Olga Korchazhkina

Elevated urinary excretion of aluminium and iron in multiple sclerosis

Multiple sclerosis (MS) is a chronic, immune-mediated, demyelinating disease of the central nervous system of as yet unknown aetiology. A consensus of opinion has suggested that the disorder is the result of an interplay between environmental factors and susceptibility genes. We have used a battery of analytical techniques to determine if the urinary excretion of i) markers of oxidative damage; ii) iron and iii) the environmental toxin aluminium and its antagonist, silicon, are altered in relapsing remitting (RRMS) and secondary progressive MS (SPMS). Urinary concentrations of oxidative biomarkers, MDA and TBARS, were not found to be useful indicators of inflammatory disease in MS. However, urinary concentrations of another potential marker for inflammation and oxidative stress, iron, were significantly increased in SPMS (P<0.01) and insignificantly increased in RRMS (P>0.05). Urinary concentrations of aluminium were also significantly increased in RRMS (P<0.001) and SPMS (P<0.05) such that the levels of aluminium excretion in the former were similar to those observed in individuals undergoing metal chelation therapy. The excretion of silicon was lower in MS and significantly so in SPMS (P<0.05). Increased excretion of iron in urine supported a role for iron dysmetabolism in MS. Levels of urinary aluminium excretion similar to those seen in aluminium intoxication suggested that aluminium may be a hitherto unrecognized environmental factor associated with the aetiology of MS. If aluminium is involved in MS then an increased dietary intake of its natural antagonist, silicon, might be a therapeutic option.

Measurement by reversed-phase high-performance liquid chromatography of malondialdehyde in normal human urine following derivatisation with 2,4-dinitrophenylhydrazine

A selective and sensitive method based on derivatisation with 2,4-dinitrophenylhydrazine (DNPH) and consecutive HPLC gradient separation is described for the determination of malondialdehyde (MDA) in urine. Preparation of urine samples involved a one-step derivatisation/extraction procedure. Separation was achieved using a Waters SymmetryC(18) column (3.9 x 150 mm) and linear gradient of acetonitrile in water (from 30% to 70% in 30 min). The overall detection limit of the method was 56 nM of MDA in urine. The recovery of MDA was 94.3+/-8.6%. MDA in urine of healthy volunteers, measured using the method of standard additions, was 0.019+/-0.012 microM/mmol creatinine. MDA in the same samples measured using the 2-thiobarbituric acid (TBA) assay was 0.181+/-0.063 microM/mmol creatinine. We demonstrate that the commonly used TBA assay in conjunction with HPLC may overestimate the MDA concentration in human urine by almost 10-fold.

Promotion of formation of amyloid fibrils by aluminium adenosine triphosphate (AlATP).

The formation of amyloid fibrils is considered to be an important step in the aetiology of Alzheimers disease and other amyloidoses. Fibril formation in vitro has been shown to depend on many different factors including modifications to the amino acid profile of fibrillogenic peptides and interactions with both large and small molecules of physiological significance. How these factors might contribute to amyloid fibril formation in vivo is not clear as very little is known about the promotion of fibril formation in undersaturated solutions of amyloidogenic peptides. We have used thioflavin T fluorescence and reverse phase high performance liquid chromatography to show that ATP, and in particular AlATP, promoted the formation of thioflavin T-reactive fibrils of beta amyloid and, an unrelated amyloidogenic peptide, amylin. Evidence is presented that induction of fibril formation followed the complexation of AIATP by one or more monomers of the respective peptide. However, the complex formed could not be identified directly and it is suggested that AlATP might be acting as a chaperone in the assembly of amyloid fibrils. The effect of AlATP was not mimicked by either AlADP or AlAMP. However, it was blocked by suramin, a P2 ATP receptor antagonist, and this has prompted us to speculate that the precursor proteins to beta amyloid and amylin may be substrates or receptors for ATP in vivo.

Non-invasive therapy to reduce the body burden of aluminium in Alzheimer's disease

There are unexplained links between human exposure to aluminium and the incidence, progression and aetiology of Alzheimers disease. The null hypothesis which underlies any link is that there would be no Alzheimers disease in the effective absence of a body burden of aluminium. To test this the latter would have to be reduced to and retained at a level that was commensurate with an Alzheimers disease-free population. In the absence of recent human interference in the biogeochemical cycle of aluminium the reaction of silicic acid with aluminium has acted as a geochemical control of the biological availability of aluminium. This same mechanism might now be applied to both the removal of aluminium from the body and the reduced entry of aluminium into the body while ensuring that essential metals, such as iron, are unaffected. Based upon the premise that urinary aluminium is the best non-invasive estimate of body burden of aluminium patients with Alzheimers disease were asked to drink 1.5 L of a silicic acid-rich mineral water each day for five days and, by comparison of their urinary excretion of aluminium pre-and post this simple procedure, the influence upon their body burden of aluminium was determined. Drinking the mineral water increased significantly (P<0.001) their urinary excretion of silicic acid (34.3 +/- 15.2 to 55.7 +/- 14.2 micromol/mmol creatinine) and concomitantly reduced significantly P=0.037) their urinary excretion of aluminium (86.0 +/- 24.3 to 62.2 +/- 23.2 nmol/mmol creatinine). The latter was achieved without any significant (P>0.05) influence upon the urinary excretion of iron (20.7 +/- 9.5 to 21.7 +/- 13.8 nmol/mmol creatinine). The reduction in urinary aluminium supported the future longer-term use of silicic acid as non-invasive therapy for reducing the body burden of aluminium in Alzheimers disease.

Plasmin cleaves Aβ42 in vitro and prevents its aggregation into β-pleated sheet structures

The formation, aggregation and deposition of amyloid β peptide (Aβ) is implicated in the aetiology of Alzheimers disease. Impairment of proteolytic degradation of Aβ may be a key factor in the progression of the disease. We have used RP-HPLC and thioflavin T fluorescence to demonstrate that Aβ42 is rapidly cleaved by the protease plasmin and that cleavage prevented the aggregation of Aβ42, and its cleavage products, into β-pleated sheet structures. Plasmin may fulfil a similar role in vivo.

Intravascular ATP and coronary vasodilation in the isolated working rat heart

Adenosine‐5′‐triphosphate (ATP) is a potent coronary vasodilator. Because of the efficient hydrolysis of ATP, adenosine‐5′‐diphosphate (ADP) and adenosine‐5′‐monophosphate (AMP) by ectonucleotidases located in the coronary endothelium ATP‐induced vasodilation may be mediated via both P1 (AMP and adenosine) and P2Y (ATP and ADP) receptors. We have used the change in total coronary resistance (TCR) induced by intravascular ATP in the isolated working rat heart to determine both the component of the vasodilation mediated via P2Y receptors and the identity of the subclass of receptor involved. The dose response for ATP revealed a half maximal effect at an apparent ATP concentration of 0.08±0.009 μM. The response was saturated at apparent ATP concentrations greater than 0.23 μM. Contrary to much of the current literature, the perfusion of a 0.25 μM concentration of adenosine resulted in the identical response to an equimolar concentration of ATP suggesting a significant role for adenosine in coronary vasodilation. The non‐selective P1 receptor antagonist 8‐(p‐Sulfophenyl)theophylline (8‐SPT) was used to show that the response to ATP was mediated via both P1 and P2Y receptors. Whilst 8‐SPT abolished the effect of adenosine it reduced the effect of ATP by only 50%. Thus, at a saturating concentration of ATP, P1 and P2Y receptors were shown to contribute equally to the observed vasodilation. Uridine‐5′‐triphosphate (UTP), ADP and adenosine‐5′‐O‐thiotriphosphate (ATPγS) were used to characterize the component of coronary vasodilation that was mediated via P2Y receptors. UTP at 0.25 μM was ineffective and did not induce vasodilation. Perfusion with 0.25 μM ADP resulted in a vasodilation that was identical to 0.25 μM ATP. In the absence of 8‐SPT the perfusion of 0.25 μM ATPγS produced a vasodilation that was significantly (P<0.05) less than ATP. However, the vasodilation due to ATPγS, like that of adenosine, but unlike that of both ATP and ADP, was abolished in the presence of 8‐SPT. The ability of ADP to induce vasodilation combined with both the lack of response to UTP and the ability of 8‐SPT to abolish the vasodilation induced by ATPγS suggested very strongly that the component of ATP‐induced coronary vasodilation in the isolated working rat heart that was mediated via P2Y receptors was achieved by the action of ADP (and not ATP) at P2Y1 receptors. These results suggest that the vasodilatory action of intravascular ATP in the coronary circulation should be attributed to the dual and equal activities of adenosine and ADP acting at P1 and P2Y1 receptors respectively.

Effects of exclusive formula or breast milk feeding on oxidative stress in healthy preterm infants

Background: Compared to formula, breast milk is considered to have superior antioxidant properties and consequently may reduce the occurrence of a number of diseases of prematurity associated with oxidative stress. Aims: To test whether the antioxidant properties of breast milk in healthy premature infants are different to those of formula milk by comparing vitamin E levels in milk and determining the excretion of malondialdehyde (MDA) in urine. Methods: Vitamin E was measured in the breast milk of 20 mothers who had given birth prematurely. Urinary MDA was measured in 10 exclusively breast milk fed and 10 exclusively formula fed healthy preterm infants receiving no vitamin supplements. MDA was measured after derivatisation with 2,4-dinitrophenylhydrazine and consecutive HPLC with UV detection. Results: Urinary MDA concentrations were consistently very low (0.074±0.033 μM/mM Cr and 0.078±0.026 μM/mM Cr in breast and formula fed infants respectively) and not significantly different between healthy breast milk and formula fed infants. Both breast and formula milk contained satisfactory levels (0.3–3.0 mg/100 ml) of vitamin E. Conclusion: Antioxidant properties of both breast milk and formulae are sufficient to prevent significant lipid peroxidation in healthy premature infants.

The degradation of Aβ25–35 by the serine protease plasmin is inhibited by aluminium

The catabolism of amyloid beta peptides (Abeta) may be important in their accumulation in the brain in both early and late-onset Alzheimers disease (AD). The serine protease plasmin is one of a suite of proteases implicated in AD. It is a promoter of alpha-cleavage of the amyloid beta precursor protein (AbetaPP) and will degrade Abeta in vitro. Herein we have demonstrated cleavage of the amyloidogenic Abeta(25-35) by plasmin to produce the non-amyloidogenic fragment Abeta(29-35). The activity of plasmin was halved by pre-mixing it with aluminium (Al) prior to its addition to the peptide. An interaction between Al and proteases involved in the catabolism of Abeta might define the putative link between Al and AD.

Action of Al-ATP on the isolated working rat heart

ATP is an important extracellular messenger in the coronary vasculature of the heart. To be effective its extracellular concentration must be tightly controlled and this is achieved via ectonucleotidases located in the luminal surface of the coronary endothelium. Al-ATP is a potent inhibitor of the hydrolysis of ATP and we speculated that Al-ATP released by cells into the blood would disrupt the signalling function of extracellular ATP. We tested this hypothesis by perfusing isolated working Wistar rat hearts with buffers containing either ATP or Al-ATP. The functional parameters measured were, coronary flow, heart rate and pulsatile power. A number of control perfusions including adenosine, ATP-gamma-S and Al were used to identify those effects which might be specific to ATP and Al-ATP. Al-ATP did not appear to inhibit the function of the endothelial ectonucleotidases. Both ATP and Al-ATP produced a significant increase in coronary flow and this could be attributed to a coronary vasodilation. Interestingly, whilst the effect of ATP was reversible that of Al-ATP was not. ATP caused a reduction in heart rate which was potentiated by aluminium. The negatively chronotropic effect of Al-ATP was mediated via a mechanism which was either distinct from or in addition to the similar response known to be caused by adenosine. We have demonstrated for the first time an influence of Al-ATP on heart function. Perhaps more pertinently we present the first evidence that Al-ATP may influence the function of ATP-specific receptors.

Chapter 22 – The Association of Aluminium and β Amyloid in Alzheimer’s Disease

The proteolytic cleavage of the amyloid precursor protein and the production of the neurotoxic and amyloidogenic beta amyloid peptide are believed to be critical events in the aetiology of Alzheimer’s disease. Aluminium is bound by beta amyloid and is found co-localised with beta amyloid in the Alzheimer’s disease brain. The possibility that this association between beta amyloid and aluminium might be involved in the aetiology of Alzheimer’s disease is discussed and a number of mechanisms whereby physiologically significant concentrations of aluminium could instigate or accelerate the formation of neurotoxic amyloid fibrils are explored.