observed to affect the concentration, composition and sulphation pattern of heparan sulphate in a rat model (Kalea et al. 2006).Ascorbate and retinoic acid seem to boost heparan sulphate systhesis but toxic agents such as Pb and Cd (Cardenas et al.) or excess glucose in diabetes, diminish it.There has also been one report that liver-derived amyloid directly alters GAG synthesis (Palmoski et al., 1975), apparently by boosting hyaluronic acid probably by a simultaneous diminution of heparan sulphate biosynthesis.

Many of the current blood assays which use commercial heparin anticoagulation for sample preparation (e.g., as reported in JAAS) and other findings of the current researchers working on the biochemistry of heparin/heparan sulphate may have to be revised by future workers because trace metal ions in heparin/heparan sulphate giving false blood metal assays or inadvertently affecting the biological activities of these polysaccharides as well as the effect of unacknowledged required metal ions to allow these polysaccharides to function correctly.

It would be helpful if an inorganic mass spectroscopic analysis of heparin or heparan sulphate were published by some laboratory in the USA.

Similar metal ion-water cluster binding of sulphonated ionomers and sulphated polysaccharides suggest related ion conduction including proton conductivity roles.A further possible hint at early functions of heparan sulphate is that polysuphated ionomers have found commercial employment of sulphonated organic polymers as industrial membranes, e.g. for use in electrolysis and in fuel cells; proton conduction is believed to occur via interaction of polymer–SO3

- (H2O)n clusters. The numbers of molecules in these clusters varies with the counterions. Heparin films were found to show the same relationship between hydration and counterions to that exhibited by the commercial sulphonated ionomers (James et al., 2000).The addition of SiO2 particles to such industrial membranes seems to improve stability (Adjemain 2002). (Si is an essential element for animals, the association of inorganic Si with GAGs may also improve their stability). It has also been suggested on the basis of epidemiological evidence interlinking Si nutriture to physiological effects attributable to heparan sulphate that Si somehow also modulates the synthesis of heparan sulphate (McCarty, 1997).

References & Further Notes

Some of the Following references were kindly provided by Graham Steel (formerly of the CJD Alliance Glasgow)

Adjemian KT et al., J Electrochem Soc. 2002; 149 A256-61(Silicon oxide Nafion composite membranes for proton-exchange membrane fuel cell operation at 80-140oC)

Albertini R et al., Int J Mol Med. 2000; 6: 129-36(The effect of glycosaminoglycans on lipid peroxidation)FEBS Lett. 1995: 377: 240-2(The effect of heparin on Cu2+-mediated oxidation of human low density lipoprotein)

Alcock NW, Elem Metab. Man Anim. Proc Int. Symp., 4th 1981(Pub 1982), p 678-80

Bae JH et al. Neurosci Lett. 2006 Jan14 (internet)(Manganese induces inducible nitric oxide synthase expression via alteration of both MAP kinase and PBK/Akt pathways in ..microglial cells;[cf., Fabrizi C et al., J Biol Chem 2001(28) (The stimulation of inducible nitric oxide synthse by the prion protein fragment 106-126 in human microglia is tumor necrosis factor-alpha-dependent and involves p38 mitogen-activated protein kinase)

Basu M et al., Mol Biol Cell. Sept 1 2007; 18(9): 3302-3312(Modulation of proteinase K-resistant prion protein in cells and infectious brain homogenate by redox iron: implications for prion replication and disease pathogenesis)

Bazin HG et al., Biochemistry. 2002; 41: 8203-11(Inhibition of apoliporotein E –related neurotoxicity by glycosaminoglycans and their oligosaccharides)

Bernfield M et al., Ann Rev Biochem. 1999; 68: 729-77

Bishop JR et al., Infect & Immunity. 2005; 73: 5395 (Graham Steel CD)(Cell surface heparan sulfate promotes replication of Toxoplasma gondii)

Bisse E et al., Anal Biochem. 2005; 337: 130-5(Reference values for serum silicon in adults)(Si concs. were det. for 1325 healthy subjects aged 18-91, medians varied with age and sex. (men 18-59 had 9.5M.L decr. to 8.5 with age; women had 10 M/L for age 18-29 but 11.1M/L for ages 30-44 but decr. with age to 9.23M/L.The variation with age of Si serum concentration may be relevant to the age-dependence of the tendencey for the occurrence of atherosclerosis (cf., McCarty, 1997, loc. cit.) a phenomenon which is believed to be directly related to alteration in heparan sulphate microstructure (cf., Feyzi et al. 1998, loc. cit.) seeming to directly link heparan sulphate biosynthesis to silicon nutriA corollary to such action of Si nutrition is that it will influence all diseases for which there is a known dependence on heparan sulphate biochemistry (which include many viral infections and cancer).

Black Sheep (Symposium) 433-447%20kkoniish.pdf file (Graham Steel CD)Situation ignored by mainstream Alzheimer’s disease researchersRole of Metal Ions“Apolipoprotein A is a black sheep in a good family..possible central role in pathogenesis of AD; a metal binding apoliprotein-protection of lipoproteins from oxidation by transition metals. Synaptic activity and acute phase response.- plausible physiological funciton of A.”“Spontaneous” A aggregation to fibrils in vitro is caused by traces of transition metals present in laboratory buffers.A is readily aggregated by transition metal Cu(II) Fe(III), Zn2+ and Al3+. “In contrast, in the absence of such metal ions A is monomeric”. (Presume the fibrils are less effective functionally). “Brain homeostasis of transition metals is heavily impaired in AD suggesting such aggregation may be a key event in the aetiology”.

{DG: metal ion uptake and control by sulphated polysaccharides may also be an underlying fault in AD}

Boeve ER et al., World J Urol. 1994; 12;(1) 43-48(Glycosaminoglycans and other sulphated polysaccharides in calculogeneis of urinary stones)(Glycosaminoglycans inhibit crystal growth and agglomeration and possibly also of nucleation)Boeve list reference to prior work which their studies confirmed which includes reported by, e.g., Fellstrom B et al.,Osswald et al, and Norman RW [Clin Sci.,1985; 68: 369-71]which had shown that PPS also strongly inhibited urolithiasis;Similar results were obtained in Aberdeen U. studies);Others (Pantazopoulos D et al., had reported the inhibition of urinary tract infection and the prevention of bacterial adhesion by PPS;Suba K et al., [Med Sci Res. 1993; 21: 233-4 had observed changes in renal tissue glycosaminoglycans in urolithiatic rats treated with PPS

Bohrer D et al., RBAC (Brasil) 2004; 36(2) 99-103The multi-element study of heparin in Aberdeen U. had used an Al sample preparation method for spark source mass spectrometry. The cation exchange efficiency of removal of Al from heparin as deduced from the information given in the Bohrer paper seems similar to that of a number of ions including Fe3+ which were found to be reduced by a factor in the range 102-103 . The above tabulation of residual Al in commercial heparins, which can be assumed to have been subjected to some kind of purification process, could indicate that unpurified heparin may naturally contain e.g. 1000 ppm Al.

Cf., Bohrer D et al., J Parenteral Enteral Nutrition. 2005; 29: 1-7(Arsenic species in solutions for parenteral nutrition)(This confirms Aberdeen U. information, that arsenic (together with aluminium etc.) commonly occurs in commercial heparin)

Bowen HJM “Trace Elements in Biochemistry” Academic, London, 1966, p. 63(Ba, Ca, Cu, Mn, Sr and Zn commonly occur in heparin)

Capila I et al., Structure (Camb.) 2001; 9: 57-64(Ca2+ ions crosslink heparin to annexin-V, suggesting that a similar crosslinking is needed for apoptosis)

Carrell R(obin) W & Bibeck Gooptu, Curr Opin Struct Biol. 1998; 8: 799-809[{This Cambridge University group could still be a possible useful UK source of opinion re prion diseases.Carrell draws attention to the occurrence of a wide range of diseases arising from misfolding of proteins with some similarity to all aspects of prion diseases} Abstract: “Some of the most perplexing disorders in medicine are each now known to arise form the conformational instability of an underlying protein. The consequence is a continuum of pathologies with typically a change in fold leading to ordered aggregation and tissue deposition. The serpins provide a structural prototype for these pathologies and give a perspective on the assessment of current proposals as to the conformational basis of both Alzheimer’s disease and the transmissible prion encephalopathies”].Work of Carrell et al. – Other Protein Misfoldimg Diseases Suggested to Give Insight Into TSEsSimilarities between pathologies leading to ordered aggregation and tissue deposition for which conformational dysfunction of the serpins was believed to provide a useful model.As to whether protein deposition and accumulation is sufficient to explain late onset dementia the suggestion from A1-antitrypsin-associated liver disease was clear: hepatocyte loss and eventual cirrhosis was a consequence of variant protein deposition and not of loss of function, as cirrhosis only developed with the conformationally unstable variant. It was further suggested that as neurons are long–lived, non-dividing, cells this would enable aberrant proteins to accumulate irreversibly in such cells.

That this may not be the case is suggested by the possible successful therapeutic intervention in such a disease by heparin-like pentosan polysulphate and also by claimed benefits of AD patients to related therapy. One can speculate that the list of diseases given by Carrell might also be treatable by PPS.

Each aggregation disease including Alzheimer’s disease and TSEs were believed to arise from misfolding of a specific protein. The onset of such misfolding diseases is insidious when this occurs with a normal protein but is sudden when it occurs with a variant protein (BSE and nvCJD). Sickle cell anaemia arises from a periodic aggregation of a variant form of haemoglobin giving rise to ordered helical fibrils which distort erythrocytes. {The list of diseases include also the TSEs ( CJD, nvCJD, Gerstmann-Straussler-Scheinker disease, fatal familial insomnia and Kuru) as well as serpins 1-antitrypsin deficiency, antithrombin deficiency, C-1-inhibition deficiency, Huntington’s disease, Down’s syndrome, tau Frontotemporal dementia and various amyloidoses, chronic inflammatory diseases, transthyretin senile systemic amyeloidosis, apolipoprotein A1 familial amyloid polyneuropathy and cystatin hereditary cerebral angiopathy}].

Cardenas A et al., Toxicology 1992; 76: 219-31 (Cd2+ impacts negatively on heparan sulphate biosynthesis)In a separate paper to be posted by D Grant on the internet is is noted thatmany other workers (e.g. those listed below) have reported a similar modulation of heparan sulphate proteoglycan biosynthesis by e.g., by negative the effects of toxic inorganic ions, of Pb (Kaji et al.) of

Hg, Ni and Mn (Templeton), fluoride (e.g., Susheela et al. Pawalowska-Goral et al.) and also similar actions of hyper-glucose (Morno et al., Kasinath et al., Kolm et al.,), apolipoprotein E (Paka et al.), oxidised low density lipoproteins (Chang et al.), hypoxia (Karlinsky et al.), hydrogen peroxide (Nishingae et al.), endotoxin (Colburn et al.,), a fibrin dimer (Yevdokimova et al.) which can be countered by ascorbate (Edward & Oliver, loc. cit, ; Kao et al.), retinoic acid (Zhang et al.), heparin* (Mason et al.); antithrombotic agents (Pinhal et al.), polyamines (Ding et al.) and mechanical stress (Lin et al.) and Mg (Jaya & Kurup) Ca, (e.g., Fujiwara et al.,)A correlation between heparan sulphate and homocysteine (Karlinsky et al.) supports the concept of redox control of heparan sulphate biosynthesis. * This could suggest that heparinoid mimetics might also act in this beneficial way. Non-anticoagulant heparin has also been reported to increase endothelial nitric oxide synthase activity (Kouretas et al.; Miriami et al.); there are also possible effects of inorganic sulphate status, via actions on the inorganic sulphate transporter which is affected by Vitamin D derivatives (with an input from UV) and thyroid factors. (Dawson et al.,)

Case F. Chemistry World. 2005; 2(2): 29-32(Metals for the mind)(This review draws attention to the similarities between Alzheimers’s, Parkinson’s Huntingdon’s, amylotropic lateral sclerosis and the transmissible spongiform encephalopathiesas regards the putative roles of metal dyshomeostasis)

Castellot JJ et al. J Cell Biol. 1986; 102: 1979-84(Structural determinants of the capacity of heparin to inhibit the proliferation of vascular smooth muscle cells).

Casu B et al., European Pat Appl 1987; 0245813(EDTA-free heparins etc.)

Celsus® “Heparin Salts” http://www.heparin.com.heparin_salts.html

Chambers RC et al., Am J Respir Cell Mol Biol. 1998; 19: 498-506(Cadmium inhibits proteoglycan and procollagen production by cultured human lung fibroblasts)[Greatest inhibition of proteoglycan synthesis occurred with the major matrix-associated proteoglycans versican, decorin and the large heparan sulphate proteoglycans as well as a lesser diminution of other heparan sulphate protoglycans]

Choay SA (a French Company), 1974 British Patent Specification 1,471,482 (Heparin Salts)

Church FC, FEBS Lett. 1988; 237: 26-30(Antithrombin action of phosvitin and other phosphate-containing polyanions is mediated by heparin cofactor II)(The HCFII anticoagulant activity of linear inorganic polyphosphates markedly increased with increased polymer chain length, showing greatest activity with chain lengths of 65 or above, but showed no anticoagulant activity via with the heparin-binding antithrombin system) [N.b. this shows that the action of polyphosphate resembles that of PPS which although under n ormal conditions is believed to function a an anticoagulant by binding to Factor VIIIa, also functions via HCFII] [A common factor may be the induction of water structuring asociated with high ionic strength]

Cohen SM et al., Carcinogenesis. 2000; 21: 783-792(Calcium phosphate-containing precipitate and the carcinogenicity of sodium salts in rats)

Colburn P Dietrich CP Buonassi V Arch Biochem Biophys. 1996; 325(1) 129-38(Alteration of heparan sulfate moieties in cultured endothelial cell exposed to endotoxin)(Endotoxin causes via a post-synthetic process, a loss of the more sulphated segments of heparan sulphate which in the absence of endotoxin were destined for the extracellular matrix; this could be prevented by blockers of reactive oxygen radical activites)

David RL et al., Nature. 1999; 401; 376-9(A familial dementia caused by polymerization of mutant neroserpin)[This is an exmple of an analogous aetiological process to that of prion diseases]

Dawson PA Markovich D, Pflugers Arch-Eur J Physiol. 2002; 444: 353-9(Regulation of the mouse Nas1 promoter by vitamin D and thyroid hormone)

Edward M Oliver RF, Biochem Soc Trans. 1984; 12: 304; ibid., 1983; 11: 304; J Cell Sci, 19896; 85: 217-9[The boosting of highly sulphated heparan sulphate biosynthesis by ascorbate][This activity of ascorbate was later confirmed by the report of Kao et al., 1990]

Elliot SJ et al., J Am Soc Nephrol. 1999; 10: 62-8(Pentosan polysulfate decreases proliferation and net extracellular matrix production in mouse mesangial cells)[cf also ibid., 2001; 12: 2086-7

Ethell IM Yamaguchi Y, J Cell Biol 1999: 144: 575-86(Cell surface heparan sulfate proteoglycan syndecan-2 induces the maturation of dendritic spines in rat hippocampal neurons)(This is thought to be the structural basis of learning and memory)(Could this also involve prions?)(The processing of this dendritic heparan sulphate by nitric oxide might contribute to cognitioncf., nitric oxide synthase is localised in the dendritic spines of hippocampal CA1 cells (Barette et al., J Neurosci. 2002; 22: 8961-70))

Ferrero ME et al., Biochem Soc Trans. 1989, 360Ateroid a heparan sulphate-containing mixture was putatively a therapeutically effective treatment for Alzheimer’s disease(Cf., Lorens SA et al., Seminar Tromb Hemost. 1991; 17 (Suppl. 2) :164

Feyzi E et al. with U Lindahl, J Biol Chem. 1998; 273: 13395-13398(Age-dependent modulation of heparan sulfate structure and function)(This paper could be of great importance in regard to the search for novel strategies to inhibit the ageing process [cf. the age-old quest for the “elixir of life”). Change in the heparan sulphate microstructure could be suggested from the work of E. Feyzi to control the ageing process throughout the whole animal organism in a similar manner to how such a change makes the vascular surface prone to atherosclerosis, as had been reported in this paper).The author had advanced an alternative hypothesis of age-related promotion of atherosclerosis which in a letter written in response to this paper which was replied to by U. Lindahl, had suggested that vascular dysfunction heparan sulphate from older organisms might depend on seeding processes which lead to the formation of pathological crystals (thought for blood vessels mainly to be Ca salts which can be inhibited by “correct” heparan sulphate microstructures).

Folkman J et al., Science. 1983; 221: 719=25(Angiogensis inhibition and tumor regression caused by heparin or a heparin fragment in the presence of cortisone)on p. 722 of this article it is noted that “the angiogenesis inhibitory activity of heparin (in the presence of cortisone) varies greatly among manufacturers…one brand “was capable of bringing about complete regression in both reticulum cell sarcoma and Lewis lung carcinoma while heparins form other suppliers were able to cause either partial or complete regression of reticulum cell sarcoma, but could not suppress the growth of Lewis lung carcinoma. The reason for this variability is unclear.”It was later found (ibid., 1985; 230: 1375-8) that a synthetic pentasaccharide fragment of heparin produced by nitrosative cleavage also could act together with corticoid as an anti-angiogenesis factor which tended to rule out “contaminants” in heparin as the source of the original variability of the reactivity of heparin in this context.

The finding that heparin fragments produced by nitrosative cleavage inhibit steroid-dependent angiogenesis could perhaps also be relevant to the modus operandi of PPS as a

heparinoid.

It is now known (cf., Bohrer) that different commercial heparins contain inorganic “contaminants” ; this may arise from differences in ‘clean-up’ procedures starting from a

highly multi-element heparin (derived from the collection of the wide range of inorganic elements present in e.g. blood serum). These elements include redox metals which are

required for oligosaccharide generation via the ‘nitrous acid’ process. This suggests therefore that the anti-tumour activity of hepairn, elicited via the actions probed by Folkman

et al., may have depended on the presence of a correct amounts in the different heparins of heparin-associated metal ions. This idea was actually discussed with Folkman at the

time and could have contributed to why he later studied copper heparin affinity chromatography for separating growth factors.

Fo-We Forschings und Verwertungs-Anstalt (Vaduz) Brit Pat 890622 (1962)(This patent describes the formation of therapeutically active complexes between metal salts and heparin (viz. complexes of heparin with NaI, CoI2, CuCl2, MnI2 and CoSO4)Since the importance of bindingto heparin of anions (e.g. in association with metal ions or independently) has not been accorded high research priority, evidence for such occurrence as that provided by this Patent disclosure assumes more significance than might otherwise have been thought appropriate.In the classical studies of the purification of heparin (Scott DA & Charles AF J Biol Chem. 1933; 437 et seq.) it was noted purified, fractionated heparin gave an ash content of 25-8-38.8% by weight which seemed to consist mainly of “NaCl”) but natural heparin also contains Ca (WE Howell Bull Johns Hopkins Hospital 1928; 42 (4) 199-206).

Fransson L-A, Carbohydr Res. 1982; 110: 127-33(Structural features of the contact zones for heparan sulphate self-association)(A situation where inorganic crosslinking, including those containing inorganic anions, can be predicted on physical chemical considerations, to be involved; i.e. this may be part of the mechanism of information transmission by heparan sulphate, e.g. by the diminution of anionic charges in selected areas of the polymer by the screeening effects of counter cations)

Fujiwara Y Kaji. T , J Health Sci. 2002; 48: 460-9cf., Toxiciology 1999; 133: 159-69

Gabizon R et al., (Hijazi N et al.), J Biol Chem. 2005; 280 (17) 17057-61(PrPSc incorporation to cells requires endogenous glycosaminoglycan expression)Gabizon R et al., (Ovadia I et al., ) J Biol Chem. 1996; 271 (28) 16856-61(Effect of scrapie infection on the activity of neuronal nitric oxide synthase in brain and neuroblastoma cells)(Nitric oxide synthase activity is markely inhibited in brains of mice and hamsters and neuroblastoma cells infected with scrapie)

Gatti G Casu B et al., Macromolecules. 1979; 12: 1001-7

Golding JC et al., Current Therapeutic Research. 1983; 33(2) 173-184(Drugs for osteoarthritis I: the effects of pentosan polysulphate (SP53) on the degradation and loss of proteoglycans from articular cartilage in a model of osteoarthritic induced in the rabbit knee joint by immobilization)[PPS was administered intra-muscularly at 10mg/kg/48h; half of this dose was ineffective] Glucosamine sulphate, which is believed to boost heparan sulphate and hyaluronan biosynthesis became a popular dietary supplement which became known to the public as a highly effective treatment for osteoarthritis and became the popular choice in place of the drugs which were offered by medical practicioners. The medical use of this drug was later sanctioned in the USA (earlier European clinical trials which had not been believed were eventually confirmed in the USA). The use of heparan sulphate related therapy may only be accepted only after a long delay, considerable scepticism etc., and eventually is permitted only following dietary supplement or semi-illegal employment of such therapies.

Gonzalez-Iglesias R et al., J Mol Biol. 2002; 319: 527-540(Prion protein interaction with glycosaminoglycan occurs with the formation of oligomeric complexes stabilized by Cu(II) bridges)[n.b., for heparin and heparan sulphate prion complexes, the formation of Cu[II] cross-linking seemed to increase the resistance to proteolysis (but this effect was more evident for Cu(II) than for Ni(II) abd Zn2+]

Grant D J Appl Chem Biotechnol 1974 24 (1/2) 49-58(Pyrolysis of chlorocarbons)[This ICI Ltd. enabled study was prompted by a prior involvement in a major study by J.R Van Wazer promoted in-house by Monsanto at the St Louis h.q. labs which attempted to redefine the nature of inorganic polymers within a system of stochastic reorganization processes which applied to all chemical systems at suffiently high temperatures where reaction mechanisms became of no practical use for predicting the outcome of chemical reactions. N.E. Aubrey and J.R. Van Wazer (J. Amer Chem Soc 1964 86 4380-4383) had discovered that sealed tube pyrolysis of a range of compounds containing only carbon and chlorine atoms led to the same three products, hexachlorobenzene, carbon tetrachloride and hexachloroethane being formed in amounts

which indicated the occurrence of a reversible equilibrium between these three molecules. The above D. Grant study (by starting from some additional chlorocarbons and also by using mixtures containing elementary chlorine) confirmed the general occurrence of the phenomenon. The outcome of such pyrolysis depended on the ratio Cl/C. The reaction mechanism was irrelevant to the outcome. N.E. Aubrey also believed that a similar system applied to single phase hydrocarbon pyrolysis]

(Duck EW,)Grant D et al. 1974 Eur Polym J. 1974; 10 (6) 481-488 (….the polymerization of ethylene with VOCl3-Et3Al2Cl3-(chlorinated activator) catalysts…) [The chlorinated organic moieties were potent boosters of catalytic effectiveness by allowing a one-pot recycling of spent (chemically reduced) vanadium catalytic sites by their efficient re-oxidation to the active oxidation state needed to perform the desired reaction; this process was facilitated by use of a hydrophobic reaction medium. The uptake of chlorinated moieties derived from pesticide residues which become concentrated in the food chain of animals being eventually transferred into the fatty tissues stores of animals could provide a reservoir of pro-oxidants for augmenting the oxidation states of numerous protein (including prion) bound transition metal sites.]

Grant D et al., 1987. Poster presented by Mike L Tait at the 1987 Vancouver Seaweed Symposium (This reported that the ability of inorganic moieties to selectively interact with different microstructures in complex polysaccharides could be of possible relevance for the development of methods for the sequencing of complex linear polysaccharides). Cf., Different alginate micsostructures have distinct influences on crystallization of BaSO4 allowing information on such microstructure to be derived from such data)

Grant D et al. Biochem J. 1987; 244:143-9(Infrared spectroscopy of heparin-cation complexes) cf. also Grant D et al.; ibid., 1992; 287: 849-53; ibid., 1992; 283: 243-6

Grant D et al., Biochem J. 1989; 259: 41-5(Inhibition by glycosaminoglycans of CaCO3 (calcite) crystallization

Grant D et al., Med Hypotheses. 1989; 28: 245-53(Comparison of antioxidant requirements of proteins with those of synthetic polymers suggests an antioxidant function for clusters of aromatic and bivalent sulphur-containing amino acid residues)

Grant D et al., Biochem Soc Trans. 1990; 18: 1283-4(The dependence on counter-cation of the degree of hydration of heparin;cf., also ibid., 1983; 11: 96 and ibid., 1984: 12: 302)

Grant D et al., Medical Hypotheses 1992a; 38: 46-8(A putative role for colloidal silicates in primitive evolution deduced in part from their relevance to modern pathological afflictions)

Grant D et al. ibid., 1992b; 38; 49-55(Degenerative and inflammatory diseases may result from defects in antimineralization mechanisms afforded by glycosaminoglycans)

Grant D et al., Biochem Soc Trans. 1992; 20: 361s(Complexation of Fe2+ based ions by heparin)

Grant D et al., Biochem Soc Trans. 1996; 24: 194s(cf., also Med Hypotheses. 28: 245-253(The antioxidant activity of heparin)

Grant D, Chemweb Preprint Server Archive. (Paper entitled: “Multi-Ion Content of Heparin”)CPS: biochem/0010002 (2000)Now available on the internet from Elsevier Was downloaded on 13/1/07 (but not more recently) from Google with search term “biochem soc trans 1996 24 1496” [Publications of articles on this internet site (which has now been discontinued) had been encouraged by the Royal Society of Chemistry; this paper had been positively but informally reviewed by users of the site; apologies for some remaining spelling errors]. (Abstract

“Spark source mass spectrometry of sodium heparin reveals the presence of 38 additional counterions, comparison of their amounts before and after cation exchange treatment allows residual binding strengths to be classified as: small amounts of relatively strongly held K, phosphorus (likely as phosphate) Ni, Co, Zn Cr and Ag as well as Pb and Sn; somewhat less strongly held were more abundant Mg, Fe and Cu. Non-physiological elements included difficult-to-remove Sr, La and Ce. Such sequestration of small amounts of large numbers of ions to heparin/heparan sulphate suggests possible physiological and pathological significance for cellular nutrition, ionic transport and detoxification.”)

The probable in vivo association of a wide range of inorganic elements with heparin/heparan sulphate is probably not a trivial phenomenon of random post synthetic contamination, but a key, functionally active, inorganic complexation process which could be highly relevant, inter alia, to how redox equilibria control heparan sulphate signalling processes (e.g., those which involve ascorbate and redox metal catalysis of the deaminative cleavage of heparan sulphate – putatively part of the signalling processes which determines how cells respond to misfolded proteins).

Grant 2000b(the instalments are found on several sites on how the 1970s Linus Pauling’s ascorbate-cancer/viral hypothesis could be explained by heparan sulphate biochemisty from a nitric oxide biochemical perspective; re-reading this (it is no longer available from Google but can be accessed via. Yahoo) I now see that it was poorly written but I think it remains scientifically sound but needs to be updated.

“Ascorbate & Cancer” files available from Yahoo.com (original ukonline files)

Grant D (current)Discussions on heparin/heparan sulphate continue with FB Williamson. Similar discussions included Ms J Grant and Prof H McColl (Glasgow U.) relating to their upper stomach cancer researchinterests [preliminary ideas derived from these discussions have been posted on the internet {D Grant, 2000}.Some field work concerned soil and plant sampling for inorganic element assays of with multiple sclerosis clusters in NE Scotland (previously identified by Shepherd) were conducted with the late Mark Purdey [the outcome of this was the putative roles of metal ion dyshomeostasis as affecting heparan sulphate controlled growth factor signalling is, in part, presented in the 2004 paper by Mark in Medical Hypothesis, loc. cit.]. Prior literature linking heparan sulphate and metal ions to prion diseases were also communicated to Mark. Discussions with Frank Williamson also for a time included Vance Spence of Dundee U. on putative roles of heparin/heparan sulphate and nitric oxide in chronic fatigue syndrome and related illnesses.

It is hoped eventually to make a more formal presentation of these literature surveys, discussions, hypotheses and the resultant suggestions for future work in a general hypothesis of disease which suggests that all degenerative and infectious diseases are at least in part the outcome of dysfunctions of tissue protection afforded by heparan sulphate proteoglycans and the roles played by metal ions and nitric oxide in such biochemistry.

Hall JG Immunology 1988; 64: 345-51(Beryllium ions interact strongly with heparin)

Haraguchi H, J Anal At Spectrom. 2004; 19: 5-14(This is a review of metallomics including the full range of inorganic elements which occur both in seawater and blood serum)[This JAAS 2004 paper by Haraguchi. which listed heparin in a table of relevant systems but did not further state why heparin had been included, might also suggest that the perhaps more fundamentally relevant animal biochemical cell surface heparan sulphate proteoglycan receptor system must be a prime example of this sort of the multi-element matrix concept. I have found it difficult to discuss these ideas via academic journals or by direct communications to academic scientists including the possible holders of the intellectual property rights at Aberdeen U. who do not accept such direct correspondence from members of the public; a notable exception was the now elderly Professor RJP Williams, Oxford University, “the father of metallomics” who has engaged in an extended correspondence but a sticking point has been the presence in blood serum and heparin of a range of “non-physiologcial elements ” which he apparently finds difficult to accept].

Hamazaki H. J Biol Chem. 1987; 262: 1456-60[Cited by Janciauskiene S et al., ibid., 1995; 270: 26041-4(Inhibition of Alzheimer beta-peptide fibril formation by serum amyloid P component)]

Harrison GE Sutton A, Nature.1963 (4869) 809

Hu W-L Reogoeczi R, Biochem Cell Biol. 1992; 70: 535-8

Iler RK, “The Chemistry of Silica” , Wiley, New York, 1979

Ingham PW, Nature. 1999, 394: 16Cf., Bernfield M et al. loc. cit. and Perrimon N & Bernfield M, Nature. 2000; 404: 725-8

Ischiropoulos H, Arch Biochem Biophys. 1998; 356 1-11(Biological tyrosine nitration: a pathological function of nitric oxide and reactive oxygen species)

Ito K, et al., J Biol Chem. 1981; 917; (Altered proteoglycan synthesis by epiphyseal cartilage in culture at low SO4

2- concentration)Cf., Silbert et al., Arch Biochem Biophys. 1991; 285 (1) 137-41(Effects of sulfate deprivation on the production of chondroitin/dermatan sulfate by cultures of skin fibroblasts from normal and diabetic individuals) [Cf., also Krijgsheld et al.,loc cit who found that the serum concentration of inorganic sulfate in mammals showed marked species differences and circadian rhythm]

Jaques LB, Science. 1978; 206: 528-33 and Amer Chem Soc Adv Chem Ser. 1980; 187; Sect 23: p 349 et seq.(Heparin strongly binds counterions of sodium, potassium, ammonium, quaternary ammonium radicals, and co-ions such as sulphate, phosphate and acetate (providing an effective ion-exchange vehicle).(The heparin-histamine-basic protein in mast cells was thought to provide an ion exchanger for control of tissue fluid composition for ions (including protein antibodies).

James PJ et al., J Materials Sci 2000; 35: 5111-9(Hydration of Nafion® studied by AFM and X-ray scattering)Cf. Grant D et al., Biochem Soc Trans. 1990; 18:1293-4 (The dependence on counter-cation of the degree of hydration of heparin).Both of these sulphate or sulphonate dependent systems show very similar metal ion dependent hydration; as this is believed to be a critical requirement for function and that function is proton conduction a corollary is that heparin/heparan sulphates are also metal ion dependent proton conductors (gated by dopant rare earths in blood serum? This observation needs looking into experimentally).

Janciauskiene S et al., J Biol Chem. 1995; 270: 26041-4(Inhibition of Alzheimer beta-peptide fibril formation by serum amyloid P component)

Jarrett DJR et al., J Clin Exper Gerontology. 1989; 11 (3&4) 145-54(Ageing as a cause of raised serum ferritin in the absence of disease){Ageing also causes a systematic change in blood vessel wall heparan sulphate fine structrure (cf. Feyzi et al., loc. cit.; the putative role of heparan sulphate (Aberdeen U. work) in protecting against iron-induced tissue damage may become compromised a diminution by heparan sulphate protection from reactive oxygen and nitrogen species in ageing}.Since exogenous heparin prompts increased blood vessel wall heparan sulphate production (as shown by the Dietrich group) a role of such heparin (and putatively heparinoid) therapeutic intervention in ageing processes might be anticipated. Cf. also Mark Purdey Document in Microsoft Internet Explorer 19/08/05 (“Metal microcrystal nucleators” Draft Abstract of Chapter for “Trends in Prion Research” Graham Steel CD)(loc. cit.)which outlines a hypothesis (PrP)-ferritin ‘fibril’ crystals, suggested to be nucleators of solid phases which may represent the true pathological entities of prion diseases.

Kerey G et al., 1986; UK Pat GB 2,176,200 (Process for the preparation of heparin salts)

Kalea AZ et al., Biometals. 2006; 19: 535-46(Dietary manganese affects the concentration, composition and sulphation pattern of heparan sulphates in the rat aorta)

Kan M et al., J Biol Chem. 1996; 271; 26143-7(Divalent cations and heparin/heparan sulfate cooperate to control assembly and activity of the fibroblast growth factor receptor complex)[Sucrose octasulphate also seems to mimic heparin for fibroblast growth factor receptor dimerization, cf., Yeh BK et al., Mol Cell Biol. 2002; 22: 7184-92;PPS also can apparently also seems able to mimic heparin for fibrolast growth factor activation effects cf., Robinson et al.,, loc. cit.]

Kao J Huey G Kao R Stern R, Exp Mol Pathol. 1990; 53: 1-10[This paper confirms the earlier work of Oliver et al [Dundee U.] which seems to have been unknown to Kao et al.; ascorbate in cell culture media enhances highly sulphated heparan sulphate biosynthesis; this could be the reason for the ascribed ascorbate (e.g. by L Pauling) anti-tumour

activities, e.g. via anti-angiogenesis, antimetastatic and pro-apoposis and altered signalling towards normal cellular development putative anti-cancer mechanisms; cf., Grant, 2000 {internet documents}]

Kojima S et al., e.g., in Eur J Nucl Med. 1983; 8: 52-9; ibid. 1984; 9: 51-6

Kornberg A (with Brown MRW), PNAS. 2004; 46: 16085(Inorganic polyphosphate in the origin and survival of species)(This is a hypothesis of the inorganic chemical origin of terrestial life;whereas heparan sulphates have been associated with all animal like for some 1000 106yr it seems that the other group of highly anionic, wholly inorganic polyphosphate system (which binds metal ions) has been associated with all forms of life for some 3.5x1000 106yr)

Kraemer PM, J Cell Physiol. 1968; 71: 109-120(Production of heparin related glycosaminoglycans by an established mammalian cell line)(The is the first report of cell cytosol heparan sulphate unlinked to protein; the author had noted that N-linked SO3

- groups are extremely labile comparted to the O- linked SO3-; at the time the possible

relevance of such lability in regard to intracellular signaling by nitric oxide was unknown since the use of this second messenger by animals was yet to be established (the C-NH2 groups become potential sites of NO-dependent oligosaccharide generation))

Krijgsheld KR et al., Comp Biochem Physiol 1980; 67A; 683-6(Serum concnetrationof inorganic sulfate in mammals: species differences and circadian rhythm)

Kuberan B et al., (with Rosenberg RD), J Biol Chem. 2004; 279: 5053-4(Light induced 3-O-sulfotransferase expression alters pineal heparan sulfate fine structure: A SURPRISING LINK TO CIRCADIAN RHYTHM){Apart from its functions which are known to include “cell-cell adhesion, cell-matrix adhesion, cell proliferation, motility and differentiation, lipoprotein metabolism, blood coagulation, inflammation, tissue regeneration, tumor progression and invasion, pathogenic infection by bacteria, protozoa and viruses”, light ( by inducing 3-O-sulfotranferase activity in pineal glands) can also change heparan sulphate fine structure (and by implication its functional activity}.

Lahiri B et al., Arch Biochem Biophys. 10992: 293: 54-68(Depolymerization of heparin by complexed ferrous ions)

Leteux C et al. (with T Feyzi). J Biol Chem. 2001; 276: 12539-45(Imperial College School of Medicine, Northwick Park Hospital, Harrow)(10E4 antigen of scrapie lesions contains an unusual nonsulphated motif)(This is the GlcNH3

+ site, the NO metabolite target which creates heparan sulphate oligosaccharides)

Liebel MA White AA, Biochem Biophys Res Commun. 1982; 104: 957-964(Inhibition of soluble guanylate cyclase from rat lung by sulphated polyanions; (the range of sulphated polyanions studied included heparin, carrageeenan as well as man-made polyanions; this action of the natural polyanions, but not the man-made ones, were potentiated by Mn2+ (but not by Ca2+ or Mg2+)) [Guanylate cylcase is thought to be involved in generation of nitric oxide]

Lima –de-Faria A, “Evolution without selection : form and function by autoevolution”Elsevier, Amsterdam,1988(“Minerals and other pure chemicals have no genes yet they already display these two basic features: constancy of pattern and ability to change it by forming a very large number of forms ….(these) can behave in a similar way to living organisms as regards replication of form which in a general way has noting to do with genes”; “water has no genes, calcite has no genes, yet they already possess mechanisms that at present are considered fundamental gene attributes”“Calcite can occur in about 600 forms and over 2000 combinations”).

[Studies at Aberdeen U. by Grant et al., loc cit., and related studies elsewhere, have demonstrated that heparin/heparan sulphate can control calcite and other types of crystal formation i.e. heparan sulphate may have originally acted as a morphogen for inorganic moieties a situation which seems to have evolved into how heparan sulphate is now a key morphogen for the assembly of animal cells into organisms and hence controls animal morpholopgy (other information holding polysaccharides such

as pectins and alginates may have similar key roles respectively in plants and algae); the different microstructures in anionic polysaccharides can be shown to translate into crystallisation kinetics which influence inorganic morphology (studied by Grant et al. for the alginate modulation of BaSO4 crystallisation)].

[The possession of crystallinity per se is also unlikely to be the sole requisite for inorganic chemicals having the ability to behave like organisms as amorphous silica also demonstrates these properties (Grant et al., 1992a)].

Linhardt RJ, Chemistry & Biology. 2004; 11: 420-3(Heparin-induced cancer cell death)(Heparin uptake into cancer cells can be promoted by conjugation to poly-amino esters. As established by DA Berry et al. ibid., 2004; 11: 487-98; related papers concern methods of intracellular delivery of DNA and polysaccharides; oversulfated glycosaminoglycans as theapeutic agents; use of proteoglycans to regulate aberant inflammatory responses etc. (cf David A Berry publications listed in his Curriculllum Vitae, available on the internet; could his methods be adapted for administration of PPS?)

Liu Z Perlin AS, Carbohydr Res. 1994; 255: 183-91(Evidence of a selective free radical degradation of heparin mediated by cupric ion)

Long WF Williamson FB, IRCS J Med Sci (Library Compendium). 1979, 7, 429-34(Glycosaminoglycans, calcium ions and the control of cell proliferation)This postulated that the major role of heparan sulphate is for modulating metal ions especially Ca2+)

Long WF Research and Scholarshiphttp://www.abdn.ac.uk/~bch118/publications2003march.doc

Lyon M Gallagher JT, Matrix Biol. 1998; 17: 485-93

Mackintosh, Gillian, “Heparin-Iron Interaction and its Possible Relevance to Antioxidant Activity”Ph.D. Thesis University of Aberdeen, 1995 (This study was principally an investigation of the antioxidative and anti-inflammatory action of heparin and heparinoids including PPS but conducted in the context of the pro-oxidant and pro-inflammatory actions of iron (II) ions which was thought to be inhibited by the direct binding of iron ions to heparin and to PPS etc. The catalysis of the oxidation of Fe(II) to Fe(III) by heparin and PPS (this is a putative mechanism by which these polysacharides could also, it was thought protect tissue by removing toxic Fe(II) by the formation of insoluble Fe(II) aggregates.In general the order of effectiveness was heparin>PPS> an in-house experimental triose sulphate.E.S.R. was used to study the effect of these sulphated polysaccharides on Fenton (iron induced free radical) oxidative damage in various in vitro and cell systems.This included the ability of the sulphated polysaccharides to protect HepG2 cells and erythrocytes from damage.This thesis also reported the anti-tumour activities of PPS.Cf on p. 157 it is stated that“The in vivo anti-inflammatory activity of xylan sulphate and a semi-synthetic saccharide, triose sulphate, were studied using the mouse tumour model. Fig 5.47 shows the leg diameter measurements for the mice in the presence and absence of xylan sulphate. Leg diameter measurements are presented as the increase in girth in the presence of the tumour, either in the presence or absence of xylan sulphate, less the normal growth of the leg, derived by measurement of the left leg of the same mouse. It can be seen that the presence of xylan sulphate can significantly reduce the growth of the tumour. By day 7, the xylan sulphate treated group have leg diameters approximately 66% of that of the group not treated with xylan sulphate” [trisose sulphate was less effective]. “Fig 5.49 shows the effect of xylan sulphate on the development of the tumour…it can be seen that the presence of xylan sulphate significantly reduces inflammation, in fact only limited leucocyte infiltration and fibroplasia can be seen. Photograph (b) shows that the tumour is unable to infiltrate into the surrounding connective tissue, and this connective tissue shows little evidence of angiogenesis, meaning that no nutrients are being provided to maintain the tumour. Photograph (c) shows the ischaemic necrosis occurring within the tumour mass. Cells on the periphery of the tumour are able to get some nutrients directly from neighbouring connective tissue cells, and hence the outer cells of the tumour are still fairly healthy.”

A sometimes dramatic putative anti-tumour effect of heparin and PPS was apparently demonstrated(FB Williamson, personal communication.)The results presented in the thesis are less dramatic but positive (using a EL4 ascites lymphoma (an established animal model (chemically induced thymus-derived lymphoma produced by 9,10-dimethyl-1,2-benzanthracene) in studies conducted in collaboration with G Pugh-Humphries (lymphoma transplants). [Cf., also the related study reported by Ross MA, Long WF Williamson FB, Biochem J. 1992; 286; 717-20 (Inhibition by heparin of Fe(II)-catlaysed free-radical peroxidation of linolenic acid)].

Mani K, et al., J Biol Chem. 2007; 282: 21934-4(How nitration of tyrosine can be linked to nitrant stress involving mis-signalling via deaminative cleavage of glypican-1 heparan sulphate; other factors can be suggested also such as metal ion intoxication and lipid (cholesterol) misprocessing;)cf. also Mani K, et al., Glycobiology. 2004: 14: 599-603(The heparan sulphate-specific epitope 10E4 is NO sensitive and partly inaccessible in glypican-1 alludes to the heparan sulphate scenario with respect to prion diseases)cf., also J Biol Chem. 2003; 278: 38956-65

Mani K et al., J Biol Chem. 2007; 282; 21934-40 and Glycobiology 2006 loc. cit.(Heparan sulphate degradation products can associate with oxidised proteins and proteosomes; cf., defective NO-dependent cleavage of glypican-1 heparan sulphate in Niemann-Pick fibroblasts)

Masters CL et al., 1993; PCT Int Appl WO 9310459. Chem Abs. 119: 136893b(Therapeutic intervention in Alzheimer’s disease. Possible treatment by heparin requires control of Zn2+ concentrationvia effect on amyloid precursor protein (APP)50nM Zn2+ promoted heparin binding to APP butZn2+ abolished the protective effect afforded by heparin re: proteolysis of APP){DG note: The presence of Al3+ and/or Cd2+ could interfere with this action of Zn2+}

Mathew R et al., J Neurosci. 2006; 26: 10636-10645(Tau nitration occurs at tyrosine 29 in the fibrillar lesions of Alzheimer’s disease and other taupathies)

Merce AL et al., J Inorg Biochem. 2002; 89: 212-8

McCarty MF, Med Hypotheses. 1997; 49: 175-6(Reported antiatherosclerotic activity of silicon may reflect increased endothelial synthesis of heparan sulfate proteoglycans)also US Pat 5707970, Jan 13 1998 (McCarty MF et al.) arginine silicate as an anti-atherosclerosis therapeutic agent cf., also, McCarty MF, ibid., 1997; 48: 245-51(Glucosamine may retard atherogenesis by promoting endothelial production of heparan sulfate proteoglycans).Glucosamine had been reported by Quastel JH & Cantero, loc cit., to be a potent anti-tumour agent in a mouse model. This action, like the anti-atherogenesis activity, can hence also be postulated to arise via promotion by glucosamine of anti-tumour heparan sulphate (which is a glucosamine containing copolymer);Glucosamine can like ascorbate (cf., Oliver et al, loc cit,; Kao et al loc cit; Parish et al loc cit.,) be a useful non-toxic self administered putative pro-heparan sulphate potential anti-tumour agent

Mishra RS et al., J Neurosci. 2004; 24: 11280=90(Protease-resistant human prion protein and ferritin are cotransported across Cao-2 epithelia cells: implication for species barrier in prion uptake from the intestine)

Moffat CF, “Synthesis Characterisation and Application of Chemically Modified Heparins”, Ph.D. Thesis, University of Aberdeen, 1987)(cf., Moffat CF et al., Arch Biochem Biophys. 1997; 338: 201-6which reported the preparation of N-propionylated heparin)

Moffat et al., 1997 is cited in Fernandez C et al., ref 30 Carbohydr Res 2006 [Semi-synthetic heparin derivative etc.])

Morano S et al., Diabetes/Metab Res Rev. 1999 15 (1) 13-20

Nader HB et al., Comp Biochem Physiol. 1983; 76; 433-6(A correlation between the sulphated glycosaminoglycan content and the degree of salinity of the “habitat” in fifteen species of the classes Crustacea, Pelecypoda and Gastropoda)The main driving force for altered heparan sulphate could be the inorganic Na+ concentrationCf. also Jyothirmayi GN et al., Res Commun Mol Pathol Pharmacol. 1995; 90 (1) 115; Chem Abs. 123: 311506j (Na depletion augments glomerular heparan sulphate proteoglycan biosynthesis in spontaneously hypertensive rats). [This could suggest that altered heparan sulphate in this organ in the rat has parallels in the other inorganic ion homeostasis mechanisms in the species studied by Nader et al.] Nader, Dietrich et al. were responsible for groundbreaking research into heparan sulphate including the theory that these polysaccharides have been strictly conserved throughout animal evolution (Cf., Carbohydr Res. 1988: 184: 292-300) and that they function as “code polymers” (Biochem Biophys Res Commun. 1983; 111 865-71)(Other workers, have reported that the biosynthesis of heparan sulpjate is modulated by the presence of specfic inorganic ions (and also by small organic molecules such as ascorbate (cf. Edward M & Oliver RF (when at Dundee U.) Biochem Soc Trans. 1983; 11: 383; 1984; 12: 304) and glucose which respectively increase and decrease sulphation and total heparan sulphate)

Nader HB et al., Int J Biol Macromol. 1981; 3: 356-60(Heterogeneity of heparin: characterisation of one hundred components with different anticoagulant activities by a combination of electrophoretic and affinity chromatographic methods.This included selective Ba2+ precipitation, i.e., selective interaction strengths of difference in heparin microstructures

Neville GA Mori F Holme KR Perlin AS, J Pharm Sci. 1988; 78 101-4

Parrish RF Fair WR, Biocchem J. 1981; 193: 407-10(Selective binding of zinc ions to heparin rather than to other glycosaminoglycans)

Parish CR & Snowden JM Wo Patent Application WO 88/05301(Sulphated polysaccharides having anti-metastatic and/or anti-inflammatory activity)(Anti-heparanase activity inhibits tumour metastasis; the patent specification includes hepairn, modified heparin, pentosan sulphate, dextran sulphate and lambda carrageenan (administered at 4mg/rat using micro osmotic pumps implanted subcutaneously in the back, to give plasma levels of heparin etc., of 10-20g/ml); heparin was found to be most effective polysaccharide followed by lambda carrageenan, pentosan sulphate (from Sigma) and fucoidan;Heparin preparations from two different sources had identical anticoagulation properties by differed by approximately 10 fold in their antimetastatic capabilities

Palmoski MJ Brandt KD, Biochem J. 1975; 148: 145-7(Synthesis of GAGs affected by the presence of amyloid which seems primarily to boost hyaluronic acid, which is augmented by the addition of amyloid fibrils to cell culture this may suggest that (aberrant?) amyloid signalling could cause diminished heparan sulphate synthesis)

Perl DP, J Neurol Neurosurg Psychiatry. 2006; 090613v1 (Graham Steel CD)(Exposure to aluminium and the subsequent development of a disorder with features of Alzheimer’s disease)

Perlin A et al., Carbohydr Res. 1994; 255: 183(Small amounts of copper ions specifically alter heparin activity etc.)

Purdey M, Med Hypotheses. 1996; 46: 429-54(The UK epidemic of BSE: slow virus or chronic pesticide initiated modification of the prion proteins (Parts 1 and 2)

Purdey M, Med Hypotheses. 2000; 54: 278-306(Ecosystems supporting clusters of sporadic TSEs demonstrate excesses of the radical-generating divalent cation manganese and deficiencies of antioxidant cofactors Cu, Se, Fe ,Zn )

Purdey, Mark, Correspondence with D Grant cf., DG letters of 26/5/01 ; 22/8/01; 14/7/03; 2/9/03; 28/10/03The anti-prion effects of heparin and related substances were brought to Mark’s notice.Reply 29/4/01 a vCJD victim’s mother had received heparin during her pregnancy.[DG comment. It is conceivable that bovine heparin (from lung mucosa) could have harboured the aberrant form of the prion. The BSE scare also stimulated the search for anticoagulant heparin production from safer sources].Mark also stated his hypothesis that Mn(III) was involved with sporadic CJD while Mn(IV) was involved with vCJD.Eco- oxidants were thought by Mark to be contributory factors to TSEs.[DG comment : possibility that chlorinated organic molecules, especially hexachlorobenzene which convert to dioxins in vivo, could be involved as an eco-oxidant for manganese ions][These ideas were discussed further in a letter from D Grant to Mark Purdey dated 14/7/03 which noted “nitric oxide is quickly oxidised by oxygen into nitrite. Highly acidic conditions convert nitrite to nitrous acid and also the C-N(H)-SO3

- groups of heparan are quickly transformed into C-NH2 groups which react rapidly with nitrous acid to cleave the heparan sulphate; the in vivo reaction is now known also to occur at pH7.4 but by an unknown mechanism. It could involve some sort of proton channelling promoted by free redox metals (maybe sensitive to environmental stresses such as acoustic shock….nitrous acid chemically alters both heparan sulphate and mutates nucleic acids. Perhaps stress from inappropriate microwave or sonic energy could have an impact here, at the initial level of promoting illness. Mechanical stress is known to alter heparan synthesis in cell culture. The input from copper in heparan sulphate nitric oxide signalling could conceivably include heparin/heparan sulphate interaction with copper prions”.

Purdey M, Med Hypoth. 2004; 62: 746-756(Chronic barium intoxication disrupts sulphated proteoglycan synthesis: a hypothesis for the origins of multiple sclerosis)The author was involved in correspondence relating to this hypothesis and also with arranging for collection of soil and plant samples from an Aberdeenshire cluster area of multiple sclerosis which had been identified by DI Shepherd (1976, Aberdeen U., Dept Medicine, Doctoral Thesis).During the visit of Mark to Aberdeenshire, a discussion was arranged with Dr Frank B Williamson in which Mark’s infrasonic acoustic shock wave paper was discussed in the context of the possible initiation of multiple sclerosis by a barium intoxication mechanism implicating heparan sulphate growth factor signalling for maintenance of the myelin sheath.Mark was apparently suggesting that infrasonic shocks generated by blasting and supersonic aircraft, etc., might have roles in the formation of various aggregated proteins which occur in various neurodegenerative diseases including prion diseases. Cf., Mark’s published article:Purdey M, Med Hypotheses. 2003; 60(6) 797-820(Does an infrasonic acoustic shock wave resonance of the Mn3+ loaded Cu depleted prion initiate the pathogenesis of TSE?)(This idea seems to have anticipated the employment of sonication technology as a critically important factor to greatly enhance protein misfolding during a process of cyclic amplification (PCMA), an ultra-efficient method developed by Saa et al., 2006, loc. cit., for the generation of infectious prion oligomers). [The concept that shock waves can generate very high temperatures and hence greatly enhance chemical reactions is a well-established concept [e.g., being the subject of an early review which is still valid (Pritchard HS, Quart Rev (Chem Soc London) 1960; 14(1) 46-61 which notes “that..strong shock waves …are associated with, for example, thunder, the motion of supersonic aircraft and projectiles, and with explosions of all kinds” molecular fragments may be formed in states of high excitation; this review contains references to earlier work on the effect of shock waves on nitrogen oxides; the nitric oxide and its metabolites are now known to constitute key biochemical second messengers in animals and be implicated in the modus operandi of heparan sulphate tissue protection, which also includes the modulation of prion biochemistry]

Purdey, MarkDraft Abstract (19/8/05) of Chapter; for ‘Trends in Prion Research’ Graham Steel CD(Metal microcrystal nucleators; the heat resistant, transmissible, piezoelectric pollutants which initiate athe pathogenesis of TSEs?)

Park PW et al., J Biol Chem 2000; 275; 29923-6

Quaglio E et al. (Graham Steel CD), J Biol Chem. 2001; 276: 11432-11438

(Copper converts the cellular prion protein into a protease-resistant species that is distinct from the scrapie isoform)(However this author noted that the proteoase cleavage pattern of PrPsc derived from the brains of patients with Creutzfeld-Jacob disease is altered by the addition of Cu and Zn suggesting that metal ions confer prion strain properties)

Quastel JH & Cantero A, Nature. 1953; 171: 252-4(Inhibition of tumour growth by D-glucosamine)(The administration of D-glucosamine to mice bearing the tumour sarcoma 37 results in a marked inhibition of the rate of growth of the tumour and in the development of cytotoxic effects in the rumour itself; although there was no observed complete regression of the tumour the results indicated that there had been a marked specificity of action of D-glucosamine on the tumour mass as compared to that on the host tissue; the survival rate of D-glucosamine treated animals was considerably greater than untreated animals).(The administration of PPS or heparin also diminishes tumours implanted in mice, cf Mackintosh, 1995, loc. cit.)

Rej RN Holme KR Perlin AS, Carbohydr Res. 1990; 207: 143-52(Marked stereoselectivity in the binding of copper ions by heparin. Contrasts with the binding of gadolinium and calcium ions)(Heparin (and heparan sulphate) was found to be exceptionally sensitive to the paramagnetic relaxation characteristics of Cu2+ especially under acidic pD=5.5 conditions (characteristic of endosomes?) but not under pD=7.5 conditions(Cf. also, Liu Z Perlin A, ibid., 1994; 285: 183-91)(Evidence of a selective free radical degradation of heparin mediated by cupric ion)

Ricard–Blum S, et al., J Biol Chem. 2004; 279: 2927-36(Characterization of endostatin binding to heparin and heparan sulfate by surface plasmon resonance and molecular modelling role of divalent cations)

Robinson GW Cho CH, Biophys J. 1999; 77 (6): 3311-18(Role of hydration water in protein unfolding)Cf., Luck WAP, Topic in Current Chemistry. 1975; 5: 115-80(Water in biological systems)[Cf. also Bernal JD, Symp Soc Exptl Biol. 1965; 19: 17-32; Chem Abs. 65; 9242f(The structure of water and its biological implications)]

Robinson CJ et al., (UK National Institute for Biological Standards & Control)Abstract 93, 644th Meeting, Biochemical Society, Glasgow, 1992Potentiation of the action of FGF by heparin & related molecules(Includes a study of PPS as a growth factor regulator)

Saa P et al., J Biol Chem. 2006; 281: 35245-52(Ultra-efficient replication of infectious prions by automated protein misfolding cyclic amplification)

Schwarz K, PNAS, USA. 1973; 70: 1698-1612A bound form of silicon in glycosaminoglycans and polyuronides

Shiba T et al., J Biol Chem. 2003; 278: 26788-92(Modulation of mitogenic activity of fibroblast growth factors by inorganic polyphosphate)A heparin/heparan sulphate-like activity is shown by inorganic polyphosphate which seems to be involved in a similar wide range of biochemical activities to those affected by heparin/heparan sulphate; both of these anionic systems probably contribute to the animal metallome. Could the inorganic metals which are required for the heparan sulphate determined fibroblast growth factor receptor dimerisation (cf., Kan et al., loc. cit.) also be involved in the modulation by inorganic polyphosphate of fibroblast growth factor activity?

Sipos P et al., J Inorg Biochem. 2003; 95: 55-63(Formation of spheroid iron(III) oxyhydroxide nanoparticles sterically stabilized by chitosan in aqueous solution)

(Putatively this is an example of the phase change mechanism of how polysaccharides can bind metal ions at high affinity; heparin appears able to act in a similar manner)

Straus AH Sant’anna OA Nader HB Dietrich CP, Biochem J. 1984; 220: 625-630(An inverse relationship between heparin content and antibody response in genetically selected mice)[This paper lists the pharmacological activities of heparin known in 1984 as: an anticoagularn, antilipaemic and antiheaemostatic agent, inhibitor of myosin ATPase, RNA-dependent DNA polymerase, hyaluronidase, elastase and renin, and is a putative anti-tumour, antibacterial and antiviral agent]

Supattapone S et al., (Deleaut NR et al.,) PNAS, 2007; 104 (23) 9741-6(Formation of native prions from minimal components in vitro) (As commented on by Lee & Coughey in this issue of PNAS on p 9551-2, this key paper seems to fully confirm the prion only hypothesis of TSEs)(Cf., also Suttapone S et al., J Biol Chem. 2005; May 24 who noted that polyanionic compounds which stimulate purified PrPres amplicfication include polyA and polydT as well as non nucleic acid polyanions such as heparan sulfate proteoglycan; Gabizon et al. (Hijazi et al.) (lo.c cit.) had demostrated that PrPsc incorporation to cells requires endogenous glycosamonoglycan expression

Supattapone S (with Nishina K & Jinks S), J Biol Chem. 2004; 279 (39) 40788-94(Ionic strength and transition metals contol PrPSc protease resistance and conversion-inducing activity)

Takeuchi T et al., Anyalusis. 1998; 28: 61-4(Ion chromatography using anion exchangers modified with heparin(This allows a surprising simultaneous separation both anions and cation on a single column which seems mainly to consist of heparinized SiO2)

Templeton DM, Proc Trace Elem Health Disease Conf Aiyo A, Ed. Proc J Nord Trace Elem Soc/IUPAC. Published in 1991 by Roy Soc Chem (Cambridge UK) Prod IUPAC Int Symp. 1990 p. 209; Chem Abs. 111: 12940121z(Metal-proteoglycan interactions in the regulation of renal mesangial cells: implications for metal induced nephropathy)(Heparan and dermatan sulphate synthesis was diminished by metal ion intoxication in the order of activity Cd2+ >> Cu2+ > Hg2+ and Ni2+, [the effects of Mn2+, Co2+, and Zn2+ intoxication were also investigated];, (Renal dysfunction was suggested to arise from effects of metal ion binding to proteoglycans causing a charge reduction by direct binding and effects of divalent metals on the biosynthesis, secretion and anti-mitogenic properties of proteoglycans which was studied in isolated glomeruli (which participate in glomerular filtration including by electrostatic selectivity) and cultured glomerular mesangial cells (which although quiescent in healthy tissue as a possible result of the growth suppressive actions of heparan sulphate ologosaccharide signalling to the cell nucleus such dysfunctional signalling (promoted by metal ion intoxication?) causes abnormal proliferation which contributes to sclerosis; the counter-ion environment also was believed to affect the hydration properties of the matrix); a separate study had shown that Ni2+ intoxication had been effective in diminishing the effect induced suppression of mitogenic activity of mesangial cells by exogenous heparin].

Treiber C Simons A Multhhaup G, Biochemistry. 2006; 45: 6647-80(Effect of copper and manganese on the de novo generation of protease-resistant prion protein in yeast cells)(Manganese ions can apparently directly promote prion misfolding)

Trevitt CR Collinge J, Brain. 2006 preprinthttp://brain.oxfordjournals,org/cgi/reprint/awl150v1.pdf(A systematic review of prion therapeutics in experimental models)

Turnbull J et al., Trends Cell Biol. 2001; 11: 75-82(Heparan sulfate: decoding a dynamic multifunctional cell regulator) cf, Bernfield M et al., Ann Rev Biochem. 1999; 68: 729-77[Reviews of heparan sulphate biochemistry]

Vilar RE et al., Biochem J. 1997; 342: 473-97(Nitric oxide degradation of heparin and heparan sulphate under physiological conditions)

Wassermann A, Annal Bot (N.S.). 1949; 13: 79-88(Alginates of brown algae occur in vivo as multi-element salts)

Whitfield DM et al., Biopolymers. 1992; 32: 585-96 ibid., 1992; 32: 597-619(Increased metal ion chelation due to the presence of iduronate in heparin/heparan sulphate)(Cf., also the work of Templeton, loc cit., who investigated the alteration of glomereular and mesangial proteoglycan synthesis and activity as a result of direct and indirect actions of metal ions which bind to iduronate containing heparan and dermatan sulphates , the prinicpal polysaccharides of renal glomerular protoglycans)

Whittle JR et al., Acta Neurochir (Wien). 2006; preprint (Graham Steel CD).(Unsuccessful intraventricular pentosan polysufate treatement of variant Creutzfeldt-Jacob disease)(The toxicity of possible pyridine derivative impurity in PPS, in the high dose used, may be the reason for the lack of success in contrast to the greater success achieved by the lower doses used, as reported by Todd NV Morrow S Doh-ura K et al., J Infect. 2005; 50: 394-6)

AddendumThe Heparan Sulphate (HS) Hypothesis of Animal Diseases. Arising from the growing awareness that all animal physiology can be affected by HS biochemistry,and also the growing list of diseases which are believed to involve dysfunction of HS-controlled systems, it can suggested that all animal diseases are in some way HS-related and could arise principally or in part from a dysfunction of HS biochemistry especially involving fragments of HS generated nitrosatively (including a contribution to this process by metal ions) leading to dysfunction of HS control mechanisms affecting protein conformation and their supramolecular assembly. The general hypothesis can also suggests roles for HS fragments and HS mimetics can act as therapeutic agents by substituting for messenger HS both directly and by induction of altered native HS biosynthesis. A general discussion of the factors which affect heparan sulphate biochemistry was given by the author on the internet [accessed recently via Yahoo.com with search term “ascorbate & cancer ukonline/dgrant” which gives 2 sites of the 6 which can be accessed by clicking on “repeat results with similar results included”. There are some spelling mistakes, The sites need to be upgraded but provide useful references].