Erythrocyte membrane Na+,K+-ATPase and Mg2+-ATPase activities in subjects with methylenetetrahydrofolate reductase (MTHFR) 677 C→T genotype and moderate hyperhomocysteinaemia. The role of L-phenylalanine and L-alanine
-
Kleopatra H. Schulpis
Abstract
Background: Increased homocysteine (Hcy) blood levels are correlated with vascular and neurological problems. The aim of our study was to investigate erythrocyte membrane Na+,K+-ATPase and Mg2+-ATPase activities in patients with methylenetetrahydrofolate reductase (MTHFR) 677 C→T genotype.
Methods: Blood was obtained from 25 patients before and after folic acid supplementation and from controls (n=30) once. Plasma folate, vitamin B12 and total antioxidant status (TAS) were measured using commercial kits, Hcy was determined by HPLC and membrane enzyme activities were measured spectrophotometrically.
Results: Mg2+-ATPase remained unaltered. Membrane Na+,K+-ATPase activity was remarkably increased in patients (0.77±0.06μmolPi/h × mg protein) and decreased to normal levels (0.52±0.05μmolPi/h × mg protein; p<0.001) after therapy. TAS did not differ significantly before and after treatment. Hcy levels were significantly higher before therapy (25.4±2.8μmol/L) than levels after therapy (12.1±2.0μmol/L; p<0.001) and in controls (10.5±2.5μmol/L, p<0.001). In vitro, L-phenylalanine (Phe) reversed to normal the stimulated enzyme from patients before therapy. In addition, Phe incubation of the Hcy activated membrane Na+,K+-ATPase from controls resulted in restoration of its activity, whereas L-alanine (Ala) incubation protected the enzyme from Hcy activation.
Conclusions: The increased membrane Na+,K+-ATPase activity may be due to high -SH group Hcy levels. In vitro, Phe reversed the increase in enzyme activity induced by Hcy in controls, as well as the stimulated membrane enzyme in untreated patients. Ala protected the enzyme from Hcy action.
References
1. Delvin EE, Rozen R, Merouani A, Genest J, Lambert M. Influence of methylenetetrahydrofolate reductase genotype, age, vitamin B-12, and folate status on plasma homocysteine in children. Am J Clin Nutr 2000; 72:1469–73.10.1093/ajcn/72.6.1469Search in Google Scholar
2. Papoutsakis C, Yiannakouris N, Manios Y, Papakonstantinou E, Magkos F, Schulpis KH, et al. Plasma homocysteine concentrations in Greek children are influenced by an interaction between the methylenetetrahydrofolate reductase C677T genotype and folate status. J Nutr 2005; 135:383–8.10.1093/jn/135.3.383Search in Google Scholar
3. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetra-hydrofolate reductase. Nat Genet 1995; 10:111–3.10.1038/ng0595-111Search in Google Scholar
4. Matthews RG, Sheppard C, Goulding C. Methylenetetrahydrofolate reductase and methionine synthase: biochemistry and molecular biology. Eur J Pediatr 1998; 157(Suppl 2):S54–9.10.1007/PL00014305Search in Google Scholar
5. Visy JM, Le Coz P, Chadefaux B, Fressinaud C, Woimant F, Marquet J, et al. Homocystinuria due to 5,10-methyl-enetetrahydrofolate reductase deficiency revealed by stroke in adult siblings. Neurology 1991; 41:1313–5.10.1212/WNL.41.8.1313Search in Google Scholar
6. Sweadner KJ, Goldin SM. Active transport of sodium and potassium ions: mechanisms, function, and regulation. N Engl J Med 1980; 302:777–83.10.1056/NEJM198004033021404Search in Google Scholar
7. Calcutt NA, Tomlison DR, Bishwas S. Coexistence of nerve conduction deficit with increased Na(+),K(+)-ATPase activity in galactive-fed mice. Implications for polyol pathway and diabetic neuropathy. Diabetes 1990; 36:663–6.10.2337/diab.39.6.663Search in Google Scholar
8. Sanui H, Rubin H. The role of magnesium in cell proliferation and transformation. In: Boynton AL, MacKeehan WL, Whitfield JP, editors. Ions, cell proliferation and cancer. New York: Academic Press, 1982:517–37.Search in Google Scholar
9. Streck EL, Lugno AI, Tagliari B, Wannmacher C, Wajner M, Wyse AT. Inhibition of Na+,K+-ATPase activity by the metabolites accumulating in homocystinuria. Metab Brain Dis 2002; 17:83–91.10.1023/A:1015594111778Search in Google Scholar
10. Streck EL, Zugno AI, Tagliari B, Franzon R, Wannmacher CM, Wajner J, et al. Inhibition of rat brain Na+,K+-ATPase activity by homocysteine is probably mediated by oxidative stress. Neurochem Res 2001; 26:1195–200.10.1023/A:1013907104585Search in Google Scholar
11. Streck E, Zugno AI, Tagliari B, Sarkis JJ, Wajner M, Wannmacher CM, et al. On the mechanism of the inhibition of Na(+),K(+)-ATPase activity caused by homocysteine. Int J Dev Neurosci 2002; 20:77–81.10.1016/S0736-5748(02)00043-6Search in Google Scholar
12. Schulpis KH, Tjamouranis J, Karikas GA, Michelakakis H, Tsakiris S. In vivo effect of high phenylalanine blood levels on Na+,K+-ATPase, Mg2+-ATPase activities and biogenic amine concentration in phenylketonuria. Clin Biochem 2002; 35:281–5.10.1016/S0009-9120(02)00311-9Search in Google Scholar
13. Tsakiris S, Kouniniotou-Krontiri P. Involvement of sulfhydryl groups in the inhibition of diaphragm Na+,K+-ATPase by lithium. Med Sci Res 1987; 15:697–8.Search in Google Scholar
14. Araki A, Sako Y. Determination of free and total homocysteine in human plasma by high-performance liquid chromatography with fluorescence detection. J Chromatogr 1987; 422:43–52.10.1016/0378-4347(87)80438-3Search in Google Scholar
15. Schneede J, Refsum H, Ueland PM. Biological and environmental determinants of plasma homocysteine. Semin Thromb Hemost 2000; 26:263–79.10.1055/s-2000-8471Search in Google Scholar
16. Saiki PK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, et al. Primer-directed enzymatic application of DNA with a thermostable DNA polymerase. Science 1988; 239:487–91.10.1126/science.2448875Search in Google Scholar
17. Schulpis KH, Tsakiris S, Karikas GA, Moukas M, Behrakis P. Effect of diet on plasma total antioxidant status in phenylketonuric patients. Eur J Clin Nutr 2003; 57:383–7.10.1038/sj.ejcn.1601529Search in Google Scholar
18. Strube M, Haenen GR, Van den Berg H, Bast A. Pitfalls in a method for assessment of total antioxidant capacity. Free Radic Res 1997; 26:515–21.10.3109/10715769709097822Search in Google Scholar
19. Galbraith DA, Watts DC. Changes in some cytoplasmic enzymes from red cells fractionated into age groups by centrifugation in Ficoll/Triosil gradients. Comparison of normal humans and patients with Duchenne muscular dystrophy. Biochem J 1980; 191:63–70.10.1042/bj1910063Search in Google Scholar
20. Kamber E, Poyiagi A, Deliconstantinos G. Modifications in the activities of membrane-bound enzymes during in vivo ageing of human and rabbit erythrocytes. Comp Biochem Physiol 1984; 77:95–9.Search in Google Scholar
21. Lowry OH, Rosebrough NJ, Farr AL, Randal RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:265–75.10.1016/S0021-9258(19)52451-6Search in Google Scholar
22. Bowler K, Tirri R. The temperature characteristics of synaptic membrane ATPases from immature and adult rat brain. J Neurochem 1974; 23:611–3.10.1111/j.1471-4159.1974.tb06068.xSearch in Google Scholar
23. Lees GJ. Contributory mechanisms in the causation of neurodegenerative disorders. Neuroscience 1993; 54:287–322.10.1016/0306-4522(93)90254-DSearch in Google Scholar
24. Welch GN, Upchurch G Jr, Loscalzo I. Homocysteine, oxidative stress and vascular disease. Hosp Pract 1997; 32:81–2, 85, 88–92.10.1080/21548331.1997.11443510Search in Google Scholar
25. Cipolla MJ, Williamson WK, Nehler ML, Taylor LM, Porter JM. The effect of elevated homocysteine levels on adrenergic vasoconstriction of human resistance arteries: the role of the endothelium and reactive oxygen species. J Vasc Surg 2000; 31:751–9.10.1067/mva.2000.103797Search in Google Scholar
26. Engbersen AM, Franken DG, Boers GH, Stevens EM, Trijbels FJ, Bloom HJ. Thermolabile, 5,10-methylenetetrahydrofolate reductase as a cause of mild hyperhomocysteinemia. Am J Hum Genet 1995; 56:142–50.Search in Google Scholar
27. Atmaca G. Antioxidant effects of sulfur-containing amino acids. Yonsei Med J 2004; 45:776–88.10.3349/ymj.2004.45.5.776Search in Google Scholar
28. Cuttormsen AB, Ueland PM, Nesthus I, Nygard O, Schneede J, Vollset SE, et al. Determinants and vitamin responsiveness of intermediate hyperhomocysteine (≥40 micromol/liter). The Hordaland Homocysteine study. J Clin Invest 1996; 98:2174–83.Search in Google Scholar
29. Malinow MR. Hyperhomocyst(e)inemia. A common and easily reversible risk factor for occlusive atherosclerosis. Circulation 1990; 81:2004–6.10.1161/01.CIR.81.6.2004Search in Google Scholar
30. Chevillard C, Cardenas ML, Cornish-Bowden A. The competition plot: a simple test of whether two reactions occur at the same active site. J Biochem 1993; 289:599–604.10.1042/bj2890599Search in Google Scholar
31. Erecinska M, Silver IA. Ions and energy in mammalian brain. Prog Neurobiol 1994; 16:37–71.10.1016/0301-0082(94)90015-9Search in Google Scholar
©2006 by Walter de Gruyter Berlin New York
Articles in the same Issue
- Natriuretic peptides and evidence-based quality specifications
- Preanalytical variability: the dark side of the moon in laboratory testing
- Clinical relevance of biological variation: the lesson of brain natriuretic peptide (BNP) and NT-proBNP assay
- Hepatorenal syndrome
- Modified Levey-Jennings charts for calculated laboratory tests
- Increased free malondialdehyde concentrations in smokers normalise with a mixed fruit and vegetable juice concentrate: a pilot study
- The exponentially weighted moving average (EWMA) rule compared with traditionally used quality control rules
- Intermethod calibration of alanine aminotransferase (ALT) and γ-glutamyltransferase (GGT) results: application to Fibrotest® and Actitest® scores
- Comparison of TEST 1 with SRS 100 and ICSH reference method for the measurement of the length of sedimentation reaction in blood
- Multicenter evaluation of the interference of hemoglobin, bilirubin and lipids on Synchron LX-20 assays
- Technical evaluation of the Beckman Coulter OV-Monitor (CA 125 antigen) immunoassay
- Erythrocyte membrane Na+,K+-ATPase and Mg2+-ATPase activities in subjects with methylenetetrahydrofolate reductase (MTHFR) 677 C→T genotype and moderate hyperhomocysteinaemia. The role of L-phenylalanine and L-alanine
- Matrix metalloproteinases and their inhibitors in different acute stroke subtypes
- Pyrosequencing protocol requiring a unique biotinylated primer
- Detection of antibodies against 60-, 65- and 70-kDa heat shock proteins in paediatric patients with various disorders using Western blotting and ELISA
- Quantitative determination of erythrocyte folate vitamer distribution by liquid chromatography-tandem mass spectrometry
- Time-level relationship between indicators of oxidative stress and Glasgow Coma Scale scores of severe head injury patients
- Stepwise strategies in analysing haematuria and leukocyturia in screening
- Elevation of serum cerebral injury markers correlates with serum choline decline after coronary artery bypass grafting surgery
- Drug screening in urine by cloned enzyme donor immunoassay (CEDIA) and kinetic interaction of microparticles in solution (KIMS): a comparative study
- Release of anandamide from blood cells
- Rapid decrease in plasma D-lactate as an early potential predictor of diminished 28-day mortality in critically ill septic shock patients
- Evaluation of an immunoassay of whole blood sirolimus in pediatric transplant patients in comparison with high-performance liquid chromatography/tandem mass spectrometry
- Sample processing and its preanalytical impact on the measurement of circulating matrix metalloproteinases
- Physiological matrix metalloproteinase (MMP) concentrations: comparison of serum and plasma specimens
- Importance of the functional sensitivity determination of a serum hyaluronic acid assay for the prediction of liver fibrosis in patients with features of the metabolic syndrome
- The dilemma of invasive and non-invasive investigations for adult and paediatric non-alcoholic fatty liver disease: has the time come for a new biochemical marker?
- Is cystatin C a reliable renal marker in trauma?
- On the independence of intraindividual reference values
- Sixth International Symposium on Molecular Diagnostics, Graz, Austria, May 25-27, 2006
Articles in the same Issue
- Natriuretic peptides and evidence-based quality specifications
- Preanalytical variability: the dark side of the moon in laboratory testing
- Clinical relevance of biological variation: the lesson of brain natriuretic peptide (BNP) and NT-proBNP assay
- Hepatorenal syndrome
- Modified Levey-Jennings charts for calculated laboratory tests
- Increased free malondialdehyde concentrations in smokers normalise with a mixed fruit and vegetable juice concentrate: a pilot study
- The exponentially weighted moving average (EWMA) rule compared with traditionally used quality control rules
- Intermethod calibration of alanine aminotransferase (ALT) and γ-glutamyltransferase (GGT) results: application to Fibrotest® and Actitest® scores
- Comparison of TEST 1 with SRS 100 and ICSH reference method for the measurement of the length of sedimentation reaction in blood
- Multicenter evaluation of the interference of hemoglobin, bilirubin and lipids on Synchron LX-20 assays
- Technical evaluation of the Beckman Coulter OV-Monitor (CA 125 antigen) immunoassay
- Erythrocyte membrane Na+,K+-ATPase and Mg2+-ATPase activities in subjects with methylenetetrahydrofolate reductase (MTHFR) 677 C→T genotype and moderate hyperhomocysteinaemia. The role of L-phenylalanine and L-alanine
- Matrix metalloproteinases and their inhibitors in different acute stroke subtypes
- Pyrosequencing protocol requiring a unique biotinylated primer
- Detection of antibodies against 60-, 65- and 70-kDa heat shock proteins in paediatric patients with various disorders using Western blotting and ELISA
- Quantitative determination of erythrocyte folate vitamer distribution by liquid chromatography-tandem mass spectrometry
- Time-level relationship between indicators of oxidative stress and Glasgow Coma Scale scores of severe head injury patients
- Stepwise strategies in analysing haematuria and leukocyturia in screening
- Elevation of serum cerebral injury markers correlates with serum choline decline after coronary artery bypass grafting surgery
- Drug screening in urine by cloned enzyme donor immunoassay (CEDIA) and kinetic interaction of microparticles in solution (KIMS): a comparative study
- Release of anandamide from blood cells
- Rapid decrease in plasma D-lactate as an early potential predictor of diminished 28-day mortality in critically ill septic shock patients
- Evaluation of an immunoassay of whole blood sirolimus in pediatric transplant patients in comparison with high-performance liquid chromatography/tandem mass spectrometry
- Sample processing and its preanalytical impact on the measurement of circulating matrix metalloproteinases
- Physiological matrix metalloproteinase (MMP) concentrations: comparison of serum and plasma specimens
- Importance of the functional sensitivity determination of a serum hyaluronic acid assay for the prediction of liver fibrosis in patients with features of the metabolic syndrome
- The dilemma of invasive and non-invasive investigations for adult and paediatric non-alcoholic fatty liver disease: has the time come for a new biochemical marker?
- Is cystatin C a reliable renal marker in trauma?
- On the independence of intraindividual reference values
- Sixth International Symposium on Molecular Diagnostics, Graz, Austria, May 25-27, 2006
