Tissue Extracts and Perfusates

Depending on the perfusion system, varying amounts of proteases and other enzymes may be present m the perfusates and these amounts determine the handling of the samples. The dialysates and perfusates are normally collected in small plastic tubes, frozen, and stored at -20°C for a limited period of time. 

One problem which is particularly critical when small quantities of ammo acids are measured is the risk of contamination with extraneous amino acids (aspartate, glutamate, serine, glycine, and alanine) from the glassware, solutions, and so forth (Lee and Drescher, 1978) Obvious precautions necessary to follow include the use of disposable gloves, freshly distilled water, and highly pure chemicals. Ammo acids in the blanks are further reduced if the glassware and plastic collection tubes are prerinsed several times with diluted phosphoric acid and distilled water. In order to avoid contamination from the injection loop, syringe, and elution buffer, the reaction mixture may be acidified after a set time (Jones et al., 1981). 

Fig 3 Correlative analysis of free amino acids m extracts from cerebral cortex, cerebellum, and brain stem with the OPA liquid chromatography and lon-exchange techniques 

As a check for correct derivatization and pipehng, it is advisable to include an internal standard with the sample (preferably as early as possible in the sample treatment), e.g., p-ammo-n-butyric acid, which is eluted in the middle of the chromatogram. Compounds that are eluted in other regions of the spectra, e.g., cysteate or cystein sulfinate, may also be used. For routine quantification we compare peak heights with known amounts of external and internal standards 

The separation and shape of some peaks are distorted after consecutive sample loading, and this may be due either to increased dead volume at the top of the column and/or to accumula- 

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