Hippuric Acid Formation Research Articles

Flavonoids-Rich Orthosiphon stamineus Extract as New Candidate for Angiotensin I-Converting Enzyme Inhibition: A Molecular Docking Study.

This study aims to evaluate the in vitro angiotensin-converting enzyme (ACE) inhibition activity of different extracts of Orthosiphon stamineus (OS) leaves and their main flavonoids, namely rosmarinic acid (RA), sinensetin (SIN), eupatorin (EUP) and 3′-hydroxy-5,6,7,4′-tetramethoxyflavone (TMF). Furthermore, to identify possible mechanisms of action based on structure–activity relationships and molecular docking. The in vitro ACE inhibition activity relied on determining hippuric acid (HA) formation from ACE-specific substrate (hippuryl-histidyl-leucine (HHL)) by the action of ACE enzyme. A High Performance Liquid Chromatography method combined with UV detection was developed and validated for measurement the concentration of produced HA. The chelation ability of OS extract and its reference compounds was evaluated by tetramethylmurexide reagent. Furthermore, molecular docking study was performed by LeadIT-FlexX: BioSolveIT’s LeadIT program. OS ethanolic extract (OS-E) exhibited highest inhibition and lowest IC50 value (45.77 ± 1.17 µg/mL) against ACE compared to the other extracts. Among the tested reference compounds, EUP with IC50 15.35 ± 4.49 µg/mL had highest inhibition against ACE and binding ability with Zn (II) (56.03% ± 1.26%) compared to RA, TMF and SIN. Molecular docking studies also confirmed that flavonoids inhibit ACE via interaction with the zinc ion and this interaction is stabilized by other interactions with amino acids in the active site. In this study, we have demonstrated that changes in flavonoids active core affect their capacity to inhibit ACE. Moreover, we showed that ACE inhibition activity of flavonoids compounds is directly related to their ability to bind with zinc ion in the active site of ACE enzyme. It was also revealed that OS extract contained high amount of flavonoids other than RA, TMF, SIN and EUP. As such, application of OS extract is useful as inhibitors of ACE.

In Vitro Study on Antihypertensive and Antihypercholesterolemic Effects of a Curcumin Nanoemulsion.

Atherosclerosis and hypertension can potentially progess into dangerous cardiovascular diseases such as myocardial infarction and stroke. Statins are widely used to lower cholesterol levels while antihypertensive agents such as captopril are widely prescribed to treat high blood pressure. Curcumin, a phenolic compound isolated from Curcuma domestica, has been proven effective for a broad spectrum of diseases, including hypertension and hypercholesterolemia. Therefore, curcumin is quite promising as an alternative therapeutic compound. Our previous studies have proven a significant increase in physical properties, bioavailability, and stability of curcumin when encapsulated in a nanoemulsion. The purpose of this study was to assess the ability of the nanoemulsion in enhancing curcumin activity as a antihypertensive and antihypercholesterolemic agent. The formulation and preparation method of the curcumin nanoemulsion have been developed in our previous study. Physical characterization was performed, including measurement of droplet size, polidispersity index, zeta potential, entrapment efficiency, and loading capacity. Antihypertensive activity of curcumin was evaluated by determining Angiotensin Converting Enzyme (ACE) inhibition in vitro. A substrate for ACE, hippuryl-L-histidyl-L-leucine was allowed to react with ACE, resulting in hippuric acid formation as the product. The degree of ACE inhibition by curcumin was represented by the amount of hippuric acid formed. Antihypercholesterolemic activity of curcumin was studied using the HMG-CoA reductase assay equipped with a 96-well UV plate. This assay was based on the spectrophotometric measurement of the decrease in absorbance which represents the oxidation of NADPH by the catalytic subunit of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) in the presence of the substrate HMG-CoA. Curcumin is known to have no significant difference in inhibiting ACE compared to Captopril, but when it was incorporated in the self-nanoemulsifying carrier, it slightly increased the inhibitory effect on ACE. In contrast, the effect of curcumin in reducing cholesterol based on the HMGR assay was more pronounced. Curcumin encapsulated in a nanoemulsion showed significant cholesterol-lowering activity compared to a standard drug, pravastatin. Therefore, we conclude that curcumin does not show ACE inhibitory effects, but has potential use as an alternative therapeutic compound to treat hyperlipidaemia. Curcumin encapsulated in a nanoemulsion increased not only the HMGR inhibition, but also ACE inhibition of curcumin. These effects are suggested to be the result of improved solubility in the nanoemulsion system.

Inhibitory Efficacy and Biological Variability of Tryptophan Containing Dipeptides on Human Plasma Angiotensin Converting Enzyme Activity

Background: Hypertension remains a major unsolved health problem with life threatening consequences. A potential innovative prophylactic approach against its development may be supplementation of daily food with natural antihypertensive compounds. In our work we tested the efficacy of novel tryptophan containing dipeptides from whey protein with respect to their inhibitory potency on the angiotensin converting enzyme (ACE) as well as their stability in human plasma. Methods: ACE activity from rabbit lung and human plasma as well as recombinant human ACE activity were measured using benzoyl-glycyl-histidyl-leucine as a substrate. Product formation of hippuric acid and histidyl-leucine was assessed by UV-HPLC. The stability of the peptides was quantified in human plasma and, in case of incubation with recombinant human angiotensin converting enzyme, in buffer. Variability of ACE inhibiting activity of peptides was assessed via analysis of coefficient of variation with particular reference to true inter-individual variability corrected for methodological and analytical errors. Results: Isoleucine-tryptophan (IW) had a stronger ACE inhibiting potency (IC50 of 1.9 and 38.8 μmol/l with rabbit lung and human plasma ACE, respectively) than other tested peptides, i.e. glutamic acid-tryptophan (EW), tryptophanleucine (WL) and tryptophan-glutamic acid (WE). However, stability of IW showed a high degree of variability in plasma of different volunteers. In contrast EW, WL, WE were relatively stable in plasma and no major inter-individual variability was observed. Inhibition of plasma proteases extended plasma half-life of IW significantly. ACE did not contribute to peptide elimination. Conclusion: Milk whey protein represents a valuable source for bioactive peptides with ACE inhibitory potency that can be used as a food supplement to prevent, or in combination with other pharmaceutical agents to treat hypertension. However, in addition to the ACE inhibitory efficacy the stability of these peptides must be considered for in vivo ACE inhibition.

Evaluation of Metabolism and Disposition of GDC-0152 in Rats Using 14C Labeling Strategy at Two Different Positions: A Novel Formation of Hippuric Acid from 4-Phenyl-5-Amino-1,2,3-Thiadiazole

The compound (S)-1-[(S)-2-cyclohexyl-2-([S]-2-[methylamino]propanamido)acetyl]-N-(4-phenyl-1,2,3-thiadiazol-5-yl)pyrrolidine-2-carboxamide (GDC-0152) is a peptidomimetic small molecule antagonist of inhibitor of apoptosis (IAP) proteins with antitumor activity. The mass balance, pharmacokinetics, tissue distribution and metabolism of GDC-0152 was investigated in rats following intravenous administration of 15 mg/kg of [(14)C]GDC-0152, labeled either at the terminal phenyl ring (A) or at the carbonyl of the 2-amino-2-cyclohexylacetyl moiety (B). In rats, 92.2%-95.1% of the radiolabeled GDC-0152 dose was recovered. Approximately 62.3% and 25.1% of A was excreted in urine and feces, respectively. By contrast, B was excreted almost equally in urine (27.2%), feces (32.2%), and expired air (27.5%). GDC-0152 underwent extensive metabolism, with less than 9% of the dose recovered as parent in excreta. Similarly, in plasma, GDC-0152 represented 16.7% and 7.5% of the area under the curve of the total radioactivity for A and B, respectively. The terminal half-life (t(1/2)) for total radioactivity was longer for B (21.2 hours) than for A (4.59 hours). GDC-0152 was highly metabolized via oxidation and amide hydrolysis, followed by subsequent sulfation and glucuronidation. The most abundant circulating metabolites were the amide hydrolyzed products, M26, M28, M30, M31, and M34, which ranged from 3.5% to 9.0% of total radioactivity. In quantitative whole-body autoradiography studies, the residence of radioactivity in tissues was longer for B than for A, which is consistent with the t(1/2) of the total radioactivity in circulation. A novel 4-phenyl-5-amino-1,2,3-thiadiazole (M28) oxidative cleavage resulted in the formation of hippuric acid (M24). This biotransformation was also observed in rat hepatocyte incubations with para-substituted M28 analogs. In addition, the formation of M24 was inhibited by 1-aminobenzotriazole, which points to the involvement of P450 enzymes.

Angiotensin II upregulates renin–angiotensin system in human isolated T lymphocytes

This study was aimed at investigating the effects of Angiotensin (Ang) II stimulation on T lymphocytes mRNA expression of angiotensinogen (AGTN), angiotensin-converting enzyme (ACE) and AT1-receptor (R) and on ACE activity and Ang II content. The effects of Ang II stimulus were studied in lipopolysaccharide (LPS)-stimulated or not stimulated lymphocytes. mRNA expression for interferon-gamma (INF-γ) was also studied to investigate whether a link between lymphocyte RAS and immunological function might occur. mRNAs for AGTN, ACE and AT1-R were obtained from peripheral blood of 18 healthy subjects and were quantified by real time quantitative transcriptase–polymerase chain reaction (PCR). ACE activity was assayed in cell pellets and supernatants by measuring the hippuric acid formation by high performance liquid chromatography (HPLC) and Ang II cell content was measured by radioimmunoassay (RIA) after HPLC separation. All determination were performed under baseline conditions and after the addition of 10(e− 13) M Ang II to LPS-stimulated or unstimulated lymphocytes. Ang II caused a significant upregulation of T subset lymphocytes gene expression of ACE and AT1-R and of INF γ, and a marked increase in ACE activity and cell Ang II concentration. AGTN gene was never expressed. All these effects were further enhanced in T lymphocytes presitmulated by LPS and completely inhibited by Irbesartan. Our findings strongly support the evidence of a positive Ang II driven autocrine loop that upregulates cell RAS of isolated lymphocytes and activates the immuno response. The immuno-potentiating effect of Ang II, specifically shown in T subset, can be deleterious when local RAS are disregulated as in cardiovascular atherosclerotic disease.

Comparative In Vitro Metabolism of 14C-Ciclesonide in Hepatocytes From the Mouse, Rat, Rabbit, Dog, and Human

The in vitro metabolism of ciclesonide, a novel inhaled nonhalogenated glucocorticoid for the treatment of asthma, was compared in cryopreserved hepatocytes from mice, rats, rabbits, dogs, and humans. Incubations of C-ciclesonide with individual hepatocyte suspensions revealed similar metabolite profiles in all 5 in vitro systems used. Ciclesonide was rapidly converted to its active metabolite, desisobutyryl-ciclesonide (des-CIC). Des-CIC was then extensively metabolized to pharmacologically inactive metabolites through oxidation and reduction, followed by glucuronidation. A total of 12 groups of metabolites derived from des-CIC were characterized and identified by liquid chromatography/radioactivity monitor/mass spectrometry. Oxidation occurred on both the cyclohexane ring and the steroid moiety. Hippuric acid formation by cleavage of the cyclohexylmethyl moiety of ciclesonide, followed by aromatization of the cyclohexane ring through multiple steps of hydroxylation, dehydration, and conjugation with glycine, was found in rat, rabbit, and human hepatocyte incubations. The results indicated that ciclesonide and its active metabolite, des-CIC, were extensively metabolized in vitro in animal and human hepatocytes and that the metabolite profiles in mouse, rat, rabbit, and dog hepatocytes were similar to the profiles in human hepatocytes.

Effects of ALDH2, CYP1A1, and CYP2E1 genetic polymorphisms and smoking and drinking habits on toluene metabolism in humans.

In this study, we evaluated the effects of ALDH2, CYP1A1, and CYP2E1 genetic polymorphisms and smoking and drinking habits on the toluene metabolism. The study subjects were 92 male workers who handle toluene in a printing factory, an electrical parts factory, and a painting workplace in Japan. Their exposure levels to toluene were monitored using the diffusion-type sampler. Benzyl alcohol concentrations in their blood and hippuric acid (HA) and creatinine concentrations in their urine at the end of a workshift were determined. The genotype of ALDH2 was classified into the homozygous genotype of a normal ALDH2 gene (NN), the homozygous genotype of an inactive ALDH2 gene (DD), and the heterozygous genotype of normal and inactive ALDH2 genes (ND). The genetic polymorphism of CYP1A1 and CYP2E1 were also determined by restriction fragment length polymorphism (RFLP). A strong correlation between the personal exposure level and the urinary HA concentration was observed. Regression lines were calculated after being divided by the five factors, i.e., ALDH2, CYP1A1, CYP2E1, smoking, and drinking. The HA formation from toluene was significantly (p < 0.001) different among the genotypes of ALDH2. The slopes of the regression lines decreased from NN to ND to DD in this order. The benzyl alcohol concentration in the blood of the DD group was significantly higher than that found in the NN and ND groups. This result demonstrates that ALDH2 polymorphism affects the oxidation of benzyl alcohol to benzoic acid. The toluene metabolism was also affected by CYP1A1 polymorphism. The slope for the Ile/Ile (the predominant homozygous allele) group was significantly lower than that for the Ile/Val (the heterozygous allele) and Val/Val (the rare homozygous allele) group after correction for creatinine. A drinking habit significantly (p < 0.05) reduced urinary HA concentration in the NN group. A smoking habit also significantly (p < 0.05) reduced urinary uncorrected HA concentration in both the NN and ND groups. In a multiple regression analysis, ALDH2 and the drinking habit were significantly (p < 0.01) associated with HA excretion after toluene exposure with and without correction for creatinine, and the corrected HA concentration was also significantly (p < 0.01) increased in the Ile/Val and Val/Val group of CYP1A1. The smoking habit reduced the corrected HA concentration (p < 0.05); however, the polymorphism in the 5'-flanking region of CYP2E1 did not affect HA appearance in urine.

Angiotensin I is converted to angiotensin II by a serine protease in human detrusor smooth muscle.

The aim of the present study was to investigate whether a pathway for conversion of angiotensin I (ANG I) to angiotensin II (ANG II) other than that via angiotensin-converting enzyme (ACE) is present in the smooth muscle of the human detrusor. Isolated detrusor strips from 11 patients were contracted by ANG I (1 microM) in the absence or presence of enalaprilat (10 microM), soybean trypsin inhibitor (STI, 200 micrograms/ml), or both. The metabolic activity in detrusor membranes from four patients was studied separately using Hip-Gly-Gly or ANG I as a substrate, with or without various protease inhibitors. The contractile response to ANG I (1 microM) was depressed by enalaprilat from 66 +/- 22 (mean +/- SD) to 39 +/- 13% of the K+ (124 mM)-induced response (P < 0.01, n = 11), and the combination of enalaprilat and STI resulted in a further reduction in contractile amplitude to 25 +/- 14% (P < 0.01 vs. K+, and P < 0.05 vs. enalaprilat alone) and a significantly slower developing contraction with a time to peak of 3.7 +/- 1.7 vs. 1.1 +/- 0.3 min for ANG I alone (P < 0.01). In detrusor membranes, a low ACE activity, inhibitable by captopril, was demonstrated by the formation of hippuric acid (0.70 nmol.min-1.mg protein-1) from the synthetic ACE substrate, Hip-Gly-Gly. However, the conversion of ANG I (166 nmol.min-1.mg protein-1) to ANG II was not affected by ACE inhibition, while serine protease inhibitors, e.g., STI and chymostatin, completely prevented ANG II formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Formation of des-Asp-angiotensin I in the hypothalamic extract of normo- and hypertensive rats.

Exogenous angiotensin I (ANG I) was degraded to mainly des-Asp-ANG I instead of ANG II in the hypothalamic homogenate of the Sprague Dawley (SD), Wistar Kyoto (WKY), left renal artery stenosed hypertensive SD (LRAS), deoxycorticosterone acetate/salt-induced hypertensive SD (DOCA-salt) and spontaneously hypertensive rats (SHR). In the same homogenate, ANG II was degraded to ANG III and ANG III remained unchanged during the first 10 min of incubation. However, all the homogenates were able to catalyse hippuryl-L-histidyl-L-leucine to hippuric acid and the catalysis was completely inhibited by 3 microM captorpil. The data show that the angiotensin converting enzyme present in the hypothalamus when extracted by the normal laboratory procedures is not able to hydrolyse ANG I to ANG II. In addition, the aminopeptidase that degraded ANG I to des-Asp-ANG I was not inhibited by amastatin, bestatin and EDTA, indicating that it is not aminopeptidase A or B. The formation of hippuric acid was significantly higher in the homogenate of the LRAS whilst the SHR and DOCA-salt showed significant higher rate of des-Asp-ANG I formation than in the normotensive control rats.

The relationship between the effect of biphenylcarboxylic acid analogs on glyceride synthesis and conformation

The inhibition of biphenylcarboxylic acid analogs on glyceride synthesis was studied in association with conformation of the chemicals. Chemicals used were benzoic acid substituted with phenyl, benzoyl and phenoxy groups, phenylphenyloxyacetic acid and p-phenylacetic acid. Twist angles were calculated by a molecular orbital method. Following oral administration of p-phenylbenzoic acid to the rats for 7 days, the decrease of the triglyceride synthesized from [ 14C]oleic acid was observed in the adipose tissue. The in vitro inhibitions on the formation of fatty acyl-CoA thioester were examined from the influence on the hippuric acid formation from [ 14C]benzoic acid using the rat liver slices. In the homologous series, high inhibition was observed in para and meta substituents. None, or low inhibitions of ortho-substituted chemicals were related to the steric hindrance of the phenyl ring against carboxyl groups. This hinderance was associated with conformation and with the geometrical relationship between substituted phenyl rings and carbon atoms of carboxylic acid. In the in vitro inhibition on overall formation of diglyceride and triglyceride from [ 14C]glycerol, o-phenylbenzoic acid showing no inhibition on hippuric acid formation did not show inhibition. However, other ortho-substituted chemicals showed higher inhibition in comparison with the inhibition on hippuric acid formation. The inhibition of para- and meta-substituted chemicals were higher than the corresponding values of ortho substituted compounds.

Liver function in rats treated individually and with a combination of xylene, toluene and methanol.

The individual and combined influences of three industrial solvents (namely xylene, toluene, and methanol) on certain parameters of liver function in laboratory rats have been studied. Although individual treatments with all three solvents elevated activities of serum enzymes (i.e., GOT, GTP, and alkaline phosphatase), and did manifest hyperbilirubinemia, nevertheless metabolic interaction occurring after their combined treatment suggested a weak antagonistic mechanism. This assumption has been supported by the fact that the detoxification ability of the liver, as indicated by the formation of hippuric acid, improved after the combined treatment.

Conjugation of Benzoic Acid with Glycine in the Human Fetal and Adult Liver and Kidney

The rate of hippuric acid formation was measured in the homogenates obtained from 26 specimens of human adult liver, 9 specimens of human mid-gestational fetal liver, 5 specimens of human adult kidney and 11 specimens of human mid-gestational fetal kidney. The average (pmol/min/mg tissue +/- SD) rate of hippuric acid formation was 322 +/- 99 (adult liver), 7.6 +/- 3.6 (fetal liver), 284 +/- 117 (adult kidney) and 6.4 +/- 6.7 (fetal kidney). One third of the fetal livers and kidneys studied were inactive in the formation of hippuric acid. These findings indicate that the conjugation of carboxylic acid with glycine is poorly developed in the mid-gestational human fetus. The kinetics of the formation of hippuric acid were studied in 3 fetal and adult livers and also in 3 fetal kidneys and in 3 specimens of the cortex and medulla of adult kidneys. The transformation of the data into Eadie-Hofstee plots generated straight lines in fetal and adult livers and kidneys. The Michaelis-Menten constant for the formation of hippuric acid (mean +/- SD, mM) was 43.4 +/- 6.6 (adult liver), 27.3 +/- 10.1 (fetal liver), 33.3 +/- 6.1 (adult renal cortex), 34.7 +/- 11.3 (adult renal medulla) and 15.3 +/- 3.5 (fetal kidney). The maximum velocity of the reaction (mean +/- SD, pmol/min/mg tissue) was 204 +/- 47.8 (adult liver), 6.0 +/- 1.3 (fetal liver), 199 +/- 40.7 (adult renal cortex), 24.2 +/- 16.9 (adult renal medulla) and 6.4 +/- 1.8 (fetal kidney). The inhibitory effect of 8 drugs containing a carboxylic acid group on the rate of hippuric acid formation was studied in 3 adult livers. Salicylic acid and diflunisal were the most powerful as inhibitors. Ibuprofen, furosemide and sodium valproate were weak inhibitors, whereas ketoprofen, naproxen and captopril did not inhibit the formation of hippuric acid. The IC50 values (mean +/- SD) of salicylic acid and diflunisal on the rate of hippuric acid formation were 0.19 +/- 0.05 and 1.18 +/- 0.19 mM, respectively.

Benzyl alcohol metabolism and elimination in neonates.

Reports on fatal benzyl alcohol poisoning in premature neonates implied that the toxicity may be due to larger doses per kilogram than for adults. It has been postulated that the load of benzoic acid (metabolite of benzyl alcohol) may exceed the capacity of the immature liver or kidney for detoxification through glycine conjugation to form hippuric acid. To test this hypothesis, 14 term and 9 preterm neonates receiving loading doses of phenobarbital containing benzyl alcohol were studied. Urine and serum benzoic and hippuric acid levels were measured by GC and HPLC methods, respectively. There was greater accumulation of benzoic acid in the serum of preterm compared to the term neonates which was reflected in higher normalized peak levels (2130.6 vs. 237.8 kg/l, p less than 0.001) and larger normalized AUCIV (1,253.2 vs. 483.0 kg.h/l, p less than 0.01). Furthermore, larger percentages of benzyl alcohol doses were found in urine as benzoic acid in preterm babies, while less hippuric acid appeared in their urine than term newborns. These results indicate that hippuric acid formation is deficient in preterm neonates. Although we did not encounter in our patients the specific toxic signs described as part of the benzyl alcohol toxicity syndrome, we cannot directly answer the issue of safety of 'low doses' of benzyl alcohol as found in some medications administered to neonates. This study confirms the immaturity of the benzoic acid detoxification process in premature newborns.

Effect of Plasmodium berghei infection on benzoic acid metabolism in mice.

The metabolism of benzoic acid was studied in Plasmodium berghei infected mice both in vitro and in vivo. Results of in vitro studies showed a considerable decrease in the ability of the infected liver to detoxify benzoic acid by hippuric acid formation. The in vivo study showed that hippuric acid formation decreases with increasing parasitemia and the emergence of benzoyl-glucuronide. This new pathway stops operating with further increase in parasitemia.

The aromatization of cyclohexanecarboxyl-CoA to hippuric acid by guinea pig liver mitochondria: submitochondrial localization.

The conversion of cyclohexanecarboxyl-CoA to hippuric acid in submitochondrial fractions from guinea pig liver was studied using a gas chromatographic-mass spectrometric method employing selected ion monitoring. Comparison of the activities of the cyclohexanecarboxyl-CoA to hippuric acid converting system (CCoAHC-system) and marker enzymes in the various submitochondrial fractions showed that the CCoAHC-system is localized in the mitochondrial matrix. Partial separation of the inner and outer membranes has been accomplished by treating mitochondria with digitonin in isotonic medium and fractionating the treated mitochondria by differential centrifugation. A digitonin-protein ratio of 2.6 mg of digitonin/10 mg of protein must be used in order to release significant amounts of amine oxidase activity (outer membrane marker) from low speed mitochondrial pellets. This pellet still contained most of the glutamate dehydrogenase activity and was insignificantly contaminated with adenylate kinase. Moderate concentrations of phenazine methosulfate (PMS) greatly stimulated the activity of the CCoAHC-system, even in intact mitochondria (optimal concentration of PMS: 1 mM) whilst higher concentrations (greater than 1 mM) decreased the activity. The formation of hippuric acid in these mitochondrial preparations was linear with time for at least 40 min and linear with respect to protein concentration up to approximately 2.0 mg mitochondrial protein X ml-1.

Reduced formation of hippuric acid after oral benzoic acid in Friedreich's ataxia.

We have observed a markedly decreased formation of hippuric acid after benzoic acid load in patients with typical Friedreich's Ataxia compared to normal control subjects. Since there is evidence for normal or even enhanced tauro-conjugation in the bile of patients with this disease, with a decreased G/T ratio, it is unlikely that co-factor or enzyme concentrations are the cause of this defect. We postulate decreased availability of the enzyme for glycine conjugation either to bile acids in the usual situation or to benzoic acid in the artefactual test condition. This could be due to the enzyme's preference for an increased amount of taurine substrate in the liver. The relationship of this observation to the other biochemical changes observed in Friedreich's Ataxia must still be established.

Metabolism of drugs by the kidney

The kidneys have many clearly defined physiologic functions. Although their role as an excretory organ for drugs and chemicals and their polar metabolites is well described, their involvement in the biotransformation of xenobiotics is relatively poorly understood. It is accurate to state that our present understanding of the metabolic processes of drugs is based largely on studies carried out in the liver. Only recently have detailed investigations into drug metabolism in the kidney been carried out. These studies have shown that the kidney is meta-bolically very active in effecting the biotransformation of a variety of chemicals and drugs and, in some cases, surpasses the liver. It is important to understand the role of the kidney in drug and chemical biotransformation for a variety of reasons. Because the kidney receives a substantial portion of the cardiac output, it is reasonable to expect that it may make a significant contribution to the total metabolic alteration of drugs in the body. Furthermore, it is now known that the toxic effects of many drugs and chemicals are attributable to their metabolic conversion to reactive electrophilic intermediates, which, on reaction with cellular nucleophiles, lead to a variety of deleterious effects. Perhaps most important is the appreciation that this interaction between reactive intermediates and critical cellular macromolecules is intimately involved in mutagenic and carcinogenic changes. In addition, necrotic changes may also be associated with cellular alkylation. Thus, a complete perspective on the role of the kidney in pharmacologic and toxicologic processes is dependent on a thorough understanding of the drug and chemical metabolic capabilities of this organ. The biotransformation of organic compounds can be conveniently divided into oxidative, reductive, hydrolytic, and synthetic or conjugation reactions. The first three reactions are based on the type of chemical change produced. Synthetic or conjugation reactions usually involve the enzyme-catalyzed combination of a chemical or a metabolite and a carbohydrate or an amino acid, and they yield highly polar, readily excretable metabolites. Furthermore, multiple metabolic alterations are very common. Williams, in his classic book, Detoxication Mechanisms, suggests that the metabolism of many xenobiotics occurs in two steps [1]. The first, termed I reactions, includes oxidative, reductive, and hydrolytic reactions; the second, termed II reactions, consists of conjugative reactions and includes, for example, glucuronide, sulfate, and hippuric acid formation. It should be noted that although the metabolites produced in phase I reactions are frequently less toxic or less active pharmacologically than the parent compound is, abundant examples can be cited where the metabolites are more toxic and more active pharmacologically. By the same token, although the conjugates produced in phase II reactions are usually very polar and less toxic, notable exceptions exist; for example, the sulfate conjugate of N-hydroxyacetyl-aminofluorene is thought to be the proximate carcinogen in the case of this compound [5].

Conjugation and urinary excretion of toluene and m-xylene metabolites in a man.

Ingestion of benzoic acid (intermediary metabolite of toluene) by one volunteer subject indicated that the maximum urinary excretion rate of hippuric acid (final metabolite of toluene) was about 190 µmol/min. An almost identical figure for the saturation kinetics of hippuric acid formation has been reported in the literature, and it has been shown to be limited by the availability of glycine for benzoic acid conjugation. The maximum excretory capacity of the kidneys for hippurate is known to be considerably larger than the observed maximum rate of benzoic acid conjugation. In addition the intravenous administration of methylhippuric acid showed that its urinary excretion is rapid, approaching an excretion rate constant of 1.3 h-1. The glycine conjugation of benzoic acid and methylbenzoic acid may well be the ratelimiting step in toluene and, respectively, xylene metabolism. During the inhalation of these solvents, uptake rate may be estimated to equal the full conjugating capacity at a toluene or xylene air concentration of about 780 ppm at rest or of about 270 ppm during moderately heavy work. These data suggest that the body has a relatively large capacity for the conjugation reactions of toluene and xylene metabolism, but the consumption of a large part of easily mobilized glycine may impair the conjugation, and hence excretion, of poorer substrates, e.g., drugs.

Species differences in the metabolism of benzoic acid by isolated hepatocytes and kidney tubule fragments

Abstract The metabolism of benzoic acid to hippuric acid and benzoyl glucuronide has been studied in viable hepatocytes and renal tubule fragments from two species of omnivores (rat, hamster) and two species of carnivores (ferret, dog). Hippuric acid formation was detected in hepatocytes and tubules from omnivores but was not detectable in hepatocytes from the two carnivore species. High levels of hippuric acid were produced in the tubules from the carnivores. A small amount of glucuronidation occured in hepatocytes of all these species tested and in the carnivores this reaction was the predominant pathway of benzoic acid metabolism. These results indicated that the marked species differences in patterns of benzoic acid conjugation are related to differences in the ability of liver and kidney cells to carry out glycine and glucuronic acid conjugation.

Post-mortem survival of hippuric acid formation in rat and human cadaver tissue samples.

1. A rapid and sensitive semi-micro method for the determination of hippuric acid formation by tissue samples in vitro is described and applied to the determination of the post-mortem survival of hippuric acid formation in rat and human cadaver tissue samples. 2. Hippuric acid formation survived in rat and human cadaver liver for at least 72 h when corpses were stored at 4 degrees. 3. Hippuric acid formation was detected in human cadaver liver and kidney samples and was absent from brain, intestine, heart and lung. 4. Post-mortem liver samples from a case of acute pancreatitis failed to form hippuric acid as did kidney samples from a case of systemic lupus erythematosus with renal involvement.