Direct monitoring of enzyme reactions using micellar electrokinetic capillary chromatography. Optimisation of drug glucuronide and sulfate conjugate hydrolysis.

This paper demonstrates the use of micellar electrokinetic capillary chromatography (MECC) to monitor enzyme reaction conditions. The hydrolysis reactions of model conjugated substrates (morphine and reduced flunixin glucuronides, napthyl sulfate), by proprietary beta-glucuronidase preparations, were studied under varied experimental conditions. Reactions were carried out in autosampler vials with incubation in a thermostatted CE autosampler tray. MECC was performed using borax buffer (17.5 mM, pH 9.3) modified with sodium dodecyl sulfate (70 mM). Repetitive injections were made from the sample vial throughout the course of the reactions at a frequency of up to 10 h-1. MECC provided a rapid and reproducible assay for the model substrates. Baseline interference from the enzymes prevented measurement of product increase, therefore substrate decrease was measured from the peak areas. Monitoring of reactions in this way has proved valuable in the optimisation of hydrolysis conditions used in sample preparation for drug analysis. beta-Glucuronidase preparations from Helix pomatia were found to give the best performance of those evaluated in terms of deconjugation efficiency.

Analysis of corticosteroids in biofluids by capillary electrochromatography with gradient elution.

Capillary electrochromatography (CEC) with gradient elution was used to separate mixtures of corticosteroids (adrenosterone, hydrocortisone, dexamethasone, fluocortolone) in extracts of equine urine and plasma. Urine samples were first purified using solid phase extraction. Two purification steps were necessary to prevent contamination of the CEC column. Plasma was purified using automated dialysis. A laboratory-built CEC interface, connected to a gradient HPLC system, delivered samples and mobile phase to the CEC column. CEC was performed in fused-silica capillaries of 50 µm i.d., 24 cm total length, and 16 cm effective length packed with Apex ODS, 3 µm particle size. The mobile phase was ammonium acetate (5 mM) in water/acetonitrile. Acetonitrile in the mobile phase was varied from 9 to 80% (v/v) using the gradient HPLC system. Detection was by UV absorbance at 240 nm. Samples, 10-250 µL, were injected into the mobile phase stream and loaded onto the CEC column under an applied field of 1.04 kV cm(-1) and a CEC column head pressure of 12 bar. Mobile phase flow rate through the sampling interface was 100 µL min(-1). The system was reproducible and could be left in unattended operation for long periods. After injection of 200 urine extracts, a broadening of peaks was observed but the CEC column was still serviceable.

Determination of phase II drug metabolites in equine urine by micellar electrokinetic capillary chromatography.

Micellar electrokinetic capillary chromatography (MECC) using diode array detection has been investigated for the determination of phase I and phase II metabolites of drugs in biofluids. Methods were optimised for the determination of morphine, morphine-3-glucuronide, morphine-6-glucuronide, normorphine, meclofenamic acid and its metabolites in equine urine. Solid-phase extraction procedure were developed to concentrate and purify the analytes from spiked and post administration urines for MECC analysis. A simple on-line procedure for monitoring the kinetics of hydrolysis of morphine-glucuronide conjugates by beta-glucuronidase was demonstrated.

Antigenic analysis of the major human phosphoglucomutase isozymes: PGM1, PGM2, PGM3 and PGM4.

The cross-reactivity of human phosphoglucomutase isozymes (PGM1, PGM2, PGM3 and PGM4) has been investigated using anti-rabbit muscle PGM polyclonal antibodies. Significant differences were revealed: an IgG fraction of the antiserum reacted with the primary and secondary PGM1 isozymes of all the common phenotypes. However, there was no reaction with the PGM2 or PGM3 isozymes; thus these latter isozymes share no major antigenic determinants with human or rabbit PGM1 and are therefore structurally distinct. In contrast, the PGM isozymes of human milk attributed to a fourth locus, PGM4, showed similar cross-reactivity as PGM1 suggesting close structural similarity. The IgG was also employed as a reagent to remove PGM1 from haemolysates so as to allow the unambiguous assessment of the PGM2 isozyme patterns by isoelectric focusing. However, no proven genetic variation was encountered in a sample of 32 individuals.

An evaluation of the polymerase chain reaction method for forensic applications.

This paper describes the use of polymerase chain reaction (PCR) for amplifying small amounts of DNA obtained from samples of interest to the forensic scientist. A region of the HLA DQalpha (DQa) locus was amplified in DNA prepared from the following: hair roots, liquid blood, blood-stains, semen and vaginal swabs (semen free and semen contaminated). A population study was conducted using DNA from 78 unrelated individuals. The observed distribution of HLA DQa alleles varied from that reported for an American population but obeyed the Hardy-Weinberg equilibrium. Interpretation problems associated with the PCR technique are discussed.

The collaborative study on typing group-specific component in casework bloodstains.

Casework bloodstains were typed for group-specific component (GC) at eight forensic laboratories. Approximately 600 bloodstains were examined of which a mean of 62.7% gave results. This is comparable to other blood grouping systems in current use. Stains that were over three-months old were successfully typed in six of the laboratories. A wide variety of substrates was examined; these included many items of clothing as well as metal blades, concrete, paint, cement, glass and grass. Of substrates that were examined several times, none consistently gave problems with GC typing. The GC system has been shown, therefore, to be an effective test in operational forensic science.

The collaborative study on typing group-specific component by eight forensic science laboratories.

This report describes a collaborative study on typing group-specific component (GC), conducted between the Central Research and Support Establishment and the forensic science laboratories of England, Wales and Northern Ireland. A population study (n=114) was performed. Fifty blood donors were selected to provide a distribution, slightly biased from normal, in favour of the GC 1F-1F and GC 1F-1S phenotypes. A protocol was devised for preparing large bloodstains. The strongest GC bands were obtained from the edge of a stain after the blood had been treated with K+/EDTA. Each laboratory received a representative portion of the large bloodstains for GC typing. Five of the eight laboratories correctly grouped all the bloodstains. No errors directly attitributable to the system were recorded in over 800 tests, indicating that GC in bloodstains can be typed reliably using the combination of isoelectric focusing in ultrathin narrow pH interval gels followed by immunofixation and silver staining.

Immunological detection of human phosphoglucomutase (PGM 1) subtypes.

Anti-phosphoglucomutase (PGM) antibodies have been produced by immunising a sheep with a purified preparation of rabbit skeletal muscle PGM and used to devise an immunological procedure for detecting PGM isozymes after isoelectric focusing. The anti-rabbit PGM antibodies cross react with human PGM and can be used to identify the PGM1 isozymes characteristic of this polymorphism. The patterns revealed by immunodetection are exactly comparable with those obtained by isozyme staining.

The use of alpha 2HS-glycoprotein and group specific component in typing forensic blood samples for discriminative and investigative purposes.

Methods are described for phenotyping alpha 2HS-glycoprotein (A2HS) and group specific component (GC) in plasma and blood-stains. The methods have been developed to be sensitive, to provide unequivocal results and to give maximal information from minimal amounts of sample. In attempting to fulfill the last criterion, a new method is described for the simultaneous typing of alpha 2 HS-glycoprotein and group-specific component from serum or plasma samples. The use of these proteins in determining the racial origin of a blood sample is also discussed.

The phenotypic frequencies of group specific component and alpha-2-HS-glycoprotein in three ethnic groups. The use of these proteins as racial markers in forensic biology.

The phenotypic frequencies of group-specific component (Gc) and alpha-2-HS-glycoprotein (A2HS) were determined in White European, Asian and Afro-Caribbean populations. Typical allele frequencies were observed for Gc, with Gc 1S being the major allele for the first two groups and Gc 1F being the major allele for Afro-Caribbeans. For all groups the dominant A2HS allele was A2HS 1, although Asians had a significantly higher proportion of this allele than the White Europeans. Gc and A2HS either singly or in combination with other blood grouping systems provide good discriminating potential. The A2HS 10 allele was detected with a very low frequency in the White European group (A2HS 10 = 0.0013) and was not detected in the Asian group, while the Afro-Caribbean group had a relatively high frequency of this allele (A2HS 10 = 0.0966). The different distribution of the Gc 1F and A2HS 10 alleles in White Europeans and Asians compared with Afro-Caribbeans, can be used to determine the likelihood of blood coming from an Afro-Caribbean.

Group-specific component: a review of the isoelectric focusing methods and auxiliary methods available for the separation of its phenotypes.

This review compares the major isoelectric focusing methods that have been published for the separation of group-specific component (Gc) phenotypes since 1978. The various parameters of gel composition, size, electrical and running conditions and sample application points are listed. More current auxiliary methods are also listed. These relate to the extraction of Gc from bloodstains and its identification after isoelectric focusing. Protocols are then recommended for the forensic analysis of Gc phenotypes.

A comparison of the sensitivity of typing group-specific component (Gc) and the phosphoglucomutase (PGM1) locus in control and casework bloodstains by three isoelectric focusing methods.

The performance of typing group-specific component (Gc) in bloodstains by two isoelectric focusing methods followed by its detection with silver staining has been compared with an established forensic system of typing phosphoglucomutase (PGM1) locus phenotypes by isoelectric focusing (IEF) in 1 mm gels. For Gc typing ultra-thin isoelectric focusing (UTIEF) gels and immobilized pH gradient (IPG) gels were used. Both laboratory prepared stains and casework stains were examined. The Gc UTIEF method is approximately eight times more sensitive than the existing PGM1 1 mm IEF method for control and casework stains. However, on average, a larger amount of stain was taken from casework stains than control stains for each typing system. A total of 53 casework stains were examined. Comparable success rates of 62% and 64% were obtained for typing Gc on UTIEF gels and PGM1 by 1 mm IEF, respectively. A success rate of 55% was obtained for typing Gc on IPGs. Bloodstains that were over 200 days old were successfully grouped by all three methods.

Role of myosin light chain kinase in muscle contraction.

In resting striated muscles of the rabbit muscle in vivo, the phosphorylatable light chain is partially phosphorylated. Tetanic stimulation increased the level of phosphorylation more rapidly in fast twitch than in slow twitch muscle. In both types of muscle the rate of dephosphorylation was relatively slow. In rabbit fast twitch muscles, phosphorylation levels persisted significantly above the resting value for some time after posttetanic potentiation had disappeared. The role of myosin light chain kinase in modulating contractile response in striated muscle is uncertain. In vertebrate smooth muscle the role of myosin phosphorylation appears to be different from that in striated muscle despite the general similarity of the actomyosin system in both tissues. Although phosphorylation in vitro increases the Mg2+ -ATPase of actomyosin, a number of features imply that a somewhat complex relationship exists between the level of phosphorylation and the actin activation of the Mg2+ -ATPase in vertebrate smooth muscle. Contrary to many earlier reports, preparations of smooth muscle actomyosin can be obtained with Mg2+ -ATPase activities comparable to those of actomyosin from skeletal muscle. Preliminary evidence is presented that suggests that phosphorylation changes the Ca2+ sensitivity of the Mg2+ -ATPase of smooth muscle actomyosin.

Phosphorylation in vivo of the P light chain of myosin in rabbit fast and slow skeletal muscles.

The P light chain of myosin is partially phosphorylated in resting slow and fast twitch skeletal muscles of the rabbit in vivo. The extent of P light-chain phosphorylation increases in both muscles on stimulation. Rabbit slow-twitch muscles contain two forms of the P light chain that migrate with the same electrophoretic mobilities as the two forms of P light chain in rabbit ventricular muscle. The rate of phosphorylation of the P light chain in slow-twitch muscle is slower than its rate of phosphorylation in fast-twitch muscles during tetanus. The rate of P light-chain dephosphorylation is slow after tetanic contraction of fast-twitch muscles in vivo. The time course of dephosphorylation does not correlate with the decline of post-tetanic potentiation of peak twitch tension in rabbit fast-twitch muscles. The frequency of stimulation is an important factor in determining the extent of P light-chain phosphorylation in fast- and slow-twitch muscles.

The effect of adrenaline on the phosphorylation of the P light chain of myosin and troponin I in the perfused rabbit heart.

1. Two-dimensional electrophoresis has been used to study the extent of phosphorylation of the P light chain of myosin and troponin I in the rabbit beating heart. 2. A procedure has been developed that eliminates endogenous protein phosphatase activity during homogenization and sample preparation for electrophoresis. 3. Evidence has been obtained for two unphosphorylated forms of the P light chain in myosin from the ventricle of the rabbit, guinea pig and cow. 4. In vivo and in the rabbit perfused beating heart about 25% of the P light-chain fraction is in the phosphorylated form. 5. Intervention with adrenaline produced a slight increase in the extent of phosphorylation that reached a maximum after the peak in inotropic response. A similar increase was obtained with ischaemia in the absence of adrenaline. 6. The changes in phosphorylation of the major forms of troponin I identified by electrophoresis occurred after the peak of response to adrenaline and were compatible with previous results.