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Rapid Responses: Rapid Responses published:
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MTHFR C677T not valuable thrombophilia test
- Joel G Ray (22 November 2002)
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Diet-induced Folate Deficiency
- Bill D. Misner (23 November 2002)
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Another cardiovascular effect of homocysteine
- Serdar KULA (23 November 2002)
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Minimising the risk of deep venous thrombosis through genetic screening
- Maritha J Kotze, Stephen Toovey (26 November 2002)
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Homocysteine causes mitochondrial dysfunction
- Richard G Fiddian-Green, Formerly Professor and Chair General Surgery Univeristy of Massachusetts. (3 December 2002)
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Observational data do not prove homocysteine is causal in cardiovascular disease
- Sagar N Doshi, Stuart J. Moat, Anil K. Madhavan, Derek Lang, Malcolm J. Lewis, Jonathan Goodfellow. (6 December 2002)
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Meta-analysis of homocyst(e)ine in CVD
- Dan G. Hackam (15 December 2002)
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Do port and red wine preserve ATP stores?
- Richard G Fiddian-Green (16 December 2002)
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Homocysteine, ATP degradation and free radical release.
- Richard G Fiddian-Green (18 December 2002)
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Homocysteine, folic acid, uric acid, ATP and free radical scavenging
- Richard G Fiddian-Green (19 December 2002)
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Exercise and plasma homocysteine
- Harpal S Randeva, Vivian Fonseca, Professor, Medicine and Pharmacol, Tulane University, USA; Gordana M Prelevic, Senior Lecturer, Reproductive Endocrinology, Royal Free&UCL, UK (20 December 2002)
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Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis
- Miranda B. Keijzer, Martin den Heijer (20 December 2002)
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Testing travellers for MTHFR TT is absurd
- Joel G Ray (22 December 2002)
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Pre-travel screening to identify genetic factors predisposing to thrombosis
- Maritha J Kotze (27 December 2002)
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Re: pre-travel screening for thromboembolism
- Richard G Fiddian-Green (30 December 2002)
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Re: Pre-travel screening may be harmful
- Joel G Ray (30 December 2002)
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"Lactic acidosis": the common denominator?
- Richard G Fiddian-Green (3 January 2003)
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Pre-test assessment to determine appropriateness of pre-travel genetic screening for thrombosis risk
- Maritha J Kotze (4 January 2003)
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Role of renal impairment.
- Dan Hackam (5 January 2003)
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Role of renal impairment.
- Dan Hackam (7 January 2003)
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Homocysteine: an adenine nucleotide trap?
- Richard G Fiddian-Green (9 January 2003)
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Biochemical definitions
- Richard G Fiddian-Green (10 January 2003)
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pH/calcium regulation of homocysteine metabolism
- Richard G Fiddian-Green (10 January 2003)
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MTHFR and meta-Analysis
- Markus Weih (16 January 2003)
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MTHFR gene mutation, methylmalonic acidosis, and exercise.
- Richard G Fiddian-Green (19 January 2003)
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The MTHFR poymorphism and screening for deep vein thrombosis
- David S Wald, Malcolm Law, Joan Morris (31 January 2003)
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Re: Observational data do not prove homocysteine is causal in cardiovascular disease
- David S Wald, Malcolm Law, Joan Morris (31 January 2003)
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Re: Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis
- David S Wald, Malcolm Law, Joan Morris (31 January 2003)
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Genetic equilibrium suggests no link between MTHFR and ischaemic heart disease.
- Gavin Willis, Rebecca Walker, Barbara A. Jennings (7 April 2003)
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Joel G Ray, Consultant and researcher Sunnybrook and Women's College Health Sciences Centre, Toronto, M6P 2N6
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Dear Editor: I cannot agree with the conclusion drawn by David Wald and colleagues that the association between the C677T polymorphism of methylenetetrahydrofolate reductase (MTHFR) and deep vein thrombosis is "highly significant" <1>. Although it may lead to higher concentrations of plasma homocysteine, the MTHFR 677TT polymorphism is, at most, a weak risk factor for venous thromboembolism (VTE) <1>. In a previously published meta-analysis of 31 studies, we evaluated the risk for VTE in the presence of the MTHFR 677TT genotype (compared to either the CC or CT genotype), and included 4,901 cases and 7,886 controls <2>. With greater statistical confidence than in the review by Wald et al <1>, we observed a very weak association between the MTHFR 677TT state and the risk of VTE (pooled odds ratio [OR] 1.2, 95% CI 1.1- 1.4) <2>. This risk estimate remained low after excluding cases with other classic thrombophilia factors, including a deficiency of either protein C, protein S or antithrombin (pooled OR 1.5, 95% CI 1.2-1.9). Although mild hyperhomocysteinemia may be a modifiable risk factor for VTE <3>, testing affected patients for the MTHFR C677T polymorphism would contribute little or no information to their clinical care. Testing for the MTHFR C677T polymorphism should be a restricted part of any thrombophiilia work-up. References <1> Wald DS, Law M, Morris JK. Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis. BMJ 2002; 325: 1202- 6. <2> Ray JG, Shmorgun D, Chan WS. Common C677T polymorphism of the methylenetetrahydrofolate reductase gene and the risk of venous thromboembolism: meta-analysis of 31 studies. Pathophysiol Haemost Thromb 2002; 32: 51-8. <3> Ray JG. Meta-analysis of hyperhomocysteinemia as a risk factor for venous thromboembolic disease. Arch Intern Med 1998; 158: 2101- 6. Competing interests: None declared |
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Bill D. Misner, Director R & D E-CAPS Inc. Spokane, Wa. USA 99205
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It would appear that an the association between the C677T polymorphism of methylenetetrahydrofolate reductase (MTHFR) and deep vein thrombosis may present a predisposition to Homocysteine-related cardiovascular disease. Grant emphasized dietary links to ischemic heart disease (IHD) and coronary heart disease (CHD) mortality from a correlation from for various age groups aged 35+. His paper presented a multi-country statistical approach involving 32 countries suggesting dietary links to IHD and CHD. For IHD, milk carbohydrates were found to have the highest statistical association for males aged 35+ and females aged 65+, while for females aged 35-64, sugar was found to have the highest association. In the case of CHD, non-fat milk was found to have the highest association for males aged 45+ and females aged 75+, while for females 65-74, milk carbohydrates and sugar had the highest associations, and for females aged 45-64, sugar had the highest association. A number of mechanisms proposed in the literature to explain the milk carbohydrate or non-fat milk association. One of the most prominent theories is that animal proteins contribute to homocysteine (Hcy) production; however, milk more than meat lacks adequate B vitamins to convert Hcy to useful products. Lactose and calcium in conjunction with Hcy from consumption of non-fat milk may also contribute to calcification of the arteries [1]. Homocysteine Hcy plays an important role in the etiology of heart disease through its role in the development of atherosclerosis. Hcy is derived from the amino acid methionine, more common in animal proteins than in vegetable proteins, and can be converted back to methionine with the help of folic acid and vitamin B12. It can also be eliminated from the body through the action of vitamin B6. Those who have elevated levels of Hcy are generally found to be deficient in the B vitamins [2, 3, 4], which can be overcome by vitamin supplementation [5, 6, 7, 8]. When dietary vitamin deficiency is factored along side of genetic predisposition, the significance may hypothetically far exceed the presented of "highly significant [9]" value. Preventative medicine supports folate supplementation for reducing the dietary and genetic influences on cardiovascular disease. References [1]-Milk and Other Dietary Influences on Coronary Heart Disease, W. B. Grant, Ph.D. In Altern Med Rev 1998;3(4):281-294. [2] Ubbink JB, Vermaak WJH, van der Merwe A, Becker PJ. Vitamin B-12, vitamin B-6, and folate nutritional status in men with hyperhomocysteinemia. Am J Clin Nutr 1993;57:47-53. [3] McCully KS. Homocysteine, folate, vitamin B6, and cardiovascular disease. JAMA 1998;279:392-393. [4] Rimm EB, Willett WC, Hu FB, et al. Folate and vitamin B6 from diet and supplements in relation to risk of coronary heart disease among women. JAMA 1998;279:359-364. [5] Barber GW, Spaeth GL. The successful treatment of homocystinuria with pyridoxine. J Pediatrics 1969;463:463-478. [6] Selhub J, Jacques PF, Wilson PWF, et al. Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. JAMA 1993;270:2693-2698. [7] Ubbink JB, Vermaak WJH, van der Merwe A, et al. Vitamin requirements for the treatment of hyperhomocysteinemia in humans. J Nutr 1994;124:1927-1933. [8] Ubbink JB, Becker PJ, Vermaak WJH, Delport R. Results of B- vitamin supplementation study used in a prediction model to define a reference range for plasma homocysteine. Clin Chem 1995;41:1033-1037. [9] Wald DS, Law M, Morris JK. Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis. BMJ 2002; 325: 1202- 6. Bill Misner Ph.D. C.S.M.T. Director R & D E-CAPS Inc. Competing interests: The author declares competing interests in exogenous supplements industry. |
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Serdar KULA, Pediatric Cardiologist Gazi University Medical School, Besevler,Ankara, TURKEY
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Dear Sir, As writen in article of Wald et al, homocysteine and thromboembolic events is well known couple. However, homocysteine has another importance in causes of cardiovascular disease. I want to touch on this side of homocystein effect. The association between folic acid deficiency and congenital malformation is especially well-known: both animal experimentation and epidemiologic studies have shown that a deficiency of folic acid may be associated with defects of neural tube closure, as weel as defects of aorticopulmonary (or conotruncal) septation(1). Furthermore folate supplementation during the pregnancy protects against heart and neural tube defects is well known. There are many paper on maternal hyperhomocysteinemia effected fetal heart (1,2,3). Kapusta et al found that women with hyperhomocysteinemia were at increased risk for bearing children with all forms of congenital heart diseases (2).Interestingly, in this study congenital heart anomalies were not limited to conotruncal (out-flow) septum as seen in folate depletion. Besides, in the study of Wenstrom et al, isolated congenital heart defects were found to be associated with either the C677T MTHFR mutation or elevated amniotic fluid homocysteine levels, or both(3). In conclusion, homocysteine is not only cause of thromboembolic events, besides one of the important cause of congenital heart defects. REFERENCES: 1. Rosenquist TH, Ratashak SA, Selhub J. Homocysteine induces congenital defects of the heart and neural tube: effect of folic acid.Proc Natl Acad Sci U S A. 1996;93(26):15227-32. 2. Kapusta L, Haagmans ML, Steegers EA, Cuypers MH, Blom HJ, Eskes TK. Congenital heart defects and maternal derangement of homocysteine metabolism. J Pediatr. 1999;135(6):773-4. 3. Wenstrom KD, Johanning GL, Owen J, Johnston KE, Acton S, Cliver S, Tamura T. Amniotic fluid homocysteine levels, 5,10- methylenetetrahydrafolate reductase genotypes, and neural tube closure sites. Am J Med Genet. 2000;90(1):6-11. Competing interests: None declared |
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Maritha J Kotze, Managing director PO Box 15734, Vlaeberg, South Africa, Stephen Toovey
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The debate around the significance of screening for mutations of the methylenetetrahydrofolate reductase (MTHFR) gene as a risk factor for deep vein thrombosis (DVT) is timely. We feel however that some important points have been overlooked in this debate, which appears to have centred on the utility of testing in confirmed DVT cases only. Testing for mutation 677C-T in the MTHFR gene forms part of our strategy to prevent DVT in long haul travellers. From October 2002, a pre- travel genetic screening service for DVT – popularly known as “Economy Class Syndrome” – has been offered through Netcare Travel Clinics in South Africa.(1) The intention is to identify genetically at risk individuals before they suffer a potentially fatal thrombosis. In the paper by Wald et al convincing evidence is provided that hyperhomocysteinaemia is an important risk factor for DVT, with mutation 677C-T in the MTHFR gene being associated with an elevation in serum homocysteine concentrations of approximately 20%.(2) The response by Ray asserts that the testing of patients with venous thromboembolism for mutation MTHFR 677C-T would not have a significant impact on clinical care.(3) It is here that we need to avoid confusion and must differentiate between individuals already diagnosed with a DVT, and those wishing to avoid one. Thus, while Ray’s argument may be applicable to the clinical management of patients with DVT, individuals possessing the implicated mutation would be denied the opportunity to adopt simple preventive measures if Ray’s argument were to be extended and screening denied them. We have a question too on the importance of providing genetic information to DVT victims, with a possible view to allowing first degree relatives to avail themselves of screening: this might be a valid indication for testing DVT victims for the MTHFR mutation. Furthermore, individuals with a genetic predisposition to DVT as a result of the presence of Factor V Leiden or prothrombin mutations would be at even greater risk if they were to also possess the MTHFR mutation. These individuals should also benefit from screening. In South Africa a population-based screening approach is used for risk assessment, since a heterogeneous distribution of MTHFR genotypes was found among different ethnic groups in South Africa.(4) Treatment tailored according to the specific genetic profile identified by screening would minimise the risk of DVT and the potentially lethal complications of this condition. References 1. http://www.genecare.co.za 2. Wald DS, Law M, Morris JK. Homocysteine and cardiovascular disease; evidence on causality from a meta-analysis. Br Med J 2002; 325: 1 -7. 3. Ray JG. MTHFR C677T not valuable thrombophilia test. Br Med J 2002; online. 4. Scholtz CL, Odendaal HJ, Thiart R, Loubser L, Hillermann R, Delport R, Vermaak WJH, Kotze MJ. Analysis of two mutations in the MTHFR gene associated with mild hyperhomocysteinaemia: Heterogeneous distribution across ethnic groups. S Afr Med J 2002; 92: 464-7. Competing interests: MK is a shareholder in Genecare Molecular Genetics and derives income from providing genetic testing. ST - none declared |
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Richard G Fiddian-Green, None None, Formerly Professor and Chair General Surgery Univeristy of Massachusetts.
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It has been proposed that an impairment of mitochondrial oxidative phosphorylation might be the cause of atherosclerosis, thromboembolism, hypertension and other chronic diseases of the aged including neurodegenerative diseases and cancers (1,2,3). A meta-analysis has, however, provided evidence of causality between elevated levels of homocysteine and ischaemic heart disease, deep vein thrombosis and stroke (4). Homocysteine, whose elevated levels may be restored towards normality by folate supplements, has also been implicated in the pathogenesis of Parkinson’s and Alzheimer’s diseases (5,6). Elevated levels of homocysteine have also been implicated in the pathogenesis of cancers. The two hypotheses can be reconciled because homocysteine appears to exert its actions by altering mitochondrial gene expression, function and structure (7-11). Homocysteine may cause mitochondrial dysfunction and even apoptosis. The homocysteine thiolactonyl derivative, thioretinaco ozonide, is believed to function as an electron acceptor in oxygen metabolism and as the binding site for adenosine triphosphate (ATP) synthesis by mitochondria, preventing damage by free radical oxidants in resting cells. During cell division, methionine is converted to homocysteine thiolactone, converting thioretinaco to thioco, increasing free radical oxidants, and oxidizing cellular glutathione and ascorbate. During cell division, methionine is converted to homocysteine thiolactone, converting thioretinaco to thioco, increasing free radical oxidants, and oxidizing cellular glutathione and ascorbate. The free base of homocysteine thiolactone produces keratinization, squamous metaplasia, dysplasia, and carcinogenesis in normal mouse tissues. The efficiency of homocysteine thiolactone metabolism declines with aging, explaining decreased formation of adenosyl methionine in aging and suggesting loss of thioretinaco ozonide from membranes of aging cells. The effects of aging on enzyme activity, connective tissues, lipid synthesis, auto-immune diseases, atherogenesis and carcinogenesis are related to these changes in homocysteine metabolism. Homocysteine acts with synergistically with H2O2 to exert some of its noxious effects (7) and may modulate the cytotoxic effects of tumor necrosis factor (TNF) (12,13,14). Cytokines such as TNF alpha may exert their cytotoxic effects by opening the permeability transition pore on the miotochondrial membrane thus uncoupling oxidative phosphorylation by dissipating the protonmotive force upon which ATP resynthesis depends. The beneficial effects of folic acid supplements in patients whose homocysteine levels are elevated can, therefore, be expected to be limited in those patients who have other causes of an impairment of oxidative phosphorylation. 1. Hypertension: product of mitochondrial dysfunction? Richard G Fiddian-Green bmj.com/cgi/eletters/325/7370/917#26634, 31 Oct 2002 2. Unreversed ATP hydrolysis: the initiating endothelial event? Richard G Fiddian-Green bmj.com/cgi/eletters/325/7369/887#26445, 22 Oct 2002 3. Iatrogenic diseases with a common cause? Richard G Fiddian-Green bmj.com/cgi/eletters/325/7370/913#26512, 25 Oct 2002 4. Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis David S Wald, Malcolm Law, and Joan K Morris BMJ 2002; 325: 1202-1206. 5. Duan W, Ladenheim B, Cutler RG, Kruman II, Cadet JL, Mattson MP. Dietary folate deficiency and elevated homocysteine levels endanger dopaminergic neurons in models of Parkinson's disease. J Neurochem. 2002 Jan;80(1):101-10 6. Kruman II, Culmsee C, Chan SL, Kruman Y, Guo Z, Penix L, Mattson MP. Homocysteine elicits a DNA damage response in neurons that promotes apoptosis and hypersensitivity to excitotoxicity. J Neurosci. 2000 Sep 15;20(18):6920-6. 7. Austin RC, Sood SK, Dorward AM, Singh G, Shaughnessy SG, Pamidi S, Outinen PA, Weitz JI. Homocysteine-dependent alterations in mitochondrial gene expression, function and structure. Homocysteine and H2O2 act synergistically to enhance mitochondrial damage. J Biol Chem. 1998 Nov 13;273(46):30808-17 8. Mercie P, Garnier O, Lascoste L, Renard M, Closse C, Durrieu F, Marit G, Boisseau RM, Belloc F. Homocysteine-thiolactone induces caspase- independent vascular endothelial cell death with apoptotic features. Apoptosis. 2000 Nov;5(5):403-11. 9. Olszewski AJ, McCully KS. Homocysteine metabolism and the oxidative modification of proteins and lipids. Free Radic Biol Med. 1993 Jun;14(6):683-93. Review. 10. McCully KS. Chemical pathology of homocysteine. III. Cellular function and aging. Ann Clin Lab Sci. 1994 Mar-Apr;24(2):134-52. Review 11. McCully KS. Chemical pathology of homocysteine. II. Carcinogenesis and homocysteine thiolactone metabolism. Ann Clin Lab Sci. 1994 Jan-Feb;24(1):27-59. Review. 12. .Ratter F, Gassner C, Shatrov V, Lehmann V. Modulation of tumor necrosis factor-alpha-mediated cytotoxicity by changes of the cellular methylation state: mechanism and in vivo relevance. Int Immunol. 1999 Apr;11(4):519-27 13. Lassus P, Opitz-Araya X, Lazebnik Y. Requirement for caspase-2 in stress-induced apoptosis before mitochondrial permeabilization. Science. 2002 Aug 23;297(5585):1352-4. 14. Reid RA, Moyle J, Mitchell P. Synthesis of adenosine triphosphate by a protonmotive force in rat liver mitochondria. Nature. 1966 Oct 15;212(59):257-8 Competing interests: None declared |
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Sagar N Doshi, Senior SpR in Cardiology The Queen Elizabeth University Hospital, Birmingham, B15 2TH, Stuart J. Moat, Anil K. Madhavan, Derek Lang, Malcolm J. Lewis, Jonathan Goodfellow.
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Editor- The meta-analysis reported by Wald et al on homocysteine and cardiovascular disease purports to show strong evidence that the association between homocysteine and cardiovascular disease is causal (1). However, analysis of observational data can never allow conclusions to be drawn on causality. The association between homocysteine and cardiovascular disease may alternatively be explained if homocysteine were related to another causal agent (confounding). For example, neopterin, a marker of immune system activation and directly pro-oxidative, is associated with atherosclerosis and has also been shown to correlate with homocysteine levels. It is plausible that the apparent association between homocysteine and atherosclerosis could be due to its linkage with neopterin or indeed to some, as yet, unknown factor. Indeed the pathological significance of the very modest plasma levels of homocysteine found in the general population with atherosclerosis is questionable. Patients with homocystinuria, an inborn error of metabolism, have total homocysteine levels >100mmol/l. Treatment of homocystinuria with folic acid and Vitamin B6 lowers homocysteine and reduces cardiovascular events to levels that may be expected in the healthy population. This reduction in events occurs despite the fact that homocysteine levels remain considerably higher than the ‘upper limit’ of normal in the general population (15mmol/l), suggesting that folate may have beneficial effects other than homocysteine lowering (2;3). Furthermore, the low plasma levels of free reduced homocysteine (the presumed pathological moiety) found in patients with cardiovascular disease have never been found to induce endothelial injury, an early phase of atherosclerosis, in experimental models. In their discussion on the evidence of risk reduction the authors cite a paper by Schnyder et al (4) and state that this randomised placebo controlled trial of treatment with B vitamins (folate, B-6 and B-12) to lower homocysteine in patients with ischaemic heart disease had shown a ‘rapid reduction in risk’. This is misleading in the context of their argument as this study demonstrated that folate appears to reduce restenosis following coronary angioplasty. Restenosis is an entirely separate pathological entity (neointimal smooth muscle proliferation) to atherosclerosis and is not related to the usual factors associated with atherosclerosis. Finally, one of the major randomised folate studies addressing homocysteine reduction in cardiovascular disease has just reported (CHAOS 2; 1882 patients) and found no significant reduction in cardiovascular endpoints after 2 years active treatment despite reducing homocysteine (5). This lack of effect of homocysteine lowering strongly suggests that homocysteine is not causal in cardiovascular disease. References 1. Wald DS, Law M, Morris JK. Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis. BMJ 2002;325(7374):1202. 2. Doshi SN, Moat SJ, McDowell IF, Lewis MJ, Goodfellow J. Lowering plasma homocysteine with folic acid in cardiovascular disease: what will the trials tell us? Atherosclerosis 2002;165(1):1-3. 3. Yap S, Naughten ER, Wilcken B, Wilcken DE, Boers GH. Vascular complications of severe hyperhomocysteinemia in patients with homocystinuria due to cystathionine beta-synthase deficiency: effects of homocysteine-lowering therapy. Semin.Thromb.Hemost. 2000;26(3):335-40. 4. Schnyder G, Roffi M, Pin R, Flammer Y, Lange H, Eberli FR et al. Decreased rate of coronary restenosis after lowering of plasma homocysteine levels. N.Engl.J.Med. 2001;345(22):1593-600. 5. Baker F, Picton D, Blackwwod S, Hunt J, Erskine M, Dyas M et al. Blinded comparison of folic acid and placebo in patients with ischaemic heart disease: an outcome trial. Circulation. 2002;106(22):Supplement 1- 3642. Competing interests: None declared |
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Dan G. Hackam, Internal Medicine McMaster University, Hamilton, Canada
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Wald and colleagues have contributed to the burgeoning body of literature on the links between hyper-homocyst(e)inemia and vascular disease. As mentioned by a previous author, this toxic amino acid has also been implicated in the pathogenesis of dementia & parkinson's disease, stroke, cancer, other non-vascular infirmities in the elderly (recent Am J Med Nov 2002 study), and venous thromboembolism. Although I agree with the previous author that observational data can never prove causality 100%, would he, on similar grounds, reject the observational evidence that smoking causes lung cancer, asbestos exposure causes mesotheliomas, and radioactive gamma emissions lead to leukemia? Obviously, an RCT in any of these areas is going to be exceedingly unethical, for human beings are not guinea pigs to be randomized to highly likely harmful measures or placebo. In the Swiss Heart study, treatment for 6 months with vitamins not only prevented post-PCI restenosis, as the previous author admits, but also prevented other coronary events such as non-fatal myocardial infarction. The one year follow-up of this 6 month trial, recently reported in the JAMA, confirmed the sustained, beneficial effects of this simple intervention. Another RCT reported in the Lancet last year showed that reduction of homocysteine with a B-vitamin complex reduced atherosclerotic burden, as measured by exercise-induced positive ECG stress tests and other objective measures, in relatives of patients with premature atherosclerosis. I screen every patient in a cardiovascular context for homocysteine level, along with the standard screen of fasting lipids and blood sugar. Until the definitive trial data is in (and there are at least 11 large, multicentre trials studying homocyst(e)ine reduction), I would prefer to later be proved wrong, than miss the boat in employing this very safe therapy for high risk vasculopathic patients with hyper-homocyst(e)inemia (Folic Acid/B6/B12). Competing interests: None declared |
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Richard G Fiddian-Green, None None
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People who drink lots of port are said to be more likely to develop gout and those who do develop gout are said to be more likely to live longer. This makes biochemical sense if most acute and chronic diseases are the product of an impairment of mitochondrial oxidative phosphorylation as has been proposed (1). The law of mass action dictates that the failure to excrete uric acid, the accumulation of which causes the symtomatic lesions in gout, should impair the rate of depletion of ATP stores in anaerobiosis. The depletion of ATP stores in anaerobiosis normally occurs by degradation into AMP, then into adenosine which is able to diffuse out of cells, and then into hypoxanthine, xanthine and ultimately into uric acid which is excreted in urine. Might, therefore, drinking port and red wine, which is said to reduce the risk of coronary artery disease, exert their beneficial effects by preserving ATP stores? 1. Fiddian-Green RG. Homocysteine causes mitochondrial dysfunction bmj.com, 2 Dec 2002 . Competing interests: None declared |
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Richard G Fiddian-Green, None None
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Homocysteine exacerbates oxidative stress, mitochondrial dysfunction and apoptosis in dopaminergic cells (1) Uric acid ameliorates these adverse effects by, it is thought, acting as an antioxidant. It might alternatively or in addition ameliorate these adverse effects by preserving ATP stores as proposed for port and red wine (2). Under normal circumstances the generation of uric acid from the degradation of ATP is catalysed by xanthine dehydrogenase. In hypoxia and in the presence of proinflammatory mediators xanthine oxidase (XO) is generated by the “posttranslational modification of xanthine dehydrogenase (XD), either through the reversible, incremental thiol oxidation of sulfhydryl residues on XD or the irreversible proteolytic cleavage of a segment of XD” (3). Xanthine oxidase increases the rate at which ATP is degraded into uric acid and in so doing generates the free radicals implicated in reoxygenation/reperfusion injury. Allopurinol inhibits the degradation of ATP into uric acid under normal circumstances by being converted into oxypurinol. The degradation catalysed by xanthine oxidase during hypoxia or the release of proinflammatory mediators is inhibited by allopurinol rather than its derivative oxypurinol. Hence the therapeutic benefits of allopurinol on uric acid concentrations in gout and in the prevention of reoxygenation/reperfusion injury. Free radicals are said to be generated by all oxidases in the body including cytochrome oxidase and xanthine oxidase but it is those generated by xanthine oxidase in hypoxia and the presence of proinflammatory mediators that appear to be primarily responsible for those free radicals believed to be responsible for reoxygenation/reperfusion injury. Stochiometric examination, however, reveals that the amount of free radicals generated by xanthine oxidase must be proportional to the degree of the accompanying depletion of the ATP pool. Guyton suggests that the depletion of ATP pools might be a particularly important cause of cellular dysfunction and observes that depletion of ATP pools is serious because the ATP pools can only be restored at a slow rate even the most optimal of circumstances (4). This raises the possibility that the depletion of ATP pools might be as or even more important than the generation of free radicals in the induction of reoxygenation/reperfusion injury. Strategies that might be considered for preventing or ameliorating reoxygenation/reperfusion injury might, therefore, include the administration of a protease inhibitor to inhibit the conversion of xanthine dehydrogenase into xanthine oxidase, allopurinol and possibly the administration of exogenous uric acid to prevent the degradation of ATP, adenosine and phosphates to aid in the replenishment of depleted ATP pools, free radical scavengers and folate especially if homocysteine levels are elevated. 1. Duan W, Ladenheim B, Cutler RG, Kruman II, Cadet JL, Mattson MP. Dietary folate deficiency and elevated homocysteine levels endanger dopaminergic neurons in models of Parkinson's disease. J Neurochem. 2002 Jan;80(1):101-10. 2. Fiddian-Green RG. Do port and red wine preserve ATP stores? bmj.com/cgi/eletters/325/7374/1202#27887, 16 Dec 2002 3. Meneshian A, Bulkley GB. The physiology of endothelial xanthine oxidase: from urate catabolism to reperfusion injury to inflammatory signal transduction. Microcirculation. 2002 Jul;9(3):161-75. Review. 4. Guyton and Hall. Textbook of medical physiology. 10th edition. WB Saunders Philadelphia. 2000 pp 261-262. Competing interests: None declared |
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Richard G Fiddian-Green, None None
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Folic acid is a potent inhibitor of xanthine oxidase (1) and is said to be a “free radical scavenger” (2) in addition to being necessary for the enzymatic catalysation of the disposal of homocysteine. Thus the beneficial effect of folic acid on outcome reported in this study might have been due to the preservation of ATP stores, to the inhibition of free radical generation, and/or to the scavenging of free radicals (3) rather than to the reduction in the concentrations of homocysteine in serum (4). The professional and especially the lay press is full of reports of the allegedly beneficial effects of antioxidants including polyphenols in red wine. Despite these enthusiastic reports the evidence suggesting that any benefical effect is due to the scavenging of free radicals generated in anaerobiosis rather than to the preservation of ATP by preventing loss of uric acid in urine is highly questionable (3). Consider further the effects of ethanol. Ethanol increases the uric acid concentrations, supposedly another free radical scavenger (5). The increasing levels of uric acid are accompanied by a decrease in 8-OHdG levels, a marker of DNA damage. This evidence is consistent with that from patients with acute ischemic strokes in whom there is a 12% increase in the odds of good clinical outcome for each milligram per deciliter increase of serum uric acid (6). Whilst these data are consistent with the view that beneficial effects of red wine might be due to free radical scavenging of with polyphenols or uric acid, the law of mass action dictates that any increase in uric acid concentration in anerobiosis must be accompanied by a decrease in the rate of ATP degradation into uric acid being catalysed by the conversion of xanthine dehydrogenase to xanthine oxidase. This should be especially so if the increase in uric acid is due to an impairment of renal excretion. Indeed familial gout, which may be due to an impairment of uric acid secretion, has lower serum triglyceride, total cholesterol, and percentage of hypertension than nonfamilial gout controls (7). The recent identification of the F2-isoprostanes as oxidative products of arachidonic acid has provided a reliable measure of in vivo lipid peroxidation, or free radical damage (8). As no reduction in lipid peroxidation occurs following red or white wine consumption any protective effects of wine drinking on cardiovascular disease would seem unlikely to be related to inhibition of lipid oxidation by the scavenging of free radicals. Antioxidants reduce the incidence of organ failure and shorten ICU length of stay in critically ill surgical patients (9). The development of organ dysfunction and extended ICU stay appears, however, to be accompanied by an impairment ATP resynthesis by oxidative phosphorylation and can be prevented by preventing its development (10). Given the very large number of compounds said to be anitoxidants and the multiplicity of their actions, the possibility that any clinical benefit induced by the taking of antioxidants might be due to an accompanied and unappreciated preservation of ATP stores rather than the scavenging free radicals needs to be seriously considered. Cytochrome oxidase, complex IV in the respiratory chain in mitochondria, does not appear to cause any appreciable damage in healthy subjects even though it too is said in textbooks of biochemistry to generate free radicals in the absence of aneobiosis during healthy resynthesis of ATP by oxidative phosphorylation. Indeed the damage occurring in rexoygenation/reperfusion would appear to be always accompanied by some degree of ATP degradation from unreversed ATP hydrolysis which is turn is accompanied by an abnormal fall in tissue pH and rise in cytosolic calcium. The free radicals released, the fall in tissue pH and the rise in cytosolic calcium seen in anaerobiosis may all open the permeability transition pores. This dissipates the protonmotive force upon which ATP resynthesis depends and thereby uncouples oxidative phosphorylation compounding the depletion of ATP stores present and hence the likelihood of cellular apoptosis and necrosis, organ dysfunctions and failures. Before it can be concluded that free radicals are even partly the cause of the cellular damage seen in ischaemia/reperfusion it would seem that it would have to be shown that their allegedly harmful effects are independent of those induced by the fall in pH and rise in cytosolic calcium. The harmful effects of homocysteine need to be reconsidered in the same context. The prevailing view that free radicals cause tissue damage and that antioxidants, and even catalase, superoxide dismutase and glutathione perioxidases, prevents free radical-induced injury might be invalid. 1. Spector T, Ferone R. Folic acid does not inactivate xanthine oxidase. J Biol Chem. 1984 Sep 10;259(17):10784-6. 2. Joshi R, Adhikari S, Patro BS, Chattopadhyay S, Mukherjee T. Free radical scavenging behavior of folic acid: evidence for possible antioxidant activity. Free Radic Biol Med. 2001 Jun 15;30(12):1390-9. 3. Fiddian-Green RG. Homocysteine, ATP degradation and free radical release. bmj.com/cgi/eletters/325/7374/1202#27916, 17 Dec 2002 4. David S Wald, Malcolm Law, and Joan K Morris Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis. BMJ 2002; 325: 1202-1206 5. Yoshida R, Shioji I, Kishida A, Ogawa Y. Moderate alcohol consumption reduces urinary 8-hydroxydeoxyguanosine by inducing of uric acid. Ind Health. 2001 Oct;39(4):322-9. 6. Chamorro A, Obach V, Cervera A, Revilla M, Deulofeu R, Aponte JH. Prognostic significance of uric acid serum concentration in patients with acute ischemic stroke. Stroke. 2002 Apr;33(4):1048-52. 7. Chen SY, Chen CL, Shen ML, Kamatani N. Clinical features of familial gout and effects of probable genetic association between gout and its related disorders. Metabolism. 2001 Oct;50(10):1203-7. 8. Abu-Amsha Caccetta R, Burke V, Mori TA, Beilin LJ, Puddey IB, Croft KD. Red wine polyphenols, in the absence of alcohol, reduce lipid peroxidative stress in smoking subjects. Free Radic Biol Med. 2001 Mar 15;30(6):636-42. 9. Nathens AB, Neff MJ, Jurkovich GJ, Klotz P, Farver K, Ruzinski JT, Radella F, Garcia I, Maier RV. Randomized, prospective trial of antioxidant supplementation in critically ill surgical patients. Ann Surg. 2002 Dec;236(6):814-22 10. Fiddian-Green RG. Gastric intramucosal pH, tissue oxygenation and acid -base balance. Br J Anaesth. 1995 May;74(5):591-606. Review. Competing interests: None declared |
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Harpal S Randeva, Senior Lecturer in Medicine Molecular Medicine Research Group, University of Warwick, CV4 7AL, United Kingdom., Vivian Fonseca, Professor, Medicine and Pharmacol, Tulane University, USA; Gordana M Prelevic, Senior Lecturer, Reproductive Endocrinology, Royal Free&UCL, UK
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Wald and colleagues present their findings of a causal association between serum homocysteine and cardiovascular disease (CVD)(1), based on a meta-analysis of the methylenetetrahydrofolate reductase (MTHFR) studies and prospective studies of serum homocysteine and disease risk. Furthermore, they suggest that by increasing folic acid intake this cardiovascular risk could be reduced. Although a mutation in the MTHFR enzyme may lead to fasting hyperhomocysteinaemia (HHcy), increased cardiovascular risk is not associated with the mutation per se. The high frequency (~30%) of post- methionine load HHcy that occurs in patients with CVD would suggest non- genetic factors as a major cause of HHcy. However, the risk of cardiovascular risk may increase in those individuals with a genetic cause of HHcy with concomitantly low folate levels, an example of a gene- environment interaction. Several 'environmental' factors determine serum homocysteine levels and cardiovascular risk, including for example, chronic disease states, hormonal factors, and physical inactivity, the latter being identified more so especially in adolescence (2). Diabetes and obesity, are major risk factors for CVD, with individuals being insulin resistant and having a relative HHcy; improving insulin resistance lowers serum homocysteine. Lifestyle intervention, such as exercise improves insulin sensitivity and has many benefits for health and CVD morbidity and mortality. Recently, walking (150 minutes per week) has been shown to be associated with substantial reduction (58%) in the incidence of type 2 diabetes mellitus(3). More recently, prospective data indicate that both walking and vigorous exercise are associated with substantial reductions in the incidence of cardiovascular events (4). The exact mechanism(s) remain unclear. We recently reported the effects of a six-month exercise programme on plasma total homocysteine levels (5),in young overweight or obese women [age (mean ± SD) 30.6 ± 6.6 years, body mass index (BMI): 35.49 ± 7.57 kg/m²] with polycystic ovary syndrome (PCOS), who have a clustering of cardiovascular risk factors. Subjects were invited to take up sustained brisk walking, and by the sixth week walked an equivalent to 30 min per day. All walking was in addition to any normal habitual activity. Compared to baseline, there was a statistically significant decrease in plasma total homocysteine concentrations (27%; p<0.001) and WHR, and a significant increase in aerobic capacity (VO2max)(5). Wald et al(1), conclude that "a decrease in serum homocysteine of 3 micromol/L (achievable by daily intake of about 0.8 mg folic acid) should reduce risk of ischaemic heart disease by 16%, deep vein thrombosis by 25%, and stroke by 24%." The clinical translation of our findings (5), that brisk walking (30 min per day) decreased plasma homocysteine levels by an average of 2.7 micromol/L, could enhance the conclusion by Wald et al. It is also important to emphasise that exercise has health benefits beyond a reduction in plasma homocysteine and is particularly relevant given physical inactivity is increasing and diabetes and obesity are becoming a global health problem. References 1. Wald DS, Law M, Morris JK. Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis. BMJ 2002; 325:1202-9. 2. Kimm SY, Glynn NW, Kriska AM, Barton BA, Kronsberg SS, Daniels SR, et al. Decline in physical activity in black girls and white girls during adolescence. N Engl J Med 2002; 347:709-15. 3. Knowler WC, Barrett-Connor E, Fowler SE, Hamman RF, Lachin JM, Walker EA, Nathan DM. Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin. N Engl J Med 2002; 346:393-403. 4. Manson JE, Greenland P, LaCroix AZ, Stefanick ML, Mouton CP, Oberman A, et al. Walking compared with vigorous exercise for the prevention of cardiovascular events in women. N Engl J Med 2002; 347:716- 25. 5. Randeva HS, Lewandowski KC, Drzewoski J, Brooke-Wavell K, O'Callaghan C, Czupryniak L, Hillhouse EW, Prelevic GM. Exercise decreases plasma total homocysteine in overweight young women with polycystic ovary syndrome. J Clin Endocrinol Metab 2002; 87:4496-501. Competing interests: None declared |
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Miranda B. Keijzer, Department of Endocrinology University Medical Center Nijmegen, PO-Box 9101, 6500 HB Nijmegen, Martin den Heijer
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To the editor, Wald et al. (1) in their large meta-analysis reported the MTHFR 677TT polymorphism to be associated with a 1.29-fold (95%CI 1.08 to 1.54) increased risk on venous thrombosis, based on a meta-analysis of 26 studies. We would like to make some additional remarks on this finding. In our opinion, the overview of these studies is not completely correct. The data that represent the control group of the study of Salden et al. (2) are not those of the controls but data of another patient group. Using this original control group yielded a crude odds ratio of 1.17 instead of 1.09 found by Wald et al. (1). Also the study of Fujimura et al. (3) for which Wald et al. selected a subgroup of patients. This in our opinion should have been the total patient population, yielding an odds ratio of 1.88 instead of 2.00. Furthermore, there might have been some studies included that had better been left out (4-6) while other articles that were not included in our opinion are eligible (7-12). The study by Legnani et al. (4), for example, uses only patients with thrombophilic conditions and the study by Rintelen et al. (6) uses subjects with factor V Leiden as cases. The estimated odds ratios in our opinion, to much reflect interactions of risk factors instead of the isolated effect of MTHFR on the risk of venous thrombosis. The main point we want to make is to be careful for the interpretation that MTHFR is indeed a risk factor for venous thrombosis. Although Wald et al. mentioned the possibility of publication bias, we feel it is important to realise that the largest studies in this meta- analysis (Brown et al.(13), Kluijtmans et al. (14)) did not find the MTHFR 677TT polymorphism to be a risk factor for venous thrombosis. Therefore, we find that the risk for venous thrombosis associated with this MTHFR polymorphism needs further investigation, preferably by conducting large individual studies. References 1. Wald DS, Law M, Morris J. Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis. BMJ 2002; 325: 1-7. 2. Salden A, Keeney S, Hay CRM, Cumming AM. The C677T MTHFR variant and the risk of venous thrombosis. Br J Haematol 1997; 99: 472. 3. Fujimura H, Kawasaki T, Sakata T, Ariyoshi H, Kato H, Monden M et al. Common C677T polymorphism in the methylenetetrahydrofolate reductase gene increases the risk for deep vein thrombosis in patients with predisposition of thrombophilia. Thromb Res 2000;98:1-8. 4. Legnani C, Palareti G, Grauso F, Sassi S, Grossi G, Piazzi S, Bernardi F, Marchetti G, Ferraresi P, Coccheri S. Hyperhomocyst(e)inemia and a common methylenetetrahydrofolate reductase mutation (Ala223Val MTHFR) in patients with inherited thrombophilic coagulation defects. Arterioscler Thromb Vasc Biol 1997;17:2924-9. 5. Rintelen C, Mannhalter C, Lechner K, Eichinger S, Kyrle PA, Papagiannopoulos M, Schneider B, Pabinger I. No evidence for an increased risk of venous thrombosis in patients with factor V Leiden by the homozygous 677 C to T mutation in the methylenetetrahydrofolate-reductase gene. Blood Coagul Fibrinolysis 1999;10:101-5. 6. Philipp CS, Dilley A, Saidi P, Evatt B, Austin H, Zawadsky J, et al. Deletion polymorphism in the angiotensin-converting enzyme gene as a thrombophilic risk factor after hip arthroplasty. Thromb Haemost 1998;80:869-873. 7. De Stefano V, Chiusolo P, Paciaroni K, Serra FG, Voso MT, Casorelli I et al. Prevalence of the 677C to T mutation in the methylenetetrahydrofolate reductase gene in Italian patients with venous thrombotic disease. Thromb Haemost 1998; 79: 686-687. 8. Gaustadnes M, Rüdiger N, Møller J, Rasmussen K, Bjerregaard Larsen T, Ingerslev J. Thrombophilic predisposition in stroke and venous thromboembolism in Danish patients. Blood Coag Fibrinol 1999; 10: 251-259. 9. Gemmati D, Serino ML, Trivellato C, Fiorini S, Scapoli GL. C677T substitution in the methylenetetrahydrofolate reductase gene as a risk factor for venous thrombosis and arterial disease in selected patients. Haematologica 1999; 84:824-828. 10. Lin J-S, Shen M-C, Tsai W, Lin B. The prevalence of C677T mutation in the methylenetetrahydrofolate reductase gene and its association with venous thrombophilia in Taiwanese Chinese. Thromb Res 2000; 97: 89-94. 11. Hsu L-A, Ko Y-L , Wang S-M, Chang C-J, Hsu T-S, Chiang C-W et al. The C677T mutation of the methylenetetrahydrofolate reductase gene is not associated with the risk of coronary artery disease or venous thrombosis among Chinese in Taiwan. Hum Hered 2001; 51: 41-45. 12. Hanson NQ, Aras Ö, Yang F, Tsai MY. C677T and A1298C polymorphisms of the methylenetetrahydrofolate reductase gene: Incidence and effect of combined genotypes on plasma fasting and post-methionine load homocysteine in vascular disease. Clin Chem 2001; 47: 661-666. 13. Brown K, Luddington R, Baglin T. Effect of the MTHFRC677T variant on risk of venous thromboembolism: interaction with factor V Leiden and prothrombin (F2G20210A) mutations. Br J Haematol 1998; 103: 42-44. 14. Kluijtmans LAJ, den Heijer M, Reitsma PH, Heil SG, Blom HJ, Rosendaal FR. Thermolabile methylenetetrahydrofolate reductase and factor V Leiden in the risk of deep-vein thrombosis. Thromb Haemost 1998; 79: 254 -258. Competing interests: None declared |
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Joel G Ray, Clinician and researcher Sunnybrook and Women's College Health Sciences Centre
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Dear Editor: I read the BMJ electronic letter by Maritha J Kotze, in response to my letter stating that MTHFR testing among most individuals with VTE is not necessary. I do not agree with Dr. Kotze that individuals planning to travel long distance should be screened for 1 or more thrombophilia factors. This is a dangerous concept because: 1) The absolute risk of VTE with travel is very very low. 2) The studies that examined the risk of VTE with travel often included those who had also traveled by land -- should they too be screened? Would we then include truck drivers as well? What distance defines a "greater danger"? 3) The MTHFR TT polymorphism is very common (10% prevalence), so that, with screening, one would generate many false-positives, in the sense that most of these individuals will not develop VTE, even if they are not prophylaxed. 4) One opens up a whole can of worms about testing family members if an individual is found to be MTHFR TT -- what counseling should they receive? Might that that be psychologically harmful, by creating unnecessary worry? 5) There are many thrombophilia factors that have yet to be discovered, so that one might give a falase sense of assurance to those whose thrombophilia screen is negative -- that they are somehow at "lower risk" of VTE during a long-haul flight. 6) Should we not simply encourage ALL individuals planning long-haul travel to periodically stretch their legs, remain hydrated, stand up, and even walk? The answer is: "Of course." 7) Among those with Factor V Leiden and/or MTHFR 677 TT, what therapy should they receive if long-haul travel is planned: warfarin, ASA, heparin, LMWH, compression stockings? What evidence is there to support such an expensive and invasive approach? 7) I am concerned that a privately run company, which Dr. Kotze represents, stands to gain much by promoting ideas that have not yet been supported by even decent quality scientific evidence. Her company's tests can together cost several hundred US dollars for each customer ("Blood: DNA Extraction -- 270.90 Rand" and "Blood: Genotype analysis per PCR -- 361.20 Rand"), as listed on their website (http://www.genecare.co.za/other.html) Joel G. Ray, MD FRCPC MSc Department of Medicine Sunnybrook and Women's College Health Sciences Centre, University of Toronto Competing interests: None declared |
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Maritha J Kotze, Managing director Christiaan Barnard Memorial Hospital, Cape Town, 8000
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Dear Editor:
Dr Ray states in his response to my letter of 26 November 2002 that the absolute risk of thrombosis in travellers is very low. Nevertheless, individuals with hypercoagulability are at increased risk of deep vein thrombosis and its potentially lethal complications. Forewarned is forearmed, and persons in whom risk factors have been identified will be empowered to make choices concerning personal preventative measures. This information may simply reinforce the importance of periodic stretching during long-distance travel, or the avoidance of excess alcohol and sedation, and the maintenance of adequate hydration. Awareness of a potential risk and attention to these avoidance techniques may be lifesaving.
Dr Ray expresses concern about the financial aspects of the screening service, stating that Dr Kotze's "company's tests can together cost several hundred US dollars". This is not the case since we screen simultaneously for the three gene mutations included in our test procedure by using a multiplex polymerase chain reaction-based assay which costs R993.30 (approximately US$114.00 at the current rand-dollar exchange rate), with the result available within 24 hours. This is less than half the cost for a less comprehensive pre-travel screening service offered at a laboratory in the USA at a fee of US $245, with a turn around time of 4-10 working days. Our test result is provided within the context of other environmental and genetic risk factors that may be present or absent.
Class action litigation by air travellers who have suffered deep vein thrombosis is pending in Australia. The findings of the court may well be relevant to the ongoing debate on the issue of hypercoagulability screening.
Competing interests: MK is a shareholder in Genecare Molecular Genetics and derives income from providing genetic testing |
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Richard G Fiddian-Green, None None
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It is the expression of a rogue gene rather than the presence of a rogue gene per se that would appear to be of most importance in the pathogenesis of diseases. Our data suggests that a fall in endothelial pH caused by unreversed ATP hydrolysis or more likely a rise in endothelial ADP are likely to be the critical events most likely to induce rogue gene expression in the genesis of deep vein thrombosis (1). A fall in ADP is a late event and indicates the presence of a severe inadequacy of mitochondrial oxidative phosphorylation. It is accompanied by a proportionally large fall in tissue pH. Factors most likely to induce thromboembolism during a long flight are, therefore, likely to be in order of importance a fall in ambient pO2, dehydration and immobility. Pre-flight screening, if it is appropriate (2) might be best achieved by including an exercise (3) or better yet a decompression hypoxia stress test using either gastric intramucosal measurements or possibly the endothelial pH derived in a representative leg vein as end- points. It is in theory possible to measure the intra-endothelial pCO2 and indeed pO2 indirectly and to derive the intra-endothelial pH from this pCO2 and the arterial or possibly the venous bicarbonate as in gastric tonometry. A formal prospective determination of the risk factors for thromboembolism in a study incorporating these measurements in addition to screening for gene mutations (4) might generate the information necessary to determine the most appropriate screening tests, if any, to use for pre -travel screening. 1. Unreversed ATP hydrolysis: the initiating endothelial event? Richard G Fiddian-Green bmj.com/cgi/eletters/325/7369/887#26445, 22 Oct 2002 2. Testing travellers for MTHFR TT is absurd Joel G Ray bmj.com, 21 Dec 2002 3. Kolkman JJ, Groeneveld AB, van der Berg FG, Rauwerda JA, Meuwissen SG. Increased gastric PCO2 during exercise is indicative of gastric ischaemia: a tonometric study. Gut. 1999 Feb;44(2):163-7. 4. Pre-travel screening to identify genetic factors predisposing to thrombosis Maritha J Kotze bmj.com, 27 Dec 2002 Competing interests: None declared |
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Joel G Ray, Clinician and researcher Sunnybrook and Women's College Health Sciences Centre
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Dear Editor: Re. Dr. Kotze's appeal to my letter arguing that "preventive" thrombophilia screening among travelers can be life saving, and that pending litigation in Australia may drive us to consider this even more. I respond with the following: 1) Civil litigation about cramped seating on airplanes is another matter altogether, and it does not provide us with the onus to consider thrombophilia screening; 2) there is no evidence to screen prospective long-haul travelers for venous thromboembolism or thrombophilia markers -- in fact, it may be dangerous, as mentioned in my last letter. Once there is decent evidence to support doing so, I will reconsider my stance; 3) "primum non nocere" is meant to be an inner guiding voice among clinicians, whose main message includes not creating a way of making money off of patients, however cheaply or efficiently, without first proving to them (and to ourselves) that we can improve their lives, or prevent misery. Joel G Ray, MD FRCPC MSc Competing interests: None declared |
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Richard G Fiddian-Green, None None
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Wald et al report a significantly higher risk of both ischaemic heart disease and deep vein thrombosis (with or without pulmonary embolism) in people with the MTHFR mutation (1). It has also been reported that the risk of death in patients with coronary artery disease (2) and that the severity of pulmonary embolism (3) increase as the level of uric acid in serum increases. A common denominator appears to be a rise in blood lactate caused by a systemic anaerobiosis. Dysoxia may be defined as a state in which ATP pools are being partly or wholly maintained by anaerobic metabolism (4,5). It is a state in which protons accumulate in proportion with the number of molecules of ATP whose hydrolysis cannot reversed by ATP resynthesis by oxidative phosphorylation. It is also a state in which protons accumulate in proportion with the degree of ATP degradation. The generation of ATP in anaerobic metabolism occurs in part by residual oxidative phosphorylation. It is also generated in the Embden-Meyerhoff pathway to the degree it is able to run in the absence of oxidative phosphorylation by the conversion of pyruvate to lactate and by the resynthesis of ATP from ADP catalysed by adenylate kinase. The severity of the tissue acidosis produced from the unreversed ATP hydrolysis is offset by the proton needed to covert pyruvate into lactate. As the development of a tissue acidosis is not inhibited by inhibiting the generation of lactate with iodoacetate in vitro the intracellular accumulation of lactate per se does not appear to be the principle cause of the tissue acidosis as often supposed. The blood lactate rises and arterial pH falls with reperfusion/oxygenation such as that occurring when the aortic cross clamp is removed in the course of a cardiovascular operation. The blood lactate rises and the pH falls because of the washout of lactate and the tissue carbon dioxide generated by the buffering of protons released by unreversed ATP hydrolysis. The fall in arterial pH may be compounded by conversion of lactate back into pyruvate in preparation for the resynthesis of ATP by mitochondrial oxidative phosphorylation. Hence the misleading term “lactic acidosis”. The fall in tissue pH caused by the unreversed ATP hydrolysis in anaerobiosis is in effect a measure of a fall in adenylate energy charge or more specifically a fall in protonmotive energy charge for ATP resynthesis is driven by the protonmotive force established by the pH gradient generated by the electron transport chain across the mitochondrial membrane (6). [ATP]/[ADP] is maintained by the adenylate kinase reaction long after the pH falls and [AMP] the lactate rise and is, thus, an insensitive index of the presence or absence and degree of anaerobiosis present.. The fall in tissue pH, and by inference the accompanying rise in ionised calcium, accounts for some 90% of the enzymatic adaptation to anaerobiosis including translational, membrane channel and neuronal spike arrest (7). The degree of this enzymatic adaptation in anaerobiosis, which incorporates the enzymatic reactions catalysing ATP regeneration in addition to those catalysing ATP utilisation, change inversely as the adenylate energy charge falls from 1.0 to 0 intersecting at a “metabolic equilibrium” point. By inference a similar relationship exists between the protonmotive energy charge or the degree of dysoxia present and the enzymatic adaptation to anaerobiosis. One of the enzymatic adaptations to anaerobiosis and its accomanying fall in tissue pH and rise in ionised calcium is the conversion of xanthine dehydrogenase to xanthine oxidase catalysed by the calcium- induced activation of protease and accompanying increase in serum uric acid (8). The lactate that rises in blood upon reperfusion/oxygenation is said to compete with uric acid for secretion into the renal tubules thus increasing the likelihood of a rise in uric acid concentration in serum. The metabolic degradation products of alcohol appear to cause a rise in uric acid concentration in serum by the same mechanism (6,8). Since a rise in serum uric acid will, in accordance with the law of mass action impair the degradation of AMP into adenosine, hyoxanthine and xanthine adenosine concentrations can be expected to also rise. In which case the degradation of adenosylhomecysteine into adenosine and homocysteine will be impaired and the concentration of adenylhomocysteine in blood may rise. With the resumption of mitochondrial oxidative phosphorylation homocysteine levels in blood can be expected , therefore, to rise from the increased levels in adenylhomocysteine. The efficiency of homocysteine thiolactone metabolism declines with age and may explain the decreased formation of adenosyl methionine in aging and loss of thioretinaco ozonide from membranes of aging cells (9). It is possible that this putative cause of an increase in homocysteine levels is one of the many enzymatic adaptations to anerobiosis. The free base of homocysteine thiolactone appears to be responsible for many of the noxious effects of homocysteine including mitochondrial dysfunction and apoptosis. Thus anaerobiosis might induce a rise homocysteine in addition to inducing a rise in blood lactate and uric acid. The stress of a systemic anaerobiosis may induce a release of catecholamines, CRH from the hypothalamus, ACTH from the pituitary and cortisol from the adrenals. This may be accompanied by an increase in glycogenolsis, lipolysis, blood cholesterol and low density lipoproteins, and insulin resistance. (80% of the cholesterol from which steroid hormones including cortisol are synthesised are contained with the low density lipoproteins). It has also been proposed that thromboembolism might be the product of a systemic anaerobiosis (10). The possibility exists, therefore, that many of the risk factors for cardiovascular diseases might be products of a systemic anaeobiosis and not the causes of the diseases as is commonly supposed (11). 1. Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis David S Wald, Malcolm Law, and Joan K Morris BMJ 2002; 325: 1202-1206 2. Bickel C, Rupprecht HJ, Blankenberg S, Rippin G, Hafner G, Daunhauer A, Hofmann KP, Meyer J. Serum uric acid as an independent predictor of mortality in patients with angiographically proven coronary artery disease. Am J Cardiol. 2002 Jan 1;89(1):12-7. 3. Shimizu Y, Nagaya N, Satoh T, Uematsu M, Kyotani S, Sakamaki F, Nakanishi N, Miyatake K. Serum uric acid level increases in proportion to the severity of pulmonary thromboembolism. Circ J. 2002 Jun;66(6):571-5. 4. Fiddian-Green RG. Gastric intramucosal pH, tissue oxygenation and acid- base balance. Br J Anaesth. 1995 May;74(5):591-606. Review. 5. Fiddian-Green RG. Monitoring of tissue pH: the critical measurement. Chest. 1999 Dec;116(6):1839-41. 6. Garrett, Anderson. Biochemistry, Second edition, Saunders College Publishing, Orlando, Florida, 1999. 7. Hochachka PA, Somero GW. Biochemical adaptation. Oxford University Press, New York, NY, 2002. 8. Halliwell B, Gutteridge JMC. Free radicals in biology and medicine. Oxford University Press, Oxford, Third edition, 1999. 9. Homocysteine causes mitochondrial dysfunction Richard G Fiddian-Green bmj.com/cgi/eletters/325/7374/1202#27538, 2 Dec 2002 10. Re: pre-travel screening for thromboembolism Richard G Fiddian-Green bmj.com/cgi/eletters/325/7374/1202#28242, 28 Dec 2002 11. Homocysteine, folic acid, uric acid, ATP and free radical scavenging Richard G Fiddian-Green (19 December 2002) Competing interests: None declared |
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Maritha J Kotze, Managing Director Christiaan Barnard Memorial Hospital, Cape Town 8000, South Africa
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Dear Editor I agree with Dr Richard Fiddian-Green that additional measurements indicating the likelihood of expression of a genetic alteration associated with thromboembolism would greatly improve the predictive value of genetic testing (1). At present the co-existence of genetic and environmental risk factors, together with a family history or previous deep vein thrombosis (DVT) episode, are considered when individuals are categorized in low, moderate, high, or very high risk subgroups, similar to the risk stratification applying to patients under assessment for surgery (2). Inclusion of an exercise and decompression hypoxia stress test would be valuable for interpretation of genetic test results and might indeed determine the most appropriate preventative strategy. This approach will be valuable in a formal research program but due to logistical constraints incorporation in a large scale screening program would be difficult. To prevent possible psychological harm as a consequence of pre- clinical genetic testing for thrombosis risk (3), we designed a pre-test assessment form to determine the appropriateness of genetic testing in healthy individuals. This is done in the form of a questionnaire-based survey (table 1) and those individuals who agree with any of the first two statements are strongly advised against genetic testing. TABLE 1. Questionnaire-based survey - agree / uncertain / disagree? Pre-test assessment 1.Having a genetic predisposition for a modifiable genetic condition, in this case DVT, will make me anxious 2.I will fear the future if I have an increased risk of thrombosis due to the presence of both genetic and environmental risk factors 3.I believe that people should be told of their risk of disease 4.A positive test result does not mean that I will develop DVT, because the expression of a specific gene is influenced by external factors 5.The test procedure includes only the most prevalent risk factors and therefore a negative test result does not exclude the possibility of a future episode of DVT Positive test result 1.I wish I did not know that I have a genetic predisposition for thrombosis 2.My well-being has diminished after gene mutations implicated in DVT have been identified in my genetic material 3.I am satisfied to know that I have a genetic predisposition for DVT, because I can do something to reduce the risk 4.I believe that my family should be screened for genetic risk factors of DVT Negative test result 1.I am convinced that I will not suffer DVT in future 2.I wish I did not know that I test negative for the most common genetic causes of DVT, because it does not exclude the possibility of other risk factors and gives me a false sense of assurance 3.I would like to participate in a research project aimed at the identification of other known or unknown risk factors for DVT and will provide written consent for further analysis To date genetic screening for thrombosis risk using our multiplex assay including three mutations (4) has only been performed in patients with strong clinical indications of hypercoagulability referred by their doctors, and not in healthy individuals. In fact, even in these cases we have not reported on the presence or absence of the MTHFR 677C/T polymorphism although it is included in the multiplex assay, because of the fact that the role of this mutation in DVT was controversial (5). We accepted that this was clarified in the study of Wald et al. (6) and can now be routinely included as part of a thrombophilia assessment, as indicated by Ray et al. (5) provided that more compelling data become available on the role of the MTHFR mutation. The debate that followed further emphasised the importance of providing a clear interpretation of the genetic test result with reference to data available in the literature. The number of travellers who suffer DVT is relatively small, but individual cases remain an important consideration. Just yesterday an example came under my attention of a 23-year old female who started to use oral contraceptives before she got married and died of thromboembolism on her honeymoon shortly after leaving the airplane. I believe that increased awareness of the association between prolonged immobility and venous thromboembolism is of utmost importance if mortality from this cause is to be decreased. The availability of a genetic screening service for thrombosis risk contributes to increased awareness among the general population, irrespective of whether genetic testing is requested or not. References 1.Fiddian-Green FG. Pre-travel screening for thromboembolism. BMJ online, 30 Dec 2002. 2.Turpie AGG, Chin BSP, Lip GYH. Venous thromboembolism: pathophysiology, clinical features, and prevention. BMJ 2002; 325: 887-890. 3.Ray JG. Pre-travel screening may be harmful. BMJ online, 30 Dec 2002 4.Kotze MJ. Pre-travel screening to identify genetic factors predisposing to thrombosis. BMJ online, 27 Dec 2002. 5.Ray JG, Shmorgun D, Chan WS. Common C677T polymorphism of the methylenetetrahydrofolate reductase gene and the risk of venous thromboembolism: meta-analysis of 31 studies. Pathophysiol Haemost Thromb 2002; 32: 51-58. 6.Wald DS, Law M, Morris JK. Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis. BMJ 2002; 325: 1-7. Competing interests: MK is a shareholder in Genecare Molecular Genetics and derives income from providing genetic testing |
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Dan Hackam, Senior Medical Resident McMaster University
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It has been stated that the MTHFR mutation is an important contributor to an elevated homocysteine in the population. Actually there are at least 3 gene culprits and 3 vitamin deficiency states that can lead to hyperhomocysteinemia in addition to a multitude of drugs.
However, anecdotally, I must say that far and away the single most important contributor to very high homocysteines as seen in my practice has been renal impairment (mild to severe degrees). It is well known that patients with renal impairment (mild to severe degrees) have greatly increased risks of atherosclerosis (on the order of 10-20-fold increases), and that the kidney clears homocysteine from the body (hence the term homozygous homocystinuria). Perhaps, homocysteine is one of the contributors to vascular disease in this population.
On lowering homocysteine in the renally impaired, the literature conflicts. There are some studies that showed that very high doses of B vitamins are necessary, and others show partial and incomplete lowering with megadoses. A colleague in London Ontario has suggested Betaine and is currently studying its effect in the dialysis population. I personally use high doses of B vitamins: Folic acid 10 mg, B6 50 mg, and B12 1000 mcg daily, in those with renal impairment, particularly those with advanced CRF.
Competing interests: I invariably order fasting homocysteines in my patients. |
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Dan Hackam, Senior Medical Resident McMaster University
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It has been stated that the MTHFR mutation is an important contributor to an elevated homocysteine in the population. Actually there are at least 3 gene culprits and 3 vitamin deficiency states that can lead to hyperhomocysteinemia in addition to a multitude of drugs.
However, anecdotally, I must say that far and away the single most important contributor to very high homocysteines as seen in my practice has been renal impairment (mild to severe degrees). It is well known that patients with renal impairment (mild to severe degrees) have greatly increased risks of atherosclerosis (on the order of 10-20-fold increases), and that the kidney clears homocysteine from the body (hence the term homozygous homocystinuria). Perhaps, homocysteine is one of the contributors to vascular disease in this population. On lowering homocysteine in the renally impaired, the literature conflicts. There are some studies that showed that very high doses of B vitamins are necessary, and others show partial and incomplete lowering with megadoses. A colleague in London Ontario has suggested Betaine and is currently studying its effect in the dialysis population. I personally use high doses of B vitamins: Folic acid 10 mg, B6 50 mg, and B12 1000 mcg daily, in those with renal impairment, particularly those with advanced CRF. Competing interests: I invariably order fasting homocysteines in my patients. |
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Richard G Fiddian-Green, None None
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Homocysteine may cause mitcohondrial dysfunction and apoptosis (1). Upon reflection it might do so principally by creating an adenosine trap during anaerobiosis which compounds the depletion of ATP pools induced by the degradation of ATP. In an animal model an intravenous infusion of homocysteine decreased the plasma and tissue concentrations of adenosine (2). In an ischaemia/reperfusion model in dogs an intraatrial infusion of L-homocysteine (100 mg/kg/h) reduced the accumulation of adenine nucleosides and oxypurines accumulating during ischaemia by 50% (3). A high proportion of the adenosine was recovered as S-adenosyl-L- homocysteine. After 3 hours of reperfusion some 50% of the accumulated S- adenosyl-L-homocysteine were still present in the tissue. The infusion of L-homocysteine did not cause an accumulation of S-adenosyl-L-homocysteine in the nonischemic myocardial tissue. The investigators concluded that L- homocysteine was able to trap adenosine produced by ATP breakdown and that the reaction was not readily reversible. Homeocysteine may be converted to homocysteine thiolactone by aminoacyl-tRNA synthetases (4). Like homocysteine homocysteine- thiolactone causes mitochondrial dysfunction and apoptosis (5). In rat hippocampal slices both basal release of adenosine and that released by electrical stimulation or energy depletion were reduced 70% to 85% by the trapping of adenosine with L-homocysteine thiolactone (0.1- 1.0 mM (6). This effect of L-homocysteine thiolactone also occurred in the presence of adenosine uptake inhibitors. The investigators conclude that adenosine is formed intracellularly in hippocampal slices and is released as adenosine in response to either tissue depolarisation or energy depletion. The adenine nucleotide degradation products of ATP include ADP, AMP, inosine monophosphate (IMP) in muscle, adenosine in tissues and plasma, hypoxanthine and xanthine in plasma and uric acid in plasma and urine(7). ATP, ADP and AMP may not change significantly even with severe anaerobiosis induced by exercise but IMP and uric acid concentrations do increase significantly. Intracellular adenosine was not measured in this study. Being able to diffuse out of cells it is assumed, however, that it combines with homocysteine to produce S-adenosyl-L-homocysteine (3). In may in addition be trapped by homocysteine thiolactone. In which case a significant part of the adenine nucleotide pool generated in anaerobiosis may be trapped in forms that are not readily reconverted to AMP, ADP and ATP. That converted to uric acid cannot be reconverted. The harmful effects of both homocysteine and homocysteine thiolactone might be partly or wholly due to the depletion of ATP pools. This depletion might not be appreciated if the adenylate energy charge were to be measured by nuclear resonance spectroscopy unless all the compounds that can be synthesised from adenosine are included. The depletion should still be appreciated if the protonmotive energy charge were to be measured with the tissue pH or hydrogen ion concentration. 1. Homocysteine causes mitochondrial dysfunction Richard G Fiddian-Green bmj.com/cgi/eletters/325/7374/1202#27538, 2 Dec 2002 2. Chen YF, Li PL, Zou AP. Effect of hyperhomocysteinemia on plasma or tissue adenosine levels and renal function. Circulation. 2002 Sep 3;106(10):1275-81. 3. Henrichs KJ, Matsuoka H, Schaper W. Intracellular trapping of adenosine during myocardial ischemia by L-homocysteine. Basic Res Cardiol. 1986 May-Jun;81(3):267-75. 4. Jakubowski H, Zhang L, Bardeguez A, Aviv A. Homocysteine thiolactone and protein homocysteinylation in human endothelial cells: implications for atherosclerosis. Circ Res. 2000 Jul 7;87(1):45-51 5. Mercie P, Garnier O, Lascoste L, Renard M, Closse C, Durrieu F, Marit G, Boisseau RM, Belloc F. Homocysteine-thiolactone induces caspase- independent vascular endothelial cell death with apoptotic features. Apoptosis. 2000 Nov;5(5):403-11. 6. Lloyd HG, Lindstrom K, Fredholm BB. Intracellular formation and release of adenosine from rat hippocampal slices evoked by electrical stimulation or energy depletion. Neurochem Int. 1993 Aug;23(2):173-85. 7. Essen-Gustavsson B, Gottlieb-Vedi M, Lindholm A. Muscle adenine nucleotide degradation during submaximal treadmill exercise to fatigue. Equine Vet J Suppl. 1999 Jul;30:298-302. Competing interests: None declared |
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Richard G Fiddian-Green, None None
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In the course of the discussion of this paper (1) the terms adenosine, adenylate, adenine nucleotide, DNA, RNA and the prefix adenosyl- have been used. For those whose knowledge of biochemistry might be as rusty as mine the biochemical definitions, in the New Oxford Dictionary of English, are as follows. Adenine: compound which is one of the four constituent bases of nucleic acids. A purine derivative, it is paired with thymine in double- stranded DNA. Adenosine: a compound consisting of adenine combined with ribose, present in all living tissue in combined form as nucleotides. Nucleotide: a compound consisting of a nucleoside linked to a phosphate group. Nucleotides form the basic structural unit of nucleic acids such as DNA. Nucleoside: a compound (e.g adenosine or cytidine) consisting of a purine or pyrimidine base linked to a sugar. Adenylic acid: a compound consisting of an adenosine molecule bonded to one acidic phosphate group, present in most DNA and RNA. It typically exists in a cyclic form with the phosphate bonded to the nucleoside at two points. Thus adenosine is a nucleoside, adenylate a nucleoside with a phosphate bond and therefore a nucleotide, and ATP, ADP, AMP, and IMP are nucleotides. DNA and RNA are nucleic acids. 1. Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis David S Wald, Malcolm Law, and Joan K Morris BMJ 2002; 325: 1202-1206 Competing interests: None declared |
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Richard G Fiddian-Green, None None
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It has been proposed that hyperhomocysteinemia might function as an adenine nucleotide trap and in so doing deplete ATP pools (1). On further consideration it would seem that there might exist a finely balanced system for preserving substrate in anaerobiosis for ATP resynthesis with reperfusion/oxygenation. This may be regulated by the changes in pH and ionised calcium concentrations that occur with changes in the availability of oxygen and nutrients. Disorders of such a system might account for clinical findings in patients with hyperhomocysteinemia and homocysteinuria. It would further seem that the changes are intimately related to haematopoiesis. The disorders might also account for the megaloblastic anaemias seen in patients with related enzyme abnormalities. In anaerobiosis ATP is degraded into IMP, adenosine, hypoxanthine, xanthine and ultimately uric acid (2). Thiolactonase would seem to increase the availability of homocysteine in hippocampal slices by catalysing the hydrolysis of homocysteine-thiolactone in anerobiosis (3). Thiolactronase is an enzyme that is activated by calcium whose ionised concentrations probably increase with the fall in tissue pH induced by the unreversed ATP hydrolysis (4). In this respect the activation of thiolactonase in anaerobiosis would appear to be similar to the activation of the protease that converts xanthine dehydrogenase to xanthine oxidase in hypoxia (5). The presence of homocysteine provide an adenosine trap in anaerobiosis by combining with the adenosine to form adenosylhomocysteine (2). The more efficient the adenosine trapping the less likelihood that ATP degradation products will be irreversibly converted into uric acid. In aerobic conditions ATP converts methionine into adenosylmethionine, which forms adenosylhomocysteine, which in turn hydrolyses into homocysteine and adenosine. The adenosine is thus made available for the resynthesis of ATP by oxidative phosphorylation and the replenishment of the depleted ATP pools. The accumulation of homocysteine favors the formation of homocysteine-thiolactone which appears to function as an adenosine trap (2). In effect, therefore, the formation of homocysteine-thiolactone which appears to function as both an homocysteine and an adenosine trap. This biochemical scenario is consistent with the finding that during energy depletion in hippocampal slices, (i.e anaerobiosis) more adenosine was released than could be accounted for by the degradation of ATP alone. The law of mass action dictates that homocysteine formed will be metabolised to form cysteine which in turn will be metabolised to form porphyrins which are said to be incorporated into the synthesis of adenosylcobalamin in mitochondria (6,7). To synthesize DNA, deoxyribonucleotides are required as building blocks. These are formed from the corresponding ribonucleotides through the enzymatic action of ribonucleotide reductases(8). Ribonucleotide reductases are uniquely responsible for converting nucleotides to deoxynucleotides in all dividing cells. Class I enzymes depend on oxygen for radical generation, class II uses adenosylcobalamin, and the anaerobic class III requires S-adenosylmethionine and an iron-sulfur cluster(9). Homocysteine is also used to synthesise methionine, a pathway what is dependent upon the availability of folate and B12 and the enzyme methionine synthase. Methionine synthase knockout mice have been bred to examine the relevance of methionine synthase (10). Heterozygous mice were found to have elevated plasma homocysteine and methionine relative to wild -type mice but were otherwise indistinguishable. Homozygous knockout embryos survived through implantation but died soon thereafter. Nutritional supplementation during pregnancy was unable to rescue embryos that were completely deficient in methionine synthase. Human patients with methionine synthase deficiency exhibit homocysteinemia, homocysteinuria, and hypomethioninemia. They suffer from megaloblastic anemia with or without some degree of neural dysfunction and mental retardation. From this discussion it would seem that the conversion of methionine to cysteine, the synthesis of prophyrins and methionine from homocysteine, the synthesis of DNA catalysed by adenosylcobalamin, the resynthesis of ATP from adenosine and the trapping of residual adenosine by homocysteine- thiolactone are all favoured in aerobic conditions. It would further seem that the formation of adenosylhomocysteine from homocysteine and the adenosine released both by ATP degradation and possibly by the hydrolysis of homocysteine-thiolactone, the synthesis of DNA catalysed by S- adenosylmethionine, and the conversion of xanthine dehydrogenase to xanthine oxidase and synthesis of uric acid, are all favoured by anaerobiosis. In which case enzyme abnormalities that impair the conversion of homocysteine to methionine may be expected to increase adenosine trapping by conversion to adenosylhomocysteine in both aerobic and anaerobic conditions. Conversely those that impair the conversion of methionine to cysteine may be expected to impair the release of adenosine. The net effect should be an impairment of ATP resynthesis for both classes of enzyme abnormalities, the subdivision of the seven enzyme abnormalities said to cause homocysteinuria in the newborn. Enzyme abnormalities that impair the conversion of homocysteine to methionine may also be expected to cause a functional folate and B12 deficiency analogous to that caused by the “methylfolate trap”(11). They might also impair the synthesis of DNA in anaerobic conditions if the synthesis of S-adenosylmethionine is impaired. On the other hand those impairing the conversion of methionine to cysteine may be expected to impair the formation of porphyrin and hence adenosylcobalamin and the DNA synthesis it helps to catalyse under aerobic conditions. 1. Homocysteine: an adenine nucleotide trap? Richard G Fiddian-Green (9 January 2003) 2. Henrichs KJ, Matsuoka H, Schaper W. Intracellular trapping of adenosine during myocardial ischemia by L-homocysteine. Basic Res Cardiol. 1986 May-Jun;81(3):267-75. 3. Lloyd HG, Lindstrom K, Fredholm BB. Intracellular formation and release of adenosine from rat hippocampal slices evoked by electrical stimulation or energy depletion. Neurochem Int. 1993 Aug;23(2):173-85 4. Jakubowski H. Calcium-dependent human serum homocysteine thiolactone hydrolase. A protective mechanism against protein N-homocysteinylation. J Biol Chem. 2000 Feb 11;275(6):3957-62. 5. "Lactic acidosis": the common denominator? Richard G Fiddian-Green bmj.com/cgi/eletters/325/7374/1202#28322, 2 Jan 2003 6. Fenton WA, Rosenberg LE. Mitochondrial metabolism of hydroxocobalamin: synthesis of adenosylcobalamin by intact rat liver mitochondria. Arch Biochem Biophys. 1978 Aug;189(2):441-7. 7. Warren MJ, Raux E, Schubert HL, Escalante-Semerena JC. The biosynthesis of adenosylcobalamin (vitamin B12). Nat Prod Rep. 2002 Aug;19(4):390-412. Review. 8. Reichard P. From RNA to DNA, why so many ribonucleotide reductases? Science. 1993 Jun 18;260(5115):1773-7. 9. Torrents E, Aloy P, Gibert I, Rodriguez-Trelles F. Ribonucleotide reductases: divergent evolution of an ancient enzyme. J Mol Evol. 2002 Aug;55(2):138-52. 10. Swanson DA, Liu ML, Baker PJ, Garrett L, Stitzel M, Wu J, Harris M, Banerjee R, Shane B, Brody LC. Targeted disruption of the methionine synthase gene in mice. Mol Cell Biol. 2001 Feb;21(4):1058-65. 11. Tefferi A, Pruthi RK. The biochemical basis of cobalamin deficiency. Mayo Clin Proc. 1994 Feb;69(2):181-6. Review. Competing interests: None declared |
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Markus Weih, MD Student Deans office; medical faculty; University of Freiburg; Elsässerstr. 2m; 79085 Freiburg; Germ
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Dear Sir, I am writing in response to the paper of David Wald et al. in the BMJ (1). In their meta-analysis the authors investigated the relationship between homocysteine (genetic studies and serum levels) and three cardiovascular diseases: ischemic heart disease, stroke (ischemic or haemorhagic) and deep vein thrombosis with or without embolism. For the MTHFR 677 homozygote genotype and stroke, the authors found an odds ratio of 1.31 (CI 0.80-2.15) in seven studies. We appreciate the work of the authors in this difficult and expanding field. However their meta-analysis only partially covers the available literature. Though their literature search for the genetic studies was not restricted to any particular criteria or study types the authors did not include a large number of recent publications: In 2000 we performed a meta-analysis on the relation between the homozygote MTHFR genotype and ischemic stroke: 12 studies yielded an combined Odds Ratio of 1.5 (CI 1.3-1.8) (2). In 2002 Kelly et al. (3) found an Odds Ratio for ischemic stroke and the MTHFR 677 TT genotype of 1.23 (CI 0.96-1.58) in their meta analysis of 19 studies. For the homozygous C677T genotype, it seems now that this common polymorphism is probably not significantly or very weakly associated with ischemic stroke, but the available evidence is better than the data of Wald et al suggested. Reference List 1. Wald DS, Law M, Morris JK. Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis. BMJ 2002;325:1202 2. Weih M, Junge-Hulsing J, Mehraein S, Ziemer S, Einhaupl KM. Hereditary thrombophilia with ischemic stroke and sinus thrombosis. Diagnosis, therapy and meta-analysis. Nervenarzt 2000;71:936-945. 3. Kelly PJ, Rosand J, Kistler JP, et al. Homocysteine, MTHFR 677C-T polymorphism, and risk of ischemic stroke: results of a meta-analysis. Neurology 2002;59:529-536. Competing interests: None declared |
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Richard G Fiddian-Green, None None
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It is difficult to understand why a mutation in the gene coding for the enzyme methylenetetrahydrofolate reductase (MTHFR) should be associated with homocysteinaemia let alone an increased risk of cardiovascular and neurological disorders (1) unless there is another cause such as “lactic acidosis” (2). In the first place methionine requirements can easily obtained from a normal diet. In the second place the beneficial effect of folic acid supplements may not be related to folic acid per se but to an additive which inhibits xanthine oxidase (3,4). Consider this and other enzyme abnormalities in a broader metabolic context including the effects of exercise. The availability of homocysteine appears to be increased in anaerobiosis by the hydrolysis of homocysteine-thiolactone. The accompanying release of adenosine trapped by the homocysteine –thiolactone increases the availability of adenosine for trapping as adenosylhomeocysteine (5,6) and hence for ATP resynthesis and the replenishment of adenine nucleotide pools once oxidative phosphorylation resumes. An increase in availability of homocystine promotes the synthesis of methionine, cysteine and succinyl CoA. Succinyl CoA is also generated from alpha ketoglutatate in the Krebs cycle and from that proprionic acid which is synthesised from other aminioacids and/or from the beta oxidation of fatty acids. The likelihood of succinyl CoA being synthesised from the degradation product of homocysteine, methylmalonyl CoA, depends upon the concentration of methylmalonyl CoA, the concentration of the enzyme methylmalonyl CoA mutase, and the availability of the co-factor adenosylcobalamin. The adenosylcobalamin is synthesised from uroporphyrogen III, which is also the substrate for porphyrin and haeme synthesis, and which in turn is synthesised from succinyl CoA. In the anaerobiosis induced by haemorrhagic shock myocardial tissue lactate and succinyl CoA increase (7). The increase in succinyl CoA is caused by an impairment of its utilisation as substrate in the Krebs cycle to a degree that must be proportional with the degree of anaerobiosis or dysoxia present (8). Tissue lactate increases not only because of an impairment of mitochondrial oxidative phosphorylation but also because of the catabolism of the cysteine derived from the catabolism of methionine and homocysteine into pyruvate. The rise in blood lactate and accompanying rise in the secretion of lactate into renal tubules appears to impair the secretion of uric acid and may thus limit the rate and degree of adenine nucleotide loss in urine. Hence the potential for an increase in both uric acid and homocysteine in serum whenever the degree of anaerobiosis or dysoxia becomes excessive. Methylmalonic acidosis may cause the death of neonates and cause neurological and mental disorders and even organ failures in adults. The methylmalonic acidosis is caused by an impairment of the activity of the enzyme methylmalonyl CoA mutase which catalyses the conversion of methylmalonyl CoA into succinyl CoA. The dysfunction may either be reversed by adenosylcobalamin or not. In those in whom it is not reversed by adenosylcobalamin death occurs in infancy. In those in whom the dysfunction is reversed by adenosylcobalamin neurological and mental consequences and even organ failures may develop . The methylmalonic acidosis is almost certainly primarily the product of unreversed ATP hydrolysis induced by a fall in the availability of succinyl CoA not only for the generation of ATP from oxidative phosphorylation synthesis but also for the synthesis of the coenzyme adenosylcobalamin. Porphyria may also cause neurological and mental diseases, such as that in King George III, abdominal pain and organ failures and death from the organ failures. In these conditions there is an accumulation of aminolevulinic acid (ALA) in the mitochondria because it is not able to pass into the cytosol to form uroporphyrogen III a substrate for adenosylcobalamin synthesis. Thus the conversion of methylmalonyl CoA to succinyl CoA may be impaired and methylmalonic acidosis develop. This impairment of succinyl CoA synthesis is accompanied by an impairment of porphyrin and haem synthesis. The law of mass action dictates that the degradation of homocysteine will also be impaired thus favouring the accumulation of homocysteine its conversion to methionine and the trapping of adenine nucleotides as homocysteione-thiolactone and/or adenosylhomocysteine. Thus hyperhomocysteinemia, hyperuricaemia, and the inappropriate depletion of ATP pools are all favoured in the methylmalonic acidosis caused by impaired function of the enzymes methylmalonyl mutase and those responsible for porphyria. Patients with the Lesch-Nyman syndrome develop hyperuricaemia, gout, and neurological problems including spacticity, mental retardation and self mutilation. The syndrome is caused by any one of some one hundred abnormalities of the enzyme hyoxanthine-guanine-phosphoribosyl transferase (HGPRTase) which catalyses adenine nucleotide salvage by combining with phosphoribosylpyrophosphate (PRPP) to form IMP and Ppi. On occasions the enzyme defect may reduce activity as much as 98% or even 100%. A relatively benign variant of the syndrome was caused by a change in Km (9). The hyperuricaemia in the Lesch-Nyman syndrome is caused by an excessive production of uric acid induced by the degradation of ATP. Hyperhomocystienaemia may also occur because of the impairment of adenine nucleotide salvage. This syndrome is, therefore, also characterised by an inappropriate depletion of ATP pools and hence of metabolic energy for cellular activities, organ dysfunctions and failures. If the secretion of uric acid is indeed limited by the secretion of lactate into renal tubules the generation of lactate in severe exercise, both from the Emden-Meyerhoff pathway and from the degradation of the cysteine derived from the homocystiene presumably released from the homocysteine-thiolactone trap, may be one means of preserving adenine nucleotide pools in athletes. The training of athletes is known to increase the efficiency of mitochondrial oxidative phosphorylation (10) as indeed may in ingestion of modest amounts of alcohol (11). From a teleological perspective one would presume that any increase in efficiency of mitochondrial oxidative phosphorylation might be accompanied by an enhancement of the capacity to limit the losses of substrate for ATP resynthesis in urine. By impairing the conversion of homocysteine into methionine the mutation in the MTHFR gene might cause an increase in homocysteine by impairing its degradation into methionine. On the other hand homocysteine synthesis from endogenous methionine is impaired. A rise in homocysteine levels is, therefore, more likely to occur during anaerobiosis in these patients. In addition to the release of homocysteine from the homocysteine -thiolactone pool homocysteine catabolism into succinyl CoA is impaired in anaerobiosis for the utilisation of succinyl CoA as a substrate in the Krebs cycle is impaired t0o a degree determined by the degree of dysoxia present. The beneficial effects of folic acid, the additive in which is a potent inhibitor of xanthine oxidase, is consistent with this view for xanthine oxidase appears to increase the rate of adenine nucleotide depletion. But xanthine oxidase is only activated under abnormal conditions including hypoxia and cytokine release. That folic acid has a beneficial effect in patients with the mutation in the MTHFR gene implies, therefore, that abnormal conditions prevail and that the increase in homocysteine may indeed be the result rather than the primary or sole cause of the cardiovascular and neurological diseases. Folic acid rather than its additive might exert a beneficial effect in patients with the mutation in the MTHFR gene by enhancing methionine synthesis and hence the rate at which it is catabolised into homocysteine and succinyl CoA. The increase in availability of succinyl CoA would, in the presence of normal oxidative phosphorylation, tend to enhance the salvage of adenine nucleotide. If, however, anaerobiosis were present pure folic acid could not be expected to enhance ATP resynthesis or limit further the adenine nucleotide in urine. Hence the suggestion that folic acid can only be beneficial in patients who have an anaerobic energy deficit (12). B12, on the other hand could be expected to be of benefit in any patient who had developed a methylmalonic acidosis secondary to competitive inhibition of the conversion of methylmalonyl CoA to succinyl CoA by methylmalonyl Co A mutase by enhancing the synthesis of adenosylcobalamin. A dietary deficiency in B12 but not in folic acid may cause neurological disorders. The explanation for this may be that a B12 deficiency, but not a folic acid deficiency, may impair the resynthesis of ATP by oxidative phosphorylation by limiting the availability of succinyl CoA in the Krebs cycle and thereby cause a methylmalonic acidosis. The beneficial effect which folic acid supplements have in preventing neurological abnormalities in the newborn may be related to the ability of the additive to inhibit xanthine oxidase and preserve adenine nucleotide stores rather than an enhancement of methionine synthesis from homocysteine. An metabolic energy deficit whatever its causes may indeed be the primary cause of not only neurological disorders but also of psychiatric disorders (13). The HGPRTase gene is located on the Y chromosome and so the Lesch- Nyman syndrome is confined to males. This is the second of two salvage pathway for adenine nucleotides. That is the combining of adenine and PRPP to form AMP. It is not clear from my readings whether adenosine has to be converted into adenine to accommodate this pathway or whether it can as I have presumed simply diffuse back into cells and recombine with Pi to form AMP and hence ATP. In the absence of the HGPRT gene these must presumably be the sole adenine nucleotide salvage pathways available to women. It would seem, therefore, that men might be better adapted than women to withstanding periodic exposures to severe anaerobiosis such as that occurring with severe exercise. Men who exercise and drink in moderation regularly may owe their protection against cardiovascular and neurological diseases to their sex in addition to an acquired enhancement in their capacity to preserve adenine nucleotide stores and replenish ATP by mitochondrial oxidative phosphorylation (14,15). 1. Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis David S Wald, Malcolm Law, and Joan K Morris BMJ 2002; 325: 1202-1206 2. "Lactic acidosis": the common denominator? Richard G Fiddian-Green bmj.com/cgi/eletters/325/7374/1202#28322, 2 Jan 2003 3. Spector T, Ferone R. Folic acid does not inactivate xanthine oxidase. J Biol Chem. 1984 Sep 10;259(17):10784-6. 4. Homocysteine, folic acid, uric acid, ATP and free radical scavenging Richard G Fiddian-Green bmj.com/cgi/eletters/325/7374/1202#27955, 19 Dec 2002 5. Homocysteine: an adenine nucleotide trap? Richard G Fiddian-Green (9 January 2003) 6. pH/calcium regulation of homocysteine metabolism Richard G Fiddian- Green (10 January 2003) 7. Kline JA, Thornton LR, Lopaschuk GD, Barbee RW, Watts JA. Lactate improves cardiac efficiency after hemorrhagic shock. Shock. 2000 Aug;14(2):215-21. 8. Fiddian-Green RG. Gastric intramucosal pH, tissue oxygenation and acid- base balance. Br J Anaesth. 1995 May;74(5):591-606. Review. 9. Sorenson L, Benke PJ. Biochemical evidence of a distinct type of primary gout. Nature 313:1122 n1967. 10. Baar K, Wende AR, Jones TE, Marison M, Nolte LA, Chen M, Kelly DP, Holloszy JO. Adaptations of skeletal muscle to exercise: rapid increase in the transcriptional coactivator PGC-1. FASEB J. 2002 Dec;16(14):1879-86 11. Piquet MA, Nogueira V, Devin A, Sibille B, Filippi C, Fontaine E, Roulet M, Rigoulet M, Leverve XM. Chronic ethanol ingestion increases efficiency of oxidative phosphorylation in rat liver mitochondria. FEBS Lett. 2000 Feb 25;468(2-3):239-42. 12. Homocysteine causes mitochondrial dysfunction Richard G Fiddian-Green bmj.com/cgi/eletters/325/7374/1202#27538, 2 Dec 2002 13. Madness, hyperhomocysteinemia, metabolic rate and body temperature Richard G Fiddian-Green bmj.com/cgi/eletters/325/7378/1433#28469, 6 Jan 2003 14. Tiger L. Nunc est bibendum. Wall Street Journal Europe, January 13, 2003 15. Do port and red wine preserve ATP stores? Richard G Fiddian-Green bmj.com/cgi/eletters/325/7374/1202#27887, 16 Dec 2002 16. Exercise and plasma homocysteine Harpal S Randeva, Vivian Fonseca, Professor, Medicine and Pharmacol, Tulane University, USA; Gordana M Prelevic, Senior Lecturer, Reproductive Endocrinology, Royal Free&UCL, UK (20 December 2002) Competing interests: None declared |
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David S Wald, Specialist Registrar in Cardiology Southampton General Hospital, UK SO17 1PJ, Malcolm Law, Joan Morris
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We agree with Joel Ray that the MTHFR C677T polymorphism is a weak marker of the risk of deep vein thrombosis (it identifies about 10% of the population who have a relative risk of 1.29)¹, and that its measurement has no place in clinical or screening practice. We did not claim that it did; it is a useless screening test. Our results which are highly statistically significant, are important because they help establish cause and effect between homocysteine and cardiovascular disease. References <1> Wald DS, Law M, Morris JK. Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis. BMJ 2002; 325: 1202- 6. Competing interests: None declared |
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David S Wald, Specialist Registrar in Cardiology Southampton General Hospital, UK SO17 1PJ, Malcolm Law, Joan Morris
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Doshi and his colleagues state that observational data can never allow conclusions to be drawn on causality, in which case they cannot accept that smoking causes ischaemic heart disease or even that drunken driving causes road traffic accidents. Of course, observational data alone may not be sufficient to establish causality. Judgement is needed as to whether an association might be explained in a non-causal way – in which case doubt would remain. To explain the association between homocysteine and ischaemic heart disease, deep venous thrombosis and stroke as not being cause and effect one would have to postulate that the association with the MTHFR C677T polymorphism arises through linkage with a gene controlling some other factor that causes all three diseases, and that would have to be an unknown causal factor (since there is no association with the known causes). It would also be necessary to postulate that the association in the prospective studies arose through confounding with a cause of all three diseases, and that cause would again have to be an unknown. Moreover it would have to coincidentally have the same indirect effect as the MTHFR indirect effects for a given change in serum homocysteine, even though the serum homocysteine had no effect on risk. The simultaneous occurrence of both postulations is so unlikely that it can be rejected, leaving only the causal explanation; namely that increases in the concentration of serum homocysteine directly increase the risk of cardiovascular disease. Competing interests: None declared |
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David S Wald, Specialist Registrar in Cardiology Southampton General Hospital, UK SO17 1PJ, Malcolm Law, Joan Morris
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Miranda Keijzer suggested modifications to the studies included in the analysis of MTHFR and deep venous thrombosis (1), including using different controls in two studies (2-3), omitting two studies (4-5) and including an additional five studies (6-11). We re-analysed the data incorporating these changes (except that we did not include one of the five additional studies because the controls were neonates (7)). These changes had a negligible effect; the original odds ratio was 1.29 (1.08 to 1.54) and the revised one was 1.29 (1.10 to 1.51). References 1. Wald DS, Law M, Morris J. Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis. BMJ 2002; 325: 1-7. 2. Salden A, Keeney S, Hay CRM, Cumming AM. The C677T MTHFR variant and the risk of venous thrombosis. Br J Haematol 1997; 99: 472. 3. Fujimura H, Kawasaki T, Sakata T, Ariyoshi H, Kato H, Monden M et al. Common C677T polymorphism in the methylenetetrahydrofolate reductase gene increases the risk for deep vein thrombosis in patients with predisposition of thrombophilia. Thromb Res 2000;98:1-8. 4. Legnani C, Palareti G, Grauso F, Sassi S, Grossi G, Piazzi S, Bernardi F, Marchetti G, Ferraresi P, Coccheri S. Hyperhomocyst(e)inemia and a common methylenetetrahydrofolate reductase mutation (Ala223Val MTHFR) in patients with inherited thrombophilic coagulation defects. Arterioscler Thromb Vasc Biol 1997;17:2924-9. 5. Rintelen C, Mannhalter C, Lechner K, Eichinger S, Kyrle PA, Papagiannopoulos M, Schneider B, Pabinger I. No evidence for an increased risk of venous thrombosis in patients with factor V Leiden by the homozygous 677 C to T mutation in the methylenetetrahydrofolate-reductase gene. Blood Coagul Fibrinolysis 1999;10:101-5. 6. De Stefano V, Chiusolo P, Paciaroni K, Serra FG, Voso MT, Casorelli I et al. Prevalence of the 677C to T mutation in the methylenetetrahydrofolate reductase gene in Italian patients with venous thrombotic disease. Thromb Haemost 1998; 79: 686-687. 7. Gaustadnes M, Rüdiger N, Møller J, Rasmussen K, Bjerregaard Larsen T, Ingerslev J. Thrombophilic predisposition in stroke and venous thromboembolism in Danish patients. Blood Coag Fibrinol 1999; 10: 251-259. 8. Gemmati D, Serino ML, Trivellato C, Fiorini S, Scapoli GL. C677T substitution in the methylenetetrahydrofolate reductase gene as a risk factor for venous thrombosis and arterial disease in selected patients. Haematologica 1999; 84:824-828. 9. Lin J-S, Shen M-C, Tsai W, Lin B. The prevalence of C677T mutation in the methylenetetrahydrofolate reductase gene and its association with venous thrombophilia in Taiwanese Chinese. Thromb Res 2000; 97: 89-94. 10. Hsu L-A, Ko Y-L , Wang S-M, Chang C-J, Hsu T-S, Chiang C-W et al. The C677T mutation of the methylenetetrahydrofolate reductase gene is not associated with the risk of coronary artery disease or venous thrombosis among Chinese in Taiwan. Hum Hered 2001; 51: 41-45. 11. Hanson NQ, Aras Ö, Yang F, Tsai MY. C677T and A1298C polymorphisms of the methylenetetrahydrofolate reductase gene: Incidence and effect of combined genotypes on plasma fasting and post-methionine load homocysteine in vascular disease. Clin Chem 2001; 47: 661-666. Competing interests: None declared |
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Gavin Willis, Clinical Scientist Department of Molecular Genetics, Norwich University Hospital, Colney Lane, Norwich NR4 7UY, UK., Rebecca Walker, Barbara A. Jennings
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We have re-analysed the data presented in Wald et al.1 (see Figure 1 therein) on the association between the MTHFR TT genotype and ischaemic heart disease using the Hardy-Weinberg equation to obtain expected numbers of TT homozygotes with disease rather than normal controls used originally. Our analysis has the advantage that demographic differences between cases and controls are eliminated. Surprisingly the summed expected and observed numbers of TT homozygotes from the 46 separate studies, including 12,193 cases, are essentially identical, giving an overall odds ratio of 1.01; by contrast in the controls there are fewer than the expected number of TT homozygotes. This analysis appears to weaken the case for the association between the TT genotype and heart disease. However it is possible that the populations from which the cases were drawn were already depleted of homozygotes because of genotype-specific fatal disease. This is supported by some studies2,3 of MTHFR in elderly populations, including our own unpublished data. However there is no apparent trend in the different studies used in Wald et al. 1 towards a declining ratio of observed to expected homozygotes with age, although the available data is limited. Another potential source of error in Hardy- Weinberg analysis derives from mixed race of the subjects but this would produce an excess of homozygotes and so is not of concern here. A similar analysis gives a greatly reduced odds ratio of 1.06 for the association of 3439 deep vein thrombosis cases with the TT genotype (see Fig 3 in Wald et al1). We are equivocal about the precise meaning of our results but they clearly show a strong potential for systematic error in association studies of the type collected by Wald et al.1 because of population differences between cases and controls. We suggest that only long term prospective folate supplementation / control studies supported by genotyping can address the ultimate issues of whether disease can be prevented by a simple intervention and whether any benefit is restricted to individuals with particular genotypes. 1. Wald DS, Law M, Morris JK. Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis. BMJ 2002 325:1202- 1209 2. Heijmans BT, Gussekloo J, Kluft C, Droog S, Lagaay AM, Knook DL et al. Mortality risk in men is associated with a common mutation in the methylene-tetrahydrofolate reductase gene (MTHFR). Eur J Hum Genet 1999 7:197-204 3. Faure-Delanef L, Quere I, Chasse JF, Guerassimenko O, Lesaulnier M, Bellet H et al. Methylenetetrahydrofolate reductase thermolabile variant and human longevity. Am J Hum Genet 1997 60:999-1001 Competing interests: None declared |
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