Action of Sulfanilamide on Ochromonas malhamensis

SYNOPSIS. Sulfanilamide inhibited the growth of O. malhamensis. Sulfanilamide growth inhibition was reversed competitively by PABA and by very high concentrations of folic acid. Folic acid at low concentrations, however, accentuated sulfa inhibition of growth. Vitamin B₁₂, methionine, p -aminobenzoylglutamic acid and pteroic add were effective to some extent as antagonists of sulfa. A marked reduction in the folate synthesis was accompanied by sulfa growth inhibition. This was restored on growth restoration by PABA, folic acid, vitamin B₁₂ and methionine. The reduction in folate synthesis held for all the folate fractions except one derivative—a formyl poly-glutamate. Sulfanilamide-inhibited cells had a considerable activity for in vitro synthesis of folate activity from precursors. ∼75% activity being retained at the 90% growth inhibition level. There was no change in chlorophyll, RNA and DNA contents as a result of sulfa growth inhibition.     

Mutagenesis of folylpolyglutamate synthetase indicates that dihydropteroate and tetrahydrofolate bind to the same site

The folylpolyglutamate synthetase (FPGS) enzyme of Escherichia coli differs from that of Lactobacillus casei in having dihydrofolate synthetase activity, which catalyzes the production of dihydrofolate from dihydropteroate. The present study undertook mutagenesis to identify structural elements that are directly responsible for the functional differences between the two enzymes. The amino terminal domain (residues 1-287) of the E. coli FPGS was found to bind tetrahydrofolate and dihydropteroate with the same affinity as the intact enzyme. The domain-swap chimera proteins between the E. coli and the L. casei enzymes possess both folate or pteroate binding properties and enzymatic activities of their amino terminal portion, suggesting that the N-terminal domain determines the folate substrate specificity. Recent structural studies have identified two unique folate binding sites, the omega loop in L. casei FPGS and the dihydropteroate binding loop in the E. coli enzyme. Mutants with swapped omega loops retained the activities and folate or pteroate binding properties of the rest of the enzyme. Mutating L. casei FPGS to contain an E. coli FPGS dihydropteroate binding loop did not alter its substrate specificity to using dihydropteroate as a substrate. The mutant D154A, a residue specific for the dihydropteroate binding site in E. coli FPGS, and D151A, the corresponding mutant in the L. casei enzyme, were both defective in using tetrahydrofolate as their substrate, suggesting that the binding site corresponding to the E. coli pteroate binding site is also the tetrahydrofolate binding site for both enzymes. Tetrahydrofolate diglutamate was a slightly less effective substrate than the monoglutamate with the wild-type enzyme but was a 40-fold more effective substrate with the D151A mutant. This suggests that the 5,10-methylenetetrahydrofolate binding site identified in the L. casei ternary structure may bind diglutamate and polyglutamate folate derivatives.     

In vivo characterization of the absorption and biotransformation of pteroylmonoglutamic acid in man: a model for future studies

HPLC-EC has been used to measure the appearance of 5-CH3-H4 folic acid in human plasma following oral administration of folic acid. The process was found to be saturable in accordance with Michaelis-Menten kinetics. The apparent Km for this enzyme system indicates that low doses of oral folic acid are rapidly converted into 5-CH3-H4 folic acid, an observation consistent with the needs of intestinal absorption of essential trace nutrients. The appearance of L. casei active folate in plasma was not rate-limited and showed a biphasic relationship to dose. Preparative HPLC combined with L. casei bioassay demonstrated that most of the L. casei active folate appearing in plasma following a 20,000-micrograms dose of folic acid was due to the unmodified vitamin, only 5.6% being due to 5-CH3-H4 folic acid and with no detectable contribution from 5-CHO-H4 folic acid. The absorption characteristics of the system seem consistent between and within subject(s). No relationship could be demonstrated between predose levels of plasma 5-CH3-H4 folic acid and total folate in erythrocytes, which reflect the status of transport and storage forms of the vitamin, respectively.     

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-liposome-mediated antisense oligodeoxynucleotide targeting to cancer cells: evaluation in vitro and in vivo

The objective of this study was to investigate the use of folate-targeted liposomes for the delivery of encapsulated oligonucleotides to folate receptor (FR)-positive tumor cells in vitro and in vivo. This project involved the synthesis and biological evaluation of many folate-PEG-lipid conjugates, where the chemical form of the folate moiety (pteroate) and the length of the PEG linker chain were varied widely. Folate-targeted oligonucleotide-containing liposomes were prepared using conventional methods, and the extent of cell uptake was evaluated using, among others, the FR positive KB cell line. Oligonucleotide-loaded folate-targeted liposomes were found to rapidly associate with the KB cells, and saturation was typically reached within the first hour of incubation at 37 degrees C. Nearly 100,000 liposomes per cell were bound or internalized at saturation. Importantly, cell association was blocked by a large excess folic acid, thus reflecting the FR-specific nature of the cell interaction. Full targeting potential was achieved with PEG linkers as low as 1000 in molecular weight, and pteroates bearing glycine or gamma-aminobutyryl residues juxtaposed to the pteroic acid moiety were also effective for targeting, provided that a terminal cysteine moiety was present at the distal end of the PEG chain for added hydrophilicity. When tested in vivo, folate-targeted liposomes were found to deliver approximately 1.8-fold more oligonucleotide to the livers of nude mice (relative to the nontargeted PEG-containing formulations); however, no improvement in KB tumor uptake was observed. We conclude from these results that folate liposomes can effectively deliver oligonucleotides into folate receptor-bearing cells in vitro, but additional barriers exist in vivo that prevent or decrease effective tumor uptake and retention.     

Acute anemic hypoxemia produces a transient depression in fetal respiratory activity

Isovolemic anemia was produced in 11 unanesthetized fetal sheep by withdrawal of blood and replacement with saline-dextran. Fetal hematocrit fell from 36 +/- 1 to 19 +/- 1% (SE). Fetal breathing movements, which were present during 34.4 +/- 5.5% of 3 h before the anemia, occurred 10.1 +/- 5.3, 14.8 +/- 4.4, and 27.1 +/- 6.7% in the 3 h following. The anemia caused a fall in arterial O2 concentration from 8.4 +/- 0.3 to 3.6 +/- 0.1 vol% and sagittal vein PO2 fell from 15.4 +/- 0.5 to 12.4 +/- 0.3 Torr. Cerebral metabolic rate during the period of anemia was 2.9 +/- 0.1 ml.100 g-1.min-1, which was unchanged from the control value of 3.0 +/- 0.2 ml.100 g-1.min-1. Sagittal vein PCO2 (54.2 +/- 1.4 Torr) remained constant after the fetus was made anemic. We conclude that respiratory activity in the sheep fetus is depressed by anemic hypoxemia but that the effect is transient.     

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.     

Effect of hematocrit reduction on hormonal and metabolic responses to exercise

We wished to determine the effect of a 25% hematocrit reduction on glucoregulatory hormone release and glucose fluxes during exercise. In five anemic dogs, plasma glucose fell by 21 mg/dl and in five controls by 7 mg/dl by the end of the 90-min exercise period. After 50 min of exercise, hepatic glucose production (Ra) and glucose metabolic clearance rate (MCR) began to rise disproportionately in anemics compared with controls. By the end of exercise, the increase in Ra was almost threefold higher (delta 15.1 +/- 3.4 vs. delta 5.2 +/- 1.3 mg X kg-1 X min-1) and MCR nearly fourfold (delta 24.6 +/- 8.8 vs. delta 6.5 +/- 1.3 ml X kg-1 X min-1). Exercise with anemia, in relation to controls resulted in elevated levels of glucagon [immunoreactive glucagon (IRG) delta 1,283 +/- 507 vs delta 514 +/- 99 pg/ml], norepinephrine (delta 1,592 +/- 280 vs. delta 590 +/- 155 pg/ml), epinephrine (delta 2,293 +/- 994 vs. delta 385 +/- 186 pg/ml), cortisol (delta 6.7 +/- 2.2 vs. delta 2.1 +/- 1.0 micrograms/dl) and lactate (delta 12.1 +/- 2.2 vs. delta 4.2 +/- 1.8 mg/dl) after 90 min. Immunoreactive insulin and free fatty acids were similar in both groups. In conclusion, exercise with a 25% hematocrit reduction results in 1) elevated lactate, norepinephrine, epinephrine, cortisol, and IRG levels, 2) an increased Ra which is likely related to the increased counterregulatory response, and 3) we speculate that a near fourfold increase in MCR is related to metabolic changes due to hypoxia in working muscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute anemia results in an increased glucose dependence during sustained exercise

We evaluated whether acute anemia results in altered blood glucose utilization during sustained exercise at 26.8 m/min on 0% grade, which elicited approximately 60-70% maximal O2 consumption. Acute anemia was induced in female Sprague-Dawley rats by isovolumic plasma exchange transfusion. Hemoglobin and hematocrit were reduced 33% by exchange transfusion to 8.6 +/- 0.4 g/dl and 26.5 +/- 1%, respectively. Glucose kinetics were determined by primed continuous infusion of [6-3H]glucose. Rates of O2 consumption were similar during rest (pooled means 25.1 +/- 1.8 ml.kg-1.min-1) and exercise (pooled means 46.8 +/- 3.0 ml.kg-1.min-1). Resting blood glucose and lactate concentrations were not different in anemic animals (pooled means 5.1 +/- 0.2 and 0.9 +/- 0.02 mM, respectively). Exercise resulted in significantly decreased blood glucose (4.0 +/- 0.2 vs. 4.6 +/- 0.1 mM) and elevated lactate (6.1 +/- 0.4 vs. 2.3 +/- 0.5 mM) concentrations in anemic animals. Glucose turnover rates (Rt) were not different between anemic and control animals at rest and averaged 58.8 +/- 3.6 mumol.kg-1.min-1. Exercise resulted in a 30% greater increase in Rt in anemic (141.7 +/- 3.2 mumol.kg-1.min-1) than in control animals (111.2 +/- 5.2 mumol.kg-1.min-1). Metabolic clearance rates (MCR = Rt/[glucose]) were not different at rest (11.6 +/- 7.4) but were significantly greater in anemic (55.2 +/- 5.7 ml.kg-1.min-1) than in control animals (24.3 +/- 1.4 ml.kg-1.min-1) during exercise.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of Folates in Ochromonas malhamensis

SYNOPSIS. Ochromonas malhamensis contains folate derivatives active for Streptococcus faecalis, Pediococcus cerevisiae and Lactobacillus casei. These activities increase about 6-, 4- and 3-fold, respectively, on treatment with chicken liver conjugase, establishing the presence of polyglutamyl folates in the organism. The nature of the folate derivatives as well as their functional groups has been ascertained by chromatography on DEAE cellulose columns and assay of the activities of the eluted fractions, before and after the conjugase treatment, using differential microbiologic assay technic. The folate complex has been resolved into 7 fractions corresponding to N¹⁰-formyltetrahydrofolic acid, N⁵-formyltetrahydrofolic acid, unsubstituted tetrahydrofolic acid, and 4 polyglutamyl folates. The implications of these findings are indicated.     

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.     

Prospective evaluation of folic acid supplementation on plasma homocysteine concentrations during weight reduction: a randomized, double-blinded, placebo-controlled study in obese women

Elevated total homocysteine concentrations and obesity are both associated with an increased risk of cardiovascular disease. However, previous studies of weight reduction on serum homocysteine concentrations have obtained inconsistent reports. We investigated the effect of folic acid supplementation on serum homocysteine concentrations via a randomized, double-blinded, placebo-controlled study. Seventy-four obese women [age (mean +/- SEM) 41 +/- 1 years; body mass index, 29.6 +/- 0.5 kgs/m2] completed a 12 weeks weight reduction program with dietary advice and light exercise. They were also randomized to take either folic acid supplementation (5 mg daily, n = 36) or placebo (n = 38) groups. This program led to a weight reduction of 7.7% and 8.9% of initial weight for folic acid supplementation and placebo groups, respectively. Serum folate concentrations increased for 3 folds (p < 0.001) in the folic acid group. In the folic acid group, there was a trend of lower fasting serum homocysteine concentrations (7.6 +/- 0.2 vs. 7.3 +/- 0.3 micromol/L), but it did not reach statistical significance (p = 0.170). However, we found that serum homocysteine concentrations decreased significantly in those with higher baseline homocysteine concentrations (8.7 +/- 1.3 vs. 7.8 +/- 1.5 micromol/L, p = 0.004), while it did not change in those with lower baseline homocysteine concentrations (6.6 +/- 0.6 vs. 6.8 +/- 1.2 micromol/L, p = 0.334). Reduction of serum homocysteine concentrations did not correlate with elevation of serum folate concentrations (p = 0.646) in obese women with higher baseline homocysteine concentrations. In conclusion, serum homocysteine concentrations can be maintained in obese women during mild to moderate weight loss. Folic acid supplementation decreased serum homocysteine concentrations in those women who had higher serum homocysteine concentrations before participating in the weight reduction program.     

Acute anemia increases lactate production and decreases clearance during exercise

We evaluated whether elevated blood lactate concentration during exercise in anemia is the result of elevated production or reduced clearance. Female Sprague-Dawley rats were made acutely anemic by exchange transfusion of plasma for whole blood. Hemoglobin and hematocrit were reduced 33%, to 8.6 +/- 0.4 mg/dl and 26.5 +/- 1.1%, respectively. Blood lactate kinetics were studied by primed continuous infusion of [U-14C]lactate. Blood flow distribution during rest and exercise was determined from injection of 153Gd- and 113Sn-labeled microspheres. Resting blood glucose (5.1 +/- 0.2 mM) and lactate (1.9 +/- 0.02 mM) concentrations were not different in anemic animals. However, during exercise blood glucose was lower in anemic animals (4.0 +/- 0.2 vs. 4.6 +/- 0.1 mM) and lactate was higher (6.1 +/- 0.4 vs. 2.3 +/- 0.5 mM). Blood lactate disposal rates (turnover measured with recyclable tracer, Ri) were not different at rest and averaged 136 +/- 5.8 mumol.kg-1.min-1. Ri was significantly elevated in both control (260.9 +/- 7.1 mumol.kg-1.min-1) and anemic animals (372.6 +/- 8.6) during exercise. Metabolic clearance rate (MCR = Ri/[lactate]) did not differ during rest (151 +/- 8.2 ml.kg-1.min-1); MCR was reduced more by exercise in anemic animals (64.3 +/- 3.8) than in controls (129.2 +/- 4.1). Plasma catecholamine levels were not different in resting rats, with pooled mean values of 0.45 +/- 0.1 and 0.48 +/- 0.1 ng/ml for epinephrine (E) and norepinephrine (NE), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

An endogenous carrier-mediated uptake system for folate in oocytes of Xenopus laevis.

We investigated the existence of an endogenous uptake system for folate in Xenopus laevis oocytes. This was done by performing uptake measurements using [3H]folic acid. Uptake of folic acid was linear with time for 4 h of incubation, and was similar in collagenase-treated and non-treated oocytes. The uptake process was carrier-mediated, as suggested by the saturation of folic acid uptake with concentration, and by the ability of unlabelled folic acid and its related compounds to significantly inhibit the uptake of [3H]folic acid. The apparent Km and Vmax of the uptake process were 42 +/- 7 nM and 10.56 +/- 0.46 fmol per oocyte per 2 h, respectively. The uptake of folic acid was independent of the presence of Na+ in the incubation medium, but was highly pH dependent with severe inhibition occurring at pH lower than 6.5. Folic acid uptake was energy- and temperature-dependent, and was significantly inhibited by the anion transport inhibitors DIDS and SITS. These results demonstrate the existence of an endogenous carrier-mediated system for folic acid uptake in Xenopus oocytes. Further characterization of the molecular mechanism of folic acid uptake and its regulation in this non mammalian in vitro unicellular system may prove useful in furthering our understanding of folate movement across biological membranes.     

Stimulated efflux of folic acid from the mucosal epithelium of the rat in vitro

Everted sacs of rat intestine were used to observe the efflux of preloaded folic acid in the presence and absence of external folic acid and related compounds. Pteroyl-L-glutamic acid, 10-formyl folic acid and methotrexate were found to significantly stimulate efflux, whereas pteroyl-D-glutamic acid was significantly less effective and pteroic acid gave zero stimulation. These results suggest either the presence of intracellular binding sites for folic acid, or a specific membrane carrier system.     

Cardiac responsiveness to beta-adrenergic stimulation in experimental anemia.

Cardiac reactivity of beta-adrenergic stimulation was assessed by isoproterenol dose-reponse curves (dose range 0.025-0.4 mug/kg) before and 1 h after the rapid induction of anemia in dogs anesthetized with halothane:N2O:O2. Anemai (hematocrit = 16 +/- 4%) was induced by an isovolumic exchange transfusion with Dextran 70, and was followed by significant increments in cardiac output (+57 +/- 9%), max dP/dt of the left ventricle (+37 +/- 7%), and in peak acceleration of blood flow in the ascending aorta (+46 +/- 13%). Anemia was associated with a significant reduction of the chronotropic responses to all but the lowest dose of isoproterenol. The simultaneously determined inotropic responses (max dP/dt) where the same before and after the induction of anemia. The responses in terms of peak acceleration of aortic blood flow tended to be greater in the anemic than in the control phase, at all dose levels used. These findings indicate that in rapidly induced experimental anemia the heart is capable of responding to marked degrees of beta-adrenergic stimulation, representing a more than two-fold increase in the dP/dt.     

Biosynthesis of folic acid

Summary It has been attempted to isolate and characterize the folate precursors in the culture filtrates of two folate-requiring organisms, Streptococcus faecalis R and Lactobacillus casei. On the basis of paper chromatography, bioautography, ultra violet absorption spectra, chemical reactions, and differential microbiological responses it has been concluded that L. casei cultures contain a compound similar to pteroic acid which can be utilized by S. faecalis R. The S. faecalis R cultures on the other hand appear to accumulate a pteridine derivative active for Crithidia fasciculata It has been confirmed that this pteridine is not derived from the folic acid usually added to the growth medium.Abbreviations PGA Pterolyglutamic acid - PABA p-aminobenzoic acid     

Fetal anemia leads to augmented contractile response to hypoxic stress in adulthood

In response to chronic fetal anemia, coronary blood flow, maximal coronary conductance, and coronary reserve increase. We sought to determine whether chronic fetal anemia alters left ventricular (LV) function in adulthood. We studied adult sheep that had been made anemic for 20 days in utero by phlebotomy. They were transfused just before birth. At 7 mo of age, LV function was measured by pressure-volume loops at rest and during hypoxic stress. The in utero anemia group (n = 8) did not differ from controls (n = 5) with respect to hematocrit, heart and body weight, or baseline hemodynamic parameters. However, the effect of hypoxia (relative to baseline) on multiple indexes of systolic function was different between the two groups. End-systolic elastance increased in the in utero anemia group (baseline to hypoxia) by 4.15 +/- 3.47 mmHg/ml (mean +/- SD) but changed little in controls (0.24 +/- 0.45), which shows that the response to hypoxia was significantly different (P < 0.01) between groups. Similarly, the maximum derivative of LV pressure with respect to time increased in the in utero anemia group (486 +/- 340 mmHg/s,) but on average fell in the controls (-503 +/- 211 mmHg/s) with the response again being significantly different (P < 0.03). We conclude that in sheep, perinatal anemia can alter cardiac responses to hypoxic stress in the adult long after restoration of normocythemia.