Effects of Sample Pretreatment in Amino Acid Analysis

Kim, Moon Hee; Moon, Hae Ran

Abstract

BACKGROUND
Measurements of the concentrations of free amino acids in the blood are used as useful biochemical indicators. The sample pretreatments, including anticoagulant selection and deproteinization, are important steps in plasma-free amino acid analysis for accurate and stable results. Heparin and EDTA venous plasma in a frozen state are most commonly applied sample sources in our laboratory. Therefore, we investigated the effects of the anticoagulant and delayed deproteinization in amino acid measurement using ion-exchange chromatography.
METHODS
We used Biochrom 20 amino acid analyzer (Biochrom, U.K). Blood samples were taken from 3 healthy adults after a minimum of 8 hours fasting. Two different types of vacutainer tubes, including sodium heparin and EDTA were used. To investigate variations by heparin volume, 3 mL and 6 mL of blood were drawn in 10 mL heparin tubes. We used an aqueous solution of SSA for deproteinization. To investigate variations through delayed deproteinization, we deproteinized the samples immediately and 24 hours later after plasma separation.
RESULTS
There were no significant differences in concentrations except for cystine, glutamic acid and taurine, and the retention time between the 6 sample groups. The concentration of taurine was higher in the groups of late deproteinized plasma. In the groups of the same deproteinization time, there were no significant differences in concentration by different heparin concentrations. When we compared the results of 3 mL EDTA plasma with that of heparin-treated 6 mL of blood, the most widely used sample type, there was a significant difference in cystine concentration in the delayed deproteinized group but there were no differences in the immediately deproteinized group.
CONCLUSIONS
Both 3 mL EDTA blood and 6 mL heparin-treated blood can be used commonly in case of using high-resolution ion-exchange chromatography and an immediately deproteinized sample. But, the results in amino acids can be affected in delayed pretreatment samples. Their effects should always be considered when interpreting laboratory results. The laboratories should standardize adequate sample preparation for the accurate analysis of amino acids.