BIOCHEMICAL METHOD FOR MONITORING FREE FATTY ACIDS

A METHOD HAS BEEN DEVELOPED THAT FOR THE FIRST TIME ALLOWS THE DIRECT MEASUREMENT OF THE CONCENTRATION OF LONG CHAIN FREE FATTY ACIDS (FFA), THE MONOMER FORM OF FATTY ACIDS (FA) IN AQUEOUS SOLUTION. DETERMINATION OF THE FFA CONCENTRATION IS ACCOMPLISHED BY MEANS OF A FLUORESCENTLY MODIFIED FATTY ACID BINDING PROTEIN (FABP) THAT EXHIBITS DISTINCTLY DIFFERENT FLUORESCENCE WHEN FA IS BOUND AS COMPARED TO THE APO FORM OF THE PROTEIN. ACRYLODAN MODIFIED RECOMBINANT FABP FROM RAT INTESTINE FLUORESCES AT 432 NM IN THE APO FORM AND AT 505 NM IN THE HOLO STATE. THUS THE RATIO OF 505 TO 432 NM INTENSITIES PROVIDES A DIRECT MEASUREMENT OF [FFA]. BECAUSE THE RATIO OF FLUORESCENCE IS DETERMINED, THE MEASUREMENT IS VIRTUALLY INSTANTANEOUS, REQUIRES NO CALIBRATION (ONCE THE PARAMETERS CHARACTERISTIC OF EACH FA HAVE BEEN DETERMINED) AND IS EXTREMELY SENSITIVE. OUR RESULTS SHOW THAT THE PROBE (DENOTED ADIFAB) IS SPECIFIC FOR LONG CHAIN FFA AND HAS A USABLE RANGE OF LESS THAN 1 NM TO GREATER THAN 50MM, DEPENDING UPON THE FA. PRELIMINARY STUDIES HAVE DEMONSTRATED THAT ACCURATE DETERMINATIONS OF [FFA] CAN BE MADE IN AQUEOUS MEDIA IN THE PRESENCE OF CELLS AND ALBUMIN. MOREOVER, MEASUREMENTS IN HUMAN SERA AND SHOW THAT UNDER NORMAL PHYSIOLOGICAL CONDITIONS FFA LEVELS ARE LINEARLY RELATED TO TOTAL FA.