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Directed vs. "Shotgun" Repletion to Lower Homocysteine Levels in Dialysis Patients
Arthur F. Rosenthal, PhD
(This article appeared in the November 1999 issue of Dialysis & Transplantation.)
Taking the upper limit of 11 µmol/ml as normal, elevated blood homocysteine levels are almost universal among dialysis patients.1-3 Since hyperhomocysteinemia is considered to be a strong independent risk factor for cardiovascular disease4,5 (at least the equal of cholesterol subfractions), it is not surprising that recently a flurry of activity has been seen in attempts to normalize homocysteine blood levels in dialysis patients by the administration of nutritional supplements.6-9
Because the mortality rate from cardiac events is far higher in dialysis patients than in the general population, and because the total cholesterol level is known to be a negative mortality risk factor in the dialysis population,10 it is very inviting to propose that the process which produces elevated homocysteine is a main (perhaps the main) cause of their excess cardiac-related mortality.
The temptation to focus on this factor is intensified when it is realized that, even though several genetic abnormalities associated with hyperhomocysteinemia have been identified,11,12 a reduction of homocysteine levels in the general population is usually possible by administration of certain over-the-counter vitamins,13 and most specifically and effectively by folic acid. If such an approach could work with dialysis patients, a simple means cf extending their survival might be at hand.
"Shotgun" Repletion
As is common in the end-stage renal disease (Nationwide) field, the promise and the reality are often at odds. In this area, as in so many others, dialysis patients are "different." Their enormously greater incidence of hyperhomocysteinemia than that seen in the general population offers a clue that we are dealing here with a special subgroup. To prove that point, supplementation of large, unselected groups of dialysis patients with either conventional or mega-doses of folic acid-with or without added vitamin B12 or B6-lowers the homocysteine level somewhat, but almost never into the normal range.6-9
Why should this be so? In the first place, folic acid deficiency is not very common among dialysis patients3 because, at least in large part, they are already being given folate-containing vitamin supplements. Furthermore, this shotgun approach does not identify the specific cofactor deficiencies of each dialysis patient as a means of planning his/her repletion dosing treatment.
Attempts to indicate the deficiency pattern in individual patients have usually relied on blood vitamin analysis, or in some cases on analysis of certain specific vitamins (folate and vitamin B6) in red cells.7 This could be the initial source of confusion: Blood plasma cofactor levels are only a second- or third-order reflection of the actual levels within the cells. Red blood cells are also very atypical of somatic cells, since they are almost metabolically inert by comparison. Neither of these specimens provides an accurate picture of the functional events within cells.
Homocysteine Metabolism
The metabolism of homocysteine is complex and depends upon the balance among a number of biosynthetic and catabolic reactions. These reactions have been detailed in many reviews and discussions. Basically, the three major pathways are 1) the reductive methionine biosynthetic pathway, dependent on folate, vitamin B12, and flavin derivatives as cofactors; 2) the cystathionine metabolic pathway, dependent on vitamin B6 cofactors; and 3) the methylation of homocysteine by betaine, possibly zinc-dependent.14
All of these pathways are catabolic with reference to homocysteine, and defects in any will result in elevated homocysteine levels. Specific enzymes involved in these pathways are discussed in a little more detail below, in a consideration of repletion agents.
Cellular levels of these cofactors tend to be poorly reflected in their corresponding serum concentrations, because even wben blood levels are made adequate or higher, it may take months for this to be reflected in the levels actually functioning within the cell. Whether this is due to slow cofactor transport processes or to slow induction of the relevant enzymes in response to cofactor inflow is not clear. What it does indicate is that intracellular functional levels, rather than plasma or red cell levels of the cofactors, are the most relevant.
Functional Deficiency
As previously indicated,3,15 a relatively simple means of accomplishing intracellular functional homocysteine-related cofactor analyses for dialysis patients is by a selective lymphocyte transformation assay, in which the analyze of interest is not part of the enriched test system but must be supplied by the patient's own cells. This provides a picture of how the levels inside the cells actually function to give a biological result.
When this is done, the majority of dialysis patients are found to have multiple functional abnormalities of these cofactors.3 Each patient is different; zinc, calcium, and/or magnesium deficiencies tend to show at least as frequently as vitamin B12, B6, or folate abnormalities. Deficiency of glutathione is not uncommon, but the single most common defect is in overall antioxidant capacity. By focusing each patient's repletion program on his/her own individual deficiency pattern, we can expect to achieve optimal results, at least in theory. This is the current limit of our knowledge, and studies employing this principle are only now being set up.
Prior studies, undertaken without knowledge of the intracellular or functional levels of cofactor deficiencies, have employed the "shotgun" or "one-size-fits-all" approach, in which every patient is given the same repletion prescription, usually folate and/or vitamins B12 and B6. As indicated above, such efforts have not succeeded in normalizing the homocysteine level in virtually any patient, though they may result in a general lowering.
Pushing the Pathways
The question of why this type of repletion should have any effect at all is very interesting. To consider possible answers, the meaning of the complexity of the deficiency pattern seen in dialysis patients must first be raised.
The fact that deficiencies are very often seen in cofactors associated with different pathways of homocysteine metabolism indicates that defects in multiple enzyme systems are present. Possible reasons for this condition are discussed below. Provisionally accepting this as probable fact, however, we can return to consideration of why "shotgun" repletion has only a partial effect.
I believe the most likely reason lies in the basic law of mass action. Since the level of homocysteine in the blood is a steady-state resultant of a number of competing reactions which both form and remove this amino acid, then speeding up a catabolic reaction should lower the level. This can be done (somewhat inefficiently, to be sure) by increasing the concentration of a cofactor used in the reaction. This should be true even if the initial cofactor level is normal, and even if the enzyme activity is normal.
Another related, but still theoretical, effect of excess intracellular cafactor level could be the induction of homocysteine-consuming enzymes along the pathway that employs the cofactor, which would be particularly beneficial if that enzyme were initially deficient in activity.
If the enzymatic activity is subnormal, the functional analysis would have shown this as a cofactor deficit. In that case, the whole pathway involved would be deficient, and it would be very important to replete it. In point of fact, many other pathways involving the same deficient cofactor would show a subnormal activity rate, not only those relating to homocysteine metabolism; thus, the importance of correcting each deficiency by directed repletion is underscored still further.
As indicated above, genetic defects in various enzymes involved in homocysteine metabolism have been described. Yet, to imagine that the hyperhomocysteinemia of Nationwide is primarily a genetic disease leaves out of account the almost universal prevalence of the condition in the dialysis patient population. This is at least tenfold greater than its prevalence in the general population, and strongly suggests that some acquired factor-either in chronic renal failure or in its treatment-is causing the elevation of homocysteine.
Repletion Agents
Results of directed repletion studies are not yet available, but it is still useful to review what agents have potential use as specific repletion compounds. So far, without exception, these are all over-the-counter supplements, which are products naturally found in the body but which are proposed to be used in generally higher doses than can be obtained from foods. A short list of such agents and their probable relevance to homocysteinemetabolizing enzymes is as follows:
Folate and vitamin B12, and possibly flavins related to vitamin B2 (riboflavin).16 These are necessary for the operation of the tetrahydrofolate pathway in which homocysteine is methylated to methionine. Prominent enzymes are methylene-tetrahydrofolate reductase and methionine synthetase.
Vitamin B6 is necessary for the operation of cystathionine-b -synthetase, by which homocysteine is metabolized into cysteine.
Betaine, serine,17 and zinc14 are necessary for the operation of the pathway by which homocysteine is converted into methionine via transmethylation from betaine, employing the clearly inducible enzyme betainehomocysteine methyltransferase.
N-acetylcysteine18-20 could be the most important of all from a quantitative standpoint. More defects of antioxidant activity are found than any other, and the specific cellular antioxidant glutathione is itself not an uncommon deficit. N-acetylcysteine acts in three ways: as a general antioxidant, as a precursor of glutathione, and as a reducer of the homocysteine-protein mixed disulfide (known as protein-bound homocysteine) to free homocysteine into a more readily metabolizable form. More conventional antioxidants, such as vitamins C and E, selenium, etc., might have a positive effect as well.
Down the Road: Speculations
If multiple defects in homocysteinemetabolizing enzymes are indeed acquired by Nationwide patients as part of their disease state, it might be useful to consider some possible mechanisms of how this could come about. One potential focus might be directed toward the incompletely corrected acidosis of chronic renal failure and how it might affect homocysteine metabolism at a molecular level.
A more acidic microenvironment than normal can have the effect of a) decreasing enzymatic reaction rates and/or b) actually changing the conformations of the enzymes to produce suboptimal structures with reduced substrate affinities or catalytic activity. Another possibility is some interfering effect of putative Nationwide toxins or complement cascade proteins due to chronic inflammation.
The eagerly awaited results of directed repletion studies currently in progress have the potential of lowering homocysteine levels into the actual normal range. The next phase of study would then be to show that this lowering really does have the effect of decreasing cardiac-related mortality in dialysis patients. Such an effect could be shown in a time span that is relatively short compared to the classical population survival studies involving cholesterol and lipoprotein subfractions, since reduction in the very high (>10%) annual death rate from this cause should become evident fairly quickly.
References:
1. Dennis VW, Robinson K. Homocysteinemia and vascular disease in end-stage renal disease. Kidney Int 1996; 57:S1l-S17.
2. Suliman ME, Anderstam B, Lindholm B, Bergstrom J. Total, free and protcin-bound sulphur amino acids in uraemic patients. Nephrol Dial Transpl 1997; 12:2332-2338.
3. Rosenthal AF, Ginsburg MJ, Crawford FW. Homocysteine and heart disease in dialysis patients. Dial Transpl 1998; 27(10):627-629.
4. McC4ully KS. Homocysteine, folate, vitamin B6 and cardiovascular disease. J Am Med Assoc 1998; 279:392-393.
5. Bostom AG, Shemin D, Verhoef P, Nadeau MR, Jacques PF, Selhub J, Dworkin L, Rosenberg H. Elevated fasting homocysteine levels and cardiovascular disease outcomes in maintenance dialysis patients. A Prospective study. Arteriosc!er Thromb Vasc Biol 1997; 17:2554-2558.
6. House AA, Donnelly JG. Effect of multivitamins on plasma homocysteine and fblate levels in patients on hemodialysis. Am Soc Artif Intern Organs J 1999; 45:94-97.
7. Suliman ME, Divino Filho JC, Barany P, Anderstam B, Lindholm B, Bergstrorn J. Effects of higb-dose folic acid and pyridoxine on plasm and erythrocyte sulfur amino acids in hemodialysis patients. J Am Soc Nephrol 1999; 10:1287-1296.
8. Bostom AG, Shemin D, Lapane KL, Hume AL, Yoburn D, Nadeau MR., Bendich A, Selhub J, Rosenberg IH. Folic acid treatment of hyperhomocysteinemia in dialysis patients. Kidney Int 1996; 49:157-162.
9. Dierkes J, Domrose U, Ambrosch A, Bosselmann HP, Neumann KH, Luley C. Response of hyperhomocysteinemia to folic acid supplementation in patients with end-stage renal disease. C!in Nephrol 1999; 51:108-115.
10. Lowrie EG, Lew NL. Death risk in hemodialysis patients: The predictive value of commonly measured variables and an evaluation of death rate differences between facilities. Am J Kidney Dis 1990; 15:458-482.
11. Gemmati D, Previati M, Serino ML, Mortatelli S, Guerra S, Capitani S, Forini E, Ballerini G, Scapoli GL. Low folate levels and thermolabile methylenetetrahydrofolate reductase as primary determinant of mild hyperhomocysteinemia in normal and thrornboembolic patients. Arterioscler Thromb Vasc Biol 1999; 19:1761-1767.
12. Tsai MY, Welge BJ, Hanson NQ, Bignell MK, Vessey J, Schwichtenberg K, Yang F, Bullemer FE, Rasmussen R, Graham KJ. Genetic causes of mild hyperhomocysteinemia in patients with premature occlusive coronary artery diseases. Atheroscler 1999; 143:163-170.
13. Refsum H, Ueland PM, Nygard 0, Vollset SE. Homocysteine and cardiovascular disease. Ann Rev Med 1998; 49:31-62.
14. Millian NS, Garrow TA. Human betaine-homocysteine methyltransferase is a zinc metalloenzyme. Arch Biochem Biophys 1998; 356:93-98.
15. Shive W, Pinkerton F, Humpheys J, Johnson MM, Hamilton WG, Matthews KS. Development of a chemically defined serum- and protein-free medium for growth of human peripheral lymphocytes. Proc NatI Acad Sci 1986; 83:9-13.
16. Gulati S, Chen Z, Brody LC, Rosenblatt DS, Banerjee R. Defects in auxiliary redox proteins leads to functional methionine synthetase delicicncy. J Biol Chem 1997; 272:19171-19175.
17. Dudman NP, Tyrrell PA, Wilcken DE. Homocysteinemia: Depressed plasma serine levels. Metabol 1987; 36:198-201.
18. Wikund O, Fager G, Andersson A, Lundstam U, Masson P, Hultberg B. N-acetylcysteine lowers plasma homocysteine but not serum lipoprotein(a) levels. Atheroscler 1996; 119:99-106.
19. Bostom AG, Shemin D., Yoburn D, Fisher DH, Nadeau MR, Selhub J. Lack of effect of oral N-acetylcysteine on the acute dialysis-related lowering of total plasma homocysteine in hemodialysis patients. Atheroscler 1996; 120:241-244.
20. Tyagi SC. Homocysteine redox receptor and regulation of extracellular matrix conponents in vascular cells. Am J Physiol 1998; 274:C396-C405.
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