Kraemer T.. Pflugmann, T., Peters F.T., Maurer H.H.
Department of Experimental and Clinical Toxicology, University of Saarland, D-66421 Homburg (Saar), Germany
The Dark Agouti (DA) rat strain serves as an animal model for the human cytochrome P450 2D6 poor metabolizer (PM), whereas Wistar (WI) rats correspond to the human extensive metabolizer type. There still is need of elucidating the role of 2D6 polymorphism for the high interindividual differences in amphe-tamine (AM) and/or methampheamphe-tamine (MA) findings after application of AM/MA precursor drugs. The aim of our study was to test whether the DA model was suitable also for understanding those forensically important differences. Urinary excretion and plasma levels of AM and MA after intake of the model sub-stance selegiline (N-propinyl-methamphetamine) should be investigated in DA and Wl rats. Forensic implications of corresponding differences whicht might occur should be discussed.
DA and WI rats received a single oral dose of selegiline (3 mg/kg bm) by gastric intubation (n=5, each group). 24 h urine samples were collected and tested using the Abbott TDx immunoassay Amphetamine/Methamphetamine II. For quantification of AM and MA in the urine samples, GC-MS was used in SIM mode (ions m/z 240, 254 and 244, 258 for deuterated IS) after solid-phase extraction (HCX) and heptafluorobutyrylation. Blood samples were taken 2, 4 and 6 h after application. For quantification of AM and MA in these samples, GC-MS was used in NICI mode (SIM ions m/z 388, 402 and 399, 407 for deuterated IS) after solid-phase extraction (HCX) and derivatization with heptafluorobutyrylprolyl chloride. Quantification was performed using calibration curves.
The TDx results in the 24 h urine samples of the two rat strains were significantly different (P value 0.0003). The medians of the measured concentrations were 10,000 ng/mL in the DA group and 2,065 ng/mL in the WI group. GC-MS quantification confirmed these results. AM and MA concentrations were significantly (P values 0.004, each) higher in the urine samples of the DA group. The medians of the urine MA concentrations were 14,081 ng/mL for the DA rats and 1,165 ng/mL for the Wl rats. The medians of the AM concentrations were 22,710 ng/mL (DA) and 2,430 ng/mL (WI). The AM plasma levels differed significantly (P<0,05) between the two groups in the 2, 4 and 6 h blood samples, with medians being 63, 45.8 and 30.9 ng/mL in the respective 2, 4 and 6 h samples of the DA rats and 41.8, 26.5, 19.6 ng/mL in the corresponding samples of the WI rats. The MA plasma levels differed significantly between the two groups (P<0.05) in the 2 and 4 h blood samples (medians 38.0 and 15.7 ng/mL for DA rats; medians 28.6 and 8.9 ng/mL for WI rats).
The DA rats showed significantly higher AM/MA plasma and urine concentrations after selegiline inges-tion. 2D6 polymorphism seems to be of importance for that. As shown in former in-vitro studies, N-deal-kylation is not catalyzed by 2D6. Besides, this would have resulted in lower AM/MA levels for the DA group. Therefore, other metabolic steps (e.g. hydroxylation) might be catalyzed by the polymorphic 2D6, which could explain the higher AM/MA levels in the PM model by the lack of hepatic elimination.
Corresponding in-vitro studies are in progress.
Annales de Toxicologie Analytique, vol. XIV, n° 3, 2002
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Kinetics of kavain and its metabolites after oral application
Tarbah F.( 1 ). Mahler H.( l >, Kardel B.( 1 ), Weinmann W.(2), Daldrup Th,( ! )(1) Institute of Legal Medicine, Heinrich-Heine University, P.O. Box 10 10 07, D-40001 Duesseldorf, Germany
(2) Institute of Legal Medicine, University Hospital Freiburg, Albertstrasse 9, D-79104 Freiburg, Germany The metabolism of the wide-spread used phytopharmaceutical kavain is performed by the human liver cell-line Hep-G2 resulting in 13 kavain metabolites1. In the present study a high performance liquid chroma-tography (HPLC-DAD) assay method for the simultaneous determination of kavain and its main metabo-lites (p-hydroxykavain, p-hydroxy-5,6-dehydrokavain and p-hydroxy-7,8-dihydrokavain) in serum and urine was developed and validated.
The main metabolic pathways were hydroxylation of the phenyl ring and conjugation with glucuronic acid and sulphate, reduction of the 7,8-double bond, hydroxylation with subsequent dehydration and opening of the lactone ring. The metabolites were mainly excreted in the form of their conjugates. All kavain meta-bolites were detectable in serum and urine except for phydroxy-7,8-dihydrokavain which was found in urine only.
Confirmation of the results and identification of the metabolites were carried out by LC/MS or LC/MS/MS. Kinetics of kavain and its metabolites in serum were investigated after administration of a single oral dose (800 mg d,l-kavain). Within time periods between 1 and 4 hours after uptake, the serum concentrations ranged between 10 ng/ml and 40 ng/ml for kavain, 125 ng/ml and 300 ng/ml for p-hydroxy-kavain, 40 ng/ml and 90 ng/ml for phydroxydehydrokavain as well as 30 ng/ml and 50 ng/ml for dehy-drokavain.
Reference :
1- Tarbah F. et al., Problems of Forensic Sciences, Special issue: 37t h TIAFT triennial meeting XLII : 173-180.
65
Kava (Piper methysticum Forst. f.) side effects and toxicity : study of 29 heavy kava drin-kers and 2 cases of acute hepatitis in occasional kava drindrin-kers in New Caledonia
Barguil Y.w. Kritsanida M.( ,-2 ), Cabalion P.( 2\ Duhet D.<2\ Mandeau A.( , ), Poncet C.( , )
(1) Laboratoire de Biochimie, Centre Hospitalier de Nouvelle-Calédonie, Av. Paul Doumer, BP J5, 98849 Nouméa, Nouvelle-Calédonie
(2) IRD, Laboratoire des substances naturelles terrestres, Promenade Roger Laroque, BP A5, 98848 Nouméa, Nouvelle-Calédonie
New Caledonia is a South Pacific island where kava beverage (aqueous extract of the root of Piper methys-ticum Forst. f.) was introduced from Vanuatu around 20 years ago. It is used for its sedative properties due to lipophylic cc-pyrones (kavalactones). Following recent accidents with liver failure in Europe after oral intake of kava medications, we studied the biological parameters of 29 heavy kava drinkers. We compared them to those of 2 New Caledonian cases of acute hepatitis after light oral intake of kava.
29 volunteers (19 men and 10 women), heavy kava drinkers (mean: 6.56 units (cups prepared by the mela-nesian way: «70mL of kava, =650mg of kavalactones) /day, 6.04 days/week, nearly 25g of total kavalac-tones/week, since more than 5 years), participated. The presence of kavalactones in their urine was veri-fied by HPLC/DAD. 2 women who developed an acute hepatitis, light kava drinkers (<5 units/day, for less than one month) took part in the study. Blood and urine biochemistry, tests for hepatitis A, B, C, serum protein electrophoresis and ultrasonography were performed.
Among the 29 heavy kava drinkers, 17 men and 8 women had an isolated increase of gamma-glutamyl-transferase, 3 times the reference range. The only important clinical sign was skin dryness (ichtyosis in 17 volonteers). After significant diminution of kava consumption, GGT returned to normal values, skin dry-ness disappeared. Ultrasonography, blood tests for hepatitis A, B, C and serum protein electrophoresis gave normal results for all the volunteers. In the 2 cases of acute hepatitis, all biological signs of liver func-tion alterafunc-tion were present. Hepatitis in New Caledonia and in Europe occurred after intake of small amounts of kavalactones (<10g/week and <2g/week in each case). Clinical signs occurred both within 1 -2 months. On the contrary, heavy drinkers had only an isolated increase of GGT, which may suggest an hepatic enzymatic induction with no other clinical sign than a skin dryness.
Kava consumption may lead to an enzymatic induction of the cytP450, by which kavalactones are possi-bly metabolized. This could explain kava hepatotoxicity: a formation of a toxic metabolite may occur, which would be either the cause of an immuno-allergic reaction or/and accumulated in poor metabolizers (idiosyncratic cases) (1). Russmann et al. have already showed a CYP2D6 deficiency in one case of kava hepatotoxicity in Switzerland (1). Deficiency of retinoids (metabolized by the cyt. P450) in ichtyosis would be explained as well.
Reference
1- Russmann S. et al. Annals of Internal Medicine 2001 ; 135 : 1
Annales de Toxicologie Analytique, vol. XIV, n° 3, 2002