Determination of triacylglycerols in serum by capillary gas chromatography with trinonadecanoylglycerol as internal standard

ANALYTICAL

BIOCHEMISTRY

171,366-372

(1988)

Determination of Triacylglycerols in Serum by Capillary Gas Chromatography with Trinonadecanoylglycerol as Internal Standard ALFRED LOHNINGER, LEOPOLD LINHART, MICHAEL LANDAU, DIETMAR GLOGAR,* CHRISTOPHKRATOCHWIL,* AND ERICH KAISER Department qfkfedical Chemistry and *Department of Cardiology* University of Vienna, Austria Received October 22, 1987 An accurate capillary gas-chromatographic method with trinonadecanoylglycerol as internal standard for determining triacylglycerols in human serum and other biological sources is described. After serum extraction. total triacylglycerol and triacylglycerol species (differing in the number of carbon atoms in the acyl radicals) are directly determined without any further sample manipulation. In addition, from the same gas-chromatographic run the data obtained by the integrator record are compared with those of a computer data acquisition system. Evaluation of the triacylglycerol values resulted in a coefficient of variation (CV) of 2.08% (computer evaluation). Simultaneous evaluation of data obtained from tripalmitoylglycerol and tristearoylglycerol standards resulted in CV of 2.04 and 1.99%, respectively (computer evaluation), and 6.63 and 4.84%, respectively (integrator evaluation). Gas chromatography at lower elution temperature resulted in better separations but enhanced CV values up to about 4%. Triacylglycerol values were not influenced by storage of plasma at -20°C up to 4 days prior to extraction. 0 1988 Academic Press. Inc. KEY WORDS: gas-liquid chromatography, lipids; triglycerides; nutrition; computer methods, integration: instrumentation, programmed temperature vaporizer injector; clinicai chemistry.

In most retrospective studies a univariate relation has been reported between serum triacylglycerol level and coronary heart disease. Recently (1) it was found that when cholesterol, triacylglycerol, and their interaction term are introduced in a regression equation all variables contribute significantly to the risk. In patients with a serum cholesterol level lower than 220 mg/ 100 ml, serum triacylglycerol level is an independent predictor of risk ( 1). Dietary variations resulted in significant changes of molecular weight or carbon number profiles of the serum triacylglycerols. On an unsaturated fat diet there was a decrease in the lower molecular weight species and an increase in the higher molecular weight species in comparison to a saturated fat diet in all lipoprotein classes (2). It has been shown that in about 25% of hypertriglyceridemia cases there is an anomaly of plasma triacyl0003-2697188 $3.00 Copyright 0 1988 by Academic Press. Inc. All rights of reproduction in any form reserved.

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glycerol composition (3). The knowledge of the triacylglycerol species composition in patients suffering from coronary heart disease is therefore of interest when these patients are kept on a diet. Rapid profiling of triacylglycerols is preferably done by high temperature gas-liquid chromatography with short capillary columns and nonpolar phases (4-6). Of components with higher molecular weight, however, a discrimination during injection has been reported (7). For quantitative determinations the integrator record is known to be quite erratic for certain slope sensitivity settings requiring a systematic examination for errors in baseline resetting (8). The aim of the present study was to determine triacylglycerols from body fluids and fat emulsions by gas-chromatography injection of the Folch extract without any further sample manipulation and to compare the re-

CAPILLARY

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TRIACYLGLYCEROLS

367

maximum detector response. Before use the column was conditioned at 350°C for about 3 h. The gas chromatography system was subsequently calibrated with an appropriate mixture of triacylglycerols. The oven temperature was programmed from 320 to 350°C at a rate of 4”C/min (“short” proMATERIALS AND METHODS gram) or from 186 to 276°C at a rate of 8”C/ min and from 276 to 340°C at a rate of 6”C/ Reagents and standards. Chloroform and min (“two-ramp” program). methanol were obtained from E. Merck The chromatograms were recorded on an (Darmstadt, FRG). Tripalmitoylglycerol and LDC computing integrator (Laboratory Data tristearoylglycerol were obtained from Serva Control, Division of Milton Roy C. Inc., (Heidelberg, FRG), and trinonadecanoylglyRiviera Beach, FL) and on a Lingo PC-88 cerol was obtained from Sigma Chemical Co. XT computer (Taiwan) running under MS(St. Louis, MO). Purity of the synthetic triacDOS equipped with a 20 MByte hard disk ylglycerols was confirmed by thin-layer chro(for data storage) and a 12-bit analog-to-digimatography (high-performance thin-layer tal converter device. The digitized FID signal chromatography plates, 10 X 20 cm, Silica was digitally filtered to eliminate the baseline gel 60, from E. Merck) and by fatty acid analnoise. The peaks were marked manually by yses as corresponding methyl esters, by gas means of a keyboard-controlled cursor on chromatography. Enzymatic triacylglycerol the computer screen. Subsequently, the peak uv test (Kit No. 148 270) and enzymatic colareas were calculated by standard algorithms orimetric triacylglycerol test (Kit No. 842 and directly processed by database software 24 1) were obtained from Boehringer-Mannto avoid transcription errors. heim (Mannheim, FRG). The correction factors were calculated at Apparatus. The analyses were carried out four different sample:standard ratios (0.5: 1, on a Dani Model 6500 gas chromatograph 1: 1, 1.5: 1, and 2: 1~respectively) by multiply(Dani S.p.A., Monza, Italy) equipped with a ing the determined peak area ratios by the programmed temperature vaporizer (PTV)’ reciprocal value of the ratio of the weight of injector. A capillary glass liner was inserted the test component and the internal staninto the vaporizer block of the PTV injection dard. These correction factors were highly resystem. A small amount of quartz wool was producible on a given column and under a inserted into the liner. The low thermal mass specific set of experimental conditions. A of the vaporizer block allowed the temperastandard run was done before and after each ture to rise from 70 to 400°C in 1.5-20 s. series of analyses. With the injection system A 5 m, a 0.9-pm i.d. fused silica capillary described, no significant difference of the column with chemically bonded OV-1 data between a split or a splitless sample in(0.1 -pm coating thickness) (J&W Scientific, jection mode was found. Folsom, CA) was used for all analyses. The The enzymatic calorimetric triacylglycerol approximate column life was 400 h. Hydrogen was used as carrier gas with a flow rate of determinations were carried out on a Hitachi 705 autoanalyzer. 10 ml/min and nitrogen as the make-up gas. Sample preparation. Stock solutions (1 The detector temperature was kept at 380°C; standards were air and hydrogen flows were adjusted to give mg/ml) of triacylglycerol prepared in chloroform. The analytical samples were prepared by mixing appropriate volumes. Twenty different experimental fat ’ Abbreviation used: PTV. programmed temperature vaporizer. emulsions composed of soy bean oil, egg suits with those of enzymatic methods. In addition, the data obtained by the integrator record should be compared with those of a computer data acquisition system, simultaneously recorded from the same run of the same sample.

368

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phosphatidylcholine, and glycerol (comparable to commercially available fat emulsions) were prepared. Blood was taken from 20 patients in observation at the Department of Cardiology of the University of Vienna, all younger than 50 years, and all having had a heart attack about a year ago. After an overnight fast the patients’ blood was collected into tubes containing EDTA. Aliquots of the samples were mixed with 20 vol of chloroform-methanol (2: 1). The lipid extracts were washed as described by Folch et al. (9) and diluted with chloroform to a defined volume. The chloroform solutions were used directly for subsequent analyses. Day-to-day variation was determined by dividing a serum pool into three equal portions. One portion was extracted immediately as described above; two portions were stored at -20°C and extracted on Days 1 and 4 (10). The absolute amounts of plasma and fat emulsion triacylglycerols were quantitated by means of an internal standard (trinonadecanoylglycerol), using appropriate response factors (as described above). The results were expressed in milligrams per 100 milliliters because generally, determination of the exact molecular weight of a complex species composition of serum triacylglycerols is not possible. Other methods. The total triacylglycerol content of the fat emulsions was determined by means of the triacylglycerol uv test. The total triacylglycerol content of the plasma samples was determined by an enzymatic calorimetric test, using the autoanalyzer method. RESULTS

A plot of a representative chromatogram of the triacylglycerol species of a fat emulsion and a plasma sample are shown in Fig. 1. In the fat emulsion the TGL 54 species made up the main triacylglycerol fraction, while in the human plasma sample the TGL 52 species were dominating; the carbon number was in-

ET AL.

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sa-

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FIG. 1. Gas chromatograms of human plasma and fat emulsion. Peak 27, cholesterol (27 carbon atoms); peaks 28-40, diacylglycerols of plasma and pyrolysis products of serum sphingomyelins and glycerophospholipids (28-40 carbon atoms in the acyl radicals); peaks 43,45, and 47, cholesteryl esters (43,45, and 47 carbon atoms); peaks 48-60, triacylglycerols (48,50,52,54,56,58, and 60 carbon atoms in the acyl radicals).

traduced as the total number of carbon atoms in the acyl groups, neglecting the three carbon atoms in the glycerol residue.

Linearity The linearity of the gas-chromatographic method was evaluated with different concentrations of tripalmitoylglycerol and tristearoylglycerol. The internal standard concentration was kept constant; the concentration of tripalmitoylglycerol and tristearoylglycerol was changed from 0.5: 1 to 2: 1. The area response declined with increasing concentrations when normalized on internal standard (Fig. 2). This effect was more pronounced for tristearoylglycerol than for tripalmitoylglycerol. Thus, for quantitative determinations adequate response factors

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Table 2 shows the results of the evaluated day-to-day variation. Storage of plasma portions at -20°C for neither 1 nor 4 days had ,’ any measurable influence on the data ob-‘,tained. These determinations were carried , “‘-’ _,/ ’ out with the “two-ramp” program (see Mate‘/ ‘, ~,~~’ ~ rials and Methods). The higher CV values, obtained when using the “two-ramp” program, were probably the result of the longer retention time (25 min versus 10 min) for 0 0.2 0.4 0.8 0.8 1 1.2 1.4 16 I.8 2 one run. Weight Ratio ~Andyte:hf.Std.l + triatearin Gmpalmiln

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FIG. 2. Comparison of the evaluated values of tripalmitoylglycerol/trinonadecanoylglycerol (Cl) and tristearoylglycerol/trinonadecanoylglycerol (+) in ratios of 0.5: I, I : 1, I.5 1~and 2: I, respectively.

were used considering both the number of carbon atoms in the molecule and different area ratios (sample/standard).

~~~~~~~~

The accuracy of a given method is usually evaluated by comparison with a reference method. Gas-chromatographic runs were used only with a ratio of the sample peak area to the peak area of the internal standard that was in the range of 0.5: 1 to 1S: 1. Otherwise a new mixture of sample and internal

Precision

A measure of the precision of the gas-chromatographic method was obtained by calculation of the standard deviation and coefficient of variation (CV) of 10 repeated analyses determining tripalmitoylglycerol and tristearoylglycerol standards, as well as of a human plasma pool. With regard to the determinations of the standards a comparison of both the integrator and the computer records of the same chromatograms were made (Table 1). The precision of the data evaluated by the computer-supported integration system was superior compared to that of the data of the integrator record. The CV of the triacylglycerol determinations in human plasma was only slightly higher, although three main peaks and two to three smaller peaks were integrated individually and the data were summarized to obtain the total triacylglycerol content of the sample, in contrast to the integration of a single standard peak (Table 1). For these determinations the “short” program was used (see Materials and Methods).

TABLE

1

PRECISIONOFTHEGCMETHOD:TRIACYLGLYCEROL STANDARDS(INTEGRATORANDCOMPUTER RECORDSCOMPARED)

Tripalmitin fl= 10 Theoretical value = 4.03 Mean SD cv

(To)

Tristearin n= 10 Theoretical value = 4.0 I Mean SD cv

(9%)

Integrator

Computer

4.83 0.320 6.63

4.16 0.085 2.04

4.55 0.220 4.84

4.11 0.025 1.99

Human plasma (computer data) ?l= 10

Mean (mg/lOO ml) SD cv

(90)

82.3 2.09 2.54

No/r. CC oven temperature 320 to 350°C with a rate of 4”C/min.

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standard was prepared. The amount of a single compound injected was between 0.03 and 5 pg, dependent on the split ratio. With the injection system described, no significant difference between the data of split and splitless sample injection modes was found. Table 3 shows the comparison of data obtained by the enzymatical uv test and the present gas-chromatographic method for 20 differently prepared experimental fat emulsions. The data obtained differed within a range of 0.7 to 6.3%. Figure 3 shows a plot of the gas-chromatographic values versus the values of the calorimetric test (autoanalyzer) of plasma triacylglycerols obtained from patients with a cardiac attack history. A correlation coefficient of 0.958 was obtained. In general, triacylglycerol values determined by the gas-chromatographic method were lower than the values obtained by the autoanalyzer method.

TABLE DAY-TO-DAY HUMAN

2

ET

AL. TABLE

3

COMPARISON OF RESULTS OBTAINED BY ENZYMATIC W-TEST AND CC METHOD FOR 20 DIFFERENT LOTS OF EXPERIMENTAL FAT EMULSIONS

Lot no. 1 2 3 4 5 6 7 8 9 10 I1 12 13 14 15 16 17 18 19 20

Enzymatic uv method (mg/lOO ml) 20.26 21.26 21.32 20.66 21.90 20.50 20.00 21.76 21.86 20.46 19.48 20.00 20.14 20.32 20.38 19.76 20.12 21.38 21.24 20.06

GC method (mg/ 100 ml) 20.79 2 1.40 20.62 20.65 20.75 21.66 20.83 20.9 1 20.56 20.20 20.12 19.72 20.34 19.38 20.09 20.00 19.60 20.89 21.12 20.40

Difference (%I 2.5 0.7 3.4 0.0 5.5 5.4 4.0 4.1 6.3 1.3 3.2 1.4 1.0 4.9 1.4 1.2 2.7 2.3 0.6 1.7

VARIATION OF THE GC METHOD: PLASMA (COMPUTER DATA)

DISCUSSION Within-day Immediate (n = 6) Mean (mg/lOO ml) SD cv (W)

44.5 1.76 3.96

Day 1 (n = 6) Mean (mg/lOO SD cv (%)

43.1 0.87 2.02

ml)

Day 4 (n = 6) Mean (mg/ 100 ml) SD cv (%) Day-to-day

(n = 18)

Mean (mg/lOO SD cv (%) Note. I”C/min,

44.3 I .66 3.75

ml)

GC oven temperature 186 to 276°C 276 to 340°C at a rate of 6”C/min.

44.0 1.51 3.44 at a rate of

Chromatographic analyses of body fluid lipids are extensively utilized in biomedical research but only few of them have become established as routine methods in clinical chemistry. Recently, Brunnekreeft and Leijnse (11) reported a method for determining triacylglycerols after serum extraction and chemical hydrolysis with subsequent direct measurement of liberated glycerol by gas chromatography. The advantage of the present gas-chromatographic method is that after serum extraction triacylglycerols are directly determined and quantitated by means of an internal standard without any further sample manipulation. Gas-chromatographic analyses of intact long-chain triacylglycerols and other neutral plasma lipids have been described previously (3,4,8,12,13). Mostly,

CAPILLARY

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GAS CHROMATOGRAPHY

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400

600

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FIG. 3. Comparison of results (mg/lOO ml) for total plasma triacylglycerols as obtained by gas-chromatography (x) and autoanalyzer (y) methods; n = 19, y = 1.077 x, r = 0.9584, x = 132.6 mg/lOO ml, and y = 150.7 mg/lOO ml.

these compounds have been separated on packed columns with poor resolution. With the introduction of capillary columns (14) better resolution within shorter analysis time could be achieved (15) and capillary columns have become routine tools in characterizing oils and fats (16). On nonpolar phases the lipids are separated according to the number of carbon atoms in the molecule. One problem associated with capillary gas chromatography is the quantitative aspect of triacylglycerol analysis. Using classical vaporizing injectors, the vapor pressures are too low to allow reasonable evaporation in the injector. Grob (7) reported coefficients of variation of data obtained following splitless injections of 9- 13%, 15-30% following split sampling, and l-3% following cold on-column injection, normalized on internal standard. Therefore, the cold on-column injection has been recommended (5,7,17). Using the PTV injector comparably low CV values determining standard triacylglycerols and plasma triacylglycerols were obtained (Table 1). The advantages of using a PTV injection system are (a) the capillary column can be of any diameter, (b) the capillary column temperature can be adjusted at any value, (c)

OF SERUM TRIACYLGLYCEROLS

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nonvolatile residues are retained by the quartz wool in the precolumn, (d) most of the solvent can be eliminated via the split (at a lower temperature) prior to the vaporization of the components of the sample, and (e) large sample volumes can be injected. During cold on-column injection, all sample components enter the column and either unfavorable impurities can irreversibly contaminate the stationary phase or the top of the column must be removed quite frequently. Using the PTV injector, only a clean glass liner (acting as precolumn) must be inserted. Generally, for gas-chromatographic methods the “internal standard method” is considered to be superior to external calibration. We selected trinonadecanoylglycerol as internal standard for two reasons: trinonadecanoylglycerol is a triacylglycerol species not present in human plasma, differing from naturally occurring triacylglycerol species by only one to nine carbon atoms in its acyl radicals, in contrast to tridecanoylglycerol, frequently used by other investigators (4,8). The two different temperature programs used each resulted in a different elution temperature for a given triacylglycerol. The advantages of the “short” program are an analysis time of only 10 min (versus 25 min for the “two-ramp” program) and, probably, better recovery of the different triacylglycerol species (5) and lower CV values. However, the separation efficiency was better with the “two-ramp” program with regard to overlapping of TGL 48 with cholesteryl esters with 20 carbon atoms in the acyl residue. It has been found that the carrier gas flow rate decreases with increasing temperature at constant inlet pressure (5). Thus a decrease in the elution temperature is associated with an increase in the carrier gas flow-rate which can effect the& (weight correction factor) for a given substance analyzed at different rates of temperature change. Also, a different response with respect to increasing concentrations was obtained for tripalmitoylglycerol and tristearoylglycerol (Fig. 2) indicating

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different response factors for triacylglycerols with different numbers of carbon atoms in the molecule. Thus, adequate response factors for each triacylglycerol species for both gas-chromatographic conditions (“short” and “two-ramp” program) were used considering different area ratios (sample/standard) as well. In accordance with other reports triacylglycerol values determined by the gas-chromatographic method were lower than those obtained by the autoanalyzer method for the same plasma sample (8). The mean value of total plasma triacylglycerols (determined by the gas-chromatographic method described in this paper) was about 18 mg/ 100 ml lower than the mean value obtained by the autoanalyzer method. Generally, autoanalyzer values are not routinely corrected for the presence of free glycerol and mono- and diacylglycerols which may together lead to an overestimation of up to 10% of normal triacylglycerol content. In addition, falsely high results for triacylglycerols in patients receiving intravenous nitroglycerin has been reported ( 18). Also, the presence of nonspecific chromogens can contribute to higher triacylglycerol values. Furthermore, TGL 54 is used as standard for the autoanalyzer method, while for human plasma triacylglycerols an average carbon number of 5 1.8 has been determined (8). In contrast to the autoanalyzer method the data obtained by the gas-chromatographic method are not influenced by interfering components, although lower cutoff values must be established for diagnosing hyperlipidemia. In the present study less precise results were obtained by integrator recording compared to computer integration with manual baseline setting for processing comparatively simple chromatograms. With increasing complexity we found a progressively decreasing precision of the integrator record in contrast to computer integration (data not shown). The present method is supposed to be suitable for rapid and accurate quantitation of

ET AL.

triacylglycerols in body fluids with the advantage of giving additional information about species composition. This is of interest in patients kept on a special diet and probably in patients on total parenteral nutrition, determining the clearance rate of fat emulsions. ACKNOWLEDGMENTS This work was supported by a grant from the “&terreichischer Herzfonds.” The authors thank H. Staniek and Barbara Lohninger for technical assistance.

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18. Ng, R. H., Guilmet, R., Altaffer, M., Statland, B. E. ( 1986) Clin. Chem. 32,2098-2099.