High-resolution mass spectrometric investigation of the phase I and II metabolites of finasteride in pig plasma, urine and bile.

1. The metabolite profile of the 5α-reductase type II inhibitor finasteride has been studied in pig plasma, urine and bile using high-resolution mass spectrometry. The porcine biotransformation products were compared to those formed by human liver microsomes and to literature data of recently identified human in vivo metabolites. The objective of this study was to gain further evidence for the validity of using pigs for advanced, invasive drug-drug interaction studies that are not possible to perform in humans. 2. The use of high-resolution mass spectrometry with accurate mass measurements enabled identification of the metabolites by calculation of their elemental compositions as well as their fragmentation patterns. 3. There was an excellent match between the porcine and human metabolic profiles, corroborating the pig as a model of human drug metabolism. The glucuronides of the two recently described human hydroxylated metabolites MX and MY and the carboxylated metabolite M3 were identified as the major biotransformation products of finasteride in pig urine and bile. 4. Furthermore, the CYP enzymes involved in the formation of the hydroxylated metabolites were characterized. Human recombinant CYP3A4 could produce the two major hydroxylated metabolites MX and MY, whereas human recombinant CYP2D6 formed MY only.

Life cycle assessment of segregating fattening pig urine and feces compared to conventional liquid manure management.

Gaseous emissions from in-house storage of liquid animal manure remain a major contributor to the environmental impact of manure management. Our aim was to assess the life cycle environmental consequences and reduction potential of segregating fattening pig urine and feces with an innovative V-belt system and to compare it to conventional liquid manure management, that is, the reference. Moreover, we aimed at analyzing the uncertainty of the outcomes related to applied emission factors. We compared a reference with two scenarios: segregation with solid, aerobically, stored feces and with liquid, anaerobically, stored feces. Results showed that, compared to the reference, segregation reduced climate change (CC) up to 82%, due to lower methane emission, reduced terrestrial acidification (TA) and particulate matter formation (PMF) up to 49%, through lower ammonia emission, but increased marine eutrophication up to 11% through nitrogen oxide emission from storage and nitrate leaching after field application. Fossil fuel depletion did not change. Segregation with liquid feces revealed lower environmental impact than segregation with solid feces. Uncertainty analysis supported the conclusion that segregating fattening pig urine and feces significantly reduced CC and additionally segregation with liquid feces significantly reduced TA and PMF compared to the reference.

Analysis of naturally occurring zearalenone in feeding stuffs and urine of farm animals in Croatia.

The aim of this study was to determine feed and urinary levels of zearalenone. A total of 114 samples, 64 feeding stuffs (commodities, pig and cattle feed), and 50 urine samples were analyzed by the use of enzyme-linked immunosorbent assay (ELISA). Zearalenone was detected in 68.7% of feeding stuffs, while all urine samples except for four yearling samples were positive for zearalenone. The maximum zearalenone concentration in feeding stuffs and urine was 577 ng/g and 241.1 ng/mL, respectively. Although zearalenone concentrations in some samples were high, the risk for humans was negligible since the calculated concentrations in meat were below the tolerable daily intake (TDI).

Urinary Kinetics of Heroin Metabolites in Pigs Shortly After Intake.

In previous experimental studies on heroin metabolites excretion in urine, the first sample was often collected a few hours after intake. In forensic cases, it is sometimes questioned if a positive urine result is expected e.g., 30 min after intake. The aim of this study was to investigate urinary excretion of heroin metabolites (morphine, 6-monoacetylmorphine (6-MAM) and morphine-3-glucuronide (M3G)) every 30 min until 330 min after injection of a 20 mg heroin dose in six pigs. Samples were analyzed using a previously published, fully validated liquid chromatography-tandem mass spectrometry method. All metabolites were detected after 30 min in all pigs. The time to maximum concentration (Tmax) median (range) for 6-MAM and morphine was 30 min (first sample) (30-120), and 90 min (30-330) for M3G. In four of the six pigs, the Tmax of 6-MAM and morphine was reached within 30 min. All analytes were still detectable at the end of study. This study showed that positive results in urine are expected to be seen shortly after use of heroin in pigs. Detection times were longer than previously indicated, especially for 6-MAM, but previous studies used lower doses. As the physiology of these animals resembles that of the humans, transferability to man is expected.

Urinary Hepcidin Levels in Iron-Deficient and Iron-Supplemented Piglets Correlate with Hepcidin Hepatic mRNA and Serum Levels and with Body Iron Status.

Among livestock, domestic pig (Sus scrofa) is a species, in which iron metabolism has been most intensively examined during last decade. The obvious reason for studying the regulation of iron homeostasis especially in young pigs is neonatal iron deficiency anemia commonly occurring in these animals. Moreover, supplementation of essentially all commercially reared piglets with iron entails a need for monitoring the efficacy of this routine practice followed in the swine industry for several decades. Since the discovery of hepcidin many studies confirmed its role as key regulator of iron metabolism and pointed out the assessment of its concentrations in biological fluids as diagnostic tool for iron-related disorder. Here we demonstrate that urine hepcidin-25 levels measured by a combination of weak cation exchange chromatography and time-of-flight mass spectrometry (WCX-TOF MS) are highly correlated with mRNA hepcidin expression in the liver and plasma hepcidin-25 concentrations in anemic and iron-supplemented 28-day old piglets. We also found a high correlation between urine hepcidin level and hepatic non-heme iron content. Our results show that similarly to previously described transgenic mouse models of iron disorders, young pigs constitute a convenient animal model to explore accuracy and relationship between indicators for assessing systemic iron status.

Development and application of salting-out assisted liquid/liquid extraction for multi-mycotoxin biomarkers analysis in pig urine with high performance liquid chromatography/tandem mass spectrometry.

Direct determination of urinary mycotoxins is a better approach to assess individual's exposure than the indirect estimation from average dietary intakes. In this study, a new analytical method was developed and validated for simultaneous analysis of aflatoxin B1, deoxynivalenol, fumonisin B1, ochratoxin A, zearalenone and T2 toxin and their metabolites in pig urine. In total 12 analytes were selected. A salting-out assisted liquid-liquid extraction procedure was used for sample preparation. High performance liquid chromatography/tandem mass spectrometry was used for the separation and detection of all the analytes. The extraction recoveries were in a range of 70-108%, with the intra-day relative standard deviation and inter-day relative standard deviation lower than 25% for most of the compounds at 3 different concentration levels. Meanwhile the method bias for all the analytes did not exceed 20%. The limits of quantification ranged from 0.07ngmL(-1) for ochratoxin A to 3.3ngmL(-1) for deoxynivalenol. Matrix effect was evaluated in this study and matrix-matched calibration was used for quantification. The developed method was also validated for human urine as an extension of its application. Finally, the developed method was applied in a pilot study to analyze 28 pig urine samples. Deoxynivalenol, aflatoxin B1, fumonisin B1 and ochratoxin A were detected in these samples.

Metabolic fate of intravenously administered N-acetylneuraminic acid-6-14C in newborn piglets.

BACKGROUND: Sialic acid (N-acetylneuraminic acid), a component of gangliosides and sialylglycoproteins, may be a conditional nutrient in early life because endogenous synthesis is limited. The aim of this study was to investigate the metabolic fate of intravenously administrated N-acetylneuraminic acid-6-14C (sialic acid) in piglets. METHOD: Three-day-old male domestic piglets (Sus scrofa) were injected via the jugular vein with 5 microCi (11-12 x 10(6) cpm) of N-acetylneuraminic acid-6-14C (specific activity of 55 mCi/mmol). Blood samples were collected at regular intervals over the next 120 min. The organs were then removed and the urine collected for determination of residual radioactivity. RESULTS: Within 2 min of injection, 80% of the activity was removed from the blood and by 120 min the remaining activity approached 8%. At 120 min, the brain contained significantly more radioactivity (cpm/g tissue) than the liver, pancreas, heart and spleen, but less than the kidneys. Within the brain, the percentage of total injected activity was highest in the cerebrum (0.175 +/-0.008) followed by the cerebellum (0.0295 +/-0.006, p=0.00006) and the thalamus (0.029 +/- 0.006, p =0.00003). CONCLUSIONS: An exogenous source of sialic acid is capable of crossing the blood-brain barrier and being taken up into various tissues. The findings suggest that dietary sources of sialic acid may contribute to early brain development in newborn mammals.