Differential binding of folates by rat renal cortex brush border and basolateral membrane preparations

The binding of radioactive 5-methyltetrahydrofolate and folic acid was found to be greater in brush border than in basolateral membrane preparations of rat renal cortex. This appeared to be due to an increased amount of a specific folate binding protein in the brush border membrane preparations as compared to those of the basolateral membrane. The binding was saturable and inhibited by nonradioactive folic acid and, therefore, a specific, rather than nonspecific process. The Km's for folic acid binding in brush border and basolateral membrane preparations were similar and involved a single high-affinity binding site. In contrast, methotrexate was found to bind equally well to both brush border and basolateral membrane preparations. Moreover, folic acid binding was not inhibited by an equimolar amount of methotrexate. A folate binding protein could be extracted from either membrane preparation with 1% Triton X-100 and, to a lesser extent, with 0.6 M NaCl. These different extraction procedures resulted in different apparent molecular weights for folate binding protein (greater than 160,000 for Triton X-100-extracted samples and 40,000 for NaCl-extracted samples). The membrane preparation pellets remaining after NaCl extraction were able to rebind tritiated folic acid and also the 40,000-Da folate binding protein. On the other hand, membrane preparations extracted with Triton X-100 lost the ability to bind folic acid or the 40,000-Da folate binding protein. These differences in molecular weight and rebinding capacity may be explained by the existence of a receptor for folate binding protein which was extracted by Triton X-100, but not by NaCl. The greater concentration of folate binding protein in the renal tubule cell brush border membrane preparations as compared to those from basolateral membranes ascribes, for the first time, a functional role for folate binding protein in the renal reabsorption of folates which is required to prevent loss of folate in the urine and perhaps in the membrane transport of folates in general.     

Neurologic disorders responsive to folic acid therapy.

Six women aged 31 to 70 years had folate deficiency and neuropsychiatric disorders. The three with acquired folate deficiency were depressed and had permanent muscular and intellectual fatigue, mild symptoms of restless legs, depressed ankle jerks, diminution of vibration sensation in the legs, stocking-type hypoesthesia and long-lasting constipation; D-xylos absorption was abnormal. The bone marrow was megaloblastic in only one patient, and she and one other had atrophy of the jejunal mucosa. The third was a vegan. All three recovered after folic acid therapy. The other three were members of a family with the restless legs syndrome, fatigability and diffuse muscular pain. One also had subacute combined degeneration of the spinal cord and kidney disease but no megaloblastosis; she improved spectacularly after receiving large daily doses of folic acid. The other two also had minor neurologic signs, controlled with 5 to 10 mg of folic acid daily. Unrecognized and treatable folate deficiency (with low serum folic acid values but normal erythrocyte folate values) may be the basis of a well defined syndrome of neurologic, psychiatric and gastroenterologic disorders, and the restless legs syndrome may represent the main clinical expression of acquired and familial (or inborn) folate deficiency in adults.     

Effect of genetic polymorphisms involved in folate metabolism on the concentration of serum folate and plasma total homocysteine (p-tHcy) in healthy subjects after short-term folic acid supplementation: a randomized,double blind,crossover study

Data on the effect of combined genetic polymorphisms, involved in folate metabolism, on the concentration of serum folate after folic acid supplementation are scarce. Therefore, we investigated the impact of seven gene polymorphisms on the concentration of serum folate and p-tHcy in healthy subjects after short-term folic acid supplementation. In a randomized, double blind, crossover study, apparently healthy subjects were given either 0.8 mg folic acid per day (n = 46) or placebo (n = 45) for 14 days. The washout period was 14 days. Fasting blood samples were collected on day 1, 15, 30 and 45. Data on subjects on folic acid supplementation (n = 91) and on placebo (n = 45) were used for the statistical analysis. The concentration of serum folate increased higher in subjects with higher age (53.5 ± 7.0 years) than in subjects with lower age (24.3 ± 3.2 years) after folic acid supplementation (p = 0.006). The baseline concentration of serum folate in subjects with polymorphism combination, reduced folate carrier protein, RFC1-80 GA and methylenetetrahydrofolate reductase, MTHFR677 CT+TT, was lower than RFC1-80 AA and MTHFR677 CT+TT (p = 0.002). After folic acid supplementation, a higher increase in the concentration of serum folate was detected in subjects with polymorphism combination RFC1-80 GA and MTHFR677 CC than RFC1-80 GG and MTHFR CT+TT combination (p < 0.0001). The baseline concentration of plasma total homocysteine (p-tHcy) was altered by combined polymorphisms in genes associated with folate metabolism. After folic acid supplementation, in subjects with combined polymorphisms in methylenetetrahydrofolate dehydrogenase, MTHFD1-1958 and MTHFR-677 genes, the concentration of p-tHcy was changed (p = 0.002). The combination of RFC1-80 and MTHFR-677 polymorphisms had a profound affect on the concentration of serum folate in healthy subjects before and after folic acid supplementation.     

Malabsorption of Folate Polyglutamates in Tropical Sprue

Malabsorption of folate polyglutamates prepared from yeast has been shown in eight patients with untreated tropical sprue and in three out of six patients receiving therapy for sprue. The absorptive defect for folate polyglutamates among these 14 patients occurred more frequently and in all but one patient more severely than for folic acid.Folate polyglutamates, the principal dietary form of folate, probably require deconjugation by the jejunal enzyme, folate conjugase, before absorption. The mean concentration of jejunal folate conjugase of 21 patients with untreated sprue and of 13 patients with sprue receiving therapy were both significantly less than the mean concentration in a control group. Nevertheless, all but five of the 34 patients had jejunal folate concentrations within the control range. There was no correlation in the individual patients between the jejunal folate conjugase concentration measured in vitro and the ability to absorb folate polyglutamates—nine patients having normal jejunal folate conjugase levels despite showing malabsorption of folate polyglutamates.     

Effectiveness of a free folic acid supplement program in family planning clinics

BACKGROUND: Adequate periconceptional folic acid consumption lowers the risk for neural tube defects. We report the results of an evaluation of a folic acid intervention in Georgia family planning clinics that provided free folic acid supplements or fortified breakfast cereal. METHODS: Six family planning clinics participated in the evaluation. Three clinics provided folic acid pills and educational materials to clients, two provided super-fortified cereal and educational materials, and one clinic provided educational materials only. Participants between the ages of 18 and 45 who visited the clinics in 2000 completed a brief survey and provided a blood sample. Of the 1093 women who participated, we evaluated the 165 women who had returned to the clinic at least once during the study period. We compared participants' survey and serum folate data from their first and subsequent visits. RESULTS: Participation in the intervention was associated with increased knowledge about folic acid, (odds ratio, 1.94; 95% confidence interval, 1.37-2.76), but was not directly associated with increased self-reported folic acid consumption or increased serum folate levels. Reported use of folic acid supplements or cereal within two days of a visit was associated with higher serum folate levels. Knowledge about folic acid was one of the best predictors of self-reported folic acid consumption. CONCLUSIONS: Participation in the intervention increased clients' knowledge about folic acid but did not directly increase reported folic acid consumption. Because knowledge predicted folic acid consumption, the intervention may be indirectly associated with increased consumption of folic acid.     

Follow up Study of Coeliac Disease

In 9 out of 10 adults in whom a diagnosis of coeliac disease had been made in childhood, the diagnosis was confirmed by the finding of a flat intestinal mucosa. None showed abnormal physical signs, but three had a haemoglobin concentration below 10 g./100 ml. and all those not receiving folic acid supplements showed low serum folate levels. Five had moderate to severe symptoms at the time of investigation, but none was receiving treatment with a gluten-free diet.Periodic investigation of these patients may be necessary throughout life, and if they are found to have malnutrition they should be treated with a gluten-free diet.     

Studying the mechanism of sperm DNA damage caused by folate deficiency

Sperm DNA injury is one of the common causes of male infertility. Folic acid deficiency would increase the methylation level of the important genes, including those involved in DNA double‐strand break (DSB) repair pathway. In the early stages, we analysed the correlation between seminal plasma folic acid concentration and semen parameters in 157 infertility patients and 91 sperm donor volunteers, and found that there was a significant negative correlation between seminal folic acid concentration and sperm DNA Fragmentation Index (DFI; r = −0.495, p < 0.01). Then through reduced representation bisulphite sequencing, global DNA methylation of sperm of patients in the low folic acid group and the high folic acid group was analysed, it was found that the methylation level in Rad54 promoter region increased in the folic acid deficiency group compared with the normal folic acid group. Meanwhile, the results of animal model and spermatocyte line (GC‐2) also found that folic acid deficiency can increase the methylation level in Rad54 promoter region, increased sperm DFI in mice, increased the expression of γ‐H2AX, that is, DNA injury marker protein, and increased sensitivity of GC‐2 to external damage and stimulation. The study indicates that the expression of Rad54 is downregulated by folic acid deficiency via DNA methylation. This may be one of the mechanisms of sperm DNA damage caused by folate deficiency.     

Nutritional Anemia and Megaloblastosis in Pregnancy

Macrogranulocytic and/or erythroid megaloblastic bone marrow changes which could not be accurately predicted from the hematologic findings in the blood were present in 25% of 305 mildly to moderately anemic pregnant women attending a public antepartum clinic in Montreal. Iron deficiency was the primary cause of anemia in most instances. Serum folate activity of less than 4.1 ng./ml. and/or serum vitamin B₁₂ levels of less than 100 pg./ml. were present in 90% of the 77 patients having these bone marrow changes, whereas approximately one-third of 228 patients with normoblastic marrow had these low values. Red cell folate did not correlate as well as serum folate activity with bone marrow changes. After treatment with oral folic acid in the range of 0.2 mg. to 0.8 mg., daily, for seven to 14 days, the megaloblastic and macrogranulocytic changes in patients with low serum folate activity and normal serum vitamin B₁₂ values disappeared in 15 of 21 patients. Of five women having both low folate and vitamin B₁₂ values, three failed to respond and two showed only partial improvement after 0.4 mg. of folic acid daily, per os, for 10 days. The average diet of these anemic women was suboptimal in folate and in iron.     

Folate Status Throughout Pregnancy and in Postpartum Period

The serial trends of the whole blood folate level in two groups of patients have been followed throughout pregnancy and up to six weeks postpartum. In those receiving iron alone the whole blood folate remained normal until the test at six weeks after delivery, at which time over half were in the deficient range. There appears to be a delay before this test reflects the current folate status when this changes rapidly. In those receiving iron plus 330 μg. of folic acid a day the results at this time were close to those at the beginning of pregnancy. Subnormal whole blood folate, red cell folate, and serum folate values occurred close to term in patients receiving iron alone, but were not found in those also receiving folic acid. Megaloblastic changes occurred at term in three patients receiving iron alone in whom the whole blood folate had repeatedly been low in early pregnancy.The observations are consistent with the previous suggestion that 300 μg. of folic acid daily is a suitable supplement to prevent deficiency in late pregnancy and the puerperium.     

Folate-responsive neuropathy: report of 10 cases.

Ten patients with severe neurological disease that was clinically indistinguishable from subacute combined degeneration of the spinal cord were found to have normal serum vitamin B12 levels. All were folate deficient. Specific folate treatment led to significant reversal of the neuropathy. These findings indicate the need to review orthodox concepts of the role of folic acid in maintaining the integrity of the nervous system.     

Preparation of a stable folate-sepharose complex for affinity chromatography.

A stable folic acid affinity gel has been developed for the purification of nanograms of protein that bind folic acid or its derivatives. The affinity gel was prepared by first coupling folic acid covalently to bovine serum albumin, followed by covalent coupling of the albumin to p-benzoquinone-activated Sepharose. After the albumin-folic acid complex was formed, it was treated with charcoal to remove ionically bound folate which would otherwise elute from the gel and decrease the recovery of the binding protein. The p-benzoquinone activation resulted in a more stable binding of the albumin to the Sepharose.     

Effect of insulin on folic-acid transport in cultured fibroblasts

Uptake of folic acid was measured in secondary cultures of skin fibroblasts from fetal rats. The cultures were made quiescent by 24 hours preincubation in medium containing 1% serum and subsequent 3 hours preincubation in phosphate buffered saline. The uptake of ³H-folic acid was linear with time during 15 seconds and reached a plateau level at 2–3 minutes. There was no further increase in the intracellular radioactivity until the end of the experiments at 10 minutes. The uptake of folic acid in fibroblasts was not concentrative and proceeded until equilibration with the extracellular concentration. Intracellular metabolic conversion of folic acid was not significant during the time of the experiments (up to 10 minutes). Insulin caused a two-fold increase in the initial rate of folate uptake as determined from the 15 second uptake values. The dose response curves for the insulin effect showed that 85% of the maximal effect was exerted by 1 m?M insulin. A lag period of 7–10 minutes was observed after the addition of insulin and before the effect on folic acid uptake was manifested. Thereafter the effect increased with the time of preincubation with insulin. The concentration dependence of folate uptake yielded non homogeneous curves. At low concentrations of substrate, saturable components were observed while at high concentration (above 5 × 10^?6 M) a linear component was observed. Insulin increased the slope of the linear component and the V_max of the saturable component while the K_m remained unaltered.     

Uric acid and urea in human sweat

The present study investigated whether thermal sweating may relieve elevated concentrations of serum uric acid or urea. Concentrations of uric acid and urea were measured in the sweat of sixteen male volunteers, who were treated with external heat after one hour of intense physical exercise. The same analytes were also measured in their urine and serum samples. Furthermore, creatinine and some electrolytes were determined in these specimens. The results show that the concentration of uric acid in the sweat is 24.5 micromol/L, which is only 6.3% of that in serum. The concentration of urea in the sweat is 22.2 mmol/L, which is 3.6 times that in serum. The results indicate that sweat uric acid concentration is quite minimal, and the estimated total uric acid excretion per day in normal physiological range is insignificant. However, the level of sweat urea was found at a much higher concentration than the serum level. No correlation could be established between the level of uric acid in sweat and in serum. There was also no correlation between the level of urea in sweat and that in serum. These results suggest it would not be effective to relieve the elevated serum uric acid concentration by thermal sweating when the renal excretion of uric acid is partly compromised. Nevertheless, the potential of urea excretion via profuse sweating is apparent particularly when the kidneys are damaged or their function is impaired. These findings also suggest that persons who take vigorous exercise or are exposed to hot environments should be well advised to drink adequate fluids since heavy sweating excretes only minimal uric acid, accompanied by significant diminution of urinary output and diminished urinary excretions of uric acid, which may induce elevated levels of serum uric acid.     

The impact of folate status and folic acid supplementation on the micronucleus frequency in human erythrocytes

Folic acid has a well-documented stabilising effect on chromosomes. A correlation between folate status and chromosome stability in humans has been reported in studies that were restricted to certain subpopulations, e.g., folate-deficient persons. The goal of the present investigation was to clarify if there also is a correlation between folate status and chromosome stability among individuals without any folate deficiency. The method used here is the recently developed flow cytometry-based micronucleus assay in human transferrin-positive reticulocytes (MN-Trf-Ret). In a blood sample, separation of the very young reticulocytes from the mature erythrocytes makes this micronucleus assay possible. This investigation comprises three studies (cross-sectional, giving baseline data), two of which are connected to an intervention study. In the three cross-sectional studies (total number of subjects, 99) the frequency of MN-Trf-Ret (fMN-Trf-Ret) was measured and compared with the serum folate status. In two of the studies also serum homocysteine and Vitamin B12 were measured and compared with the baseline fMN-Trf-Ret. Combining the results from the three cross-sectional studies, a negative correlation between folate status and fMN-Trf-Ret was obtained (p<0.05). The goal of the intervention studies was to clarify if different nutritional supplementations had any effect on the fMN-Trf-Ret and the cell proliferation (percentage polychromatic erythrocytes, PCE). Each of the two studies involved two groups, one placebo and one supplemented group. In one of the studies the supplementation was folic acid, 1000 microg/day during 1 week (n=30, both sexes); in the other intervention study, folic acid (800 microg/day), B12 (20 microg/day) and B6 (4 mg/day) were taken during 1 week (n=29, both sexes). No significant difference in %PCE or fMN-Trf-Ret between the two groups was found in either of the two intervention studies.     

The influence of smoking on plasma folate and lipoproteins in Alzheimer disease, mild cognitive impairment and depression

The risk for Alzheimer's disease (AD) is associated with lifestyle factors, especially cigarette smoking. In this study we investigated the influence of smoking on the serum levels of folic acid, LDL and HDL in AD patients, patients with minimal cognitive impairment (MCI) and patients with major depression. We investigated a total of n = 374 patients in the diagnostic categories:, AD: n = 272, MCI: n = 60, Major depression: n = 42. We found significantly lower HDL levels in smokers and previous smokers in comparison to non-smokers, p<0,05. The LDL: HDL ratio in smokers was significant higher (+20%) compared to previous smokers and non-smokers, p < 0.05. The mean levels of folic acid were statistically significant (p<0.05) lower (-24%) in smokers compared to non-smokers. Patients with MCI and Alzheimer;s disease (and also major depression) who are "smokers" show serum levels of HDL and folic acid that are known to be strong risk factors for vascular damage and increased risk for vascular brain damage and impaired cognitive function. Therefore cessation of smoking, substitution with folate or statin therapy of smoking patients with MCI or AD might be beneficial to slow down further cognitive decline.     

Demonstration of vitamin B12 deficiency by a simple cytochemical reaction in the peripheral blood smear

A simple, rapid and inexpensive cytochemical method for the detection of vitamin B 12 deficiency was applied in several types of anemias and matched with the levels of vitamin B12 and folic acid in the serum of the patients. It was found that in patients with low vitamin B12 levels the stained erythrocytes and the erythroid precursors showed a yellowish brown discoloration, which was not detected in folic acid deficiency and all other types of anemias. This test therefore may be used for differentiation between B12 and folate deficiency whenever megaloblastic anemia is diagnosed.     

Folic acid fortification: why not vitamin B12 also?

Folic acid fortification of cereal grains was introduced in many countries to prevent neural tube defect occurrence. The metabolism of folic acid and vitamin B12 intersect during the transfer of the methyl group from 5-methyltetrahydrofolate to homocysteine catalyzed by B12-dependent methioine synthase. Regeneration of tetrahydrofolate via this reaction makes it available for synthesis of nucleotide precursors. Thus either folate or vitamin B12 deficiency can result in impaired cell division and anemia. Exposure to extra folic acid through fortification may be detrimental to those with vitamin B12 deficiency. Among participants of National Health And Nutrition Examination Survey with low vitamin B12 status, high serum folate (>59 nmol/L) was associated with higher prevalence of anemia and cognitive impairment when compared with normal serum folate. We also observed an increase in the plasma concentrations of total homocysteine and methylmalonic acid (MMA), two functional indicators of vitamin B12 status, with increase in plasma folate under low vitamin B12 status. These data strongly imply that high plasma folate is associated with the exacerbation of both the biochemical and clinical status of vitamin B12 deficiency. Hence any food fortification policy that includes folic acid should also include vitamin B12.     

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.     

Plasma Trace Elements, Vitamin B12, Folate, and Homocysteine Levels in Cirrhotic Patients Compared to Healthy Controls

Increased serum homocysteine (Hcy) can induce liver diseases and can play a remarkable role in hepatic disorders. The purpose of the present study therefore was to investigate the relationship between serum vitamin B(12), folate, zinc and copper, cysteine, and Hcy level differences between cirrhotic patients and healthy subjects. We studied 32 cirrhotic patients (12 females and 20 males) aged 45 +/- 11 years and 32 control subjects (12 females and 20 males) aged 39 +/- 9 years. There was an inverse correlation between Hcy and vitamin B(12) in controls (r = -0.442, p < 0.011) but not in cirrhotic patients (r = -0.147, not significant). Also, mean plasma folate was decreased in cirrhotic patients compared to controls (p < 0.001). Copper increased whereas zinc decreased significantly in cirrhotic patients. A positive correlation was seen between the Cu/Zn ratio and Cu in controls (r = 0.690, p < 0.01), but the correlation between the Cu/Zn ratio and Cu was not significant in the cirrhotic group. Negative correlations were seen between plasma concentration of zinc and the Cu/Zn ratio in controls and cirrhotic patients (r = -0.618, p < 0.01 and r = -0.670, p < 0.01, respectively). Cirrhotic patients displayed multiple abnormalities, including changes in cysteine metabolism and in zinc and copper levels. Although hyperhomocysteinemia is known as an atherogenic and thrombogenic risk factor for cardiovascular disease, it might also be a risk factor for cirrhotic patients. Plasma Hcy, vitamin B(12), and folic acid measurement may be useful in the evaluation of cirrhotic patients.