Effect of vitamin D deficiency on D-glucose and citrate transport in rat renal basolateral membrane vesicles.

We had previously reported that feeding rats on Steenbock and Black's rickets-inducing diet markedly influences the metabolic picture of the kidney and the transmembrane transport systems of D-glucose and citrate in renal brush-border membrane vesicles. We have now studied D-glucose and citrate transport into basolateral membrane vesicles prepared from kidney cortex of control and rachitic rats and the effect of 1,25-dihydroxyvitamin D3 on these transport systems was also investigated. D-glucose and citrate uptake, determined in the presence of a Na(+)-gradient, was lowered in rachitic animals and 1,25-dihydroxyvitamin D3 administration proved to be ineffective in restoring normal values. Citrate transport, determined in the presence of a K(+)-gradient, was not influenced by both rickets and 1,25-dihydroxyvitamin D3 supply. The in vitro addition to vesicle preparations of calcium or phosphate or citrate or 1,25-dihydroxyvitamin D3 did not show a selective influence on D-glucose and citrate uptake.     

Influence of a rachitogenic regime on hepatic metabolism in the rat

We have previously reported that rats fed on the Steenbock and Black's rickets-inducing diet (deficient in vitamin D and with an altered Ca/P ratio) show metabolic modifications in kidney and intestinal mucosa. We have therefore decided to investigate if also in liver, seat of vitamin D hydroxylation, changes in the metabolic pattern occur. An increase of mitochondrial NAD+-dependent isocitrate dehydrogenase and a decrease of citrate and ATP content was demonstrated in liver of rachitic rats, together with changes in ATP-citrate lyase and glucose-6-phosphate dehydrogenase activity. The inhibitory effect of ATP on liver mitochondria NAD+-dependent isocitrate dehydrogenase was also studied.     

Parvalbumin in rat kidney. Purification and localization

The Ca2+-binding parvalbumin has been purified for the first time from rat kidney. Its biochemical and immunological properties were indistinguishable from the muscle counterpart. By immunohistochemical methods parvalbumin was localized in part of the distal tubule and proximal collecting duct, similar to the vitamin D-dependent Ca2+-binding protein, calbindin-28K. Parvalbumin was found to be independent of the vitamin D status of the animal since its concentration remained unchanged in kidney extracts of normal, rachitic and vitamin D-replete rats. Both proteins may be involved in the regulation of intracellular Ca2+ in kidney.     

Factors affecting Ca2+ transport in mouse islet and kidney mitochondria

The transport of Ca2+ in islet and kidney mitochondria respiring on succinate was inhibited by atractylate and fluorocitrate, and stimulated by pyruvate, isocitrate, alpha-ketoglutarate, dibutyryl cAMP, oligomycin and bongkrekate, and by in vivo administration of glucagon, glyceraldehyde or glucose. The kidney [beta-hydroxybutyrate]/[acetoacetate] ratio was increased in glyceraldehyde treated mice. The data suggest a relationship, which might be influenced by cAMP, between activity of pyruvate, isocitrate and alpha-ketoglutarate dehydrogenases and transport of Ca2+ in islet and kidney mitochondria. A contributory role of reductive carboxylation for Ca2+ uptake, and a role of citrate for Ca2+ retention are discussed.     

Citrate content of liver and kidney of rat in various metabolic states and in fluoracetate poisoning

The citrate content of rat liver changes little when normal rats are starved, when starved rats are re-fed with various diets and when normal animals are made diabetic with alloxan. The citrate content of rat kidney changes little on starvation, but it doubles on induction of diabetes. Fluoroacetate poisoning has relatively little effect on the citrate content of liver under a variety of conditions except that normal female rats show a 2·4-fold increase. Fluoroacetate poisoning leads to increases in the citrate content of kidney under all conditions. The relevance of these observations to the regulation of fatty acid synthesis is discussed. The acetic anhydride–pyridine method and the pentabromoacetone method for the estimation of citrate are compared.     

Effect of 25-hydroxyvitamin D3 on Na+-dependent D-glucose transport in rachitic rats

We have previously reported that feeding rats on Steenbock and Black's rickets-inducing diet, deficient in vitamin D and with an altered Ca/P ratio, leads to metabolic consequences and a marked decrease of Na+-dependent D-glucose uptake at the jejunum-ileum level. To clarify the relationship between experimental rickets and D-glucose uptake, 25-hydroxyvitamin D3 (25-OH-D3) was given to rats fed on the rickets inducing diet. In the jejunum-ileum of these animals Na+-dependent D-glucose uptake returned to the values of the controls while the decrease in D-glucose uptake in the brush-border membrane vesicles prepared from kidney cortex of rachitic animals was not corrected by the administration of 25-OH-D3.     

Mechanism of citrinin-induced dysfunction of mitochondria. IV—Effect on Ca2+ transport

The effect of citrinin on Ca²⁺ transport was studied in isolated kidney cortex and liver mitochondria, and baby hamster kidney cultured cells. The mycotoxin significantly inhibited the activity of 2-oxoglutarate and pyruvate dehydrogenases in both kidney cortex and liver mitochondria. Citrinin promoted a decrease in the velocity and in the total capacity of Ca²⁺ uptake, in both mitochondria. Apparently, citrinin acts by a mechanism similar to ruthenium red. In intact cultured cells, citrinin also had a preferential effect on mitochondrial Ca²⁺ fluxes. Citrinin promoted a marked decrease in the Ca²⁺ level in the mitochondrial matrix, whereas that of the extramitochondiral fraction became less affected. All the observed effects were dependent on the citrinin concentration.     

Effect of oral aluminum and aluminum citrate on blood level and short-term tissue distribution of aluminum in the rat

Aluminum (Al) absorption seems to be very low, but many factors can enhance it in animals and humans. In the present study, we investigated the acute effect of Na citrate on Al absorption by monitoring Al levels in blood and several tissues. For this purpose, 18 Wistar male rats were divided into 3 groups: control, Al, and Al + Na citrate. After a 14-h fasting period, animals were dosed orally with deionized water, or 2 mmol Al chloride, or 2 mmol Al chloride plus 2 mmol Na citrate. Blood samples were taken before and 1, 2, 4, and 6 h after the gavage. Al concentrations in blood, liver, tibia, kidney, and intestinal wall were determined by ICP-OES. In the Al and Al + citrate groups, Al blood concentrations peaked at 1 h and 2 h with higher levels in the Al + citrate group. Al gavage resulted in an increase in Al level in intestinal wall, but not in the other investigated tissues. Simultaneous gavage of citrate with Al significantly increased its tissue levels in tibia, kidney, and in intestinal wall. Our data show clearly that Al as chloride can be absorbed, but not well retained by the organism tissues. Furthermore, the model used in the present study is appropriate for acute studies to investigate the effect of various compounds on Al absorption in the rat.     

Purification of cofactor-dependent enzymes by affinity chromatography

Several 8-(6-aminohexyl)-amino adenine nucleotide derivatives, including ATP, 2′,5′-ADP, 3′,5′-ADP and desulfo-CoA (CoA, reduced coenzyme A), were prepared and immobilized on Sepharose by cyanogen bromide activation. 8-(6-Aminohexyl)-amino-ATP-Sepharose was found to exhibit good affinity for both NAD⁺-dependent dehydrogenases and kinases. Sequential biospecific elutions with NADH and ATP resulted in a good separation of dehydrogenases from kinases. As many as eight different dehydrogenases and kinases could be substantially purified from both porcine muscle and mouse kidney extracts by this new procedure. 8-(6-Aminohexyl)-amino-2′,5′-ADP- and −3′,5′-ADP-Sepharose were shown to exhibit good affinity for many NADP⁺-dependent dehydrogenases from yeast extracts and CoA-dependent enzymes, respectively. Purification of citrate synthases from pig heart and Eschericia coli extracts by means of these 8-substituted adenine nucleotide affinity columns was also presented.     

D-glucose uptake in intestinal brush-border membrane vesicles of rachitic rats

We have previously reported the metabolic consequences of feeding rats Steenbock and Black's rickets-inducing diet, deficient in vitamin D and with an altered Ca/P ratio. Using isolated brush-border membrane vesicles prepared from the jejunum, ileum and duodenum of control and rachitic rats, we have demonstrated a marked decrease of Na+-dependent D-glucose uptake at jejunum-ileum level of rachitic rats. At duodenum level Na+-dependent D-glucose transport was not influenced by rickets. A lack of any significant difference between the two animal groups was observed studying the facilitated transport of D-glucose, the diffusion of L-glucose and the Na+-dependent uptake of phenylalanine and aspartate.     

Vitamin D-dependent calcium-binding protein synthesis by chick kidney and duodenal polysomes

The hormonally active form of vitamin D, 1,25-dihydroxy vitamin D₃, is known to induce in the intestine and kidney of chicks the synthesis of a calcium-binding protein (CaBP). Here we report a correlation between the tissue levels of CaBP and the levels of apparent messenger RNA in total polysomes as determined by the vitamin D and dietary calcium status. Polysomes from pooled duodenal mucosa and kidney were prepared by the Mg²⁺ precipitation method. After translation in a heterologous, rabbit nuclease-treated reticulocyte system, the immunoprecipitated pellet of CaBP was dissolved and the proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gels. When 13 nmol of D₃ was given to 4-week-old rachitic chicks which were sacrificed 48 h later, it was found that the duodenum had eightfold more apparent mRNA for CaBP in the polysomes than the kidney. This was also reflected in the values of CaBP/mg protein in these tissues (duodenum, 7 μg/mg vs kidney, 0.9 μ/mg). Also, after giving D₃, there was a twofold increase in both apparent mRNA levels in the polysomes and in CaBP levels in the duodena of chicks which were raised on low-calcium diets versus chicks raised on high-calcium diets. While apparent mRNA for CaBP was present in polysomes from rachitic chick kidney, it was not detectable in the duodenum. From these studies it appears that the induction of CaBP by 1,25(OH)₂D₃ in both the intestine and kidney is determined by similar control mechanisms.     

The mode of toxic action of the pesticide gliftor: the metabolism of 1,3-difluoroacetone to (-)-erythro-fluorocitrate

The biochemical toxicology of 1,3-difluoroacetone, a known metabolite of the major ingredient of the pesticide Gliftor (1,3-difluoro-2-propanol), was investigated in vivo and in vitro. Rat kidney homogenates supplemented with coenzyme A, ATP, oxaloacetate, and Mg2+ converted 1,3-difluoroacetone to (-)-erythro-fluorocitrate in vitro. Administration of 1,3-difluoroacetone (100 mg kg(-1) body weight) to rats in vivo resulted in (-)-erythro-fluorocitrate synthesis in the kidney, which was preceded by an elevation in fluoride levels and followed by citrate accumulation. Animals dosed with 1,3-difluoroacetone did not display the 2-3 hour lag phase in either (-)-erythro-fluorocitrate synthesis or in citrate and fluoride accumulation characteristic of animals dosed with 1,3-difluoro-2-propanol. We demonstrate that the conversion of 1,3-difluoro-2-propanol to 1,3-difluoroacetone by an NAD+-dependent oxidation is the rate-limiting step in the synthesis of the toxic product, (-)-erythro-fluorocitrate from 1,3-difluoro-2-propanol. Prior administration of 4-methylpyrazole (90 mg kg(-1) body weight) was shown to prevent the conversion of 1,3-difluoro-2-propanol (100 mg kg(-1) body weight) to (-)-erythro-fluorocitrate in vivo and to eliminate the fluoride and citrate elevations seen in 1,3-difluoro-2-propanol-intoxicated animals. However, administration of 4-methylpyrazole (90 mg kg(-1) body weight) to rats 2 hours prior to 1,3-difluoroacetone (100 mg kg(-1) body weight) was ineffective in preventing (-)-erythro-fluorocitrate synthesis and did not diminish fluoride or citrate accumulation in vivo. We conclude that the prophylactic and antidotal properties of 4-methylpyrazole seen in animals treated with 1,3-difluoro-2-propanol derive from its capacity to inhibit the NAD+-dependent oxidation responsible for converting 1,3-difluoro-2-propanol to 1,3-difluoroacetone in the committed step of the toxic pathway.     

Activities of citrate synthase and NAD+-linked and NADP+-linked isocitrate dehydrogenase in muscle from vertebrates and invertebrates.

1. The activities of citrate synthase, NAD+-linked and NADP+-linked isocitrate dehydrogenase were measured in muscles from a large number of animals, in order to provide some indication of the importance of the citric acid cycle in these muscles. According to the differences in enzyme activities, the muscles can be divided into three classes. First, in a number of both vertebrate and invertebrate muscles, the activities of all three enzymes are very low. It is suggested that either the muscles use energy at a very low rate or they rely largely on anaerobic glycolysis for higher rates of energy formation. Second, most insect flight muscles contain high activities of citrate synthase and NAD+-linked isocitrate dehydrogenase, but the activities of the NADP+-linked enzyme are very low. The high activities indicate the dependence of insect flight on energy generated via the citric acid cycle. The flight muscles of the beetles investigated contain high activities of both isocitrate dehydrogenases. Third, other muscles of both vertebrates and invertebrates contain high activities of citrate synthase and NADP+-liniked isocitrate dehydrogenase. Many, if not all, of these muscles are capable of sustained periods of mechanical activity (e.g. heart muscle, pectoral muscles of some birds). Consequently, to support this activity fuel must be supplied continually to the muscle via the circulatory system which, in most animals, also transports oxygen so that energy can be generated by complete oxidation of the fuel. It is suggested that the low activities of NAD+-linked isocitrate dehydrogenase in these muscles may be involved in oxidation of isocitrate in the cycle when the muscles are at rest. 2. A comparison of the maximal activities of the enzymes with the maximal flux through the cycle suggests that, in insect flight muscle, NAD+-linked isocitrate dehydrogenase catalyses a non-equilibrium reaction and citrate synthease catalyses a near-equilibrium reaction. In other muscles, the enzyme-activity data suggest that both citrate synthase and the isocitrate dehydrogenase reactions are near-equilibrium.     

Distribution of control of oxidative phosphorylation in mitochondria oxidizing NAD-linked substrates.

The flux control distribution of the net rate of state 3 respiration was determined in heart and kidney mitochondria incubated with low concentrations of pyruvate (0.5 mM) or 2-oxoglutarate (1 mM), and in conditions that led to activation of NAD-linked dehydrogenases, i.e., high substrate or Ca2+ concentrations. Control of flux was exerted by the ATP/ADP carrier (flux control coefficient, ci = 0.37) and Site 1 of the respiratory chain (ci = 0.28) when dehydrogenase activity was low. Control of the process shifted to the ATP synthase (ci = 0.32) and the Pi carrier (Ci = 0.27) when dehydrogenases were activated by high pyruvate and high Ca2+. The changes in the control exerted by the ATP/ADP carrier and the ATP synthase were not due to changes in the transmembrane potential, nor to a modification of intramitochondrial ATP/ADP ratios. Applying the summation theorem of the control analysis, it was found that at low Ca2+ and pyruvate concentrations the dehydrogenases shared the control of state 3 respiration with other steps. The NAD-linked dehydrogenases did not exert any significant control at high Ca2+ or high pyruvate concentrations.     

Aldosterone stimulates Na+ transport without affecting citrate synthase activity in cultured cells

Aldosterone increases citrate synthase activity in toad urinary bladder and mammalian kidney. It has been suggested that this action is important to aldosterone stimulation of Na+ transport, and it has been used as a marker of those epithelia which are stimulated by aldosterone. We describe three continuous lines of cultured cells derived from toad urinary bladder and toad kidney in which aldosterone increases active Na+ transport but does not increase the activity of citrate synthase. Therefore, in cultured cells at least, citrate synthase is not a critical enzyme for, or a suitable marker of, aldosterone stimulation of Na+ transport.     

Alterations of the gamma-carboxyglutamic acid and osteocalcin concentrations in vitamin D-deficient chick bone

The content of osteocalcin and protein bound gamma-carboxyglutamic acid (Gla) was studied as a function of bone maturation and mineralization in normal and vitamin D-deficient, rachitic chickens. The Gla/Ca2+ ratio was elevated in rachitic bone, particularly in the most undermineralized regions. For example, there is a 10- to 20-fold elevation in Gla/Ca2+ in the newly synthesized, least mineralized rachitic bone fraction, which progressively decreases to a 1.5-fold elevation in the most highly mineralized areas of rachitic tissue. Osteocalcin, which is the principal Gla-containing protein of mature bone, was quantitated by radioimmunoassay using specific antiserum to the 5670-dalton chicken protein. Surprisingly, the osteocalcin concentration is decreased 50% in vitamin D-deficient bone. From this we infer that accumulated Gla-containing protein in vitamin D-deficient and poorly mineralized bone may possibly represent a precursor of osteocalcin.     

The activation of rabbit muscle, liver, and kidney fructose bisphosphatases by histidine and citrate

Purified fructose 1,6-bisphosphatases from rabbit muscle, liver, and kidney require a metal chelator for optimal activity at neutral pH. This requirement is satisfied by physiological concentrations of histidine and citrate, and at pH 7 their effects are additive. In the presence of both histidine and citrate the optimum activity is shifted from about pH 8 to pH 7.2, and the activity is greater than that obtained with optimal concentrations of EDTA. Carnosine, anserine, and 1-methyl histidine are also effective, but only at much higher concentrations, while 3-methyl histidine is effective in the same concentration range as is histidine. Isocitrate can replace citrate. The results suggest that fructose bisphosphatases possess distinct binding sites for divalent cations (Mg²⁺ or Mn²⁺) and also for histidine and citrate complexes of these cations.     

A complex effect of arsenite on the formation of alpha-ketoglutarate in rat liver mitochondria

This investigation presents disturbances of the mitochondrial metabolism by arsenite, a hydrophilic dithiol reagent known as an inhibitor of mitochondrial alpha-keto acid dehydrogenases. Arsenite at concentrations of 0.1-1.0 mM was shown to induce a considerable oxidation of intramitochondrial NADPH, NADH, and glutathione without decreasing the mitochondrial membrane potential. The oxidation of NAD(P)H required the presence of phosphate and was sensitive to ruthenium red, but occurred without the addition of calcium salts. Mitochondrial reactions producing alpha-ketoglutarate from glutamate and isocitrate were modulated by arsenite through various mechanisms: (i) both glutamate transaminations, with oxaloacetate and with pyruvate, were inhibited by accumulating alpha-ketoglutarate; however, at low concentrations of alpha-ketoglutarate the aspartate aminotransferase reaction was stimulated due to the increase of NAD+ content; (ii) the oxidation of isocitrate was stimulated at its low concentration only, due to the oxidation of NADPH and NADH; this oxidation was prevented by concentrations of citrate or isocitrate greater than 1 mM; (iii) the conversion of isocitrate to citrate was suppressed, presumably as a result of the decrease of Mg2+ concentration in mitochondria. Thus the depletion of mitochondrial vicinal thiol groups in hydrophilic domains disturbs the mitochondrial metabolism not only by the inhibition of alpha-keto acid dehydrogenases but also by the oxidation of NAD(P)H and, possibly, by the change in the ion concentrations.     

Correlation between the malate dependent progesterone and citrate biosynthesis in the mitochondrial fraction of human term placenta. The stimulatory effect of ADP and ATP

The relationship between malate dependent conversion of cholesterol to progesterone and citrate biosynthesis in human term placental mitochondria has been investigated. It has been shown that ADP and ATP (but not AMP) stimulate, significantly, both progesterone and citrate formation. The stimulatory effect of these adenine nucleotides was dependent on the presence of Mn2+ in the incubation medium. When Mn2+ was omitted or replaced by Mg2+ only negligible stimulatory effect of ADP and ATP was observed. Atractyloside and oligomycin were without effect on ADP and ATP stimulated progesterone and citrate production. Other dinucleotides tested as: GDP, UDP and CDP stimulated both progesterone and citrate formation only slightly. In all the experiments presented the rate of progesterone biosynthesis was found to be significantly correlated with the rate of citrate production. The experimental results presented in this paper suggest that the stimulatory effect of ADP and ATP on malate dependent progesterone biosynthesis is a consequence of an increased conversion of malate to tricarboxylic Krebs cycle intermediates. The possible mechanism by which ATP and ADP stimulate the citrate formation in human placental mitochondria is discussed.     

Effects of dietary supplementation with aluminum and citrate on iron metabolism in the rat

The metabolism of iron (Fe) has been shown to interact with that of aluminum (Al) in relation to intestinal absorption, transport in the blood plasma, and the induction of lipid peroxidation and cellular damage. Also, dietary supplementation with citrate has been shown to increase the absorption of both metals and, in the presence of high intakes of Fe and Al, leads to excessive accumulation of both metals in the body. In this study, the likely interaction between Al and internal Fe metabolism was investigated using rats fed diets that were either deficient, sufficient, or loaded with Fe, with or without the addition of Al and sodium citrate. These diets commenced when the rats were 4 wk old and were continued for 9–11 wk. At that time, Fe metabolism as assessed by measurement of organ uptake of⁵⁹Fe and¹²⁵I-transferrin, after iv injection of transferrin labeled with both isotopes, plus measurement of tissue concentrations of nonheme Fe and Al. The Fedeficient diet and Fe-loaded diet led to states of Fe deficiency and Fe overload in the rats, and supplementation of the diet with Al increased Al levels in the kidneys, liver, and femurs, but, generally, only when the diet also contained citrate. Neither Al nor citrate supplementation of the diet had any effect on nonheme Fe concentrations in the liver, kidney, or brain, or on the uptake of⁵⁹Fe or¹²⁵I-transferrin by liver, kidney, brain, or spleen. Only with the femurs was a significant effect observed: increased⁵⁹Fe uptake in association with increased Al intake. Therefore, using this animal model, there was little evidence for interaction between Fe and Al metabolism, and no support was obtained for the hypothesis that dietary supplementation with Fe and citrate can lead to excessive Fe absorption and deposition in the tissues.