ANALYTICALBIOCHEMISTRY70, 156-166 (1976)

Quantitative Determination of Co:o-Cls:3 Serum Nonesterified Fatty Acids by Gas- Liquid Chromatography GEORGE H . DEVRIES, PETER MAMUNES, CHARLES D . MILLER, AND DONALD M . HAYWARD

Department of Biochemistry and Department of Pediatrics. Health Sciences Division, Virginia Commonwealth University, Richmond, Virginia 23298 Received December 16, 1974; accepted August 8, 1975

A rapid gas-liquid chromatographic method for the quantitation of nonesterified fatty acids (C6:0-C18:3) is described which does not require derivatization and allows separation and quantitation of all fatty acid species in a single analysis. Quantitation of the serum of normal, overnight-fasted children showed that palmitic, oleic, and linoleic acids are the predominant nonesterified fatty acids, and about one-half of the nonesterified fatty acids are unsaturated. The level of the toal serum nonesterified fatty acids quantitated by this method is about two-thirds of the total level as determined by colorimetric analysis of identical serum samples. The possible reasons for the discrepancy between the two methods in the quantitation of the total nonesterified fatty acids of serum are discussed.

The nonesterified fatty acids (NEFA) of serum constitute a small but metabolically active fraction of the total fatty acids of serum (1,2). Quantitative estimation of total N E F A has been carried out by extraction of N E F A into an organic phase, followed by titrimetric determination of total acidity or colorimetric analysis based on the transfer of N E F A soaps from a copper or cobalt nitrate triethanolamine reagent to the chloroform phase, followed by determination of the metal in this phase (3). The major N E F A in the serum of normal children have been quantitated by gas-liquid chromatography (glc) of the N E F A after conversion to their methyl esters in studies of essential fatty acid deficiency (4) as well as acrodermatitis enteropathica (5-7). There is only one report of the complete spectrum of N E F A from Cs:0 to C22:6 analyzed as their methyl esters by glc (8). Individual serum N E F A from acetic through hexanoic acid have also been quantitated by glc in metabolic derangements which cause accumulation of certain short chain fatty acids, (i.e., having six carbons or less), which are normally present only in minimal quantities (9, I0). In this paper we describe a method for quantitation of the individual N E F A from C6:0 to C18:3 which has the following advantages: (a) glc analysis is performed directly on the N E F A extracted by the Dole 156 Copyright© 1976by AcademicPress, Inc. Allrightsof reproduchonm any formreserved

QUANTITATION OF SERUM NEFA BY GLC

157

procedure avoiding the time and losses associated with a derivatization step, and (b) the majority of the total serum N E F A can be quantitatively determined on an individual basis in a single glc analysis.

METHODS

Extraction of NEFA Ten microliters of internal standard (IS) 1 was added to 1 ml of serum, followed by brief mixing. All mixing was done with a Vortex Geni (Scientific Industries, Inc., Queens Village, New York)for at least 15 sec. The serum was extracted by the method of Dole (1) as follows. All organic solvents used were redistilled. Six milliliters of Dole extraction medium (isopropanol/heptane/concentrated H2SO4, 40:10:1, v/v/v) were added to the serum, followed by mixing. Hexane (2 ml) was added, the solution mixed, followed by the addition of 1 ml of water with mixing. The lipid-containing upper phase which resulted was transferred to a clean tube with a Pasteur pipet and extracted four times with 1 ml of 4% Na2CO3. The lower phase from each extraction which contained the fatty acids as their sodium salts, together with the emulsion at the interface, was transferred to another tube and washed twice with 2 ml of hexane. The two-phase systems were centrifuged briefly in a clinical tabletop centrifuge to break up the emulsion at the interface. The upper organic phases which contained all serum lipids other than N E F A were discarded, and the lower aqueous phase was slowly acidified by cautious addition of 1 ml of concentrated H2SO4. The N E F A were extracted from the acidified aqueous solution once with 2 ml ofhexane and twice with 1 ml of hexane. The upper organic phases were collected in a conical tube and evaporated to approximately 20 /.d under a stream of nitrogen. For analysis, 2-4/zl of the final concentrated extract was injected with a microliter syringe into the glc apparatus.

Gas-Liquid Chromatography Chromatography was performed using matched, 6-ft glass (4 mm i.d.) columns packed with 10% SP-216-PS on 100-120 mesh Supelcoport (Supelco, Inc., Bellefonte, Pennsylvania) in a Beckman GC-65 gas chromatograph equipped with a flame ionization detector. An Autolab Minigrator computing integrator (Spectra-Physics, Mountain View, California) was used to determine retention times and peak areas. Nitrogen was the carrier gas at a flow rate of 40 ml/min. Column temperature was 160°C at the beginning of every run and was increased by 2°C/min for 19 min following injection of the sample. The temperature was held at 198°C ~The internal standard, pentadecanoic acid, was purchased from Sigma Chemical Company, St. Louis, Missouri. It was diluted in chloroformto a final concentrationof 10 mg/ml.

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until the linolenic acid peak was eluted at 35 min after the initial injection. The glc analysis was performed at a range of 10 and attenuation of 1 (5 × 10-~ A for full-scale deflection) with a further 64-fold attenuation between the integrator and recorder. Standard&ation

The N E F A standards were purchased from the following vendors: 6:0, Aldrich Chemical Co. (Milwaukee, Wisconsin); 8:0, 15:0, 16:0, 16:1, 18:0, 18:1, Sigma Chemical Co. (St. Louis, Missouri); 10:0, 12:0, 14:0, 18:1, 18:2to6, 18:3to3, 18:3to6, 20:3to3, 22:1, Supelco, Inc. (Bellefonte, Pennsylvania). At least 10/xg of each standard N E F A used was analyzed by glc, and all standards had purity of 87% or greater. Corrections were made for contamination using the following formula: Area NEFAsTD/tOtal area all peaks × micrograms of NEFAsTo = corrected micrograms of N E F A . IS (10/zl) was then added to individual N E F A standards in about the same proportion relative to the N E F A as expected in the physiological standard used for calibration (vide infra). The ratio (R) of area NEFAsTD/area IS was then calculated, and this ratio together with the corrected value for the weight (micrograms) of the N E F A standard was used to calculate the actual amount of N E F A standard in the physiological standard using the following formula: (Rphys.STD/RsTD)(corrected micrograms of NEFAsxD) = micrograms of NEFAphys. STD. The physiological standard contained N E F A at the approximate average concentrations found in normal serum of children who had been fasted overnight, and it had the following composition (all values are miligrams per 100 milliliters in the standard and were calculated as previously described): 6:0, 0.071; 8:0, 0.061 ; 10:0, 0.069; 12:0, 0.092; 14:0, 0.183; 16:0, 1.674; 16:l, 0.232; 18:0, 0.290; 18:l, 2.601; 18:2to6, 3.075; 18:3to6, 0.109. It was determined that the response of the flame ionization detector was not linear over the wide range of the N E F A found in s.erum but would yield higher or lower values of N E F A than were actually present when the concentration of N E F A differed significantly from the physiological standard. For example, increasing the concentration of the physiological standard five-fold while holding the amount of IS constant, and calculating the N E F A using Formula I, as follows, (Rphys. STD/Rphys.STD5×) (micrograms of NEFAphys.STD),would yield values which were 35% too high. This discrepancy required calculation of a variable correction factor (CF). This was found to be a function of the ratio (F) of the actual level of the individual N E F A to the level of the analogous N E F A in the physiological standard. This CF was derived as follows: A 1 = micrograms of N E F A in physiological standard; A 2 = micrograms of N E F A determined by Formula I; A3 = actual micrograms of N E F A present in serum sample; F = A3/A1 = (Formula II); A3 = (A2) (CF). From (II) A3 = F/A1. Substituting: A2 (CF) = F / A I . Rearrangement leads to: A2/A1 = F / C F .

QUANTITATION OF SERUM NEFA BY GLC

159

Holding the amount of IS constant, the level of physiological standard was varied using F values of 0.5, 1.0, 2.5, and 5.0, allowing calculation of the CF. A plot of CF against the log (A JA 1) yielded a straight line with a correlation coefficient of 0.9886, The best straight line was determined by the method of least squares so that the slope andy intercept in the equation, C F = (slope) log A 2/A1 + y intercept, could be calculated. Calculation of the value log (AJA 1) for a particular N E F A and substitution in this equation allowed calculation of CF, which was then used to calculate A from the formulaA3 = (A3) (CF). The values for the NEFA in serum were all within the range o f F values used for the calculations of the correction factor, i.e., from 0.5 to 5 times the level of N E F A in the physiological standard. In order to test the validity of the quantitation the following incremental addition experiment was performed. The N E F A in 1 ml of normal adult serum were quantitated. An increment of N E F A of known quantities was added to another aliquot of the serum and quantitation of the N E F A was repeated. The difference in the values before and after addition of the increment were then calculated and compared with the known increment which was added. Qualitative identification of the individual serum N E F A was based on isothermal glc (192°C) of N E F A standards. A plot of the logarithm of the retention times versus the number of carbons yielded a series of parallel straight lines for each similar family of N E F A : saturated; monounsaturated, diunsaturated to6; triunsaturated to6, and triunsaturated to3. The extracted serum N E F A were also analyzed isothermally (192°C) immediately after glc analysis of the N E F A standards, and the retention time of each peak was determined. Qualitative identification was then made on the basis of the N E F A standard plots. The peaks in the glc chromatogram were further identified as N E F A by treating the extracted N E F A with boron trifluoride and methanol to convert the N E F A to their methyl esters (11). The hexane extracts containing the methyl esters were then concentrated to 50/~1 under a stream of nitrogen. Aliquots were then analyzed by glc using appropriate temperature programs and utilizing the previously described 10% SP 216 PS column as well as 6-ft stainless-steel (1/8 in. i.d.) columns packed with 3% OV-1 on 100-200 Chromsorb W H P and 15% DEGS on 80/100 Chromsorb WAW. Both column packings were purchased from Supelco, Inc. (Bellefonte, Pennsylvania). The peaks were identified by comparison with a standard mixture of fatty acid methyl esters purchased from Applied Science Laboratories (State College, Pennsylvania).

Analysis of Extracted Lipid The lipids initially extracted by the Dole procedure, as well as the final washed extract from 1-ml serum samples, were evaporated to 10/zl under a stream of nitrogen and applied to 2-cm lanes of a 0.250-ram silica gel G plate

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purchased from Analtech, Inc. (Newark, Delaware). The following lipid standards (10 ~xg of each) purchased from Supelco, Inc. (Bellefonte, Pennsylvania), were also applied to the plate: monolein, cholesterol, oleic acid, triolein, and cholesterol oleate. The plate was developed for 15 cm in hexane/anhydrous ethyl ether/glacial acetic acid, 80:20:1 (v/v/v), and visualized by charring at 150°C after spraying with 0.6% K2Cr207 in 50% H2SO4. RESULTS

Thin-layer chromatographic analysis of the Dole solvent mixture extract of the serum revealed that in addition to N E F A it contained phospholipid, cholesterol ester, triglycerides, cholesterol, and some monoglyceride. However, the final extract used for glc analysis contained only N E F A . Figure 1 illustrates a typical glc chromatogram which results from analysis of N E F A in normal serum of a normal, overnight-fasted child with added internal standard (10 /xl). It was ascertained that the internal standard chromatographed in an area in which no detectable peaks are seen in normal serum. The major N E F A labeled in Fig. 1 are well separated, allowing ready calculation of peak areas. There are a number of small peaks due to impurities in the solvent, which persist even after redistillation, at the beginning of the chromatographic run. It should be realized that the peaks shown in Fig. 1 have been attenuated between the integrator and recorder 16 times at the beginning of the run and 64 times after C12:0 is eluted, so that although a peak may appear as insignificant on the chart recording it can be accurately integrated by the electronic integrator. The even-numbered fatty acids predominate; palmitic (Cj6:0), oleic (C18:1), linoleic (C1s:2), and stearic (C18:0) acids are clearly the major N E F A . The relative areas of the peaks which resulted from glc analysis of the hexane extract containing the fatty acid methyl esters were similar to those observed when the extracted N E F A of the same serum was subjected to glc analysis on the SP-216-PS column. The area of the fatty acid methyl esters of 14-carbon chain-length and less was generally smaller due to the greater volatility and concomitant losses of these methyl esters. When analyzed on three different glc columns the calculated retention times of the serum methyl esters relative to methyl stearate compared closely with standard methyl esters analyzed on the same columns under the same conditions. Addition of the standard methyl ester mixture to an aliquot of the hexane extract of the serum fatty acid methyl esters did not produce new peaks in the glc fatty acid methyl ester profile but rather gave greater areas to the peaks already present, confirming the identity of the peaks in the serum glc chromatogram as fatty acid methyl esters. The results of the incremental addition experiment are shown in Table 1. Note that the duplicate values agreed to within 5% of each other or better. The values determined by difference are within 10% of the actual increment

Q U A N T I T A T I O N OF S E R U M N E F A BY GLC

f

161

I.S.

16

18"1

li /

10

182

12

I x16

Ix64

,v~r~u~,--o

FIG. 1. Typical glc analysis of serum from normal child (female, 12 yr). A 1-ml serum sample was analyzed as described in test. The attenuation figures on the abscissa refer to the attenuation of the electrometer and integrator, respectively, while the range was held constant at 10 for the entire chromatographic analysis.

added for N E F A with 14 or more carbon chain-length. These fatty acids constitute 97% of the fatty acids analyzed by this procedure; therefore, this experiment demonstrates that the majority of serum N E F A can be quantitated with an accuracy of ___10%. The poorer agreement in the N E F A with a chain-length of 12 or fewer carbons is probably related to the difficulty in accurately quantitating the small amounts present in both the serum and increment added. The increment of each N E F A from C6:0 to Ca0:0 was kept small, while the increment from Ca2:0 to C18:2 was considerably larger so that the distribution of N E F A in the serum plus increment sample would still reflect a physiological distribution o f N E F A .

162

DE VRIES ET AL. TABLE 1 INCREMENTAL ADDITION OF N E F A TO SERUM

Fatty acid 6:0 8:0 10:0 12:0 14:0 16:0 16:1 18:0 18:1 18:2

Serum only a 0.016 0.005 0.046 0.060 0.108 1.779 0.104 0.449 1.637 1.279

± 0.003 SV ___ 0.004 ± 0.001 ± 0.001 ± 0.043 ± 0.001 ± 0.014 ± 0.031 ± 0.026

Serum plus increment 0.029 0.036 0.074 0.128 0.263 3.159 0.305 0.676 3.671 3.460

± 0.001 + 0.004 SV ± 0.004 ± 0.003 ± 0.040 --- 0.013 ± 0.007 ± 0.083 ± 0.071

Difference

Incmment

Difference/ increment added (%)

0.013 0.031 0.028 0.068 0.155 1.380 0.201 0.227 2.034 2.181

0.053 0.049 0.038 0.081 0.172 1.306 0.201 0.252 2.218 2.408

24.5 63.3 73.7 84.4 90.1 105.7 100.0 90.1 91.7 90.6

a All values are reported as milligrams per 100 milliliters ± S D and are based on two determinations except where noted (SV = single value). F o r further details see text.

The average values of N E F A for overnight-fasted, normal children are s h o w n in Table 2. The data s h o w n are the average o f at least duplicate determinations o f individual serum samples from seven males and seven females ranging in age from 1.5 to 15 yr. A s can be expected from a serum TABLE2 NONESTER1FIED FATTY ACIDS (C6:0-C18:3) OF NORMAL CHILDREN'S SERUMa PREPARED FROM VENOUS WHOLE BLOOD TAKEN FROM OVERNIGHT-FASTED CHILDREN b Range Fatty acid

Mean (/~equiv/liter)

6:0 8:0 10:0 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3

1.40 0.60 2.96 4.78 10.20 132.75 9.26 29. I0 91.43 67.33 3.93

Total

353.74

SD

Mole percent

High

Low

0.54 0.82 2.71 3.30 5.19 74.32 6.48 18.56 72.35 31.29 3.45

1.98 2.43 9.23 14.23 18.56 298.40 21.20 53.98 272.13 137.16 11.92

ND c ND ND 0.86 2.76 36.64 1.88 6.41 37.03 22.45 ND

0.4 0.2 0.8 1.4 2.9 37.4 2.6 8.3 25.8 19.1 1.1

211.57

830.50

95.46

100.0

Based on 1 ml serum sample. b Range in age: 1.5-15 yr (seven males and seven females): analyzed as described in text. c N D , not detected; less than 0.3/zequiv/liter.

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lipid constituent such as N E F A , which are metabolically labile, the value for each N E F A is quite variable as shown by the large standard deviations; nevertheless, the following trends were evident in all cases: C16:0 is the major NEFA, usually followed by C,8:1, then C~8:2. Another major N E F A is C18:0 , while C~4:0, C16.1, C14:0, and C,8:3, although nearly always present, only contribute a few percent to the overall molar composition. The combined N E F A containing 6-12 carbons contribute less than 3% to the N E F A measured and are not always detectable in normal children's serum. Unsaturated fatty acids constitute about 50% of the total N E F A present. The total microequivalent/liter was quite variable with a standard deviation which is 60% of the average value. There were no obvious correlations which could be made between levels of total N E F A and age or sex of the child whose serum was analyzed. The mean total microequivalents/liter of the identical serum samples as determined by the Novak colorimetric procedure (12) was 540 _ 225 ~equiv/liter with a range from 219 to 930/zequiv/liter. Thus the mean glc value for total N E F A is 65.5% of the value determined by the colorimetric procedure, the agreement between the two values ranging from 95.6 to 40.3%. There were no obvious correlations which could be made between the agreement of the colorimetric and glc analysis with the percentage of unsaturated N E F A , with the total N E F A , or with the distribution of the N E F A . The molar absorbance index of the serum N E F A colorimetric procedure was not influenced by (a) prior dialysis of serum against distilled water, (b) dialysis of serum against 50 mM ethylenediaminetetraacetic acid, followed by dialysis against distilled water, or (c) absence or presence of the following factors at physiological concentrations in serum: calcium, phosphate, ammonia, amino acids, triglycerides, and lecithin.

DISCUSSION This method is able to quantitate the majority of the normal serum N E F A , which range from 6 to 18 carbons in length. The more volatile fatty acids with fewer than 6 carbons cannot be accurately determined due to significant and variable losses which occur when the extracted N E F A are concentrated. Fatty acids 20 carbons or greater in chain-length, such as arachidonic acid (C20:4) and eicosatrienoic acid (C20:3), are not volatile enough in the flee-acid form to give peaks of sufficient magnitude to allow precise quantitation. Tic analysis showed that the Dole extraction procedure was not selective for N E F A , since in addition to N E F A it also contained phospholipid, cholesterol, triglyceride, and cholesterol ester. Evaporation and direct analysis of this extract by glc was not satisfactory since there was a large interfering peak present in the chromatogram which eluted after linolenic acid and required an additional 20-30 min for complete elution. This peak was completely eliminated by converting the acids to their sodium salts and extracting into aqueous phase, which

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effectively separated the N E F A from the other serum lipids which were extracted. Quantitation of the total N E F A by this procedure using either the concentrated Dole extract or the concentrated final extract of the standard procedure gave essentially the same results, showing that there was no loss of N E F A due to the conversion of N E F A to their sodium salts, aqueous extraction, acidification, and extraction with hexane. Control experiments indicate that the peaks identified as N E F A in the glc chromatogram of the serum extract are due to N E F A and not to any other serum constituent which is also extracted by the procedure. The identity of the peaks as N E F A is confirmed both by their conversion to methyl esters and by identical chromatographic behavior on three different glc column packings compared with standards, as well as by reinforcement of the identified peaks by added N E F A standards. Because this glc method does not measure acetic through hexanoic acids nor the fatty acids of 20 carbons or more, it is reasonable to assume that the total N E F A measured by this procedure would be less than the total N E F A measured by the colorimetric method. From the data of Tanaka et al. (10) it can be calculated that the level of propionic through beta methyl crotonic acid in normal children's serum is about 14 /zequiv/liter as determined by glc. The levels of N E F A of 20 carbon and more chain-length in normal children's serum amounts to about 18/xequiv/liter as calculated from other glc data (5,6). Therefore, with the addition of the 32/zequiv/liter of N E F A not measured by our technique, the total serum N E F A by glc is approximately 385 /zequiv/liter in contrast to the 540 tzequiv/liter determined by the colorimetric procedure of Novak. Other colorimetric procedures of normal serum yielded mean values of 400-500 tzequiv/liter with a range of 100-1210 ~equiv/liter (3). The difference in the total normal serum N E F A determined by the colorimetric and the present glc method cannot be accounted for by the N E F A not quantitated in our glc procedure. One explanation for this discrepancy is that there is a factor in serum which yields a false chromogen in the N E F A colorimetric procedure and causes the total N E F A as determined colorimetrically to be higher than the comparable value determined by glc. Even though the total N E F A as determined by glc are only about 70% of the value which is determined colorimetrically, due to the variability of the total N E F A , the values do overlap when the standard deviations are considered. The agreement between the two methods was also quite variable, which suggests that there may be a variable level of the false chromogen in the serum. A number of compounds with structures related to N E F A (amino acids, lecithin, triglyceride) or which might influence the colorimetric assay (presence or absence of divalent ions) did not influence the molar absorbance index of the colorimetric assay for N E F A performed according to Novak. Our results indicate that previous colorimetric determinations of total N E F A should be interpreted with caution.

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The distribution of N E F A in normal serum shown in Table 2 is in agreement with Dole, who reported that palmitic and oleic acids were the major N E F A in serum (1). Our results are qualitatively similar to those found in normal children's serum (5-8) by glc analysis, in that palmitic, oleic, and linoleic acids are the major N E F A . The only other report of normal children's N E F A from C8:0to C14:0is that of Salvioli (8), who found that the quantitative contribution of these N E F A to the total N E F A is only about an eighth of what is shown in Table 2, with a concomitant rise in the quantitative contribution of the longer-chain N EFA. This variation could be due to differences in the age of children whose serum was analyzed. It could also be due to a selective loss of the shorter-chain N E F A during the derivatization step. This procedure has several features which recommend it over the currently used glc procedures for serum NEFA. First, the absence of a derivatization step coupled with the use of an internal standard makes the method rapid and reliable, and it avoids the time and loss of material associated with a derivatization step. In addition to measuring 92% of the total serum N E F A on a molar basis, this method makes possible evaluation of the quantitative contribution of each N E F A to the total serum N E F A in a single glc analysis. Finally, this method allows accurate and sensitive quantitations of serum N E F A , with confidence that what is being measured is indeed NEFA. This method was developed to quantitate the individual serum N E F A present in diseases in which elevations only in total N E F A (by colorimetric techniques) had been described (13). We have observed striking alterations in the distribution of the serum N E F A using this method to analyze serum at various stages of Reye's syndrome (14). With the ability to quantitate rapidly and accurately a spectrum of individual N E F A which comprise the majority of the total serum N E F A , new insights should be gained into the metabolism of these important serum constituents and into derangements of their metabolism in various disease states.

ACKNOWLEDGMENTS These studies were supported by N I H Grant No. 2101 PE 001020 8 and by the Medical College of Virginia Pediatric Metabolic Fund.

REFERENCES 1. 2. 3. 4. 5. 6. 7.

Dole, V. P. (1956)J. Clin. Invest. 35, 150-154. Bierman, E. L., Schwartz, I. L., and Dole, V. P. (1957)Amer.J. Physiol. 191, 359-362, Falholt, K., Lund, B., and Falholt, W. (1973) Clin. Chim. Acta 46, 105-111. Caldwell, M. D,, Jonsson, H. T., and Othersen, H. B. (1972)J. Pedit. 81, 894-898. Julius, R., Schulkind, M., Sprinkle, T., and Rennert, O. (1973)J. Pediat 83, 1007-1011. Cash, R., and Berger, C. K. (1969)J. Pediat 74, 717-279. Woodruff, C. W., Bailey, M. C., Davis, J. T., Rogers, N., and Coniglio, J. G. (1964) Amer. J. Clin. Nutr. 14, 83-90.

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8. Salvioli, G. P., Ambrosioni, G., and Cacciari, E. (1967) Boll. Soc. Ital. Biol. Sper. 43, 1427-1429. 9. Kurtz, D. J., Levy, H. L., Plotkin, W., and Kishimoto, Y. (1971) Chim. Acta 34, 463 -466. 10. Tanaka, K., Budd, M. A., Efron, M. L., and Isselbacher, K. J. (1966)Proc. Nat. Acad. Sci. USA 56, 236-242. 11. Metcalfe, L. D , and Schmitz, A. A. (1961)Anal. Chem. 33, 363-364. 12. Novak, M. (1965)J. Lipid Res. 6 431-433. 13. Schubert, W, K., Partin, J. C., and Partin, J. S. (1972) Progr. Liver Dis. 4, 489-510. 14. Mamunes, P., DeVries, G. H., Miller, D. C,, and David, R. B., (1975) Fatty acid quantitation in Reye' s syndrome in Reye" s Syndrome (J. D, Pollack, ed.) pp. 245-254. Grune and Stratton, New York.

Quantitative determination of C6:0-C18:3 serum nonesterified fatty acids by gas-liquid chromatography.

ANALYTICALBIOCHEMISTRY70, 156-166 (1976) Quantitative Determination of Co:o-Cls:3 Serum Nonesterified Fatty Acids by Gas- Liquid Chromatography GEORG...
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