P.P. van Jaarsveld

Beta-sitosterol and beta-sitosterol glucoside stimulate human peripheral blood lymphocyte proliferation : implications for their use as an immunomodulatory vitamin combination

The phytosterols, beta-sitosterol (BSS), and its glucoside (BSSG) enhance the in vitro proliferative response of T-cells stimulated by sub-optimal concentrations of phytohaemagglutinin (PHA) several fold at extremely low concentrations (femtogram level). A 100:1 (mass:mass) ratio of BSS:BSSG (termed essential sterolin formulation, ESF) showed higher stimulation than the individual sterols at the same concentration. In vivo activity of ESF was also demonstrated when volunteers ingested ESF for 4 weeks. Proliferation of their T-cells, stimulated maximally with PHA, was significantly enhanced (20-920%) when compared to baseline values. In vitro, ESF (1 microgram.ml) was able to significantly enhance the expression of CD25 and HLA-Dr activation antigens on T-cells and increased the secretion, into the medium, of IL-2 and gamma interferon. NK-cell activity was also increased by BSS and BSSG alone, but with EST a higher activity was always found at different effector:target ratios (100:1 12:1).

High-performance liquid chromatographic determination of isoniazid, acetylisoniazid and hydrazine in biological fluids

The basic principle of derivatization of a hydrazide moiety with an aldehyde as applied in the method developed by Lacroix et al. [J. Chromatogr., 307 (1984) 137-144] for the quantitation of isoniazid and acetylisoniazid was improved by modification, standardization and extension to allow quantitation of hydrazine in patient samples. It could be shown that 40 microliters of 1% methanolic cinnamaldehyde per 200 microliters of deproteinized analysate gave maximal chromophoric isoniazid-cinnamaldehyde conjugate, read at 340 nm. The hydrolytic loss of isoniazid, crucial to the quantitation of acetylisoniazid, could be compensated for by introduction of an appropriate set of calibration curves. Although the method described here allows quantitation of monoacetylhydrazine and diacetylhydrazine, in addition to hydrazine, in mono-spiked samples, the method cannot be used for the quantitation of the acetylated metabolites of hydrazine in patient samples because of a lack of specificity. Linear calibration curves in the range 1-25 micrograms/ml for isoniazid and acetylisoniazid, 10-400 ng/ml for hydrazine and 50-1000 ng/ml for monoacetylhydrazine and diacetylhydrazine, could be constructed; analyte recoveries approaching 100% could be achieved in all instances.

Therapeutic monitoring of antituberculosis drugs by direct in-line extraction on a high-performance liquid chromatography system

A direct in-line pre-column extraction technique in which guanidinium and ammonium sulfate are used, followed by column switching, was employed to analyze serum, plasma and cerebrospinal fluid samples of patients treated for tuberculous meningitis. Resolution of a wide range of polar to non-polar xenobiotics was obtained on a C8 silica column by using a linear gradient from a binary system consisting of solvent A (0.05 M KH2PO4) and solvent B (acetonitrile-isopropanol, 4:1, v/v). Apart from the antituberculosis drugs (isoniazid, pyrazinamide, ethionamide and rifampicin) the patients received up to sixteen different medicines for prevention of complications and the treatment of symptoms. Qualitative resolution of all the drugs was obtained by the chromatographic system. Quantitation of pyrazinamide and ethionamide was achieved with high precision and low inter-sample variation.

Evaluation of a high-performance thin-layer chromatographic technique for the determination of salbutamol serum levels in clinical trials☆

Salbutamol concentrations were determined by high-performance thin-layer chromatography in the sera of two sets of ten volunteers at hourly intervals for 6 h after taking one 8-mg slow-release tablet. The influence of time lapse in processing of serum samples, i.e. centrifugation, extraction and chromatography, was studied. A statistical significant instability of salbutamol in the sera of patients was found which was not present in standard drug-free serum samples spiked with salbutamol and used for construction of standard curves.

Bovine 37S iodoprotein: Isolation and characterization

Abstract A thyroglobulin-like protein with a sedimentation constant of 36.6S has been isolated from bovine thyroid glands and further characterized. Its molecular weight, 2 × 106, is close to three times that of 19S thyroglobulin, the major thyroid iodoprotein. The amino acid composition of this iodoprotein also closely resembled that of 19S thyroglobulin and of the 27S iodoprotein. A reaction of identity in a double diffusion system with 19S, 27S and 36.6S has also been found. The iodine content of the 36.6S iodoprotein was greater than that of 19S thyroglobulin, but similar to that of the 27S species isolated from the same thyroid extract.

Use of guanidine hydrochloride and ammonium sulfate in comprehensive in-line sorption enrichment of xenobiotics in biological fluids by high-performance liquid chromatography

A novel approach has been developed for direct injection of physiological fluids on an in-line extraction pre-column followed by column switching in order to introduce the adsorbed xenobiotic onto the analytical column. The physiological fluid is pre-treated with guanidinium solution in water (200 microliters of fluid plus 300 microliters of a reagent containing 8.05 M guanidinium and 1.02 M ammonium sulfate) in order to denature protein binding sites and to serve as a universal solvent for a divergent range of polar to non-polar xenobiotics in a hydrophilic medium. A 0.5 M ammonium sulfate solution (500 microliters) is used as a pre- and post-flush reagent for the extraction pre-column (30 mm x 2.1 mm I.D.). The pre-flush reagent prepares the sorbent environment of the C18 pre-column for the hydrophobic retention of analytes. The post-flush reagent flushes non-retained sample proteins and salts to waste prior to switching the pre-column in-line with the analytical column. Universal chromatographic conditions for the analytical phase allows elution of a range of polar to non-polar xenobiotics within 20 min from an end-capped C8 silica analytical column (250 mm x 4.6 mm I.D.). This is effected by a linear gradient from a binary system consisting of solvent A (0.05 M KH2PO4) and solvent B (acetonitrile-isopropanol, 80:20, v/v).

A radioimmunoassay for metoclopramide

A radioimmunoassay for the anti-emetic drug, metoclopramide, in the pmol range was developed. The immunogen was prepared by photolytic coupling of metoclopramide to bovine serum albumin. A crosslinking reagent, N-hydroxy-succinimidyl-4-azidobenzoate, was first reacted with serum albumin through nucleophilic substitution. Ultraviolet irradiation (lambda greater than 300 nm) of the photoactive serum albumin conjugate in the presence of metoclopramide resulted in covalent attachment of the drug to the protein. An 125I-labelled metoclopramide derivative was prepared by diazotisation of the aromatic amine group and substitution of the resultant diazo group with 125I-. Binding data of the antibody with radioiodinated metoclopramide gave a linear Scatchard plot indicative of a homogeneous antibody population. A dissociation constant of 3 X 10(-11) mol/l was calculated for the antigen-antibody interaction. The antibodies showed negligible cross-reactivity with lignocaine which is structurally closely related to metoclopramide.

Bovine thyroxine-binding globulin: Purification and comparison of molecular weight and amino acid composition with human thyroxine-binding globulin

Abstract Bovine and human thyroxine-binding globulin were purified from serum by a three-step purification procedure which comprised affinity chromatography consecutively on thyroxine- and Concanavalin A-Sepharose and finally preparative polyacrylamide gel electrophoresis. The molecular weights of the two proteins were similar (54 000) as well as their carbohydrate contents while some differences in amino acid composition were found. Rabbit antiserum against bovine thyroxine-binding globulin reacted with human thyroxine-binding globulin with no sign of spur formation.

Inhaled and Oral Salbutamol: How Effective in the Prophylaxis of Asthma?

Inhaled and oral salbutamol were compared in 12 asthmatic patients for prophylaxis in antigen-induced asthma. The patients were pretreated with 0.2- and 1.0-mg doses of inhaled salbutamol and with the standard oral 4- and 8-mg slow-release (SR) salbutamol preparations. Bronchodilatation was monitored over the ensuing 3 h and protection against antigen challenge at the end of the period. On each study day the degree of baseline airway hyperreactivity was determined by histamine challenge. Precautions were taken during the antigen challenge to ensure a reproducible response. Blood levels of salbutamol were monitored at hourly intervals for the 3 h after treatment and during the asthmatic reaction subsequent to challenge. Both the 0.2- and 1.0-mg inhalations caused immediate bronchodilation as compared to a placebo (p less than 0.05), but only the 1.0-mg dose protected subjects against antigen challenge (p less than 0.05). In comparison to the placebo, no bronchodilatation was achieved with the standard 4-mg oral preparation in spite of measurable blood levels, nor were the patients protected against antigen challenge at 3 h after pretreatment. However, the 8-mg SR salbutamol caused significant bronchodilatation within 2 h and suppressed antigen challenge responses as compared to placebo (p less than 0.05). It can be concluded that doses of inhaled salbutamol higher than the conventional 0.2- or the standard 4-mg oral preparations are required to protect asthmatics against inadvertent antigen exposure. In patients who are unable to use inhalers effectively, the SR preparation can be considered as an alternative.

Isolation and structure elucidation of xanthotoxin, a phototoxic furanocoumarin, from Peucedanum galbanum

The major vesicant principal of Peucedanum galbanum has been isolated for the first time and its structure confirmed as xanthotoxin on the basis of melting-point and spectroscopic evidence.