H-folic Acid Research Articles

A DIRECT MEASUREMENT OF FOLIC ACID ABSORPTION IN HEALTHY CHILDREN DETERMINED BY A SINGLE STOOL SAMPLE TEST- A DOUBLE ISOTOPE TECHNIQUE SPECIALLY ADAPTED TO THE USE IN CHILDREN

The fractional folic acid absorption (FAFol) was determined in 66 patients with various gastrointestinal diseases by a double isotope technique, employing a single stool sample test (SSST), as well as a complete stool collection. The age of the patients ranged from 2.5 mo to 16.8 yrs(mean 6.3). The test dose was administered orally and consisted of 50 mikrog. of 3H folic acid (app. 20 mikroCi), carmine powder, and 2 mg 51CrCl3 (app.1.25 mikroCi)as the inabsorbable tracer. The wholebody radiation given to a 1-year-old child averaged 4.8 mrad, ie a negligible radiation dose. The stool and napkin contents were collected and homogenized by the addition of chromium sulfuric acid. The content of 51Cr was measured in a broad-based well counter and the quantity of 3H folic acid by liquid scintillation, after duplicate destillation. Estimated by SSST, the FAFol, which employs the stool with the highest content of 51Cr corresponding to the most carmine-colored stool, correlated closely with the FAFol based on complete stool collection (r=0.96, N=39, p 0.0001).The reproducibility of FAFol determined by SSST was assessed from double assays in 18 patients. For a mean of 81%, the SD was 4.6% which corresponded to a CV of 5.7%. The mean FAFol in 45 healthy children aged 9 mo to 16.8 yrs (mean 6.4) was 83% (range 66-95%). The FAFol levels showed no correlation with age. This study is the first attempt of direct measurement of folic acid absorption in healthy children.

Intestinal absorption, liver uptake, and excretion of 3H-folic acid in folic acid-deficient, alcohol-consuming nonhuman primates

Nonhuman primates fed folic acid-deficient diets +/- 30% kcal ethanol were used to determine alcohol effects on megaloblastic anemia development and folate bioavailability. Lower hemoglobin (Hb) and red blood cell (RBC) counts and higher mean corpuscular volume (MCV) occurred after 13 wk in alcohol-fed monkeys, later in controls. Plasma, RBC, and liver folate declined and urinary formiminoglutamic acid (FIGLU) was elevated in both groups with FIGLU increasing more among alcohol-fed monkeys at 38 wk. After 40 wk, the bioavailability of oral 3H-folic acid was investigated and showed increased fecal and reduced urinary tritium excretion in alcohol-fed monkeys compared with controls while plasma uptake and liver and whole body tritium retention were similar in both groups. These observations demonstrate that chronic alcohol consumption impairs folate coenzymes, accelerates appearance of hematologic indices of megaloblastic anemia, and causes possible malabsorption of enterohepatically circulated folates in folate deficiency even when other essential nutrients are provided.

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 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 microM 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 concentrations (above 5 X 10(-6) M) a linear component was observed. Insulin increased the slope of the linear component and the Vmax of the saturable component while the Km remained unaltered.

Half-life and metabolism of 3H-folic acid in oral contraceptive treated rats.

Half-life and tissue distribution of injected tritium labelled folate was examined in oral contraceptive (OCA) treated and control rats to elucidate the mechanism by which OCA treated rats show increased urinary excretion as well as tissue levels of folate. Urinary excretion of folate within the first 12 hours of injection was higher but subsequent excretion lower in OCA treated rats. Faecal excretion of folate was also lower in treated rats. Thus, the half-life of the rapid turnover labile pool of folate appears to be reduced, whereas that of the slow turnover stable pool is raised by OCA treatment. Concentration of labelled folate was higher in the liver and kidneys of treated rats but it was not affected in tissues such as intestine, bone and brain. The concentration of soluble folate binders in the kidney was raised by OCA treatment and the activity of dihydrofolate reductase in the liver (a folate binder) also tended to be higher though the difference was not significant. The observation suggest that OCA may alter folate turnover by changing the concentration of folate binders in the tissues.

Specific and non-specific folate binding protein in normal and malignant human tissues.

Binding of tritiated folic acid by supernatants prepared from extracts of normal and leukaemic leucocytes, normal mucosa, and malignant tumours from different parts of the gastrointestinal tract has been measured using Sephadex-gel filtration and albumin-coated charcoal techniques. Non-specific binding (measured by Sephadex G-75 gel filtration) was almost invariably greater than specific binding measured by albumin-coated charcoal separation of bound and unbound folate. In nine normal leucocyte extracts, binding measured by Sephadex G-75 filtration ranged from 1.3 to 18.2 (mean 8.2) pg/mg protein and by albumin-coated charcoal from 1.0 to 14.8 (mean 6.7) pg/mg protein. Raised specific binding was found in the extracts from leucocytes of eight of 14 patients with chronic granulocytic leukaemia, in four substantially so (389, 121, 108, 59.7 pg/mg protein), but was only marginally increased in one of eight cases of acute myeloid leukaemia and in two of five cases of chronic lymphocytic leukaemia. Binding was normal in the extracts of all three cases of acute lymphoblastic leukaemia tested. Among the tissues of the gastrointestinal tract binding was greatest by the duodenal mucosa and liver. Extracts of carcinoma of the stomach and colon bound greater amounts of (3)H-folic acid than the corresponding normal mucosal extracts but the differences were not large. Sephadex G-200 gel chromatography showed more than one binding peak in the extracts of liver and duodenum but only one peak in the other tissues of the gastrointestinal tract, and only one peak, of molecular weight either about 50 000 or over 200 000, in the leucocyte extracts.

Vitamin B12 deprivation in the rat: effects on folate metabolism with emphasis on the nervous system.

Rats were subjected to deprivation of vitamin B12 in order to induce neuropathy and to study effect on folate metabolism. Plasma vitamin B12 concentrations were maintained at about 100 pg/ml for 5 months. Neurological testing failed to reveal signs of neuropathy. Vitamin B12 deprivation induced high plasma folate levels but had no effects on the levels of total folate in the liver or the nervous system, probably because of an adequate methionine supply. If the rats were starved, a smaller proportion of 5-methyltetrahydrofolate was found both in liver and brain of the vitamin-B12-deprived animals. The folate concentration in the cerebrospinal fluid was lower than in the plasma but the values were correlated. There was a striking similarity between the liver and the brain in the way of handling 3H-folic acid and 14C-methyltetrahydrofolic acid. However, both substances showed a higher specific activity in the brain than in the liver, indicating a more dynamic state of brain folate.

A Modified Fecal Excretion Test for Assaying Intestinal Absorption of 3H-Folic Acid

A method for determination of the intestinal absorption of folic acid is described. It is based on the principle of double tracer technique in assay of fecal samples using gas proportional counting for 3H determinations. As the procedure is simple, reliable, and inexpensive, it is considered to be of value for laboratories without advanced equipment for radiochemical assay.

Binding of folic acid to serum proteins. V. Tritiated folic acid in the liver.

Sephadex G-200 chromatographies of the liver cell sap of rabbits, 1, 3 and 6 days after an injection of 3H-folic acid activity (FAA) into the circulation, were used in an attempt to study the binding of this labelled FAA to liver proteins. The labelled FAA was quickly accumulated in the liver, and in the cell sap it was grouped chromatographically to two maxima, which corresponded to the two maxima of microbiological FAA (L. casei). The maxima were eluated in the chromatography together with cell sap proteins. The possible coupling of FAA to proteins in the liver is discussed.

Jejunal Uptake of Tritiated Folic Acid in the Rat Studied by in vivo Perfusion

The jejunal uptake of tritiated folie acid was measured in male Wistar rats by in vivo perfusion. The uptake of 3H-folic acid was linear relative to concentra tions between 4 nM and l /UM,but between concentrations of l UM and 4.6 MM,uptake was similar, suggesting a saturable uptake process. A double reciprocal plot ( Lineweaver- Burk) yielded a fC„, value of 1.57 /IM with a V„„ value of 0.654 itmoles/cm/hour. Ex posure of the lumen to 11 /UMmethotrexate, an inhibitor of dihydrofolate reducA­ase, prior to and during perfusion of 1.1 /AM 3H-folic acid resulted in a significant decrease of jejunal dihydrofolate reducA­ase (folie acid reducA­ase, EC 1.5.1.3) with no change in the jejunal uptake of H-folic acid. There was no observable difference in jejunal dihy drofolate reducA­ase nor of the uptake of H-folic acid among rats fed normal diet, normal diet plus ethanol, a protein-deficient diet, or a protein-deficient diet plus ethanol. These studies suggest that the initial uptake of 3H-folic acid by the jejunal mucosa is rate limited between concentrations of 1 and 4.6 /IM, does not require metabolic reduc tion, and is not affected by protein deficiency or elhanol. J. Nulr. 104: 1674-1680, 1974.

The effect of vitamin B 12 and methionine on folic acid uptake by rat liver.

SummaryThe liver uptake of intravenously administered 3H folic acid in rats fed a vitamin B12-methionine diet was only 55% of that found in normal rats. Supplementation of the deficient diet for 30 hr with 100 μg of B12/kg did not alter folate uptake significantly, but if vitamin B12 was fed together with 0.7% methionine, the rate of uptake was rapidly increased to control values. When uptake was stimulated in this way it was observed that plasma folate levels decreased from 72 to 45 ng/ml and liver folate concentrations increased from 5 to 8 μg/g—an increment of 48 μg of folate/liver in 30 hr. Intraperitoneal injection of 2.5 μg of B12 and 40 mg of methionine together in deficient animals, demonstrated that the depressed rate of folate uptake could be restored to control values even within 10 hr. Injection of 225 mg of methionine stimulated 3H folic acid uptake in the absence of any additional vitamin B12. It is suggested that vitamin B12 and or methionine can influence folate metabolism in rat liver by ...

A macromolecular factor in some leukemic cells which binds folic acid.

SummaryThe reduction of 0.57 μμmoles of 3H-folic acid to tetrahydrofolic acid by folate reductase in the cell lysates from two patients with chronic myelogenous leukemia was not apparent until the total substrate concentration of the reaction mixture was raised by the addition of stable folic acid. In addition, these leukemic cell lysates inhibited the reduction of tracer 3H-folic acid by a leukemic cell lysate containing “normal” folate reductase activity. Gel-filtration experiments demonstrated that this phenomenon was due to binding of the tracer folate to a large molecule because 3H-folic acid preincu-bated with these peculiar leukemic cell lysates and filtered through Sephadex G-75 appeared in the early or excluded volume indicating that the substrate was bound to a factor with a molecular weight of at least 50,000. This binding of 3H-folic acid could be competitively inhibited by stable folate.I thank Carol Schreiber for her technical assistance for part of these studies, and Drs. Robert Goldstein a...

Increased urinary excretion of 3H-folic acid injected to rats with riboflavin deficiency or excessive dietary methionine.

Increased urinary excretion of 3H-folic acid injected to rats with riboflavin deficiency or excessive dietary methionine.