A comparison of different methods of determination of amino acid profiles in mammalian tissues: a study of teratogenic effects of vigabatrin and alcohol

Abstract

Amino acid analysis has been used extensively as a biochemical tool for the diagnosis and monitoring of amino acid levels in normal and diseased conditions resulting from inborn errors of metabolism. Normaly, a volume of 1 ml of venous blood is often recommended but this is a serious problem when blood has to be taken from new born and small children who have low blood volume. Moreover, in the treatment of such disease as epilepsy with anti-epileptic drugs (AED) and in the incidence of foetal alcohol syndrome (FAS), it is of paramOunt importance to monitor the levels of amino acids in both the mother and their babies in order to determine any possible inborn error which may occur as a result of either drug treatment or alcohol intake.
One of the primary aims of this study was to develop a new micro-capillary technique employing a small volume of human blood for amino acid analysis using ion-exchange chromatography. Blood was collected from healthy adult subjects and newborn babies and infants of the United Arab Emirates. Three methods for the collection and preparation of the amino acid profile by ion-exchange chromatography were compared; a new micro-capillary method, a standard filter paper technique and the standard venous blood methodl In addition, the project investigated the teratogenic effects of the AED, vigabatrin (VGB) and acute alcohol administration on amino acid metabolism in maternal blood, placenta and embryo of Theiler Outbred (TO) mice. The mice were injected intraperitoneally with either 400 mg kg VOB on gestation day 10 or with 70 mg kj' -150 mg kg' methionine and 0.03 ml kg' absolute alcohol on gestation days 7-12.
Following injection, blood samples, placentas and embryos were collected at 3.5, 6.0 and 9.0 hours after treatment for the analysis of amino acids.
The results have shown that intra-sample reproducibility in the filter paper and capillary blood from the same individual was less than 20% and 5%, respectively. The greatest variability in amino acid concentrations was in cystine and methionine. There was no significant difference between results obtained from capillary blood and from venous blood, but there was asignificant (Pc 0.05) difference between amino acid concentrations in venous and capillary blood on the one hand and filter paper blood on the other. Amino acid profiles of newborn babies, obtained using the micro-apillary technique, show an
overall increase in most of the amino acids compared to venous blood samples, especially that of serine, ornithine and glycine where the increase was approximately 400%, 200% and 150%, respectively. Administration of VGB to the TO mice resulted in an overall decrease in all amino acids following 3.5 hours of treatment with the drug. The most significant decrease was in methionine concentration (p <0.05 in maternal blood; p <0.00 1 in embryo and p< 0.01 in placenta). Methionine levels remained low even 6.0 and 9.0 hours after VGB treatment. The AED drug had no significant effect on cation levels in either the placentas or embryos of TO mice. Supplementation of alcohol and methionine to TO mice resulted in a marked reduction in plasma concentrations of several amino acids (including threonine, serine, glutamine, glycine, alanine) and methionine compared to saline-treated (control).
In conclusion, this study presented a new micro-capillary technique for the analysis of amino acid concentrations from small children. A comparison of this technique with standard methods revealed a significant difference in concentration of several amino acids between capillary and venous blood and blood collected on filter paper blood on the other. Studies on animal models revealed that methionine concentration is severely decreased in the maternal blood, placentas and embryos of VOB treated TO mice compared to control. Moreover, plasma levels of several amino acids including methionine are significantly reduced by alcohol. However, supplementation with methionine helped to enhance the concentration of these amino acids.