Microassay for N-acetyltransferase activity using high-performance liquid chromatography with electrochemical detection

A rapid, sensitive procedure has been developed for determination of N-acetyltransferase activity against octopamine, dopamine and 5-hydroxytryptamine. The assay, which is performed in a volume of 10 microliters, is based upon the separation and detection of monoamine substrates and their N-acetylated derivatives using high-performance liquid chromatography with electrochemical detection. The method has been used to measure N-acetyltransferase activity against octopamine, dopamine and 5-hydroxytryptamine in the cerebral ganglion of the American cockroach, Periplaneta americana and to study bi-substrate kinetics of the enzyme.     

Analysis of lipoprotein lipase activity using high-performance liquid chromatography

Lipoprotein lipase (LPL) is a key enzyme which regulates the plasma triglyceride concentration by hydrolyzing triglycerides in chylomicrons and very-low-density lipoprotein (VLDL). The activity of LPL was conventionally analyzed using radio-labeled residues or direct sandwich-ELISA. An assay for lipoprotein lipase activity which used a nonradioactive substrate, tri-olein, is described. In this method, LPL activity was detected fluorometrically by reacting 9-anthryldiazomethane (ADAM) with the oleic acid generated from tri-olein by enzyme activity and separated by reversed-phase HPLC. This method has been optimized and the optimum enzyme incubation time and reaction time of the generated oleic acid with ADAM were both at 20 min. The method correlated well with the conventional method.     

Determination of beta-galactosidase activity in the intestinal tract of mice by ion-exchange high-performance liquid chromatography using epsilon-N-1-(1-deoxylactulosyl)-L-lysine as substrate

epsilon-N-1-(1-Deoxylactulosyl)-L-lysine was synthesized and used as a substrate to assay beta-galactosidase activity. epsilon-N-1-(1-Deoxylactulosyl)-L-lysine and its degradation product epsilon-N-1-(1-deoxyfructosyl)-L-lysine were detected by high-voltage paper electrophoresis and ion-exchange high-performance liquid chromatography. The beta-galactosidase activity in different parts of the intestinal tract of germ-free and control mice was determined and compared with a beta-galactosidase activity which degrades lactose at pH 8.5 and 5.0 and which corresponded with bacterial and host enzymatic activities, respectively.     

Microassay for the simultaneous determination of cocaine, norcocaine, benzoylecgonine and benzoylnorecgonine by high-performance liquid chromatography

An improved method for the simultaneous determination of cocaine, norcocaine, benzoylecgonine and benzoylnorecgonine using reversed-phase high-performance liquid chromatography with ultraviolet detection is described. Following solid-phase extraction, chromatography was performed using a column containing an octadecylsilica-coated packing, eluted with 6% acetonitrile in phosphate buffer, pH 2.1, and detected at 233 nm. Using 80-microliters samples, the detection limit is 18 ng/ml for benzoylecgonine and benzoylenorecgonine and 35 ng/ml for cocaine and norcocaine. The coefficients of variation range from 3.5% (benzoylecgonine) to 7.0% (norcocaine). The procedure has been applied to samples of guinea pig plasma, urine and amniotic fluid and human urine.     

Detection of Nepsilon-monomethyllysine using high-performance liquid chromatography and high-performance liquid chromatography-mass spectrometry

Nepsilon-Monomethyllysine was identified in the serum, urine, brain, and liver samples of rats treated per os with L-deprenyl. The identification procedure included reaction with Fmoc chloride, clean-up, and analysis using HPLC-UV-MS. Oral administration of (-)-N-14C-methyl-N-propynyl(2-phenyl-1-methyl)ethylammonium hydrochloride L-deprenyl) to rats resulted in transfer of the radiolabelled methyl group to the Nepsilon-amino group of the endogenous lysine. The radiolabelled Nepsilon-monomethyllysine was urinary eliminated together with the other radiolabelled deprenyl metabolites, such as deprenyl-N-oxide and methamphetamine. The presence of Nepsilon-monomethyllysine has also been traced, and its concentrations were compared in the serum, liver and brain of rats subjected to L-deprenyl treatment. Methyl group transfer from the L-deprenyl to endogenous compounds; and the urinary elimination of their products may offer a vital way to eliminate or to decrease the degree of drug transmethylation to the lysine constituents of blood vessels' proteins.     

Highly sensitive assay for PZ-peptidase activity by high-performance liquid chromatography

A rapid and highly sensitive assay method for measuring PZ-peptidase activity in newborn rat brain is described. The method is based on monitoring the absorption at 320 nm of PZ-Pro-Leu enzymatically formed from the substrate, PZ-L-Pro-L-Leu-Gly-L-Pro-D-Arg, after separation by high-performance liquid chromatography using a reversed-phase column. This method is sensitive enough to measure PZ-Pro-Leu at concentrations as low as 5 pmol, and is able to make the column ready for the next injection within 10 min after the preceding injection. By using this method, PZ-peptidase activity was discovered in clonal osteoblastic cells derived from newborn mouse calvaria.     

Rapid assay for tryptophanase using reversed-phase high-performance liquid chromatography.

A rapid and sensitive high-performance liquid-chromatographic assay for tryptophanase based upon the fluorometric measurement of the enzymatically liberated indole was developed. The total incubation time is 20 min, and the reversed-phase separation is fast (elution time of indole in 8 min) and reproducible. The sensitivity of the method is in the nanomole range. This method was tested in the assay of tryptophanase activity in E. coli, giving an average activity of 6589.6 U/g of cells. Because of its speed, high sensitivity and minimal sample preparation, this method circumvents several problems commonly encountered in standard spectrophotometric methods of analysis.     

Fluorimetric assay for measuring Dns-His-Lys-Arg-His-Lys cleaving enzyme using high-performance liquid chromatography

A rapid and sensitive assay for the determination of Dns-His-Lys-Arg-His-Lys cleaving enzyme activity is reported. This assay is based on fluorimetric detection of a dansylated dipeptide, 5-dimethylaminonaphthalene-1-sulfonyl-His-Lys, enzymatically formed from the substrate 5-dimethylaminonaphthalene-1-sulfonyl-His-Lys-Arg-His-d-Lys, after separation by high-performance liquid chromatography (HPLC) using a C-18 reversed-phase column by isocratic elution. This assay is sensitive enough to measure 5-dimethylaminonaphthalene-1-sulfonyl-His-Lys at concentrations as low as 7 pmol, and yields highly reproducible results and requires less than 9.0 min per sample for separation and quantitation. The optimum pH for Dns-His-Lys-Arg-His-Lys cleaving enzyme activity was 7.5-8.0. The Michaelis constant (K_m) and the maximum velocity (V_max) values were 33.3 μM and 47.07 pmol/(μg h), respectively with the use of enzyme extract obtained from bovine pituitary. By using this assay, axonal transport of this enzyme activity was observed 48 h after double ligations of rat sciatic nerves. The high sensitivity and selectivity of this assay would be useful for clarification of the physiological role of this enzyme.     

Determination of plasma lactic acid concentration and specific activity using high-performance liquid chromatography.

Assessment of lactate metabolism is of particular interest during exercise and in disease states such as diabetes, shock, and absorptive abnormalities of short-chain fatty acids by the colon. We describe an analytical method that introduces radio-active tracers and high-performance liquid chromatography (HPLC) to simultaneously analyze concentrations and specific activities (SAs) of plasma lactate. The HPLC conditions included separation on a reversed-phase column (octadecylsilane) and an isocratic buffer (30% acetonitrile in water). [3H]Acetate served as an internal standard. Lactate and acetate were extracted from plasma samples with diethyl ether following a pH adjustment to less than 1.0 and back-extracted into a hydrophilic phase with sodium carbonate (2 mM, pH greater than 10.0). Lactate is detected in the ultraviolet range (242 and 320 nm) by derivatization with alpha-bromoacetophenone. Control plasma samples were studied after an overnight fast for precision and analytical recovery. Calibration curves were linear in the range 0.18-6.0 mM (r = 0.92). The precision was 3% and the analytical recovery was 87%. The detection limit of the method was 36 pmol. Determination of lactate metabolism was performed in a patient with chronic congestive heart failure who was administered primed-continuous L-[U-14C]lactate (10 microCi bolus and 0.3 microCi/min continuously) during a 60-min rest period. Mean arterial lactate concentration and SA were 1.69 +/- 0.2 mM and 253.8 +/- 22 dpm/mumol, respectively. Systemic lactate turnover was 25.65 mumol/kg per min. Lactic acid systemic turnover, organ uptake and release rates can be accurately determined by isocratic HPLC.     

Feasibility of enhancing high-performance liquid chromatography using microwave radiation

High-performance liquid chromatography (HPLC) is the most versatile of the chromatographic techniques because it is applicable to a wide variety of analytes. Unfortunately, liquid mobile phases have relatively low diffusivities. A novel approach is presented for increasing the diffusivity of a liquid mobile phase. The method advocated in this study is the first process that has been described by which it is possible to increase the diffusivity of a liquid while having essentially no effect on other physical properties of the liquid, such as the temperature or solvating power. It is demonstrated that it is possible to sharpen peaks, in an HPLC separation, by the application of microwave radiation.