Effect of nitrous oxide and methionine on hepatic S-adenosylmethionine levels and in vivo histidine oxidation in rats.

Nitrous oxide (N2O) decreased in vivo oxidation of histidine in rats fed a basal diet marginally deficient in methionine, although hepatic levels of S-adenosylmethionine (AdoMet) were not significantly altered. Excess dietary methionine increased hepatic levels of AdoMet and increased histidine oxidation. However, it did not protect histidine oxidation when the rats were treated with N2O. Parenteral administration of methionine greatly increased hepatic levels of AdoMet and increased histidine oxidation in normal and N2O treated rats. This indicates that when hepatic levels of AdoMet are greatly elevated by administration of methionine, N2O does not affect in vivo histidine oxidation.

Association of folic acid with rat liver microsomes.

Shin et al. (Biochim Biophys Acta 444: 794-801, 1976) described the subcellular location of [3H]folic acid after injection into rats. The microsomal fraction of the liver contained relatively large amounts of tracer initially but lower amounts at later times. Because of the heterogeneous nature of the microsomal fraction of the liver we re-examined the nature of the folate binding fraction. The location of injected [3H]folic acid resembled that of the microsomes derived from the plasma membrane, where ultracentrifugal analysis was conducted in the presence and absence of cesium ions. The location of the folate did not resemble that of microsomes derived from the endoplasmic reticulum (ER). One of the marker enzymes of the ER was the vitamin K-dependent carboxylase. A simple method for reducing vitamin K is described.

Nitrous oxide provokes changes in folylpenta- and hexaglutamates.

Liver folates isolated from rats fed a control diet consisted of H4PteGlu5 (44% of total) and 5-methyl-H4PteGlu5 (19%) with smaller amounts of 10-formyl-H4PteGlu5 (13%), H4PteGlu6 (13%), 10-formyl-H4PteGlu6 (4%) and 5-methyl-H4PteGlu6 (2%). Folates from methionine-deficient rats contained more nearly equal amounts, on a molar basis, of folylpenta- and hexaglutamates, and hence these were more suitable subjects for comparing the metabolism of these compounds in vivo. Livers from methionine-deficient rats contained H4PteGlu5 (30%), 5-methyl-H4PteGlu5 (13%), 10-formyl-H4PteGlu5 (10%), H4PteGlu6 (22%), 5-methyl-H4PteGlu6 (9%) and 10-formyl-H4PteGlu6 (11%). With exposure to N2O, 5-methyl-H4PteGlu5 and 6 increased to 27% and 13%, respectively, whereas H4PteGlu5 and 6 decreased to 20% and 17%, respectively. Nitrous oxide (N2O) perturbed both the penta- and hexaglutamates; the effect was somewhat more pronounced with the pentaglutamates. The partial depletion in tissue H4PteGlun with N2O treatment helps explain the concomitant inhibition of the oxidation of [ring-14C]histidine, an event dependent on tetrahydrofolates.

Identification of 10-formyltetrahydrofolate dehydrogenase-hydrolase as a major folate binding protein in liver cytosol.

10-Formyltetrahydrofolate dehydrogenase (10-formyltetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.6) purified from pig liver contained bound tetrahydropteroylhexa-gamma-glutamate, a potent product inhibitor. Dehydrogenase purified from rat liver had chromatographic properties indistinguishable from those of a previously described major cytosolic folate binding protein of unknown function (Zamierowski, M.M. and Wagner, C. (1977) J. Biol. Chem. 252, 933-938; Cook, R.J. and Wagner, C. (1982) Biochemistry 21, 4427-4434). The dehydrogenase catalyzes the oxidative deformylation of 10-formyltetrahydrofolate to carbon dioxide and tetrahydrofolate. The tight binding of product to the enzyme suggests that oxidation of one-carbon moieties is regulated by the ratio of formyltetrahydrofolate to tetrahydrofolate in liver.

Effect of hypothyroidism on methylmalonate excretion and hepatic vitamin B-12 levels in rats.

The effect of hypo- and hyperthyroidism on vitamin B-12 metabolism in the rat was studied by measuring methylmalonic acid excretion, B-12 content of liver and oxidation of 2-[14C]histidine. Ten percent pectin was added to increase severity of B-12 deficiency. The addition of thiouracil to a diet containing 10% pectin decreased the excretion of methylmalonic acid suggesting an amelioration of the B-12 deficiency. It was found that part of this decreased methylmalonic acid excretion was due to a decreased food consumption with a correspondingly decreased intake of branched-chain amino acids which are precursors of methylmalonic acid. When attempts were made to increase the protein intake of animals receiving thiouracil so their amino acid intake was equal to that of the control animals, methylmalonic acid excretion was still lower than that of the controls. It was also found that the vitamin B-12 content of the liver was higher in the animals receiving thiouracil than in the controls. Thyroidectomy had the same effect as feeding thiouracil. Liver B-12 levels are rapidly depleted on a B-12 deficient diet containing 10% pectin. It appears that hypothyroidism, induced either by thyroidectomy or by feeding thiouracil, slows the rate of depletion of hepatic B-12 which in turn facilitates the metabolism of methylmalonic acid and decreases its excretion in the urine.

Effect of thiouracil in modifying folate function in severe vitamin B12 deficiency.

The effects of thiouracil in correcting defects in folic acid function produced by B12 deficiency were studied. Addition of the thyroid inhibitor, thiouracil, to a low methionine diet containing B12, increased the oxidation of [2-14C]histidine to carbon dioxide, and increased liver folate levels. Addition of 10% pectin to the diet accentuated B12 deficiency as evidenced by a greatly decreased rate of histidine oxidation (0.19%) and an increased excretion of methylmalonic acid. Addition of thiouracil to the diet restored folate function as measured by increased histidine oxidation and increased liver folate levels similar to that produced by addition of methionine to a B12-deficient diet. Thiouracil decreased methylmalonate excretion, and increased hepatic levels of B12 in animals on both B12-deficient and -supplemented diets. Hepatic methionine synthase was increased by thiouracil, which may be the result of the elevated B12 levels. S-Adenosylmethionine and the enzyme methionine adenosyltransferase were also increased by thiouracil. Thus it is possible that the effect of thiouracil in increasing folate function consists both in the effect of thiouracil in decreasing levels of methylenetetrahydrofolate reductase, and also in its action in increasing S-adenosylmethionine which exerts a feedback inhibition of this enzyme.

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.

Pharmacokinetics of leucovorin calcium after intravenous, intramuscular, and oral administration.

The pharmacokinetics of leucovorin was evaluated after intravenous, intramuscular, and oral administration in a randomized crossover study of 37 healthy men. A single 25-mg dose of leucovorin calcium was administered intravenously, intramuscularly, or orally to the subjects. Blood samples were obtained immediately before and at 13 time points up to 24 hours after the leucovorin dose. The three treatment phases were separated by one-week intervals. Bioavailability was assessed by measuring over 24 hours the blood concentrations of total folates, the parent compound 5-formyltetrahydrofolate, and the metabolite 5-methyltetrahydrofolate, using differential microbiologic assays with Lactobacillus casei and Streptococcus faecalis. Both intravenous and intramuscular administration produced rapid increases in serum concentrations of biologically active folates; these rises were sustained over time and were still detectable at 24 hours after drug administration. The bioavailability of intravenous and intramuscular doses was comparable based on area under the serum concentration-time curve, although for intramuscular administration, the peak concentration was lower and the time to peak concentration was longer. The initial rise in serum folate with intravenous and intramuscular dosing represented 5-formyltetrahydrofolate; this fell concomitantly with the appearance of 5-methyltetrahydrofolate. Oral leucovorin was 92% bioavailable compared with intravenous administration and produced a predictably different pattern of circulating folates, 5-methyltetrahydrofolate being the predominant form. Terminal elimination half-life, apparent volume of distribution, and clearance of total folate were not significantly different among the three treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

Folic acid: effect on zinc absorption in humans and in the rat.

The effect of folic acid on zinc absorption and utilization was studied in humans and in rats. Serum Zn response curves after a 25-mg oral dose of Zn was measured for 4 h with and without 10 mg folic acid. The increase in serum Zn expressed as AUC (area under the curve) was 57 mumol/L for Zn alone and 65 mumol/L with folate. Zn intragastric intubation experiments with rats showed that adding 4.4 or 176 micrograms of folate to test meals containing 13 micrograms Zn did not affect the bioavailability of Zn as measured by the retention of 65Zn by the liver and kidney. A 3-wk feeding test carried out with diets containing either 6 or 12 mg Zn/kg diet showed that adding either 2 or 160 mg of folic acid had no effect on growth or Zn uptake by the femur. These results do not indicate any inhibition of Zn utilization by folic acid.

Increased methionine synthetase activity in zinc-deficient rat liver.

To study the effect of zinc deficiency on folate metabolism, three groups of male Sprague-Dawley rats (zinc deficient (ZD), restricted-fed (RF + Zn), and ad libitum-fed control (control] were given a semipurified 25% egg white protein diet. The ZD group received less than 10.3 nmol zinc/g of diet, while the RF + Zn and control groups were given 1620 nmol zinc/g of diet. After 6-7 weeks of feeding, severe zinc deficiency developed in ZD rats. Hepatic methionine synthetase activity was increased in the ZD group compared to both the RF + Zn and control groups, but hepatic 5,10-CH2-H4folate reductase activity was similar in all groups. This increased methionine synthetase activity found in zinc-deficient rats might induce secondary alterations in folate metabolism. These changes include significantly lowered plasma folate levels, decreased 5-CH3-H4folate in liver, and increased rates of histidine and formate oxidation. The latter two findings suggest that the available non-5-CH3-H4folate is increased in zinc deficiency.

Absorption kinetics of orally administered leucovorin calcium.

Oral dose proportionality and pharmacokinetics of leucovorin [(d,l)-5-formyltetrahydrofolate (5-formyl-THF)] were studied in 30 healthy male subjects. In a randomized cross-over design, 24 fasted subjects were given 4 of a series of 5 single test doses between 20 and 100 mg, at 1-week intervals, of 5-formyl-THF as an oral solution of leucovorin calcium. Six separate subjects received 200 mg iv and po in a 2-way crossover. Blood and urine samples were collected over 24 hours for differential microbiological folate assays using Lactobacillus casei and Streptococcus faecalis. Using L casei activity to measure total serum folates, the area under the concentration-time curve from 0 to infinite time (AUC[0-infinity]) was calculated. Relative bioavailabilities were 78%, 62%, 49%, and 42% for the 40-, 60-, 80-, and 100-mg doses, respectively. Both the AUC and peak concentration (CPEAK) of total folates (consisting predominantly of the major metabolite, 5-methyltetrahydrofolate (5-methyl-THF], displayed significant deviation from linearity consistent with a saturation of folate absorption. Absolute bioavailability of the 200-mg oral dose of leucovorin based on AUC was 31% compared with that of the iv dose (6,848 vs. 22,298 ng.hr/ml, respectively). Total clearance, terminal half-life, and apparent volume of distribution of total folate at the 200-mg dose were not significantly different between the two routes of administration. Eighty-three percent of the biologically active iv dose was recovered in the urine within 24 hours, 31% as 5-methyl-THF. Twenty percent of the same oral dose was excreted in 24 hours, 16% as 5-methyl-THF. In contrast to the nondose-proportionality observed in total serum folates, AUC of the small component of S faecalis activity, which appeared earlier than 5-methyl-THF, displayed linear kinetics, suggestive of a distinct mechanism of uptake. As dose increased, S faecalis activity increased in relative proportion to L casei, indicating that saturation of the enzymatic bioconversion to 5-methyl-THF may also be occurring. In light of the demonstrated nondose-proportionality of total folates with oral leucovorin in this dose range, consideration should be given to parenteral administration in regimens employing higher doses. Oral administration should be at a level consistent with the capacity for efficient folate uptake.

Experiments on the biological activities of various fractions of the folic acid antagonist "X-methyl" folic acid.

A crude synthetic preparation called crude "X-methyl" folate has previously been shown to function as a folate antagonist for rats and chicks. This product has been shown to contain two folate antagonists: 9-methyl folate, present as 6% by weight of the product and which has low activity as a folate antagonist for Streptococcus faecalis, and pyrrofolic acid, a compound present in small amounts (0.04%), but having high anti-folate biological activity for S. faecalis. These experiments deal with the antifolate activity of these two fractions for the rat as measured by their effects on histidine oxidation. Rats were fed a purified diet based on 20% vitamin-free casein and containing 1.0% sulfasuxidine. When this diet was supplemented with a marginal amount of folic acid (0.3 mg per kg diet), the addition of 4 g of crude antagonist decreased histidine oxidation and decreased liver folate levels. The addition of 240 mg of pure 9-methyl folic acid (amount of 9-methyl folic acid in 4 g of crude) produced similar decreases in histidine oxidation and liver folate levels. A concentrate of pyrrofolic acid (equivalent to 4 g of crude) free of 9-methyl folic acid produced no decrease in histidine oxidation and minimal changes in liver folate. This indicates that the folate antagonist activity observed previously with animals is probably due to the 9-methyl folic acid component rather than to the pyrrofolic acid activity.

Comparison of folic acid coenzyme distribution patterns in patients with methylenetetrahydrofolate reductase and methionine synthetase deficiencies.

Folic acid coenzyme distribution patterns were examined in the liver and kidney of two patients with homocystinuria due to different inborn errors of metabolism affecting the remethylation of homocysteine to methionine. One patient, with severe mental retardation (and death at 3 1/2 yr), had greatly reduced levels of methylenetetrahydrofolic acid (THF) reductase in fibroblasts as well as in liver and kidney. Chromatographic separation of folate coenzymes in liver showed an abnormal pattern with THF as the main component and almost no methyl-THF but total folate was normal. The other patient, who was dystrophic, microcephalic, and had megaloblastic anemia died at age 4 months. He had reduced levels of methionine synthetase in liver and kidney due to a defect of intracellular cobalamin metabolism. Chromatographic analysis of his tissues showed methyl-THF to be the principal folate form and a markedly reduced total folate. These results support the "methyl-THF trap" hypothesis and offer information with respect to the possible therapy of these two disorders.

Prolonged vaginal cornification and other changes in mice treated neonatally with coumestrol, a plant estrogen.

This study used the neonatal mouse model to determine if early exposure of female mice to coumestrol, a plant estrogen, would result in reproductive-tract alterations similar to those seen after neonatal exposure to diethylstilbestrol (DES). Newborn female C57BL/Crgl mice were given daily subcutaneous injections of 0.08 micrograms DES or 100 micrograms coumestrol in 0.005 ml dimethyl sulfoxide (DMSO), or DMSO alone, or were untreated, for the first 5 d of life. The doses chosen were equivalent in biological activity based on published uterine bioassay data (using young adult mice). Observations were made twice daily for 1.5 mo to determine the times of eye and complete vaginal opening. Half of the animals were ovariectomized at 40 d of age. Vaginal lavages were examined for 30 consecutive d beginning both at 2 and at 5 mo of age. DES and coumestrol significantly advanced the time of complete vaginal opening and induced a comparable degree of ovary-independent persistent vaginal cornification. In addition, coumestrol resulted in the occurrence of hemorrhagic ovarian follicles.

Vitamin B12-folate interrelationships.

The studies discussed in this review support the view that biochemical and clinical symptoms common to both folate and vitamin B12 deficiency are due to the induction of a functional folate deficiency, which in turn is induced by cobalamin deprivation. The interrelationship between these two vitamins is best explained by the methyl trap hypothesis stating that vitamin B12 deficiency can lead to lowered levels of methionine synthetase, which results in a functional folate deficiency by trapping an increased proportion of folate as the 5-methyl derivative. In addition, as 5-methyl-H4PteGlu is a poor substrate for folylpolyglutamate synthetase, there is a decreased synthesis of folylpolyglutamates and consequently a decreased retention of folates by tissues. The real folate deficiency that ensues because of decreased tissue folate levels is probably as important physiologically as the functional deficiency caused by the methyl trap. The sparing effect of methionine can be explained by adenosylmethionine inhibition of methylenetetrahydrofolate reductase, which would prevent the buildup of 5-methyl-H4PteGlun. A deficiency in vitamin B12 would not, in itself, be sufficient to cause a disturbance in folate metabolism. The deficiency would have to result in lowered methyltransferase levels before any such disturbance would be manifest.

Folate oligoglutamate:amino acid transpeptidase.

A new and previously unrecognized enzyme activity folate oligoglutamate:amino acid transpeptidase (folate transpeptidase), was detected in rat liver extracts. This activity involves the exchange of the terminal gamma-glutamyl residue (where PteGlu represents pteroylglutamic acid) of H4PteGlu-gamma-Glu, PteGlu-gamma-Glu, or p-aminobenzoyl-Glu-gamma-Glu for an amino acid such as [14C]glutamic acid, [14C]methionine, [14C]glutamine, or [14C]glycine. In the case of substrates PteGlu2 and [14C]Glu, the product is PteGlu-gamma-[14C]Glu. The enzyme responsible for catalyzing this activity was purified 39,000-fold by precipitation of inactive protein, fractionation on anionic and molecular sieve columns, and chromatography on diaminohexane-Sepharose. The enzyme functions maximally at pH 7.9, shows an absolute requirement for sulfhydryl reductants, and appears to have a molecular weight of 65,000. The function of folate transpeptidase is not known. A second, though much weaker, enzyme activity, tetrahydrofolic acid:amino acid ligase (folate ligase), co-purified with folate transpeptidase through all steps. Folate transpeptidase and the associated folate ligase activity were separated from folate polyglutamate synthetase during the initial steps of the purification procedure.

Nitrous oxide inactivates methionine synthetase in human liver.

Activity of methionine synthetase was measured in liver biopsies of seven patients who had received 50% to 70% nitrous oxide supplemented by a combination of a narcotic and/or barbiturate with or without a volatile anesthetic, and from seven patients who were anesthetized without nitrous oxide (control group). Methionine synthetase activity (+/- SE) averaged 219 +/- 28 nmol of methionine per hour per gran of liver in patients given nitrous oxide, and 414 +/- 29 in control patients. Inactivation of methionine synthetase progressively increased as the product of the concentration of nitrous oxide and the exposure time increased. These results in humans are similar to those in animals and suggest that inactivation of methionine synthetase may play a role in the development of the pathologic effects seen in patients and medical personnel after exposure to nitrous oxide.