Scandinavian Journal of Clinical and Laboratory Investigation
ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: http://www.tandfonline.com/loi/iclb20
Enzymatic microdetermination of plasma and serum free fatty acids E. Jebens & O. M. Sejersted To cite this article: E. Jebens & O. M. Sejersted (1992) Enzymatic microdetermination of plasma and serum free fatty acids, Scandinavian Journal of Clinical and Laboratory Investigation, 52:7, 717-724, DOI: 10.3109/00365519209115517 To link to this article: http://dx.doi.org/10.3109/00365519209115517
Published online: 08 Jul 2009.
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Date: 10 April 2016, At: 23:08
Scand J Clin Lab Invest 1992; 52: 717-724
Enzymatic microdetermination of plasma and serum free fatty acids E . J E B E N S & 0. M. S E J E R S T E D
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Department of Physiology, National Institute of Occupational Health, Oslo 1, Norway
Jebens E , Sejersted OM. Enzymatic microdetermination of plasma and serum free fatty acids. Scand J Clin Lab Invest 1992; 52: 717-724. A simple and sensitive enzymatic method for determination of plasma and serum fatty acids (FAs) is described. The method is based on acylation of long chain FAs by a bacterial acyl-CoA synthetase (ACS) producing equivalent amounts of acyl-CoA and AMP. AMP production was measured using the coupled reaction of myokinase (MK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) allowing fluorinate detection of NADH. Two moles of NAD were produced per mole of FA acylated. Concentrations of substrates and enzymes were kept as low as possible maintaining the ACS reaction as rate limiting. Addition of fat-free human serum albumin (HSA) to standards reduced initial reaction rates but did not affect end-point fluorescence levels. Triton X-100 partly counteracted the inhibition by HSA. To keep albumin concentration low, plasma or serum samples were diluted by 1:400. Duplicate measurements of plasma or serum FA concentrations between 0 and 2 mmol I-' can then be performed on 5 PI samples with intra- and inter-assay variation coefficients of 1.7 and 4% respectively.
Key words: acyl-CoA synthetase, clinical chemistry, fluorometry, free fatty acids Einar Jebens, Department of Physiology, National Institute of Occupational Health, P O Box 8149 Dep, N-0033 Oslo, Norway.
The enzyme acyl-CoA synthetase (ACS) (EC 6.2.1.3) from Pseudomonas aeruginosa IFD 3919 [l] has recently been introduced [ l , 2, 31 for quantitative enzymatic determinations of FA in serum and plasma. The enzyme catalyses activation of FA in the presence of ATP and CoA . ACS: FA+ ATP+CoA+ AMP+acyl-CoA+ PP,
The equilibrium of this reaction favours acylCoA production [4] and the reaction can be followed fluorimetrically using the following reactions:
(1)
Myokinase (MK). AMP+ATP+2 A D P
(2)
Pyruvatekinase (PK) 2 ADP+2 PEP+2 ATP+2 pyruvate.
Lactate dehydrogenase (LDH) (3) 2 pyruvate+2 NADH-+2 lactate+2 NADt
Activation of one mole of FA is equivalent to oxidation of two moles of NADH. When comparing reported methods [ l , 2, 31 we found great variations regarding duration of reaction and amounts of substrates and enzymes used. The reported effects of possible activators 717
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and inhibitors e.g. Triton X-100 and serum albumin were also conflicting. Shimizu et ul. [ 1, 2) assumed that the assay conditions were insignificantly affected by the albumin concentration. However, Bar-Tana et ul. 141 showed decreased specific activity of ACS from liver microsomes with concentrations of bovine serum albumin (BSA) higher than 0.1% (wIY). Miles et ul. (31 showed a dose dependent reduction of specific fluorescence (fluorescence ymol-' FA) when fatty acid free human serum albumin (HSA) was added to the standard samples. They ascribed this to an irreversible binding of a fraction of FA to albumin. Alternatively, if albumin slows the rate of the ACS reaction, the reaction in presence of albumin as described by Miles et ul. (31 may not have been completed. Therefore, the FA concentration in serum or plasma might be underestimated. We optimized reaction conditions and investigated the effect of albumin on the kinetic variables of the ACS reaction. Jt became evident that commercial preparations of auxiliary enzymes and substrates contain substantial amounts of NADH-consuming impurities. Therefore, concentrations of enzymes and substrates were varied to find the best compromise between the demand for high reaction rates and low concentrations of contaminating chemicals.
of analytical grade. Plasma and serum samples were obtained from volunteers. The fluorescence of N A D H at excitation 361.5 nm and emission 458 nm was read with an Aminco filter microfluorimeter. The reagent medium was prepared from concentrated stock solutions of CoA, N A D H and the other reagents immediately before use. The stock solution of N A D H was approximately 10 mmol I-' dissolved in carbonate buffer (80 mmol I-' Na2C03,20 mmol I-' NaHCO?). The calculation of the N A D H concentration in the stock solution was based on a molar extinction coefficient of 6270 cm-' at 340 nm. This stock solution had to be standardized at least once a week. Standards of N A D H were prepared from the stock solution diluted with the reagent medium containing no L D H . Final concentrations in the cuvette were: 25 mmol I-' triethanolamine buffer p H 8.0, 75 mmol I-' KCI, 10 mmol I-', MgC12, 1 mmol I-', EDTA, 0.085 mmol I-', ATP, 0.018 mmol I-' N A D H , 0.030 mmol I-', PEP, 0.225 mmol I-' CoA, 0.05% (vlv) Triton X-100, 300 U I-' MK, 750 U I-' PK, 400 U I-' L D H and ACS 5 U I-'. Blank and standards contained in addition 0.01% (w/v) fatty acid free HSA. The stock solution of CoA (43 mmol I-') was stabilized with SO mmol I-' dithiotreitol. FAstandards were made by dissolving the Na+salts in isopropanol and 0.25% Triton X-100 giving a final concentration about 100 ymol I-'.
MATERIALS AND METHODS ACS (EC6.2. I .3) from pseudomonas aeruginosa was purchased from Sigma Chemical Company St Louis, MO, USA. A stock was made from a solution of 10 mmol I-' phosphate buffer (K2HP04/KH2P04), 5 mmol I-' dithiotreitol and 50% ( d v ) glycerol adjusted to pH 8.0 to give an ACS concentration of 1000 U I-'. It was stored in batches of 1-3 ml at -80 "C. MK ( E C 2.7.4.3) and PK ( E C 2.7.1.40) from rabbit skeletal muscle were purchased from Boerhinger Mannheim GnibH. L D H ( E C 1.1.1.27) from bovine heart, CoA as sodium salt, ATP as disodium salt (grade l ) , PEP as trisodium salt, N A D H (grade I I I ) , fatty acid free human serum albumin (HSA) and the sodium salt of all the used FFAs were purchased from Sigma Chemical Company. SeronormK Lipid was from Nycomed Pharma AS, Oslo, Norway. All other chemicals are commercially available and
Procedure All reagents and samples were kept on ice before the analysis started. Plasma was prepared immediately from heparinized blood (30 IU ml-I) and serum was obtained by allowing blood to coagulate at room temperature for 25 min before centrifugation (250Oxg for 10 min at 20 "C). Samples were stored frozen for no longer than 12 months at -80 "C 151. Samples were thawed at 4 "C, centrifuged at 3000xg for 5 min at 4 "C and diluted 1:20 with 0.25% (v/v) Triton X-100. Finally 55 yl of the diluted samples was added to 1 ml of the reagent medium which gave a final dilution of 1:400. The assay was performed at 25 "C. After a 1 0 min pre-incubation of the reagent medium the reaction was started by adding the samples. Initial reaction rates were estimated to find optimal conditions for routine estimates of
Determination of free fatty acids
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endpoint fluorescence. Initial reaction rates were measured by manually fitting a straight line to the first linear part of the fluorescence decay curves. Optimal substrate concentrations were obtained with a high palmitate concentration (18.5 pmol I-') and a low ACS concentration (1 U I-'). Endpoint fluorescence was read after 50 min i.e. when there was no further decrease in fluorescence. If the FA concentration in plasma or serum was higher than 2 mmol I-', the samples were further diluted and the albumin concentration in blank and standards reduced accordingly.
Polmitote
!A
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Assay of standards in optimized reaction medium
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We chose to perform the FA assay as an endpoint assay. Figure 1 shows that addition of C o A to a standardized incubation medium caused a reduction in fluorescence that must have been due to impurities in the reagents. Therefore, in all assays the fatty acid samples were added when the fluorescence stabilized after about 10 min. Addition of palmitate in a concentration of 1.9 pmol I-' caused a further reduction in fluorescence at an initial rate that was dependent on the amount of ACS in the cuvette. Therefore, it is likely that the overall reactionrate wascontrolled bythe ACSreaction.
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FIG. I . Changes in fluorescence of the incubation medium containing I or 5 U I-' ACS following sequential addition of CoA (225 pmol I-') and palmitate ( 1 . 9 pmoI I-'). Original tracings of single samples are shown.
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FIG.2. Time course of decrease in fluorescence at addition of ACS (5 U I-') to the cuvette containing 0, 1 , 3 , and 5 pnol I-' (a) palmitate or (b) stearate. In both cases the standard contained 0.01% USA. Original tracings of single samples are shown.
Figure 2, a and b shows the time course for formation of acyl-CoA esters of palmitate and stearate measured as decrease in fluorescence units. The fatty acid range was 1-5 pmol I-' in the cuvette. After 30 min there was a continued slow decrease in fluorescence. However, that was paralleled by the decrease in fluorescence of the blank. Comparison of Figs 2a and 2b also reveals that initial reaction rate of palmitate and stearate were not very different. Figure 3, a and b shows standard curves of palmitate and stearate. The curves were linear within the range of 0-5 pmol I-' corresponding to a range of standards from 0 to 2 mmol I-' with a dilution of 1:400.This range was limited by the amount of N A D H remaining in blank after incubation. About 2.5 pmol I-' N A D H was oxidized in the blank during incubation. Therefore, samples that oxidized more than 10 pmol I-' N A D H had to be further diluted before the analysis was performed again. Recovery of palmitate added to plasma was
E. Jebens & 0. M . Sejersted
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104.6+6.9%. The standard deviation for 16 samples within a concentration range of 0.1-2.7 pmol I-' analysed on two occasions with dilutions 1:400 and 1:SOO respectively was 0.1 p o l I-' [6]. Removal of particles from the samples by centrifugation reduced the possibility for quenching and improved parallel readings. No significant differences were observed for 64 thawed plasma samples analysed before and after centrifugation (range 0.01-2.1 p o l I-', r=0.991, slope= 1.016) and the standard deviation was 0.05 p o l I-' [6]. The intra- and inter-assay variation coefficients equalled 1.7 and 4% respectively. A comparison was made with the titrimetric method described by Dole [7]. Figure 4 shows a correlation with r2=0.76. The slope was significantly less than unity and the Dole method showed significantly higher FA values in the low concentration range as compared with the present method. Figure 5 shows that various treatments of blood samples had profound effect on FA concentrations. Plasma obtained rapidly from cooled heparinized blood and kept cool or frozen gave the lowest values. Lipolytic activity caused serum samples to have significantly elevated FA concentrations compared with plasma. Furthermore, leaving samples at room temperature also caused the FA concentration to rise, but only in samples collected during physical activity. Hence, plasma obtained rapidly from chilled heparinized blood and stored frozen seems to be the best samples for analysis.
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P o l m i t o t e p m o l I-'
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1 2 3 4 5 S t e a r a t e p m o l I-'
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Fic. 3. Standard curves for (a) palmitate and (b) stearate, based on decrease in fluorescence after 30 min incubation. Blank values were subtracted. The standards contained 0.01%HSA and 0.075% Triton. The means of three parallels are shown.
Enzyme concentrations
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Compared to recommended amounts of enzymes for AMP analysis described by Lowry and Passonneau [S] MK could be reduced by 17% from 360 to 300 U I-', PK had to be increased 2.5 fold from 300 to 750 U I-', whereas LDH could be maintained at 400 U I-', to obtain maximal rates for the ACS reaction. These experiments were performed using a saturated solution of palmitate as substrate.
FA p m o l I-'(Enzymotic)
FIG. 4. Comparison between plasma samples analysed by the present enzymatic method (abscissa) and the Dole method (ordinate). The values are means of two replicates.
Background fluorescence Some reagents contain NADH oxidizing compounds that increase the unspecific fluor-
Determination of free fatty acids
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0Frozen at -80°C Frozen at -20°C
hzsl 1 h at 0% frozen at -20°C 1 h at 24°C. frozen at -20°C
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FIG.5. Comparison of FA content in plasma and serum at rest and after a bout of physical exercise] After preparation the samples were instantly frozen at -80 "C, instantly frozen at -20 "C, kept 1 h at 0 "C and'then frozen at -20 "C, kept 1 h at 24 "C and then frozen at -20 "C.
escence and reduce the sensitivity and precision of the assay. CoA and ATP are contaminated with ADP and AMP, PEP with pyruvate, and NADH with AMP. The concentrations of these reagents were therefore minimized with the least possible effect on overall reaction rate. To define the required amount of CoA and ATP their Km values for the ACS reaction were determined to 85 and 27 kmol I-' respectively. Reaction rate was maintained with concentrations of CoA and ATP 3 times Km in the reagent medium. The concentration of PEP was chosen equal to the reported Km for its reaction with PK. This makes ADP the rate limiting substrate in the PK reaction [8]. Finally, ACS contains some minor NADH oxidizing impurities, which could be FA. The concentration of NADH was adjusted to 18 p o l I-' that would leave about 15 kmol I-' for reaction with added FA since fluorescence of the blank due to impurities decreased by 14% during a 10 min incubation.
tration. Figure 6 shows a pH optimum at 7.9. Initial rate was lower by 11% at pH 8.0. However, since previous investigators performed their assays at pH 8, we chose the same value for the standard incubation.
I
.-C
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.-mC A-
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FIG.6. Effect of pH on the initial reaction rate after Effect of p H Initial reaction rates at various pH was determined in 25 mmol I-' imidazole buffer using a high palmitate and low ACS concen-
addition of ACS. The reactions were performed in 25 mmol I-' imidazolelHC1 buffer at 23.5 "C. Concentrations were of Triton X-100 0.075% (v/v), ACS 1 U I-', Na-palmitate 18.5 pmol I-' and other reagents as described in methods. The means of two parallels are shown.
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Effect of albumin and Triton X-100 Serum albumin has several binding sites for FA [9, lo]. HSA added to the reagent medium will therefore compete with ACS for the FAs. Figure 7 shows the effect of HSA on the rate of formation of palmitoyl-CoA. Increasing concentrations of HSA between 0.004 and 0.1YO (w/v) reduced the initial reaction rate for a given concentration of FA in a concentration dependent manner. The solubility of F A in aqueous medium will increase by adding a detergent. In addition Triton X-100 is described to increase the specific activity of ACS [4].With 0.05% Triton the reaction rate was nearly two fold higher as compared with 0.025% Triton. Increasing the Triton concentration further depressed palmitoyl-CoA formation rate slightly. With albumin present at 0.1%,Triton again doubled the formation rate of palmitoyl-CoA with little effect of higher concentrations (Fig. 8). However, Triton could not counteract the reduction in formation rate of palmitoyl-CoA caused by albumin. The effect of Triton was striking using SeronomTM Lipid as substrate. N o FAs could be detected without Triton whereas dilution to a final concentration of 0.05% allowed quantification of FA with an accuracy of 102% of the reported standard FA content of Seronorm.
Normal plasma and serum contain approximately 4% (w/v) albumin. As a large fraction of FAs are bound to albumin the samples cannot be deproteinized without removing some of the FAs. Therefore, the alternative approach to reduced concentration of albumin in the cuvette was to dilute the samples about 400 times giving a final albumin concentration of approximately 0.01%. Figure 9 shows the effect on the initial reaction rate with plasma and serum samples diluted 200 and 400 times compared to standards
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FIG.8. Effect of Triton X-100 on the initial reaction rate of palmitate acylation at pH 8.0 in the presence of 0.01% HSA. The means of three parallels are 045% (0).0.1% shown: Triton X-100: 0.025% (0). (A),0.125"/0 (0),0.25% (W).
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FIG.7. Effect of addition of fatty acid free HSA on the initial reaction rate of paimitate acylation at pH 8.0. The reagent medium was pre-incubated for 40 min before the reaction was started by adding Napalmitate. The means of three parallels are shown. HSA: 0% (0). 0.004% (0).0.01% ( A ) , 0.02'X ( A ) 0.1% (0) added to the standards.
0
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FIG.9. Initial reaction rate at different FA conccntrations. Standards with (0)and without (0)0.01'% HSA. Plasma at dilution 1:200 (A)and 1:400 ( 7 ) and serum at dilutions of 1:200 (0) and 1:400 (0). ACS concentration was 5 U I-'. The means o f three parallels are shown.
Determination of free fatty acids TABLE I. Effect of HSA on reduction in fluorescence measured after 40, 80, and 120 min. Standards of palmitate (0, 1 , 3, 5 pmol I-') were analysed with 0, 0.01, and 0.02% HSA added. Concentration of ACS was 1.5 U I-'. The means of three parallels are shown. A Fluorescence units Time min 40
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80 I20
HSA
Palmitate pmol I-' 3 5
% wlv
1
0 0.01 0.02 0 0.01 0.02 0 0.01 0.02
1.3 1.2 0.6 1.4 1.5 0.9 1.4 1.5 1.4
3.6 3.7 2.3 3.8 4.6 3.1 4.0 4.3 3.7
7.0 5.9 4.3 7.0 6.8 5.6 7.3 7.3 6.8
with no or 0.01% HSA. The initial reaction rate at a dilution of 400 is close to the rate observed after adding 0.01% HSA to standards. FAs do not bind irreversibly to HSA, but its presence reduces the reaction rate. This is evident from the complete, quantitative reaction of FA with CoA that could be obtained by extending the incubation time (Table I). In the standard incubation medium reaction rate was increased by using an ACS concentration of 5 mU m1-I as compared with the above rates obtained with 1 mU ml-' (Fig. 1). Endpoint recovery of FA in standards with and without added HSA was not significantly different. In separate experiments it was found out that 30 mmol I-' lactate and 1 mmol I-' P-OHbutyrate did not affect recovery.
DISCUSSION The present modification of an enzymatic fluorimetric method for determination of FAs in biological samples was carried out with the purpose of minimizing use of costly reagents and identify conditions that would affect reaction rate. Optimum conditions for the formation of acyl-CoA esters could be determined when the ACS reaction was the rate limiting step. In both the original spectrophotometric method [ l , 21 as well as with the modifications made by Miles et al. [31 who adjusted the
723
method to fluorimetry, a large excessof auxilliary enzymes and substrates were used. We have presently shown that reducing MK, PK, and LDH by 91%, 5070, and 67% compared to Miles et al's recommendations did not affect reaction rate. Kather & Wieland [ l l ] have published a luminescence method for FA determination. The use of additional reagents as well as little or no improvement in sensitivity and precision with their method, favours the use of a fluorimetric method. Fluorimetry is known to be sensitive to impurities of reagents. In order to gain high sensitivity it is important to keep the blank values low [8]. The described method and many other fluorimetric methods are based upon the fluorescence of NADH. Impurities that lead to oxidation of NADH will increase blank values and reduce sensitivity. We found that the total amount of impurities in commercial lots of CoA, NADH, ATP, and PEP (probably AMP, ADP, and pyruvate formed by decomposition) and in ACS (probably contaminating FAs) caused substantial decrease in the blank fluorescence. Since the ACS reaction follows Michaelis-Menten kinetics 141 with regard to CoA and ATP the conentration of these substrates could be kept at 3 times Km which gave acceptable blank values and maintained a reaction rate of about 75% of Vmax. Bar-Tana et al. [4] found that albumin had both a stimulatory and an inhibitory effect on the ACS reaction. Unlike Bar-Tana et al. we found that albumin reduced the initial reaction rate at all concentrations tested in a concentration dependent manner. Supposedly only unbound FAs act as substrate for ACS. Therefore, reduced reaction rate is easily explained by FA binding to albumin. The binding sites on albumin have different affinities for FAs (9, 121. Therefore, at high FA concentrations rapid initial reaction rates are obtained since FA'S are easily detached from albumin. At lower concentrations release will be slower from the higher affinity sites. Therefore, the relationship between FA concentration and initial reactionrate in the presence of albumin tends to be sigmoidal (see Fig. 8). We were unable to detect any irreversible binding of FA to albumin as proposed by Miles et al. [3). Increasing ACS and/or prolonging incubation time always resulted in close to 100% recovery. Therefore, it is possible to obtain complete release of FA
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from albumin with the present modification of the method. It is not quite clear why Triton X-100 facilitates the formation of acyl-CoA esters. Reduced hydrophobic character of the free FAs is probably important. However, the inhibitory effect of albumin could also partly be reversed by Triton X-100. This effect may be due t o increased dissociation of FA from albumin. Since Triton X-100 alone had a biphasic effect in that reaction rate increased at low concentrations, but fell again at high concentrations, it seems likely that excess Triton X-100 actually inhibits ACS. With albumin the inhibitory effect of Triton X-100 could not be detected which could imply that an increased rate of FA release from albumin outweighs the enzyme inhibition. Since Triton X-100 was unable to restore full activity with albumin present the assay was carried out with as low an ovalbumin concentration as possible. Therefore, full recovery of FAs is highly dependent on the incubation time which in turn is influenced by the amount of ACS and albumin present. The low concentration of H S A makes initial reaction rates sensitive to differences in protein concentration in the samples. This can be avoided by decreased dilution of the samples, but at the expense of reduced reaction rate and increased time to endpoint. From a practical point of view this would be unfortunate. If plasma albumin concentrations a r e higher than normal in the samples it is possible to reduce the concentration by increased dilution. The recommended or even further dilution of the samples will also reduce occurrence of samples out of assay range. The Dole method overestimated the FA concentrations in the lower concentration range. This method includes an extraction with inorganic solvents that could make the method sensitive to high concentrations of non-FA organic acids like lactic acid and P-OH-butyric acid. Those acids had no effect upon recovery in the present enzymatic method. The ACS enzyme reacts with FAs with carbon chain length from 6 to 18 [ 11, which accounts for more than 95% of the FAs in serum and plasma 19,121. Therefore, the specificity of the present assay is sufficient for determination of FA concentration in plasma and serum samples. It is clear that serum is not a suitable source for correct
FA determination unless lipolysis during preparation can be prevented [17]. The present modifications of an enzymatic determination of FAs in plasma and serum makes the method simpler and inexpensive and well suited for automation.
ACKNOWLEDGMENT We are grateful to Astrid Bolling for her excellent technical assistance and for valuable help from our colleague Odd Vaague.
REFERENCES I Shimizu S, Inoue K, Tani Y , Yamada H. Enzymatic microdetermination of serum free fatty acids. Anal Biochem 1979; 98: 341-5. 2 Shimizu S, Tani Y, Yamada H, Tahata M , Murachi T. Enzymatic determination of serumfree fatty acids: A colorimetric method. Anal Biochem 1980; 107: 193-8. 3 Miles J , Glasscock R, Aikens J , Gerich J , Haymond M. Microfluorimetric method for the determination of free fatty acids in plasma. J Lipid Res 1983; 24: 96- 100. 4 Bar-Tana J, Rose G , Shapiro B. The purification and properties of microsomal palmitoyl-coenzyme A synthetase. Biochem J 1971; 122: 353-62. 5 Kallner A , Broughton PMG, Magid E, editors. Improvement of comparability and compatibility of laboratory assay results in life sciences. Scand J Clin Lab Invest 1990; 50: 92. 6 Fenstad GU, Kjaernes M, Wallcw L. Robust estimation of standard deviation. J Statist Comput Simul 1980; 10: 113-32. 7 Dole VP. A relation between non-esterified fatty acids in plasma and the metabolism of glucose. J Clin Invest 1957; 35: 150-4. 8 Lowry OH, Passonneau JV. A flexible system of enzymatic analysis. Academic Press 1972. 9 Spector AA. Plasma lipid transport. Clin Physiol Biochem 1984; 2: 123-34. 10 Ashbrook JD, Spector AA, Santos EC, Fletcher JE. Long chain fatty acid binding to human plasma albumin. J Biol Chem 1975; 250: 2333-8. I 1 Kather H, Wieland E. Bioluminiscent determination of free fatty acids. Anal Biochem 1984; 140: 349-53. 12 Spector AA. Fatty acid binding to albumin. J Lipid Res 1975; 16: 165-79. 13 Degen AJM, Vies J van der. Enzymatic microdetermination of free fatty acids in plasma o f animals using paraoxon to prcvent lipolysis. J Clin Lab Invest 1985: 45: 283-5. Received 5 August 1991 Accepted 18 May 1992
ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: http://www.tandfonline.com/loi/iclb20
Enzymatic microdetermination of plasma and serum free fatty acids E. Jebens & O. M. Sejersted To cite this article: E. Jebens & O. M. Sejersted (1992) Enzymatic microdetermination of plasma and serum free fatty acids, Scandinavian Journal of Clinical and Laboratory Investigation, 52:7, 717-724, DOI: 10.3109/00365519209115517 To link to this article: http://dx.doi.org/10.3109/00365519209115517
Published online: 08 Jul 2009.
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Article views: 15
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Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iclb20 Download by: [RMIT University Library]
Date: 10 April 2016, At: 23:08
Scand J Clin Lab Invest 1992; 52: 717-724
Enzymatic microdetermination of plasma and serum free fatty acids E . J E B E N S & 0. M. S E J E R S T E D
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Department of Physiology, National Institute of Occupational Health, Oslo 1, Norway
Jebens E , Sejersted OM. Enzymatic microdetermination of plasma and serum free fatty acids. Scand J Clin Lab Invest 1992; 52: 717-724. A simple and sensitive enzymatic method for determination of plasma and serum fatty acids (FAs) is described. The method is based on acylation of long chain FAs by a bacterial acyl-CoA synthetase (ACS) producing equivalent amounts of acyl-CoA and AMP. AMP production was measured using the coupled reaction of myokinase (MK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) allowing fluorinate detection of NADH. Two moles of NAD were produced per mole of FA acylated. Concentrations of substrates and enzymes were kept as low as possible maintaining the ACS reaction as rate limiting. Addition of fat-free human serum albumin (HSA) to standards reduced initial reaction rates but did not affect end-point fluorescence levels. Triton X-100 partly counteracted the inhibition by HSA. To keep albumin concentration low, plasma or serum samples were diluted by 1:400. Duplicate measurements of plasma or serum FA concentrations between 0 and 2 mmol I-' can then be performed on 5 PI samples with intra- and inter-assay variation coefficients of 1.7 and 4% respectively.
Key words: acyl-CoA synthetase, clinical chemistry, fluorometry, free fatty acids Einar Jebens, Department of Physiology, National Institute of Occupational Health, P O Box 8149 Dep, N-0033 Oslo, Norway.
The enzyme acyl-CoA synthetase (ACS) (EC 6.2.1.3) from Pseudomonas aeruginosa IFD 3919 [l] has recently been introduced [ l , 2, 31 for quantitative enzymatic determinations of FA in serum and plasma. The enzyme catalyses activation of FA in the presence of ATP and CoA . ACS: FA+ ATP+CoA+ AMP+acyl-CoA+ PP,
The equilibrium of this reaction favours acylCoA production [4] and the reaction can be followed fluorimetrically using the following reactions:
(1)
Myokinase (MK). AMP+ATP+2 A D P
(2)
Pyruvatekinase (PK) 2 ADP+2 PEP+2 ATP+2 pyruvate.
Lactate dehydrogenase (LDH) (3) 2 pyruvate+2 NADH-+2 lactate+2 NADt
Activation of one mole of FA is equivalent to oxidation of two moles of NADH. When comparing reported methods [ l , 2, 31 we found great variations regarding duration of reaction and amounts of substrates and enzymes used. The reported effects of possible activators 717
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and inhibitors e.g. Triton X-100 and serum albumin were also conflicting. Shimizu et ul. [ 1, 2) assumed that the assay conditions were insignificantly affected by the albumin concentration. However, Bar-Tana et ul. 141 showed decreased specific activity of ACS from liver microsomes with concentrations of bovine serum albumin (BSA) higher than 0.1% (wIY). Miles et ul. (31 showed a dose dependent reduction of specific fluorescence (fluorescence ymol-' FA) when fatty acid free human serum albumin (HSA) was added to the standard samples. They ascribed this to an irreversible binding of a fraction of FA to albumin. Alternatively, if albumin slows the rate of the ACS reaction, the reaction in presence of albumin as described by Miles et ul. (31 may not have been completed. Therefore, the FA concentration in serum or plasma might be underestimated. We optimized reaction conditions and investigated the effect of albumin on the kinetic variables of the ACS reaction. Jt became evident that commercial preparations of auxiliary enzymes and substrates contain substantial amounts of NADH-consuming impurities. Therefore, concentrations of enzymes and substrates were varied to find the best compromise between the demand for high reaction rates and low concentrations of contaminating chemicals.
of analytical grade. Plasma and serum samples were obtained from volunteers. The fluorescence of N A D H at excitation 361.5 nm and emission 458 nm was read with an Aminco filter microfluorimeter. The reagent medium was prepared from concentrated stock solutions of CoA, N A D H and the other reagents immediately before use. The stock solution of N A D H was approximately 10 mmol I-' dissolved in carbonate buffer (80 mmol I-' Na2C03,20 mmol I-' NaHCO?). The calculation of the N A D H concentration in the stock solution was based on a molar extinction coefficient of 6270 cm-' at 340 nm. This stock solution had to be standardized at least once a week. Standards of N A D H were prepared from the stock solution diluted with the reagent medium containing no L D H . Final concentrations in the cuvette were: 25 mmol I-' triethanolamine buffer p H 8.0, 75 mmol I-' KCI, 10 mmol I-', MgC12, 1 mmol I-', EDTA, 0.085 mmol I-', ATP, 0.018 mmol I-' N A D H , 0.030 mmol I-', PEP, 0.225 mmol I-' CoA, 0.05% (vlv) Triton X-100, 300 U I-' MK, 750 U I-' PK, 400 U I-' L D H and ACS 5 U I-'. Blank and standards contained in addition 0.01% (w/v) fatty acid free HSA. The stock solution of CoA (43 mmol I-') was stabilized with SO mmol I-' dithiotreitol. FAstandards were made by dissolving the Na+salts in isopropanol and 0.25% Triton X-100 giving a final concentration about 100 ymol I-'.
MATERIALS AND METHODS ACS (EC6.2. I .3) from pseudomonas aeruginosa was purchased from Sigma Chemical Company St Louis, MO, USA. A stock was made from a solution of 10 mmol I-' phosphate buffer (K2HP04/KH2P04), 5 mmol I-' dithiotreitol and 50% ( d v ) glycerol adjusted to pH 8.0 to give an ACS concentration of 1000 U I-'. It was stored in batches of 1-3 ml at -80 "C. MK ( E C 2.7.4.3) and PK ( E C 2.7.1.40) from rabbit skeletal muscle were purchased from Boerhinger Mannheim GnibH. L D H ( E C 1.1.1.27) from bovine heart, CoA as sodium salt, ATP as disodium salt (grade l ) , PEP as trisodium salt, N A D H (grade I I I ) , fatty acid free human serum albumin (HSA) and the sodium salt of all the used FFAs were purchased from Sigma Chemical Company. SeronormK Lipid was from Nycomed Pharma AS, Oslo, Norway. All other chemicals are commercially available and
Procedure All reagents and samples were kept on ice before the analysis started. Plasma was prepared immediately from heparinized blood (30 IU ml-I) and serum was obtained by allowing blood to coagulate at room temperature for 25 min before centrifugation (250Oxg for 10 min at 20 "C). Samples were stored frozen for no longer than 12 months at -80 "C 151. Samples were thawed at 4 "C, centrifuged at 3000xg for 5 min at 4 "C and diluted 1:20 with 0.25% (v/v) Triton X-100. Finally 55 yl of the diluted samples was added to 1 ml of the reagent medium which gave a final dilution of 1:400. The assay was performed at 25 "C. After a 1 0 min pre-incubation of the reagent medium the reaction was started by adding the samples. Initial reaction rates were estimated to find optimal conditions for routine estimates of
Determination of free fatty acids
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endpoint fluorescence. Initial reaction rates were measured by manually fitting a straight line to the first linear part of the fluorescence decay curves. Optimal substrate concentrations were obtained with a high palmitate concentration (18.5 pmol I-') and a low ACS concentration (1 U I-'). Endpoint fluorescence was read after 50 min i.e. when there was no further decrease in fluorescence. If the FA concentration in plasma or serum was higher than 2 mmol I-', the samples were further diluted and the albumin concentration in blank and standards reduced accordingly.
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We chose to perform the FA assay as an endpoint assay. Figure 1 shows that addition of C o A to a standardized incubation medium caused a reduction in fluorescence that must have been due to impurities in the reagents. Therefore, in all assays the fatty acid samples were added when the fluorescence stabilized after about 10 min. Addition of palmitate in a concentration of 1.9 pmol I-' caused a further reduction in fluorescence at an initial rate that was dependent on the amount of ACS in the cuvette. Therefore, it is likely that the overall reactionrate wascontrolled bythe ACSreaction.
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FIG. I . Changes in fluorescence of the incubation medium containing I or 5 U I-' ACS following sequential addition of CoA (225 pmol I-') and palmitate ( 1 . 9 pmoI I-'). Original tracings of single samples are shown.
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FIG.2. Time course of decrease in fluorescence at addition of ACS (5 U I-') to the cuvette containing 0, 1 , 3 , and 5 pnol I-' (a) palmitate or (b) stearate. In both cases the standard contained 0.01% USA. Original tracings of single samples are shown.
Figure 2, a and b shows the time course for formation of acyl-CoA esters of palmitate and stearate measured as decrease in fluorescence units. The fatty acid range was 1-5 pmol I-' in the cuvette. After 30 min there was a continued slow decrease in fluorescence. However, that was paralleled by the decrease in fluorescence of the blank. Comparison of Figs 2a and 2b also reveals that initial reaction rate of palmitate and stearate were not very different. Figure 3, a and b shows standard curves of palmitate and stearate. The curves were linear within the range of 0-5 pmol I-' corresponding to a range of standards from 0 to 2 mmol I-' with a dilution of 1:400.This range was limited by the amount of N A D H remaining in blank after incubation. About 2.5 pmol I-' N A D H was oxidized in the blank during incubation. Therefore, samples that oxidized more than 10 pmol I-' N A D H had to be further diluted before the analysis was performed again. Recovery of palmitate added to plasma was
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104.6+6.9%. The standard deviation for 16 samples within a concentration range of 0.1-2.7 pmol I-' analysed on two occasions with dilutions 1:400 and 1:SOO respectively was 0.1 p o l I-' [6]. Removal of particles from the samples by centrifugation reduced the possibility for quenching and improved parallel readings. No significant differences were observed for 64 thawed plasma samples analysed before and after centrifugation (range 0.01-2.1 p o l I-', r=0.991, slope= 1.016) and the standard deviation was 0.05 p o l I-' [6]. The intra- and inter-assay variation coefficients equalled 1.7 and 4% respectively. A comparison was made with the titrimetric method described by Dole [7]. Figure 4 shows a correlation with r2=0.76. The slope was significantly less than unity and the Dole method showed significantly higher FA values in the low concentration range as compared with the present method. Figure 5 shows that various treatments of blood samples had profound effect on FA concentrations. Plasma obtained rapidly from cooled heparinized blood and kept cool or frozen gave the lowest values. Lipolytic activity caused serum samples to have significantly elevated FA concentrations compared with plasma. Furthermore, leaving samples at room temperature also caused the FA concentration to rise, but only in samples collected during physical activity. Hence, plasma obtained rapidly from chilled heparinized blood and stored frozen seems to be the best samples for analysis.
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Fic. 3. Standard curves for (a) palmitate and (b) stearate, based on decrease in fluorescence after 30 min incubation. Blank values were subtracted. The standards contained 0.01%HSA and 0.075% Triton. The means of three parallels are shown.
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Compared to recommended amounts of enzymes for AMP analysis described by Lowry and Passonneau [S] MK could be reduced by 17% from 360 to 300 U I-', PK had to be increased 2.5 fold from 300 to 750 U I-', whereas LDH could be maintained at 400 U I-', to obtain maximal rates for the ACS reaction. These experiments were performed using a saturated solution of palmitate as substrate.
FA p m o l I-'(Enzymotic)
FIG. 4. Comparison between plasma samples analysed by the present enzymatic method (abscissa) and the Dole method (ordinate). The values are means of two replicates.
Background fluorescence Some reagents contain NADH oxidizing compounds that increase the unspecific fluor-
Determination of free fatty acids
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FIG.5. Comparison of FA content in plasma and serum at rest and after a bout of physical exercise] After preparation the samples were instantly frozen at -80 "C, instantly frozen at -20 "C, kept 1 h at 0 "C and'then frozen at -20 "C, kept 1 h at 24 "C and then frozen at -20 "C.
escence and reduce the sensitivity and precision of the assay. CoA and ATP are contaminated with ADP and AMP, PEP with pyruvate, and NADH with AMP. The concentrations of these reagents were therefore minimized with the least possible effect on overall reaction rate. To define the required amount of CoA and ATP their Km values for the ACS reaction were determined to 85 and 27 kmol I-' respectively. Reaction rate was maintained with concentrations of CoA and ATP 3 times Km in the reagent medium. The concentration of PEP was chosen equal to the reported Km for its reaction with PK. This makes ADP the rate limiting substrate in the PK reaction [8]. Finally, ACS contains some minor NADH oxidizing impurities, which could be FA. The concentration of NADH was adjusted to 18 p o l I-' that would leave about 15 kmol I-' for reaction with added FA since fluorescence of the blank due to impurities decreased by 14% during a 10 min incubation.
tration. Figure 6 shows a pH optimum at 7.9. Initial rate was lower by 11% at pH 8.0. However, since previous investigators performed their assays at pH 8, we chose the same value for the standard incubation.
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FIG.6. Effect of pH on the initial reaction rate after Effect of p H Initial reaction rates at various pH was determined in 25 mmol I-' imidazole buffer using a high palmitate and low ACS concen-
addition of ACS. The reactions were performed in 25 mmol I-' imidazolelHC1 buffer at 23.5 "C. Concentrations were of Triton X-100 0.075% (v/v), ACS 1 U I-', Na-palmitate 18.5 pmol I-' and other reagents as described in methods. The means of two parallels are shown.
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Effect of albumin and Triton X-100 Serum albumin has several binding sites for FA [9, lo]. HSA added to the reagent medium will therefore compete with ACS for the FAs. Figure 7 shows the effect of HSA on the rate of formation of palmitoyl-CoA. Increasing concentrations of HSA between 0.004 and 0.1YO (w/v) reduced the initial reaction rate for a given concentration of FA in a concentration dependent manner. The solubility of F A in aqueous medium will increase by adding a detergent. In addition Triton X-100 is described to increase the specific activity of ACS [4].With 0.05% Triton the reaction rate was nearly two fold higher as compared with 0.025% Triton. Increasing the Triton concentration further depressed palmitoyl-CoA formation rate slightly. With albumin present at 0.1%,Triton again doubled the formation rate of palmitoyl-CoA with little effect of higher concentrations (Fig. 8). However, Triton could not counteract the reduction in formation rate of palmitoyl-CoA caused by albumin. The effect of Triton was striking using SeronomTM Lipid as substrate. N o FAs could be detected without Triton whereas dilution to a final concentration of 0.05% allowed quantification of FA with an accuracy of 102% of the reported standard FA content of Seronorm.
Normal plasma and serum contain approximately 4% (w/v) albumin. As a large fraction of FAs are bound to albumin the samples cannot be deproteinized without removing some of the FAs. Therefore, the alternative approach to reduced concentration of albumin in the cuvette was to dilute the samples about 400 times giving a final albumin concentration of approximately 0.01%. Figure 9 shows the effect on the initial reaction rate with plasma and serum samples diluted 200 and 400 times compared to standards
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FIG.8. Effect of Triton X-100 on the initial reaction rate of palmitate acylation at pH 8.0 in the presence of 0.01% HSA. The means of three parallels are 045% (0).0.1% shown: Triton X-100: 0.025% (0). (A),0.125"/0 (0),0.25% (W).
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FIG.7. Effect of addition of fatty acid free HSA on the initial reaction rate of paimitate acylation at pH 8.0. The reagent medium was pre-incubated for 40 min before the reaction was started by adding Napalmitate. The means of three parallels are shown. HSA: 0% (0). 0.004% (0).0.01% ( A ) , 0.02'X ( A ) 0.1% (0) added to the standards.
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FIG.9. Initial reaction rate at different FA conccntrations. Standards with (0)and without (0)0.01'% HSA. Plasma at dilution 1:200 (A)and 1:400 ( 7 ) and serum at dilutions of 1:200 (0) and 1:400 (0). ACS concentration was 5 U I-'. The means o f three parallels are shown.
Determination of free fatty acids TABLE I. Effect of HSA on reduction in fluorescence measured after 40, 80, and 120 min. Standards of palmitate (0, 1 , 3, 5 pmol I-') were analysed with 0, 0.01, and 0.02% HSA added. Concentration of ACS was 1.5 U I-'. The means of three parallels are shown. A Fluorescence units Time min 40
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% wlv
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0 0.01 0.02 0 0.01 0.02 0 0.01 0.02
1.3 1.2 0.6 1.4 1.5 0.9 1.4 1.5 1.4
3.6 3.7 2.3 3.8 4.6 3.1 4.0 4.3 3.7
7.0 5.9 4.3 7.0 6.8 5.6 7.3 7.3 6.8
with no or 0.01% HSA. The initial reaction rate at a dilution of 400 is close to the rate observed after adding 0.01% HSA to standards. FAs do not bind irreversibly to HSA, but its presence reduces the reaction rate. This is evident from the complete, quantitative reaction of FA with CoA that could be obtained by extending the incubation time (Table I). In the standard incubation medium reaction rate was increased by using an ACS concentration of 5 mU m1-I as compared with the above rates obtained with 1 mU ml-' (Fig. 1). Endpoint recovery of FA in standards with and without added HSA was not significantly different. In separate experiments it was found out that 30 mmol I-' lactate and 1 mmol I-' P-OHbutyrate did not affect recovery.
DISCUSSION The present modification of an enzymatic fluorimetric method for determination of FAs in biological samples was carried out with the purpose of minimizing use of costly reagents and identify conditions that would affect reaction rate. Optimum conditions for the formation of acyl-CoA esters could be determined when the ACS reaction was the rate limiting step. In both the original spectrophotometric method [ l , 21 as well as with the modifications made by Miles et al. [31 who adjusted the
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method to fluorimetry, a large excessof auxilliary enzymes and substrates were used. We have presently shown that reducing MK, PK, and LDH by 91%, 5070, and 67% compared to Miles et al's recommendations did not affect reaction rate. Kather & Wieland [ l l ] have published a luminescence method for FA determination. The use of additional reagents as well as little or no improvement in sensitivity and precision with their method, favours the use of a fluorimetric method. Fluorimetry is known to be sensitive to impurities of reagents. In order to gain high sensitivity it is important to keep the blank values low [8]. The described method and many other fluorimetric methods are based upon the fluorescence of NADH. Impurities that lead to oxidation of NADH will increase blank values and reduce sensitivity. We found that the total amount of impurities in commercial lots of CoA, NADH, ATP, and PEP (probably AMP, ADP, and pyruvate formed by decomposition) and in ACS (probably contaminating FAs) caused substantial decrease in the blank fluorescence. Since the ACS reaction follows Michaelis-Menten kinetics 141 with regard to CoA and ATP the conentration of these substrates could be kept at 3 times Km which gave acceptable blank values and maintained a reaction rate of about 75% of Vmax. Bar-Tana et al. [4] found that albumin had both a stimulatory and an inhibitory effect on the ACS reaction. Unlike Bar-Tana et al. we found that albumin reduced the initial reaction rate at all concentrations tested in a concentration dependent manner. Supposedly only unbound FAs act as substrate for ACS. Therefore, reduced reaction rate is easily explained by FA binding to albumin. The binding sites on albumin have different affinities for FAs (9, 121. Therefore, at high FA concentrations rapid initial reaction rates are obtained since FA'S are easily detached from albumin. At lower concentrations release will be slower from the higher affinity sites. Therefore, the relationship between FA concentration and initial reactionrate in the presence of albumin tends to be sigmoidal (see Fig. 8). We were unable to detect any irreversible binding of FA to albumin as proposed by Miles et al. [3). Increasing ACS and/or prolonging incubation time always resulted in close to 100% recovery. Therefore, it is possible to obtain complete release of FA
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from albumin with the present modification of the method. It is not quite clear why Triton X-100 facilitates the formation of acyl-CoA esters. Reduced hydrophobic character of the free FAs is probably important. However, the inhibitory effect of albumin could also partly be reversed by Triton X-100. This effect may be due t o increased dissociation of FA from albumin. Since Triton X-100 alone had a biphasic effect in that reaction rate increased at low concentrations, but fell again at high concentrations, it seems likely that excess Triton X-100 actually inhibits ACS. With albumin the inhibitory effect of Triton X-100 could not be detected which could imply that an increased rate of FA release from albumin outweighs the enzyme inhibition. Since Triton X-100 was unable to restore full activity with albumin present the assay was carried out with as low an ovalbumin concentration as possible. Therefore, full recovery of FAs is highly dependent on the incubation time which in turn is influenced by the amount of ACS and albumin present. The low concentration of H S A makes initial reaction rates sensitive to differences in protein concentration in the samples. This can be avoided by decreased dilution of the samples, but at the expense of reduced reaction rate and increased time to endpoint. From a practical point of view this would be unfortunate. If plasma albumin concentrations a r e higher than normal in the samples it is possible to reduce the concentration by increased dilution. The recommended or even further dilution of the samples will also reduce occurrence of samples out of assay range. The Dole method overestimated the FA concentrations in the lower concentration range. This method includes an extraction with inorganic solvents that could make the method sensitive to high concentrations of non-FA organic acids like lactic acid and P-OH-butyric acid. Those acids had no effect upon recovery in the present enzymatic method. The ACS enzyme reacts with FAs with carbon chain length from 6 to 18 [ 11, which accounts for more than 95% of the FAs in serum and plasma 19,121. Therefore, the specificity of the present assay is sufficient for determination of FA concentration in plasma and serum samples. It is clear that serum is not a suitable source for correct
FA determination unless lipolysis during preparation can be prevented [17]. The present modifications of an enzymatic determination of FAs in plasma and serum makes the method simpler and inexpensive and well suited for automation.
ACKNOWLEDGMENT We are grateful to Astrid Bolling for her excellent technical assistance and for valuable help from our colleague Odd Vaague.
REFERENCES I Shimizu S, Inoue K, Tani Y , Yamada H. Enzymatic microdetermination of serum free fatty acids. Anal Biochem 1979; 98: 341-5. 2 Shimizu S, Tani Y, Yamada H, Tahata M , Murachi T. Enzymatic determination of serumfree fatty acids: A colorimetric method. Anal Biochem 1980; 107: 193-8. 3 Miles J , Glasscock R, Aikens J , Gerich J , Haymond M. Microfluorimetric method for the determination of free fatty acids in plasma. J Lipid Res 1983; 24: 96- 100. 4 Bar-Tana J, Rose G , Shapiro B. The purification and properties of microsomal palmitoyl-coenzyme A synthetase. Biochem J 1971; 122: 353-62. 5 Kallner A , Broughton PMG, Magid E, editors. Improvement of comparability and compatibility of laboratory assay results in life sciences. Scand J Clin Lab Invest 1990; 50: 92. 6 Fenstad GU, Kjaernes M, Wallcw L. Robust estimation of standard deviation. J Statist Comput Simul 1980; 10: 113-32. 7 Dole VP. A relation between non-esterified fatty acids in plasma and the metabolism of glucose. J Clin Invest 1957; 35: 150-4. 8 Lowry OH, Passonneau JV. A flexible system of enzymatic analysis. Academic Press 1972. 9 Spector AA. Plasma lipid transport. Clin Physiol Biochem 1984; 2: 123-34. 10 Ashbrook JD, Spector AA, Santos EC, Fletcher JE. Long chain fatty acid binding to human plasma albumin. J Biol Chem 1975; 250: 2333-8. I 1 Kather H, Wieland E. Bioluminiscent determination of free fatty acids. Anal Biochem 1984; 140: 349-53. 12 Spector AA. Fatty acid binding to albumin. J Lipid Res 1975; 16: 165-79. 13 Degen AJM, Vies J van der. Enzymatic microdetermination of free fatty acids in plasma o f animals using paraoxon to prcvent lipolysis. J Clin Lab Invest 1985: 45: 283-5. Received 5 August 1991 Accepted 18 May 1992
