High-affinity folate binding in human prostate

Binding of³H-folate in Triton X-100 solubilized human prostate homogenate was of a high-affinity type and displayed apparent positive cooperativity typical of specific folate binding. Radioligand dissociation was slow at pH 7.4, but rapid at pH 3.5. Gel chromatography reveled two major folate binding proteins (M_r100 and 25kDa), but only one single band (M_r65–70 kDa) was detectable on SDS-PAGE and immunoblotting with rabbit-anti human milk folate binding protein. Concentration of folate binding protein in prostate homogenate expressed as maximum³H-folate binding was 1.10 nmol/g protein, and the cross-reactivity with rabbit-anti human milk folate binding protein serum was 15% as determined by an enzyme-linked immunosorbent assay (median values; n=6).     

Simple method for the quantitative analysis of endogenous folate catabolites p-aminobenzoylglutamate (pABG) and its acetamido (apABG) derivative in human serum and urine by liquid chromatography-tandem mass spectrometry

OBJECTIVE: To develop a routine method for quantitative measurement of the folate catabolites p-aminobenzoylglutamate (pABG) and acetamidobenzoylglutamate (apABG) in serum and urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS). DESIGN AND METHODS: Urine, serum and aqueous standards were thawed. Two microliters of d3-glutamic acid (d3-Glu; 1 mmol/L) was added to 200 uL of specimen as internal standard. The samples were acidified with 4 uL 6N HCL, and aliquots were precipitated with 2 volumes (412 uL) of acetonitrile. For urine specimens 30 volumes (6.18 mL) of acetonitrile was used. Samples were centrifuged at 1900 x g for 10 min and the supernatant (10 microL) injected into a Biorad CAT/MET analytical column fitted to the LC-MS/MS. Detection of the catabolites was by selective multiple ion monitoring (multiple SRM) of the respective transitions. Urine and serum samples were analysed in a group of healthy volunteers and in anonymous samples from patients being tested for PTH and urinary catecholamines. RESULTS: pABG and apABG eluted at 5.2 and 4.74 min, respectively while the d3-glutamic acid eluted at around 7 min. Limit of quantitation (LOQ) for both catabolites was 10 nmol/L (which is equivalent to 33.3 fmol for a 10 microL injection). Limit of detection (LOD) was 1 nmol/L based on a signal to noise ratio of 5:1. A linear calibration curve was obtained from 10 to 100 nmol/L for serum specimens and from 10 to 200 micromol/L for urines. Imprecision for spiked serum samples (n=10) was between 2.5 and 20% for apABG and 4.5 and 21% for pABG (at 10 and 100 nmol/L, respectively). Imprecision for spiked urine samples (n=10) was between 2.9 and 4.0% for apABG and 6.0-12.7% for pABG. Recoveries were between 80 and 122% for serum samples and between 92 and 102% for urine specimens. Total folate catabolites in random urine samples from volunteers (n=5) are 2.9+/-2.3 umol/L (mean+/-S.D.). This group also had total serum catabolites of 11.9+/-7.6 nmol/L and serum folate of 35.3+/-5.8 nmol/L. Serum from patients being tested for PTH (n=11) had serum folate levels of 27.0+/-10.4 nmol/L with total serum catabolites of 20.4+/-23.8 nmol/L. Levels of serum folate and total catabolites in pregnant women (n=18) were 33.9+/-22.7 and 11.4+/-8.7 nmol/L, respectively. Mean urinary folate catabolites in patients being tested for urinary catecholamines (n=19) was 581.8+/-368.4 nmol/L. CONCLUSION: A simple, reliable and highly specific method by LC-MS/MS for detecting and quantifying the folate catabolites pABG and apABG was developed. This enables, for the first time, the routine clinical analysis of folate utilization in patients.     

Additional food folate derived exclusively from natural sources improves folate status in young women with the MTHFR 677 CC or TT genotype

The effectiveness of additional food folate in improving folate status in humans is uncertain particularly in people with the common genetic variant (677 C-->T) in the methylenetetrahydrofolate reductase (MTHFR) gene. To examine the effect of a doubling of food folate consumption on folate status response variables, women (n=32; 18-46 years) with the MTHFR 677 CC or TT genotype consumed either 400 (n=15; 7 CC and 8 TT) or 800 (n=17; 8 CC and 9 TT) microg/day of dietary folate equivalents (DFE) derived exclusively from naturally occurring food folate for 12 weeks. A repeated measures two-factor ANOVA was used to examine the effect of the dietary treatment, the MTHFR C677T genotype and their interactions on serum folate, RBC folate and plasma total homocysteine (tHcy) during the last 3 weeks of the study. Consumption of 800 microg DFE/day resulted in serum folate concentrations that were 67% (P=.005) higher than consumption of 400 microg DFE/day (18.6+/-2.9 vs. 31.0+/-2.7 nmol/L, respectively) and RBC folate concentrations that were 33% (P=.001) higher (1172+/-75 vs. 1559+/-70 nmol/L, respectively). Serum folate (P=.065) and RBC folate (P=.022) concentrations were lower and plasma tHcy was higher (P=.039) in women with the MTHFR 677 TT genotype relative to the CC genotype. However, no genotype by dietary treatment interaction was detected. These data suggest that a doubling of food folate intake will lead to marked improvements in folate status in women with the MTHFR 677 CC or TT genotype.     

Quantification of key folate forms in serum using stable-isotope dilution ultra performance liquid chromatography–tandem mass spectrometry

Folates act as essential coenzymes in many biological pathways. Alteration in folate form distribution might have biological significance, especially in relation to certain genetic polymorphisms. We developed a stable-isotope dilution ultra performance liquid chromatography–mass spectrometry (UPLC–MS/MS) method for quantification of the folate forms 5-methyltetrahydrofolate (5-methylTHF), 5-formylTHF, 5,10-methenylTHF, THF, and folic acid in serum. After extraction using an ion exchange and mixed mode solid-phase, samples were separated and detected using an UPLC–MS/MS system. The quantification limits were between 0.17 nmol/L (5-formylTHF) and 1.79 nmol/L (THF), and the assay was linear up to 100 nmol/L (5-methylTHF) and 10 nmol/L (5-formylTHF, 5,10-methenylTHF, THF, and folic acid). The intraassay CVs for 5-methylTHF and 5-formylTHF were 2.0% and 7.2%, respectively. Mean recoveries were between 82.3% for THF and 110.8% for 5,10-methenylTHF. Concentrations of total folate measured by the new method showed a strong correlation with those measured by an immunologic assay (r = 0.939; p < 0.001). The mean total folate from 32 apparently healthy subjects was 18.09 nmol/L, of which 87.23% was 5-methylTHF. Concentrations of homocysteine showed a better correlation to the total folate measured by the new method compared to that obtained by an immunologic assay. We also confirmed that MTHFR polymorphism has a significant effect on folate distribution in this small population of non-supplemented subjects.     

Complex interaction between serum folate levels and genetic polymorphisms in folate pathway genes: biomarkers of prostate cancer aggressiveness

Little is known about the role of folate and polymorphisms associated with folate metabolism on prostate cancer risk in populations of African origin. We examined the relationship between serum folate and prostate cancer and whether any association was modified by genetic polymorphisms for folate metabolism. The study was case–control in design and consisted of 218 men 40–80 years old with newly diagnosed, histologically confirmed prostate cancer and 236 cancer-free men attending the same urology clinics in Jamaica, March 2005–July 2007. Serum folate was measured by an immunoassay method and genomic DNA evaluated for MTHR (C677T and A1298C), MTRR A66G, and MTR A2756G polymorphisms. Mean serum folate concentration was higher among cases (12.3 ± 4.1 nmol/L) than controls (9.7 ± 4.2 nmol/L). Serum folate concentration showed a positive association with prostate cancer (OR, 4.41; CI, 2.52–7.72 per 10 nmol/L) regardless of grade. No interactions were observed between genotype and folate concentration, but a weak gene effect was observed for MTHFR A1298C and low-grade prostate cancer. Larger studies to investigate the role of gene–gene/gene–diet interactions in Black men are needed.     

High-affinity folate binding in human mammary gland

High-affinity³H-folate binding in Triton X-100 solubilized human mammary gland tissue displayed characteristics, e.g. apparent positive cooperativity and increasing affinity with decreasing concentration of folate binding protein, shown to be typical of specific folate binding. Radioligand dissociation was slow at pH 7.4. A major fraction of the bound radioligand dissociated rapidly at pH 3.5, while a residual binding of 20% persisted even after prolonged dialysis at pH 3.5. Gel chromatography revealed two major folate binding proteins (M_r100 kDa and 25 kDa). However, only one single band was detectable on SDS-PAGE immunoblotting. The highest folate binding activity per g protein was associated with the upper triglyceride-containing layer of the 1000 g supernatant of the homogenate. The folate binding protein extracted from this layer had a low cross-reactivity (<5%) with rabbit antibodies against 25 kDa human milk folate binding protein. The folate binding protein in the 1000 g pellet and the aqueous phase of the 1000 g supernatant was present at a low concentration and had a cross-reactivity of 100%.     

High-pressure liquid chromatography separation and determination of rat liver folates

The endogenous levels of the various folate compounds in rat liver were determined using high-pressure liquid chromatography for the rapid separation of folate monoglutamate forms with specific quantitation of the folates by microbiological analysis of eluted fractions. The eight folate derivatives that were assayable were tetrahydrofolic acid (H₄PteGlu), 5-methyl-H₄PteGlu, 10-formyl-H₄PteGlu, 5-formyl-H₄PteGlu, 5,10-methenyl-H₄PteGlu, 5,10-methylene-H₄PteGlu, H₂PteGlu, and PteGlu. New techniques for the preparation of tissues were developed in order to reduce the degradation of the folates. Tissue folates were converted to the monoglutamate form by a partially purified hog kidney polyglutamate hydrolase preparation and incubations were carried out at pH 6.0. This minimized folate degradation but still allowed for maximal polyglutamate hydrolase activity. Rapid removal of tissues was compared with freeze-clamping techniques. The major folates in rat liver were H₄PteGlu and 5-methyl-H₄PteGlu, comprising 42 and 39%, respectively, of the total liver folate pool of 27.30 nmol/g liver (about 13 μg/g liver). In addition, 10-formyl-H₄PteGlu and 5-formyl-H₄PteGlu each comprised 10% of the total folate pool. No endogenous PteGlu, H₂PteGlu, or 5,10-methylene-H₄PteGlu was detected in rat liver samples under our conditions. Distribution of ¹⁴C derived from a previous [¹⁴C]folic acid injection paralleled the distribution of folate as determined microbiologically after high-pressure liquid chromatography separation. The importance of these methods for the direct determination and estimation of flux of H₄PteGlu, 5-methyl-H₄PteGlu, and 10-formyl-H₄PteGlu in studies dealing with the folate system was emphasized.     

Abnormal folate metabolism in feed-related anemia of cultured channel catfish

"No-blood disease" is a severe, feed-related anemia of channel catfish. Pathological features in advanced cases include almost total absence of circulating red blood cells, hepatic fatty change, megaloblastic arrest of hematopoiesis, and intussusceptions of the small intestine. In view of similarities to folate deficiency in man studies were made regarding the intake and metabolism of folic acid. When a bacterial culture medium, containing inorganic salts and folic acid as the sole carbon source, was inoculated with a small sample of anemia-producing feed, over 95% of the folate was destroyed. A yellow precipitate formed and had the characteristic uv spectrum of pteroic acid; it represented 32% of the folate originally present. Differential microbiological assays revealed that the same anemia-producing feed contained 20 times as much folate activity for Streptococcus fecalis as for Lactobacillus casei (59 micrograms/g vs 2.6 micrograms/g). This growth response is compatible with an excess of pteroic acid and/or formyl-pteroic acid which support the former but not the latter organism. Plasma folate activity (mean +/- SD, ng/ml) assayed with L. casei and S. fecalis in 22 normal catfish (hematocrit range 32-43) was 17.2 +/- 6.2 and 23.4 +/- 13.8, respectively. Comparable values in 15 anemic catfish (hematocrit range 0 to 30) were 35.5 +/- 33.7 and 58.7 +/- 76.5. The mean plasma pteroate activity, estimated by subtraction, was 6.2 and 23.2 ng/ml, respectively, in normal and anemic fish. Fingerling catfish raised under controlled conditions on feed containing 130 mg/kg of pteroic acid failed to gain weight and developed anemia with the characteristic red cell morphologic features that are seen in the naturally occurring disease. We conclude that severe anemia in channel catfish can be caused by abnormal folate metabolism and may be due to ingestion of folic acid-breakdown products, such as pteroic acid. It is postulated that microorganisms in contaminated feed synthesize folate which, in turn, is converted to pteroate by a pseudomonad or similar organism.     

Gene-nutrient and gene-gene interactions of controlled folate intake by Japanese women

Elevated serum total homocysteine (tHcy) levels are associated with increased risk for cardiovascular disease and dementia. The prevalence rates of homozygous mutants among Japanese women (n = 300) were 17.3%, 1.3%, 18.6%, and 5.3% for methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C, reduced folate carrier (RFC-1) A80G, and methionine synthase (MS) A2756G, respectively. The tHcy value was significantly lower (p < 0.001) in young women with CC or CT of MTHFR than with TT (10.9+/-4.7 micromol/L) (n =250). Diversities of serum folate and tHcy in women with 23 combinations of different alleles at low folate intake converged to the highest (34.0+/-8.6 nmol/L) and lowest (7.6+/-1.5 micromol/L) levels, respectively, after folic acid (400 microg/day) supplementation. In the regression equation ( y= ax + b) of serum folate ( y nmol/L) plotted against mean folate intake ( x microg/day), the values of "a" were 0.032, 0.037, and 0.045 for individuals with CC, CT, and TT alleles, respectively, of MTHFR.     

Comparison of folate quantification in foods by high-performance liquid chromatography-fluorescence detection to that by stable isotope dilution assays using high-performance liquid chromatography-tandem mass spectrometry

A comparison study on folate quantitation was carried out between the recently developed stable isotope dilution assay using liquid chromatography-tandem mass spectrometry (LC-MS-MS) and the frequently used HPLC with fluorimetric detection (LC-FD). By applying LC-MS-MS, spinach, wheat bread, beef, and blood plasma were found to contain 159.2, 19.8, 1.2, and 5.6 microg/100 g total folates, respectively, whereas the respective quantitative data obtained by LC-FD were 95.5, 16.2, 0.7, and 6.8 microg/100 g. In all samples, LC-MS-MS revealed superior selectivity and precision and circumvented the shortcomings of conventional LC techniques, i.e., ambiguous peak assignment as well as high detection limits for 5-formyltetrahydrofolate, 10-formylfolic acid, and folic acid. The affinity chromatography columns used in this study showed excellent cleanup performance and permitted detection limits as low as 0.1, 0.5, 0.1, 0.08, and 0.1 microg/100 g for tetrahydrofolate (H(4)folate), 5-methyl-H(4)folate, 5-formyl-H(4)folate, 10-formylfolate, and pteroylglutamic acid, respectively. Thus, a 10-fold higher sensitivity compared to solid-phase anion-exchange cartridges was achieved. However, affinity chromatography columns revealed a significantly higher affinity toward the natural vitamers than to the racemic isotopomeric standards, which has to be considered when applying the latter in stable isotope dilution assays.     

Folate concentrations and folic acid supplementation among women in their first trimester of pregnancy in a rural area with a high prevalence of neural tube defects in Shanxi, China

BACKGROUND: Although an information campaign concerning periconceptional folic acid supplementation was launched in 1998 in Shanxi Province, China, the prevalence of neural tube defects in rural areas was reported as high as 140 per 10,000 births in 2002. The blood folate concentrations and the practice of folic acid supplementation among pregnant women in rural areas of the province are described. METHODS: A total of 483 pregnant women (mean gestation, 8.1 weeks) in a rural area of Shanxi were interviewed. Nonfasting blood samples and information on folic acid supplementation were collected. Folate concentrations in plasma and erythrocytes were determined by a microbiological assay. RESULTS: The mean concentrations of plasma and erythrocyte folate for pregnant women was 10.4 nmol/liter and 375.8 nmol/liter, respectively. Deficiencies of plasma and erythrocyte folate were observed in 20.9% and 47.6% of women, respectively. Seasonal variations were noted in the prevalence of folate deficiency, with significantly lower plasma folate concentrations in spring and summer and lower erythrocyte folate concentrations in seasons other than summer. Among pregnant women, <10% reported having taken or currently taking folic acid, and virtually no women (0.6%) took folic acid as recommended. CONCLUSIONS: Women in rural areas had low plasma and erythrocyte folate levels, and folate deficiency was highly prevalent in the area. Few women followed the recommendations regarding folic acid supplementation, and the information campaign in Shanxi was unsuccessful. These findings suggest the urgent need for combined strategies in rural areas to fortify grain with folic acid and promote folic acid supplements for childbearing-age women.     

Application of stable isotope dilution assays based on liquid chromatography-tandem mass spectrometry for the assessment of folate bioavailability

A pilot study was performed to prove the suitability of stable isotope dilution assays for assessing the bioavailability of endogenous folates in foods. By using [2H(4)]folic acid, [2H(4)]tetrahydrofolate, [2H(4)]5-methyltetrahydrofolate, [2H(4)]5-formyltetrahydrofolate and [2H(4)]10-formylfolic acid as internal standards, folates in spinach, apple juice and blood plasma were quantified by liquid chromatography coupled to tandem mass spectrometry. To liberate the pteroyl monoglutamates, sample extracts of foods were treated by rat plasma. Sample clean-up was achieved by solid-phase extraction on anion-exchange cartridges, which proved to be sufficient to obtain mass chromatograms devoid of matrix interferences. The bioavailability study was designed as a short-time protocol with three meals, the first consisting of 600 g spinach (meal A), the second consisting of 600 g apple sauce with additionally 400 microg synthetic folic acid (meal B) and the third consisting solely of 600 g apple sauce (meal C). Prior to the meals, the participating volunteer's tissue was saturated with folates to achieve a significant response of plasma folate to the meals. After consumption of meals A and B a significant rise in folate plasma level compared to meal C (mean level at 28 microg/ml) was observed. The relative bioavailability of folate following meal A exceeded significantly the suggested value of 50% for food folates by taking the dose-normalized area under the curve (AUC) following ingestion of meal B as reference.     

Solid-phase extraction-electrospray ionization mass spectrometry for the quantification of folate in human plasma or serum

The measurement of 5-methyltetrahydrofolic acid (5 MT) blood levels is one of several factors used to diagnose folate deficiency in humans. 5 can be selectively purified from either human plasma or human serum via solid-phase extraction procedures and specifically detected and quantified in the extracts with liquid chromatography/isotope-dilution electrospray-ionization mass spectrometry. Two different, yet complementary, solid-phase extraction-liquid chromatography/mass spectrometry methods have been developed and applied to the quantification of 5 MT from such extracts. One method utilizes the high-affinity folate-binding protein from cow's milk coupled with multiple-reaction-monitoring-mode tandem mass spectrometry while the other method utilizes reversed-phase C(18) extraction followed by selected-ion-monitoring-mode mass spectrometry. The accuracy of each method is assessed through a comparative determination of 5 MT levels in homogenous plasma and serum pools. Additionally, each method is compared and evaluated against the "total folate" results provided by routine radioassay and microbiological assay determinations. On the basis of the experimental data presented in this report, it is suggested that both methods have the capacity to serve as potential reference methods for the quantification of circulating 5MT in plasma or serum.     

Effects of dietary zinc deficiency on homocysteine and folate metabolism in rats

In rats, zinc deficiency has been reported to result in elevated hepatic methionine synthase activity and alterations in folate metabolism. We investigated the effect of zinc deficiency on plasma homocysteine concentrations and the distribution of hepatic folates. Weanling male rats were fed ad libitum a zinc-sufficient control diet (382.0 nmol zinc/g diet), a low-zinc diet (7.5 nmol zinc/g diet), or a control diet pair-fed to the intake of the zinc-deficient rats. After 6 weeks, the body weights of the zinc-deficient and pair-fed control groups were lower than those of controls, and plasma zinc concentrations were lowest in the zinc-deficient group. Plasma homocysteine concentrations in the zinc-deficient group (2.3 +/- 0.2 micromol/L) were significantly lower than those in the ad libitum-fed and pair-fed control groups (6.7 +/- 0.5 and 3.2 +/- 0.4 micromol/L, respectively). Hepatic methionine synthase activity in the zinc-deficient group was higher than in the other two groups. Low mean percentage of 5-methyltetrahydrofolate in total hepatic folates and low plasma folate concentration were observed in the zinc-deficient group compared with the ad libitum-fed and pair-fed control groups. The reduced plasma homocysteine and folate concentrations and reduced percentage of hepatic 5-methyltetrahydrofolate are probably secondary to the increased activity of hepatic methionine synthase in zinc deficiency.     

Genetic determinants of folate status in Central Bohemia

Although several genetic factors have been implicated as determinants of blood folate concentration in various populations, their effect on folate status in the Czech population has not yet been examined. We explored whether blood folate concentrations in healthy Czech population are associated with polymorphisms in 5,10-methylenetetrahydrofolate reductase (MTHFR), folate hydrolase 1 (FOLH1), reduced folate carrier (RFC), and folate receptor (FOLR1) genes. In a cross-sectional study of 591 control subjects we determined genotypes by PCR-RFLP or ARMS-PCR methods, and plasma and erythrocyte folates by MEIA. The effect of different genotypes on folate status was examined by non-parametric tests and by regression analysis. The prevalence of the MTHFR 677C>T, MTHFR 1298A>C, FOLH1 1561C>T, RFC 80G>A and FOLR1 480G>C variant alleles was 0.34, 0.33, 0.05, 0.44 and 0.00, respectively. Only the MTHFR 677C>T variant was significantly associated with plasma folate concentrations (median 14.7, 14.0 and 12.2 nmol/l for the CC, CT and TT genotypes, respectively). Our study showed that among the five studied allelic variants, only the 677C>T polymorphism in the MTHFR gene is a significant genetic determinant of plasma folate concentrations in Czech population.     

Characterization of a High-Affinity Folate Receptor in Normal and Malignant Human Testicular Tissue

We have characterized the folate receptor in normal and malignant tissue from male gonads. Radioligand binding displayed characteristics typical of other folate receptors. Those included a high-affinity type of binding (K = 10^{10 M–1}), apparent positive cooperativity changing into non-cooperativity at low receptor concentrations, a tendency to increased binding affinity with decreasing receptor concentrations, a slow dissociation at pH 7.4 becoming rapid at pH 3.5 and inhibition by folates, in particular oxidized forms. The gel filtration profile of Triton X-100 solubilized tissue contained a 25 and 100 kDa peak of radioligand-receptor. The latter peak could represent receptor equipped with a hydrophobic membrane anchor that inserts into Triton X-100 micelles. The concentration of radiolabelled receptor ranged from 0.41 nmol/g protein to 1.68 nmol/g protein in specimens of normal testicular tissue from patients with prostatic carcinomas and from 1.54 nmol/g protein to 3.82 nmol/g protein in testicular tissue from young individuals. Compared to normal testicular tissue the concentration of receptor in seminoma tissue was low (0.38–1.27 nmol/g protein) but showed a higher degree of immunoreactivity in the presence of antibodies against human milk folate binding protein as evidenced by ELISA and immunohistochemistry data. Hence a folate receptor isoform homologous to human milk folate binding protein is apparently expressed in seminomas where the total expression of receptor, however, seems to be lower than in normal testicles.     

On the nature of folate derivatives in neurospora crassa and the effect of sulphanilamide

Summary The folic acid complex of Neurospora crassa has been separated by chromatography on DEAE-cellulose column and the nature of the individual derivatives ascertained from microbiological response. The functional groups of the polyglutamyl folates were ascertained.Major portion of Neurospora folates is composed of the polyglutamyl derivatives, as seen from the increase in activities for Streptococcus faecalis R, Pediococcus cerevisiae, and Lactobacillus casei after conjugase treatment.The presence of N¹⁰ and N⁵-formyl and N⁵-methyl tetrahydrofolic acid, N⁵-methyl diglutamyl tetrahydrofolic acid, two formylated derivatives of polyglutamates, and two N⁵-methyl polyglutamyl folates was ascertained in the folate complex of Neurospora crassa.Sulphanilamide growth inhibition results in the lowering of all the Neurospora folate derivatives.     

Endogenous folate of normal fibroblasts using high-performance liquid chromatography and modified extraction procedure

The endogenous levels of the various folate monoglutamate compounds in cultured human fibroblasts were determined using high-performance liquid chromatography for the separation of folate monoglutamate. Endogenous folates were converted to monoglutamate forms using conjugase enzyme present in rat serum and incubation was carried out at pH 6.5. This minimized folate coenzyme interconversion during processing. Using methanol for precipitation of protein instead of heat minimized degradation of labile folates. Recovery of all folates except 10-formyltetrahydrofolic acid (10-CHO H4PteGlu) using this procedure was more than 90%. Disruption of cells by boiling appeared to cause less postextraction changes of cell folates than did freezing and thawing or sonication. When heat to release endogenous folate, conjugase treatment with rat serum at pH 6.5, and precipitation of protein with methanol were used, more than half of the intracellular folate of normal fibroblasts in confluent growth was 5-methyltetrahydrofolic acid (5-CH3 H4PteGlu), and 10-CHO H4PteGlu and tetrahydrofolic acid (H4PteGlu) comprised 29 and 6%, respectively.     

Effect of high levels of dietary folic acid on folate metabolism in vitamin B12 deficiency

The effect of administering high levels of folic acid to vitamin B12-deficient animals was studied. In B12 deficiency histidine oxidation is decreased. This is the result of both decreased liver folate levels and increases in the proportion of methyltetrahydrofolates. The purpose of this study was to determine if the addition of very high levels of folic acid to B12-deficient diets could increase liver folates and thereby restore histidine oxidation. Rats were fed a soy protein B12-deficient diet containing 10% pectin which has been shown previously to accelerate B12 depletion. When this diet was supplemented with B12 and folic acid, histidine oxidation was 5.4% in 2 h and the livers contained 3.49 micrograms of folate/g. In the absence of B12, the histidine oxidation rate was 0.34% and the liver folate level was 1.33 micrograms/g. When 200 mg/kg of folic acid was added to the B12-deficient diet there was no increase in histidine oxidation (0.35%) but the liver folates were increased to 3.68 micrograms which is about the same as that with B12 supplementation. The percentage tetrahydrofolate of the total liver folates was the same with and without a high level of dietary folic acid. Thus there was an increase in the absolute level of tetrahydrofolate without any increase in folate function as measured by histidine oxidation. Red cell folate levels were the same with and without B12, which is in contrast to the markedly lower liver folate levels in B12 deficiency. These data suggest a difference between B12 regulation of folate metabolism in the liver and in the bone marrow.     

The impact of plasma folate levels of mothers and newborns on intrauterine growth retardation and birth weight

Folate plays an important role in the genomic stability of human cells. In our studies of the impact of environmental pollution on human health, we have found that air pollution can affect pregnancy outcome. As it may be also affected by nutrition, we examined the effect of plasma folate levels of mothers and newborns on intrauterine growth retardation (IUGR) and birth weight (BW) in cohorts from Prague (N=319) and Teplice (N=444). The lower plasma levels (<6.1 nmol/L) were observed in 7.1% of mothers in Prague, and in 9.6% of mothers in Teplice. The higher plasma levels (>36.5 nmol/L) were observed in 28.4% of mothers in Prague, and in 15.7% of mothers in Teplice. The higher plasma levels were observed in 75.4% of newborns in Prague, and 73.2% of newborns in Teplice. When comparing maternal high versus low folate plasma levels and IUGR by logistic regression, the risk of IUGR was significantly decreased for European cohort (according to ethnicity) with gestation age >36 weeks [N=536, OR=0.44, P=0.026], and even more pronounced in the group of European smokers [N=157, OR=0.14, P=0.015]. Using multiple regression analysis, plasma folate levels of mothers and newborns significantly affected the birth weight of newborns of smoking mothers (P<0.05). The obtained results seem to indicate a positive effect of folate on pregnancy outcome, especially its potential to decrease the risk of IUGR in European population and lower birth weight in smoking European mothers. It would be warranted to study the effect of folate levels on pregnancy outcomes in the relationship to different environmental pollution and life styles of mothers.