Cerebrospinal fluid kynurenic acid in male patients with schizophrenia – correlation with monoamine metabolites

Background:The tryptophan metabolite kynurenic acid (KYNA) is an endogenous glutamate/nicotinic receptor antagonist. Previous studies have shown that the concentration of the compound is increased in cerebrospinal fluid (CSF) of patients with schizophrenia. Furthermore, it has been found that the CSF concentration of KYNA is positively correlated to CSF concentrations of the monoamine metabolites homovanillic acid (HVA) and 5-hydroxy indoleacetic acid (5-HIAA) in healthy control subjects.Objectives:To study the correlations between KYNA and the monoamine metabolites HVA, 5-HIAA and 4-hydroxy-3-methoxyphenylglycol (HMPG) in CSF of male patients (n= 53, ranging from 20 to 48 years of age) with verified schizophrenia.Methods:CSF was obtained by lumbar puncture, and KYNA analysis was performed with an isocratic reversed-phase high-performance liquid chromatography system connected to a fluorescence detector. HVA, 5-HIAA and HMPG concentrations were measured by mass fragmentography with deuterium-labelled internal standards.Results:Positive intercorrelations were found between CSF KYNA, HVA and 5-HIAA, while CSF content of HMPG did not correlate to KYNA or any of the monoamine metabolites in CSF.Conclusion:The results of this study suggest that increased KYNA formation is associated with an increased dopamine and serotonin turnover in male patients with schizophrenia.

Mass Fragmentography of Cortisol and Cortisone: Preliminary Studies on the Development of a Reference Method

Cortisol and cortisone are the two main glucocorticoids found in human plasma. Cortisol is commonly measured as a means of assessing adrenal function, usually by immunoassay, but such procedures must be subject to proper quality control which ideally uses analyte target values measured by isotope dilution mass fragmentography (GC-MS). Three derivatives of these steroids have been investigated for their chromatographic and mass spectral characteristics in order to develop a simple and rapid GC-MS method for this purpose. Trimethylsilyl ether-O-methyloxime derivatives gave poor sensitivity and thesyn- andanti-isomers were resolved into two peaks. Cyclic methylboronate-O-methyloxime derivatives gave excellent sensitivity but were difficult to prepare, occasionally giving rise, by some unknown mechanism, to 11-oxidation of cortisol. The underivatised 11-hydroxyl of this derivative of cortisol also gave rise to chromatographic problems. Enol-trimethylsilyl ethers however were easy to prepare and gave excellent sensitivity (10-17 times that achieved with the trimethylsilyl ether-O-methyloxime derivatives). Straight line calibration graphs were obtained and the method gave clean traces allowing high specificity and sensitive measurement of cortisol and cortisone in human plasma using tri-deuterated cortisol as an internal standard.

Routine analysis of plasma busulfan by gas chromatography–mass fragmentography

Busulfan (BU) is a widely used alkylating agent for antineoplastic therapy and marrow ablation in preparation for bone marrow transplantation (BMT). High-dose BU often leads to successful preparation and low relapse but is associated with veno-occlusive disease of liver. We established a protocol to determine postdosage plasma BU concentrations by gas chromatography–mass fragmentography in an attempt to relate clinical outcome to plasma BU concentrations. We used nonisotopic pusulfan as the internal standard. After extraction into ethyl acetate, BU and pusulfan were iodinated into 1,4-diiodobutane and 1,5-diiodopentane, respectively. Gas chromatography–mass spectrometry (GC–MS) analysis was carried out on an Hewlett–Packard (HP) 5890II gas chromatograph with a 30-m 100% methyl silicon narrow bore, fused-silica capillary column interfaced with an HP 5970A mass spectrometer. Helium was the carrier gas. The sample molecules were identified by total ion monitoring and quantified by selective ion monitoring of m/z 183 and 197. The calibration curve was linear to 4 mg/L. The limit of quantification was 0.04 mg/L, and the analytical recovery was ∼97%. The within-day and between-day imprecision (CV) was <6% and 9%, respectively. In a preliminary study of 12 children, the BU areas under the BU-time curve were 616–949 μmol · min/L after the first dose and 793-1143 μmol · min/L after the fifth dose. We conclude that the GC–MS procedure is suitable for routine analysis of plasma BU.

Oral administration of deuterium-labelled polyamines to sucking rat pups: luminal uptake, metabolic fate and effects on gastrointestinal maturation

Non-physiological amounts of oral polyamines have been reported to induce precocious gut maturation in rat pups. The aim of the present study was to investigate organ distribution and metabolic fate of orally administered stable-isotopically labelled polyamines in rat pups. Pups received tetradeuterium-labelled putrescine (Pu-d4; 3 μmol), spermidine (Sd-d4; 5 μmol), spermine (Sp-d4; 3 μmol), or physiological saline twice daily on postnatal days 7–10 or 12–15. They were killed on days 10 and 15. We determined activities of ileal lactase (EC3.2.1.23), maltase (EC3.2.1.20), sucrase (EC3.2.1.48) and diamine oxidase (EC1.4.3.6) and established villus and crypt lengths. Polyamines and their labelling percentages in organs were determined by GC and mass fragmentography. Treatments did not affect growth rate, but caused lower weights of liver, kidneys and heart. Maltase activity increased, lactase decreased, whereas sucrase and diamine oxidase did not change. Villus and crypt lengths increased. Organ polyamine pools were labelled to different extents. Irrespective of the orally administered polyamine, all organs contained Pu-d4, Sd-d4 and Sp-d4. Administered Pu-d4 and Sd-d4 were recovered mainly as Sd-d4, whereas Sp-d4 was recovered as Sp-d4 and Sd-d4. Total polyamines in a caecum, colon and erythrocytes increased, but increases were only to a minor extent with regard to labelled polyamines. Our data confirm precocious gut maturation by exogenous polyamines. Putrescine appears to be the limiting factor. The exogenous polyamines were distributed among all investigated organs. They are not only used for the synthesis of higher polyamines, but also retroconverted to their precursors. Changes in erythrocyte polyamine contents suggest precocious stimulation of erythropoiesis.