Analysis of femtomole concentrations of alpha-ketoisocaproic acid in brain tissue by precolumn fluorescence derivatization with 4,5-dimethoxy-1,2-diaminobenzene.
An extremely sensitive fluorimetric HPLC method used to determine in vivo cerebral alpha-ketoisocaproic acid (KIC) concentration with less than 2 g of tissue (e.g., a single rat brain, approximately 1.8 g) is reported. Removal of unwanted lipids and amino acids and isolation of alpha-ketoacids in an optimal derivatization buffer are achieved by C18 solid-phase extraction of the acid-soluble fraction of brain tissue. Quantitation of KIC to the femtomole level is accomplished by reversed-phase HPLC using 4,5-dimethoxy-1,2-diaminobenzene precolumn fluorescence derivatization and on-line fluorescence detection. These techniques are applicable to femtomole quantitation of other alpha-ketoacids in various tissue and blood matrices. Combination of this fluorimetric method with simple nonchromatographic procedures to measure in vivo cerebral [1-14C]alpha-ketoisocaproic acid radioactivities in tissue provides estimates of specific activities.
Keen
R E
RE
Department of Pharmacology, UCLA School of Medicine.
Nissenson
C H
CH
Barrio
J R
JR
D000818:Animals; D001923:Brain Chemistry; D002250:Carbon Radioisotopes; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005456:Fluorescent Dyes; D005470:Fluorometry; D007651:Keto Acids / Q000032:analysis* / Q000097:blood / Q000302:isolation & purification; D007930:Leucine / Q000378:metabolism; D008297:Male; D008832:Microchemistry; D010655:Phenylenediamines / Q000737:chemistry*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013552:Swine
D002250:Carbon Radioisotopes; D005456:Fluorescent Dyes; D007651:Keto Acids; D010655:Phenylenediamines; C047826:1,2-diamino-4,5-dimethoxybenzene; C013082:alpha-ketoisocaproic acid; D007930:Leucine