Folate-homocysteine interrelations: potential new markers of folate status.

We report a transient drop in plasma Hcy and Cys following a single oral dose of PteGlu. The thiol change was concomitant with both the peak plasma 5CH3H4PteGlu1 level (by HPLC) and the maximum plasma Lactobacillus casei activity which reflects absorption of unmodified PteGlu. The significant reciprocal association of Hcy with radioassay RBC folate (r = -0.28, 99% CI -0.48, -0.05, P = 0.0016), serum folate (r = -0.37, 99% CI -0.56, -16, P = 0.0001), and vitamin B12 (r = -0.42, 99% CI -0.59, -21, P = 0.0001) is shown and reflects the long-term nutritional effect of B vitamins on this important, potentially atherogenic thiol. These are now well-established associations. We extend the potential for investigation of folate metabolism in health and disease by evaluating a range of new folate indices which are based on erythrocyte coenzymes. These have been looked at independently and in association with established parameters. Erythrocyte methylfolates (mono- to hexaglutamate-5CH3H4PteGlu1-6), formylfolates (tri- to pentaglutamate-5CHOH4PteGlu3-5),formiminotetrahydrofolate (formiminoH4PteGlu1), unsubstituted tetrahydrofolate (H4PteGlu1), andpara-aminobenzoylglutamate (P-ABG) have been measured by HPLC with fluorescence detection. A positive linear association exists between (i) H4PteGlu1 and radioassay RBC folate (r = 0.50, 99% CI 0. 07, 0.77, P = 0.0036), and (ii) H4PteGlu1 and tetraglutamates of both formyl- and methylfolate (r = 0.52, 99% CI 0.10, 0.78, P = 0. 0022, and r = 0.56, 99% CI 0.15, 0.80, P = 0.0009, respectively). Since, in addition, a reciprocal linear association exists between Hcy and tetraglutamyl formylfolate (r = -0.41, 99% CI -0.73, 0.05, P = 0.0206), erythrocyte tetraglutamates may be a good reflection of the bodies' active coenzyme pools. Pentaglutamyl formylfolate, the longest oligo-gamma-glutamyl chain form of this coenzyme may be a good indicator of folate depletion. The abundance of this coenzyme both increases with increasing Hcy (r = 0.55, 99% CI 0.13, 0.80, P = 0.0014) and increases as H4PteGlu1, the principle folate congener, decreases (r = -0.59, 99% CI -0.82, -0.20, P = 0.0004). Furthermore, the apparent equilibrium between substrate (5CH3H4PteGlu1) and product (H4PteGlu1) of methionine synthase is significantly associated with the abundance of 5CHOH4PteGlu5 (r = -0.53, 99% CI -0. 79, -0.11, P = 0.0018). This suggests that low methionine synthase activity for whatever reason (metabolic or dietary) may lead to an increase in the relative abundance of 5CHOH4PteGlu5. Like 5CHOH4PteGlu5, evidence is given that 5CH3H4PteGlu6, the longest oligo-gamma-glutamyl chain form of this particular coenzyme pool, may also be a good indicator of folate depletion. This is shown by a change in the relative proportion of erythrocyte methylfolate polyglutamates following supplementation with 400 microg/day PteGlu. Short-chain polyglutamates of methylfolate (5CH3H4PteGlu1--> 5CH3H4PteGlu4) increase in proportion to the total methylfolate pool, while long-chain polyglutamates of methylfolate (5CH3H4PteGlu5 and particularly 5CH3H4PteGlu6) decrease in their relative abundance.

Impaired regeneration of monoglutamyl tetrahydrofolate leads to cellular folate depletion in mothers affected by a spina bifida pregnancy.

Periconceptional folate prevents neural tube defects (NTD) by a mechanism which is unclear. The present study found significant changes in the equilibrium of the homocysteine remethylation cycle in NTD affected mothers, possibly involving B12-dependent methionine synthase or 5,10-methylenetetrahydrofolate reductase. Data were consistent with impaired Hcy remethylation leading to poor regeneration of H4PteGlu1, the main intracellular precursor of all folates. This lesion leads to cellular folate deficiency indicated by a significantly lower radioassay RBC folate and 5CH3H4PteGlu4 in affected mothers. The drop in this tetraglutamate is associated with an increase in the abundance of longer chain oligo-gamma-glutamyl folate, again reflecting the underlying folate deficiency. This effect may compromise purine, DNA-thymine, and methionine production, particularly during embryogenesis when folate demand is high. At this time serine hydroxymethyltransferase may play a critical role in conserving H4PteGlu1 for purine synthesis. Many of these depletion effects were corrected with folate supplementation for 1 month.

Risk of neural tube defect-affected pregnancy is associated with a block in maternal one-carbon metabolism at the level of N-5-methyltetrahydrofolate:homocysteine methyltransferase.

The disposition of whole blood mono-to hexaglutamyl methylfolate and plasma homocysteine (HCY) was used to evaluate potential lesion sites in one-carbon metabolism which could be responsible for neural tube defect(NTD)-affected pregnancies. An isocratic high-performance liquid chromatographic system (HPLC) with photodiode array detection was used to quantify and speciate whole-blood methylfolate into mono-, di-, tri-, tetra-, penta-, and hexaglutamate forms. This technique was also used with off-line radioassay to identify nonmethyl whole-blood folates. Isocratic HPLC with fluorescence detection was used to quantify SBDF derivatized homocysteine in plasma. The study investigated blood from 11 women who had experienced a previous NTD-affected pregnancy and 11 controls of similar age and social class. No subjects were pregnant. HCY levels were significantly higher in NTD subjects (P = 0.0486, 95% CI-2.799,0.001 using the Mann-Whitney test), as was the ratio of known intracellular (tri-to hexaglutamyl) methylfolate compared to extracellular (mono- and diglutamyl) methylfolate (P = 0.0062 95% CI-0.543, 3.862 using the Mann-Whitney test). Vitamin B12, red cell folate, circulating total methylfolate, and circulating mono-to hexaglutamyl methylfolates showed no difference between population groups. The disposition between individual and cumulative glutamate chain lengths of methylfolate showed significant trends which differed between population groups: (i) total blood methylfolate (Glu1-6) appears to be utilized by N-5-methyltetrahydrofolate:homocysteine methyltransferase (MS) in control blood but not NTD blood, where it appears to accumulate following a 45-min incubation; (ii) whole-blood hexaglutamyl methylfolate (5CH3-H4PteGlus) becomes a larger proportion of the total blood methylfolate in NTD than in control populations; and (iii) the intermediate glutamate chains of methylfolate (Glu1-5) remain relatively constant as 5CH3-H4PteGlu6 accumulates in NTD but appear to increase linearly with 5CH3-H4PteGlu6 in controls. The significant elevation of HCY in the NTD population is associated with the increasing proportion of 5CH3-H4PteGlu6 relative to the total methylfolate, since, when corrected for HCY level, the proportion of 5CH3-H4PteGlus to total methylfolate is similar in NTD and control populations. These trends are consistent with a defect at the level of vitamin B12 dependent MS which "traps" folate at the 5CH3-H4PteGlus level.