Analysis of anabolic steroids in hair by GC/MS/MS.

A simple and sensitive gas chromatography/tandem mass spectrometry (GC/MS/MS) method is described for the detection of anabolic steroids, usually found in keratin matrix at very low concentrations. Hair samples from seven athletes who spontaneously reported their abuse of anabolic steroids, and in a single case cocaine, were analyzed for methyltestosterone, nandrolone, boldenone, fluoxymesterolone, cocaine and its metabolite benzoylecgonine. Anabolic steroids were determinate by digestion of hair samples in 1 m NaOH for 15 min at 95 degrees C. After cooling, samples were purificated by solid-phase and liquid-liquid extraction, then anabolic steroids were converted to their trimethylsilyl derivative and finally analyzed by GC/MS/MS. For detection of cocaine and benzoylecgonine, hair samples were extracted with methanol in an ultrasonic bath for 2 h at 56 degrees C then overnight in a thermostatic bath at the same temperature. After the incubation, methanol was evaporated to dryness, and benzoylecgonine was converted to its trimethylsilyl derivative prior of GC/MS/MS analysis. Results obtained are in agreement with the athletes' reports, confirming that hair is a valid biological matrix to establish long-term intake of drugs.

Effects of light physical exercise on sleep in middle-aged rats.

The effects of physical activity on sleep were evaluated in 12-month-old rats. The animals (n = 18) were induced to walk or run for 45 min in a rota-rod treadmill while control mates remained in their home cages. Immediately after the trial, they were left free to sleep for four hours, during which their electroencephalographic activity was recorded. Baseline electroencephalogram showed no differences among groups in sleep parameters and spike wave discharges during wakefulness in all rats. Sleep variables and spike wave discharges remained constant in the controls over times. On the contrary, Student's t-test for paired data indicated a decrease in spike wave discharges in both walking and running rats while paradoxical sleep rose parallel with slow wave sleep in walking animals but declined in running rats, in spite of an increment in slow wave sleep. The results seem to indicate that: i) light exercise improves sleep quality in middle aged rats, provided it is not stressful and ii) physical activity supplies important benefits to waking brain by reducing spike wave discharges.

Absinthe: enjoying a new popularity among young people?

Absinthe, an alcoholic drink used in certain artistic circles and considered the inspiring muse of many famous artists because it was reputed to stimulate creativity and possess exciting, aphrodisiacal and healing properties, in the past enjoyed enormous popularity so much so that it led to a real collective abuse so causing its prohibition in many countries, is again enjoying a new period of popularity. Also in Italy there is increasing information about the use and abuse of this drink. We received a request to analyse and determine the nature of two samples of alcoholic drinks, obtained by macerating Artemisia absinthium leaves in ethanol. Analyses of extracts by gas chromatography/mass spectrometry (GC/MS) identified beta-thujone, which is responsible for the activity and toxic effects on the CNS of absinthe, in both alcohol samples.

Physical exercise intensity can be related to plasma glutathione levels.

The aim of the present study was to examine the effect of different kinds of physical exercise on plasma glutathione levels. Male Wistar rats were randomly divided into four groups: In walking group (W; n=6), rats were trained to walk 0.8 m/min for 45 min; slow running group (SR; n=6) were trained to run 4 m/min for 45 min; fast running group (FR; n=6) ran 8m/min for 60 min and control rats (C; n=6) remained in their home cages. All animals were sacrificed after exercise and the levels of reduced glutathione (GSH) in plasma samples determined by high performance liquid chromatography (HPLC) with a fluorescent detector. Compared to controls, exercise did not change GSH plasma levels of the W group. A tendency to decrease blood GSH was observed in plasma samples of the SR group and in the FR group, physical exercise resulted in a dramatic decrease in GSH plasma levels. These data suggest that during light physical exercise there is a low production of reactive oxygen species (ROS) with a low request for antioxidant defence such as oxidation of GSH. The dramatic decrease observed in GSH levels in FR rats would indicate the presence of oxidative stress able to modify blood antioxidant profiles. Our results suggest that GSH plays a central antioxidant role in blood during intensive physical exercise and that its modifications are closely related to exercise intensity.

Physical exercise intensity can be related to plasma glutathione levels.

The aim of the present study was to examine the effect of different kinds of physical exercise on plasma glutathione levels. Male Wistar rats were randomly divided into four groups: In walking group (W; n=6), rats were trained to walk 0.8 m/min for 45 min; slow running group (SR; n=6) were trained to run 4 m/min for 45 min; fast running group (FR; n=6) ran 8 m/min for 60 min and control rats (C; n=6) remained in their home cages. All animals were sacrificed after exercise and the levels of reduced glutathione (GSH) in plasma samples determined by high performance liquid chromatography (HPLC) with a fluorescent detector. Compared to controls, exercise did not change GSH plasma levels of the W group. A tendency to decrease blood GSH was observed in plasma samples of the SR group and in the FR group, physical exercise resulted in a dramatic decrease in GSH plasma levels. These data suggest that during light physical exercise there is a low production of reactive oxygen species (ROS) with a low request for antioxidant defence such as oxidation of GSH. The dramatic decrease observed in GSH levels in FR rats would indicate the presence of oxidative stress able to modify blood antioxidant profiles. Our results suggest that GSH plays a central antioxidant role in blood during intensive physical exercise and that its modifications are closely related to exercise intensity.

Effects of light physical exercise on sleep regulation in rats.

PURPOSE: Acute physical exercise is known to enhance slow-wave sleep (SWS) and reduce paradoxical sleep (PS) in humans. In this study, we examined the effects of moderate physical exercise on sleep in rats. METHOD: Young adult Wistar rats underwent a 4-h baseline electroencephalographic (EEG) recording session. The following day, they were induced to walk (0.8 m x min(-1)) or run (4 m x min(-1)) for 45 min in a rota-rod treadmill. Active control rats (ACR) were placed on the locked rota-rod for 45 min, whereas passive control rats (PCR) remained in their home cages. They were then left free to sleep for 4 h during which EEG activity was recorded. Rectal temperature (Tre) was monitored before and after exercise in ACR, walking and running rats (WR and RR, respectively) and at 45 min intervals in PCR. RESULTS: WR were able to walk for 45 min consecutively whereas in RR performances differed. Posttraining Tre was unchanged in ACR, PCR, and WR and resulted about 1.8 degrees C above baseline in RR. In both WR and RR after exercise i) length of SWS and PS, ii) intensity of SWS (spectral power density in 1-4 Hz range), and iii) propensity for falling asleep were enhanced. Interestingly, there was a more conspicuous increment in PS than SWS. In ACR and PCR there were no changes in sleep. CONCLUSIONS: Due to the complexity of sleep regulation, the interaction of several factors might underlie the observed increment in SWS and PS. Nevertheless, it is interesting that light physical exercise favors sleep and above all a harmonic enhancement of both sleep phases.

Beta-N-acetylhexosaminidase activity and isoenzyme profile in the kidney and urine of trained rats.

Lysosomes play an important role in the immune system functioning and are involved in different aspects of inflammatory reaction, repair processes and tissue damage at various levels. Among various effects, it is known that physical exercise influences the release of different lysosomal components. The aim of this study was to evaluate enzyme activity and isoenzymatic profile of beta-N-acetylhexosaminidase both in kidney and urine of normal and trained rats. Enzyme activity was measured by fluorimetric assay while beta-N-acetylhexosaminidase isoenzymes were separated using DEAE-cellulose chromatography. Hexosaminidase specific activity was significantly increased in urine of trained rats whereas there was no increase in the kidneys of trained rats. Indeed, no significant differences were observed in the isoenzyme profile of kidney and urine extracts from normal and trained rats. Our findings suggest the exercise-induced release of lysosomal enzymes is a functional effect and not due to disruption of lysosomal membranes.

N-acetylcysteine increases apoptosis induced by H(2)O(2) and mo-antiFas triggering in a 3DO hybridoma cell line.

N-Acetylcysteine (NAC) has been used as an antioxidant to prevent apoptosis triggered by different stimuli in different cell types. It is common opinion that cellular redox, which is largely determined by the ratio of oxidized and reduced glutathione (GSH), plays a significant role in the propensity of cells to undergo apoptosis. However, there are also contrasting opinions stating that intracellular GSH depletion or supplemented GSH alone are not sufficient to lead cells to apoptosis or conversely protect them. Unexpectedly, this study shows that NAC, even if it maintains the peculiar characteristics of an agent capable of reducing cell proliferation and increasing intracellular GSH content, increases apoptosis induced by H(2)O(2) treatment and mo-antiFas triggering in a 3DO cell line. We found that 24 h of NAC pre-treatment can shift cellular death from necrotic to apoptotic and determine an early expression of FasL in a 3DO cell line treated with H(2)O(2).

Stem cell mobilization in normal donors.

We studied peripheral blood and apheresis samples from 39 consecutive normal donors who were parents or siblings of patients who received matched or mismatched bone marrow transplants using a combination of rhG-CSF-mobilized peripheral blood stem cells (PBSCs) and bone marrow (BM). BM was harvested from donors 1-7 days before starting rhG-CSF treatment: 12 micrograms/kg/day rhG-CSF was administered by continuous s.c. infusion for 4-7 days. Peripheral blood progenitor cells were harvested by leukapheresis using an automated continuous-flow blood cell separator, beginning on day 4 of rhG/CSF, for 1-4 consecutive days. Peak peripheral blood CD34+ cell and CFU-GM levels were reached simultaneously on day 5 or 6 of rhG-CSF administration. Median peak levels were 1.65% for CD34+ cells (range 0.34%-4.7%) and 142 CFU-GM/10(5) plated cells (range 16-700). The greatest numbers of CD34+ cells and CFU-GM, expressed per liter of blood volume processed, were harvested during the second and third leukapheresis: CD34+ cells 37.77 +/- 25.48 x 10(6) and CFU-GM 3.32 +/- 2.51 x 10(6) during the second leukapheresis, and CD34+ cells 37.01 +/- 16.33 x 10(6) and CFU-GM 3.82 +/- 4.36 x 10(6) during the third. The number of CD34+ cells and CFU-GM did not correlate with the sex, age, or body weight of the donors. This study indicates that this protocol for administration of rhG-CSF mobilizes large numbers of hematopoietic progenitor cells into the peripheral blood and that bone marrow harvesting before G-CSF administration does not impair stem cell mobilization.

High-performance liquid chromatographic determination of orotic acid as its methyl derivative in human urine.

Described is a method for the determination of orotic acid as its methyl ester in human urine. The method involves the use of solid-phase extraction to isolate pyrimidines from urine and derivatization with methanol and sulfuric acid, followed by isocratic high-performance liquid chromatography on a reversed-phase C18 column with UV absorbance detection. The assay is shown to be sufficiently sensitive for use in clinical investigations where elevated orotic acid excretion is suspected.

Response variability to ischemic injury in the Mongolian gerbil: an electroencephalographic and behavioral study.

The early effects of 5 or 10 min global cerebral ischemia, sham operation and halothane anesthesia were evaluated in Mongolian gerbils by means of electroencephalography (EEG), neurological examination and passive avoidance training. The "ischemia-sensitive" gerbils (33% and 64% of the 5 and 10 min ischemic groups, respectively) died during carotid ligation or within 24 h; the "ischemia-resistant" gerbils showed variable behavioral responses. Six hours after ischemia, all of the animals presented EEG activity characterized by increased delta (1-4 Hz) activity and a decreased theta 2 (6-9 Hz) band, with a tendency to recovery at 24 h. Learning impairment was observed in 5 of the 5 min ischemic animals (83%) and in 1 sham (17%) and 1 halothane (17%) control. Fourteen days after ischemia, histologic damage was observed in 4 ischemic gerbils and 1 sham control. On the whole, this study confirms the widely variable susceptibility of gerbils to cerebral ischemia. Moreover, although the variable effects of carotid occlusion have been attributed to multiple factors involving the cerebrovascular system, our data suggest that endogenous cellular mechanisms might protect against ischemia. In view of this consideration, it would be useful to investigate the molecular causes of the variable cerebral ischemic tolerance shown by Mongolian gerbils.

Variable response of the Mongolian gerbil to unilateral carotid occlusion: magnetic resonance imaging and neuropathological characterization.

In the present investigation, we estimated both the evolution and the severity of ischemic damage following unilateral carotid occlusion (UCO) in Mongolian gerbils by using conventional magnetic resonance imaging (MRI, i.e. T2 weighted imaging) and histological techniques. Immediately after UCO, the animals showed different clinical effects. The mortality (46%) detected within the first 48h was considered an "stroke-sensitivity", the "stroke-resistant" animals showed wide variability in terms of both temporal evolution and the extent of ischemic damage. The signal hyperintensity and negative MRI observed during the first 30h after UCO did not always correlate with the cerebral damage presented after 14 days, although a close correlation was established between the T2 weighted images taken more than 30h after UCO and neuropathology: the gerbils negative to imaging showed no morphological changes, whereas an enhanced signal was always prognostic of ischemic injury. Moreover, late MRI documented ventricular dilatation. Histopathology showed that the ischemic damage differed among the stroke-resistant gerbils and was often bilateral. The present study confirms the differences in gerbil susceptibility to hemispheric infarction after permanent UCO and suggests that conventional MRI may be a useful non-invasive method for i) identifying the stroke-resistant animals prone to mature ischemic injury and ii) monitoring the evolution of therapeutic efficacy without sacrificing animals.

The HLA class I-CML association revisited taking into account the two forms of gene fusion in the Philadelphia chromosome. A multicenter study.

CML patients possess either a b3-a2 or a b2-a2 fusion between the BCR and ABL genes. Depending on the type of fusion, two different series of non-self potentially immunogenic peptides may be produced. If they are presented by HLA class I molecules and recognized by cytotoxic CD8 lymphocytes, individuals could be more susceptible or resistant to leukemic cells bearing one or the other form of fusion according to their HLA class I phenotype. To test this point, the frequencies of HLA-A and HLA-B alleles were compared between b3-a2 and the b2-a2 CML patients. In essence, no difference was found whose significance could withstand correction for multiple comparisons.

Successful engraftment of T-cell-depleted haploidentical "three-loci" incompatible transplants in leukemia patients by addition of recombinant human granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells to bone marrow inoculum.

Patients who undergo transplantation with haploidentical "three-loci" mismatched T-cell-depleted bone marrow (BM) are at high risk for graft failure. To overcome the host-versus-graft barrier, we increased the size of the graft inoculum, which has been shown to be a major factor in controlling both immune rejection and stem cell competition in murine models. Seventeen patients (mean age, 23.2 years; range, 6 to 51 years) with end-stage chemoresistant leukemia were received transplants of a combination of BM with recombinant human granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells from HLA-haploidentical "three-loci" incompatible family members. The average concentration of colony-forming unit-granulocyte-macrophage in the final inoculum was sevenfold to 10-fold greater than that found in BM alone. The sole graft-versus-host disease (GVHD) prophylaxis consisted of T-cell depletion of the graft by the soybean agglutination and E-rosetting technique. The conditioning regimen included total body irradiation in a single fraction at a fast dose rate, antithymocyte globulin, cyclophosphamide and thiotepa to provide both immunosuppression and myeloablation. One patient rejected the graft and the other 16 had early and sustained full donor-type engraftment. One patient who received a much greater quantity of T lymphocytes than any other patient died from grade IV acute GVHD. There were no other cases of GVHD > or = grade II. Nine patients died from transplant-related toxicity, 2 relapsed, and 6 patients are alive and event-free at a median follow-up of 230 days (range, 100 to 485 days). Our results show that a highly immunosuppressive and myeloablative conditioning followed by transplantation of a large number of stem cells depleted of T lymphocytes by soybean agglutination and E-rosetting technique has made transplantation of three HLA-antigen disparate grafts possible, with only rare cases of GVHD.

Sleep-wake variables and EEG power spectra in Mongolian gerbils and Wistar rats.

Using electroencephalographic methods (EEG), we have analyzed the basal sleep structure and the EEG power spectra of gerbils and rats during periods of wakefulness (W), synchronized sleep (SS) and paradoxical sleep (PS). During the 6 hr light period examined, duration of sleep was similar for rats and gerbils, but gerbils showed fewer PS episodes and a longer amount of SS episodes followed by wakefulness. In addition, SS episodes preceding PS were of longer duration in gerbils than in rats. EEG power spectral analysis indicated a higher relative output in the 1-4 Hz range in gerbils in comparison with rats. On the whole, the data indicate the existence of significant differences in the basal sleep structure and EEG power spectra of gerbils and rats. This background information might be useful in the comparison of the effects of a given experimental treatment, such as cerebral ischemia, on the EEG activity of these two animal species.