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Serum cholesterol determination by gas-liquid chromatography
Clinica Chimica Acta, 43 (1973) 257-265 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed in The Netherlands
257
CCA 5344
SER’CTMCHOLESTEROL
DETERMINATION
BY GAS-LIQUID
CHROMATOGRAPHY
JAN PETTER Medical (Received
BLO~HOFF
Department July 14,
A, lJndue:rsity Hospital, Rikshospa’talet.
Oslo I (Norway)
1972)
SUMMARY
A gas-liquid chromatographic (GLC) method adapted from Schmit and Mater is described for the measurement of total and free cholesterol in serum. The method has been found to be simple, sensitive and precise. The results have been compared with the calorimetric method of Carr and Drekter for total cholesterol and the method of Sperry and Webb for free cholesterol. For total cholesterol lower values were obtained by the GLC method both in sera from patients without signs of liver disease and in sera with high total bilirubin. There was a tendency for somewhat higher values for free cholesterol by the GLC method than by the calorimetric method. This difference was not significant. The present study has demonstrated that the GLC method for total and free cholesterol determination is more specific than calorimetric methods. Gas-liquid chromatography should be given consideration as a reference method for the estimation of free cholesterol and total cholesterol.
INTRODUCTION
Several methods have been developed for the estimation of cholesterol in serum. The majority of these are photometric methods based on colour reactions of cholesterol in the Liebermann-Burchard reaction or with other acid reagents’. As pointed out by Tonksl and Vanzettis these methods have several disadvantages; in particular they lack specificity, as other serum constituents may interfere with the colour reactions .
Gas-liquid chromatography (GLC) is one of the most valuable technics in sterol analysis. Surprisingly little information has, however, been derived by this method for routine nleasurement of serum cholestero13-g. This paper presents details of GLC methods adapted from Schmit and Mater5 for the measurement of total cholesterol and free cholesterol in serum. The results are compared with the calorimetric method of Carr and Drekter7 for total cholesterol and the method of Sperry and Webb8 for free cholesterol.
258 MATERIAL
BLOMHOFF AND
METHODS
Materials After an overnight fast, serum was collected from healthy volunteers, from a group of patients without liver disease and from patients who suffered from a variety of liver disorders. Serum was analysed within a few hours or stored at -15’ until cholesterol determination. [7-3Hlcholesterol (spec. act. 9.4 C/mmole) from Radiochemical Center, Amersham, England was dispersed in albumin and esterified in vitro by incubating in human serum at 37’ for about 20 ha, using lecithin cholesterol acyl transferase. Routine
laboratory procedures Total cholesterol was estimated by the Carr and Drekter method which includes deproteinization, and the Liebermann-Burchard reaction without saponification. A serum blank was used as correction for bilirubin. This correction was performed only when jaundiced serum samples were discovered. Free cholesterol was estimated by the digitonin precipitation method of Sperry and Webba. Cholesterol “standard for chromatography grade 9g%+” (Sigma Chem. Corp., St. Louis, Miss., U.S.A.) was used as reference standard in both method+‘. Preparation for gas-liquid chromatography For total cholesterol estimation 4.7 ml 95% ethanol and 0.3 ml 33% KOH in aqueous solution were added to 0.2 ml serum. After thorough mixing for about 15 set on a Whirlimixer (Fisons Scientific App., Loughborough, England) hydrolysis was performed for 15 min at 55” in a waterbath. As the columns used separate free cholesterol and not cholesteryl esters, the hydrolysis step is rather important. This step was tested using [7-3H]cholesteryl ester added to serum. After hydrolysis the lipids were separated by thin-layer chromatography, whereafter fractions containing free and esterified cholesterol were quantitated by liquid scintillation ll. From 14 min up to 40 min of the test a plateau of 99% free cholesterol was obtained. After briefly cooling, 5 ml distilled water and IO ml hexane (n-hexane pure, E. Merck, Darmstadt, Germany) were added. The solution was mixed for 30 set on a Whirlimixer and allowed to stand until complete separation of the two phases, usually IO min. From the upper phase 5 ml was removed and evaporated under a stream of nitrogen. To the dry residue 200 ~1 of IOO mg/Ioo ml 5a-cholestane (Sigma Chem. Corp., St. Louis, U.S.A.) in chloroform was added. For free cholesterol estimation g ml 95% ethanol and 3 ml diethyl ether was added to 0.2 ml serum. After mixing for about 15 set on a Whirlimixer the solution was allowed to stand for 5 min and then centrifuged at 2000 rev./min for IO min. After decanting the solvent phase this was evaporated to dryness with nitrogen in a waterbath at 55”. To the dry residue 200 ~1 of IOO mg/Ioo ml or 50 mg/Ioo ml 5acholestane in chloroform was added. Schmit and Mater5 propose only one extraction to be performed. However, an average of 3.5% higher values was obtained after a second extraction with 9 ml 95% ethanol and 3 ml diethyl ether in 20 different serum samples. Extraction has therefore been performed twice in this study. The analyses were performed in glass-stoppered test tubes.
SERUM CHOLESTEROL DETERMINATION
Gas-liquid chromatography GLC was performed
BY GLC
on F&M Scientific
259
Instruments’
High Efficiency
Chroma-
tograph 402 or Research Chromatograph 5750 G, both equipped with hydrogen flame ionization detector. The column was 18 inch by 114 inch o.d. glass packed with 3.8 wt.% silicone gum rubber (Methyl GE SE-30, F&M Scientific Corp., Avondale, U.S.A.) on 80-100 mesh Gas-Chrom Q (Applied Science Lab., State College, Pa. (U.S.A.) The carrier gas was argon at the flow rate of 60 ml/min and the column temperature was 225’. The liquid phase was dissolved in chloroform and applied to the solid support using a solution-coating technique with a rotation evaporator. The columns were noflow conditioned at 275” from 2 up to 12 h and then conditioned with flow at operating temperature until the baseline was stable. Approximately 3,ul in a Hamilton syringe was injected. A solution of known amount Sigma cholesterol “for chromatography, grade 99% +” and Sa-cholestane in chloroform used as standard was chromatographed for about every IO samples. The peak heights of 5cr-cholestane and cholesterol of the standard and the unknown extract were measured in millimeters and used for the calculation of cholesterollz. RESULTS Chromatograms Fig. I shows typical chromatograms of cholestane and cholesterol. The peaks are almost symmetrical without tailing. No additional peaks were detected in the chromatograms of the standard solutions.
2
2
2 1
i A
J
k
B I
0
5
IO
15
ITI”
20
Fig. 1. Chromatograms of cholestane (I) and cholesterol (2) for standard solution (A), total cholesterol (B) and free cholesterol (C). Conditions as described in the text.
260
BLOMHOFF
Response ratio linearity The response ratio curve is linear and goes through zero as shown in Fig. 2, curve A. After operating a column for one month the response ratio curve B, Fig. 2, was obtained indicating a substantial loss of cholesterol relative to cholestane over a wide range but proportionally greater for small samples. Reconditioning or injecting
xx)
Km
3m CHCLESTEMX
mQ/lmti
Fig. 2. Response ratio linearity curve for A: a new column, B: a concentration of the internal standard is constant.
after extensive use. The
of 20 ~1 hexamethyl-disilazane13 (Applied Science Lab., State College, Pa. U.S.A.) was not effective in restoring the linearity. Overloading the column or the detector shown by a curve plateau on the response curves did not occur up to the tested level of 2000 mg/roo ml cholesterol indicating a wide linear range of the system. The ratio of cholestane to cholesterol was sometimes somewhat higher when starting the analysis, especially when the columns had not been used for some days. This absorption phenomenon was also observed by others3*6. Before the start of the analysis cholesterol has therefore to be injected to saturate the absorptive sites. It was very difficult to predict the life of a column even with the same batch column support material. The column life was usually longer than one month. Conditioning for from 2 h up to 12 h at 275” did not seem to be of major importance, nor treatment of the glass with 5% hexamethyl-disilazane in toluene13 before packing the column. Extensive usage and high temperature are thought to be the most important factors determining the lifetime of the columns5. Recovery The recovery
of serum cholesterol
and cholesterol
added to a known serum
SERUM CHOLESTEROL DETERMINATION BY GLC
sample was tested
261
both for the total and the free cholesterol
procedure.
Thirty-one
separate analyses were performed. The recovery was equal for free and total cholesterol and was in the range of 95-10676 (SD 3.2). Some of the results are given in Table I, indicating linear standard curves and complete recovery over a wide range. TABLE
I
RECOVERYSTUDIESOF CHOLESTEROL
BY
GAS-LIQUID
CHROMATOGRAPHY
Serum values are means of 5 replicates. Cholesterol
in samfile
swum cholesterol
I
0 0
(pg)
500 250 100
TC 25 IO0 200
FC
25 50 IO0 IO
30 TC = total cholesterol,
Cholesterol
recovered
added free cholesterol 101
101
245 518 299 376
98 104 99 104 97
25 49
100 98
210
101
160 I74
IO1 102
98
FC = free cholesterol.
Cholesterol extraction The following experiments were performed to test the completeness of the cholesterol extraction procedures. In IO different samples the ethanol-water phase was removed after extraction with hexane. Cholestane was added, and extraction performed with IO ml petroleum ether. In every IO samples there were small amounts of cholesterol but less than ho/oof the initial total cholesterol value. To the precipitate after the second ethanoldiethyl ether extraction cholestane was added to IO different samples and a third extraction was performed with chloroform-methanol 2 :I v/v. Only trace amounts of cholesterol were found in 8 samples, and less than 1.2% of the initial free cholesterol value in 2 samples. Precision The precision of the methods was calculated from 82 duplicate different serum samples for total cholesterol and 47 duplicate analyses
analyses of of different
serum samples for free cholesterol according to the formula: s = dZ(d2)/zn where d is the difference between duplicates and n is the number of duplicates. The precision for total and free cholesterol at different cholesterol levels are given in Table II. The precision for free cholesterol is not as good as for total cholesterol. This depends probably on some difficulties in measuring the cholestane peak as usually this was eluted before reaching the baseline (Fig. I c). The day-to-day variability of the GLC system was determined by single daily analyses of the same cholesterol-cholestane solution for 13 days. During this time there was also a change of the SE-30 column to a new one. The mean and standard deviation for the specimen tested during these days was 348 f 6.7 mg/roo ml with a coefficient of variation of 1.9%.
262 TABLE PRECISION
BLOMHOFF II OF THE
GLC
METHOD
AT DIFFERENT
LEVELS
Mean
Standard deviation mglroo ml
Coejicient variation y0
36 28 18
58-199 zoo--295 3oo--529
160 226 384
4.0 5.6 10.6
2.5 2.5 2.8
28 I9
26-70 71-267
52 II4
I.9 4.0
3.7 3.5
mg/100 ml
FC
CHOLESTEROL
Range
n
TC
SERUM
of
n = number of duplicates. TC = total cholesterol, FC = free cholesterol.
Comparison with calorimetric methods Total cholesterol. The results of the comparison of the Carr and Drekter method’ and the GLC method for total cholesterol are given in Table III. Both serum samples with normal and raised total bilirubin content were included. Lower values were obtained with the GLC method than with the calorimetric method in serum samples from persons without liver disease. There was no difference between the two methods in samples from patients with liver disease with normal or slightly elevated total bilirubin, while there was a marked difference at high total bilirubin levels. TABLE
III
COMPARISONBETWEEN DIFFERENTMETHODSFOR TOTAL CHOLESTEROL Cholesterol values are mean f S.D. The statistical differences between pairs of results obtained by the two methods were evaluated by Student’s t-test. Percent CE was calculated by subtraction of FC from TC (both values obtained by GLC).
No liver disease
Total bilirubin mglzoo ml
Carr and Drekter
-
211 & 81
I95 zt 54
1.0-1.9
Total cholesterol mg/Ioo
210 + 48
ml
% CE
GLC n = 36 n = 15
I94 f
83**
74 f
2
Ig4 & 61 ns
7o f
6
Ig8 & 52 ns
68 & 6
231 4 6g ns
65 f
I2
219 5 71**
39 *
I4
168 * 88**
27 k I5
n = 20 Liver disease
2.0-4.9 I
237 i
65
5.0-9.9
246 + 83
>I0
234 i
IoI
I TC = total cholesterol, FC = free chromatography, ** = P ~0.01, ns
=
n = 15 n=g $2 = II
cholesterol, CE = cholesteryl no significant difference.
esters,
GLC = gas-liquid
No hydrolysis is included in the Carr and Drekter method’. Hydrolysis was therefore performed in 13 different serum samples before the Carr and Drekter analysis. After hydrolysis and hexane extraction, 4 ml of the hexane phase was analyzed by the GLC method and 4 ml by the Carr and Drekter method. The mean and standard deviation for total cholesterol obtained by the GLC method and the Carr and
SERUM CHOLESTEROL DETERMINATION BY
GLC
263
Drekter method with this procedure were respectively 346 f 84 and 362 f 85 mg/roo ml ($J< 0.01). Seronorm (Nyegaard & Co., Oslo, Norway) is a reference serum from horse used for control of clinical chemical analysis. The values obtained with this serum (Batch 116) with the Carr and Drekter method’, the direct method of Huang14 modified by Ness?, the Liebermann-Burchard reaction after hydrolysis and the GLC method for total cholesterol are compared in Table IV. There is a marked difference between the calorimetric methods and the GLC method. The value for bilirubin in this batch was 1.99 mg/Ioo ml and cholesteryl esters 80% of total cholesterol determined by the proposed GLC method. Free cholesterol. The results are given in Table V. There is a tendency to higher values with the GLC method than with the calorimetric method, but this difference was not significant. TABLE TOTAL
IV CHOLESTEROL
VALUES
FOR SERONORM
WITH
DIFFERENT
METHODS
Values are tneans of 3 replicates. Methods
Total cholesterol mgl100 ml
Huang et aZ.14*15 Carr and Drekter’ L-B after hydrolysis GLC
94 91 92 71
L-B
= Liebermann-Burchard
TABLE
reaction.
V
COMPARISON
BETWEEN
DIFFERENT
Cholesterol values are mean f
METHODS
FOR FREE
S.D. The statistical
CHOLESTEROL
evaluation
Total bilirubin mg/Ioo ml
Free cholesterol mglroo ml Sfierry and Webb
GLC
No liver disease
-
53 * 19
57 * 23 ns
Liver disease
0.4-47 (mean 6.3)
79 * 46
n = 17
as in Table III.
84 + 53 ns fl = 44
Digitonin precipitation To determine the completeness of the free cholesterol precipitation by digitoning the supernatant after digitonin precipitation was extracted with hexane and free cholesterol estimated in the hexane phase by GLC as earlier described. There was free cholesterol in all of the 13 different samples investigated. The per cent of this free cholesterol of the initial free cholesterol value varied from 1.4% to 5.6%, mean 2.2%. There was no difference in the free cholesterol values nor in the high total bilirubin values in the serum sample. DISCUSSION
The results demonstrate clearly that the proposed GLC method is sensitive,
264
BLOMHOFF
precise and more specific than calorimetric methods. The method is not laborious. The precision of the GLC method will be even better using the integrated areas of the peaks for calculation rather than the ratios of the peak heightslO that were used in the present study. Driscoll et al.8 have developed a rather rapid method for GLC analysis of cholesterol, but their method is not as precise as the one presented. In this study the purity of cholesterol standard was only tested by the GLC method described. Although no additional peaks were detected on the chromatogram, the SE-30 column used will not separate cholestanol from cholestero116~17. But Williams et aZ.ls, in a study on the purity of commercial cholesterol standards, were not able to detect any of the usual companions of cholesterol in the Sigma preparation. The excellent correspondence between the composition by weight and the composition found by peak height measurements, as shown in the recovery experiments and in the response ratio curve A, Fig. 2, indicates very little loss of cholesterol in new columns. With longer use of the columns there is probably an irreversible or slowly reversible absorption of cholesterol relative to cholestane on the solid support material, as shown by the response ratio curve B, Fig. 2. This is indicated on the chromatograms by slightly tailing peaks. Small samples are more affected by absorption than larger sampleslg. Using the initial response ratio curve as a standard curve for the calculation of cholesterol leads to substantial error. The results have to be calculated from the standard solution run several times daily and will be correct as long as the response ratio the column must be changed.
curve is linear.
When greater
absorption
loss occurs,
The present GLC method used for total cholesterol estimation gave somewhat lower values than the Carr and Drekter method7 in patients without liver disease. When hydrolysis was performed before the Liebermann-Burchard reaction there was still a difference. This is in accordance with the findings of others4F6 using different GLC methods for cholesterol determination, and depends probably on serum constituents other than cholesterol reacting in the Liebermann-Burchard reactionl. In patients with liver diseases with normal and slightly elevated serum total bilirubin there was no difference between the compared methods for total cholesterol. The reason for this is not exactly known. WebsterzO observed that a procedure using the Liebermann-Burchard type reagent but not including hydrolysis will give falsely high values for total cholesterol and cholesteryl esters when pure cholesterol standard is used, since the Liebermann-Burchard reaction gives more colour with cholesteryl esters than with free cholesterol. These patients have reduced amount of cholesteryl esters which therefore could be an explanation for the relatively lower values obtained with the Carr and Drekter method’. The present study shows rather great differences for total cholesterol between the two methods in samples with high total bilirubin content, probably because of interference of bilirubin with the Liebermann-Burchard reagentl. A serum blank as correction for high total bilirubin values in this calorimetric method is therefore insufficient. That GLC is a more specific method than the calorimetric methods using the Liebermann-Burchard reaction was more clearly demonstrated with Seronorm (Table III). There is no difference between the two methods for free cholesterol determina-
SERUM
CHOLESTEROL
DETERMINATION
BY
tion even with high serum total bilirubin. tion that
bilirubin
interference
GLC
2%
This is in accordance
is completely
eliminated
with Tonks’l observa-
by digitonin
precipitation.
The method by Sperry and Webb” is recommended as reference method for free cholesteroll92. In this study it has been shown that some free cholesterol remains in the supernatant after the standard centrifugation procedure. The present study has demonstrated that GLC methods for total and free cholesterol determination are more specific than calorimetric methods. Gas-liquid chromatography should therefore be given consideration as a reference method for the estimation of free cholesterol and total cholesterol. ACKNOWLEDGEMENTS
The
Department
of Clinical
Chemistry,
Rikshospitalet,
Oslo,
is gratefully
acknowledged for performing the routine laboratory procedures. This work has been supported by the Norwegian Research Council for Science and the Humanities. REFERENCES I D. B. TONKS, C&z. Biochem., I (1967) 12. 2 G. VANZETTI, Clin. Chim. Acta, IO (1964) 389. 3 L. P. CAWLEY, B. 0. MUSSER, S. CAMPBELL AND W.
FAUCETTE, Amer.
J. Clin. Pathol., 39
(1963) 4.50.
4 VON H.-CH. CURTIUS AND W. BORGI, 2. Klin. Chews., 4 (1966) 38. 5 J. A. SCHMIT AND A. MATER, F&M Scienti$c Corporation B&l. No. 116 (1964). 6 J. L. DRISCOLL, D. AUBUCIION, M. DESCOTEAUX AND H. F. MARTIN, Anal. Chem., 43 (1971) 1196.
7 J. 8 W. g K. IO J, II K. 12 B. 13 E. 14 15 16
17 18 Ig 20
J. CARR AND I. J. DREKTER, Clin. Chem., 2 (1956) 353. M. SPERRY AND M. WEBB, .I. Biol. Chem., 187 (1950) g7. T. STOKKE, Biochim. Biophis. Acta, 270 (1972) 156. H. WILLIAMS, M. KUCHMAK AND R. F. WITTER, J. Lipid Res., 6 (1965) 461. T. STOKKE AND K. R. NORUM, &and. I. Clin. Lab. Invest., 27 (1971) 21. A. VELA AND H. F. AcEVEDo,.Steroids,“Iq (1969) 4gg. C. HORNING, W. J. A. VANDENHEUVEL AND B. G. CREECH, Methods Biochem,
Anal.,
II
(‘963) 69. T. C. HUANG, C. P. CHEN, V. WEFLER AND A. RAFTERY, Anal. Chem., 33 (1961) 1405. A. T. NESS, J. V. PASTEWKA AND A. C. PEACOCK, C&n. Chim. Acta, IO (1964) zzg. R. FUMAGALLI, P. CAPPELLA AND W. J. A. VANDENHEUVEL, Anal. Biochem., IO (1965) 377. M. KANAI, J. Biochem., 56 (1964) 266. J. H. WILLIAMS, M. KUCHMAK AP*‘DR. F. WITTER, CZin. Chem., 16 (1970) 423. G. R. UMBRETT in H. S. KROMAN AND S. R. BENDER (Eds.), Theory and Application of Gas Chromatography, Grune and Stratton, New York, 1968, p. 54-67. D. WEBSTER, CZilz. Chim. Acta, 8 (1963) 731.
Amsterdam
- Printed in The Netherlands
257
CCA 5344
SER’CTMCHOLESTEROL
DETERMINATION
BY GAS-LIQUID
CHROMATOGRAPHY
JAN PETTER Medical (Received
BLO~HOFF
Department July 14,
A, lJndue:rsity Hospital, Rikshospa’talet.
Oslo I (Norway)
1972)
SUMMARY
A gas-liquid chromatographic (GLC) method adapted from Schmit and Mater is described for the measurement of total and free cholesterol in serum. The method has been found to be simple, sensitive and precise. The results have been compared with the calorimetric method of Carr and Drekter for total cholesterol and the method of Sperry and Webb for free cholesterol. For total cholesterol lower values were obtained by the GLC method both in sera from patients without signs of liver disease and in sera with high total bilirubin. There was a tendency for somewhat higher values for free cholesterol by the GLC method than by the calorimetric method. This difference was not significant. The present study has demonstrated that the GLC method for total and free cholesterol determination is more specific than calorimetric methods. Gas-liquid chromatography should be given consideration as a reference method for the estimation of free cholesterol and total cholesterol.
INTRODUCTION
Several methods have been developed for the estimation of cholesterol in serum. The majority of these are photometric methods based on colour reactions of cholesterol in the Liebermann-Burchard reaction or with other acid reagents’. As pointed out by Tonksl and Vanzettis these methods have several disadvantages; in particular they lack specificity, as other serum constituents may interfere with the colour reactions .
Gas-liquid chromatography (GLC) is one of the most valuable technics in sterol analysis. Surprisingly little information has, however, been derived by this method for routine nleasurement of serum cholestero13-g. This paper presents details of GLC methods adapted from Schmit and Mater5 for the measurement of total cholesterol and free cholesterol in serum. The results are compared with the calorimetric method of Carr and Drekter7 for total cholesterol and the method of Sperry and Webb8 for free cholesterol.
258 MATERIAL
BLOMHOFF AND
METHODS
Materials After an overnight fast, serum was collected from healthy volunteers, from a group of patients without liver disease and from patients who suffered from a variety of liver disorders. Serum was analysed within a few hours or stored at -15’ until cholesterol determination. [7-3Hlcholesterol (spec. act. 9.4 C/mmole) from Radiochemical Center, Amersham, England was dispersed in albumin and esterified in vitro by incubating in human serum at 37’ for about 20 ha, using lecithin cholesterol acyl transferase. Routine
laboratory procedures Total cholesterol was estimated by the Carr and Drekter method which includes deproteinization, and the Liebermann-Burchard reaction without saponification. A serum blank was used as correction for bilirubin. This correction was performed only when jaundiced serum samples were discovered. Free cholesterol was estimated by the digitonin precipitation method of Sperry and Webba. Cholesterol “standard for chromatography grade 9g%+” (Sigma Chem. Corp., St. Louis, Miss., U.S.A.) was used as reference standard in both method+‘. Preparation for gas-liquid chromatography For total cholesterol estimation 4.7 ml 95% ethanol and 0.3 ml 33% KOH in aqueous solution were added to 0.2 ml serum. After thorough mixing for about 15 set on a Whirlimixer (Fisons Scientific App., Loughborough, England) hydrolysis was performed for 15 min at 55” in a waterbath. As the columns used separate free cholesterol and not cholesteryl esters, the hydrolysis step is rather important. This step was tested using [7-3H]cholesteryl ester added to serum. After hydrolysis the lipids were separated by thin-layer chromatography, whereafter fractions containing free and esterified cholesterol were quantitated by liquid scintillation ll. From 14 min up to 40 min of the test a plateau of 99% free cholesterol was obtained. After briefly cooling, 5 ml distilled water and IO ml hexane (n-hexane pure, E. Merck, Darmstadt, Germany) were added. The solution was mixed for 30 set on a Whirlimixer and allowed to stand until complete separation of the two phases, usually IO min. From the upper phase 5 ml was removed and evaporated under a stream of nitrogen. To the dry residue 200 ~1 of IOO mg/Ioo ml 5a-cholestane (Sigma Chem. Corp., St. Louis, U.S.A.) in chloroform was added. For free cholesterol estimation g ml 95% ethanol and 3 ml diethyl ether was added to 0.2 ml serum. After mixing for about 15 set on a Whirlimixer the solution was allowed to stand for 5 min and then centrifuged at 2000 rev./min for IO min. After decanting the solvent phase this was evaporated to dryness with nitrogen in a waterbath at 55”. To the dry residue 200 ~1 of IOO mg/Ioo ml or 50 mg/Ioo ml 5acholestane in chloroform was added. Schmit and Mater5 propose only one extraction to be performed. However, an average of 3.5% higher values was obtained after a second extraction with 9 ml 95% ethanol and 3 ml diethyl ether in 20 different serum samples. Extraction has therefore been performed twice in this study. The analyses were performed in glass-stoppered test tubes.
SERUM CHOLESTEROL DETERMINATION
Gas-liquid chromatography GLC was performed
BY GLC
on F&M Scientific
259
Instruments’
High Efficiency
Chroma-
tograph 402 or Research Chromatograph 5750 G, both equipped with hydrogen flame ionization detector. The column was 18 inch by 114 inch o.d. glass packed with 3.8 wt.% silicone gum rubber (Methyl GE SE-30, F&M Scientific Corp., Avondale, U.S.A.) on 80-100 mesh Gas-Chrom Q (Applied Science Lab., State College, Pa. (U.S.A.) The carrier gas was argon at the flow rate of 60 ml/min and the column temperature was 225’. The liquid phase was dissolved in chloroform and applied to the solid support using a solution-coating technique with a rotation evaporator. The columns were noflow conditioned at 275” from 2 up to 12 h and then conditioned with flow at operating temperature until the baseline was stable. Approximately 3,ul in a Hamilton syringe was injected. A solution of known amount Sigma cholesterol “for chromatography, grade 99% +” and Sa-cholestane in chloroform used as standard was chromatographed for about every IO samples. The peak heights of 5cr-cholestane and cholesterol of the standard and the unknown extract were measured in millimeters and used for the calculation of cholesterollz. RESULTS Chromatograms Fig. I shows typical chromatograms of cholestane and cholesterol. The peaks are almost symmetrical without tailing. No additional peaks were detected in the chromatograms of the standard solutions.
2
2
2 1
i A
J
k
B I
0
5
IO
15
ITI”
20
Fig. 1. Chromatograms of cholestane (I) and cholesterol (2) for standard solution (A), total cholesterol (B) and free cholesterol (C). Conditions as described in the text.
260
BLOMHOFF
Response ratio linearity The response ratio curve is linear and goes through zero as shown in Fig. 2, curve A. After operating a column for one month the response ratio curve B, Fig. 2, was obtained indicating a substantial loss of cholesterol relative to cholestane over a wide range but proportionally greater for small samples. Reconditioning or injecting
xx)
Km
3m CHCLESTEMX
mQ/lmti
Fig. 2. Response ratio linearity curve for A: a new column, B: a concentration of the internal standard is constant.
after extensive use. The
of 20 ~1 hexamethyl-disilazane13 (Applied Science Lab., State College, Pa. U.S.A.) was not effective in restoring the linearity. Overloading the column or the detector shown by a curve plateau on the response curves did not occur up to the tested level of 2000 mg/roo ml cholesterol indicating a wide linear range of the system. The ratio of cholestane to cholesterol was sometimes somewhat higher when starting the analysis, especially when the columns had not been used for some days. This absorption phenomenon was also observed by others3*6. Before the start of the analysis cholesterol has therefore to be injected to saturate the absorptive sites. It was very difficult to predict the life of a column even with the same batch column support material. The column life was usually longer than one month. Conditioning for from 2 h up to 12 h at 275” did not seem to be of major importance, nor treatment of the glass with 5% hexamethyl-disilazane in toluene13 before packing the column. Extensive usage and high temperature are thought to be the most important factors determining the lifetime of the columns5. Recovery The recovery
of serum cholesterol
and cholesterol
added to a known serum
SERUM CHOLESTEROL DETERMINATION BY GLC
sample was tested
261
both for the total and the free cholesterol
procedure.
Thirty-one
separate analyses were performed. The recovery was equal for free and total cholesterol and was in the range of 95-10676 (SD 3.2). Some of the results are given in Table I, indicating linear standard curves and complete recovery over a wide range. TABLE
I
RECOVERYSTUDIESOF CHOLESTEROL
BY
GAS-LIQUID
CHROMATOGRAPHY
Serum values are means of 5 replicates. Cholesterol
in samfile
swum cholesterol
I
0 0
(pg)
500 250 100
TC 25 IO0 200
FC
25 50 IO0 IO
30 TC = total cholesterol,
Cholesterol
recovered
added free cholesterol 101
101
245 518 299 376
98 104 99 104 97
25 49
100 98
210
101
160 I74
IO1 102
98
FC = free cholesterol.
Cholesterol extraction The following experiments were performed to test the completeness of the cholesterol extraction procedures. In IO different samples the ethanol-water phase was removed after extraction with hexane. Cholestane was added, and extraction performed with IO ml petroleum ether. In every IO samples there were small amounts of cholesterol but less than ho/oof the initial total cholesterol value. To the precipitate after the second ethanoldiethyl ether extraction cholestane was added to IO different samples and a third extraction was performed with chloroform-methanol 2 :I v/v. Only trace amounts of cholesterol were found in 8 samples, and less than 1.2% of the initial free cholesterol value in 2 samples. Precision The precision of the methods was calculated from 82 duplicate different serum samples for total cholesterol and 47 duplicate analyses
analyses of of different
serum samples for free cholesterol according to the formula: s = dZ(d2)/zn where d is the difference between duplicates and n is the number of duplicates. The precision for total and free cholesterol at different cholesterol levels are given in Table II. The precision for free cholesterol is not as good as for total cholesterol. This depends probably on some difficulties in measuring the cholestane peak as usually this was eluted before reaching the baseline (Fig. I c). The day-to-day variability of the GLC system was determined by single daily analyses of the same cholesterol-cholestane solution for 13 days. During this time there was also a change of the SE-30 column to a new one. The mean and standard deviation for the specimen tested during these days was 348 f 6.7 mg/roo ml with a coefficient of variation of 1.9%.
262 TABLE PRECISION
BLOMHOFF II OF THE
GLC
METHOD
AT DIFFERENT
LEVELS
Mean
Standard deviation mglroo ml
Coejicient variation y0
36 28 18
58-199 zoo--295 3oo--529
160 226 384
4.0 5.6 10.6
2.5 2.5 2.8
28 I9
26-70 71-267
52 II4
I.9 4.0
3.7 3.5
mg/100 ml
FC
CHOLESTEROL
Range
n
TC
SERUM
of
n = number of duplicates. TC = total cholesterol, FC = free cholesterol.
Comparison with calorimetric methods Total cholesterol. The results of the comparison of the Carr and Drekter method’ and the GLC method for total cholesterol are given in Table III. Both serum samples with normal and raised total bilirubin content were included. Lower values were obtained with the GLC method than with the calorimetric method in serum samples from persons without liver disease. There was no difference between the two methods in samples from patients with liver disease with normal or slightly elevated total bilirubin, while there was a marked difference at high total bilirubin levels. TABLE
III
COMPARISONBETWEEN DIFFERENTMETHODSFOR TOTAL CHOLESTEROL Cholesterol values are mean f S.D. The statistical differences between pairs of results obtained by the two methods were evaluated by Student’s t-test. Percent CE was calculated by subtraction of FC from TC (both values obtained by GLC).
No liver disease
Total bilirubin mglzoo ml
Carr and Drekter
-
211 & 81
I95 zt 54
1.0-1.9
Total cholesterol mg/Ioo
210 + 48
ml
% CE
GLC n = 36 n = 15
I94 f
83**
74 f
2
Ig4 & 61 ns
7o f
6
Ig8 & 52 ns
68 & 6
231 4 6g ns
65 f
I2
219 5 71**
39 *
I4
168 * 88**
27 k I5
n = 20 Liver disease
2.0-4.9 I
237 i
65
5.0-9.9
246 + 83
>I0
234 i
IoI
I TC = total cholesterol, FC = free chromatography, ** = P ~0.01, ns
=
n = 15 n=g $2 = II
cholesterol, CE = cholesteryl no significant difference.
esters,
GLC = gas-liquid
No hydrolysis is included in the Carr and Drekter method’. Hydrolysis was therefore performed in 13 different serum samples before the Carr and Drekter analysis. After hydrolysis and hexane extraction, 4 ml of the hexane phase was analyzed by the GLC method and 4 ml by the Carr and Drekter method. The mean and standard deviation for total cholesterol obtained by the GLC method and the Carr and
SERUM CHOLESTEROL DETERMINATION BY
GLC
263
Drekter method with this procedure were respectively 346 f 84 and 362 f 85 mg/roo ml ($J< 0.01). Seronorm (Nyegaard & Co., Oslo, Norway) is a reference serum from horse used for control of clinical chemical analysis. The values obtained with this serum (Batch 116) with the Carr and Drekter method’, the direct method of Huang14 modified by Ness?, the Liebermann-Burchard reaction after hydrolysis and the GLC method for total cholesterol are compared in Table IV. There is a marked difference between the calorimetric methods and the GLC method. The value for bilirubin in this batch was 1.99 mg/Ioo ml and cholesteryl esters 80% of total cholesterol determined by the proposed GLC method. Free cholesterol. The results are given in Table V. There is a tendency to higher values with the GLC method than with the calorimetric method, but this difference was not significant. TABLE TOTAL
IV CHOLESTEROL
VALUES
FOR SERONORM
WITH
DIFFERENT
METHODS
Values are tneans of 3 replicates. Methods
Total cholesterol mgl100 ml
Huang et aZ.14*15 Carr and Drekter’ L-B after hydrolysis GLC
94 91 92 71
L-B
= Liebermann-Burchard
TABLE
reaction.
V
COMPARISON
BETWEEN
DIFFERENT
Cholesterol values are mean f
METHODS
FOR FREE
S.D. The statistical
CHOLESTEROL
evaluation
Total bilirubin mg/Ioo ml
Free cholesterol mglroo ml Sfierry and Webb
GLC
No liver disease
-
53 * 19
57 * 23 ns
Liver disease
0.4-47 (mean 6.3)
79 * 46
n = 17
as in Table III.
84 + 53 ns fl = 44
Digitonin precipitation To determine the completeness of the free cholesterol precipitation by digitoning the supernatant after digitonin precipitation was extracted with hexane and free cholesterol estimated in the hexane phase by GLC as earlier described. There was free cholesterol in all of the 13 different samples investigated. The per cent of this free cholesterol of the initial free cholesterol value varied from 1.4% to 5.6%, mean 2.2%. There was no difference in the free cholesterol values nor in the high total bilirubin values in the serum sample. DISCUSSION
The results demonstrate clearly that the proposed GLC method is sensitive,
264
BLOMHOFF
precise and more specific than calorimetric methods. The method is not laborious. The precision of the GLC method will be even better using the integrated areas of the peaks for calculation rather than the ratios of the peak heightslO that were used in the present study. Driscoll et al.8 have developed a rather rapid method for GLC analysis of cholesterol, but their method is not as precise as the one presented. In this study the purity of cholesterol standard was only tested by the GLC method described. Although no additional peaks were detected on the chromatogram, the SE-30 column used will not separate cholestanol from cholestero116~17. But Williams et aZ.ls, in a study on the purity of commercial cholesterol standards, were not able to detect any of the usual companions of cholesterol in the Sigma preparation. The excellent correspondence between the composition by weight and the composition found by peak height measurements, as shown in the recovery experiments and in the response ratio curve A, Fig. 2, indicates very little loss of cholesterol in new columns. With longer use of the columns there is probably an irreversible or slowly reversible absorption of cholesterol relative to cholestane on the solid support material, as shown by the response ratio curve B, Fig. 2. This is indicated on the chromatograms by slightly tailing peaks. Small samples are more affected by absorption than larger sampleslg. Using the initial response ratio curve as a standard curve for the calculation of cholesterol leads to substantial error. The results have to be calculated from the standard solution run several times daily and will be correct as long as the response ratio the column must be changed.
curve is linear.
When greater
absorption
loss occurs,
The present GLC method used for total cholesterol estimation gave somewhat lower values than the Carr and Drekter method7 in patients without liver disease. When hydrolysis was performed before the Liebermann-Burchard reaction there was still a difference. This is in accordance with the findings of others4F6 using different GLC methods for cholesterol determination, and depends probably on serum constituents other than cholesterol reacting in the Liebermann-Burchard reactionl. In patients with liver diseases with normal and slightly elevated serum total bilirubin there was no difference between the compared methods for total cholesterol. The reason for this is not exactly known. WebsterzO observed that a procedure using the Liebermann-Burchard type reagent but not including hydrolysis will give falsely high values for total cholesterol and cholesteryl esters when pure cholesterol standard is used, since the Liebermann-Burchard reaction gives more colour with cholesteryl esters than with free cholesterol. These patients have reduced amount of cholesteryl esters which therefore could be an explanation for the relatively lower values obtained with the Carr and Drekter method’. The present study shows rather great differences for total cholesterol between the two methods in samples with high total bilirubin content, probably because of interference of bilirubin with the Liebermann-Burchard reagentl. A serum blank as correction for high total bilirubin values in this calorimetric method is therefore insufficient. That GLC is a more specific method than the calorimetric methods using the Liebermann-Burchard reaction was more clearly demonstrated with Seronorm (Table III). There is no difference between the two methods for free cholesterol determina-
SERUM
CHOLESTEROL
DETERMINATION
BY
tion even with high serum total bilirubin. tion that
bilirubin
interference
GLC
2%
This is in accordance
is completely
eliminated
with Tonks’l observa-
by digitonin
precipitation.
The method by Sperry and Webb” is recommended as reference method for free cholesteroll92. In this study it has been shown that some free cholesterol remains in the supernatant after the standard centrifugation procedure. The present study has demonstrated that GLC methods for total and free cholesterol determination are more specific than calorimetric methods. Gas-liquid chromatography should therefore be given consideration as a reference method for the estimation of free cholesterol and total cholesterol. ACKNOWLEDGEMENTS
The
Department
of Clinical
Chemistry,
Rikshospitalet,
Oslo,
is gratefully
acknowledged for performing the routine laboratory procedures. This work has been supported by the Norwegian Research Council for Science and the Humanities. REFERENCES I D. B. TONKS, C&z. Biochem., I (1967) 12. 2 G. VANZETTI, Clin. Chim. Acta, IO (1964) 389. 3 L. P. CAWLEY, B. 0. MUSSER, S. CAMPBELL AND W.
FAUCETTE, Amer.
J. Clin. Pathol., 39
(1963) 4.50.
4 VON H.-CH. CURTIUS AND W. BORGI, 2. Klin. Chews., 4 (1966) 38. 5 J. A. SCHMIT AND A. MATER, F&M Scienti$c Corporation B&l. No. 116 (1964). 6 J. L. DRISCOLL, D. AUBUCIION, M. DESCOTEAUX AND H. F. MARTIN, Anal. Chem., 43 (1971) 1196.
7 J. 8 W. g K. IO J, II K. 12 B. 13 E. 14 15 16
17 18 Ig 20
J. CARR AND I. J. DREKTER, Clin. Chem., 2 (1956) 353. M. SPERRY AND M. WEBB, .I. Biol. Chem., 187 (1950) g7. T. STOKKE, Biochim. Biophis. Acta, 270 (1972) 156. H. WILLIAMS, M. KUCHMAK AND R. F. WITTER, J. Lipid Res., 6 (1965) 461. T. STOKKE AND K. R. NORUM, &and. I. Clin. Lab. Invest., 27 (1971) 21. A. VELA AND H. F. AcEVEDo,.Steroids,“Iq (1969) 4gg. C. HORNING, W. J. A. VANDENHEUVEL AND B. G. CREECH, Methods Biochem,
Anal.,
II
(‘963) 69. T. C. HUANG, C. P. CHEN, V. WEFLER AND A. RAFTERY, Anal. Chem., 33 (1961) 1405. A. T. NESS, J. V. PASTEWKA AND A. C. PEACOCK, C&n. Chim. Acta, IO (1964) zzg. R. FUMAGALLI, P. CAPPELLA AND W. J. A. VANDENHEUVEL, Anal. Biochem., IO (1965) 377. M. KANAI, J. Biochem., 56 (1964) 266. J. H. WILLIAMS, M. KUCHMAK AP*‘DR. F. WITTER, CZin. Chem., 16 (1970) 423. G. R. UMBRETT in H. S. KROMAN AND S. R. BENDER (Eds.), Theory and Application of Gas Chromatography, Grune and Stratton, New York, 1968, p. 54-67. D. WEBSTER, CZilz. Chim. Acta, 8 (1963) 731.