Automated enzymatic determination of plasma free fatty acids by centrifugal analysis

Simple and specific measurement of free fatty acid (FFA) levels in plasma and serum is of practical value to the clinical biochemist and, in the authors’ laboratory, in veterinary nutritional and metabolic studies. Most early methods, such as that of Duncombe [1], relied on the colorimetric determination of metal-FFA complexes in the organic phase of solvent extracted plasma. These techniques are relatively non-specific and, in addition, are extremely laborious and time-consuming. Recently, alternative assays for FFA, based on specific enzymic oxidation by acyl-coenzyme-A-synthetase (ACS) and acyl-coenzyme-A-oxidase (ACOD), have been developed (Shimizu et al. [2]). Hydrogen peroxide generated by these reactions can be quantitated colorimetrically (Mizuno et al. [3]; Shimizu et al. [4]; Matsubara et al. [5]) and is directly related to the FFA content of the test sample. One such procedure involving specific peroxidase (POD)-dependent quinone-dye formation is now available commercially. This article reports on the adaptation of the method .for routine automated determination ofFFA using an I.L. Multistat III microcentrifugal analyser.


Introduction
Simple and specific measurement of free fatty acid (FFA) levels in plasma and serum is of practical value to the clinical biochemist and, in the authors' laboratory, in veterinary nutritional and metabolic studies. Most early methods, such as that of Duncombe [1], relied on the colorimetric determination of metal-FFA complexes in the organic phase of solvent extracted plasma. These techniques are relatively non-specific and, in addition, are extremely laborious and time-consuming.
Recently, alternative assays for FFA, based on specific enzymic oxidation by acyl-coenzyme-A-synthetase (ACS) and acyl-coenzyme-A-oxidase (ACOD), have been developed (Shimizu et al. [2]). Hydrogen peroxide generated by these reactions can be quantitated colorimetrically (Mizuno et al. [3]; Shimizu et al. [4]; Matsubara et al. [5]) and is directly related to the FFA content of the test sample. One such procedure involving specific peroxidase (POD)-dependent quinone-dye formation is now available commercially.
This article reports on the adaptation of the method .for routine automated determination of FFA using an I.L. Multistat III microcentrifugal analyser.

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Manual FFA determination FFA were estimated colorimetrically by the method of Duncombe [1] based on the extraction procedure proposed earlier by Dole [6].
Application of ACS-ACOD method to the MCA Reagents were prepared according to the manufacturers' instructions. Assay volumes were then adjusted to suit the capacity of the MCA analytical rotor. For routine analysis, sample (or distilled water as reference) and reagent A (containing ACS, coenzyme A, adenosine triphosphate and 4-aminoantipyrine in 0.05 M phosphate buffer pH 6.9) were added to the inner sample-well using the loader 'sample' and '2nd reagent' settings indicated in table (a). Rotors were preincubated for 10 min at 37C in a hot-air oven, then 160#1 (60 reagent syringe capacity) of reagent B (containing ACOD, POD and 3-methyl-N-ethyl-N- [6-hydroxyethyl] aniline, MEHA) was added to the outer reagent-well with the loader reprogrammed as in table (b). The rotor was analysed using the 'Substrate II' tape with parameters set as shown in table 2, except in some preliminary studies where the general absorbance program was used.

Statistics
Results for the method comparison were evaluated by simple regression analysis and by calculating the mean +standard deviation (S.D.) difference between values obtained by the two procedures, as recommended by Altman [7].

Optimization of run conditions
The change in absorbance for aqueous solutions containing 0" to 2.5 mmol/litre oleic acid was monitored for 10min at 550 nm.
Maximum colour formation was not reached until 240s.
However, initial reaction rates, particularly for higher concentrations, were very rapid and it was impossible to obtain reliable initial absorbance readings for the assay mixtures. A bichromatic technique was therefore applied using a blanking wavelength of 690nm, and a reading wavelength of 550nm at 300 s.
For plasma or serum samples, preincubation at 37C with reagent A was found to be essential for optimal colour formation     For each species, figures quoted are the means for the same 10 plasmas run on 10 different occasions over a four-day period.

Recovery of FFA and assay precision
The

Discussion
Reported here, for the first time as far as the authors are aware, is an automated procedure for the specific enzymatic determination of serum and plasma FFA by centrifugal analysis. The method, based on commercially formulated reagents, is rapid (18 tests every 5rain), sensitive (down to 0.1 mmol/litre FFA), reproducible (see table 3), and requires only small reagent volumes (less than 250 #l/test). Further, the incorporation of within-sample blanking eliminates the need to run separate sample blanks, a time-consuming and expensive necessity for the manual kit procedure. These modifications not only improve the efficiency of FFA determination, but also reduce the cost per test to approximately one tenth of that for the manual enzymatic method. 5 lower. These results are consistent with previous comparative studies between enzymatic and extraction-based techniques described by Shimizu et al. [2], who attributed the differences to the fact that only about 90 of normal human FFA are within the C6-C18 chain length limits for the ACS used in the enzymatic method. The sensitivity of the procedure described is sufficient to allow accurate measurement of FFA even in fasting human patients, whose levels are usually between 0.13 and 0.45 mmol/ litre (Kushiro et al. [8]). As suggested by the manufacturers, heparinized plasma was found to give FFA values which were falsely elevated by about 10. However, it should be mentioned that Shimizu et al. [-2] reported that heparin added to human serum had no effect on the estimated FFA concentration. FFA in ovine plasma were highly unstable at room temperature, but could be preserved for at least 24 h at 4C. Therefore it is essential that samples should be kept cool after collection and during preparation for assay. When frozen at 20C, FFA were stable for at least two weeks. Rat plasma FFA have been reported stable for over a week at 15C, although values rose sharply thereafter (Hron and Menahan [9]). Similar traits have been observed even at ultra-low(-196C)storage temperatures (Trichopoulou et al. [10]; Bergmann et al. [11]). The method described provides a cost-effective routine FFA analysis, which can be practicably applied to large numbers of samples, making it a potentially valuable tool in both a clinical and research context.