Reduction of pantethine in rabbit ocular lens homogenate.

In several animal models, preliminary studies have indicated that pantethine may inhibit cataract formation. Therefore, preclinical trials need to be conducted to study the pharmacology of pantethine in the ocular lens and to establish its efficacy. Since pantethine, which is a disulfide, can undergo a variety of chemical modifications such as reduction and formation of mixed disulfides, a detailed study was first conducted to determine the stability of pantethine in rabbit lens homogenate. A knowledge of the stability of pantethine in lens homogenate was necessary to establish if pantethine could be metabolized in the time it takes to harvest and homogenize a lens. The results of this study will be used to establish a protocol for harvesting and homogenizing lens samples. Pantethine (100 microM) is completely reduced to pantetheine in rabbit lens homogenate in about 16 min. About 1.5% of the pantethine added to lens homogenate forms a mixed disulfide with lens proteins, and the remainder is found in the supernatant. The supernatant pantethine concentration decreases exponentially as a function of time, and the terminal half-life for this process is 3.3 min. The free supernatant pantetheine concentration increases in pseudo first order manner as a function of time with a rate constant of 4.3 min. Pantethinase activity is not significant, because the free supernatant pantetheine concentration did not decrease. The exact mechanism of pantethine reduction in rabbit lens homogenate remains to be determined.

Immunodetection approaches and high-performance immunoaffinity chromatography for an analogue of bovine growth hormone releasing factor at trace levels.

An indirect detection method using high-performance immunoaffinity chromatography (HPIAC) was used to measure low levels of an analogue of bovine Growth Hormone Releasing Factor (bGHRF). An antibody (Ab) labelled with alkaline phosphate (ALP) was incubated with the bGHRF analogue to perform a complex between the antigen (Ag) and the antibody-enzyme (Ab-En) conjugate. The complex was then injected onto a cartridge containing an immobilized Ag affinity support. Species which were not recognized by the affinity cartridge, i.e. eluted, were then directly combined, via a connecting tee, with a buffer containing a substrate. Incubation proceeded on-line, inside a knitted reactor coil, under conditions of constant flow. The subsequent generation of a fluorescently active substrate product was detected by conventional means. The assay described has a linear response region from 1.0 to 25 ng of the bGHRF analogue and a limit of detection of 0.60 ng (1.7 x 10(2) femtomole, 30 p.p.b.). This approach was compared against a method in the antigen/Ab-En complex was injected onto a immobilized Ab affinity cartridge to form an antibody-antigen conjugate sandwich and subsequent stop-flow incubation with substrate.

Immunochromatographic analysis of bovine growth hormone releasing factor involving reversed-phase high-performance liquid chromatography-immunodetection.

We have developed high-performance immunoaffinity chromatography (HPIAC) methods for the detection and quantitation of bovine growth hormone releasing factor (GHRF), which could also be applicable to its metabolites in biofluids. These approaches have involved a combination of IAC using immobilized antibody (Ab) to GHRF, together with reversed-phase high-performance liquid chromatography (RP-HPLC) separations of initially isolated and concentrated protein, followed by selective detection, involving on-line immunodetection (ID) schemes. ID methods involved HPIAC supports of the Ab, together with synthesized Ab-fluorescein isothiocyanate conjugates. We have demonstrated optimization methods for each step of the entire hyphenated technique (IAC-HPLC-ID), and then actually quantitated GHRF using this overall system. The minimum detectable concentration was about 1 ng/5 ml (200 ppt) with fluorescence detection (excitation wavelength, 490 nm; emission wavelength, 510-650 nm). We have also tested a single blind, spiked biological sample (bovine plasma), spiked with a known level of GHRF. Accuracy (7.4%) and precision (S.D. = +/- 22%) were quite acceptable for a double immunoassay method.

Quantification of 9-carboxy-11-nor-delta 9-tetrahydrocannabinol in urine using brominated 9-carboxy-11-nor-delta 9-tetrahydrocannabinol as the internal standard and high-performance liquid chromatography with electrochemical detection.

A method was developed for quantitating 9-carboxy-11-nor-delta 9-tetrahydrocannabinol in human urine as part of the process for validating an automated enzyme immunoassay for marijuana metabolites. Sample cleanup was accomplished using a mixed-mode solid-phase extraction. 9-Carboxy-11-nor-delta 9-tetrahydrocannabinol and the internal standard, brominated 9-carboxy-11-nor-delta 9-tetrahydrocannabinol, were quantified using high-performance liquid chromatography with electrochemical detection (+ 0.85 V). The linear range for this method is 0.012-0.20 microgram/mL. No interference was seen for 22 drugs and metabolites. The pooled relative standard deviation is 4.1% (n = 27) for the quality control samples. This method was compared to gas chromatography with mass spectrometry by linear regression analysis. The slope of the line is 1.00 +/- 0.05 (standard error), the intercept is approximately zero, the coefficient of determination is 0.994, and the standard error of the estimate is 0.006 microgram/mL.

Lack of effect of oral N-acetylcysteine on the acute dialysis-related lowering of total plasma homocysteine in hemodialysis patients.

Hyperhomocysteinemia refractory to standard B-vitamin supplementation treatment persists in > or = 75% of maintenance dialysis patients, potentially increasing their risk for atherothrombotic sequelae. We examined whether predialysis administration of oral N-acetylcysteine (NAC), which acutely increases the non-protein bound, dialyzable fraction of plasma homocysteine, might augment the homocysteine-lowering effect of dialysis therapy. Predialysis and postdialysis total plasma homocysteine levels were determined on a control day, and on a day in which oral NAC (1200 mg) was administered predialysis in n = 11 maintenance hemodialysis patients. Although NAC treatment had no significant effect on hemodialysis removal of plasma homocysteine (P = 0.594), we observed a 16% reduction (P = 0.033) in non-fasting prehemodialysis total plasma homocysteine on the NAC treatment vs. non-treatment day. Longer term, placebo-controlled confirmation of this finding will be required to evaluate the possible chronic homocysteine-lowering efficacy of NAC treatment in hemodialysis patients.

Quantification of amphetamine in urine: solid-phase extraction, polymeric-reagent derivatization and reversed-phase high-performance liquid chromatography.

The concentration of amphetamine was determined in urine using solid-phase extraction, polymeric-reagent derivatization, and reversed-phase high-performance liquid chromatography with ultraviolet detection. To remove a majority of acidic and neutral compounds in urine, a solid-phase extraction was first performed on a sample spiked with the internal standard, 1-methyl-3-phenyl-propylamine. Because amphetamine has a relatively low molar absorptivity, the base was derivatized with a polymeric 1-hydroxybenzotriazole reagent containing a 3,5-dinitrobenzoate active ester. The limit of detection is 14 ng/ml, and the limit of quantification is 47 ng/ml. The calibration curve is linear from 0.01 to 4.0 micrograms/ml. The pooled relative standard deviation is +/- 5.5% for eight urine samples measured in duplicate. The average relative error (bias) is +2.2% when compared to gas chromatography-mass spectrometry.

Superoxide chemical transformation of diolepoxide polyaromatic hydrocarbon DNA adducts. Determination of benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol by gas chromatography.

Benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol (100 pg, 342 fmol) was measured using the following sequence of steps: (1) chemical transformation with potassium superoxide to 2,3-pyrenedicarboxylic acid; (2) electrophore derivatization with pentafluorobenzyl bromide; (3) sample clean-up by high-performance liquid chromatography and (4) measurement by gas chromatography with electron-capture detection and by gas chromatography with electron-capture negative-ion mass spectrometry. The overall, absolute yields obtained by the two procedures were 69% and 60%, respectively. This work completes the first stage towards the establishment of a general method for detecting diolepoxide polyaromatic hydrocarbon DNA adducts by gas chromatography.

Determination of 5-methylcytosine in DNA by gas chromatography-electron-capture detection.

The following sequence of analytical steps was used to determine the amount of 5-methylcytosine (mol-%) in calf thymus and human lymphocyte DNA:acid hydrolysis of the DNA, derivatization (pentafluorobenzyl bromide, solid phase extraction, pivalic anhydride), internal standard addition, solid phase extraction, high-performance liquid chromatography, and gas chromatography with electron-capture detection. The steps were carefully optimized, leading to a recovery of 30 +/- 1.0% starting with a nucleobase standard containing 1.25 ng of 5-methylcytosine. A second analysis of this sample gave a 30 +/- 0.3%, demonstrating a high precision for the method. In good agreement with earlier work by others, 1.2 +/- 0.10 mol-% of 5-methylcytosine was then found in a 350 ng sample of calf thymus DNA, and values of 0.9 +/- 0.07 and 0.8 +/- 0.04 mol-% (two runs) were found in hyman lymphocyte DNA.

Trace derivatization of cytosine with pentafluorobenzoyl chloride and dimethyl sulfate.

Methods are being developed for derivatizing trace amounts of DNA adducts for ultimate determination by gas chromatographic techniques. High-pressure liquid chromatography is used to optimize appropriate derivatization reactions for the determination of cytosine. A single vial reaction scheme involves acylation with electrophoric pentafluorobenzoylchloride followed by alkylation with dimethyl sulfate. Currently, the overall yield for this reaction starting with 50 nmole of cytosine is 59 +/- 4.6%.