C. A. Benassi

Surface modification of proteins activation of monomethoxy-polyethylene glycols by phenylchloroformates and modification of ribonuclease and superoxide dismutase

A single-step method of activation of monomethoxy-polyethylene glycols suitable for its binding to polypeptides and proteins is proposed. Based on the reaction with 2,4,5-trichloro-phenylchloroformate orp-nitrophenylchloroformate, it gives reactive PEG-phenylcarbonate derivatives. The PEG intermediate is stable on storage, the activating group is easily quantified, and the reaction with amino acid and proteins proceeds rapidly at pH near neutrality. The PEG derivatization of enzymes with this procedure is less inactivating than those previously reported. Ribonuclease and super-oxide dismutase were modified and the effect of (a) bound polymer on clearance time in rats, (b) antibody recognition, and (c) on the enzymatic activity toward low and high molecular weight substrates were studied.

High-performance liquid chromatographic determination of free formaldehyde in cosmetics

An improved, sensitive method for the determination of formaldehyde in cosmetics and other commercial products is reported. The procedure is based on dilution of the sample with tetrahydrofuran-water (9:1), followed by precolumn derivatization with 2,4-dinitrophenylhydrazine and direct reversed-phase high-performance liquid chromatography. The formaldehyde derivative is stabilized in the reaction medium by addition of phosphate buffer and neutralization and detected in less than 10 min by the standard additions methods. The method also appears to be suitable for the direct evaluation of the formaldehyde donors used in cosmetics as preservatives.

The metabolism of tryptophan in patients with bladder cancer and other urological diseases

Abstract The excretion of tryptophan metabolites was followed in the urine of 201 patients with bladder tumour, of 55 subjects with cancer of genito-urinary organs (ex. tra-bladder), and of 112 patients with various urological diseases (non-neoplastic). All 3 groups contained patients excreting abnormal amounts of kynurenine, 3-hydroxykynurenine and 3-hydroxyanthranilic acid. The urinary kynurenine excretion was followed for several days in 9 bladder tumour patients during simultaneous administration of daily large doses of vitamin B 6 . In some cases pyridoxine slightly decreased, but never completely inhibited the abnormal excretion of kynurenine. In addition to these investigations on spontaneous excretion, l -tryptophan was administered (100 mg/kg body weight) to 4 bladder tumour patients. Kynurenine, N -α-acetylkynurenine, 3-hydroxykynurenine, kynurenic acid, xanthurenic acid and 3-hydroxyanthranilic acid were measured after loading. Small differences in the sum of compounds excreted were found between these patients and normal controls. The data presented are difficult to reconcile with the hypothesis that the excretion of abnormal quantities of tryptophan metabolites, suggested as endogenous carcinogens, might have caused the induction of cancer only when excreted by bladder tumour patients. In fact such an excretion occurs in patients with extra-bladder tumours and with various urological diseases, as well as in other human pathological conditions.

Excretion and isolation of kynurenine and 3-hydroxykynurenine from human pathological urine

Abstract The urines of 346 subjects have been examined for the presence of products of tryptophan metabolism, using paper chromatography. 314 of the subjects were patients of the different Departments of the Medical School of Padua and the remaining 32 were healthy control subjects. Neither group received a special diet or supplements of tryptophan. go subjects suffering from different forms of haemoblastotic diseases excreted considerable quantities of kynurenine and 3-hvdroxy-kynurenine. From the urine of four of these leukaemia patients kynurenine (as the sulfate) and 3-hydroxykynurenine were isolated.

On the determination of small amounts of tryptophan metabolites and their occurrence in normal human urine

Abstract A technique has been developed for the determination of the main products of tryptophan metabolism occurring usually in small amounts in the urine of healthy persons on a free diet. With a single column of cation exchange resin (Amberlite IR-120) and volatile buffers, metabolites present in 1–2% of 24-h urine were fractionated by elution with 100 ml of 0.1 M pyridine-formic acid buffer pH 2.60, and 140 ml of same buffer 0.3 M, pH. 5.60. The effluent was pooled in 6 large fractions which were lyophilized. Each residue was subjected to two-dimensional paper chromatography for a further purification. The spots corresponding to kynurenine, 3-hydroxykynurenine, Nα-acetylkynurenine, Nα-acetyl-3-hydroxykynurenine, kynurenic acid, xanthurenic acid, xanthurenic acid 8-methyl ether, anthranilic acid, 3-hydroxyanthranilic acid and 3-methoxyanthranilic acid were eluted and determined fluorometrically or colorimetrically. Since o-aminohippuric acid is partially destroyed, the method of Price and Brown was preferred for the first step of elution followed by paper chromatography of the residue to achieve complete specificity. Recoveries were quantitative over a range of 10 μg to 100 μg for all tested metabolites in each sample. Normal occurrence of the derivatives was determined in urines from 20 healthy young men and 17 healthy young women. No significant difference was noted between the two groups, where excretion showed similar values. The spontaneous excretion of tryptophan metabolites by healthy human beings ranges from trace amounts to a few milligrams daily.

High-performance liquid chromatographic determination of free formaldehyde in cosmetics preserved with Dowicil 200

We recently reported a rapid and reliable method for the determination of free formaldehyde in cosmetics; the procedure is based on sample dilution with a tetrahydrofuran (THF) -water solvent mixture, followed by precolumn derivatization with 2,4-dinitrophenylhydrazine (2,4-DNPH) and direct high-performance liquid chromatographic (HPLC) analysis. Our studies showed the applicability of this method when preservative donors are present in the cosmetic formula: only Dowicil 200 [cis-1-(chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride] is not compatible with the procedure, because of its instability in the acidic media

Reversed-phase high-performance liquid chromatography applied to the direct analsis of untreated heterophasic systems

Abstract A simple and rapid approach for the direct analysis of multi-component heterophasic matrices is reported. The procedure is based on sample dilution with tetrahydrofuran—water (9:1) followed by direct reversed-phase high-performance liquid chromatography. The method is suitable for quality control and stability studies of drugs, food and cosmetic products.

TRYPTOPHAN METABOLISM IN SPECIAL PAIRS OF TWINS.

Abstract In 4 twin pairs, each consisting of a schizophrenic sibling and a normal sibling, tryptophan metabolism was studied after a loading test. Seven out of 8 subjects showed an abnormal metabolites excretion, higher than that observed in the chronic schizophrenic patients previously studied. Such an urinary pattern was independent of the genetic character of monozygous or dizygous twins. A possible relationship between schizophrenia, genetics and tryptophan metabolism is suggested.

A rapid HPLC technique for determining levels of histamine in tears from normal and inflamed human eyes

Because of the important role of histamine in the inflammatory process, the measurement of histamine levels in tears has been considered as a possible index of eye irritancy. Histamine levels have been measured using an HPLC procedure involving fluorimetric detection after fluorescamine derivatization. Analyses, at picomole levels, were carried out in less than 5 minutes using 10 microliter of tear samples collected from either normal or inflamed human eyes.

Chemical stability and microbiological reply of preservative systems in cosmetics: a direct correlation.

The results of a chemical/microbiological comparative study on different preservative mixtures are reported. The aim of the investigation was to ascertain whether there was a correlation between chemical stability and microbiological activity.