Howard Ko

A simplified semiautomated assay for plasma triglycerides

Abstract A simpler, semiautomated, fluorescence method for the determination of triglycerides has been developed for the AutoAnalyzer (Technicon). The method involves manual plasma extraction and then the following automated procedure: ( a ) transesterification of triglycerides (sodium methylate in isopropanol) to release free glycerol, ( b ) oxidation of glycerol to formaldehyde, and ( c ) reaction (Hantzsch) of formaldehyde with acetylacetone and ammonia to form the fluorescent product 3,5-diacetyl-1,4-dihydrolutidine. Triglyceride is extracted from human serum under conditions that maximize extraction (quantitative) of triglyceride, minimize extraction of glycerol, glucose, and phospholipid, and provide good separation of phases. Interferences from phospholipid in human serum are sufficiently low that the usual step (in other methods) of adsorbing phospholipid (onto silicic acid, Florisil, zeolite, or Doucil) is not required. To minimize evaporation, nonane is used as nonpolar solvent in the extraction mixture. The amounts of reagents, the temperature and time required to carry out the reaction steps ( a ) through ( c ) were varied stepwise until maximal or near-maximal response was obtained. The automated portion of the assay is run at 50 samples an hour. With “normal” human serum, the relative standard deviation of triplicate determinations ranged from 0.71% for 83 mg % of triglyceride (milligrams of tripalmitin equivalents per 100 ml of serum) and 0.35% for triplicate determinations of 228 mg %. The lowest detectable amount of triglyceride for which the 95% confidence limits did not include zero was 1.6 mg %.

A submicromolar assay for nonpolar acids in plasma and depot fat

Abstract A simple titrimetric assay, requiring about a third less time than that required for the Dole procedure (2), has been developed for determining submicromolar amounts of nonpolar acids (e.g., long-chain fatty acids) in depot fat and plasma. The method has the following capabilities: 1. 1. 0.03 and 0.06 μeq of added palmitic acid are detected with standard deviations of a single determination of 12–14 and 6–7%, respectively; these amounts of nonpolar acid correspond to those present in 0.1 and 0.2 ml of serum, respectively. 2. 2. About (95–97)% of added palmitic acid is recovered in the heptane phase by the extraction procedure. 3. 3. The common interfering materials in plasma, lactic acid, β-hydroxybutyric acid, and pyruvic acid gave positive interference to the extent on only about 0.3 to 0.5%, values which are about one-fourth the interference of the regular Dole extraction method. 4. 4. The presence or absence of Krebs-Ringer buffer or serum components has no significant effect on the recovery of palmitic acid by the modified method or by Doles extraction method (1). Thus, standard curves for the esterified fatty acids may be constructed with or without the presence of these substances.

A gas-liquid chromatographic assay for plasma free fatty acids.

Abstract An operationally simple extraction-gas-liquid chromatographic assay has been developed for determining the quantity and composition of individual free fatty acids (FFAs, C 14:0 –C 18:2 ) in plasma. FFAs in a dried extract of plasma are reacted with N,N′-carbonyldiimidazole and methanol to form methyl esters. The methyl esters are washed with base, analyzed by gas-liquid chromatography (GLC), and plasma concentrations of individual and total FFAs calculated. Values for total FFAs obtained by the thin-layer chromatographic (TLC) method did not differ from those where the samples were initially purified by TLC and then measured by the GLC method. Extraction-titration assays for FFAs in plasma were 17–30% higher than the GLC values; but after TLC, the average titratable acidity from eluted FFA zones was not significantly different from total FFA determined by the TLC method. The data support the hypothesis that the extraction procedures used extract significant quantities of titratable acids (or bases) from plasma which are not the “normal” fatty acids (C 14:0 –C 20:4 ).

Purification and properties of a tween-hydrolyzing enzyme from rat adipose tissue.

Abstract A method is described for the preparation with good yields and approx. 75-fold purification of a hydrolase preparation from rat adipose tissue. Ultracentrifuge studies indicate that the best preparations are approx. 90% pure enzyme protein. The purified enzyme can be electrophoretically resolved into at least two components which appear to be very similar molecular forms of the same enzyme. Provisional substrate-specificity studies demonstrate that the enzyme will hydrolyze a number of different ester bonds including simple aliphatic esters, “Tween” esters, and certain triglyceride esters. With simple aliphatic esters, under the conditions employed, a marked preference is shown by the enzyme for esters in which the alcohol component is small, and the fatty acid moiety is 5–6 carbon atoms in length.

Radioimmunoassay for Glyburide in Human Serum

Abstract A radioimnunoassay (RIA) has been developed to measure nanogram amounts of drug-related materials in human serum, after oral administration of 1.25, 2.5, 3.75 or 5.0 mg glyburide, G, Micronase®, (Micronase® is the registered trademark of The Upjohn Company for 1-[[p-[2-(5-chloro-o-anisamido)ethyl]phenyl]-sulfonyl] 3-cyclohexylurea). Of the compounds tested, only the known hydroxy metabolites of G cross-reacted significantly. Using 20 μl specimens, containing 25.6 ng G/ml serum, the within-day and between-day coefficients of variation for the assay were 3.47% and 3.18%, respectively. Recoveries were quantitative (100.6%). In normal human volunteers, peak serum drug concentrations were observed at 4.3 ± 1.4 hrs (S.D., M = 32). After single oral doses

Radioimmunoassay of triazolam

Abstract Two radioimmunoassay (RIA) assay methods (I, II) have been developed for triazolam (T, U-33,030) in serum and plasma. The parameters of equilibration time, serum blank, antibody specificity, extraction efficiency, and drug-binding to glass were studied. Of the various triazolam analogs tested for cross reactivity, only the hydroxy metabolites interfered significantly. At the levels normally found in plasma or serum, a background blank was encountered from constituents such as fatty acids, lysolecithin, lecithin, and cholesterol. However, serum or plasma samples could be analyzed with (I) by constructing standard curves in which blank serum from the same subject was used. An alternate method (II) was found which simultaneously extracted and precipitated the interferences. Both methods could be employed for analysis of plasma or serum samples. However, II detects T metabolites less efficiently than I. The within day and between day coefficients of variation for method I were found to be 5.7% and 3....

Automated determination of glycerol in plasma

Abstract Two automated fluorometric procedures using the Technicon Auto-Analyzer, one for plasma (Method I) and the second for plasma (or solutions) deproteinized by perchloric acid (Method II), have been developed. In Method I, the amount of glycerol in plasma is stabilized by adding triethylamine to increase the pH of the plasma. Thirty analyses (sample or sample blank) per hour can be run with a relative standard deviation of 5% at glycerol levels of 10 μg/ml (ca. 0.1 μmole/ml) in rat plasma. The lowest detectable concentration of glycerol for which the 95% confidence limit did not include zero was 0.008 μmole/ml. Since a sample blank must be run for each sample, the effective rate is 15 plasma samples per hour. The use of triethylamine also permits titration of plasma for nonpolar acids (e.g., fatty acids) should this be desired. Recovery (±S.D.) of glycerol added to rat plasma by Method I was (97.7 ± 1.1)%. The results suggests glycerol binding by plasma proteins or perhaps fluorescence quenching. In Method II, the samples of plasma or solution containing glycerol are deproteinized with perchloric acid. Sixty or 70 deproteinized solutions or their blanks are analyzed in an hour with a relative standard deviation of 2.3% for 10 μg/ml (ca. 0.1 μmole/ml). The lowest concentration of glycerol for which the 95% confidence limits did not include zero was ca. 0.005 μmole/ml. Recovery (±S.D.) of glycerol added to rat plasma by Method II was (105.0 ± 0.3)%. The result is consistent with the “volume displacement error” resulting from the concentration of solutes when plasma proteins are precipitated from solution. With a decrease in deproteinization time presently required, Method II having a greater sampling rate and precision would be more rapid and convenient than Method I. Under different storage conditions, glycerol levels in rat plasma differed in the time periods during which they are stable. Frozen samples with triethylamine as inhibitor were stable for as long as ten weeks, while those that were simply frozen or were deproteinized with perchloric acid and refrigerated were stable for two weeks.

Manufacture of antithymocyte globulin (ATGAM) for clinical trials.

Methods are described for preparing large amounts of horse anti-human thymocyte globulin (ATG, ATGAM; The Upjohn Company) for clinical use. These methods have been used since 1968 to provide material for clinical trials. Characteristics of 40 lots of ATG are summarized.

Automatic extraction and analysis of serum triglycerides

Abstract A simple automated procedure and equipment for extracting and quantifying serum or plasma triglycerides has been developed. Fifty samples or standards per hour can be analyzed with a relative standard deviation of 3% at triglyceride concentrations of 125 mg%%. The lowest detectable concentration of triglyceride, for which the 95% confidence limit did not include zero, was 7 mg %.

A GC-MS Procedure for the Measurement of Dopamine in Mouse Striatal Tissue

Abstract A gas chromatographic-mass spectrometric (GC-MS) method for measuring dopamine levels in brain tissue has been developed. Deuterated dopamine was prepared by exchange of the aromatic hydrogens with deuterium. The method involves adding a known amount of deuterated dopamine to the extraction medium or vessel and extracting the brain sample. The sample is acylated with pentafluoropropionic anhydride, and an aliquot of the derivative in benzene injected into a gas chromatography-mass spectrometry-computer system. The ion fragments, m/e = 428 and 431 respectively, of the protium and deuterium form of dopamine pentafluoro-propionate are detected. From the relative intensities of the two forms the concentration of dopamine in the brain samples is determined. Trace amounts of selenium in biological samples were estimated by atomic absorption utilizing a Varian carbon rod flameless atomizer. Samples were digested with nitric perchloric acids, selenium was separated from the interfering sample matrices by...