Urine Of Human Volunteers Research Articles

Translating dosimetry of Dibenzo[def,p]chrysene (DBC) and metabolites across dose and species using physiologically based pharmacokinetic (PBPK) modeling

Dibenzo[def,p]chrysene (DBC) is an environmental polycyclic aromatic hydrocarbon (PAH) that causes tumors in mice and has been classified as a probable human carcinogen by the International Agency for Research on Cancer. Animal toxicity studies often utilize higher doses than are found in relevant human exposures. Additionally, like many PAHs, DBC requires metabolic bioactivation to form the ultimate toxicant, and species differences in DBC and DBC metabolite metabolism have been observed. To understand the implications of dose and species differences, a physiologically based pharmacokinetic model (PBPK) for DBC and major metabolites was developed in mice and humans. Metabolism parameters used in the model were obtained from experimental in vitro metabolism assays using mice and human hepatic microsomes. PBPK model simulations were evaluated against mice dosed with 15 mg/kg DBC by oral gavage and human volunteers orally microdosed with 29 ng of DBC. DBC and its primary metabolite DBC-11,12-diol were measured in blood of mice and humans, while in urine, the majority of DBC metabolites were obeserved as conjugated DBC-11,12-diol, conjugated DBC tetrols, and unconjugated DBC tetrols. The PBPK model was able to predict the time course concentrations of DBC, DBC-11,12-diol, and other DBC metabolites in blood and urine of human volunteers and mice with reasonable accuracy. Agreement between model simulations and measured pharmacokinetic data in mice and human studies demonstrate the success and versatility of our model for interspecies extrapolation and applicability for different doses. Furthermore, our simulations show that internal dose metrics used for risk assessment do not necessarily scale allometrically, and that PBPK modeling provides a reliable approach to appropriately account for interspecies differences in metabolism and physiology.

HPLC ANALYSIS OF HUMAN URINE FOR OXALATE CONTENT

<p><strong>Objective: </strong>In the present communication, development and validation of reverse phase-high performance liquid chromatography method have been carried out for estimation of oxalate content in the urine of human volunteers with recurrent kidney stone disease and healthy status.</p><p><strong>Methods: </strong>The analysis of oxalic acid has been carried out on KYA TECH HiQ Sil C<sub>18</sub>HS column using a mobile phase of methanol: 0.001 N acetic acid in water (50:50, v/v) with a flow rate of 1 ml/min and detection wavelength, 237 nm.</p><p><strong>Results: </strong>Analysis of oxalate content was carried out using single point calibration method with retention at 2.705 min with good resolution parameters. Urine sample collected from kidney stone patients and healthy volunteers over the period of 24 h were analyzed and it has been found that concentration of oxalate in healthy volunteers is less than 12 µg/ml whereas that in case of kidney stone patients is in the range of 39-151 µg/ml and this data can be utilized for further interpretations about oxalate content in healthy and kidney stone diseased volunteers. This method was validated as per united states food and drug administration (USFDA) guidelines by the study of accuracy, precision, linearity, range, selectivity, the lower limit of quantitation, extraction recovery studies and stability studies for determining oxalate content in the urine of human volunteers. As relative standard deviations of oxalate content estimated are less than 5 percent, the method can be claimed accurate, precise, sensitive and selective for determining oxalate content in the urine of human volunteers.</p><p><strong>Conclusion: </strong>The results are satisfactory, proving the effectiveness of the method for analysis of oxalate content from other biological fluids with few optimizations.</p>

Determination of bioactive, free isothiocyanates from a glucosinolate-containing phytotherapeutic agent: A pilot study with in vitro models and human intervention

Isothiocyanates (ITCs) derived from plants of the order Brassicales are known for their antibacterial, anti-inflammatory or anticarcinogenic potential. Although only the free ITCs exert bioactivity, quantification in vivo is almost exclusively performed on total ITC/metabolite content. We therefore investigated in a pilot study the amount of free ITC at different steps critical for therapeutic efficacy. A sensitive and specific GC–MS/MS method for the simultaneous quantification of individual free ITC after solid-phase extraction (SPE) was developed. We show here that release of biologically active ITC from plants occurs at not only alkaline but also acidic pH. Furthermore, in human urine conversion of the ultimate, inactive mercapturic acid conjugate back into its corresponding bioactive form is increased at alkaline as compared to neutral pH. This was also observed in the urine of human volunteers, where – in correlation with the pH value – a mean of 0.16 to 1.03μmol ITC was detected after oral application of a phytotherapeutic agent containing 30.4μmol of the initial pro-drugs. The amounts of free ITC being necessary for bioactivity in vitro were found to be indeed achieved in vivo. These data might be helpful to better understand the beneficial effects of ITC observed in vivo.

Quantitation of trans-resveratrol and detection of its metabolites in human plasma and urine by high performance liquid chromatography

We describe a reversed-phase HPLC method that uses gradient elution and UV detection (325 nm) to determine levels of resveratrol and identify six major conjugated metabolites in the plasma and urine of human volunteers after administration of a single oral dose of 1 g. Waters Atlantis C 18 3 μm served as the stationary phase. The gradient was formed using ammonium acetate and methanol, containing 2% propan-2-ol. Detection is linear between 5 ng/mL and 500 ng/mL in plasma (5–1000 ng/mL in urine). The coefficient of variation for intra- and inter-day variation is <10%. The average recovery of resveratrol from plasma and urine is 58 ± 3%. The data presented in this report demonstrate a rapid, sensitive and accurate method for the analysis of resveratrol and its metabolites in human plasma and urine for pharmacokinetic studies.

Increased Salicylate Concentrations in Urine of Human Volunteers after Consumption of Cranberry Juice

The aim of this study was to assess whether regular consumption of cranberry juice results in elevations in urinary salicylate concentrations in persons not taking salicylate drugs. Two groups of healthy female subjects (11/group) matched for age, weight, and height consumed 250 mL of either cranberry juice or a placebo solution three times a day (i.e., 750 mL/day) for 2 weeks. At weekly intervals, salicylic acid and salicyluric acid (the major urinary metabolite of salicylic acid) concentrations were determined in urine by HPLC with electrochemical detection. Concentrations of salicylic acid in plasma were also determined. Consumption of cranberry juice was associated with a marked increase (p < 0.001) of salicyluric and salicylic acids in urine within 1 week of the intervention. After 2 weeks, there was also a small but significant (p < 0.05) increase in salicylic acid in plasma. The regular consumption of cranberry juice results in the increased absorption of salicylic acid, an anti-inflammatory compound that may benefit health.

Formation of a Mutagenic Heterocyclic Aromatic Amine from Creatinine in Urine of Meat Eaters and Vegetarians

Liquid chromatography electrospray ionization mass spectrometry (MS) with a triple quadrupole MS was used to identify known and novel heterocyclic aromatic amines (HAAs) in human urine. The identities of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were confirmed by their product ion spectra. The constant neutral loss scan mode was employed to probe for other analytes in urine that display the transition [M+H]+-->[M+H-CH3*]+*, which is common to HAAs containing an N-methylimidazo moiety, and led to the detection of a previously unreported isomer of 8-MeIQx [Holland, R., et al. (2004) Chem. Res. Toxicol. 17, 1121-1136]. We now report the identification of another novel HAA, 2-amino-1-methylimidazo[4,5-b]quinoline (IQ[4,5-b]), an isomer of the powerful animal carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). The amounts of IQ[4,5-b] measured in the urine of human volunteers who consumed grilled beef ranged from 15 to 135% of the ingested dose, while the amounts of 8-MeIQx and PhIP excreted in urine were on average <2% of the ingested dose. Base treatment of urine at 70 degrees C increased the concentrations of 8-MeIQx and PhIP by as much as 6-fold, indicating the presence of phase II conjugates; however, the amount of IQ[4,5-b] increased by more than 100-fold. IQ[4,5-b] was also detected in the urine of vegetarians following base hydrolysis. The formation of IQ[4,5-b], but not IQ, 8-MeIQx, or PhIP, also occurred in urine incubated at 37 degrees C. Creatinine and 2-aminobenzaldehyde are likely precursors of IQ[4,5-b]. The detection of IQ[4,5-b] in the urine of both meat eaters and vegetarians suggests that this HAA may be present in nonmeat staples or that IQ[4,5-b] formation may occur endogenously within the urinary bladder or other biological fluids.

Clinical analysis of sampatrilat, a combined renal endopeptidase and angiotensin-converting enzyme inhibitor: II: Assay in the plasma and urine of human volunteers by dissociation enhanced lanthanide fluorescence immunoassay (DELFIA)

Sampatrilat is a dual inhibitor of angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) under development for the treatment of hypertension and congestive heart failure. In order to support the early clinical development (with oral administration and an expected low bioavailability), a sensitive and selective assay was required. An HPLC-atmospheric-pressure chemical ionisation mass-spectrometric (HPLC-APCI-MS-MS) assay had been already validated (R.F. Venn et al., J. Pharm. Biomed. Anal., in press), but due to its low throughput an alternative method was sought. As the molecule is peptide-like and not metabolised, we believed the immunoassay approach was appropriate. Thus we developed an immunoassay for the compound using time-resolved fluorescence as an end-point (DELFIA®) with lower limits of quantification of 0.2 ng ml −1 for the plasma assay and 5 ng ml −1 for the assay in urine. This assay is a 96-well plate based competitive immunoassay; the end-point is the determination of a (non-radioactive) europium label by time-resolved fluorimetry. Sampatrilat is labelled with chelated europium via isothiocyanate chemistry. The advantage of this assay is its extremely high throughput, allowing rapid analysis of many thousands of samples. The DELFIA method was successfully cross-validated with the HPLC-APCI-MS-MS method.

HUMAN EXPOSURE TO NATURALLY OCCURRING HYDROQUINONE

Hydroquinone (HQ) is a nonvolatile chemical used in the photographic, rubber, chemical, and cosmetic industries. HQ is also known to occur in nature as the beta-D-glucopyranoside conjugate (arbutin), and free HQ is a known component of cigarette smoke. Low concentrations of HQ have been detected in the urine and plasma of humans with no occupational or other known exposure to HQ. The studies reported here investigate dietary and other potential sources of HQ and their contribution to HQ concentrations in the plasma and urine of human volunteers. Analysis of possible food sources of HQ by GC indicated significant amounts of arbutin in wheat products (1-10 ppm), pears (4-15 ppm), and coffee and tea (0.1 ppm). Free HQ was found in coffee (0.2 ppm), red wine (0.5 ppm), wheat cereals (0.2-0.4 ppm), and broccoli (0.1 ppm). After consuming a meal including arbutin- and HQ-containing foods, volunteers showed significant increases in plasma and urinary levels of HQ and its conjugated metabolites (total HQ). Mean plasma concentrations of total HQ peaked at 5 times background levels at 2 h after the completion of the meal, and mean urinary excretion rates of total HQ peaked at 12 times background at 2-3 h after the meal. Immediately after smoking four cigarettes in approximately 30 min, mean plasma concentrations of total HQ were maximally 1.5 times background levels; mean urinary excretion rates of total HQ peaked at 2.5 times background at 1-3 h after smoking. These data indicate that considerable human exposure to HQ can result from plant-derived dietary sources and, to a lesser extent, from cigarette smoke.

Biotransformation of a new pyrrolidinone cognition-enhancing agent: isolation and identification of metabolites in human urine

1. The metabolites of N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl)acetamide (DMPPA; MH-1), in the urine of human volunteers have been investigated. 2. Ten metabolites together with the unchanged drug (MH-1) were isolated by h.p.l.c. and identified by n.m.r. and mass spectrometry as: three metabolites hydroxylated in the pyrrolidine ring of MH-1 (MH-2, MH-3 and MH-4), three metabolites hydroxylated in the dimethylphenyl ring of MH-1 (MH-6, MH-7 and MH-8), N-[(2,6-dimethylphenylcarbamoyl)methyl]-4-hydroxybutyrylamide++ + (MH-5), N-[(2,6-dimethylphenyl-carbamoyl)methyl]succinamic acid (MH-9), the 3-O-sulphate of MH-6 (MH-10) and the 3-O-sulphate of N-(2,6-dimethyl-3-hydroxyphenyl)-2-(5-hydroxy-2-oxo-1-pyrrolidinyl)aceta mide (MH-11). 3. DMPPA was extensively metabolized. The principal metabolic transformations were hydroxylation of the pyrrolidine ring at the C5 carbon followed by oxidative C-N cleavage, and hydroxylation of the phenyl ring followed by sulphate conjugation.

Determination of the sulphoxide metabolites of S-carboxymethyl-L-cysteine by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic method has been developed for the determination of the mucolytic drug S-carboxymethyl-L-cysteine (SCMC) and its major metabolites S-methyl-L-cysteine (SMC), SCMC-sulphoxide and SMC-sulphoxide. Pre-column derivatisation with o-phthalaldehyde ethanethiol and electrochemical detection were employed. The mobile phase was methanol-phosphate buffer of various compositions. SCMC and its sulphoxide were determined simultaneously but, for optimum resolution, they were determined separately using a different mobile phase composition for each. The method, which was convenient and rapid, allowed the resolution of the sulphoxide diastereoisomers. Good recoveries of SCMC and its metabolites were obtained from spiked urine samples. All four compounds were detected in the urine of human volunteers during a 24-h period following a single dose of the drug. The major constituent recovered was unchanged SCMC.

Kinetics of 3- tert-butyl-4-hydroxyanisole (BHA) in man

High-resolution capillary gas chromatography-mass spectrometry with selective ion monitoring and using deuterated 3- tert-butyl-4-hydroxyanisole (BHA) as an internal standard was used to measure BHA in the plasma and urine of human volunteers after oral administration of 30 or 5 mg of the compound in olive oil. Pharmacokinetic studies showed similar plasma-concentration profiles in subjects treated with either level of BHA. About 20% of the administered dose was excreted as BHA glucuronide in the urine within the first 24 hr.

The metabolism of S-carboxymethyl-L-cysteine in man: isolation of an ester glucuronic acid conjugate from urine.

A conjugate isolated from urine of human volunteers after an oral dose of S-carboxymethyl-L-cysteine was characterized as a carboxylic acid ester glucuronide. Of the 166 volunteers investigated, 61 gave no detectable drug glucuronide (less than 0.5% administered dose); the remaining 105 showed a unimodal distribution of drug glucuronide excretion accounting for 0.5-11.5% (mean 4.1%, median 3.4%) of the total dose recovered in the 0-8 h urine. No significant variation in subject age, sex or urinary pH was observed between the two groups, but those not excreting urinary glucuronide had significantly higher 0-8 h urine volumes (P less than 0.001), although notable exceptions occurred.

TLC determination of griseofulvin in plasma and 6-demethylgriseofulvin in urine

Fluorometric TLC procedures are described for the determination of griseofulvin in human plasma and 6-demethylgriseofulvin in human urine. Griseofulvin is extracted from plasma with ether, and its metabolite, 6-demethylgriseofulvin, is extracted from urine with benzene. Both compounds are subjected to TLC on silica gel plates. The plates are scanned using the fluorescent mode of a spectrodensitometer. For griseofulvin, the quantitation limit is 20ng/ml of plasma and the recovery is 100%; for 6-demethylgriseofulvin, the limit is 1 μ/ml of urine and the recovery is 96%. The methods were used to determine the plasma levels of griseofulvin and the amount of 6-demethylgriseofulvin excreted in the urine of human volunteers after a single oral dose of griseofulvin.

Quantitative analysis of hydralazine pyruvic acid hydrazone, the major plasma metabolite of hydralazine

A specific, high-performance liquid chromatographic techinique for the measurement of hydralazine pyruvic acid hydrazone is described. This method utilized reversed-phase chromatography for the separation of this hydrophilic metabolite of hydralazine from other fluid constituents present in serum, plasma, or urine of human volunteers and rabbits receiving hydralazine. Detection of the compound of interest is accomplished spectrophotometrically at 250 nm.

Concentrations of nitrate in normal human urine and the effect of nitrate ingestion

A method suitable for the analysis of nitrate in human urine was developed. Normal urinary concentrations of nitrate in urine of human volunteers in Dade County, Florida, where the drinking water contains negligible amounts of nitrate, averaged 47.6 ppm of NO 3 − (SD = 17.3). On a vegetable and preserved-meat-free diet, the nitrate concentration was reduced (10 to 30 ppm of NO 3 − ), but, on nitrate-supplemented drinking water, the urinary concentration rose to a range of 34–87 ppm of NO 3 − . A high vegetable diet resulted in peak urinary nitrate concentrations of 270–425 ppm. These results indicated that nitrate in drinking water is a factor in determining urinary nitrate concentration, but that vegetable ingestion is of greater significance.

Species variations in the N-oxidation of chlorphentermine

The metabolism and excretion of orally administered or injected [ 14C]chlorphentermine has been studied in man, rhesus monkey, marmoset, rabbit, guinea-pig and rat. These species excreted 55–95% of the administered radioactivity in the urine over 5 days. Two metabolites were characterised by thin-layer and paper chromatography, gas-liquid chromatography and g.c.-m.s. and these were N-hydroxychlorphentermine and 1-(4'-chlorophenyl)-2-methyl-2-nitropropane. There are marked species differences in the excretion of N-oxidation products which were found in the urine of human volunteers. rhesus monkeys, rabbits and guinea-pigs, but not in the urine of marmosets or rats. The rat, rabbit and marmoset also excreted an unidentified unstable acid-labile precursor of chlorphentermine. The results are discussed in relation to the toxicity of the drug and to the metabolism of amphetamines in general.

Excretion of bephenium salts in urine of human volunteers.

Excretion of bephenium salts in urine of human volunteers.