Automated LC-MS method for the fast stereoselective determination of methadone in plasma.

Methadone (MTD) is a chiral drug widely used for the treatment of opioid dependence for which a rapid analytical method for the determination of each enantiomer would be advantageous. In order to improve method sensitivity and to automate the entire analytical process, a column-switching configuration has been developed. An online extraction system coupled to a cellulose-based chiral stationary phase (CSP), namely Chiralcel OJ-R, was used and detection was performed by mass spectrometry. Fifty microl of plasma were injected into the liquid chromatography-mass spectrometry (LC-MS) system after addition of acetonitrile (ACN) containing methadone deuterated D9 (MTD-D9) (internal standard) and centrifugation. For the rapid extraction step, a large particle size support was selected. A baseline separation of MTD enantiomers was obtained in less than 12 min. Trueness and precision were evaluated with control samples at 500 ng/ml of (R,S)-methadone. Trueness values were 106.6% and 103.0% for (R)-MTD and (S)-MTD, respectively, with a coefficient of variation inferior to 2.5% for both compounds. Finally, a good concordance was found using this method for analysis of plasma samples from patients in maintenance treatment as compared to a previously described HPLC-UV method after liquid-liquid extraction.     

Immunoassays for proteins alkylated by nicotine-derived N-nitrosamines

Polyclonal antibodies recognizing the pyridyloxobutyl (POB) moiety of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were produced in rabbits immunized either with POB-bovine albumin or POB-Sepharose. The POB intermediates necessary to modify the protein were generated by alkaline (pH 9.0) treatment of the synthetic precursor 4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone. In a competitive enzyme linked immunoabsorbent assay (ELISA), 70 pmole NNK inhibited 50% of the binding of the anti-POB antibodies to POB-protein absorbed on microtiterplates. This 50% inhibition varied from 70 pmole to 200 nmole using a series of NNK analogues, depending on the integrity of the POB moiety. Immunological techniques initiated in this study detect NNK-protein conjugates or measure the quantity of POB groups liberated upon alkaline or acid treatment of NNK modified protein.     

Apomorphine disrupts the inhibition of acoustic startle induced by weak prepulses in rats

Separate experiments conducted in two different laboratories assessed the importance of the prepulse intensity in the ability of apomorphine to reduce prepulse inhibition of acoustic startle responses. Rats were presented with noise bursts alone or noise bursts 100 ms after presentation of prepulse stimuli ranging from 70 to 85 or 90 dB. Throughout testing, the background noise was maintained at 65 dB. In both laboratories, apomorphine markedly decreased the absolute magnitude of prepulse inhibition when the prepulse stimuli were no more than 10 dB above the background. With more intense prepulse stimuli, apomorphine had no significant effect on prepulse inhibition. Hence, apomorphine does not interfere with the inhibitory process which actually mediates prepulse inhibition, but appears to affect the detectability of the prepulse.     

Childhood fractures are associated with decreased bone mass gain during puberty: an early marker of persistent bone fragility?

Whether peak bone mass is low among children with fractures remains uncertain. In a cohort of 125 girls followed over 8.5 years, 42 subjects reported 58 fractures. Among those, BMC gain at multiple sites and vertebral bone size at pubertal maturity were significantly decreased. Hence, childhood fractures may be markers of low peak bone mass acquisition and persistent skeletal fragility. INTRODUCTION: Fractures in childhood may result from a deficit in bone mass accrual during rapid longitudinal growth. Whether low bone mass persists beyond this period however remains unknown. MATERIALS AND METHODS: BMC at the spine, radius, hip, and femur diaphysis was prospectively measured over 8.5 years in 125 girls using DXA. Differences in bone mass and size between girls with and without fractures were analyzed using nonparametric tests. The contribution of genetic factors was evaluated by mother-daughter correlations and that of calcium intake by Cox proportional hazard models. RESULTS: Fifty-eight fractures occurred in 42 among 125 girls (cumulative incidence, 46.4%), one-half of all fractures affecting the forearm and wrist. Girls with and without fractures had similar age, height, weight. and calcium intake at all time-points. Before and during early puberty, BMC and width of the radius diaphysis was lower in the fracture compared with no-fracture group (p < 0.05), whereas aBMD and BMAD were similar in the two groups. At pubertal maturity (Tanner's stage 5, mean age +/- SD, 16.4 +/- 0.5 years), BMC at the ultradistal radius (UD Rad.), femur trochanter, and lumbar spine (LS), and LS projected bone area were all significantly lower in girls with fractures. Throughout puberty, BMC gain at these sites was also decreased in the fracture group (LS, -8.0%, p = 0.015; UD Rad., -12.0%, p = 0.004; trochanter, -8.4%, p = 0.05 versus no fractures). BMC was highly correlated between prepuberty and pubertal maturity (R = 0.54-0.81) and between mature daughters and their mothers (R = 0.32-0.46). Calcium intake was not related to fracture risk. CONCLUSIONS: Girls with fractures have decreased bone mass gain in the axial and appendicular skeleton and reduced vertebral bone size when reaching pubertal maturity. Taken together with the evidence of tracking and heritability for BMC, these observations indicate that childhood fractures may be markers for low peak bone mass and persistent bone fragility.     

Glucocorticoids Decrease Hippocampal and Prefrontal Activation during Declarative Memory Retrieval in Young Men

Glucocorticoids (GCs, cortisol in human) are associated with impairments in declarative memory retrieval. Brain regions hypothesized to mediate these effects are the hippocampus and prefrontal cortex (PFC). Our aim was to use fMRI in localizing the effects of GCs during declarative memory retrieval. Therefore, we tested memory retrieval in 21 young healthy males in a randomized placebo-controlled crossover design. Participants encoded word lists containing neutral and emotional words 1 h prior to ingestion of 20 mg hydrocortisone. Memory retrieval was tested using an old/new recognition paradigm in a rapid event-related design. It was found that hydrocortisone decreased brain activity in both the hippocampus and PFC during successful retrieval of neutral words. These observations are consistent with previous animal and human studies suggesting that glucocorticoids modulate both hippocampal and prefrontal brain regions that are crucially involved in memory processing. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cephalometric measurements in snorers, non-snorers, and patients with sleep apnoea.

Cephalometry is often used to assess patients with sleep apnoea but whether these measurements differ from those in non-apnoeic snorers and how they are influenced by age is not clear. Cephalometric radiographs of patients with sleep apnoea were compared with those of snorers without sleep apnoea and those of non-snorers. Fifty two snorers with suspected sleep apnoea had a conventional sleep study and were divided into two groups: those with an apnoea-hypopnoea index greater than 10/h (n = 40, sleep apnoea group) and those whose apnoea-hypopnoea index was 10/h or less (n = 12, snorer group). The cephalometric measurements in these patients were compared with those of 34 non-snoring control subjects. Controls were subdivided into two groups: control group 1 included 17 subjects similar in age to the sleep apnoea and snorer groups (mean (SD) age 50.0 (10.9), 50.7 (9.4), and 50.6 (9.7) years); control group 2 included 15 young men (25.4 (2.6) years). The distance from the mandibular plane to the hyoid bone (MP-H) and the length of the soft palate were greater in the patients with sleep apnoea (28.7 (7.8) and 43.6 (5.0) mm) than in the snorers (23.7 (4.2) and 40.3 (4.9 mm). The MP-H was similar in snorers and age matched control subjects, but was significantly greater in the older than in the younger control subjects (22.1 (6.1) vs 17.0 (6.8]. The soft palate was longer in subjects who snored (both sleep apnoea patients and snorers) than in control subjects. The MP-H distance significantly correlated with age for all subjects (snorers and controls) and for the control subjects alone. This study shows that non-apnoeic snorers have cephalometric abnormalities that differ from those of patients with sleep apnoea and that cephalometric values are influenced by the subject's age.     

Sclerosed liver hemangioma mimicking malignant tumor at MR imaging: Pathologic correlation

This case report illustrates atypical magnetic resonance (MR) imaging findings in a liver hemangioma mimicking a malignant lesion—lower signal intensity than cerebrospinal fluid on T2-weighted spin-echo images and lack of early enhancement on dynamic contrast material—enhanced gradient-echo images. Pathologic analysis demonstrated nearly total replacement of the vascular cavities by dense fibrous tissue. In this rare, sclerosed form, this lesion could not be defined as a hemangioma with MR imaging.     

Quantification of oxidative DNA modifications in mitochondria

Specific repair endonucleases were used to quantify oxidativemodifications in mitochondrial DNA (mtDNA) from rat liver andfrom porcine liver and kidney by means of a relaxation assay.In rat liver mitochondria the number of modifications sensitiveto formamidopyrimidine-DNA glycosylase (FPG protein), whichinclude 8-hydroxyguanine (8-oxo-7, 8-dihydro-guanine) residues,was only 0.8±0.2 per 10⁵ base pairs (bp). Even lowervalues were observed in porcine kidney (0.5±0.3 per 10⁵bp) and liver (0.4±0.2 per 10⁵ bp). The numbers of sitesof base loss (AP sites) sensitive to T4 endonuclease V and of5,6-dihydropyrimidines sensitive to endonuclease III were lessthan 0.2 per 10⁵ bp in all cases. The data provide evidencethat the steady-state levels of oxidative mtDNA modificationsare low under physiological conditions, either because reactiveoxygen species generated in the mitochondria are instantly inactivatedor because of efficient DNA repair processes inside mitochondria.     

Stimulation of tumor growthin vitro andin vivo by suramin on the VX2 model

Suramin is an antitrypanosomal compound with confirmed efficacy against several human malignancies. It is generally assumed that its mechanism of action includes the interaction with different growth factors, unlike most of the anticancer drugs. Its anticancer activity has not been testedin vivo against squamous cell carcinoma. The purpose of this study was to assess the efficacy and toxicity of suraminin vivo andin vitro on the VX2 tumor model at therapeutic monitored plasma concentrations. We determined the pharmacokinetics of suramin in rabbits, and modelized its administration in order to obtain plasma concentrations between 150 and 300 μg/ml throughout the treatment course of 3 weeks. Under these conditions, antitumor effects of suramin were evaluatedin vivo by comparing liver tumor involvement in suramin-treated and control rabbits. Liver involvement was quantified by image analysis andin vitro effects were also determined at the same concentrations.In vivo, suramin promoted liver tumor growth significantly (p<0.05), compared to untreated controls.In vitro, suramin significantly stimulated tumor cell growth at concentrations above 200 μg/ml (p<0.01). Suramin may have stimulatory effects on tumor growth in squamous cell carcinoma at relevant plasma drug concentrations. Caution should be taken in further trials in patients with squamous cell carcinomas.