Metachromatic leucodystrophy: study of the free amino acids in blood, urine, saliva and cerebrospinal fluid

Abstract

In the study of 2 cases of metachromatic leucodystrophy, late juvenile form, an increase of the total amino acid content in urine was found. The results of the study of the free amino acids in blood, urine, saliva, and cerebrospinal fluid (CSF) by means of circular and two-dimensional chromatography, and high voltage electrophoresis, are the subject of this communication. MATERIAL AND METHODS MATERIAL-Two familial cases (cousins) of the late juvenile type of metachromatic leucodystrophy were studied. The diagnosis was based on the clinical picture plus the finding of metachromatic bodies in urine (Austin test 1 ), saliva, and CSF (Canelas et al. 12 ), as well as in biopsies of peripheral nerve, liver, and kidney. The clinical and laboratorial data on these cases are extensively reported elsewhere ".The following samples were used:(a) recently emitted or 24-hour urine preserved with thymol; (b) 10 ml of recent blood serum; (c) whole saliva obtained by mechanic stimulus (rubber ball chewing) for periods from 8 to 12 hours; (d) 10 ml of recent CSF collected by lumbar puncture.The fluids were, kept in a refrigerator at 5°C. METHODS-(a) Deproteination, according to Awapara 3 , Troll and Cannan 57 , Stein and Moore 54 , and Campos et al. 10 . Two milliliters of each sample are added to 6 ml of ethanol and then transferred to a glass-stoppered tube and left at room temperature for one hour. The volume now is centrifuged for 10 minutes at 2,000 rpm; 2.5 ml of the supernatant are transferred to a graduated cylinder and completed up to 10 ml with chloroform. The cylinder is shaken and then left in rest until the layers are separated. The acqueous upper layer contains amino acids (A). (b)Hydrolysis of the biologic fluids: 0.5 ml of the upper layer (A) are transferred to a 10 ml ampoule; 0.5 ml of 6N hydrochloric acid are added; the ampoule is closed and put in a stove at 100-110°C for 24 hours. The ampoule is then opened From the