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C-S lyase enzymes catalyse the generation of mutagenic and/or cytotoxic thiols from cysteine conjugated xenobiotics. These cysteine conjugates are produced subsequent to glutathione conjugations as a metabolic step in the mercapturic acid pathway, traditionally thought of as a pathway solely associated with detoxification. Human Chang liver (HCL) cells were challenged with a range of cysteine conjugates demonstrated to be substrates for human hepatic C-S lyases. The cellular toxicity of these compounds was determined and it was observed that the rank order of substrate toxicity obtained for the HCL cells followed the rank order of C-S lyase activity of the substrates in a freshly isolated mitochondrial fraction of human tissue. The presence of C-S lyase activity was also established in this cell line.

A new, simple and rapid extraction procedure coupled with a combined coulometric-fluorescence HPLC assay is described for the simultaneous determination of morphine (M) and morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G), and normorphine (NM) in plasma. The effect of concentration and pH of selected ion-pairing agents on the extraction of these compounds from plasma by solid-phase extraction was investigated. The extraction procedure was optimized in terms of recovery, reproducibility and lack of interference from endogenous materials. The optimized method uses tetrabutylammonium hydrogen sulphate (TBAHS) at pH 10 followed by separation on a single C18 solid-phase extraction cartridge. For routine analysis the procedure provides high and reproducible recoveries over a concentration range of 1.0-1000 ng ml-1 for morphine, M6G and normorphine and 20-1000 ng ml-1 for M3G. The method was used successfully to analyse plasma samples from a pharmacokinetic study in which sheep had received an intravenous dose of 0.015 mg kg-1 of M6G.

Objective To examine the functional and skeletal effects of 30 months of steroid treatment in boys with Duchenne muscular dystrophy. Study design Lumbar spine (L 2 L 4 ) and subcranial dual energy X-ray absorptiometry scanning was performed on 25 boys (mean age 7.4 years) at baseline and after 30 months of steroid treatment. Results At baseline, L 2 L 4 bone mineral content (BMC) was significantly low for projected bone area although appropriate for reduced lean body mass (LBM). Subcranial bone area for height and subcranial BMC for area and LBM were all significantly reduced. After 30 months of steroid therapy there was a significant increase in subcranial bone area for height but a significant reduction of subcranial BMC for area. At the lumbar spine there were no significant changes in bone area but small increases in L 2 L 4 BMC both for bone area and LBM. Conclusion At baseline reduced mechanical load from diminished muscle function results in narrow light bones more noticeable in the subcranial region than the lumbar spine. Increases observed in subcranial bone area at 30 months suggest a gradual adaptation to increased gravitational load whereas at the spine there were no apparent detrimental effects on bone after 30 months of steroid therapy.

The C-S lyase enzymes are responsible for the generation of mutagenic and cytotoxic metabolites via aberrant drug-metabolising pathways in mammalian tissues. We have examined human hepatic cytosolic, mitochondrial and microsomal fractions for evidence of C-S lyase activity. The cytosolic enzyme was purified using fast protein liquid chromatography over FFQ Sepharose, Mono P and Superose 12. An homogeneous protein (monitored by SDS-PAGE) was obtained following purification, and an 11-fold increase in C-S lyase specific activity was observed. The molecular weight of the enzyme was found to be 37 kDa in denaturing conditions, 82.3 kDa in non-denaturing conditions, and the C-S lyase activity was shown to co-purify with kynurenine aminotransferase activity when the transaminase activity of the enzyme was examined with kynurenine as the substrate.

The enzyme cysteine conjugate /.I-lyase (C-S lyase, EC 4.4.1.13) from rat renal tissue has been identified as a transaminase: glutamine transaminase K (GTK) [l]. This is one member of a family of enzymes which effect a transamination reaction on amino acid substrates. Other members of this family include glutamine transaminase L (GTL, isolated from rat liver, but which has a structure-activity profile broadly overlapping that of GTK), kynurenine aminotransferase (KAT), and aspartate aminotransferase. Rat hepatic cytosolic C-S lyase co-purifies as kynureninase, an enzyme which is involved in anthranilic acid biosynthesis [2], but antibodies raised against this purified protein did not crossreact with the renal cytosolic enzyme (i.e. with GTK). In one study with human hepatic cytosolic C-S lyase [3], kynurenine was not a substrate for the enzyme. These workers did not establish whether the C-S lyase enzyme had a physiological role, and if it co-purified with a protein which had been previously characterized. We have recently established that human hepatic cytosolic C-S lyase co-purifies as an aminotransferase: KAT [4]. In this article, we discuss the structureactivity relationships displayed by human C-S lyases obtained from both the cytosolic and mitochondrial sub-cellular fractions of kidney, liver and lung tissue. Elsewhere

Children with cerebral palsy are recognised to be at risk of osteopenia but it's extent and aetiology are unclear. Nine children aged 2 to 13 years with cerebral palsy who were non-ambulant had measurements of bone mineral density of the lumbar spine. Assessments of 25 OH vitamin D status, parathyroid hormone activity and urinary calcium excretion were also performed. All had evidence of severe osteopenia with bone mineral density standard deviation scores ranging from −1.7 to −4.2. Three, children had gross hypercalciuria. Parathyroid hormone activity was not increased. Although 25 OH vitamin D2 levels were universally low, all but one had adequate 25 OH vitamin D3 levels. Three children with recurrent long bone fractures who have been treated over a 12 to 18 month period with bisphosphonate drugs have shown a marked increase in bone mineral density ranging from 16 to 40%. It appears that osteopenia in severe cerebral palsy is common but there is no consistent abnormality of vitamin D or parathyroid hormone status. Bisphosphonates may well be a useful therapeutic option in such children with recurrent fractures.

A simple model simulating the kinetics of drug metabolism inhibition interaction is investigated. The relative sensitivity of drug and metabolite concentration-time profiles as indices of inhibition is assessed. Under steady-state conditions where inhibitor concentrations are maintained constant, the determination of metabolite in addition to drug kinetics provides little additional information when inhibition is nonselective in nature. However metabolite profiles do offer increased sensitivity when parallel routes of metabolism exist and there is selectivity of inhibitory action. Non-steady-state conditions are also investigated as they often apply in inhibition studies; the inhibitor is often administered as a single dose or as a multiple dosing regimen rather than by a constant rate intravenous infusion. Under the former conditions, when inhibitor concentrations in the body fluctuate during the study, metabolite kinetics can be more useful than drug kinetics. The changes evident in the metabolite concentration-time profiles are substantial due to both the inhibition per se and the nonlinearity in the system arising from inhibitor elimination. It is concluded that metabolite kinetics provide a useful aid in the detection of drug metabolism inhibition interactions.

To explore a pharmacy school curriculum for opportunities for student engagement and to determine how these might shape student identity as pharmacists.The learning aims and objectives and methods of assessment from the curriculum of a bachelor of pharmacy (BPharm) program were collected and a concept map was generated. The concept map was interpreted using Barnett and Coates' curricular domains of knowing, acting and being.The key concepts within the intended curriculum that were identified from the concept map were drugs, pharmacy, understanding, practice, and skills. Concepts such as patient and consumer, which would indicate a patient-centered approach to the curriculum, were limited. The main form of assessment used in the curriculum was multiple-choice and short-answer examinations.There was an emphasis in the curriculum on student acquisition of knowledge and this was reinforced by the use of theoretical examinations. The content of the curriculum was drug-centered rather than patient-centered and the emergence of students' identity as pharmacists may be fragmented as a result.

Capillary electrochromatography (CEC) has proved to be applicable to the separation of neutral and acidic (chromatographed in their ion-suppressed mode) compounds but a major factor holding back its further development has been its perceived inability to analyse basic compounds. In this paper we demonstrate that the addition of a competing base, such as triethylamine (TEA) or triethanolamine (TEOA), to a low pH buffer can achieve excellent CEC analysis of a range of strong pharmaceutical bases. Acceptable peak symmetry and efficiencies of up to 510000 plates m–1 were obtained for bases such as procainamide, nortriptyline and diphenhydramine on CEC Hypersil C18, C8 and phenyl reversed-phase packing materials. The effect of TEA or TEOA concentration and mobile phase pH on the separation behaviour of the bases is discussed. In addition, the paper reports the first example of the simultaneous CEC analysis of acids, bases and neutral compounds by using a mobile phase containing TEOA phosphate at pH 2.5.

In the process of a liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay development for two antimicrobial drugs using their stable isotope-labeled (SIL) analogues as internal standards to correct for matrix–ion suppression effects, an unusually large scatter of data was observed. A systematic observation revealed that the analytes and their SIL analogues (internal standards) were not completely coeluted; therefore, the analytes were not experiencing matrix effects similarly. When a column with lower resolution ability was used to achieve complete overlapping of the analyte and internal standard peaks, the scatter of LC–MS/MS data was minimized, indicating that the maximum correction of matrix effects by internal standards occurs when they are completely coeluted with the analytes. This study highlights the importance of ensuring complete overlapping of analyte and internal standard peaks in eliminating matrix effects when using SIL analogues as internal standards in LC–MS.