Plasma levels of copper, manganese and selenium in an adult population in southern Spain: influence of age, obesity and lifestyle factors.

Copper, manganese and selenium are elements involved in protecting the body against oxidative stress. Determining their plasma level may contribute to assessing the health and nutritional status of populations. The aim of this study was to assess factors influencing copper, manganese and selenium plasma levels in an adult Mediterranean population and to identify groups at risk of deficiency. A cross-sectional survey was carried out in Andalusia, a region in southern Spain. Blood samples were obtained in a random subsample of 340 subjects. Food consumption was assessed by 48-h recall. Height, weight, skinfolds, waist and hip circumferences were measured. Copper, manganese and selenium were measured in plasma. Information about physical exercise, educational level, alcohol and smoking habits was obtained with a structured questionnaire. Plasma copper was found to be higher in women than among men. Hypocupraemia was found in 4.4% of the population, while 9.7% presented hypomanganesemia. Moreover, 86.5% presented plasma selenium values below 125microg/L (cutoff for optimal glutathione peroxidase activity). No association was found between plasma elements, anthropometric indices and lifestyle factors; there were tendencies, no more. Copper tended to decrease in obese and increase in sedentary, while selenium tended to decrease among smokers. Plasma Cu was positively correlated with the consumption of monounsaturated and polyunsaturated fats. Plasma Mn was directly correlated with the consumption of dairy products. Levels of Se were positively correlated with age, the consumption of fruit, vegetables, energy obtained from carbohydrates, and the consumption of fibre, and inversely correlated with the consumption of meat and sweets. Our results provide an estimate of the copper, manganese and selenium status in the adult population of southern Spain. The correlations found for Se suggest that there is a tendency for Se levels to be better maintained among the population that shows a stronger preference for the traditional diet.

Plasma kinetics of procarbazine and azo-procarbazine in humans.

The plasma kinetics of procarbazine (PCB) and its major metabolite azo-procarbazine (azo-PCB) were systematically investigated in humans for the first time. Eight therapy-refractory tumor patients with normal liver and renal function were given a single oral dose of 300 mg PCB hydrochloride as a drinking solution under fasting conditions. With the exception of the single i.v. administration of 10 mg ondansetron hydrochloride immediately before the administration of PCB, the patients were free of any co-medication 4 weeks before and during the study. PCB and azo-PCB were determined by a specially developed HPLC-UV method. PCB was absorbed very rapidly. Mean maximum plasma concentration was 12.5 min. A high elimination rate of PCB from plasma was found. The mean apparent oral systemic clearance and the plasma elimination half-life were estimated at 35.8 l/min and 9.2 min, respectively. Considerable amounts of azo-PCB are found in the plasma of the eight tumor patients. The mean Cmax and AUC ratios of azo-PCB/PCB were estimated at 5.5 and 45.2. Azo-PCB is formed very rapidly from PCB, but eliminated much more slowly from plasma than PCB. Considerable interindividual differences in the conversion rate of azo-PCB to its further metabolites were observed which should have consequences for the individual tumor therapeutic efficiency of PCB. No toxic side-effects or symptoms such as nausea or vomiting were observed during the entire study.

Determination of procarbazine in human plasma by liquid chromatography with electrospray ionization mass spectrometry.

Procarbazine is a cytotoxic chemotherapeutic agent used in the treatment of lymphomas and brain tumors. Its pharmacokinetic behavior remains poorly understood even though more than 30 years have elapsed since the drug was approved for clinical use. To characterize the pharmacokinetics of procarbazine in brain cancer patients during a phase I trial, a method for determining the drug in human plasma by reversed-phase high-performance liquid chromatography (HPLC) with electrospray ionization mass spectrometry (ESI-MS) was developed and thoroughly validated. Plasma samples were prepared for analysis by precipitating proteins with trichloroacetic acid and washing the protein-free supernatant with methyl tert-butyl ether to remove excess acid. The solution was separated on a Luna C-18 analytical column using methanol-25 mM ammonium acetate buffer, pH 5.1 (22:78, v/v) as the mobile phase at 1.0 ml/min. A single-quadrupole mass spectrometer with an electrospray interface was operated in the selected-ion monitoring mode to detect the [M+H](+) ions at m/z 222.2 for procarbazine and at m/z 192.1 for the internal standard (3-dimethylamino-2-methylpropiophenone). Procarbazine and the internal standard eluted as sharp, symmetrical peaks with retention times (mean+/-S.D.) of 6.3+/-0.1 and 9.9+/-0.3 min, respectively. Calibration curves of procarbazine hydrochloride in human plasma at concentrations ranging from 0.5 to 50 ng/ml exhibited excellent linearity. The mean absolute recovery of the drug from plasma was 102.9+/-1.0%. Using a sample volume of 150 microl, procarbazine was determined at the 0.5 ng/ml (1.9 nM) lower limit of quantitation with a mean accuracy of 105.2% and an interday precision of 3.60% R.S.D. on 11 different days over 5 weeks. During this same time interval, the between-day accuracy for determining quality control solutions of the drug in plasma at concentrations of 2.0, 15 and 40 ng/ml ranged from 97.5 to 98.2% (mean+/-S.D., 97.9+/-0.4%) and the precision was 3.8-6.2% (mean+/-S.D., 5.1+/-1.2%). Stability characteristics of the drug were thoroughly evaluated to establish appropriate conditions to process, store and prepare clinical specimens for chromatographic analysis without inducing significant chemical degradation. The sensitivity achieved with this assay permitted the plasma concentration-time profile of the parent drug to be accurately defined following oral administration of standard doses to brain cancer patients.

Profile of procarbazine-induced embryotoxicity in an embryo hepatocyte co-culture system and after in utero glutathione depletion.

Procarbazine (PCZ) is an antineoplastic agent useful in the treatment of Hodgkin's disease, brain tumors, and chronic leukemia. PCZ is dysmorphogenic to developing embryos exposed in vivo or cultured in the serum of PCZ-treated rats. However, embryos directly cultured with PCZ (up to 400 micrograms/ml) or PCZ plus S-9 liver fractions are unaffected. Since intact liver cells provide several advantages over hepatic subcellular fractions for in vitro bioactivation, we exposed rat embryos to PCZ in an embryo/hepatocyte co-culture system. Gestation day (GD) 9.5 rat embryos exposed to 0, 200, 300, or 400 micrograms PCZ/ml in the presence of untreated or phenobarbital induced male rat hepatocytes failed to display toxicity. However, in a companion study GD 9.5 rat embryos cultured in the serum from PCZ-treated rats exhibited developmental deficiencies. Studies have shown that the formation of toxic metabolites can result from glutathione (GSH) conjugation of toxicants in the liver. Therefore, in a second set of experiments, rat embryos were cultured in serum from rats pretreated with two GSH depleters (phorone and buthionine sulfoximine) and subsequently dosed with PCZ. Effects on development were enhanced when embryos were cultured in the serum from PCZ-treated/GSH depleted rats. These data indicate that PCZ requires in vivo activation to be dysmorphogenic and further suggest that the metabolite(s) responsible for procarbazine embryo-toxicity are formed readily under conditions of low GSH levels. This argues against a glutathione conjugate as the ultimate toxicant.

Studies on the metabolism of procarbazine by mass spectrometry.

The mass spectrometric properties of procarbazine and some of its breakdown products were examined using isotopic labeling and high resolution mass spectrometry. A specific intramolecular methyl group transfer, induced by electron impact, was used diagnostically to distinguish between two isomeric derivatives. The mass spectrometric information was used for identification of the in vivo metabolites of procarbazine in rat. Minimum sample manipulation was used due to the inherent instability of the suspected metabolites. The azo compound N-isopropyl- alpha -(2-methylazo)-p-toluamide was identified as the principal circulating metabolite of procarbazine.

Teratogenicity induced in cultured rat embryos by the serum of procarbazine treated rats.

Male rats were injected with single intraperitoneal doses of 50-200 mg/kg of procarbazine hydrochloride (P). Their undiluted serum was then used as the culture medium for 9.5-day-old embryos to evaluate its teratogenic and toxic potential in vitro. A 48-h exposure to this medium resulted in a variety of dysmorphogenic effects. Somite, limb bud and otic vesicle deformities were seen at all dose levels, and neural tube and optic vesicle abnormalities were frequently observed in the 150 and 200 mg/kg groups. Embryonic growth and differentiation were only moderately affected at any dose levels. In a second set of experiments, embryos were directly exposed to 100-150 micrograms/ml P combined with a liver enzymatic activating system. No abnormalities were detected but toxicity occurred within narrow concentration ranges; 125 micrograms/ml affected growth and differentiation slightly, and 150 micrograms/ml suppressed the embryonic development severely. Exposure to 125 micrograms/ml P without the liver enzymatic activating system had no effect on embryonic growth and differentiation, suggesting that the metabolites responsible for the toxic effect were generated by the liver enzymatic preparation. Our results indicate that stable metabolites of P, responsible for its teratogenic action, are formed only within body compartments, whereas the formation of these metabolites does not take place in vitro in the presence of hepatic enzyme preparations.

Analysis of procarbazine and metabolites by gas chromatography-mass spectrometry.

Twelve compounds representing procarbazine, seven metabolites, and an internal standard were analyzed by gas chromatography-mass spectrometry on a 3% OV-1 column. Procarbazine and four metabolites were derivatized with acetic anhydride. A sensitive, specific and quantitative assay was established by selected ion monitoring using a synthetic analogue of the drug as an internal standard. The limits of detection were approximately 1 ng/ml of plasma while the limits of quantitation were 10 ng/ml of plasma. Studies of the degradation of procarbazine . HCl in 0.05 M phosphate buffer (pH 7.4) were compared to in vivo studies. At 1 h after incubation of procarbazine . HCl in buffer, the azo and aldehyde metabolites were detected in the highest concentrations representing 27.2% and 20.3% of total drug and metabolites. In the in vivo studies, analyses of rat plasmas indicated that 1 h after an oral dose of procarbazine . HCl, the aldehyde metabolite represented 72% of the total drug and metabolites, and that relatively little of the azo metabolite was present.

Application of an electrochemical detector to the determination of procarbazine hydrochloride by high-performance liquid chromatography.

An amperometric flow-through detector with a carbon paste working electrode was utilized as a high-performance liquid chromatographic (HPLC) detector to determine procarbazine hydrochloride, an antineoplastic agent, in both buffer solution and biological fluids. The HPLC system included an amino-cyano stationary phase and an aqueous (pH 7)-methanolic mobile phase which enabled the separation of procarbazine from its only electroactive degradation product, N-isopropyl-alpha-(2-methylhydrazono)-p-toluamide. The electrochemical detector, with an approximate limit of detection of 2 ng procarbazine injected, was 20 times more sensitive to procarbazine than a typical UV detector. The low dead volume (1 microliter) and superior selectivity of the electrochemical detector enabled the HPLC determination of procarbazine in untreated human urine and plasma.