The Major Emerging Paradigm of Heparan Sulphate Related Therapy

THE HEPARAN SULPHATE PARADIGM

THE HEPARANOME MAY BE THE PRIME MASTER ANIMAL TISSUE PROTECTIVE SYSTEM
WHICH
INFLUENCES ALL ASPECTS OF HUMAN HEALTH

Progress Report
On a Continued Literature Survey on Heparin/Heparan Sulphate Following Employment as a Fixed Term Research Fellow
in Polysaccharide Research at Aberdeen University
+ Information in Graham Steel’s CD Literature Collection

by

David Grant, PhD, New Deer, Turriff, AB53 6SX, 30/7/07

Modified slightly July 10, 2010

Current studies in human subjects of the therapeutic potential of pentosan polysulphate

(PPS), a heparinoid drug, could lead to a wider employment of the powerful therapeutic
potential inherent in the current awareness of how heparan sulphate biochemistry is a master
system which influences health, but only if a greater understanding of this is achieved by the
wider scientific community.

An extensive laboratory research programme and associated literature survey* into the biochemistry of

heparin/heparan sulphate (the heparanome, the major animal biochemical information processing

system consisting of anionic polysaccharides) suggests that such research could lead to the

development of highly effective therapeutic agents for many diseases because the heparanome seems to

act as a prime “master” system, i.e., a “driver” of a wide range of “slave” biochemistry systems which

can have a major potential influences over virtually all aspects of human and animal health

[e.g., via the controls which are exerted by the heparanome over all major signalling

processes used for combatting pathological organisms, prions, other toxins, the control of

normal and pathological mineralisation, cellular immune survelillance, wound healing as well as for

embryo assembly, and ageing (which can suggest that therapeutic benefit can be attained by

replendishing heparan sulphate biosynthetic and post-biosynthetic polymer microstructure altering

pathways which have been rendered defective as a result of various pathological conditions (this

includes the pathological alteration of heparan sulphate microstructure by reactive oxygen and nitrogen

molecules during autoimmune processes).

The heparome is currently known to regulate the activities of a wide range of proteins (e.g., proteases,

anticoagulant serpins, lipoprotein lipases, growth factors, apoptosis factors, antioxidants and prions)

by interactions which are seemingly influenced by a system of fuzzy logic operated systemically in

which messenger heparan sulphate units and nitric oxide interlink cell surface heparan sulphate
polysaccharides with the environement, the extracellular matrix, intracellular compartments and the

nucleus apparently via a dual system of controlled microstructure generation and its alteration in

response to stress via an associatied process of polymer scission feedback with input from, e.g.,

ascorbate, metal ions and polyamines, affecting primary biosynthesis and by postsynthetic alteration by

non-redox-metal dependent (post synthetic heparanase, sulphotransferase and sulphatase) enzymic

action acting in concert with a non-enzymic postsynthetic redox-metal-ion-dependent system

employing nitric oxide to generate heparan sulphate oligosaccharides which function as messengers

which apparently engage in servo-feedback loops which can potentially be interrupted and boosted by

the exogenous administration of signalling oligosaccharides or their mimetics which can potentially

permit a simply-achieved medically directly augmentation of tissue protection afforded by the heparan

sulphate-based tissue protective systems.

Owing to the ability of the heparanome to influence essentially all aspects of animal biochemistry

including the ageing process, it can be suggested that it could actually eventually become

possible for all diseases and ageing to be directly combattable by heparin-like therapeutic agents.

Use of heparan sulphate mimetics of heparan sulphate fragments, e.g., designer polysaccharides-

based therapeutics or even enabled by dietary modifications may initially permit the successful

intervention in a range of currently poorly-understood illnesses for which conventional therapies are

relatively unsuccessful (this includes most degenerative diseases, most notably cancer and some

intractable diseases: Alzheimer’s disease, multiple sclerosis, all kinds of infection by antibiotic-

resistsant pathological organisms, aberrant prions, and other misfolded proteins, diabetes, various

arthritic conditions, malaria, viral infections and perhaps also major psychoses, autism and chronic

fatigue syndrome-like illnesses].

Heparin/heparan sulphate while lacking the ability to replcate by a template mechanism otherwise resembles DNA
in that it consists of a linear polymer sequence-holding encoded information systems with an additional built-in
higher level of flexibility allowing a rapid signalling sequence alteration in response to extracellular and
environmental conditions. This could also include a major input from inorganic ions. Disease processes involving
dyshomeostasis of this heparan sulpahte signalling seem to include the ammeliortion of the or the pathological
diminution of heparan sulphate protection by the direct effects of toxic metal ions such as lead, cadmum and
mercury, the effects of iron-overload, defective glucose homeostsis or certain oxidised lipids as well as augmented
generation of inappropriate heparan sulphate fragments by the presence of excessive amounts redox active iron,
manganese and copper ions.
The heparan sulphate animal-tissue-protection and protein folding control system may also be, at least partly,
redundant in the more evolved higher animal species where alternative protein-based systems have evolved which
have substituted for the roles which had been played in early animal species by the polysaccharides but
nevertheless are retained as backup systems in adult animals but which revert to their original roles during
development in embryogeneis and remodelling in wound healing.
It can be suggested on the basis of much research effort that heparan sulphate biochemistry seems to be centrally
invovled in embryology as well as in the aetilogy of cancer.
Animal evolution might also depend on the processes of crosstalk between the heparanome and the genome.
The relatively un-researched role of heparin/heparan sulphate-associated inorganic profiles (the metallome) might
allow a greater 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 surprisingly wide range of diseases can respond to heparin therapy, even in the unsophisticated current mode of
employment, and apparent relatvely poor quality control measures which seem currently to exist which can affect
the aspects of this drug which could depend on inorganic cofactors.

[The anticoagulation activity of heparin [discovered by J McLean in 1913] found wide medical use (starting
around 1937). The molecular basis of such activity (the heparin pentasaccharide antithrombin signalling sequence)
was established by Lindahl et al., Rosenberg et al. (and by 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 blood anticoagulants by substituting into the heparin cofactor II system, as well as interacting
strongly with Factor VIIIa. That algal or plant-derived polysaccharides have a useful pharmacological role in
animals confirms an evolutionary-ancient employment of such polysaccharides throughout biota.]

*[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. the 2003 internet listing of WFL’s publications (loc. cit.) which include
most of the former polysaccharide research group’s output). The pro-health strategy based on heparan sulphate biochemistry is
of an 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 the 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 which, 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 a cure-all as aspirin.

Is Heparin/Heparan Sulphate Biochemistry Relevant to Pion Diseases?

Contents
1. Introduction
Role of Polyanions as Cofactors for Protein Misfolding
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 in prion diseases of reduced nitric oxide heparan sulphate signalling
4. The metallome
4-1 Seawater & “Seawater-Related” Matrices
The Complexity of the Inorganic Profiles of Biological Fluids
4-1-1 Inorganic Ion Homeostasis in Seawater
4-2 The Heparanome & the Metallome Must Mutually Interact in Health & Disease
4-2-1 Dietary factors and heparan sulphate biosynthesis
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 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
5-3-2-1 Heparan sulphate & β Fe(II)O(OH) [akaganeite] fibril formation

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 Sulphate

6. Some of Mark Purdey’s Contributions to Medical Science

7. Promotion of Neurological Diseases by Pesticide Exposure

7 8. Roles of Inorganic Ions in Heparan Sulphate Signalling
8
9 8-1 Perturbation of Nitrosative Heparan Sulphate Signalling by Metal (e.g. Mn & Fe Ions)
10 8-2 Metal ions which are known to crosslink heparan sulphate to target proteins

8.3 Spark Source Mass Spectra of Heparin

8-3-1-1 Cation exchange resin replacement of multi-elements from heparin
8-3-1-2 Other reports of the multi-inorganic ion nature of heparin
8-3-2 The Occurrence of Thallium, Strontium & Gallium in Heparin
8-4 Phase Change Model of Heavy Metal Binding to Heparin/Heparan Sulphate
8-5 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
11 10. References & Further Notes
11. Addendum

1. Introduction
Role of Polyanions as Cofactors for Protein Misfolding.

Supattopone et al., using a seed of only one scrapie factor, thought to consist of an aggregate of ca. 20
of misfolded prion (PrPSc) monomers, was successsful in achieving a very high yield of these scrapie
factors starting from prions (PrPC) by using the in vitro prion amplification (PMCA) method (Saa et
al., 2006); since high yields were achieved by using only pure compounds (ultrasonication of the pure
synthetic protein with lipids + a synthetic polyanion which acted as a catalysts for the conformational
conversion); this experiment, as noted by Lee & Coughey, confirms the protein-only hypothesis of
scrapie factor infectvity which previously was slightly in doubt since natural nucleic acids had possibly
co-purified with the natural infective scrapie factor proteins. It should however be noted that the
various types of polyanion found by Supattopoen et al to be capable of catalysing the conversion into
aberrantly folded proteins included synthetic polynucleotides as well as heparan sulphate, so while it
still remains possible that natural nucleic acids might catalyse the formation of aberrant prion proteins
in vivo, the actual natural polyanion cofactors required for this conversion seem more likely to be the
cell surface heparan sulphates which are also known to control prion metabolism and enable prion
entry into the cell. It is however surprising that heparan sulphate can both potentiate scrapie infectivity
and also act as an anti-scrapie therapeutic agent.

If the polyanion present naturally as the necessary cofactor in the aetiolgy of TSEs is heparan sulphate,
then the complex nature of this polyanionic system needs to be addressed to fully understand the
aetiology of TSEs, and the mechanisms by which therapeutic intervention might be achieved.

It can be suggeted that the highly anionic heparan sulphate polysaccharide reactivities depends on the
microstructure of the polysaccharide, especially the anionic density which varies directly in response to
the presence of environmental metal ions; a further critical factor required for prion infectivity sould,
however be the direct association of such ions with heparan sulphate which may change the mode of
binding to proteins including prions; this could be why the toxicity of aberrant prion aggregates have
been associated with the presence of toxic ratios of metal ions, e.g. putatively involve a dyshomeostasis
of copper, iron and manganese (as well perhaps as nickel, chromium, silver, cadmium and mercury
other redox-active metal ions: barium, strontium, aluminium, silicon and perhaps also beryllium)
which, attached to heparan sulphate ligands, might directly creating infectious polymers or promote
their further aggregations into toxic plaques, processes which is known to incorporte heparan sulphate
proteoglycans. It can be suggested that the formation of amyloids is actually controlled by the heparan
sulphate system which if this becomes defective, leads directly to toxic plaque formation and it would
then follow that the actual central aetiological feature linking all amyloidoses is the dependence of
amyloid deposition on the microstructure of the heparan sulphate deaminative fragmentation processes
by which amyloidoses can be inhibited (especially those which are nitrosatively directed). Defects in
such heparan sulphate tissue protection will also be the central aetiological feature linking prion TSEs
with the other non-prion neurodegenerative processes such as Alzheimer’s disease, Parkinson’s
disease, amyotrophic lateral sclerosis, 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 extent of 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 e.g. employing the model proposed by
Fransson et al., group at Lund U.

2. All Natural Polyanionic Molecules Show Related Metal Ion Binding Properties
The 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 include
inorganic polysilicates, inorganic polyphosphate, poly-β hydroxy-butryate, nucleic acids (DNA, RNA
and polyadenylated RNA), 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 an 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 (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 for functional activity.

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 (cf Grant et al. 1992a) and this ability may be
retained in modern oranisms

The presence of variable amounts of aluminium and other toxic elements (e.g. lead, arsenic thallium
and cadmium) and large amounts of silicon 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

2.1 The Unique Roles of Heparin/Heparan Sulphate & Glycosaminoglycans in Animal Organisms
The 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 U. 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 thirty plus 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 all heparan sulphate proteoglycans (e.g., syndecan, glipcan, agrin, collagen
XVIII, cerebroglycan, 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 representative of the complexity different sequences which
seem to exist in 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 includes key 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 both a potential major quality control problem but with possible
consequences for nitric oxide mis-signalling. The ability of heparin to bind Al3+ was confirmed by
(unpublished) studies at Aberden U. [An unusual feature of aluminium heparinate is that it provides a
non-physiological acidic pH buffer (other heparinates containing physiological metals as principal
counterions create a physiological pH buffer). It can be hypothesised that Al3+ might generate acidic
conditions in the neighbourhood of heparin/heparan sulpahte chains capable of catalysing the
C-N-SO3- -> C-NH2 reaction, which primes for subsequent nitrous acid cleavage].
Recent studies have shown (Bohrer et al.) that this element occurrs consistently, but in variable
amounts, in commercial heparin, from which it may, however, be effectively removed by use of a
cation exchange resin column.

3. Role of Nitric Oxide in Animal Biochemistry

This had become firmly established by about 1992 (when nitric oxide was “the molecule of the year”).

3-1 Role of tyrosine nitration in nitrosative stress

The 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 sulpahte).
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 Biochemistry

A 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 the author in 2000, which suggested that
Fe, Mn, Ni, Cr, Ag and Ce ions might contribute to nitric oxide-dependent heparin/heparan sulphate
signalling and its disturbance.
A convenient method of establishing the presence and quantifying heparan sulphate in mixtures with
other GAGs is by 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 this assumption has needed to be revised following the discoveries 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.
The process seems also to putatively be augmented by redox active iron (and under iron-overload
conditions a major disruption by this metal of heparan suphate signalling can be anticipated).
Heparan sulphate is apparently 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, amyotrophic lateral sclerosis, 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 Role in Prion Diseases of diminished nitric oxide/heparan sulphate signalling

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)).
Pentosan poylysulfate may, like heparin exhibit tissue protection by a number of separate,
complimentary mechanisms including alteration of osmolyte balance, and metal ion activties, direct
binding which alters of protein conformation, alteration of nitric oxide signalling, binding and
deactivation of rective oxygen containing free radcials, alteration of the nucleation of phase change
including that which causes protein aggregation as well as acting as substitutes for the oligomeric
heparin/heparan sulphate fragments which could have intracellular and intercellular messenger
functions (derived inter alia from nitric oxide/redox metal ion scission of the polysaccharide side
chains of heparan sulphate proteoglycans).

4. The Metallome
This 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 originally been proposed by Williams to naturally occur in animal fluids and be essential
nutrient elements. H 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 his table of bio-topics for which his metallomics concept was relevant.
The metallome can be regarded as a key sub-system of the “phenome” (Varki, Graham Steel CD)
which also includes the geneome, the proteome, the reactome and the heparanome.

4.1 Seawater & “Seawater-Related” Metallomic Matrices

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

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.
This could correspond to a requirement of multi-elements to create the correct osmolyte balance
and related water supramolecular structure which influences protein hydration and folding.

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. Those polyanions which bind by mainly an
electrostatic mechanism (which is thought to include the sulphated polysacsharides) can be predicted to
bind a wider variety of counterions than those polyanions like polyphosphates which are able to form
liganded complexes thus having greater selectivity for counterion binding.

4-1-1 Inorganic Ion Homeostasis in Seawater

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 the results of in vitro crystallisation experiments conducted by the author at Aberdeen U
(unpublished, but now confirmed by reports from other laboratories) is of sufficient amount to allow natural humic
matter to inhibit and regulate the formation of inorganic crystallisation (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 (Grant, 1977) 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).

4-1-2 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]).
The Heparanome & the Metallome Must Mutually Interact in Health & Disease
The 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., Kodima 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)].

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 (Arthur 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, unpublished). Heparin also removes material from such columns
producing stable colloidal suspension of a heparin-Ca-hydoxyapatite (This was studied by infrared spectroscopy the author,
unpublished data Aberdeen U.).
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 sulphate 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.)

4-2-1 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, Japan or the EU.

Note added later. Information which confrims the multi-inorganic element nature of heparin had been
postec on the internet in 2005 it being deducible from a tabulation of ICP-MS data reported by a major
international analytical services laboratory for leachates from blood collection vessels (ALS) (cf. also
www.scribd.com/doc/3420554/).

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.).
[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 (loc. cit.) 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 (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 homology
with hydrolases generally and also apparently with some heat shock proteins, suggesting a more ancient common
ancestor of both (suggested by a home-based comparison of published amino acid sequences conducted by the
author).

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 glycosylation
due 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

Sulkowski (1992) proposed that the conversion of PrPC into PrPSc involved the co-ordination
of transition metal ions.
Morante et al. (2003) studies inter and intra-octarepeat Cu(II) site geometries and found evidence for
cooperativity of the metal ion binding process of prions. Such mechanism may be relevant to how
metal ions could increase aberrant prion toxicity.

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 transition metal 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 Manganese
Manganese 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 supported by
epidemiologal data collected by Mark Purdey) and the work of Quagli 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 (II oxidation state, redox-active) has recently been reported (Basu et al., 2007) to modulate
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.

5-3-2-1 Heparan sulphate & β Fe(II)O(OH) [akaganeite] fibril formation

Ferrous iron containing β FeO(OH)[akaganeite] fibrils were oobserved to be formed at heparin and putatively at
heparan sulphate surfaces by FB Williamson et al. (unpublished results of Aberdeen U.) (akaganeite crystals were
clearly identifiable by X-ray diffraction in aged Fe(II) heparin dispersions). The fibrous form of ferrous hydroxy
oxide akaganeite was previously known to be formed under conditions of high ionic strengths. Such environments
which will exist around heparin and the heparin-like segements of heparan sulphate.
Heparan sulpahte is apparently required to allow the generation of the infections poly-prion metal ion aggregates,
now known to putatively also include fibrils generated from prions by ferrous iron (Basu et al., 2007) and such
prion ferrous fibrils could constitute the proteolytically resistant toxic fibril infectious agents in TSEs in partial
agreement with the ferritin related piezoelectric crystals hypothesis of the late Mark Purdey.
The findings by that ferrous iron is a key ingredient in the generation of prion infectious particles further suggests
that the Aberdeen U. observation of the induction of alageneite fibrils by heparin might per se be relevant to the
aetilogy of TSEs and could further suggest that a dyshomeostsis of iron biochemistry, perhaps defects in Fe(II)
detoxification, could also be relevant to the aetiology of these diseases.

5-3-3 Aluminium & Beryllium

The promotion of dementia by aluminium intoxication is a well established phenomenon.
It is conceivable, from a pure-chemistry perspective, and environmental protection agency internet-available
documentation, that that beryllium, a toxic aluminium-like element apparently can be detected in ground waters in
Guamand could have entered the food chain. This may have arisen from dumpted copper beryllium bearings from
a major military aircraft engine servicing facility. This 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 of the author) 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 Newcastele General Hospital) discussed)).
There are numerous more recent references to this problem, e.g., Perl, 2006.

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]

Unpurified heparin could naturally contain e.g. 1000 ppm Al.

The cation exchange efficiency of removal of Al from heparin as deduced from the information given by Bohrer et al.
seems similar to the results of Moffat who found that several inorganic ion in heparin including Fe3+ were easily reduced by a
factor in the range 102-103 folowing percolation through an ion exchange column.

5-3-4 Cadmium
Cadmium 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 used
Heparan/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 Sulphate

The 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 (unpublished) 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 suphation of
xenobiotics. Silbert et al., reported that human skin fibroblasts cultured under conditions of inorganic sulphate
depletion showed a markly 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 Medical Science
The independent “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 hypotheses of the
origins of scrapie and bovine spongiform encephalitis and chronic wasting disease and other prion
diseases, the non-prion diseases bovine tuberculosis and human multiple sclerosis and a ferritin-
related mechanism of pathogenic particle generation which he proposed to be of relevance to the
aetiology of all TSEs, diseases which were also influenced by intoxication by insecticides and a related
manganese and copper ion binding to prion proteins.
Environmental exposure to toxic anthopogenic metal micorcrystal pollutants (containing barium
strontium and silver) were suggested to generate neuroglogically toxic fibrils via a ferritin
dyshomestasis process. A similar idea was later supported by research by Samoylov & Vodyanoy of
Auburn U. Numerous authors have supported the notion of toxic metal ions being involved in prion
diseases, e.g., as discussed in the 2005 review by Case in, “Chemistry World” (a Royal Soc Chem)
[loc.cit.]and by the 2006 research studies of the Multhaup group which supported the role
manganese/copper induction of prion misfolding in yeast cytosol and by Basu et al, 2007, who
reported that the self replicating β sheet-rich isoform agent of infectivity in TSEs is similar to a
proteinase-K resistant prion protein modulated by redox iron so that the pathological agent in all
prion diseases might similarly be subject to modulation by redox iron (which binds to PrPC and
transforms it into a PrPSc like form *PrPSc).
Mark Purdey has also found epidemiolgical evidence for key roles of of barium and strontium
intoxication in the induction of multiple sclerosis and further discussed how this effect might be
explciable in terms of heparan sulphate biochemistry which could further point to novel therapeutics
for this disease.
The general 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 was convinced of the relevance of inorganic element intoxication in prion and other
neurodegenerative diseases. These include 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 seem to be supported by experimental results reported by 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 toxic 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.

A novel low-cost therapy for bovine tuberculosis was based on an apparent role of iron intoxication in
this disease.

7 Promotion of Neurological Diseases by Pesticide Exposure

Mark Purdey’s original BSE hypothesis (Med Hypotheses. 1996; 46: 429-54) had been suggestion that chronic
pesticide [e.g. organophosphate] 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 hexachalorobenzene
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, poysaccharide 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 with Monsanto, USA; ICI and ISR, UK,
which had included research into the formation of hexachlorobenzene by the pyrolysis of chlorinated organic
substances 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 were utilised.
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 chlorinated pesticide residues 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).

8. Roles of Inorganic Ions in Heparan Sulphate Signalling

8-1
Perturbation of Nitrosative Heparan Sulphate Signalling by Metal (e.g., Mn & Fe) 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 enzymic reactions involving heparan sulphate including fibroblast growth factor actovatopm
by control of its 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). TSEs seem to involve diminution by the infective agent of nitric oxide supply. Other
degenerative diseases such as arthritis, on the other hand, seem to invovle augmentation of nitric oxide
supply.

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 Nieman-Pick disease (Mani et al,. 2007).
Manganese ions liberated from glycocalyx stores could produce excess primed sites for nitric oxide
scission.

Manganese present within iron aggregate structures could alter the magnetic character of such
aggregates.
This heterogeneity of iron-like inorganic aggregates may allow the formation of the sort of
piezoelectric fibrils suggested by Mark Purdey (loc. cit.) to be the true infectious agent in TSEs.

A general property of the most anionic natural polymers which include inorganic polyphosphate,
ubiquiteously present at cell surfaces throughtout biota (Kornberg, 2004) and heparan sulphate present
at the surfaces of all aberrant animal cells, could be to inhbit pathological solid formation both of
misfolded protein aggregates as well as inorganic crystals (Grant et al 1992b). Although most
documented for Ca-rich crystals this general principle seems also to apply to Fe-rich crystals.
The occurrence of such crystals or related pathological forms in biological tissues then could result
from defects in such anti-crystal protection mechanism especially that provided by the heparin/heparan
sulphate tissue protective system.

[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 jump
between selected carboxylate groups alone the heparan chains (a process discovered to occur with
heparin by Rey et al. in 1994)].

8-2 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)

8-3 Inorganic Elements Including Heavy Metal Ions in Heparin

The original impure form of heparin, (originally discovered and shown to be a blood anticoagulant by J
McLean in 1913) 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 Chelex 100 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; similar 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.
HJM Bowen (1966) in a standard classical text on inorganic biochemical analysis, warned against
using heparin during the assay of inorganic elements in blood due to the presence of numerous
inorganic elements in the commercial heparin then available. Previous use of heparin during assays for
manganese in blood had giving highly misleading results owing to the major input of manganese from
the heparin used.

Possible Inorganic Element Contamination of Heparin During Manufacture

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 arise from continued use of classical
methods for the preparation of commercial heparin, which employed asbestos or similar filtration aids.

8-3-1 Spark Source Mass Spectra (SSMS) of Heparin

CF Moffat (1985) was the first to systematically measure the inorganic elements associated with
heparin. He measured the amounts of 38 inorganic elements (excluding aluminium, which had been
employed to form a SSMS electrode) present at >1ppm in a standard pharmaceutical industry research
grade sodium heparin (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 by
CF Moffat and also the inclusion of heparin in a list of biological systems for which the metallomic
(all-element) concept of H Haraguchi (2004) is applicable.
37 elements were found to be simultaneously held by heparin in amounts which seemed to be
reminiscent of a list of required nutrients for animal cell culture media (but with the presence
of a number of additional elements). It was later found that this list could be correlated in
log-log plots similar to those shown by Haraguchi (2004) who had reviewed his hypothesis of
the relevance of an extended all-element metallomic science [a concept originated by RJP
Williams of Oxford U., but restricted to some 30 elements] where all [e.g., 50] seawater
inorganic elements should, he proposed, show up in biolgical tissues including human tissue.
The inorganic profile of heparin was evidently a naturally occurring inorganic array similar
to that predicted by Haraguchi. The heparin multi-elements are also correlated with those of
human hair and seaweed as well as some naturally occurring ethnic medically emplyed
substances (fulvate salt deposits).

The multi-elements in seawater and seaweed

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 (of the Institute of Seaweed Research Musselburgh) and RL Mitchell (of the
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].

8-3-1-1
Cation Exchange Resin Replacement of Multi-Elements from Heparin
Studies by the Aberdeen group (initiated by CF Moffat, [at that time a postgraduate student] and
directed as regards the selection of the analytical method, spark source mass spectrometry used, by FB
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 the 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.

Other reports of the multi-inorganic ion nature of heparin

Comparison of the spark source mass spectroscopic results obtained by Moffat [loc. cit.] with previous
reports lends some support for the idea that all heparins could be related multi-element matrix systems.
[A comparison of the Aberdeen U. 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 U. 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].
Other studies [e.g. by Alcock, 1983] also reported the occurrence of varying amounts of a range of
inorganic elements in commercial heparin.
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)].

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), the regression of tumours via inhibition angiogenisis (Folkman,
1983) and the antitumour, anti-metastatic activities of different brands of heparin (Parish & Snowden,
1988). This possibility may arise since the correct amounts of specific inorganic elements, absent in
some ‘overpurified’ could be absolutely required for the enactment of the full biological activity of
heparin/heparan sulphates. An example of this is that in absence of sufficient zinc due to
overpurification of the biopolymers involved, binding to the in vivo receptors of for collagen XVIII-
endostatin heparan fails to take place (Ricard-Blum et al., 2004); a similar scenario can be anticpated
for bivalent calcium etc. ions which 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) and the Ca2+ potentiation of the binding of heparan
sulphate and dermatan sulphate (Hamaguchi, 1992) to human serum amyloid P (this binding was
suggested (Janciauskiene et al., 1995) to normally protect against aberrant Alzheimer β peptide fibril
formation demonstrating how dyshomeostis of metal ions could potentiate this pathological action; a
related process requiring for its prevention the presence of sufficient Cu2+, ascorbate and Zn2+ to
facilitate the Cu 2+/+ -nitric oxide heparan sulphate soluble oligomer formation by the deaminative
cleavage process (cf., Mani et al., 2007) may lead to the pathological deposition of amyloid and other
misfolded proteins. [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 (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 opposite condition of lack of nitric oxide will
cause a diminution of heparan sulphate fragments [similar to PPS] which can function apparently in a
variety of tissue protective modes which includes the direction of aberrant prions into an extracellular
clearance pathway as well as the inhbition of the nucleation of fibril formation].

8-3-2 The Occurrence of Thallium, Strontium & Gallium in Heparin

Moffat (1987) had found that thallium occurred in “pharmaceutical heparin” and assumed that this
situation applied to all heparins; he also noted that there had been no reports in the literature of
thallium poisoning associated with heparin anticoagululation therapy, therefore it seemed possible 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 which it occurs in a similar amount to that found in heparin)
where it had evidently been sequestered from the trace amount present in seawater; there are no reports
of any thallium intoxication from the use of kelp as a major food ingredients in Japan and China. It
should be noted that gallium is also present in simiar amounts in kelp and in heparin – this is perhaps a
contributory factor in the anti-tumour action of heparin which might be at least partly attributable to an
effect of Ga3+ alone since administration of inorganic salts of this element is well known to have a
potent anti-tumour effect.
Sr2+ is also present in both heparin and in kelp and analogous to the situation with Ga3+ , Sr2+
administered in the form of an inorganic salt or with an organic ligand and putatively with
polysaccharide matrices apparently is able to assist in bone formation.
Traditional medicine in India, China and America is reported to employ apparently benficial medicinal
properties of similar multi-element-containing geological organic polyanionic fulvates which, like
heparin 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 contents (viz., acting as sources of required trace inorganic nutrients).

8-4 Phase Change Model of Heavy Metal Binding to Heparin/Heparan Sulphate

This 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, PhD Thesis, Edinburgh University, 1969).

8-5 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 (cf., 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).

9 Some Aberdeen U., Heparin, Heparan Sulphate & Pentosan Polysulphate Research
9-1 Heparin/Heparan Sulphate Research
A list of relevant publications was posted in 2004 on the internet by Professor WF Long (loc. cit.)

9-2 PPS Research

A 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 was
to 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 but
from 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 unpublished 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 author’s 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 which was
probably of greater activity than that provided by commercial pentosan sulfates.

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 GH 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 seeded to be involved. As
with many illnesses heparin therapy had been reported to be of major benefit to some mystery illness
patients.
References & Further Notes

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)
[M Ross et al and G Mackintosh at Aberdeen U. found related effects; this work is only partly
published]

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

Added later
ALS
(ALS Laboratory Group)
web.alsglobal.se/hem2005/pdf/blood_collection_tubes_eng.pdf

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 protien 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 replicationand 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

[A review of heparan sulpahte biochemistry]

Bishop JR et al., Infect & Immunity. 2005; 73: 5395 (Graham Steet CD)
(Cell surface heparan sulfate promotes replication of Toxoplasma gondii)
[Cf. also Sinnis et al., loc. cit.]

Bisse E et al., Anal Biochem. 2005; 337: 130-5

(Reference values for serum silicon in adults)
(Silicon concs. (i.e. soluble silicon, most likely silicic acid Si(OH)4) were det. for 1325 healthy subjects
aged 18-91, medians varied with age and sex. (men 18-59 had 9.5µ M.L decr. to 8.5 with age; women
had 10 µ M/L for age 18-29 but 11.1µ M/L for ages 30-44 but decr. with age to 9.23µ M/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 nutrition]
A 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, cancer
and prion diseases).

[Graham Steel CD file]

Black Sheep (Symposium) 433-447%20kkoniish.pdf file Situation ignored by mainstream Alzheimer’s
disease researchers
Role of Metal Ions
“Apolipoprotein Aβ 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 Aβ .”
“Spontaneous” Aβ aggregation to fibrils in vitro is caused by traces of transition metals present in laboratory buffers.
Aβ is readily aggregated by transition metal Cu(II) Fe(III), Zn2+ and Al3+. “In contrast, in the absence of such metal ions Aβ
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}

Bohrer D et al., RBAC (Brasil) 2004; 36(2) 99-103

In studies relating to the safety of substances used during hemodialysis treatments the occurrence of aluminium
[Bohrer D et al., this RBAC article] and arsenic [J Parenteral Nutr 2005: 29 (1) 1-7] in heparin are 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
30µ g/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) contains the following data:
Al (µ g/L or µ g/g) (present as an impurity in the reagents studied).
Nitric acid Merck superpure 110+1µ g/L
Merck distilled 10+1µ g/L
EDTA (sodium salt) Merck 3.37+0.7 µ g/g 3.37ppm
[data also given for sulphuric acid, trichloroacetic acid)
Sodium tungstate Merck 26.5+1.70mg/g 26.5ppm
Na2SO4 Reagen 26.5+3.77µ g/g 26.5ppm

Heparin bovine (sodium salt) Sigma 0.0 µ 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 µ g/g

Heparin porcine (ammonium salt) Sigma 0.0 µ g/g
Commercial heparins
5000 IU/ml (ca. 30mg/L) Fujisawa 732+23 µ g/L
Roche 783+51 µ g/L ca. 35ppm
Cristalia 72+6 µ g/L
25000 IU/ml (ca. 150mg/L) Eurofarma 436+40µ g/L
Roche lot1 137+2µ g/L
Roche lot2 168+3µ g/L
Roche lot3 190+5µ g/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. 15µ g/L (i.e., the column became saturated with Al3+ after three elutions).

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)

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 TSEs
Similarities 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 neurones 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. It might be speculated 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, transtyretin 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)
Many other workers 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. lopc cit.,) of Hg,
Ni and Mn (Templeton, loc cit.), 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.,)

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 lists 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

Bowen HJM “Trace Elements in Biochemistry” Academic, London, 1966, p. 63

(Ba, Ca, Cu, Mn, Sr and Zn commonly occur in heparin)

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)
Casoni F et al., J Biol Chem. 2005; 280 (16) 15295-16304
(Protein nitration in a mouse model of familial amyotrophic lateral sclerosis)

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 associated 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)
[This seems to be an example of how toxic insoluble particles can induce disease and the phenomenon
described in this paper may have some similarity to the aetiology of prion diseases. Heparan sulphate
seems normally to prevent the formation of such toxic disease inducing particles. The calcium
phosphate precipatated in the reported studies seemed to occur in conjuction with a chondroitin
sulphate SiO2 complex; a possible rationalisation of this phenomenon could be that a defect in
heparan sulphate biosynthesis induced in male but not female rats failed to prevent the formation of
the pathological pro-cancer solids; in such cases the remaining glycosaminoglycan chondroitin
sulphate rich mixture in the urine seems to have been unable to prevent precipitation]

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 cognition
cf., 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, 360

[Ateroid 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 suphate 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 agniogenesis 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 et al., loc. cit.) 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). It had also been reported that quaternary amine salts of
heparin on combusiton left no ash (this points to the easy replacement of inorganic elements from
heparin during the preparation of such salts from heparinic acid, high HCl concentrations needed to
form this (unstable) acid apparently remove metal salts as soluble chlorides)..

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: 460-9

cf., Toxiciology 1999; 133: 159-69
[Lead intoxication diminishes heparan sulphate proteoglycna biosynthesis]

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 similary become fully accepted only after a long delay,
considerable scepticism etc., and eventually be permitted only following non-medical or alternative
medical use in the context of dietary supplementation or under semi- illegal medical 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) and
Zn2+]

Grant D, Nature. 1977; 270: 709-10

(The chemical structure of humic substances)
[The key breakthrough in obtaining useful NMR spectra of these highly abundant soil polymers was
to use a separate inorganic polyphosphate chelator which removed paramagnetic metal ions from the
solution phases studies by NMR; a similar process may occur of transfer of metal ions from heparan
sulphate to inorganic polyphosphates (both polyanionic are ubiquiteously present naturally at animal
cell surfaces, and this transfer or defects in its operation may be of relevance to the aetiology of prion
diseases]
[Cf., also Proc Int Wkshop on Properties of Organic Peat Components, Braunshweig, W Germany; Cf
Absrt No 11 and full paper; and Barron PF & Wilson MA Nature. 1981; 289: 275-6
(Humic soil and coal structure study with magic-angle spinning 13C CP-NMR)
Grant D et al. Eur Polym J. 1974; 10
(….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 similarly augmenting the
oxidation states of transition metals in numerous protein (including prions aggregates)]

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)
[An update ofthis hypothesis could suggest that these tyrosine-cysteine clusters might function as anti-
nitrant agents, to mop up peroxynitrite and other damaging reactive nitrogen agents]

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
Could be ealier in the year (but not more recently) be directly downloaded 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].
(The published abstract of this article:
“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 of discussions relating to the role of heparin/heparan sulphate tissue protection as
anti- tumour agents,are found on several ukonline sites; the 1970s Linus Pauling’s ascorbate-
cancer/viral hypothesis could, it was proposed, 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 research

interests [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 Purdey.
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 who normally 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

[Heparan sulphate iron transport may be a backup system to transferrin in the liver]

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

Ingham PW, Nature. 1999, 394: 16

Cf., 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 SO42- 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 fibroblast growth factor activitaion
effects cf., Robinson et a.l, 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.; both authors found that ascorbate in cell culture media enhances the biosynthesis of
highly sulphated heparan sulphates; this now seems to be the principal reason for ascorbate induced
(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. USA, 2004; 46: 16085-7

(Inorganic polyphosphate in the origin and survival of species)
(This is a major hypothesis from a Nobel laureate 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 in comparison with the O- linked SO3-; at the time the
possible relevance of this high 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
sites of rapid NO-dependent oligosaccharide generation))

Krijgsheld KR et al., Comp Biochem Physiol 1980; 67A; 683-6

(Serum concentration of 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
nothing 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-β− 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+)
[This was the starting poiint of the interest in the Aberden U. polysaccharide research group in the
role of metal ions in hepairn.heparan sulphate biochemistry]

Long WF Research and Scholarship

http://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 o
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 of this thesis 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 discussion of the results as 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+ concentration
via effect on amyloid precursor protein (APP)
50nM Zn2+ promoted heparin binding to APP but
Zn2+ 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
[N.b., Questal was a much honoured biochemisty of his day]

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: 201-6
which reported the preparation of N-propionylated heparin)
This Moffat CF et al., 1997 article is cited in Fernandez C et al., ref 30 Carbohydr Res 2006 [Graham
Steel CD] [Semi-synthetic heparin derivative etc.])

Morante S et al., J Biol Chem., 2003; Dec 31 Manuscript M312860200 (internet)

(Inter- and intra-octarepeat Cu(II) site geometries in the prion protein: implications in Cu(II) bonding
cooperativity and Cu(II)-mediated assemblies)

Morner CT, Skand Arch Physiol., 1889, 1, 210-243

(Chemische studien uber den trachealknorpel)

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+ concentration
Cf. 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 sulphate 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 10-20µ g/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 formation could directlly 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)

Purdey M, Ecologist. 1994; 24: (3) 100-4; ibid., (5) ; ibid., 2002; 32(9) 33-7

Purdey M, J Brit Cattle Vet Assoc. 2002; 10(4): 311-35

Purdey M, Med Hypotheses. 1996; 46: 429-54; ibid.,45: 429-43

(The UK epidemic of BSE: slow virus or chronic pesticide initiated modification of the prion proteins (Parts 1 and 2)

Purdey M, Med Hypotheses 1998; 50: 91-111

(High dose exposure to systemic phosmet insecticide modifies the phosphatidylinositol anchor on the prion
protein: the origin of new variant transmissible spongiform encephalopathy?)

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 M, 2006
http://www.markpurdey.com/mark_purdey.htm
“Neuropathological survellance of CJD brain tisuue at Case Western University, Cleveland, Ohio,
recorded a ten-fold increase in manganese and 50 percent reduction in copper in CJD brain tissue in
relation to control brains.”
“Kobe University in Japan ran experiments where they beamed manganese cell cultures with ultra
violet radiation. The prions subseqently aggregated into fibril formation, which is the key
characteristic tombstone feature of the TSE diseased brain.”

Purdey M, Med Hypotheses. 2005; Oct 15

(Auburn University research substantitates the hypothesis that metal microcrystal nucleation
initiates the pathogenesis of TSEs)
Cf The Weston A Price Foundation
htttp://www.westonaprice.org/mythstruths/madcow4.html
{Purdey’s data on TSE confirmed by Auburn University data}

Purdey M, Med hypotheses. 2005; 65: 448-77

(Metal microcrystal pollutants; the heat resistant, transmissible nucleating agents that initiate the
pathogenesis of TSEs)

Purdey M, Med Hypotheses. 2004; 63 211-25

(Elevated levels of ferrimagnetic metals in foodchains supporting the Guam cluster of
neurodegeneration: do metal nucleating crystal contaminants evoke magnetic fields that initiate the
progressive pathogeneis of neurodegeneration?)

Purdey, Mark, Correspondence with the author

cf., DG letters of 26/5/01 ; 22/8/01; 14/7/03; 2/9/03; 28/10/03
The 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.
[This idea does not seem to have been thought to be credible but it has not been tested]
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 (may be 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 also
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 Purdey suggested that infrasonic shocks generated by blasting and supersonic aircraft, etc.,
might have roles in the formation of the infective agent in TSEs.
Cf.,
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 permit a greatly enhanced yield of PrPSc 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” shock waves can allow molecular fragments to 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 which are now known to constitute key biochemical second messengers in animals
and be implicated in the modus operandi of heparan sulphate during tissue protection a function which putatively also
includes the modulation of prion biochemistry, including the inhibition of prion misfolding and expulsion of misfolded prions
from the cell.]

Purdey, Mark
Draft 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

[A review of heparan sulphate biochemistry]

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 tumour (sarcoma 37) bearing mice, 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)
[“This requirement for divalent cations was discovered in an effort to understand the lack of binding
to heparin and heparan sulfate of highly purified endostatin produced by 293-EBHA cells. A single
purification step of afinity chromatography on a HiTrap heparin column of the culture medium of
2993-EBNA cells was insufficient to obtain pure endostatin as demonstrated by the presence in SDS-
PAGE of high molecular weight contaminants co-eluted with endostatin from the heparin afinity
column. A second step of purification by gel filtration was thus performed and it is likely that the
divalent ion required for binding to the glycosaminoglycans was lost during this step. In most of the
endostatin batches we tested, the addition of 5µ M zinc chloride resulted in a 35 to 50% increase in
binding to heparin and heparan sulfate. However we observed a much higher increase in binding in
some endostatin preparations, up to 175% in the presence of zinc and up to 91% in the presence of
calcium. This could indicate that loss of the the divalent cation (likely zinc…) does vary from one
preparation to another and could explain the lack of endostatin binding to heparin in some
experimenatl conditions….The variation in zinc content could also account, at least in part, for
contrasting results and lack of reproducibilty regarding the biological activites of endostatin and
attributed to incorrect folding and/or to differential post-translational modificiation between
endostatins from difffernt sources”]

Robinson GW Cho CH, Biophys J. 1999; 77 (6): 3311-18

(Role of hydration water in protein unfolding)
Cf., Luck WAP, Topics 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, 1992
Potentiation 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)
[This seems to rely on the use of sonication]

Sarrazin S et al., J Biol Chem. 2005; 280: 37538-64

(Heparan sulphate mimicry)(…the first synthetic heparan sulphate-like molecule that targets a
cytokine)
(A heparan sulphate oligosaccharide mimetic that recognizes the natural heparin-binding domain
required for the inhibition of the cytokine IFNγ )
[This paper may be a good illustration of the probable modus operandi of PPS therapy which may rely
at least partly on mimicry of a heparan sulphate domain signal]

Selleck SB
(Whilst the polysaccharide side chains are normally the active centres the heparan sulphate core protein
cana also engange in specific signalling which can be controlled by removal of the polysaccharide side
chains by heparanase action)

Schwarz K, PNAS, USA. 1973; 70: 1698-1612

(A bound form of silicon in glycosaminoglycans and polyuronides)
[This includes heparin and heparan sulphate]

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?]

Sinnis P et al., with Linhardt RL, J Biol Chem. 2007; 282 (35) 25376-84
(Mosquito heparan sulfate and its potential role in malaria infection and transmission)

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 [unpublished results from
Aberdeen U.])

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 anticoagulant,
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]

Sulkowski E, FEBS Lett. 1992; 307: 129

[Cited by Tabner et al., loc. cit.]

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 by
Supattapone et al., 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.) (loc. cit.) had
demostrated that PrPsc incorporation to cells requires endogenous glycosamonoglycan expression
[Could viroid nucleic acids promote prion misfolding, acting in a similar way to the poly A etc studied
here ?]

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)
[The ubiquitous presence of inorganic Si, putatively as SiO2 in polyuronides may point to
such a function for sorting and transport of inorganic ions in vivo]

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) Proc 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.

Tabner BJ et al., Curr Med Chem-Immu Endoc & Metab Agents. 2003; 3: 299-308 [Graham Steel CD]
(Direct production of reactive oxygen species from aggregating proteins and peptides implicated in the
pathogenesis of neurodegenerative diseases)

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 preprint

http://brain.oxfordjournals,org/cgi/reprint/awl150v1.pdf
(A systematic review of prion therapeutics in experimental models)
[Trevitt & Collinge were apparently somewhat reluctant here to acknowledge that heparan-sulphate-related-biochemistry might
be centrally implicated in prion diseases, and these authors seemed to downplay the significance of the possibility of successful
therapeutic intervention using heparinoids, e.g., pentosan polysulfate (PPS) which might confirm the importance of heparan
sulphate biochemisty in prion diseases.]

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
[A Review 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)
[Differences reported between the effect of metal containing phosphate buffers and purer
imidazole-based buffers suggest key roles for metal ions in the nitric oxide degradations reported by
these authors however they did not seem to be aware of this possibility]

Wassermann A, Annal Bot (N.S.). 1949; 13: 79-88

(Alginates of brown algae occur in vivo as multi-element salts which includes redox metals-this
seems to be a general principle which governs the reactivity of all natural anionic polysaccharides
which of course includes the animal heparan sulphates and also may influence the bio-activity of
PPS )

Whitfield DM et al., Biopolymers. 1992; 32: 585-96

ibid., 1992; 32: 597-61
[These papers suggest the occurrence of 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 protoglycan])

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)

Addendum
The 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].

Since the straightforward reporting in the mainstream literature that possibly damaging toxic elements may occur in commonly used pharmaceutical drugs cannot be approached directly for obvious reasons it was thought to be

expedient to simply briefly mention an abbreviated version of the data in the experimental section of a paper dealing more generally with infrared spectra (Grant et al., 1987) which on re-reading apparently can be taken
perhaps erroneously to suggest that multi-elements are not normally be associated with pharmaceutical heparin. This description should not be cited as evidence of the purity of heparin.