Benzoate modulates renal and extrarenal nitrogen flow: metabolic mechanisms.

The mechanism by which benzoate enhances total nitrogen excretion was investigated in-situ and in separated rat renal proximal tubules. Orally administered benzoate augmented NH4+, urea and hippurate excretion 2, 1.9 and 76 fold respectively, as compared to baseline for control. Hippurate had similar effects. Benzoate augmented renal blood flow, glutamine extraction and total NH4+ production. Arterio-venous concentration differences of glutamine, glutamate, and NH4+ across the kidney, liver and gut demonstrated an increase in glutamine uptake by the kidney despite reduced release and uptake by the liver and gut, respectively; glutamate release by the kidney and gut was increased; NH4+ handling was unchanged at these three organs. Studies in separated rat renal proximal tubules demonstrated that benzoate stimulated glutamine dependent ammonia-genesis by activation of gamma-glutamyltransferase, via the synthesis of hippurate. The results demonstrate that benzoate can modulate the interorgan partitioning of nitrogen metabolites across several organs, the net effect of which is physiologically expressed as enhanced NH4+ , urea and hippurate excretion.     

Hepatic uptake and metabolism of benzoate: a multiple indicator dilution, perfused rat liver study

Multiple, noneliminated references ((51)Cr-labeled erythrocytes, (125)I-albumin, [(14)C]- or [(3)H]sucrose, and [(2)H](2)O), together with [(3)H]hippurate or [(14)C]benzoate, were injected simultaneously into the portal vein of the perfused rat liver during single-pass delivery of benzoate (5-1,000 microM) and hippurate (5 microM) to investigate hippurate formation kinetics and transport. The outflow dilution data best fit a space-distributed model comprising vascular and cellular pools for benzoate and hippurate; there was further need to segregate the cellular pool of benzoate into shallow (cytosolic) and deep (mitochondrial) pools. Fitted values of the membrane permeability-surface area products for sinusoidal entry of unbound benzoate were fast and concentration independent (0.89 +/- 0.17 ml. s(-1). g(-1)) and greatly exceeded the plasma flow rate (0.0169 +/- 0.0018 ml. s(-1). g(-1)), whereas both the influx of benzoate into the deep pool and the formation of hippurate occurring therein appeared to be saturable. Results of the fit to the dilution data suggest rapid uptake of benzoate, with glycination occurring within the deep and not the shallow pool as the rate-determining step.     

The effect of sodium benzoate on ammonia toxicity in rats

At 9.5 mmoles/kg body weight, sodium benzoate sharply increased mortality in rats subsequently challenged with ammonia. Fasted animals were less sensitive to potentiation of ammonia toxicity by benzoate than were fed animals. At 2.5 mmoles/kg body weight, benzoate was observed to protect fasted animals against ammonia toxicity. Measurements of ammonia disappearance, urea formation, and hippurate synthesis in suspensions of isolated hepatocytes indicate that benzoate potentiates ammonia toxicity by inhibiting the urea cycle.     

Growth of the purple bacterium Rhodobacter capsulatus on the aromatic compound hippurate

The purple nonsulfur bacterium Rhodobacter capsulatus strain B10 grew phototrophically on the aromatic compound hippurate (N-benzoyl-L-glycine) and related benzoyl amino acids. Absorption spectra, extraction, and GC/MS analysis of culture supernatants showed that hippurate was stoichiometrically converted to benzoate and glycine, with the latter used as a carbon or nitrogen source for growth. This conclusion was supported by detection of the enzyme hippuricase in permeabilized intact cells. Chemotrophic growth on hippurate by Rba. capsulatus, either at full or reduced oxygen tensions, was not observed. The type strain of Rhodobacter sphaeroides as well as four strains of Rhodopseudomonas palustris also grew phototrophically on hippurate, while several other aromatic-degrading species of purple bacteria did not.     

Use of [15N]glycine in the measurement of apolipoprotein B synthesis in perfused rat liver.

Rat livers were perfused with [15N]glycine and unlabeled sodium benzoate by the single-pass technique via the portal vein or in retrograde fashion via the inferior vena cava. Perfusate [15N]hippurate enrichment was significantly greater than that of hepatic free glycine from 15 to 90 min, regardless of the direction of the perfusion. This result implies that differential labeling by periportal versus perivenous hepatocytes is not likely. When fasted animals were compared to those fed a chow diet or a sucrose-enriched diet, the labeling ratio of medium hippurate/hepatic free glycine decreased by only 9% in spite of a 5-fold decrease in the concentration of intrahepatic free glycine. Administration of nembutal to the intact animal significantly increased the enrichment of medium hippurate by 24% but did not affect the enrichment of the hepatic free glycine. We conclude that the difference between hippurate and free glycine enrichment is related to intracellular compartmentation of glycine transport. We suggest that measurement of the enrichment of hippurate after the administration of [15N]glycine with benzoate in intact animals or human subjects can therefore be used to estimate the enrichment of the intracellular precursor pool of glycine with a correction factor that does not vary appreciably under fed or fasted conditions. When uniformly labeled deuteroglycine was used as the tracer, enrichment of hepatic free glycine was decreased fivefold compared with [15N]glycine. Isotopic enrichments of apoBH and apoBL from the d less than 1.063 g/ml lipoprotein fraction isolated from the perfusion medium between 30 and 90 min averaged 3.7 and 4.1% excess, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hippurate synthesis and ammonia metabolism in isolated hepatocytes

The incorporation of benzoate into hippurate by isolated hepatocytes was limited by the availability of glycine. This limitation was unaffected by the addition of ammonia. When isolated hepatocytes were incubated in a solution containing urease, benzoate was completely ineffective in combating ammonia accumulation, while a mixture of α-keto analogues of several amino acids reduced ammonia accumulation by 50%. These findings do not support the use of benzoate to combat hyperammonemia, but they do support the current use of α-keto analogues of amino acids for that purpose.     

Effect of hippurate on glucose utilization in rat kidney cortex slices.

Hippurate action on glucose utilization was evaluated in rat kidney cortex slices. Studies have shown the following. (1) Hippurate inhibits markedly basal as well as insulin-stimulated glucose utilization and basal gluconeogenesis. (2) Ca deficiency and specific Ca channel blockers diltiazem and isradipine abolish the hippurate inhibition of glucose utilization. (3) K+ channel blockers, i.e. the increased K+ concentration in incubation medium, procaine and sulfonylurea drugs also abolish the hippurate inhibition of glucose utilization. It is concluded that hippurate and benzoate operate through the ATP-dependent K+ channel.     

The role of substrates for glycine acyltransferase in the reversal of chemically induced porphyria in the rat

Experimental porphyria in the rat induced by the porphyrogenic agent, 3,5-dicarbethoxy-1,4-dihydrocollidine, was reversed by sodium benzoate or p-aminobenzoate treatment. In porphyric rats, benzoate and p-aminobenzoate markedly decreased the urinary excretion of the heme precursors, δ-aminolevulinic acid, porphobilinogen, and porphyrins, as well as the levels of tissue and blood porphyrins. The administration of glycine prevented the reversal of the porphyria. Neither benzoate, p-aminobenzoate, nor their respective metabolites, hippurate and p-aminohippurate, had an effect on δ-aminolevulinic acid synthetase in vivo or in vitro, indicating that the reversal of porphyria could not be explained by an effect on the rate limiting enzyme for heme biosynthesis. Hippurate, administered intraperitoneally, had no effect on the porphyric state. These results indicate that benzoate and p-aminobenzoate, substrates for glycine acyltransferase (EC 2.3.1.13), promote the diversion of glycine from the heme biosynthetic pathway to hippurate biosynthesis, thereby altering the biochemical pattern associated with the porphyric state.     

The synthesis of hippurate from benzoate and glycine by rat liver mitochondria. Submitochondrial localization and kinetics.

1. Rat liver mitochondria make hippurate at up to 4 nmol/min per mg of protein. The rate of synthesis supported by oxidation of glutamate with exogenous Pi present is identical with that supported by ATP plus oligomycin. Lower rates were obtained with other respiratory substrates, and when glutamate was used without Pi. 2. A matrix localization for hippurate synthesis is indicated by the latency of benzoyl-CoA synthetase and glycine N-acyltransferase to their extramitochondrial substrates, failure of exogenous benzoyl-CoA to inhibit incorporation of [14C]hippurate and inhibition of hippurate synthesis supported by ATP, but not glutamate, by carboxyatractyloside. 3. The relative activities of the individual enzymes and the mitochondrial content of benzoyl-CoA in the presence and absence of glycine suggest that hippurate synthesis is rate-limited by formation of benzoyl-CoA. 4. The increases in rates of ATP hydrolysis and of O2 consumption on the addition of benzoate and glycine were in good agreement with those required to support hippurate synthesis. The increase in respiration indicates that State-4 respiration [Chance & Williams (1957) Adv. Enzymol 17, 65-134] is not used, with these conditions, for ATP synthesis.     

Role of chloride on carrier-mediated transport of p-aminohippurate in rat renal basolateral membrane vesicles

Effect of inorganic anions on p-amino[3H]hippurate transport in renal basolateral membranes has been studied using the vesicles preloaded with unlabeled p-aminohippurate (countertransport condition). The uptake of p-amino[3H]hippurate was stimulated by the outward gradient of unlabeled p-aminohippurate and the labeled substrate was accumulated into the vesicles against its concentration gradient in the presence of Cl-. The substitution of SCN- and SO4(2-) for Cl- in both sides of the vesicles depressed the initial rate and the overshoot magnitude of p-amino[3H]hippurate uptake. These results suggest that Cl- may play an important role for the carrier-mediated transport system of organic anion in renal basolateral membranes.     

Inhibition of pyruvate carboxylase by sequestration of coenzyme A with sodium benzoate

Pyruvate-dependent CO2 fixation by isolated mitochondria was strongly inhibited by sodium benzoate. Pyruvate carboxylase was identified as a site of inhibition by limiting flux measurements to assays of pyruvate carboxylase coupled with malate dehydrogenase. Benzoate reduced pyruvate-dependent incorporation of [14C]KHCO3 into malate and pyruvate-dependent malate accumulation by 74 and 72%, respectively. Aspartate-dependent malate accumulation was insensitive to benzoate, ruling out malate dehydrogenase as a site of action. Inhibition by benzoate was antagonized by glycine, which sharply accelerated conversion of benzoate to hippurate. Assays of coenzyme A and its acyl derivatives revealed inhibition to correlate with depletion of acetyl CoA and accumulation of benzoyl CoA. Depletion of acetyl CoA was sufficient to account for greater than 50% reduction in pyruvate carboxylase activity. Competition between acetyl CoA and benzoyl CoA for the activator site on pyruvate carboxylase was insignificant. Results support the interpretation that the observed inhibition of pyruvate carboxylase occurred primarily by depletion of the activator, acetyl CoA, through sequestration of coenzyme A during benzoate metabolism.     

Amino acids in the rat liver and plasma and some metabolites in the liver after sodium benzoate treatment

The effect of sodium benzoate administration on amino acids in the liver and plasma and various metabolites in the liver was studied. Changes in glutamine and ornithine were noted only at a higher dose (10 mmol/kg body wt) of benzoate, whereas even a lower dose caused a significant decrease in glycine, serine, and alanine levels of plasma and liver. A dose- and time-dependent decrease in glycine levels was studied. A decrease of up to 50% in the glycine concentration may limit its own transport into mitochondria and availability for the formation of hippurate. A decrease in alanine may have resulted from stimulation of gluconeogenesis from alanine, by increased ammonia. Among the metabolites studied, ATP and acetyl-CoA decreased and ammonia increased significantly even at a lower dose (5 mmol/kg body wt) of benzoate. The compounds that require ATP for their synthesis such as N-acetylglutamate and glutamine decreased significantly only at the higher dose of benzoate, whereas urea and glutathione levels were unaffected under our experimental conditions.     

A difference in the incorporation of 14C into hippurate glycine from dl-[2-14C]glutamate and dl-[5-14C]glutamate in guinea pigs

The specific radioactivity of urinary hippurate glycine was determined after injecting guinea pigs with benzoate and either dl-[2-(14)C]glutamate or dl-[5-(14)C]glutamate. The isotope dilution factor for the formation of [(14)C]glycine was significantly greater (30%) with C-2 labelled glutamate. With either form of labelled glutamate the hippurate glycine was largely carboxyl-group labelled. The observations suggest a route for the incorporation of glutamate carbon into glycine that involves C-5 but not C-2. A hypothesis for glycine biosynthesis from l-glutamate is advanced, consistent with these findings, that includes conversion of l-glutamate to 4-hydroxy-2-oxoglutarate, the scission of the latter to glyoxylate and pyruvate, and the formation of glycine by transamination.     

Effects of bile acids and their glycine conjugates on gamma-glutamyl transpeptidase

Glycine and taurine conjugates of bile acids modulate gamma-glutamyl transpeptidase by interacting with the cysteinylglycine binding site (acceptor site) of the enzyme. These compounds stimulate hydrolysis of glutamine and S-methylglutathione and the rate of the inactivation of the enzyme by the gamma-glutamyl site-directed reagent, AT-125 (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid). Transpeptidation between S-methylglutathione and methionine was inhibited by these compounds. These effects resemble those caused by hippurate; the glycine derivatives of bile acids, however, exhibit a much greater affinity for transpeptidase than hippurate. Cholate, as shown previously for benzoate, also seems to bind to a portion of the acceptor site as indicated by its effects on S-methylglutathione utilization and AT-125-dependent inactivation of the enzyme. The Kd values for cholate and benzoate are, however, at least one order of magnitude larger than those for their respective glycine derivatives. The acceptor site-directed modulators increase the affinity of the enzyme for AT-125 and kinetic and binding studies show that binding of gamma-glutamyl site-directed reagents increases the affinity of the enzyme for cholate. These results thus indicate cooperative interactions between the gamma-glutamyl donor and acceptor binding domains of the transpeptidase active center.     

Chronic Sodium Benzoate Therapy in Children with Inborn Errors of Urea Synthesis: Effect on Carnitine Metabolism and Ammonia Nitrogen Removal

Sodium benzoate (SB) therapy is known to increase ammonia (NH₃) nitrogen elimination via conjugation with glycine and excretion as urinary hippurate. In 16 children with inborn errors of urea synthesis we studied two issues: (1) the effect of chronic SB administration upon carnitine metabolism and (2) the efficacy of chronic SB therapy as measured by the molar ratio of hippurate excretion to SB intake. Measurements were performed during elective hospitalizations when the patients were in stable metabolic condition. We found that chronic SB therapy is not associated with a constant level of hippurate elimination and that interindividual and intraindividual variability may result in irregular removal of NH₃nitrogen. This variability may be due to various factors including the formation of small quantities of benzoylcarnitine, which was detected in the plasma of three of four patients receiving SB and carnitine therapy and in one of two patients on SB therapy without carnitine supplementation. The ratios of acyl to free carnitine were elevated in both plasma and urine in patients not receiving carnitine supplementation, but were normal in patients receiving supplementation.     

Significance of transported glycine in the conjugation of sodium benzoate in spf mutant mice with ornithine transcarbamylase deficiency

3H-glycine and 14C-serine were injected intraperitoneally, during treatment of spf mutant mice with 2% sodium benzoate in drinking water. Urinary hippurate was separated by thin layer chromatography and counted for 3H and 14C labels representing transported and newly synthesized glycine, respectively. The specific activity of 3H-hippurate increased significantly in mutant and normal groups, while the increase of 14C was seen only in mutants. The ratio of specific activity 3H:14C showed significant increases in normal (0.99 to 1.93; p less than 0.01) and mutant (1.53 to 3.05; p less than 0.05) groups, which shows that glycine transported from body pools played a significantly greater role in the conjugation of benzoate, compared to glycine synthesized de novo from serine. In spf mice, benzoate treatment also resulted in a decrease in orotate excretion, indicating amelioration of the hyperammonemic state. It is postulated that the elimination of glycine transported from body pools may be the primary mechanism for the reduction of ammoniagenicity in benzoate therapy, and that the de novo synthesis of glycine may have a secondary effect.     

Measurement of apolipoprotein B synthesis in perfused rat liver using stable isotopes: [15N]hippurate as a measure of the intracellular [15N]glycine precursor enrichment

Rat livers were perfused by the nonrecirculating technique with medium containing [15N]glycine and sodium benzoate. At various times, the isotopic enrichment of hepatic free glycine, hepatic glycyl-tRNA, and perfusate hippurate was measured by GLC-MS. After 60 min, these parameters had reached approximately maximal values. At 90 min, the perfusate hippurate had a 30% greater enrichment of 15N than the intracellular glycine or glycyl-tRNA. Hippurate enrichment was half that of the medium glycine. The rat livers secreted apolipoprotein B (B-100 plus B-48) at a rate of 22 micrograms/g per h. From the 15N enrichment and the secretion rate, an intrahepatic pool size of 86 micrograms/g of apoB was calculated. From the minimal intracellular transit time of 30 min, an apoB fractional synthetic rate (FSR) of 2 pools/h was indicated, whereas the FSR estimated from the 15N-enrichment was 0.26/h. A possible explanation for the discrepancy is that apoB may recycle within the hepatocyte. On the basis of the present experiments, when hippurate enrichment is used as a measure of the enrichment of intrahepatic glycine in in vivo studies with 15N-labeled glycine, a correction should be applied, under normal metabolic circumstances, of approximately 20-30%.     

Effects of Albumin, Amino Acids and Clofibrate on the Uptake of Tryptophan by the Rat Brain

Abstract: The influences of total tryptophan concentration, albumin binding and amino acid competition on the rate of tryptophan influx into rat brain were compared using a single-pass injection technique with tritiated water as a freely diffusible reference. Omission of 3% bovine albumin from a bolus containing tryptophan in Krebs–Ringer bicarbonate buffer injected into the carotid artery increased non-albumin bound (free) tryptophan concentration threefold but tryptophan uptake by only 35% and 30% into forebrain and hypothalamus, respectively. However, tryptophan uptake from injected rat plasma was more markedly elevated when free tryptophan concentration was raised. Thus, when free tryptophan was doubled, but total tryptophan unchanged, by in vitro addition of clofibrate to a plasma bolus, uptake was increased by 53% and 28% into forebrain and hypothalamus respectively. When clofibrate was injected in vivo so that plasma total tryptophan concentration was decreased by 45% but neither free tryptophan nor competing amino acid concentrations were altered, then uptake from a bolus of the rat's own plasma was unchanged. Addition of competing amino acids at physiological concentrations to tryptophan in Krebs-Ringer buffer significantly reduced tryptophan influx into both brain regions, but did not increase the effect of albumin binding. The results indicate that tryptophan uptake into rat forebrain is substantially influenced by albumin binding and competition from other amino acids, but that hypothalamic uptake is less influenced by these factors.     

Uptake of benzoate by Rhodopseudomonas palustris grown anaerobically in light.

The uptake and anaerobic metabolism of benzoate were studied in short-term experiments with phototrophic cells of Rhodopseudomonas palustris. Cells that were preincubated and assayed anaerobically in the presence of 1 mM dithiothreitol accumulated [7-14C]benzoate at a rate of at least 0.5 nmol . min-1 . mg-1 of protein. Cells that were preincubated aerobically, or anaerobically in the absence of a reducing agent or an electron donor such as succinate, took up benzoate at reduced rates. Benzoate was removed from the external medium with remarkably high efficiency; initial uptake rates were independent of substrate concentration, and uptake remained linear down to concentrations of less than 1 microM. Uptake rates were not sensitive to external pH in the range of 6.5 to 8.1, and very little free benzoate was found associated with the cells. By contrast, benzoyl coenzyme A (CoA) was formed rapidly in cells exposed to labeled benzoate. Its appearance in such cells, together with the more gradual accumulation of other compounds tentatively identified as reduction products, is consistent with the identification of benzoyl CoA as an intermediate in the anaerobic reductive metabolism of benzoate. The very effective uptake of external benzoate can be explained by its conversion to benzoyl CoA immediately after its passage across the cell membrane by simple or facilitated diffusion. Such a chemical conversion would serve to maintain a downhill concentration gradient between the cell cytoplasm and the cell surroundings, even at very low external benzoate concentrations.     

benK encodes a hydrophobic permease-like protein involved in benzoate degradation by Acinetobacter sp. strain ADP1.

The chromosomal benK gene was identified within a supraoperonic gene cluster involved in benzoate degradation by Acinetobacter sp. strain ADP1, and benK was expressed in response to a benzoate metabolite, cis,cis-muconate. The disruption of benK reduced benzoate uptake and impaired the use of benzoate or benzaldehyde as the carbon source. BenK was homologous to several aromatic compound transporters.