I. Daskalakis

Determination of Plasma Total Homocysteine and Cysteine Using HPLC with Fluorescence Detection and an Ammonium 7‐fluoro‐2,1,3‐benzoxadiazole‐4‐sulphonate (SBD‐F) Derivatization Protocol Optimized for Antioxidant Concentration, Derivatization Reagent Concentration, Temperature and Matrix pH

A sensitive HPLC-fluorescence method for determining total endogenous plasma homocysteine (Hcy), cysteine (Cys) and cysteinylglycine (Cys-Gly) following derivatization with ammonium 7-fluoro 2,1,3-benzoxadiazole-4-sulphonate (SBD-F) is described. Quantitation utilizes an internal standard, 2-mercaptoethylamine. The derivatization procedure has been optimized for concentration of SBD-F, reducing agent (tributylphosphine) and temperature. Findings indicate that values for plasma determinations vary according to the nature of the matrix in which calibration standards are made up. If quantitation is based on a peak height ratio, then standards should be made up in either pH 7.4 phosphate buffered saline or plasma taking into account the endogenous thiol concentration. These findings are based on calibration data, and 30 plasma samples quantified using thiol standards made up in plasma, pH 7.4 and pH 9.5 buffers. By defining how this matrix/pH effect influences thiol quantitation, it should be possible to make a more meaningful comparison of Hcy measurements between laboratories. The chromatographic separation was investigated at several mobile-phase pH values with the following conditions ascertained to be optimal: a mobile phase consisting of 5% (v/v) acetonitrile in 0.1 M KH2PO4, pH 2.15 was run at a flow rate of 0.5 mL/min. It was used in conjunction with a Supelco LC-18 base deactivated analytical column (150 x 4.6 cm i.d. 3 microM bonded silica). The internal standard and thiols were measured by fluorescence detection at 385 nm excitation and 515 nm emmission. Plasma levels are easily measured in a 100 microL volume. Storage for 2 months at -20 degrees C resulted in no deterioration of thiols. Furthermore, no difference in thiol levels was observed between bloods collected in lithium heparin and EDTA. Collected blood should, however, be separated as soon as possible to avoid red cell metabolism of Hcy which was observed in a case of hyperhomocysteinemia. Once derivatized, thiols are stable for at least one week at +4 degrees C.

A critical role for B-vitamin nutrition in human developmental and evolutionary biology

Several molecular mechanisms that underpin genomic integrity and function are sensitive to B-vitamin status, and in particular, are responsive to the interaction between folate nutrition and folate dependent enzyme polymorphisms. Mechanisms that may be affected include maintenance of genomic CpG methylation patterns for regulated gene expression, and proficient synthesis of nucleotides to prevent DNA strand breakage. We review some of these important molecular mechanisms that underpin the role of folate and other B-vitamins in disease processes, and discuss how they explain why this vitamin may be an important factor in human evolution. We look at three particular phenomena that link folate status/genes to evolutionary pressures: 1) The evolution of pigmentation to avert the impact of UV light on labile folates needed for developmental processes. 2) The effect of C677T-MTHFR on foetal survival, and 3) the ability of folate to mask harmful developmental mutations.

C677T MTHFR genotypes show graded response to vitamin B12 dependent regeneration of tetrahydrofolate, the main congener of all cellular folates

Abstract The cellular folate pool is influenced by the common C677T-MTHFR single nucleotide polymorphism (SNP). We show that vitamin B 12 may also be important in the context of this SNP, particularly in maintaining levels of monoglutamyl H 4 folate which is critical for all cellular folate cycles. 27 subjects were examined. Significant, but differing linear associations consistent with the known properties of methionine synthase were found to exist between serum B 12 and erythrocyte H 4 folate for MTHFR-CC genotype, MTHFR-CT genotype and MTHFR-CT+TT genotypes. Carriage of the mutant T allele was associated with reduced levels of H 4 folate for a given concentration of vitamin B 12 . This finding is consistent with the T allele conferring reduced 5,10MTHFR activity, and hence reduced availability of monoglutamyl 5methyl-H 4 folate for regeneration of H 4 folate via B 12 dependent methionine synthase. This conclusion was further substantiated by the genotype dependent relationships between vitamin B 12 and homocysteine (Hcy), vitamin B 12 and 5methyl-H 4 folate, and H 4 folate and Hcy. These findings indicate the nutritional importance of vitamin B 12, as well as folate, in the many clinical conditions where C677T-MTHFR is an aetiological factor.