Capillary electrophoretic investigation of the enantioselective metabolism of propafenone by human cytochrome P-450 SUPERSOMES: Evidence for atypical kinetics by CYP2D6 and CYP3A4

An enantioselective CE method was used to identify the ability of CYP450 enzymes and their stereoselectivity in catalyzing the transformation of propafenone (PPF) to 5-hydroxy-propafenone (5OH-PPF) and N-despropyl-propafenone (NOR-PPF). Using in vitro incubations with single CYP450 enzymes (SUPERSOMES), 5OH-PPF is shown to be selectively produced by CYP2D6 and N-dealkylation is demonstrated to be mediated by CYP2D6, CYP3A4, CYP1A2, and CYP1A1. For the elucidation of kinetic aspects of the metabolism with CYP2D6 and CYP3A4, incubations with individual PPF enantiomers and racemic PPF were investigated. With the exception of the dealkylation in presence of R-PPF only, which can be described by the Michaelis-Menten model, all CYP2D6-induced reactions were found to follow autoactivation kinetics. For CYP3A4, all NOR-PPF enantiomer formation rates as function of PPF enantiomer concentration were determined to follow substrate inhibition kinetics. The formation of NOR-PPF by the different enzymes is stereoselective and is reduced significantly when racemic PPF is incubated. Clearance values obtained for CYP3A4 dealkylation are stereoselective whereas those of CYP2D6 hydroxylation are not. This paper reports the first investigation of the PPF hydroxylation and dealkylation kinetics by the CYP2D6 enzyme and represents the first report in which enantioselective CE data provide the complete in vitro kinetics of metabolic steps of a drug.

Validated capillary electrophoresis assay for the simultaneous enantioselective determination of propafenone and its major metabolites in biological samples

A robust, inexpensive, and fully validated CE method for the simultaneous determination of the enantiomers of propafenone (PPF), 5-hydroxy-propafenone (5OH-PPF) and N-despropyl-propafenone (NOR-PPF) in serum and in in vitro media is described. It is based upon liquid-liquid extraction at alkaline pH followed by analysis of the reconstituted extract by CE in presence of a pH 2.0 running buffer composed of 100 mM sodium phosphate, 19% methanol, and 0.6% highly sulfated beta-CD. For each compound, the S-enantiomers are shown to migrate ahead of their antipodes, and the overall run time is about 30 min. Enantiomer levels between 25 and 1000 ng/mL provide linear calibration graphs, and the LOD for all enantiomers is between 10 and 12 ng/mL. The assay is shown to be suitable for the determination of the enantiomers of PPF and its metabolites in in vitro incubations comprising human liver microsomes or single CYP450 enzymes (SUPERSOMES). Incubations with CYP2D6 SUPERSOMES revealed, for the first time, the simultaneous formation of the enantiomers of 5OH-PPF and NOR-PPF with that enzyme. CE data can be used for the evaluation of the enzymatic N-dealkylation and hydroxylation rates.

Mouse lung CYP1A1 catalyzes the metabolic activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)

PhIP carcinogenesis is initiated by N(2)-hydroxylation, mediated by several cytochromes P450, including CYP1A1. However, the role of CYP1A1 in PhIP metabolic activation in vivo is unclear. In this study, Cyp1a1-null and wild-type (WT) mice were used to investigate the potential role of CYP1A1 in PhIP metabolic activation in vivo. PhIP N(2)-hydroxylation was actively catalyzed by lung homogenates of WT mice, at a rate of 14.9 +/- 5.0 pmol/min/g tissue, but < 1 pmol/min/g tissue in stomach and small intestine, and almost undetectable in mammary gland and colon. PhIP N(2)-hydroxylation catalyzed by lung homogenates of Cyp1a1-null mice was approximately 10-fold lower than that of WT mice. In contrast, PhIP N(2)-hydroxylation activity in lung homogenates of Cyp1a2-null versus WT mice was not decreased. Pretreatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increased lung Cyp1a1 mRNA and lung homogenate PhIP N(2)-hydroxylase activity approximately 50-fold in WT mice, where the activity was substantially inhibited (70%) by monoclonal antibodies against CYP1A1. In vivo, 30 min after oral treatment with PhIP, PhIP levels in lung were similar to those in liver. After a single dose of 0.1 mg/kg [(14)C]PhIP, lung PhIP-DNA adduct levels in Cyp1a1-null mice, but not in Cyp1a2-null mice, were significantly lower (P=0.0028) than in WT mice. These results reveal that mouse lung has basal and inducible PhIP N(2)-hydroxylase activity predominantly catalyzed by CYP1A1. Because of the high inducibility of human CYP1A1, especially in cigarette smokers, the role of lung CYP1A1 in PhIP carcinogenesis should be considered.

3,4-Dehydrodebrisoquine, a novel debrisoquine metabolite formed from 4-hydroxydebrisoquine that affects the CYP2D6 metabolic ratio

Considerable unexplained intersubject variability in the debrisoquine metabolic ratio (urinary debrisoquine/4-hydroxydebrisoquine) exists within individual CYP2D6 genotypes. We speculated that debrisoquine was converted to as yet undisclosed metabolites. Thirteen healthy young volunteers, nine CYP2D6*1 homozygotes [extensive metabolizers (EMs)] and four CYP2D6*4 homozygotes [poor metabolizers (PMs)] took 12.8 mg of debrisoquine hemisulfate by mouth and collected 0- to 8- and 8- to 24-h urines, which were analyzed by gas chromatography-mass spectrometry (GCMS) before and after treatment with beta-glucuronidase. Authentic 3,4-dehydrodebrisoquine was synthesized and characterized by GCMS, liquid chromatography-tandem mass spectrometry, and (1)H NMR. 3,4-Dehydrodebrisoquine is a novel metabolite of debrisoquine excreted variably in 0- to 24-h urine, both in EMs (3.1-27.6% of dose) and PMs (0-2.1% of dose). This metabolite is produced from 4-hydroxydebrisoquine in vitro by human and rat liver microsomes. A previously unstudied CYP2D6*1 homozygote was administered 10.2 mg of 4-hydroxydebrisoquine orally and also excreted 3,4-dehydrodebrisoquine. EMs excreted 6-hydroxydebrisoquine (0-4.8%) and 8-hydroxydebrisoquine (0-1.3%), but these phenolic metabolites were not detected in PM urine. Debrisoquine and 4-hydroxydebrisoquine glucuronides were excreted in a highly genotype-dependent manner. A microsomal activity that probably does not involve cytochrome P450 participates in the further metabolism of 4-hydroxydebrisoquine, which we speculate may also lead to the formation of 1- and 3-hydroxydebrisoquine and their ring-opened products. In conclusion, this study suggests that the traditional metabolic ratio is not a true measure of the debrisoquine 4-hydroxylation capacity of an individual and thus may, in part, explain the wide intragenotype variation in metabolic ratio.

The inhibition of bone resorption in rats treated with (-)-menthol is due to its metabolites

(-)-Menthol, a monoterpene from Mentha species (Lamiaceae), has been shown to inhibit bone resorption in vivo by an unknown mechanism. In the present study, plasma and urine profiling in rats determined by GC/MS demonstrate that (-)-menthol is extensively metabolized, mainly by hydroxylation and carboxylation, and excreted in the urine, in part as glucuronides. In plasma, very low concentrations of (-)-menthol metabolites were detected after a single dose of (-)-menthol, whereas after repeated treatment, several times higher concentrations and long residence times were measured. In contrast, the elimination of unchanged (-)-menthol was increased by repeated treatment. (-)-Menthol, at concentrations found in plasma, did not inhibit bone resorption in cultured mouse calvaria (skull). However, the neutral metabolites of (-)-menthol, extracted from urine of rats fed with (-)-menthol, inhibited bone resorption in vitro, the concentrations being at plasma level or higher. These results suggest that not (-)-menthol itself, but one or several of its neutral metabolites inhibit the bone resorbing cells in vivo.

Regioselective preparation of 5-hydroxypropranolol and 4'-hydroxydiclofenac with a fungal peroxygenase

An extracellular peroxygenase of Agrocybe aegerita catalyzed the H(2)O(2)-dependent hydroxylation of the multi-function beta-adrenergic blocker propranolol (1-naphthalen-1-yloxy-3-(propan-2-ylamino)propan-2-ol) and the non-steroidal anti-inflammatory drug diclofenac (2-[2-[(2,6-dichlorophenyl)amino]phenyl]acetic acid) to give the human drug metabolites 5-hydroxypropranolol (5-OHP) and 4'-hydroxydiclofenac (4'-OHD). The reactions proceeded regioselectively with high isomeric purity and gave the desired 5-OHP and 4'-OHD in yields up to 20% and 65%, respectively. (18)O-labeling experiments showed that the phenolic hydroxyl groups in 5-OHP and 4'-OHD originated from H(2)O(2), which establishes that the reaction is mechanistically a peroxygenation. Our results raise the possibility that fungal peroxygenases may be useful for versatile, cost-effective, and scalable syntheses of drug metabolites.

In vivo analysis of efavirenz metabolism in individuals with impaired CYP2A6 function

INTRODUCTION: The antiretroviral drug efavirenz (EFV) is extensively metabolized into three primary metabolites: 8-hydroxy-EFV, 7-hydroxy-EFV and N-glucuronide-EFV. There is a wide interindividual variability in EFV plasma exposure, explained to a great extent by cytochrome P450 2B6 (CYP2B6), the main isoenzyme responsible for EFV metabolism and involved in the major metabolic pathway (8-hydroxylation) and to a lesser extent in 7-hydroxylation. When CYP2B6 function is impaired, the relevance of CYP2A6, the main isoenzyme responsible for 7-hydroxylation may increase. We hypothesize that genetic variability in this gene may contribute to the particularly high, unexplained variability in EFV exposure in individuals with limited CYP2B6 function. METHODS: This study characterized CYP2A6 variation (14 alleles) in individuals (N=169) previously characterized for functional variants in CYP2B6 (18 alleles). Plasma concentrations of EFV and its primary metabolites (8-hydroxy-EFV, 7-hydroxy-EFV and N-glucuronide-EFV) were measured in different genetic backgrounds in vivo. RESULTS: The accessory metabolic pathway CYP2A6 has a critical role in limiting drug accumulation in individuals characterized as CYP2B6 slow metabolizers. CONCLUSION: Dual CYP2B6 and CYP2A6 slow metabolism occurs at significant frequency in various human populations, leading to extremely high EFV exposure.

Protein phosphatase 2A and phosphoprotein SET regulate androgen production by P450c17

Cytochrome P450c17 catalyzes 17 alpha-hydroxylation needed for cortisol synthesis and 17,20 lyase activity needed to produce sex steroids. Serine phosphorylation of P450c17 specifically increases 17,20 lyase activity, but the physiological factors regulating this effect remain unknown. Treating human adrenal NCI-H295A cells with the phosphatase inhibitors okadaic acid, fostriecin, and cantharidin increased 17,20 lyase activity, suggesting involvement of protein phosphatase 2A (PP2A) or 4 (PP4). PP2A but not PP4 inhibited 17,20 lyase activity in microsomes from cultured cells, but neither affected 17 alpha-hydroxylation. Inhibition of 17,20 lyase activity by PP2A was concentration-dependent, could be inhibited by okadaic acid, and was restored by endogenous protein kinases. PP2A but not PP4 coimmunoprecipitated with P450c17, and suppression of PP2A by small interfering RNA increased 17,20 lyase activity. Phosphoprotein SET found in adrenals inhibited PP2A, but not PP4, and fostered 17,20 lyase activity. The identification of PP2A and SET as post-translational regulators of androgen biosynthesis suggests potential additional mechanisms contributing to adrenarche and hyperandrogenic disorders such as polycystic ovary syndrome.

2-methiopropamine, a thiopene analogue of metamphetamine: studies on its metabolism abt detectability in the rat and human using

2-Methiopropamine [1-(thiophen-2-yl)-2-methylaminopropane, 2-MPA], a thiophene analogue of methamphetamine, is available from online vendors selling "Research chemicals." The first samples were seized by the German police in 2011. As it is a recreational stimulant, its inclusion in routine drug screening protocols should be required. The aims of this study were to identify the phase I and II metabolites of 2-MPA in rat and human urine and to identify the human cytochrome-P450 (CYP) isoenzymes involved in its phase I metabolism. In addition, the detectability of 2-MPA in urine samples using the authors' well-established gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-linear ion trap-mass spectrometry (LC-MS(n)) screening protocols was also evaluated. The metabolites were isolated from rat and human urine samples by solid-Phase extraction without or following enzymatic cleavage of conjugates. The phase I metabolites, following acetylation, were separated and identified by GC-MS and/or liquid chromatography-high-resolution linear ion trap mass spectrometry (LC-HR-MS(n)) and the phase II metabolites by LC-HR-MS(n). The following Major metabolic pathways were proposed: N-demethylation, hydroxylation at the side chain and at the thiophene ring, and combination of these transformations followed by glucuronidation and/or sulfation. CYP1A2, CYP2C19, CYP2D6, and CYP3A4 were identified as the major phase I metabolizing enzymes. They were also involved in the N-demethylation of the analogue methamphetamine and CYP2C19, CYP2D6, and CYP3A4 in its ring hydroxylation. Following the administration of a typical user's dose, 2-MPA and its metabolites were identified in rat urine using the authors' GC-MS and the LC-MS(n) screening approaches. Ingestion of 2-MPA could also be detected by both protocols in an authentic human urine sample.

The 17, 20-lyase activity of cytochrome p450c17 from human fetal testis favors the delta5 steroidogenic pathway.

Cytochrome P450c17 catalyzes both 17alpha-hydroxylation and 17,20-lyase conversion of 21-carbon steroids to 19-carbon precursors of sex steroids. P450c17 can mediate testosterone biosynthesis via the conversion of pregnenolone to dehydroepiandrosterone (the delta(5) pathway) or via conversion of progesterone to androstenedione (the delta(4) pathway). In many species, the 17, 20-lyase activity of P450c17 for one pathway dominates, reflecting the preferred steroidogenic pathway of that species. All studies of recombinant human P450c17 and of human adrenal microsomes have found high 17, 20-lyase activity only in the delta(5) pathway. Because the 17, 20-lyase activities in both the delta(4) and delta(5) pathways for testicular P450c17 have not been directly compared, however, it is not known if the delta(5) pathway dominates in the human testis. To resolve this issue, we assayed the conversion of 17alpha-hydroxypregnenolone to dehydroepiandrosterone (delta(5) 17, 20-lyase activity) and of 17alpha-hydroxyprogesterone to androstenedione (delta(4) 17, 20-lyase activity) by human fetal testicular microsomes. We obtained apparent Michaelis constant (K(m)) and maximum velocity (V(max)) values of 1.0 microM and 0.73 pmol.min(-1). microg(-1) for delta(5) 17, 20-lyase activity and of 3.5 microM and 0.23 pmol.min(-1). microg(-1) for delta(4) 17, 20-lyase activity. Catalytic efficiencies, expressed as the ratio V(max)/K(m), were 0.73 and 0.066 for the delta(5) and delta(4) reactions, respectively, indicating 11-fold higher preference for the delta(5) pathway. We conclude that the majority of testosterone biosynthesis in the human testis proceeds through the conversion of pregnenolone to dehydroepiandrosterone via the delta(5) pathway.

In vitro metabolism of testosterone in the horse liver and involvement of equine CYPs 3A89, 3A94 and 3A95

Testosterone (TES) 6-β-hydroxylation is a significant metabolic step in the biotransformation of TES in human liver microsomes and reflects cytochrome P450 (CYP) 3A4/5 specific metabolic activity. Several CYP3A enzymes have been annotated in the horse genome, but functional characterization is missing. This descriptive study investigates TES metabolism in the horse liver in vitro and the qualitative contribution of three CYP3A isoforms of the horse. Metabolism of TES was investigated by using equine hepatocyte primary cultures and liver microsomes. Chemical inhibitors were used to determine the CYPs involved in TES biotransformation in equine microsomes. Single CYPs 3A89, 3A94, and 3A95, recombinantly expressed in V79 hamster lung fibroblasts, were incubated with TES and the fluorescent metabolite 7-benzyloxy-4-trifluoromethylcoumarin (BFC). The effect of ketoconazole and troleandomycin was evaluated on single CYPs. Testosterone metabolites were analyzed by HPLC and confirmed by GC/MS. In hepatocyte primary cultures, the most abundant metabolite was androstenedione (AS), whereas in liver microsomes, 6-β-hydroxytestosterone showed the largest peak. Formation of 6-β-hydroxytestosterone and 11-β-hydroxytestosterone in liver microsomes was inhibited by ketoconazole, troleandomycin, and quercetin. Equine recombinant CYP3A95 catalyzed 11-β-hydroxylation of testosterone (TES). Metabolism of BFC was significantly inhibited by ketoconazole in CYP3A95, whereas troleandomycin affected the activities of CYP3A94 and CYP3A95. Both inhibitors had no significant effect on CYP3A89. Metabolic reactions and effects of inhibitors differed between the equine CYP3A isoforms investigated. This has to be considered in future in vitro studies.

Role of NonO-histone interaction in TNFalpha-suppressed prolyl-4-hydroxylase alpha1.

Inflammation is a key process in cardiovascular diseases. The extracellular matrix (ECM) of the vasculature is a major target of inflammatory cytokines, and TNFalpha regulates ECM metabolism by affecting collagen production. In this study, we have examined the pathways mediating TNFalpha-induced suppression of prolyl-4 hydroxylase alpha1 (P4Halpha1), the rate-limiting isoform of P4H responsible for procollagen hydroxylation, maturation, and organization. Using human aortic smooth muscle cells, we found that TNFalpha activated the MKK4-JNK1 pathway, which induced histone (H) 4 lysine 12 acetylation within the TNFalpha response element in the P4Halpha1 promoter. The acetylated-H4 then recruited a transcription factor, NonO, which, in turn, recruited HDACs and induced H3 lysine 9 deacetylation, thereby inhibiting transcription of the P4Halpha1 promoter. Furthermore, we found that TNFalpha oxidized DJ-1, which may be essential for the NonO-P4Halpha1 interaction because treatment with gene specific siRNA to knockout DJ-1 eliminated the TNFalpha-induced NonO-P4Halpha1 interaction and its suppression. Our findings may be relevant to aortic aneurysm and dissection and the stability of the fibrous cap of atherosclerotic plaque in which collagen metabolism is important in arterial remodeling. Defining this cytokine-mediated regulatory pathway may provide novel molecular targets for therapeutic intervention in preventing plaque rupture and acute coronary occlusion.

The roles of electrostatic charge pairing in the interaction between cytochrome P-450 and NADPH-cytochrome P-450 reductase

The cytochrome P450 enzyme catalysis requires two electrons transferred from NADPH-cytochrome P450 reductase (reductase) to P450. Electrostatic charge-pairing has been proposed to be one of the major forces in the interaction between P450 and reductase. In order to obtain further insight into the molecular basis for the protein interaction, I used two methods, chemical modification and specific anti-peptide antibodies, to study the involvement and importance of charged amino acid residues. Acetylation of lysine residues of P450c and P450b by acetic anhydride dramatically inhibited the reductase-supported P450c-dependent ethoxycoumarin hydroxylation activity, but P450 activity supported by cumene hydroperoxide is relatively unchanged. The modification of lysine residues of P450c and P450b did not grossly disturb the protein conformation as revealed by several spectral studies. This differential effect of lysine modification on the P450 activity in the system reconstituted with reductase versus the system supported by cumene hydroperoxide suggested an important role for P450 lysine residues in the interaction with reductase. Using $\rm\sp{14}C$-acetic anhydride, P450 lysine residues were labelled and further identified on P450c and P450b. Those lysine residues are at position 97, 271, 279, and 407 for P450c, and 251, 384, 422, 433, and 473 for P450b. Alignment of those identified lysine residues on P450c and P450b with amino acid residues identified in other studies indicated those residues reside in three major sequence areas. Modification of arginine residues of P450b by phenylglyoxal and 2, 3-butanedione have no significant effect on P450 activity either supported by NADPH and reductase or supported by cumene hydroperoxide. Further studies using $\rm\sp{14}C$-phenylglyoxal reveals that no incorporation of phenylglyoxal into P450b was found. These results demonstrated a predominant role of lysine residues of P450 in the electrostatic interaction with reductase. To understand the protein binding sites on each of P450 and reductase, I generated three anti-peptide antibodies against regions on reductase and five anti-peptide antibodies against five putative reductase binding sites on P450c. These anti-peptide antibodies were affinity purified and characterized on ELISA and by Western blot analysis. Inhibition experiments using these antibodies demonstrated that regions 109-120 and 204-220 of reductase are probably the two major binding sites for P450. The association of reductase with cytochromes P450 and cytochrome c may rely on different mechanisms. The data from experiments using anti-peptide (P450c) antibodies supports the important role of P450c lysine residues 271/279 and 458/460 in the interaction with reductase. ^

Cytochrome P450 2D18 in the mammalian brain: Recombinant expression, purification, and characterization of xenobiotic and endobiotic metabolism

The cytochromes P450 (P450) comprise a superfamily of hemoproteins that function in concert with NADPH-cytochrome P450 reductase (P450-reductase) to metabolize both endogenous and exogenous compounds. Many pharmacological agents undergo phase I metabolism by this P450 and P450-reductase monooxygenase system. Phase I metabolism ensures that these highly hydrophobic xenobiotics are made more hydrophilic, and hence easier to extrude from the body. While the majority of phase I metabolism occurs in the liver, metabolism in extrahepatic organ-systems like the intestine, kidney, and brain can have important roles in drug metabolism and/or efficacy. ^ While P450-mediated phase I metabolism has been well studied, investigators have only recently begun to elucidate what physiological roles P450 may have. One way to approach this question is to study P450s that are highly or specifically expressed in extrahepatic tissues. In this project I have studied the role of a recently cloned P450 family member, P450 2D18, that was previously shown to be expressed in the rat brain and kidney, but not in the liver. To this end, I have used the baculovirus expression system to over-express recombinant P450 2D18 and purified the functional enzyme using nickel and hydroxylapatite chromatography. SDS-PAGE analysis indicated that the enzyme was purified to electrophoretic homogeneity and Western analysis showed cross-reactivity with rabbit anti-human P450 2D6. Carbon monoxide difference spectra indicated that the purified protein contained no denatured P450 enzyme; this allowed for further characterization of the substrates and metabolites formed by P450 2D18-mediated metabolism. ^ Because P450 2D18 is expressed in brain, we characterized the activity toward several psychoactive drugs including the antidepressants imipramine and desipramine, and the anti-psychotic drugs chlorpromazine and haloperidol. P450 2D18 preferentially catalyzed the N-demethylation of imipramine, desipramine, and chlorpromazine. This is interesting given the fact that other P450 isoforms form multiple metabolites from such compounds. This limited metabolic profile might suggest that P450 2D18 has some unique function, or perhaps a role in endobiotic metabolism. ^ Further analysis of possible endogenous substrates for P450 2D18 led to the identification of dopamine and arachidonic acid as substrates. It was shown that P450 2D18 catalyzes the oxidation of dopamine to aminochrome, and that the enzyme binds dopamine with an apparent KS value of 678 μM, a value well within reported dopamine concentration in brain dopaminergic systems. Further, it was shown that P450 2D18 binds arachidonic acid with an apparent KS value of 148 μM, and catalyzes both the ω-hydroxylation and epoxygenation of arachidonic acid to metabolites that have been shown to have vasoactive properties in brain, kidney, and heart tissues. These data provide clues for endogenous roles of P450 within the brain, and possible involvement in the pathogenesis of Parkinson's disease. ^

Möller-Barlow disease in archaeology: Preliminary study of biochemical detection

Human bone is the most direct source for reconstructing health and living conditions of ancient populations. However, many diseases remain undetected in palaeopathology. Möller-Barlow disease (scurvy) is a historically well-documented metabolic disease and must have been common in clinical and sub-clinical severity. Due to long incubation periods and the subtle nature of bone changes osteological evidence is relatively rare (Brickley & Ives 2008). Möller-Barlow disease is caused by deficiency of dietary vitamin C (ascorbic acid) and evokes symptoms like fatigue, haemorrhage, inflammations, delayed wound healing and pain. Vitamin C is a cofactor for the hydroxylation of the amino acids proline and lysine which are essential for the production of intact connective tissue by cross-linking the propeptides in collagen. In a preliminary study we tested the detectability of Möller-Barlow disease by analysis of relative quantitative variability of hydroxylated amino acids in collagen (Pendery & Koon 2013). Samples (N=9) were taken from children with (n=3, cranium, femur, tibia) and without (n=4, cranium, femur, tibia) apparent bone reactions indicative of Möller-Barlow disease, as well as from adults with lethal traumata (n=2; negative controls). The skeletal remains originated from two early medieval cemeteries from Switzerland. Gas chromatographic (GC) analysis revealed minor differences between the samples. So far children with no pathologic alterations had fairly same values as negative controls while children with bone reactions paradoxically exhibited even slightly higher values of hydroxyproline and hydroxylysine. Future research demands for larger sample size and has to discuss sampling strategies. Beside possible misdiagnosis of Möller-Barlow disease it is arguable if only the newly built bone should be analysed even though this could lead to problems related to small sample quantity. It also remains to be seen to which extent varying turnover rates of different skeletal elements, especially in children, must be taken into account.