Multiplex quantitative PCR using self-quenched primers labeled with a single fluorophore. Nucleic Acids Res 2002;30:e37. 24. Nazarenko I, Pires R, Lowe B, Obaidy M, Rashtchian A. Effect of primary and secondary structure of oligodeoxyribonucleotides on the fluorescent proper- ties of conjugated dyes. Nucleic Acids Res 2002;30:2089 –195. 25. Collins ML, Irvine B, Tyner D, Fine E, Zayati C, Chang C, et al. A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules/ml. Nucleic Acids Res 1997;25:2979 – 84. 26. Moser MJ, Marshall DJ, Grenier JK, Kieffer CD, Killeen AA, Ptacin JL, et al. Exploiting the enzymatic recognition of an unnatural base pair to develop a universal genetic analysis system. Clin Chem 2003;49:407–14. 27. Piccirilli JA, Krauch T, Moroney SE, Benner SA. Enzymatic incorporation of a new base pair into DNA and RNA extends the genetic alphabet. Nature 1990;343:33–7. 28. Coyne SR, Craw PD, Norwood DA, Ulrich MP. Comparative analysis of the Schleicher and Schuell IsoCode Stix DNA isolation device and the Qiagen QIAamp DNA Mini Kit. J Clin Microbiol 2004;42:4859 – 62. 29. Bricker BJ, Halling SM. Differentiation of Brucella abortus bv. 1, 2, and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv. 1 by PCR. J Clin Microbiol 1994;32:2660 – 6. 30. Afonina I, Zivarts M, Kutyavin I, Lukhtanov E, Gamper H, Meyer RB. Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder. Nucleic Acids Res 1997;25:2657– 60. 31. Kutyavin IV, Lukhtanov EA, Gamper HB, Meyer RB. Oligonucleotides with conjugated dihydropyrroloindole tripeptides: base composition and back- bone effects on hybridization. Nucleic Acids Res 1997;25:3718 –23. 32. Hartman LJ, Coyne SR, Norwood DA. Development of a novel internal positive control for TaqMan based assays. Mol Cell Probes 2005;19:51–9. DOI: 10.1373/clinchem.2005.052522 Effects of Folic Acid Before and After Vitamin B 12 on Plasma Homocysteine Concentrations in Hemodialysis Patients with Known MTHFR Genotypes, Anna Pastore, 1 Sandro De Angelis, 3 Stefania Casciani, 2 Rosalba Ruggia, 2 Gianna Di Giovamberardino, 1 Annalisa Noce, 3 Giorgio Splen- diani, 3 Claudio Cortese, 2 Giorgio Federici, 2 and Mariarita Dessı ` 2* ( 1 Biochemistry Laboratory, Children’s Hospital and Research Institute “Bambino Gesu ` ”, Rome, Italy; 2 Department of Laboratory Medicine and the 3 Nephrol- ogy and Dialysis Unit, University Hospital “Tor Vergata”, Rome, Italy; * address correspondence to this author at: Department of Laboratory Medicine, University Hospital Tor Vergata“, Viale Oxford 81, 00133 Rome, Italy; fax 39-06-20902357, e-mail mariarita.dessi@ptvonline.it) Background: Treatment with folic acid and vitamin B 12 appears to be effective in lowering total plasma homo- cysteine (tHcy) concentrations, but whether vitamin B 12 alone lowers tHcy in patients with normal vitamin B 12 status is unknown. The aims of the present study were to explore the effect of individual supplementation with folic acid or vitamin B 12 on tHcy concentrations in hemodialysis (HD) patients and to compare changes in tHcy concentrations with MTHFR genotype. Methods: We recruited 200 HD patients (119 men) from the “Umberto I” Hospital (Frosinone, Italy) and the Dialysis Unit of University Hospital “Tor Vergata”. These patients were randomized blindly into 2 groups of 100 each. Unfortunately, during the study, 36 patients in the first group and 16 in the second group died. The first group was treated initially with vitamin B 12 for 2 months and with folic acid for a following 2 months. The second group was treated initially with folic acid and then with vitamin B 12 . Samples were drawn before administration of either, after the first and second peri- ods, and again 2 months after treatment. Results: The concentrations of tHcy decreased in both groups after the consecutive vitamin therapies, and the decrease was genotype-dependent. The decrease was greater for the T/T genotype (P <0.05) and was more significant when the treatment was started with folic acid (P <0.01). Conclusion: The alternating vitamin treatment demon- strated for the first time the importance of folate therapy and the secondary contribution of vitamin B 12 in lower- ing tHcy in HD patients. © 2006 American Association for Clinical Chemistry Homocysteine (Hcy) is a non–protein-forming sulfur amino acid that is synthesized from methionine. Hcy can be either remethylated to methione or catabolized through the transsulfuration pathway to form cysteine (1). Hyperhomocysteinemia has been associated with atherosclerosis and arterial thrombosis (2), and evidence suggests that metabolism of folate, vitamin B 12 , and Hcy is under genetic control. In patients undergoing hemodialysis (HD), the rate of mortality from cardiovascular disease is 10- to 20-fold greater than that seen in the general population, even after correction for age, sex, race, and the presence of diabetes (3). Hyperhomocysteinemia is common in HD patients, with 90% of dialysis patients having increased concentrations of Hcy. Increased plasma total Hcy (tHcy) concentrations result chiefly from genetic defects in the enzymes involved in Hcy metabolism (4). Recently, a common C3 T mutation at nucleotide position 677 (C677T) has been identified in the gene coding for methylenetetrahydrofolate reductase (MTHFR), which is involved in the remethylation of Hcy (5). The C677T mutation causes a valine-for-alanine sub- stitution, which decreases MTHFR activity and tends to increase tHcy concentrations in individuals who are ho- mozygous for the T/T genotype (5). In individuals with healthy renal function, the T/T genotype causes only a 25% increase in tHcy concentra- tion compared with persons with other genotypes (6), but in patients with end-stage renal disease undergoing main- tenance dialysis, the T/T genotype causes a 40% to 100% increase in tHcy (7). Folic acid is vital in humans for several metabolic reactions, including the remethylation pathway. How- ever, clinical studies have shown that folic acid therapy is not very effective in normalizing hyperhomocysteinemia in uremic patients (8). In a study by Kaplan et al. (9), vitamin B 12 supplementation alone, or in combination with folic acid, decreased tHcy concentrations, but full normalization was not achieved. Dierkes et al. (10 ) re- ported that supplementation with vitamin B 12 decreases not only tHcy but also serum folate in patients with Clinical Chemistry 52, No. 1, 2006 145 Downloaded from https://academic.oup.com/clinchem/article/52/1/145/5626623 by guest on 15 October 2022