SEMI - QUANTITATIVE ESTIMATION OF SERUM AMINO ACIDS BY PAPER CHROMATOGRAPHY

Abstract

ABSTRACT Paper chromatography is a simple         procedure performed in all Biochemistry laboratories. In our department, semi-quantitative estimation of the                       chromatographically separated amino acids was done for a case of hyperglycinemia to establish the diagnosis of non ketotic hyperglycinemia. An eight year old child who had been on Phenytion sodium for seizures with developmental delay, at the age of six years, on addition of sodium valproate for adequate control of seizures, progressively worsened and there was an increase in frequency of seizures. After Tandem Mass                  Spectrometry, the condition of Hyperglycinemia was diagnosed and organic acids were found to be normal. To prove the            condition as non Ketotic Hyperglycinemia, and to differentiate it from ketotic causes, the CSF to plasma glycine ratio was           essential. The CSF Plasma glycine was determined semi   quantitatively using paper chromatography. CSF samples by lumbar puncture, serum and urine were taken simultaneously. The samples were deproteinized, concentrated and equal           volumes were chromatographed with standard glycine of known concentration. The spots developed after spraying with              Ninhydrin were separately cut and eluted with copper               sulphate- ethanol mixture and absorbance measured                 photometrically. The results were calculated and CSF Plasma glycine was found to be 0.8. A value above 0.08 is diagnostic of Non Ketotic Hyperglycinemia. Since valproate increases glycine levels in the Central Nervous System, the drug was stopped. On follow up the child became seizure free.  Thus the procedure of chromatographic separation and quantitation of amino acids is a useful tool in diagnosing inborn errors of metabolism.