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Biomedical subjects

A Karmen

Publications and source records attributed to A Karmen.

At least 37 records · Page 2Linked to original sources
PubMed1984

High-performance liquid chromatography of amino acids in urine and cerebrospinal fluid.

Two different methods for analyzing amino acids by reversed-phase high-performance liquid chromatography (HPLC), both of which can separate D- and L- stereoisomers, have been used for studying the amino acid composition of cerebrospinal fluid (CSF) and urine. One method, by which Dns derivatives of amino acids are separated as mixed chelate complexes with Cu(II) and a single stereoisomer of a second amino acid, was used to analyze CSF. CSF contains ca. 10 mumole/l per amino acid, compared to 100 mumole/l in serum. The high sensitivity of fluorescence detection enabled complete analysis, starting with 50 microliter of fluid. The second method, which uses lower concentrations of both the copper and the second amino acid and detects amino acids by the change in absorbance of the copper complex, was used to measure the urine concentration of the lysine metabolite, pipecolic acid (piperidine-2-carboxylic acid), a secondary amino acid that is difficult to detect by the more usual detection methods. Our procedure involves passing urine through a cation-exchange column, collecting the fraction containing pipecolic acid, and chromatographing it on a reversed-phase HPLC column with a mobile phase containing L-aspartame and Cu(II). To assess the utility of the method, urine samples from a patient given loading doses of D- or L-isomers were analyzed. When either isomer was administered, both D- and L-isomers were detected, but in different proportions. Varying proportions and concentrations of both isomers were also detected in the urines of patients with hyperpipecolatemia from different metabolic abnormalities.

Amino Acid Metabolism, Inborn Errors↗
PubMed1984

Stereoselective D- and L-amino acid analysis by high-performance liquid chromatography.

This paper describes a gradient system for separating D- and L-isomers of Dns-amino acids by mixed chelate complexation through the addition of histidine methyl ester and copper sulfate to the mobile phase. Most of the biologically important amino acids were separated in a single analysis. With a simple solvent gradient consisting of increasing concentrations of acetonitrile in L-histidine methyl ester buffer all the common amino acids were resolved except cysteine and all optical isomers were resolved except those of threonine, alanine and proline. Analysis time was 90 min. Use of this system for determining non-protein amino acids in human cerebrospinal fluid showed the amino acids to be L-isomers, as expected. The pattern in fluid from a patient with bacterial meningitis was different from that of most of the others.

Amino Acids↗
PubMed1983

The cimetidine-lidocaine interaction.

Lidocaine is a widely used antiarrhythmic agent whose plasma clearance varies with changes in hepatic blood flow. Cimetidine, an H2-receptor antagonist, has been shown to decrease hepatic blood flow. To ascertain whether cimetidine affected serum lidocaine concentration, we studied 21 patients receiving lidocaine infusions and divided them into two groups. Fifteen patients received cimetidine, 300 mg every 6 hours, in addition to lidocaine; six patients received only lidocaine. In 14 of the 15 patients receiving both lidocaine and cimetidine, a rise in serum lidocaine levels was seen, whereas no change was noted in the control group. Six of the 15 patients were found to have levels in the toxic range and two had symptoms. An additional three patients on lidocaine received diphenhydramine, an H1-receptor antagonist. No elevation in serum lidocaine levels was noted after administration of diphenhydramine. We conclude that there exists an interaction between lidocaine and cimetidine and that the rise in serum lidocaine levels may be mediated by cimetidine's inhibition of the H2 receptor.

Cimetidine↗
PubMed1981

Therapeutic drug assays with gas-liquid chromatography and optical detection.

Clofibric acid (p-chlorophenoxyisobutyric acid), the major metabolite of Clofibrate, a drug used in the treatment of hyperlipemia, was assayed in blood serum using an ultraviolet absorbance monitor as a gas-liquid chromatographic detector. As in other gas-liquid chromatographic assays for this compound, an internal standard, p-chlorophenoxyacetic acid, was added, and the serum was acidified and extracted with organic solvent. The solvent was then evaporated and the acids converted into their methyl esters for analysis. The organic compounds in the effluent were scrubbed into a stream of 2-propanol, at a flow-rate of 0.5 ml/min. This was then "debubbled" and a portion drawn through the 20-microliters UV detector flow cell. With small-volume scubber and associated components, peak-broadening was minimal. Because of their moderately high extinction coefficients at 280 nm, the Clofibrate and the internal standard were detected in the submicrogram range without interference from long-chain fatty acid esters, which have similar retention times on the column used.

Chromatography, Gas↗
PubMed1980

Specific detection of primary amines in the effluent of a gas-chromatographic column by on-line measurement of fluorescence.

Compounds with primary amino groups react with o-phthalaldehyde in solution to yield highly fluorescent products. This reaction is now in wide use for detecting amino acids and amines in liquid-chromatography effluents. We report here a method for using o-phthalaldehyde to detect primary amines in the effluent of a gas-liquid-chromatographic column. The effluent gas, nominally 20 mL/min, is delivered to a scrubber consisting of a small-bore, simulated capillary chromatographic column that is simultaneously supplied with 1 mL/min of the reagent solution. The liquid effluent of the scrubber, separated from the gas, is drawn through the flow cell of a fluorometer. Short-chain amines and ammonia were quantitatively scrubbed. The response of the fluorometer was directly proportional to the number of nanomoles injected into the column. Less than a nanomole of amine was detectable. Comparison of results with those from a hydrogen flame-ionization detector showed minimal additional peak broadening or compromise of resolution. These results demonstrate the feasibility of using highly specific, as well as sensitive, liquid-chromatographic detection methods for gas-liquid chromatography.

Amines↗
PubMed1979

Detection of hepatitis B surface antigen with the miniature centrifugal fast analyzer. A modified reversed passive hemagglutination procedure.

A modified reversed passive hemagglutination test for the detection of hepatitis B surface antigen HBsAg is described. Sera and reagent cells coated with antibody to HBsAg (anti-HBs) are loaded separately into the rotor of a miniature centrifugal fast analyzer. The rotor is centrifuged briefly to transfer the components into its cuvettes. After mixing, the suspensions are allowed to stand at room temperature for 30 min, following which the rotor is again centrifuged and the absorbance of each cuvette is monitored. Cells suspended in serum containing HBsAg leave the light path more rapidly than cells suspended in sera free of antigen. The magnitude of change in absorbance varies directly with the concentration of the antigen. In 45 sera tested by the conventional V-plate technique, findings were as follows: 21 positive, 19 false positive and 5 negative. The automated procedure unequivocally differentiated the 21 positives; results for the false positive and negative specimens were identical and clearly distinguishable from the positive results. The automated procedure enhances specificity, offers equivalent sensitivity, and results that are quantitative and objective.

Absorption↗
PubMed1979

Improved method for detecting hemagglutination by centrifugal analysis.

Centrifugal analysis can be used to detect hepatitis B surface antigen, antibody to rubella virus, and fibrin-related antigen. The procedure is performed with the same reagents used in conventional hemagglutination studies. Positive and negative reactions are distinguished by the rates of erythrocyte clearance in the centrifugal field (delta A/delta time); positive cells move more rapidly than negative cells, and this difference varies directly with the concentration of detectable antigen or antibody. This phenomenon is thought to be a result of the greater adhesion of negative cells to the cuvette's surface. Sensitivity and specificity are greater in the centrifugal analysis technique than in the more conventional hemagglutination tests. False-positive reactions are eliminated and the quantitative data are accurate and reproducible.

Antibodies↗
PubMed1978

Improved approach to sequential addition immunoassay.

In the usual sequential addition enzyme immunoassays for drugs, the activity of the drug-labeled enzyme decreases continuously with time as more of it is bound to antibody. Sensitivity also decreases; the activity immediately after mixing is the most sensitive indicator of drug concentration. The reaction of enzyme-drug with antibody can be stopped by saturating the antibody with a larger quantity of unlabeled drug, which reacts with the antibody faster than does the enzyme-labeled drug. When drug is added soon after the reaction starts, the enzyme activity is stabilized and the sensitivity to small quantities of antigen is increased. This approach, with modification, should be applicable to sequential immunoassays in which other kinds of labels are used. The enzyme activity can be measured for a longer time, with the predictable increase in precision, as well as the ability to detect smaller quantities, to use less reagent, and to use end-point rather than kinetic assays.

Antigen-Antibody Reactions↗
PubMed1977

Enzyme immunoassays with the miniature centrifugal fast analyzer.

We studied the EMIT (Enzyme Multiplied Immunoassay Technique, Syva) procedures for the assay of phenytoin and phenobarbital in serum, adapting them to the miniature Centrifugal Fast Analyzer. For different concentrations of drug, each rate of reaction decreased continuously with time, tending to converge on a single common value. The rate was most affected by the concentration of drug almost immediately after the reagents were mixed, less so thereafter. The antibody evidently is present in sufficient excess to bind all the enzyme-labeled drug ordinarily present, but the antibody-bound enzyme was only 75% inhibited; this helps explain the appreciable residual activity when no drug is present. The reaction course was the same whether the serum and enzyme-labeled drug were added to the antibody sequentially or simultaneously, which suggests that antibody is bound to drug appreciably faster than to enzyme-labeled drug. The reaction rates 15 to 30 s after mixing were used as the measure of the drug concentrations. These results were confirmed by noting the rates at successive 15-s intervals. The analyzer yielded a run-to-run CV of 10% for phenobarbital at 30 mg/liter, and 9% for phenytoin at 15 mg/liter, as compared to the 15% quoted by Syva.

Centrifugation↗
PubMed1976

Chemical ionization mass spectrometry for rapid assay of drugs in serum.

A rapid procedure has been devised for analyzing anti-epileptic drugs in serum by chemical ionization mass spectrometry. An internal standard, 5-(p-methylphenyl)-5-phenylhydantoin (MPPH), is added to serum diluted in buffer at pH 12. The mixture is washed with diethyl ether to remove neutral lipids, acidified, extracted with chloroform and the chloroform extract evaporated to dryness. The residue is then dissolved in methanol and an aliquot, corresponding to approximately 2% of the original mixture, is deposited in the glass capillary sample cup of the solid probe inlet of the mass spectrometer. The sample is then volatilized by heat into the ion source of the mass spectrometer, where it reacts with ionized methane reagent gas. As the temperature of the probe is increased, quasimolecular ion peaks of the protonated anticonvulsants appear, rise and fall on the oscilloscope tracing, indicating similar but not identical rates of volatilization. We recorded these peaks photographically by opening the shutter of the oscilloscope camera for the entire heating cycle. The concentrations of the anticonvulsants were estimated from the ratio of the height of the peaks of the drug to that of the internal standard on the photograph. The peak-height ratios were proportional to concentration within, above and below the therapeutic range. Other drugs, including barbiturates, carbamazepine, nicotine and caffeine, were readily identified when present. With one solid probe inlet, an assay could be performed every 2 min.

Anticonvulsants↗