Hippuric acid test using 13C-labelling and NMR spectroscopy.

This article describes a 13C-labelling and nuclear magnetic resonance approach for hippuric acid test which is potentially useful for evaluating liver reserve. In this approach, urine samples collected after ingestion of 13C-labelled benzoic acid were directly analysed by 13C nuclear magnetic resonance spectroscopy, and the excreted 13C-labelled hippuric acid formed from the administered benzoic acid was quantitated. The amount of labelled hippuric acid excreted in a specified time can be a useful index of liver reserve. In this study, the feasibility of the nuclear magnetic resonance approach has been investigated in several healthy subjects. This approach is simple and convenient compared with conventional analytical procedures, because no chromatographic separation is required. The approach could give new insights into the liver reserve, because the benzoic acid conversion to hippuric acid intimately relates to the hepatic energy metabolism. This measurement can be conducted at a wide range of dosages without interference from endogenous hippuric acid.     

Spectrophotometric assay of angiotensin I-converting enzyme (author's transl)]

The method of chemical assay of angiotensin I-converting enzyme described is a modification of the previously published spectrophotometric assay based on quantitation of hippuric acid released from hippuryl-L-histidyl-L-leucine. The new procedure involves extraction of hippuric acid with ethylacetate, evaporation to dryness of the extract, solubilization of the residue with 1 mol/l NaCl and purification with petroleum ether before measurements of the absorbance at 228 nm of the aqueous phase. Under these conditions, hippuric acid insoluble in petroleum ether remains in the aqueous phase, whereas other A228-absorbing materials, readily soluble in the ether and able to interfere with the assay, are eliminated.     

The activity of gamma-glutamyl transferase in the serum of multiple sclerosis and other neurological diseases.

1. In the sera of 142 neurological patients, including 35 cases with multiple sclerosis (MS), and 20 normal controls the gamma-glutamyltransferase (gamma-GT) activity was determined. The result of this investigation was compared with the "combined hippuric acid test" formely often used in MS patients. 2. The MS patients are the only group which significantly differs from the normal controls. There are also small differences between them and some of the other neurological patient groups. About one-third of the MS patients show moderately pathological serum activities. On the other hand, patients with a definite liver affection, especially chronic alcoholics, regularly show an even more important elevation of the values, which makes this test a sensitive tool for the screening of alcohol-induced liver intoxications. 3. Although the gamma-GT test in not quite as sensitive in indicating organic defects in MS patients, it seems to reflected a disturbed liver function in the same way as the more complicated "combined hippuric acid test". It may therefore be used as a simple tool of finding and following up organic defects in MS patients.     

Assay of carboxypeptidase N activity in serum by liquid-chromatographic determination of hippuric acid

We describe conditions for determining carboxypeptidase N (EC 3.4.17.3) activity by liquid chromatography. Serum (10 microL) is mixed with the artificial substrates hippuryl-L-arginine (30 mmol/L) and hippuryl-L-lysine (100 mmol/L) in 50 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution at pH 8.2 and 7.8, respectively. The hippuric acid product is separated from the substrate in less than 2 min by reversed-phase "high-performance" liquid chromatography and measured spectrophotometrically. o-Methyl hippuric acid is used as internal standard. By this method, optimized for activity and sensitivity of detection, carboxypeptidase N activities are 60-fold greater than those by another procedure (J Chromatogr 266:173-177, 1983). The mean value for 80 normal control subjects was 74.8 (SD 10.3) nmol of hippuric acid released per milliliter of serum per minute for hippuryl-L-arginine substrate, 378 (SD 55) for hippuryl-L-lysine substrate. The sensitivity and precision of the method make it suitable both for routine clinical determinations and as a reference procedure.     

Improved micromethod for assay of serum angiotensin converting enzyme

We describe conditions for determining angiotensin converting enzyme (EC 3.4.15.1) in serum, by liquid chromatography. Serum (10 microL) is assayed with the artificial substrate hippuryl-glycyl-glycine (30 mmol/L) in a 50 mmol/L "HEPES" buffer solution with a high salt content (300 mmol of NaCl and 400 mmol of Na2SO4 per liter), at pH 8.0. The resulting enzymic activity is ninefold that of the currently popular spectrophotometric assay of Cushman and Cheung as modified by Lieberman (Am. J. Med. 59: 365-372, 1975). The hippuric acid end product is separated from the substrate by reversed-phase liquid chromatography and measured spectrophotometrically at 228 nm. o-Methyl hippuric acid is used as internal standard. The mean value for 100 normal control subjects was 317 (SD 96) nmol of hippuric acid released per milliliter of serum per minute. The enzyme activity is greater in newborns (p less than 0.05) and has a tendency to decrease with age. This partly automated method, which is optimized with regard to activity and detection, can be used in clinical routine.     

A liquid chromatography-assisted assay for angiotensin-converting enzyme (peptidyl dipeptidase) in serum.

I describe modification of the spectrophotometric assay described by Cushman and Cheung [biochem. Pharmacol. 20, 1637 (1971)] for serum angiotensin-converting enzyme, with use of "high-pressure" liquid chromatography to measure the hippuric acid end product. After reaction of 10 microL of untreated serum with the angiotensin-converting enzyme substrate analog hippuryl-L-histidyl-L-leucine, the hippuric acid produced is extracted into ethyl acetate and quantitated, relative to an added internal standard, by liquid chromatography. Total chromatographic running time is 3 min per sample, with a within-run CV of 4.3% and a day-to-day CV of 6.6%, for aliquots of plasma supplemented with 1 mmol of hippuric acid per liter. The measurements are linear for hippuric acid in amounts up to 20 nmol per assay. The presence of large quantities of lipid in the serum did not affect the accuracy of the determination.     

Age dependence of renal function: clearance of iohexol and p-amino hippurate in healthy males

Iohexol, a newly developed non-ionic contrast agent, has been recently documented as a reliable glomerular filtration marker. This study describes the age dependence of the single injection clearance of iohexol in a sample of healthy male volunteers ranging from 21 to 77 years of age. In parallel, renal plasma flow was studied by measuring the total clearance of p-amino hippuric acid administered as a continuous infusion. In subjects older than 50 years a negative correlation to age was found for both p-amino hippuric acid and iohexol clearance, with a reduction of 52 ml/min and 12 ml/min per decade, respectively, whereas no age dependence was found for younger subjects. Correlation between p-amino hippuric acid and iohexol clearances was 0.81. However, the filtration fraction, defined as the ratio of iohexol to p-amino hippuric acid clearance, was higher in the elderly subjects. A consistent discrepancy was found between total and renal clearances of p-amino hippuric acid, indicating significant renal metabolism. Renal clearance of creatinine was poorly correlated to iohexol clearance and did not show any relationship to age.     

The role of common urinary constituents in the precipitation of ammonium acid urate

A high proportion of the inhibitory activity shown by urine toward precipitation of ammonium acid urate is ultrafilterable and most of this can be accounted for by the common, low molecular weight components of urine. The individual inhibitory effects of sodium chloride, sodium sulphate, potassium chloride, calcium chloride, magnesium sulphate, sodium dihydrogen phosphate, sodium pyrophosphate, citric acid, hippuric acid, creatinine and urea upon the precipitation of ammonium acid urate have been quantified in an aqueous test system.     

Uricosuric agents in uremic sera. Identification of indoxyl sulfata and hippuric acid.

Serum and urine from chronically uremic patients and normal individuals were subjected to gel filtration of Sephadex-G10. The effects of the eluted fractions on the uptake of urate and para-aminohippurate by isolated cortical tubules of rabbit kidney were investigated. According to the origin of the samples, one to three major groups of fractions inhibiting both urate and para-aminohippurate transport were disclosed. The first eluted group occurred for all the samples under study. The second one was demonstrated in both sera and urines from uremic patients but only in urines from normal individuals. The third one was exclusively detected in uremic sera and urines. Among all the compounds identified, only hippuric acid, eluted in the fractions of the second group, was capable of inhibiting the uptake of urate and para-aminohippurate in vitro. The concentration for which this inhbiitory effect of hippuric acid occurred was in the range of that existing in uremic sera. Indoxyl sulfate, which accumulates to very high concentrations in uremic serum, could not be disclosed in the above-mentioned fractions. This is explained by the strong adsorption of this indole derivative to Sephadex gel. Potassium indoxyl sulfate, when tested in vitro at the concentration existing in uremic serum, substantially inhibited the uptake of both urate and para-aminohippurate. In normal subjects, ingestion of hippuric acid or potassium indoxyl sulfate significantly increased fractional urinary excretion of uric acid. On the basis of these results, it is suggested that progressive retention of hippuric acid, indoxyl sulfate, and other yet unidentified inhibitors may explain the gradual increase in urinary fractional excretion of urate observed in uremia. The present results may be viewed as an example of a mechanism in which retention of normally excreted end products is responsible for adaptation of tubular transport in uremic subjects.     

Resorption and elimination kinetics of benzoic acid during chemical necrectomy in deep burns

Abstract

Objective: Chemical necrectomy is an alternative to the surgical or sharp necrectomy for the removal of necrotic parts of the skin in the treatment of deep burns. The aim of our work was to monitor the dynamics of resorption and elimination of benzoic acid applied to the burnt skin. Methods: The set consisted of 10 patients (9 men; 1 woman) aged 25–57 years with IIb-III-degree skin burns. 40% benzoic acid in white petrolatum was applied to the burnt area to the extent of 3–5% of TBSA (total body surface area) for a period of 48 hours. The concentrations of benzoic acid, hippuric acid, and glycine in the serum was monitored at the 10th, 20th, 60th, 120th, 240th and 360th minute thereafter and further at the 12th, 24th, 48th, and 72nd hour; the excretion of hippuric acid in urine was monitored in six 12-hour intervals. Results: The highest concentration of benzoic acid in the serum was detected in the 60th minute sample (0.094±0.074 mmol/L) and of hippuric acid in the 120th minute sample (0.234±0.088 mmol/L) from the application of benzoic acid to the burnt skin. In the period between the 6th and 48th hour, the average concentration of benzoic acid in the serum ranged between 0.042 and 0.03 mmol/L. In this period there was also a significant decrease in serum glycine concentration (p<0.05). During the 48-hour application of benzoic acid to the burnt skin, 46.0–145 mmol of hippuric acid was excreted in urine. Conclusion: Chemical necrectomy with the use of 40% benzoic acid led only to a moderate increase of its concentration in the serum. After its resorption from the wound area it is transformed to hippuric acid, which is promptly excreted in urine.     

Renal excretion of total porphyrins and hippuric acid in rats

The amounts of total porphyrins, hippuric acid and creatinine, excreted in urine by adult male Wistar rats, exhibited normal distributions for hippuric acid and creatinine, but a bimodal distribution for total porphyrins. This typical distribution of total porphyrins was still observed when creatinine was used as reference parameter. In biochemical and toxicological experiments in rats, the tested parameters should be therefore be investigated for homogeneity.     

Biochemical and neurophysiological parameters in hemodialyzed patients with chronic renal failure

Serum concentrations of accumulated solutes, standard clinical biochemistry, and parameters of clinical neuropathy, were determined in hemodialyzed patients with chronic renal failure. Analyses by high-performance liquid chromatography included creatinine, pseudouridine, urate, p-hydroxyhippuric acid, hippuric acid, indoxylsulfate, tryptophan, tyrosine, 3-indoleacetic acid, and a number of as-yet unidentified solutes. Standard biochemical parameters were measured; aluminium, parathyroid hormone, serum electrolytes and enzymes, hemoglobin, bilirubin, phosphate and urea. Measures of clinical neuropathy were: maximal motor nerve conduction velocities, and Hoffmann reflex latency. Several solutes had higher concentrations when nerve function was impaired. Serum total LDH, and total calcium levels correlated positively with values of the Hoffmann reflex, as did serum hippuric acid concentrations. Concentrations of p-hydroxyhippuric acid and two fluorescent compounds correlated negatively with motor nerve conduction velocities. In principal component analysis a number of 'organic acid-like' substances, like hippuric acid and p-hydroxyhippuric acid, were shown to associate multivariately with the neurophysiological variables while urea, creatinine, urate and phosphate were not.     

Benzoylalanine: detection and identification of an alanine conjugate with benzoic acid in hyperammonemic patients treated with sodium benzoate

Urine of four hyperammonemic patients who were treated with large amounts of sodium benzoate were analyzed. Gas chromatographic and gas chromatograph-mass spectrometric analysis showed the presence of large amounts of benzoylalanine in addition to much hippuric acid. Benzoylalanine was identified by comparison with an authentic standard by means of its mass spectrum and the gas chromatographic retention time of its trimethylsilyl derivative. This abnormal metabolite is thought to be derived from the conjugation of benzoyl CoA with alanine. It is suggested that the excretion of benzoylalanine results from a reduction in the liver free glycine level caused by hippuric acid synthesis.     

Monoamine metabolites and related compounds in human amniotic fluid: assay by gas chromatography and gas chromatography-mass spectrometry.

Some catecholamine metabolites and related compounds have been identified in amniotic fluid obtained by transabdominal amniocentesis at various stages of pregnancy, including 4-hydroxy-3-methoxymandelic acid, 4-hydroxy-3-methoxyphenylglycol, 4-hydroxy-3-methoxyphenylcetic acid, p-hydroxypheny lacetic acid, p-hydroxphenyllactic acid and N-benzoylglycine (hippuric acid). Analysis was by gas chromatography with electron capture detection and by gas chromatography-mass spectrometry. Two of these compounds were determined quantitatively, free 4-hydroxy-3-methoxphenylglycol and p-hydroxyphenllactic acid: the concentration of the former increased with advancing pregnancy and that of the latter tended to decrease. Conjugated 4-hydoxy-3-methoxyphenylglycol could not be determined with accuracy as appreciable amounts of the unconjugated compound were found in the snail extract used for enzymatic hydrolysis. Assay of 4-hydroxy-3-methoxyphenylglycol in amniotic fluid is likely to be of diagnostic importance in the prenatal diagnosis of congenital neuroblastoma. Although 4-hydroxy-3-methoxyphenylethanol, 3, 4-dihydroxymandelic acid and 3, 4-dihydroxyphenylacetic acid were specifically looked for in amniotic fluid, they could not be detected.     

Simultaneous quantitation of 4-hydroxy-3-methoxymandelic (vanilmandelic) and 4-hydroxy-3-methoxyphenylacetic (homovanillic) acids in human urine.

We describe a simultaneous analysis for vanilmandelic and homovanillic acids in urine. An aliquot of a final extract, containing phenylpropriopionic acid as external standard, is dried, an internal standard (resorcinol) is added, and the mixture is silylated and chromatographed. The two compounds are identified from their characteristic retention times and from their elution behhavior in relation to four different marker compounds (the external and internal standards and two other naturally occurring compounds, p-hydroxyphenylacetic and hippuric acids) and are quantitated from their relative response factors. Within-run precision (CV) varied from 2 to 14% for vanilmandelic acid and 1 to 18% for homovanillic acid. The ratio of the amount of phenylpropionic acid extracted to the amount of added resorcinol found is a useful quality-control index. Values for vanilmandelic acid obtained by this method and by the technique of Pisano et al. [Clin. Chim. Acta 7, 285 (1962)] correlated well (r = 0.844).     

Screening of UV-absorbing solutes in uremic serum by reversed phase HPLC--change of blood levels in different therapies

In order to screen UV-absorbing solutes in large numbers of uremic serum samples, an automated liquid chromatographic method was developed. The method proved to be reliable and reproducible in more than 500 analyses. HPLC separation was performed using gradient elution on a 25-cm Ultrasphere Octyl reversed phase column, with 5 microns particles. Characteristic profiles for the uremic state were obtained in the analyses of serum samples of 43 uremic patients before and just after artificial kidney treatment; hemodialysis (n = 14), hemodiafiltration (n = 13) and hemofiltration (n = 16). In these profiles 20-40 peaks were resolved of which nine were 'quantitated' by peak height relative to a standard. Of these solutes creatinine, uracil, uric acid, hypoxanthine, indoxylsulfate, tryptophan and hippuric acid were identified. The heterogeneity of the population of uremic patients, with respect to the UV-absorbing solutes, was estimated. Significant differences of solute blood level changes during hemodialysis, hemodiafiltration and hemofiltration, were observed.     

Relationship between uptake and elimination of toluene and debrisoquin hydroxylation polymorphism

The toxicokinetics of toluene were studied in six healthy subjects. Three of the subjects were phenotyped as rapid hydroxylators of debrisoquin and three subjects were phenotyped as slow hydroxylators of debrisoquin. The subjects were exposed in an exposure chamber to toluene vapor (3.25 mmol/m3) for 4 hours. Solvent concentrations in blood and the metabolites, hippuric acid and o-cresol, in urine were measured during the exposure period and 3 hours after exposure. The capacity to metabolize debrisoquin was determined in three volunteers who had earlier experimentally been exposed to toluene. The uptake of toluene was about 3 mmol, or 50% of the inhaled dose in both rapid and slow hydroxylators. There were no significant differences between the two phenotypic groups with regard to concentrations of toluene in blood, apparent blood clearance of toluene, or excretion of hippuric acid and o-cresol.     

Organic acids in urine from human newborns.

The pattern of organic acids in urine from 15 normal newborn infants was investigated by gas chromatography/mass spectrometry of trimethylsilylated derivatives. The urine contains large amounts of succinic, fumaric, 2-ketoglutaric, and 3-hydroxy-3-methylglutaric acids, which are all fairly small components of urine from adults. On the other hand, hippuric acid is a small component in the urine from newborn infants, but a large component later in life. An additional number of differences can be seen. Some previously unrecognized aliphatic dicarboxylic acids were also observed, which are present to some extent in urine from adults.     

Determination of Urinary Hippuric Acid in Toluene Abuse

Background: Volatile substance abuse is practiced mainly by adolescents and young adults. Its effects are central nervous system excitation followed by central nervous system depression, at times accompanied by seizures. It may cause sudden death as a result of ventricular arrhythmias, reflex vagal inhibition, respiratory depression, and anoxia. Chronic toxicity may involve the nervous system, heart, kidney, and liver. Toluene-based adhesives are among the most commonly inhaled substances. Case Report: A 14-year-old female presented with confusion, hallucinations, and intermittent laughing and crying after having inhaled contact glue several times daily in the course of 5 days. Her condition improved within 3 h. Urinary hippuric acid was 93.9 g/g creatinine indicating heavy toluene exposure (biological exposure index, BEI, is 1.6 g/g creatinine). Conclusion: In this patient, urinary hippuric acid was a biomarker for her toluene abuse.     

Ultraviolet-absorbing organic anions in uremic serum separated by capillary zone electrophoresis, and quantification of hippuric acid

Organic anions accumulated in blood serum of patients with chronic renal failure were separated by a novel technique: closed-system capillary zone electrophoresis (CZE) in a pH6 carrier-electrolyte system. Hippuric acid (HA), p-hydroxyhippuric acid, and uric acid were identified by their co-elution with standards prepared in ultrafiltered normal serum and by comparison with the corresponding ultraviolet-detected peaks positively identified in the HPLC analyses. Analysis time for the entire profile is 8 min. Repeatabilities (CVs) of CZE migration times and peak areas of the three acids in serum samples were about 0.7% and 6%, respectively. We quantified HA in 10 ultrafiltered uremic serum samples and compared results with those by a previously described HPLC procedure. The very good agreement further supports the identification of hippuric acid. Accuracy and precision of the CZE method were similar to those for the HPLC gradient-elution method, but analysis time for HA (8 min) is much less than by HPLC (90 min). Our technique is very suitable for selective, rapid analysis for (ultraviolet-absorbing) anionic constituents in ultrafiltered uremic serum, without any sample pretreatment.