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Angiotensin I-converting enzyme (ACE) plays a pivotal role in blood pressure regulation. A colorimetric assay to measure hippuric acid (HA) was transformed into a rapid ACE assay wherein the released HA from the substrate hippuryl-histidyl-leucine (HHL) is mixed with pyridine and benzene sulfonyl chloride. The resulting yellow color with a lambda(max) at 410 nm is directly proportional to the released HA. The limit of detection and limit of quantitation were 1.46 x 10(-7) and 4.43 x 10(-7) M HA. ACE activities of different tissues using this method were comparable to the standard high-performance liquid chromatography (HPLC) method. Kinetic studies showed a K(m) of 30.8 +/- 0.1 x 10(-6) M for HHL and V(max) of 1.3 +/- 0.01 x 10(-6) mol/min for porcine lung ACE. This assay coupled with captopril and lisinopril showed IC(50) values of 1.1 +/- 0.05 x 10(-9) and 2.5 +/- 0.03 x 10(-9) M, respectively. A 96-well microplate format of this method was used to screen the ACE inhibitory potential of peptides fractionated from an enzymatic hydrolysate of arachin. The precision, accuracy, reproducibility, and excellent correlation demonstrated between the colorimetric and the often-used HPLC method renders this extraction-free method a powerful tool for high-throughput screening of ACE inhibitors.

Three strains of an unusual hippurate-positive Campylobacter species were isolated at 37 degrees C from caecal contents of broiler chickens and a turkey. All strains were initially identified as Campylobacter by means of genus-specific PCR, but none was further identified using specific PCRs for known thermophilic species. Phylogenetic analyses based on 16S rRNA, rpoB and groEL gene sequences revealed that these strains formed a robust clade distinct from other Campylobacter species. Amplified fragment length polymorphism analysis and whole-cell protein electrophoresis were subsequently carried out and confirmed the divergence between the avian strains and other taxa. These data indicate that the unidentified Campylobacter strains belong to a novel taxon which could be distinguished from other campylobacters through its phenotypic and genotypic characteristics. The name Campylobacter avium sp. nov., is proposed for the novel species, with the type strain 86/06T (=LMG 24591T=CCUG 56292T).

Oligomers and polymers of flavan-3-ols (proanthocyanidins) are very abundant in the Mediterranean diet, but are poorly absorbed. However, when these polyphenols reach the colon, they are metabolised by the intestinal microbiota into various phenolic acids, including phenylpropionic, phenylacetic and benzoic acid derivatives. Since the biological properties of these metabolites are not completely known, in the present study, we investigated the effect of the following microbial phenolic metabolites: 3,4-dihydroxyphenylpropionic acid (3,4-DHPPA), 3-hydroxyphenylpropionic acid, 3,4-dihydroxyphenylacetic acid (3,4-DHPAA), 3-hydroxyphenylacetic acid, 4-hydroxybenzoic acid and 4-hydroxyhippuric acid (4-HHA), on modulation of the production of the main pro-inflammatory cytokines (TNF-alpha, IL-1beta and IL-6). The production of these cytokines by lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMC) pre-treated with the phenolic metabolites was studied in six healthy volunteers. With the exception of 4-HHA for TNF-alpha secretion, only the dihydroxylated compounds, 3,4-DHPPA and 3,4-DHPAA, significantly inhibited the secretion of these pro-inflammatory cytokines in LPS-stimulated PBMC. Mean inhibition of the secretion of TNF-alpha by 3,4-DHPPA and 3,4-DHPAA was 84.9 and 86.4 %, respectively. The concentrations of IL-6 in the culture supernatant were reduced by 88.8 and 92.3 % with 3,4-DHPPA and 3,4-DHPAA pre-treatment, respectively. Finally, inhibition was slightly higher for IL-1beta, 93.1 % by 3,4-DHPPA and 97.9 % by 3,4-DHPAA. These results indicate that dihydroxylated phenolic acids derived from microbial metabolism present marked anti-inflammatory properties, providing additional information about the health benefits of dietary polyphenols and their potential value as therapeutic agents.

One possible origin of the type I hypersensitivity reaction is reaction of drugs such as acetylsalicylic acid and its metabolites being complexed with human serum albumin. Albumin, being transporting molecule abundant in blood plasma is able to bind large array of ligands varying from small single carbon particles to long hydrophobic tailed lipidic acids (e.g. myristic acid). This non specificity is possible because of multi domain scaffold and large flexibility of inter-domain loops, which results in serious reorientation of domains. Hypothesis that acetylsalicylic acid metabolites may play indirect role in activation of allergic reaction has been tested. Binding of acetylsalicylic acid metabolites in intra-domain space causes significant increase of liability of domains IIIA and IIIB. One of metabolites, salicyluric acid, once is bound causes distortion and partial unfolding of helices in domains IA, IIB and IIIB. Changed are both directions and amplitude of relative motions as well as intra-domain distances. In result albumin is able to cross-link of adjacent IgE receptors which subsequently starts allergic reaction.

Background: Protein-bound uraemic toxins provoke multiple biological changes involved in uraemia. Few if any dialytic strategies remove these compounds., Methods: In this post hoc analysis of remnant samples from a randomised controlled trial, we evaluate whether predilution haemofiltration (HF) decreases the pretreatment concentration of protein- bound uraemic solutes. Patients treated with low-flux haemodialysis (HD) were enrolled into a group continuing this strategy (group A, n=8) over 6 months, whereas group B (n=12) was switched to predilution online HF. Blood was sampled at baseline and after 6 months to determine total and free concentration and percentage binding of indoxyl sulfate (IS), indole-3-acetic acid (IAA), hippuric acid (HA), p-cresol (PC) and 3-carboxy- 4-methyl-5-propyl-2-furanpropionic acid (CMPF)., Results: Comparing concentrations at start versus 6 months of treatment by paired analysis, HD had no impact. In contrast, at the end of the HF period, we found a decrease in total and free PC, free IAA and total CMPF. In addition, the percentage protein binding of IAA increased significantly. However, unpaired analysis revealed no statistical difference between HD and HF, both at baseline and after 6 months of treatment for all compounds., Conclusions: Paired analysis showed a beneficial impact of predilution online HF for several proteinbound uraemic solutes. Unpaired analysis, however, showed no statistical difference.

Fingerprinting together with statistical analysis is often employed to compare samples in metabonomic studies of a disease. Correlation algorithms can aid by extracting information based on the variation patterns of key metabolites. This information can be linked to metabolite identification or to specific up/down-regulated biochemical pathways. Matlab-based software employing the Pearson's correlation algorithm was applied to urine electropherograms from 20 mice infected with the schistosoma parasite. The fingerprints were the sum of electropherograms analysed with normal and reverse polarity, in two different modes MEKC and CZE and with two different capillaries (uncoated and polyacrylamide coated) to provide a broad picture of the samples. Hippurate, a metabolite that was depleted in the infected group and is present in both polarities, was chosen as a test variable; it correlated with itself to a p value of <0.000. Phenylacetylglycine, a metabolite shown as over expressed in the disease, was positively correlated to three metabolites in its same pathway with a correlation coefficient of 0.7 and p<0.000 to phenylalanine, 0.7 and p<0.000 to 2-hydroxyphenylacetic and 0.55 and p<0.003 to phenylacetate. The study shows that the autocorrelation matrix is able to provide extra information from data files acquired by CE analyses. It underlined an up-regulated metabolic path by association in the schistosoma infection model.

Intestinal spirochetes of genus Brachyspira are commonly isolated from mammalian and avian hosts, and several species have been reported to cause enteric disease in pigs and birds. Except for a previous publication on three isolates from corvid birds (order Passeriformes, family Corvidae, genus Corvus), of which two are further studied in this paper, no other reports exist on Brachyspira spp. of passerine birds. In this study, cloacal and intestinal swabs of small and large intestines were collected from 116 corvid birds of three species, i.e. jackdaws (Corvus monedula), hooded crows (Corvus corone cornix) and rooks (Corvus frugilegus), from four separate geographical locations in Sweden. Isolates were obtained by selective culture from 43 of 116 birds. All isolates were weakly hemolytic, indole-negative and lacked hippurate cleavage capacity. Examination by light microscopy did not indicate association with enteric disease in necropsied birds. Pure spirochete cultures were obtained by serial dilution and subculture, and selected isolates were analyzed by PCR (n=14), randomly amplified polymorphic DNA (RAPD) (n=14), and sequencing of the almost complete 16S rRNA (n=14), and partial nox genes (n=4). Positive reactions were noticed by PCR targeting a hexa-T segment of the 16S rRNA gene, which has been previously reported as a signature characteristic of Brachyspira pilosicoli. By 16S rRNA gene sequencing, the isolates formed a separate cluster related to genus Brachyspira, but not consistent with any presently recognized or proposed Brachyspira sp. The sequence similarity of the 16S rRNA gene among the isolates from corvid birds was 99.7-100%. Compared to 16S rRNA gene sequence data from all presently recognized and several proposed Brachyspira spp. the sequence similarity of the isolates from corvid birds varied between 94.1 and 96.5%. In a radial tree based on nox gene sequences, all four analyzed isolates from corvid birds formed a separate cluster. By RAPD analysis, the banding patterns of the isolates differed from all type strains of Brachyspira spp. Based on the results presented in this paper, we propose that the described isolates from corvid birds belong to a novel species within genus Brachyspira, with the provisional name "Brachyspira corvi" (cor'vi. L gen. n. corvi, of a crow).

A total of 190 Campylobacter spp. isolates, of which 34 gave the result of very weak activity, and 156 gave the negative activity in the test for hippurate hydrolysis were characterized. The genomic DNA was isolated from a fresh culture of each isolate and the real-time PCR, targeting the hipO gene, was used to confirm the species distribution of Campylobacter isolates. The hipO gene was detected in 17 isolates (11%) within the total of 156 negative isolates for hippurate hydrolysis. Out of 34 isolates with very weak activity, 19 isolates (56%) were also found to be positive for hipO gene and characterized as C. jejuni. The real-time PCR assay used in this study could be employed for more accurate diagnosis of Campylobacter infections at species level after the biochemical characterization based on hippuricase activity of the isolates. This could also provide important data for the epidemiology of infections associated with these zoonotic pathogens.

Hippurate hydrolysis test results of 240 Campylobacter strains were compared with those of two multiplex polymerase chain reaction (PCR) assays. Of the 152 strains identified in Finnish clinical microbiology routine laboratories as C. coli (hippurate-negative), 11% were C. jejuni (hippurate-positive) by standardized hippurate test and 39% by PCR in the reference laboratory. Two of the 81 hippurate-positive strains were identified as C. coli. Standardizing the hippurate test by determining minimum and maximum turbidity limits (McFarland 6 and McFarland 10, OD(450) values 0.8 and 1.4, respectively) for the bacterial cell suspension eliminated the false-positive results, but 32% of the 145 hippurate-negative strains were still identified as C. jejuni by PCR. The species identification of Campylobacter isolates in Finland could be improved by using a standardized hippurate hydrolysis test to identify hippurate-positive C. jejuni and testing hippurate-negative strains by molecular methods. This would also improve the epidemiological data on this important zoonotic pathogen.

Background: We have studied the possibility that low-dose treatment utilizing the inhibition that may occur between two drugs at the same site of human serum albumin (HSA) improves the pharmacological effects. The purpose is to elucidate the differences in the binding capacities of sites I and II of HSA between pre-haemodialysis (HD) and post-HD in patients with end-stage renal disease., Methods: We evaluated free fractions of site probes, (14)C-warfarin (site I) and (14)C-diazepam (site II), by ultrafiltration in serum between pre-HD and post-HD. To investigate effects on the binding capacities of HSA sites, free fractions of site probes were calculated from the radioactivities measured with a liquid scintillation counter. Endogenous uraemic toxins, 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), indoxyl sulphate (IS) and hippurate (HA), were determined by HPLC. Free fatty acid (FFA) as an endogenous substance was determined with an automatic multi-item simultaneous analyser., Results: The concentrations of HSA and FFA increased significantly (post-HD/pre-HD ratio: 1.18 +/- 0.10, 5.46 +/- 4.91), the concentrations of IS and HA decreased significantly (post-HD/pre-HD ratio: 0.69 +/- 0.10, 0.33 +/- 0.15) and CMPF concentrations did not alter significantly (post-HD/pre-HD ratio: 0.97 +/- 0.12, P = 0.471). The free fractions of (14)C-warfarin decreased in all 14 patients at site I at post-HD compared to pre-HD (post-HD/pre-HD ratio: 0.59 +/- 0.13). The free fractions of (14)C-diazepam at site II remarkably decreased in 10 of 14 patients (post-HD/pre-HD ratio: 0.61 +/- 0.17) and unexpectedly increased in 4 (post-HD/pre-HD ratio: 1.08 +/- 0.06) post-HD compared to pre-HD. In these four patients, when we investigated the influences of these variation factors on the reduction of the binding capacities of site II, [FFA]/[HSA] increased significantly post-HD, compared to pre-HD (post-HD/pre-HD ratio: 6.91 +/- 6.58). ([FFA]/[HSA] ratios of the 4 patients were from 1.22 to 3.55, the highest for the 14 patients post-HD, but the ratios of the other 10 were below 1.2 post-HD.), Conclusion: The binding capacity of site II was unexpectedly decremented by the effects of the remarkable elevation of FFA. Therefore, monitoring the binding capacity of site II in HD is important for patients with end-stage renal disease in the efficacious administration plan using the binding inhibition of HSA.

The dipeptides Ala-Trp, Val-Phe, and Val-Tyr inhibit the angiotensin-I-converting enzyme. They are encrypted within the primary sequences of different food proteins, e.g. milk proteins. The angiotensin-I-converting enzyme inhibitory potency of these synthetic dipeptides was quantified using a spectrophotometric assay. The dipeptides showed no adverse effects on differentiated Caco-2 cells (model for human intestinal epithelium), as confirmed by transepithelial electrical resistance, microscopy and the activity of the brush-border enzyme dipeptidyl aminopeptidase IV. Furthermore, the transport of these bioactive dipeptides through intact Caco-2 monolayers and their stability to incubation in human blood serum has been demonstrated for the first time. Low molecular mass peptides represent the minimal structures required for angiotensin-I-converting enzyme inhibition which have a high potential bioavailability. Therefore, they may act as target peptides in enriched hydrolysates for the preparation of an angiotensin-I-converting enzyme inhibitory peptide and for the use in special formulations as functional foods/foods of specified health use.

Exposure to aromatic organic solvents may induce hearing loss in rats, the cochlea being the primary target. The aim of this study which was carried out in rat, was to evaluate the impact of the hepatic metabolism of toluene on its ototoxic potency. To this end, the solvent hepatic metabolism was shifted by treating the rats with 50 mg/kg/d of phenobarbital (PhB), a potent inducer of the microsomal cytochromes P450 system, alcohol and aldehyde dehydrogenases, and glutathione-S-transferases. The two main urinary metabolites of the oxidative and conjugate pathways [hippuric (HA) and benzyl mercapturic acids (BMA) respectively] confirmed the efficacy of the PhB treatment. For the PhB-induced rats, the amount of excreted HA increased by 43% and the amount of BMA by 35%. Auditory function impairments were assessed using auditory-evoked potentials. On completion of the auditory tests, the organs of Corti were dissected to evaluate hair cell losses. The permanent auditory threshold shifts were approximately 15 dB greater in the toluene-exposed rats than in the PhB-induced rats. Both the functional and morphological data confirmed that PhB treatment can decrease the ototoxic potency of toluene.

Numerous experiments have shown that the aromatic solvents can affect the auditory system in the rat, the cochlea being targeted first. Solvents differ in cochleotoxic potency: for example, styrene is more ototoxic than toluene or xylenes. The goal of this study was to determine the relative ototoxicity of the three isomers of xylene (o-, m- or p-xylene). Moreover, by dosing with the two urinary metabolites of xylene, methylhippuric (MHAs) and mercapturic acids (MBAs), this study points toward a causal relationship between the cochleotoxic effects and potential reactive intermediates arising from the biotransformation of the parent molecules. Separate groups of rats were exposed by inhalation to one isomer following this schedule: 1800 ppm, 6 h/d, 5 d/wk for 3 wk. Auditory thresholds were determined with brainstem-auditory evoked potentials. Morphological analysis of the organ of Corti was performed by counting both sensory and spiral ganglion cells. Among the three isomers, only p-xylene was cochleotoxic. A 39-dB permanent threshold shift was obtained over the tested frequencies range from 8 to 20 kHz. Whereas outer hair cells were largely injured, no significant morphological change was observed within spiral ganglia. The concentrations of urinary p-, o- or m-MHA were greater (p-MHA: 33.2 g/g; o-MHA: 7.8 g/g; m-MHA: 20.4 g/g) than those obtained for MBAs (p-MBA: 0.04 g/g; o-MBA: 6.2 g/g; m-MBA: 0.03 g/g). Besides, there is a large difference between o-MBA (6.2 g/g) and p-MBA (0.04 g/g). As a result, since the cysteine conjugates are not determinant in the ototoxic process of xylenes, the location of the methyl groups around the benzene nucleus could play a key role.

The competitive binding of diflunisal and three well-known uraemic toxins (3-indoxyl sulfate, indole-3-acetic acid and hippuric acid) to bovine serum albumin (BSA), human serum albumin (HSA) and human plasma was studied by direct potentiometry. The method used the potentiometric drug ion-probe technique with a home-made ion sensor (electrode) selective to the drug anion. The site-oriented Scatchard model was used to describe the binding of diflunisal to BSA, HSA and human plasma, while the general competitive binding model was used to calculate the binding parameters of the three uraemic toxins to BSA. Diflunisal binding parameters, number of binding sites, n(i) and association constants for each class of binding site, K(i), were calculated in the absence and presence of uraemic toxins. Although diflunisal exhibits high binding affinity for site I of HSA and the three uraemic toxins bind primarily to site II, strong interaction was observed between the drug and the three toxins, which were found to affect the binding of diflunisal on its primary class of binding sites on both BSA and HSA molecules and on human plasma. These results are strong evidence that the decreased binding of diflunisal that occurs in uraemic plasma may not be solely attributed to the lower albumin concentration observed in many patients with renal failure. The uraemic toxins that accumulate in uraemic plasma may displace the drug from its specific binding sites on plasma proteins, resulting in increased free drug plasma concentration in uraemic patients.

Genetic drift in animal populations has been a recognized concern for many years. Less understood is the potential for phenotypic "drift" or variation that is not related to any genetic change. Recently, stock Sprague-Dawley (Crl:CD(SD)) rats obtained from the Charles River Raleigh facility demonstrated a distinct endogenous urinary metabonomic profile that differed from historical control SD urine spectral profiles obtained over the past several years in our laboratory. In follow-up studies, the origin of the variant phenotype was narrowed down to animals of both sexes that were housed in one specific room (Room 9) in the Raleigh facility. It is likely that the two phenotypes are related to distinct populations of gut flora that particularly impact the metabolism of aromatic molecules. The most pronounced difference between the two phenotypes is the relative amounts of hippuric acid versus other aromatic acid metabolites of chlorogenic acid. Though both molecular species are present in either phenotype, the marked variation in levels of these molecules between the two phenotypes has led to the designation of high hippuric acid (HIP) and high chlorogenic acid metabolites (CA) phenotypes. Specific urinary components that distinguish the phenotypes have been thoroughly characterized by NMR spectroscopy with additional, limited characterization by LC-MS (high performance liquid chromatography coupled with mass spectrometry). Co-habitation of rats from the two phenotypes rapidly facilitated a switch of the CA phenotype to the historical Sprague-Dawley phenotype (HIP). The impact of these variant phenotypes on drug metabolism and long-term safety assessment studies (e.g., carcinogenicity bioassays) is unknown.

Background: The tubular excretion of creatinine significantly contributes to its clearance. Administration of an angtiotensin-converting enzyme (ACE) inhibitor is associated with increased organic ion clearance in experimental diabetes. This study examines the effect and implications of chronic ACE inhibition on renal organic ion excretion in patients with type 1 diabetes., Methods: Samples were obtained from the Melbourne Diabetic Nephropathy Study Group (MDNSG) that randomized patients to receive perindopril (N= 11), nifedipine (N= 11), or placebo (N= 8). Albumin excretion rate, creatinine clearance, and isotopic glomerular filtration rate (GFR) were assessed at baseline and after 24 months. In addition, the clearance of the endogenous cations N-methylynicotinamide (NMN), creatinine, and the anion hippurate were determined by high-performance liquid chromatography (HPLC)., Results: Following treatment with the ACE inhibitor, perindopril, renal clearance of NMN was increased (+96%) (P < 0.05). There was no difference in patients treated with nifedipine (P= 0.25) and NMN clearance fell in the placebo-treated patients (-26%) (P < 0.05). Changes in NMN clearance were unaffected after adjusting for the effects of perindopril on GFR. However, they were attenuated after adjusting for hippurate clearance, a marker of renal blood flow. This effect of perindopril on NMN clearance was seen in both men and women, regardless of baseline clearance and was correlated with reduced albuminuria following perindopril treatment., Conclusion: Organic ion clearance is increased in patients with diabetes following chronic ACE inhibition. This is consistent with experimental models showing increased ion transporter expression and improved tubular blood flow, following blockade of the renin-angiotensin system (RAS). These findings may have implications for the interpretation of creatinine-based indices in patients with diabetes.

A high-performance liquid chromatographic method for indirect determination of enalapril in human plasma, was developed and validated. An exogenous angiotensin converting enzyme after drug inhibition was determined by reacting with hippuryl-histidyl-leucine to produce hippuric acid (HA) which was inversely proportional to the amount of enalaprilat in plasma. The HPLC was carried out on a Lichrosphere 60RP-select B, C18, 5 microm (125 mm x 4.0 mm i.d.) column at flow rate of 1.0 ml/min. The analysis time per injection was within 6.5 min. The lowest concentration of enalaprilat to be quantitated was 3.0 ng/ml with the acceptable accuracy and precision. This successfully developed method was practically and accurately used for pharmacokinetics and bioequivalent study of enalapril.

We describe here the implementation of the statistical total correlation spectroscopy (STOCSY) analysis method for aiding the identification of potential biomarker molecules in metabonomic studies based on NMR spectroscopic data. STOCSY takes advantage of the multicollinearity of the intensity variables in a set of spectra (in this case 1H NMR spectra) to generate a pseudo-two-dimensional NMR spectrum that displays the correlation among the intensities of the various peaks across the whole sample. This method is not limited to the usual connectivities that are deducible from more standard two-dimensional NMR spectroscopic methods, such as TOCSY. Moreover, two or more molecules involved in the same pathway can also present high intermolecular correlations because of biological covariance or can even be anticorrelated. This combination of STOCSY with supervised pattern recognition and particularly orthogonal projection on latent structure-discriminant analysis (O-PLS-DA) offers a new powerful framework for analysis of metabonomic data. In a first step O-PLS-DA extracts the part of NMR spectra related to discrimination. This information is then cross-combined with the STOCSY results to help identify the molecules responsible for the metabolic variation. To illustrate the applicability of the method, it has been applied to 1H NMR spectra of urine from a metabonomic study of a model of insulin resistance based on the administration of a carbohydrate diet to three different mice strains (C57BL/6Oxjr, BALB/cOxjr, and 129S6/SvEvOxjr) in which a series of metabolites of biological importance can be conclusively assigned and identified by use of the STOCSY approach.

A hippurate-negative biovariant of Brachyspira pilosicoli (B. pilosicolihipp-) is occasionally isolated in diarrhoeic pigs in Finland, often concomitantly with hippurate-positive B. pilosicoli or Lawsonia intracellularis. We studied pathogenicity of B. pilosicolihipp- with special attention paid to avoiding co-infection with other enteric pathogens. Pigs were weaned and moved to barrier facilities at the age of 11 days. At 46 days, 8 pigs were inoculated with B. pilosicolihipp- strain Br1622, 8 pigs were inoculated with B. pilosicoli type strain P43/6/78 and 7 pigs were sham-inoculated. No signs of spirochaetal diarrhoea were detected; only one pig, inoculated with P43/6/78, had soft faeces from day 9 to 10 post inoculation. The pigs were necropsied between days 7 and 23 after inoculation. Live pigs were culture-negative for Brachyspira spp., but B. pilosicolihipp- was reisolated from necropsy samples of two pigs. The lesions on large colons were minor and did not significantly differ between the three trial groups. In silver-stained sections, invasive spirochaetes were detected in colonic mucosae of several pigs in all groups. Fluorescent in situ hybridisation for genus Brachyspira, B. pilosicoli and strain Br1622 was negative. However, in situ detection for members of the genus Leptospira was positive for spirochaete-like bacteria in the colonic epithelium of several pigs in both infected groups as well as in the control group. L. intracellularis, Salmonella spp., Yersinia spp. and intestinal parasites were not detected. The failure of B. pilosicoli strains to cause diarrhoea is discussed with respect to infectivity of the challenge strains, absence of certain intestinal pathogens and feed and management factors.

Two new complexes [Cu(Imz)(4)Cl(2)][Cu(Imz)(4)Cl] (2)(2-OH-Hip)(2) (1) and [Co(2-OH-Hip)(Imz)(3)].H(2)O (2) (with Imz=Imidazol and 2-OH-Hip=2-hydroxyhippuric acid) were prepared and characterized. The molecular structures and the solution and solid state behavior of the complexes were investigated. Complex 1 crystallizes in the monoclinic space group P2(1)/c with a=16.880(1), b=8.046(1), c=24.683(1) A, beta=107.88(1) degrees, and Z=2, while complex 2 crystallizes in the orthorhombic space group Pbca with a=11.712(2), b=15.741(4), c=22.254(4) A, and Z=8. The [Cu(Imz)(4)Cl(2)][Cu(Imz)(4)Cl](2)(2-OH-Hip)(2) solid consists in two distinct monomeric Cu(II) complexes: one of them is neutral octahedral [Cu(Imz)(4)Cl(2)] and the other, charged square basis pyramida [Cu(Imz)(4)Cl](+). The 2-hydroxyhippuric acid, which here acts as a counter ion, is deprotonated at its carboxylic group. Cobalt(III) ion in [Co(2-OH-Hip)(Imz)(3)].H(2)O is at the center of an octahedral environment, coordinated to three Imidazol ligands and to a triply deprotonated 2-hydroxyhippuric acid molecule acting as a tridentate ligand. Aqueous solution equilibrium of the quaternary system Cu(2+)/2-OH-Hip/Imz/H(+) was studied by potentiometric titrations.

Unlabelled: The aim of the present study was to compare the effect produced by juvenile rheumatoid arthritis (JRA) or rheumatic fever (RF) on the pharmacokinetics of acetyl salicylic acid (ASA) and its metabolites in children with autoimmune diseases (AD)., Methods: A prospective, open labelled study was performed in 17 children with JRA and 17 with RF who received a single dose of 25 mg ASA/kg orally. The pharmacokinetics of ASA and its metabolites were determined. The blood and urine levels of each salicylate collected during 24 h were measured by HPLC. A group of 15 healthy teenage volunteers was included as a control group., Results: The maximum plasma concentration, half-life time, area under the curve and the amount of salicylates excreted were statistically different between the JRA and the RF groups, as well as between the RF group and the controls, however, there were no significant differences between the JRA group and the controls., Conclusions: Dosage schemes must be adjusted for JRA patients, since the half life in these patients is longer than in RF patients. However, due to ample variability of pharmacokinetic parameters it is recommended that dose schemes are individualized on the type of autoimmune disease considered., (Copyright 2004 John Wiley & Sons, Ltd.)

The Campylobacter detection is performed by conventional culture methods and the identification of Campylobacter jejuni and Campylobacter coli is principally based on the hippurate hydrolysis test. The two major drawbacks of this biochemical test for species identification include the inconsistency of the results and the presence of atypical strains, which can lead to the misidentification of an isolate. As an alternative, multiplex polymerase chain reaction (mPCR) protocols for the simultaneous detection and identification of different Campylobacter species have been developed. This study examined the performances of an experimental BAX System assay for the C. jejuni and C. coli identification in comparison to a multiplex PCR protocol recently published. The samples tested were represented by 106 environmental swabs collected on Teflon strips and tables, stainless steel saws, hooks and trays, ceramic floors and walls, as well as equipment surfaces, located in a swine (N=50) and a poultry (N=56) slaughterhouse. The highest Campylobacter detection rate was obtained after 48 h of enrichment by using both the PCR procedures. After 24 h, the BAX System provides a more rapid and accurate Campylobacter detection and identification assay than the multiplex PCR. Except for two samples, all the broths where Campylobacter cells were detected after 24 or 48 h of enrichment, with at least one of the PCR protocols, gave Campylobacter colonies using the culture method.

Protein-bound uremic retention solutes are molecules with low molecular weight (MW) but should be considered middle or high MW substances. This article describes the best known substances of this group, which include p-cresol, indoxyl sulfate, hippuric acid, 3-carboxy-4-methyl-5-propyl-2-furan-propionic acid (CMPF), and homocysteine. At concentrations encountered during uremia, p-cresol inhibits phagocyte function and decreases leukocyte adhesion to cytokine-stimulated endothelial cells. CMPF has been implicated in anemia and neurologic abnormalities of uremia. CMPF could alter the metabolism of drugs of inhibiting their binding to albumin and their tubular excretion. Indoxyl sulfate administrated to uremic rats increases the rate of progression of renal failure. Hippuric acid inhibits glucose utilization in the muscle, and its serum concentration is correlated with neurologic symptoms of uremia. Homocysteine predisposes uremic patients to cardiovascular disease through impairment of endothelial and smooth muscle cell functions. The removal of protein-bound compounds by conventional hemodialysis is low. Other strategies to decrease their concentrations include increase in dialyze pore size, daily hemodialysis, peritoneal dialysis, reduction of production or acceleration of degradation, and preservation of residual renal function.

Aim: To compare amounts of salicyluric acid (SU) and salicylic acid (SA) excreted daily in the urine of non-vegetarians and vegetarians not taking salicylate drugs, and patients taking 75 or 150 mg aspirin/day., Methods: Urine excreted over 24 hours was collected from volunteers in the four groups. The volumes were recorded and the concentrations of SU and SA were determined electrochemically after separation by high performance liquid chromatography., Results: Significantly more SU was excreted daily by vegetarians (median, 11.01; range, 4.98-26.60 micro mol/24 hours) than by non-vegetarians (median, 3.91; range, 0.87-12.23 micro mol/24 hours), although amounts were significantly lower than those excreted by patients taking aspirin. Median amounts of SU excreted by patients taking 75 and 150 mg/day of low dose aspirin were 170.69 (range, 13.15-377.18) micro mol/24 hours and 165.17 (range, 5.61-429.12) micro mol/24 hours, respectively. The amount of SU excreted by patients taking either 75 or 150 mg of aspirin/day was not significantly different. Significantly more SA was excreted by vegetarians (median, 1.19; range, 0.02-3.55 micro mol/24 hours) than by non-vegetarians (median, 0.31; range, 0.01-2.01 micro mol/24 hours). The median amounts of SA excreted by vegetarians and the patients taking aspirin were not significantly different., Conclusions: More SU and SA is excreted in the urine of vegetarians than in non-vegetarians, consistent with the observation that fruits and vegetables are important sources of dietary salicylates. However, significantly less SU was excreted by vegetarians than patients taking aspirin, indicating that the daily intake of bioavailable salicylates by vegetarians is considerably lower than that supplied by a single 75 or 150 mg dose of aspirin.

Dietary phenols are antioxidants, and their consumption might contribute to the prevention of cardiovascular disease. Coffee and tea are major dietary sources of phenols. Dietary phenols are metabolized extensively in the body. Lack of quantitative data on their metabolites hinders a proper evaluation of the potential biological effects of dietary phenols in vivo. The aim of this study was to identify and quantify the phenolic acid metabolites of chlorogenic acid (major phenol in coffee), quercetin-3-rutinoside (major flavonol in tea) and black tea phenols in humans, and determine the site of metabolism. Healthy humans (n = 20) with an intact colon participated in a dietary controlled crossover study, and we identified and quantified approximately 60 potential phenolic acid metabolites in urine. Half of the ingested chlorogenic acid and 43% of the tea phenols were metabolized to hippuric acid. Quercetin-3-rutinoside was metabolized mainly to phenylacetic acids, i.e., 3-hydroxyphenylacetic acid (36%), 3-methoxy-4-hydroxyphenylacetic acid (8%) and 3,4-dihydroxyphenylacetic acid (5%). In contrast, in seven humans without a colon, we found only traces of phenolic acid metabolites in urine after they had ingested chlorogenic acid and quercetin-3-rutinoside. This implies that the colonic microflora convert most of these dietary phenols into metabolites that then reach the circulation. Metabolites of dietary phenols have lower antioxidant activity than their parent compounds; therefore, the contribution of dietary phenols to antioxidant activity in vivo might be lower than expected from in vitro tests.

Metabolic zonation was assessed with the multiple indicator dilution (MID) technique in the single-pass perfused rat liver with use of moment analysis of the formed metabolite (M) data. During single-pass, retrograde rat liver perfusion with 17 Awake M benzoate, a bolus containing tracer preformed metabolite (PM) [(3)H]hippurate was injected rapidly into the hepatic vein at 20 min postperfusion, followed by injection of a second bolus containing [(14)C]benzoate at 30 min. Both doses also contained noneliminated reference indicators ((51)Cr-labeled RBCs, (125)I-labeled albumin, [(14)C]- or [(3)H]sucrose, and (2)H(2)O). The steady-state extraction ratio of benzoate, the area under the curve (AUC) and its mean transit time (MTT) during retrograde flow were identical to those previously observed for prograde flow. Values of AUC(PM) and MTT(PM) and AUC(M) were also similar to previously published prograde data, but the MTT(M) with retrograde perfusion was smaller than that for prograde perfusion. This, according to theory based on the tubes-in-series model, was consistent with perivenous enrichment of glycination activity when transport of drug was even and when the ratio of drug influx/efflux coefficient exceeded that for metabolite. Similar benzoate transport in periportal, homogeneous and perivenous isolated rat hepatocytes existed, and the influx/efflux coefficients (partition ratio) of benzoate from MID indeed exceeded that of hippurate. However, metabolism by zonal hepatocytes failed to reveal the anticipated metabolic zonation, and this is likely due to the shallow gradient of metabolic activity. The study demonstrates that moment theory is useful in delineating the perivenous enrichment of glycine conjugation activity.

A micellar electrokinetic chromatography (MEKC) method was developed for estimating the angiotensin-I converting enzyme (ACE) inhibitory activity by separating the hippuric acid liberated in the ACE reaction mixture in the presence of an inhibitor, captopril. The hippuric acid was successfully separated and detected by MEKC with a 25 mM sodium dodecyl sulfate solution in a 25 mM phosphate-50 mM borate buffer at pH 7.0; the total analysis took about 5 min. A good linear relationship was observed between the inhibitor and the peak area of hippuric acid release. No significant difference in the ACE inhibitory activity (IC50) of captopril (an antihypertensive medicine) or autolyzed-mushrooms (functional foods) was observed between the conventional method and the MEKC method. The MEKC method was found to be a useful technique for a rapid assay of the ACE inhibitory activity.

Aims: To validate a phenotypic Campylobacter species identification method employed to identify campylobacters in broilers by comparison with campylobacterial species identification using various species-specific PCR analyses., Methods and Results: From a collection of 2733 phenotypically identified campylobacterial cultures, 108 Campylobacter jejuni cultures and 351 campylobacterial cultures other than Camp. jejuni were subjected to various species-specific PCR assays. On the basis of the genotypic tests, it was demonstrated that Camp. jejuni and Camp. coli constituted approx. 99% of all cultures, while other species identified were Helicobacter pullorum, Camp. lari and Camp. upsaliensis. However, 29% of the 309 Camp. coli cultures identified by phenotypic tests were hippurate-variable or negative Camp. jejuni cultures, whereas some Camp. lari cultures and unspeciated campylobacter cultures belonged to H. pullorum. It was also notable that 2-6% of the cultures were, in fact, mixed cultures., Conclusions: The phenotypic identification scheme employed failed to appropriately differentiate Campylobacter species and particularly to identify the closely related species, H. pullorum., Significance and Impact of the Study: Future phenotypic test schemes should be designed to allow a more accurate differentiation of Campylobacter and related species. Preferably, the phenotypic tests should be supplemented with a genotypic strategy to disclose the true campylobacterial species diversity in broilers.

Defibrinated bovine plasma (DBP) was treated with the microbial protease Flavourzyme to obtain protein hydrolysates with various degrees of hydrolysis (DH). The angiotensin I-converting enzyme (ACE) inhibiting activity of the hydrolyzed protein was assessed with hippuryl-His-Leu as the substrate. The amount of hippuric acid released, due to uninhibited ACE activity, was determined by high-performance liquid chromatography. ACE inhibiting (ACEI) activity was found to increase with increasing DH; the 43% DH hydrolysate exhibited the highest activity and had an IC(50) of 1.08 mg/mL. Peptide fractions with high ACEI activity were isolated using size exclusion chromatography. The fraction that possessed the highest ACEI activity contained peptides with GYP, HL(I), HPY, HPGH, L(I)F, SPY, and YPH sequence motifs, as determined by reversed-phase liquid chromatography-tandem mass spectrometry using a novel immonium precursor-ion scanning technique. Some of these motifs correspond to sequences found in bovine serum albumin, a potential source of ACEI peptides in bovine plasma.

We describe a novel method for the determination of three kinds of dimethylbenzylmercapturic acids (DMM) and six kinds of dimethylhippuric acids (DMH), found in urine as metabolites of trimethylbenzenes, based on liquid chromatography/electrospray ionization tandem mass spectrometry. A solid-phase extraction procedure was used for the extractions of DMM and DMH from a urine sample, and the separation was performed on a reversed-phase C(30) column. The analytes were ionized by electrospray in the positive-ion mode. Operating in the multiple reaction monitoring mode, the linearity of the relative mass spectrometric responses to the internal standard versus analyte concentrations were established in the range 0.1-100 ng ml(-1). The extraction procedure was rapid and the relative standard deviations were below 5%. The detection limits of DMM and DMH in the urine by the proposed method were in the ranges 0.26-0.41 and 0.42-2.0 ng l(-1), respectively. Furthermore, DMM and DMH were detected in a urine sample from an individual who did not suffer from occupational exposure to trimethylbenzenes, by using this method., (Copyright 2002 John Wiley & Sons, Ltd.)

A method is described for the determination of hippuric acid (HA) and o-, m-, and p-methylhippuric acids (o-, m-, p-MHAs) in urine using solid-phase extraction and gas chromatography-mass spectrometry (GC-MS). The extraction procedure uses an Empore disk, derivatized into the respective trimethyl silyl derivatives. All metabolites including the internal standard (I.S.) were clearly able to be analyzed by the DB-17 column. The calibration curves for the four acids show linearity in the range of 5-70 microg/ml. The detection limit of each acid was 1.0-2.5 microg/ml.

Urine hippuric acid determination is helpful for monitoring of group of workers at risk for exposure to toluene. However, some problems about the external source of variation are mentioned. Some studies have indicated that smoking is an important external source of variation for determination of urine hippuric acid level while the others stated the opposite findings. This study was conducted in an attempt to study the difference of urine hippuric acid between smoking and non smoking subjects in a press workers group. Urine samples were obtained from 46 workers (all male) who worked as press workers in the same press factory in Bangkok. The individuals were classified as control (non smoking, N = 26) and experimental (smoking, N = 20) according to their smoking. All samples were analyzed for hippuric acid level. The average urine hippuric acid level for the control were (0.35 +/- 0.31 mg/gCr) and experimental group (0.40 + 0.45 mg/gCr) were respectively. No significant difference was found between urine hippuric acid level between both groups. The data from the current study indicates that smoking does not influence the urinary hippuric acid levels in this study group.

The measurement of exhaled and excreted xenobiotics and their metabolites can provide accurate, non-invasive, and time-flexible measurements of internal dose. We analyzed rates of exhaled (2)H(8)-toluene and excreted urinary metabolites from 33 exposures of men to 50 ppm of (2)H(8)-toluene for 2 h at rest. The total dose was distributed as follows: exhaled (2)H(8)-toluene, 13 +/- 6.2%; (2)H(5)-hippuric acid, 75 +/- 6.4%; (2)H(7)-o-cresol, 0.31 +/- 0.22%; (2)H(7)-m-cresol, 0.53 +/- 0.44%; and (2)H(7)-p-cresol, 11 +/- 3.8%. Interindividual variability was assessed using the coefficients of variation for peak exhalation or excretion rates, and fractions of dose excreted: (2)H(8)-toluene, c.v.=60, 47%; (2)H(5)-hippuric acid, 29, 8.6%; (2)H(7)-o-cresol, 80, 73%; (2)H(7)-m-cresol, 37, 83%; and (2)H(7)-p-cresol, 38, 34%. Excretion rates of the cresols were stable over the first 5 h post-exposure, and o-cresol was determined to be the best urinary indicator of exposure, given the lower background levels of this isomer. The hippuric acid/cresol rate ratios for the first 5 h post-exposure could be described by single exponential terms, and thus provided a means for estimating time since exposure for any finite toluene duration/exposure combination.

We studied the relationship between the use of three detoxification pathways and urine pH and the tolerance of desert woodrats from two populations to a mixture of naturally occurring plant secondary metabolites (mostly phenolics) in resin from creosote bush (Larrea tridentata). The two populations of desert woodrats came from the Mojave desert (Mojave woodrats), where woodrats consume creosote bush, and from the Great Basin desert (Great Basin woodrats), where the plant species is absent. We fed woodrats alfalfa pellets containing increasing levels of the phenolic resin and measured three detoxification pathways and urine pH that are related to detoxification of allelochemicals. We found that the excretion rate of two phase II detoxification conjugates, glucuronides and sulfides. increased with increasing resin intake, whereas excretion of hippuric acid was independent of resin intake, although it differed between populations. Urine pH declined with increasing resin ingestion. The molar proportion of glucuronides in urine was three times that of the other conjugates combined. Based on an evaluation of variation in the three detoxification pathways and urine pH in relation to resin intake, we rejected the hypotheses that woodrats' tolerance to resin intake is related to capacity for amination, sulfation, or pH regulation. However, Mojave woodrats had higher maximum glucuronide excretion rates, and we accepted the hypothesis that within and between populations woodrats tolerate more resin because they have a greater capacity for glucuronide excretion.

Carboxypeptidase U (EC 3.4.17.20, CPU, TAFIa) is a novel determinant of the fibrinolytic rate. It circulates as an inactive zymogen, procarboxypeptidase U, which becomes active during the process of coagulation. We developed a high throughput method on microtiter plates for the determination of the procarboxypeptidase U concentration in human plasma samples. Following activation of procarboxypeptidase U by thrombin-thrombomodulin, the resulting enzyme activity cleaves p-OH-Hip-Arg and the generated p-OH-hippuric acid is converted by hippuricase to p-hydroxybenzoic acid and glycine. Finally, oxidative coupling of p-hydroxybenzoic acid with 4-aminoantipyrine by NaIO4 forms the quinoneimine dye. The absorbance of the latter dye is determined at 506 nm in a microtiter plate reader. A mean value of 620 U/l was found, with a CV of 3.0% within-run and 4.3% between-run. The assay showed a good correlation with the activities observed using a HPLC assay as reference method (n = 25, r = 0.979). The presented method enables the routine analysis of large sample pools in clinical setting.

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 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.

The interactions of Zn(II), Cd(II) and Hg(II) with hippuric acid (hipH) were studied and several novel compounds were synthesized and studied by NMR. Some new metal-hippuric-cytosine ternary compounds were formed and the structure of the [Cd(hip)(2)(cyt)(H(2)O)](2) ternary complex resolved. Each cadmium (II) atom has a distorted trigonal bipyramid coordination which is linked to a water molecule, a cytosine via N(3), a carboxylic oxygen atom of a hippurate moiety and two bridging dicoordinated hippurates bound through the carboxylic oxygen atoms. To these five main bonds, two longer ancillary interactions can be observed: the second oxygen of the monocoordinated hippurate group and the carboxylic oxygen of the cytosine ligand. The compound is stabilized by an intramolecular stacking between the benzene and cytosine rings and by the hydrogen bonds between the coordinated water molecules and the ligands. This is, to our knowledge, the first structure of a cadmium-amino acid derivative-natural nucleobase compound described so far.

We report here the first combined use of NMR-PR (pattern recognition) analysis and directly coupled HPLC--NMR analysis to identify metabolic subpopulations in normal laboratory animals and their discriminating endogenous urinary biomarkers. Urine samples obtained from control Sprague-Dawley rats (n = 68) were analyzed using (1)H NMR spectroscopy and principal components (PC) analysis to investigate physiological variability. Two distinct subpopulations of animals were classified based on metabolite excretion profiles. Analysis of the PC loadings established the spectral regions that were responsible for classification of the subpopulations and was used to direct the identification of biomarkers using a directly coupled HPLC--NMR analysis. One population had low urinary hippurate levels together with an increased concentration of 3-(3-hydroxyphenyl)propionic acid (3-HPPA)and 3-hydroxycinnamic acid (3-HCA). The other subpopulation excreted high levels of hippurate. Thus, we report the bimodal occurrence of hippuric acid and chlorogenic acid metabolites in a genetically homogeneous population of rats maintained under identical conditions, which may have significance in relation to the understanding of the consequences of biochemical variation in animals used for drug toxicity testing., (Copyright 2001 Academic Press.)

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.

The present concept of dialysis focuses mainly on the removal of small water-soluble compounds, and also, the currently applied kinetic parameters of dialysis adequacy are based on the behavior of water-soluble compounds. Nevertheless, many of the currently known biological effects in uremia are attributable to compounds with different physicochemical characteristics, and among these, protein-bound solutes play an important role. In this article, we review the characteristics and consequences of changes in protein binding in uremia, as well as the toxicity of the protein-bound uremic solutes 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF), indoxyl sulfate, hippuric acid, homocysteine, and p-cresol. Starting from the example of p-cresol, we then summarize the impact of protein-binding on dialytic removal, whereby it is concluded that this removal is largely hampered by this protein-binding compared with that of classic markers such as urea and creatinine. Alternative removal strategies, such as strategies to modify intestinal generation or absorption, are considered.

Hippurate (Hip), an endogenous conjugate, belongs to the group of uremic toxins. Hip stimulates P-independent glutaminase (PIG) localized at the proximal luminal membrane, desamidating glutamine with the formation of ammonia, a dominant and adaptive elimination product of H+. This appears to be important because metabolic acidosis (MAC) does not stimulate PIG. Moreover, Hip inhibits ammonia production by P-dependent mitochondrial glutaminase (PDG) that is primarily stimulated by MAC. By this mechanism, it shifts the ammonia production from mitochondria to proximal tubular lumen. MAC stimulates Hip synthesis in the liver and kidney and increases Hip plasma concentration and even fractional excretion by the kidney, which creates an effective regulatory loop of ammoniagenesis. Thus, it appears that Hip by its participation in the correction of MAC possesses the modulatory function.

Characterization of p-hydroxy-hippuric acid as an inhibitor of Ca2+-ATPase in end-stage renal failure. In patients with end-stage renal failure (ESRF), disturbances of Ca2+ metabolism are common. Besides hormonal changes, inhibition of cellular Ca2+-ATPase was postulated to contribute to uremic toxicity. We purified a potent inhibitor of the Ca2+-ATPase from the ultrafiltrate of patients with ESRF by multiple steps of high-performance liquid chromatography to homogeneity, and identified the isolated inhibitor by mass spectrometric methods as p-hydroxy-hippuric acid. The enzyme used for the Ca2+-ATPase assay system was isolated from red blood cells by cross-flow filtration. The activity of the Ca2+-ATPase was measured spectrophotometrically as the difference in hydrolysis of adenosine 5'-triphosphate (ATP) in the presence and absence of Ca2+ with different concentrations of ATP and p-hydroxyhippuric acid. The Ca2+-ATPase was found to be inhibited by p-hydroxy-hippuric acid at a concentration above 11.7 micromol/L. p-Hydroxyhippuric acid inhibited the erythrocyte Ca2+-ATPase by reducing Vmax and increasing the Km value. The EC50 (log mol/L; mean +/- SEM) for p-hydroxy-hippuric acid was calculated as 4.82 +/- 0.14. In conclusion, p-hydroxy-hippuric acid may play a role in disturbed Ca2+ metabolism in end-stage renal failure.

Hippurate (Hip) is considered to be the end product of benzoate (BA) metabolism. However, the kidney is able to metabolize Hip. Although only Hip but no BA is present in the blood, rat urine contains under normal conditions less Hip (about 0.4 mM) than BA (about 4.5 mM) and of hydroxylated derivatives of BA (hydroxy-BAs = HB and dihydroxy-BAs = DHB). Generation of HBs and DHBs is the result of radical substitution by free OH radicals (*OH). Thus, rate of synthesis of HBs and DHBs may reflect the production rate of *OH in the kidney *OH generation is elevated following ischemic stress. Therefore, production of HBs and DHBs can be expected to be elevated in postischemic injury. The validity of this assumption was tested in vitro on isolated tubular segments and in vivo in the rat. Metabolism of Hip at 0.1 mmol x l(-1 ) (0.1 mM) as well as of BA resulted in enlarged production of both HBs (especially 3-HB and 4-HB) and of DHBs (especially 2,6-DHB). Production of 2,3- and especially of 2,5-DHB was elevated in the presence of high concentration (1.0 mM) of salicylate (2-HB) only. In vivo both in acute (120 min) and in chronic (5 days) experiments ligation of one renal artery for 30 respectively 60 min resulted in enlarged excretion of HBs and DHBs, especially of 2,6- and 3,5-DHB. This finding is noteworthy since (a) formation of 2,6-DHB necessitates as precursor salicylate which could not be detected in our experiments and (b) the spontaneous attack of *OH upon the benzol ring would prefer the positions 2,3- 2,5- and 3,4-. Therefore, the existence of regulating factor(s) guiding OH groups to definite positions is a distinct possibility. These results indicate that metabolism of Hip leading to hydroxylated BAs may be a renoprotective mechanism against attack of *OH in reoxygenated renal tissue., (Copyright 2001 S. Karger AG, Basel)

Bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the copper-, ascorbate-, and O2-dependent cleavage of C-terminal glycine-extended peptides, N-acylglycines, and the bile acid glycine conjugates to the corresponding amides and glyoxylate. Two known metabolites of aspirin, salicyluric acid and gentisuric acid, are also substrates for PAM, leading to the formation of salicylamide and gentisamide. The time course for O2 consumption and glyoxylate production indicates that salicylurate amidation is a two-step reaction. Salicylurate is first converted to N-salicyl-alpha-hydroxyglycine, which is ultimately dealkylated to salicylamide and glyoxylate. The enzymatically generated salicylamide and N-salicyl-alpha-hydroxyglycine were characterized by mass spectrometry and two-dimensional 1H-13C heteronuclear multiple quantum coherence NMR.

Several commercially available immunoassay kits have been developed to detect the beta-adrenergic agonist clenbuterol HCl. Technical materials supplied with the kits do not generally report cross-reactivity with clenbuterol metabolites. Use of such kits to quantitate clenbuterol might lead to an overestimation of parent drug if metabolites were present. The objective of this study was to measure the cross-reactivity of clenbuterol metabolites with several commercially available clenbuterol immunoassays. Three clenbuterol-glucuronide conjugates, clenbuterol-sulphamate, 4-amino-3,5-dichloro-hippuric acid (clenbuterol-hippurate), and purified clenbuterol-stereoisomers were tested for cross-reactivity. The clenbuterol-sulphamate metabolite showed significant cross-reactivity (42-77%), but clenbuterol-hippurate showed very little competition (< 0.2%) towards clenbuterol. Clenbuterol-glucuronides had little (0.1-1.6%) cross-reactivity. In addition, (R)-, (S)-, and racemic clenbuterol were used to determine the stereospecificity of the kits. Both (R)- and (S)-clenbuterol competed for binding in two of the kits, however, in one kit the (S)-clenbuterol stereoisomer had an affinity 100 times greater than the (R)-stereoisomer. The presence of significant quantities of the sulphamate metabolite of clenbuterol in a biological matrix would cause an overestimation of the amount of parent clenbuterol. This study illustrates the inherent problems of using unvalidated immunoassays for quantitation purposes.

Inborn errors of urea synthesis result in hyperammonemia. Sodium benzoate (SB) therapy has been beneficial in the treatment of hyperammonemia. It conjugates with glycine to form hippurate, which is then excreted. SB has also been used to treat children with nonketotic hyperglycinemia (NKH), where glycine is removed, on conjugation, as hippurate. In mammalian liver mitochondria, SB is activated by an ATP-dependent reaction to its CoA ester, before conjugation with glycine. Pantothenic acid (PA) is the precursor of CoA. In this investigation, increasing the amounts of PA increased CoA levels in HepG2 cells. It also significantly increased formation of hippurate in SB-treated cells. These findings suggest a beneficial effect of PA on the SB therapy in children with NKH as well as hyperammonemia.

A simple synthetic route for the preparation of amino acid conjugate of 5-aminosalicylic acid (5-ASA) was exploited and prepared 5-aminosalicyl-glycine (5-ASA-Gly) in good yield. In vitro and in vivo properties of 5-ASA-Gly as a colon-specific prodrug of 5-ASA were investigated using rats as the test animal. Incubation of 5-ASA-Gly with cecal or colonic contents at 37 degrees C released 5-ASA in 65 or 27% of the dose in 8 h, respectively. No 5-ASA was detected from the incubation of 5-ASA-Gly with the homogenates of stomach or small intestine. Plasma concentration of 5-ASA-Gly decreased rapidly after intravenous administration of 5-ASA-Gly, and no 5-ASA was detected in the blood, which indicated 5-ASA-Gly was not degraded in the plasma. After oral administration of 5-ASA-Gly, about 50% of the administered dose was recovered as 5-ASA and N-acetyl-ASA and 3% as 5-ASA-Gly from feces and 14% as 5-ASA-Gly and 28% as 5-ASA and N-acetyl-ASA from urine in 24 h. These results suggested that a large fraction of 5-ASA-Gly was delivered to the large intestine and activated to liberate 5-ASA. For comparison, total recovery of 5-ASA and N-acetyl-5-ASA from feces after oral administration of 5-ASA-Gly was greater than that from sulfasalazine, which is one of the most commonly prescribed prodrugs of 5-ASA., (Copyright 2000 Wiley-Liss, Inc.)

(R)- and (S)-Hippuryl-alpha-methylphenylalanine [(R)- and (S)-Hipp-alpha-MePhe] and (S)-hippuryl-alpha-methylphenyllactic acid [(S)-Hipp-alpha-MeOPhe] were synthesized and evaluated as substrates for carboxypeptidase A (CPA) in an effort to shed further light on the catalytic mechanism of the enzyme. The rate of CPA-catalyzed hydrolysis of (S)-Hipp-alpha-MePhe was reduced by 105-fold compared with that of (S)-Hipp-Phe, but the hydrolysis rate of (S)-Hipp-OPhe was lowered by only 6.8-fold by the introduction of a methyl group at the alpha-position. (R)-Hipp-alpha-MePhe failed to be hydrolyzed initially, then started to undergo hydrolysis in about 2 h at a much reduced rate. The results of present study may be envisioned on the basis of the proposition that while peptide substrate is hydrolyzed via a tetrahedral transition state formed by the attack of the zinc-bound water molecule at the peptide carbonyl carbon, ester hydrolysis takes the path that involves an anhydride intermediate generated by the attack of the carboxylate of Glu-270 at the ester carbonyl carbon.