To elucidate physiological mechanisms underlying pathological anxiety, valid animal models are essential (Cryan and Holmes 2005). Previous studies confirmed that the high anxiety-related behavior/low anxiety-related behavior (HAB/ LAB) mouse model for trait anxiety mimics many clinically relevant features typical of psychiatric patients (Krömer et al. 2005;Landgraf et al. 2007). In this model, behavioral phenotyping on the elevated-plus-maze (EPM) test was used as selection criterion for bidirectional intra-strain (CD1) breeding. Consequently, inbreeding for more than 30 generations created hyper-anxious (HAB) and hypo-anxious (LAB) mice. These animals provide robust phenotypes for extremes in anxiety-related behavior, and show high comorbidity with depression-like behavior. Inbreeding with focus on a particular trait included a strong accumulation of specific genetic material. Therefore, HAB/LAB animals serve as a potent model to analyze candidates underlying trait anxiety. One of the first protein markers shown to be differentially regulated in extremes of trait anxiety was glyoxalase1 (GLO1; lactoylglutathione lyase, EC 4.4.1.5).Mouse GLO1 is an enzyme of 21kDa, containing 184 amino acids that is found as a dimer in the cytosol of cells. Its physiological function is the catalytic conversion of Abbreviations used: AGE, advanced glycation end; ARMS, amplification refractory mutation system; CNV, copy number variant; enpp5, ectonucleotide pyrophosphatase/phosphodiesterase 5; EPM, elevatedplus-maze; GC-MS, gas chromatography-mass spectrometry; GLO1, glyoxalase1; HAB, high anxiety-related behavior; i.c.v., intracerebroventricular; LAB, low anxiety-related behavior; LTP, long-term potentiation; MG, methylglyoxal; NAB, normal anxiety-related behavior; TST, tail suspension test; WB, western blotting. AbstractMethylglyoxal (MG) is a highly reactive metabolite that forms adducts with basic amino acid side chains in proteins. MG is degraded by glyoxalase1 (GLO1), an enzyme shown to be differentially expressed in several mouse models of anxietyrelated behavior. As yet, molecular mechanisms by which altered GLO1 expression influences emotionality have not been elucidated. Here we report that both MG concentration and protein modification are altered in brain tissue of a mouse model for trait anxiety, with elevated levels in low anxiety-related behavior relative to high anxiety-related behavior animals. Accordingly, repeated intracerebroventricular injections of MG mediated anxiolysis in inbred high anxiety-related behavior and outbred CD1 mice. We found that anxiolytic-like properties of MG were independent of GLO1 expression. In contrast, antidepressant-like properties of intracerebroventricular MG were suppressed in CD1 mice carrying extra copies of the GLO1 gene. Moreover, MG treatment increased expression of GLO1 only in CD1 mice that did not have extra copies of GLO1. Taken together, these results suggest that the MG levels in brain are negatively correlated with anxiety. Thereby, we identified a novel molecular mec...

Opiates such as hydrocodone, hydromorphone, oxycodone, noroxycodone, and oxymorphone reportedly may interfere with the analysis of morphine and codeine. The analysis of these compounds themselves also is an important issue. Thus, double derivatization approaches utilizing methoxyamine and hydroxylamine to first form oxime products with keto-opiates, followed by the derivatization with trimethylsilyl (TMS) or propionyl groups, have been developed for the simultaneous analysis of these compounds. However, these studies have not included all compounds of interest and resulted in inadequate chromatographic resolution or significant intensity cross-contribution between the ions designating the analyte and its deuterated internal standard for certain compounds. By exploring three-step derivatization approaches with the combination of various derivatization groups and orders, this study concluded that application of methoxyimino/propionyl/TMS groups, in the order listed, facilitated the simultaneous analysis of eight opiates (morphine, 6-acetylmorphine, hydromorphone, oxymorphone, codeine, hydrocodone, oxycodone and noroxycodone) in urine samples, achieving satisfactory limits of quantitation and detection. In addition, the adapted approach resulted in two usable products for morphine and codeine providing alternatives, should interferences render any of these products non-usable.

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Methadone (MTD) is widely used for detoxification of heroin addicts and also in pain management programs. Information about the distribution of methadone between blood, plasma, and alternative specimens, such as oral fluid (OF), is needed in clinical, forensic, and traffic medicine when analytical results are interpreted. We determined MTD and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in blood, plasma, blood cells, and OF by gas chromatography-mass spectrometry (GC-MS) after adding deuterium-labeled internal standards. The analytical limits of quantitation for MTD and EDDP by this method were 20 and 3 ng/mL, respectively. The amounts of MTD and EDDP were higher in plasma (80.4 % and 76.5 %) compared with blood cells (19.6 % and 23.5 %) and we found that repeated washing of blood cells with phosphate-buffered saline increased the amounts in plasma (93.6 % and 88.6 %). Mean plasma/blood concentration ratios of MTD and EDDP in spiked samples (N = 5) were 1.27 and 1.21, respectively. In clinical samples from patients (N = 46), the concentrations of MTD in plasma and whole blood were highly correlated (r = 0.92, p < 0.001) and mean (median) plasma/blood distribution ratios were 1.43 (1.41). The correlations between MTD in OF and plasma (r = 0.46) and OF and blood (r = 0.52) were also statistically significant (p < 0.001) and the mean OF/plasma and OF/blood distribution ratios were 0.55 and 0.77, respectively. The MTD concentration in OF decreased as salivary pH increased (more basic). These results will prove useful in clinical and forensic medicine when MTD concentrations in alternative specimens are compared and contrasted.

In gas chromatography-mass spectrometry methods of analysis adopting the analyte's isotopic analog as the internal standard (IS), the cross-contribution (CC) phenomenon-contribution of IS to the intensities of the ions designating the analyte, and vice versa-has been demonstrated to affect the quantitation data. A novel approach based on the deviations of the empirically observed concentrations of a set of standards was developed to assess the accuracy of the empirically derived CC data. This approach demonstrated that normalization of ion intensities derived from the analyte and the IS generates reliable CC data. It further demonstrated that an ion-pair (designating the analyte and the IS) with ϳ5% or higher CC will result in a very limited linear calibration range. (J