75
Clinica Chimica Acto, 71 (1976) 75-80 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7950
RAPID ENZYMATIC DETERMINATION OF FREE AND ESTERIFIED CHOLESTEROL CONTENT OF SERUM AND TISSUES
ROBERT
J. MORIN
Department of Pathology, Harbor General Hospital/UCLA Calif. 90509 (U.S.A.) (Received
February
13th,
School of Medicine, Torrance,
1976)
Introduction Determination of both serum free cholesterol and esterified cholesterol concentration in addition to the routine determination of total cholesterol is of value in the diagnosis of 1ecithin:cholesterol acyltransferase deficiency [l] and of liver diseases [ 2,3]. In addition, determination of free and esterified cholesterol content in serum and tissues of humans and animals is frequently required in investigations of cholesterol ester synthesis and hydrolysis and other parameters of cholesterol metabolism. Most of the earlier developed methods for analysis of free and esterified cholesterol utilize solvent extraction of the sample, followed by precipitation of free cholesterol as the digitonide, and subsequent calorimetric analysis of this fraction and also of an aliquot of the original extract for total cholesterol concentration [ 4-61. Although some recent improvements in this type of method have been proposed [ 7-91, they are still relatively time consuming and subject to inaccuracies due to the complexities of the procedures. Other investigators have separated the free and esterified cholesterol by silicic acid column chromatography [lo], Sephadex LH-20 column chromatography [ll], or thinlayer chromatography [12-141, followed by separate calorimetric or fluorimetric analysis of each fraction. These methods are also very time consuming, and usually it is necessary to concentrate the eluted fractions before the analyses can be performed. Gas chromatography has been shown to be an accurate method for quantitating serum total cholesterol, but only one investigator has reported the routine use of gas chromatography for separate quantitation of free and esterified cholesterol [ 151. The latter method does not appear convenient for routine use because the individual fatty acid esters of cholesterol are separated, and multiple peak areas must be totaled to obtain the total cholesterol ester concentration in one sample. A newly developed enzymatic method for determination of total serum cholesterol utilizes hydrolysis of the esters by the enzyme cholesterol ester hydrol-
76
ase, followed by conversion of cholesterol to cholest-4-en-3-one and H,O,! by a cholesterol oxidase [ 16-181. The H,O, is quantitated by oxidative coupling of 4-aminoantipyrine and phenol to form a colored product whose absorbance can be measured at 500 nm. This method has been shown to have equal or greater accuracy and greater specificity than the standard calorimetric methods [ 16-181 . The present study was conducted to evaluate the possible adaptation of this enzymatic method to determine both free and esterified cholesterol concentrations in serum and tissues. Results were compared to analysis of the same specimens by gas chromatography, since the latter is more accurate and specific for cholesterol than the existing calorimetric methods [ 19-221. Materials and methods Total cholesterol concentrations in human serum samples were analyzed by an enzymatic method as follows. 5 ~1 of each serum were aspirated automatically into cuvettes of an Abbott ABA-100 bichromatic analyzer, with 500/600 filters and a syringe plate ratio of l/101. The sera were automatically mixed with 500 ~1 of reagent containing: cholesterol ester hydrolase (Ames Laboratories) 0.066 I.U./ml, cholesterol oxidase (Whatman Biochemicals) 0.117 I.U./ml, peroxidase (Worthington Biochemical Corp.) 670 I.U./ml, sodium cholate 3 pmoles/ml (Sigma), 4-aminoantipyrine (J.T. Baker Chemical Co.) 0.8 pmoles/ ml, phenol (Mallinckrodt Chemical Works) 14 pmoles/ml, polyethylene glycol 0.2 pmoles/ml (Matheson, Coleman and Bell, mol. wt. 600), 50 pmoles NasHPOd and 50 pmoles NaHzPOJ/ml. The mixtures were incubated at 37°C for 10 min and absorbances printed out automatically. Serial dilutions of Serachol (General Diagnostics) containing 84-420 mg/dl were used for the cholesterol standard curve, and concentrations of cholesterol in the serum samples were calculated from this curve. For enzymatic free cholesterol determination a similar procedure was followed except that a 1:51 syringe plate was used, so as to attain a higher ratio of serum to reagent, and the cholesterol ester hydrolase was omitted from the reagent mixture. Esterified cholesterol concentrations were calculated by subtracting free cholesterol from total cholesterol values. Total serum cholesterol concentrations in the same human sera samples and in the Serachol standards were analyzed by gas chromatography as follows: One ml of alcoholic KOH (6 ml 33% KOH and 94 ml ethanol) was added to each 30+1 aliquot of serum, followed by heating at 60°C in an aluminum block for one hour. One ml of HZ0 was added, following which 2.0 ml of a stigmaster01 internal standard, 50 pg/ml in hexane was added to each. The mixtures were shaken automatically in a Vir Tis Extractomatic for 10 min, and then allowed to stand for 5 min or more to separate the phases. One ml aliquots of each top layer were evaporated to dryness, redissolved in 20 ~1 of CSI, and aliquots then injected into a Barber-Colman Model 5000 gas chromatograph with flame ionization detection, using a 3% QF-1 liquid phase on Gaschrom Q 100/120 (Applied Science Labs), at 240°C and a nitrogen carrier gas flow rate of 45 ml/min. Free cholesterol was analyzed in a similar manner except that the KOH was omitted from the reagent, and the mixture was not heated.
Portions of liver, adrenal, ovaries and kidneys were obtained from 5 female Sprague-Dawley rats, 6 months of age, and homogenized in 10 X their weight of chloroform/methanol (2 : 1, v/v) at 45 000 rpm for 2 min using a VirTis homogenizer. The homogenates were centrifuged at 2000 X g for 10 min, following which aliquots of the supernatant solutions were evaporated to dryness at 40°C under a nitrogen stream. For enzymatic analysis of free cholesterol these aliquots were dissolved iI1 dioxane, and a series of standards of cholesterol (National Bureau of Standards No. 911) were prepared in dioxane in the concentration range of 0.5-4.0 mg/ml. For enzymatic analysis of total cholesterol, the tissue extract aliquots were also dissolved in dioxane. The standard utilized was a micellar solution of one part cholesterol (NBS) one part cholesterol palmitate (applied Science Labs) and 3 parts lecithin (Calbiochem) in dioxane to achieve a concentration range of cholesterol from 0.5-4.0 mg/ ml. Addition of the lecithin was found necessary to dissolve the cholesterol ester standard in dioxane. No difficulty was encountered in dissolving the cholesterol esters of the tissue extracts in dioxane, presumably because these extracts all contained lecithin. Aliquots of these tissue extracts and standards in dioxane were then analyzed by the same procedures used to analyze free and total cholesterol concentrations in the serum samples, using the enzyme reagent mixture and the Abbott ABA 100 automatic spectrophotometer. Other aliquots of the same tissue extracts were evaporated to dryness and the free and total cholesterol contents analyzed by the same gas chromatographic methods utilized for the serum samples. Statistical analyses were done using a PDP-8 computer. Results and discusssion Results of the analysis of free and esterified cholesterol concentrations in a series of 30 human serum samples by the enzymatic method as compared to the gas chromatographic method are shown in Fig. 1. The gas-chromatographic method used was previously shown to give equal or greater accuracy and precision when compared to analyses done by the standard calorimetric method of Abel1 et al. [23]. For serum free cholesterol analysis, the present enzymatic method showed a range of 33 to 114 mg/dl and a mean + S.D. (standard deviation) of 69 f 23 mg/dl. The gas-chromatographic method showed a range of 32 to 113 mg/dl, with a mean f S.D. of 68 ?r 23. The correlation coefficient (r) for the two methods was 0.980, with a regression line slope of 0.954, a Y intercept of 2.20 and a paired t-test value of 1.17. For serum esterified cholesterol analyzed by enzymatic method, the range of values was 89-292, with a mean It S.D. of 172 t 56; for the gas-chromatographic method the range was 91-282, with a mean f S.D. of 169 f 54 mg/dl. The correlation coefficient for the two methods was 0.986, with a regression line slope of 0.955, a Y intercept of 4.84, and a paired t-test value of 1.70. In Table I are indicated the results of enzymatic and gas-chromatographic analysis of free and esterified cholesterol content as determined in aliquots of extracts of rat tissues. When all enzymatic free cholesterol analyses were plotted on an X axis vs. the results using the gas-chromatographic method coefficient between the methods was plotted on a Y axis, the correlation
. 0 FREE .
CHOLESTEROL
ESTERIFIED
CHOLESTEROL
.
. .
. l
. 2’ .. l
a*
G \
F
m1 mg/dl
SERUM
l
1
200 CHOLESTEROL
BY GAS
Fig. 1. Correlation of the present enzymatic method matographic method. Free cholesterol concentrations lesterol by closed circles.
I
250
4 I
300
CHROMATOGRAPHY for analysis of serum cholesterol with a gas-chroare represented by open circles and esterified cho-
0.979, with a regression line slope of 0.931, a Y intercept of 0.039 and a paired t-test value of 3.49. Similar regression analysis of all the tissue esterified cholesterol results indicated a correlation coefficient of 0.996, with a slope of 0.904, a Y intercept of 0.210 and a t-test value of 2.05. The precision of the enzymatic vs. gas-chromatographic method was evaluated by quadruplicate analyses by both methods of the same pooled serum sample daily for 5 consecutive days. Mean + S.D. free cholesterol in this serum for the 20‘analyses by the enzymatic method was 58 + 1.8 with a C.V. (coefficcient of variation) of 3.1%, and with the gas-chromatographic method was 57 + 1.7 (C.V. 2.9%). For the esterified cholesterol the enzymatic method gave a mean +_S.D. of 140 f 4.8 (C.V. 3.4%) and with the gas-chromatographic method 134 2 4.0 (C.V. 3.0%). Previous studies [16-181 have indicated that results of analysis of serum total cholesterol concentrations by the enzymatic method correlate well with those obtained by the manual method of Abel1 et al. [23] and with several automated calorimetric methods. Specificity for cholesterol is higher with the enzymatic method than with the calorimetric methods, and the former is not subject to interference by hemolysis, lipemia or steroid drugs [18]. Products of the Liebermann-Burchardt reaction are known to have differing extinction coefficients when derived from free cholesterol vs. esterified cholesterol [16], but this is not the case with the enzymatic method. The present study has demonstrated that an enzymatic method, utilizing a single aqueous reagent, can be employed for rapid and accurate determination of both free and esterified cholesterol content of serum and tissues. The method can be performed either
79 TABLE I ENZYMATIC
AND
GAS CHROMATOGRAPHIC
TEROL CONCENTRATIONS
Tissue cholesterol concentrations are given as n&g Free cholesterol
Liver
Mean S.D. Adrenal
Mean S.D. Ovary
Mean S.D. Kidney
Mean S.D.
ANALYSIS
OF FREE AND ESTERIFIED
CHOLES-
IN RAT TISSUE EXTRACTS wet weight of tissue. Esterified cholesterol
Enzymatic
GC
Enzymatic
1.51 1.27 1.24 1.48 1.28 1.36 0.13
1.36 1.13 1.30 1.45 1.20 1.32 0.09
2.62 3.89 3.06 3.46 3.12 3.23 0.47
2.86 3.64 2.81 3.20 2.79 3.06 0.36
2.15 2.00 2.03 2.03 2.25 2.05 2.10 0.10
1.81 2.08 1.92 1.92 2.07 1.72 1.92 0.16
5.06 4.85 4.85 5.51 5.07 4.93 5.08 0.26
4.93 4.91 4.91 5.22 5.07 4.65 4.96 0.21
2.32 2.62 2.46 2.76 2.55 2.54 0.17
2.33 2.52 2.46 2.71 2.36 2.48 0.15
1.21 1.28 1.38 1.38 1.27 1.30 0.07
1.17 1.24 1.30 1.33 1.20 1.24 0.07
2.45 1.91 2.23 2.26 1.99 2.17 0.22 32.2 34.7 36.6 34.3 27.6 33.1 3.4
GC 2.33 1.80 2.08 2.19 1.92 2.06 0.21 26.2 32.2 33.1 30.8 28.8 30.2 2.8
manually, or with an automated instrument as in the present study. The gaschromatographic method utilized for comparison purposes in this study is also a highly accurate and specific method, and if employed with an automatic sample injecting system and automatic digital integration of peak areas can also be a rapid means of routine determination of free and esterified cholesterol, with many advantages over the digitonin precipitation-calorimetric type methods. Reference8 1 Tonvik, H.. Gjone, E. and Norum, K.R. (1968) Acta, Med. Stand. 183.387 2 Jones, D.P.. Sosa, F.R., Shartsis. J.. Shah, P.T.. Skromak, E. and Beher, W.T. (1971) 3 4 5 6 7
50.259 CaIandra, S.. Martin. M.J. and McIntyre, N. (1971) J. Clin. Invest., 352 Schoenheimer. R. and Sperry, W.M. (1934) J. Biol. Chem. 106.745 Clarke, D.H. and Mamey. A.F. (1945) J. Lab. Clin. Med. 30. 615 Sperry, W.M. and Webb, M. (1950) J. Biol. Chem. 187.97 B~OWII, H.H., Zlatkis. A.. Zak, B. and Boyle. A.J. (1954) Anal. Chem. 26. 379
J. CIin. Invest.
80
8
Crawford,
9
Webster,
N. (1958)
Clin.
D. (1962)
10
Creech,
11
Cham,
B.G.
12
Zeitman,
and
B.E..
Clin. SeweU,
Hurwood,
B.B.
13
Beukers, Bondjers,
G. and
15
Ikekawa,
N.,
16
Waxman,
S. and
AlIain,
18
Witte,
C.C.,
19
Blomhoff,
20
Driscoll,
J.L.,
21
MacGee,
J.,
Med.
656
J.P.
82,
22
Morin.
R.J.
23
Abell,
L.L.,
L.S.,
Ishikawa.
Levy,
Elms. B.B.,
Res.
Ghan, Clin. T.,
N.J.
K.
Jap. Chem.
Richmond,
Wycoff,
Chim.
Acta
Descoteaux,
(1975)
Biochem.
(1971) Clin.
Miller,
G.J.M.
Anal.
C.S.G..
Brodie,
Powell,
3,
119
L.W.
(1973)
Clin.
Chim.
Acta
49.
109
6, 578
and
D.,
Biochem. and
Hooghwinkel,
Sate.
II, D.A.
(1973)
Anal. B.R.
C. (1974)
Aubuchon,
and
(1962)
and
M. and
Barrett,
3, 357 7, 277
S. (1971)
Schreiber,
Peon.
D.L..
W.A.
Bjorkerud.
Matsui,
Acta Acta
Knowles,
J. Lipid
H. Veltkamp,
17
B.W. J.J.,
(1965)
14
Chim. Chim.
Clin.
Chim.
Acta
25.
403
Clin.
Chem.
363 Med.
41,
163
725 Fu,
Clin.
P.C.
Chem.
(1974) 20,
20,
470
1282
257
Evans,
Ann.
Clin.
and
20,
(1974)
M. and
W..
B.B.
J. Exp. W. and
D.A. 43,
(1969) 42,
G., Lab.
Kendall,
Martin,
H.F.
Steiner, Sci.
5, 52
F.E.
(1952)
P.
(1971) and
Anal. Glueck,
J. Biol.
Chem.
Chem. C.J.
195,
43.1196 (1973)
357
J.
Lab.
CIin.
Clinica Chimica Acto, 71 (1976) 75-80 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7950
RAPID ENZYMATIC DETERMINATION OF FREE AND ESTERIFIED CHOLESTEROL CONTENT OF SERUM AND TISSUES
ROBERT
J. MORIN
Department of Pathology, Harbor General Hospital/UCLA Calif. 90509 (U.S.A.) (Received
February
13th,
School of Medicine, Torrance,
1976)
Introduction Determination of both serum free cholesterol and esterified cholesterol concentration in addition to the routine determination of total cholesterol is of value in the diagnosis of 1ecithin:cholesterol acyltransferase deficiency [l] and of liver diseases [ 2,3]. In addition, determination of free and esterified cholesterol content in serum and tissues of humans and animals is frequently required in investigations of cholesterol ester synthesis and hydrolysis and other parameters of cholesterol metabolism. Most of the earlier developed methods for analysis of free and esterified cholesterol utilize solvent extraction of the sample, followed by precipitation of free cholesterol as the digitonide, and subsequent calorimetric analysis of this fraction and also of an aliquot of the original extract for total cholesterol concentration [ 4-61. Although some recent improvements in this type of method have been proposed [ 7-91, they are still relatively time consuming and subject to inaccuracies due to the complexities of the procedures. Other investigators have separated the free and esterified cholesterol by silicic acid column chromatography [lo], Sephadex LH-20 column chromatography [ll], or thinlayer chromatography [12-141, followed by separate calorimetric or fluorimetric analysis of each fraction. These methods are also very time consuming, and usually it is necessary to concentrate the eluted fractions before the analyses can be performed. Gas chromatography has been shown to be an accurate method for quantitating serum total cholesterol, but only one investigator has reported the routine use of gas chromatography for separate quantitation of free and esterified cholesterol [ 151. The latter method does not appear convenient for routine use because the individual fatty acid esters of cholesterol are separated, and multiple peak areas must be totaled to obtain the total cholesterol ester concentration in one sample. A newly developed enzymatic method for determination of total serum cholesterol utilizes hydrolysis of the esters by the enzyme cholesterol ester hydrol-
76
ase, followed by conversion of cholesterol to cholest-4-en-3-one and H,O,! by a cholesterol oxidase [ 16-181. The H,O, is quantitated by oxidative coupling of 4-aminoantipyrine and phenol to form a colored product whose absorbance can be measured at 500 nm. This method has been shown to have equal or greater accuracy and greater specificity than the standard calorimetric methods [ 16-181 . The present study was conducted to evaluate the possible adaptation of this enzymatic method to determine both free and esterified cholesterol concentrations in serum and tissues. Results were compared to analysis of the same specimens by gas chromatography, since the latter is more accurate and specific for cholesterol than the existing calorimetric methods [ 19-221. Materials and methods Total cholesterol concentrations in human serum samples were analyzed by an enzymatic method as follows. 5 ~1 of each serum were aspirated automatically into cuvettes of an Abbott ABA-100 bichromatic analyzer, with 500/600 filters and a syringe plate ratio of l/101. The sera were automatically mixed with 500 ~1 of reagent containing: cholesterol ester hydrolase (Ames Laboratories) 0.066 I.U./ml, cholesterol oxidase (Whatman Biochemicals) 0.117 I.U./ml, peroxidase (Worthington Biochemical Corp.) 670 I.U./ml, sodium cholate 3 pmoles/ml (Sigma), 4-aminoantipyrine (J.T. Baker Chemical Co.) 0.8 pmoles/ ml, phenol (Mallinckrodt Chemical Works) 14 pmoles/ml, polyethylene glycol 0.2 pmoles/ml (Matheson, Coleman and Bell, mol. wt. 600), 50 pmoles NasHPOd and 50 pmoles NaHzPOJ/ml. The mixtures were incubated at 37°C for 10 min and absorbances printed out automatically. Serial dilutions of Serachol (General Diagnostics) containing 84-420 mg/dl were used for the cholesterol standard curve, and concentrations of cholesterol in the serum samples were calculated from this curve. For enzymatic free cholesterol determination a similar procedure was followed except that a 1:51 syringe plate was used, so as to attain a higher ratio of serum to reagent, and the cholesterol ester hydrolase was omitted from the reagent mixture. Esterified cholesterol concentrations were calculated by subtracting free cholesterol from total cholesterol values. Total serum cholesterol concentrations in the same human sera samples and in the Serachol standards were analyzed by gas chromatography as follows: One ml of alcoholic KOH (6 ml 33% KOH and 94 ml ethanol) was added to each 30+1 aliquot of serum, followed by heating at 60°C in an aluminum block for one hour. One ml of HZ0 was added, following which 2.0 ml of a stigmaster01 internal standard, 50 pg/ml in hexane was added to each. The mixtures were shaken automatically in a Vir Tis Extractomatic for 10 min, and then allowed to stand for 5 min or more to separate the phases. One ml aliquots of each top layer were evaporated to dryness, redissolved in 20 ~1 of CSI, and aliquots then injected into a Barber-Colman Model 5000 gas chromatograph with flame ionization detection, using a 3% QF-1 liquid phase on Gaschrom Q 100/120 (Applied Science Labs), at 240°C and a nitrogen carrier gas flow rate of 45 ml/min. Free cholesterol was analyzed in a similar manner except that the KOH was omitted from the reagent, and the mixture was not heated.
Portions of liver, adrenal, ovaries and kidneys were obtained from 5 female Sprague-Dawley rats, 6 months of age, and homogenized in 10 X their weight of chloroform/methanol (2 : 1, v/v) at 45 000 rpm for 2 min using a VirTis homogenizer. The homogenates were centrifuged at 2000 X g for 10 min, following which aliquots of the supernatant solutions were evaporated to dryness at 40°C under a nitrogen stream. For enzymatic analysis of free cholesterol these aliquots were dissolved iI1 dioxane, and a series of standards of cholesterol (National Bureau of Standards No. 911) were prepared in dioxane in the concentration range of 0.5-4.0 mg/ml. For enzymatic analysis of total cholesterol, the tissue extract aliquots were also dissolved in dioxane. The standard utilized was a micellar solution of one part cholesterol (NBS) one part cholesterol palmitate (applied Science Labs) and 3 parts lecithin (Calbiochem) in dioxane to achieve a concentration range of cholesterol from 0.5-4.0 mg/ ml. Addition of the lecithin was found necessary to dissolve the cholesterol ester standard in dioxane. No difficulty was encountered in dissolving the cholesterol esters of the tissue extracts in dioxane, presumably because these extracts all contained lecithin. Aliquots of these tissue extracts and standards in dioxane were then analyzed by the same procedures used to analyze free and total cholesterol concentrations in the serum samples, using the enzyme reagent mixture and the Abbott ABA 100 automatic spectrophotometer. Other aliquots of the same tissue extracts were evaporated to dryness and the free and total cholesterol contents analyzed by the same gas chromatographic methods utilized for the serum samples. Statistical analyses were done using a PDP-8 computer. Results and discusssion Results of the analysis of free and esterified cholesterol concentrations in a series of 30 human serum samples by the enzymatic method as compared to the gas chromatographic method are shown in Fig. 1. The gas-chromatographic method used was previously shown to give equal or greater accuracy and precision when compared to analyses done by the standard calorimetric method of Abel1 et al. [23]. For serum free cholesterol analysis, the present enzymatic method showed a range of 33 to 114 mg/dl and a mean + S.D. (standard deviation) of 69 f 23 mg/dl. The gas-chromatographic method showed a range of 32 to 113 mg/dl, with a mean f S.D. of 68 ?r 23. The correlation coefficient (r) for the two methods was 0.980, with a regression line slope of 0.954, a Y intercept of 2.20 and a paired t-test value of 1.17. For serum esterified cholesterol analyzed by enzymatic method, the range of values was 89-292, with a mean It S.D. of 172 t 56; for the gas-chromatographic method the range was 91-282, with a mean f S.D. of 169 f 54 mg/dl. The correlation coefficient for the two methods was 0.986, with a regression line slope of 0.955, a Y intercept of 4.84, and a paired t-test value of 1.70. In Table I are indicated the results of enzymatic and gas-chromatographic analysis of free and esterified cholesterol content as determined in aliquots of extracts of rat tissues. When all enzymatic free cholesterol analyses were plotted on an X axis vs. the results using the gas-chromatographic method coefficient between the methods was plotted on a Y axis, the correlation
. 0 FREE .
CHOLESTEROL
ESTERIFIED
CHOLESTEROL
.
. .
. l
. 2’ .. l
a*
G \
F
m1 mg/dl
SERUM
l
1
200 CHOLESTEROL
BY GAS
Fig. 1. Correlation of the present enzymatic method matographic method. Free cholesterol concentrations lesterol by closed circles.
I
250
4 I
300
CHROMATOGRAPHY for analysis of serum cholesterol with a gas-chroare represented by open circles and esterified cho-
0.979, with a regression line slope of 0.931, a Y intercept of 0.039 and a paired t-test value of 3.49. Similar regression analysis of all the tissue esterified cholesterol results indicated a correlation coefficient of 0.996, with a slope of 0.904, a Y intercept of 0.210 and a t-test value of 2.05. The precision of the enzymatic vs. gas-chromatographic method was evaluated by quadruplicate analyses by both methods of the same pooled serum sample daily for 5 consecutive days. Mean + S.D. free cholesterol in this serum for the 20‘analyses by the enzymatic method was 58 + 1.8 with a C.V. (coefficcient of variation) of 3.1%, and with the gas-chromatographic method was 57 + 1.7 (C.V. 2.9%). For the esterified cholesterol the enzymatic method gave a mean +_S.D. of 140 f 4.8 (C.V. 3.4%) and with the gas-chromatographic method 134 2 4.0 (C.V. 3.0%). Previous studies [16-181 have indicated that results of analysis of serum total cholesterol concentrations by the enzymatic method correlate well with those obtained by the manual method of Abel1 et al. [23] and with several automated calorimetric methods. Specificity for cholesterol is higher with the enzymatic method than with the calorimetric methods, and the former is not subject to interference by hemolysis, lipemia or steroid drugs [18]. Products of the Liebermann-Burchardt reaction are known to have differing extinction coefficients when derived from free cholesterol vs. esterified cholesterol [16], but this is not the case with the enzymatic method. The present study has demonstrated that an enzymatic method, utilizing a single aqueous reagent, can be employed for rapid and accurate determination of both free and esterified cholesterol content of serum and tissues. The method can be performed either
79 TABLE I ENZYMATIC
AND
GAS CHROMATOGRAPHIC
TEROL CONCENTRATIONS
Tissue cholesterol concentrations are given as n&g Free cholesterol
Liver
Mean S.D. Adrenal
Mean S.D. Ovary
Mean S.D. Kidney
Mean S.D.
ANALYSIS
OF FREE AND ESTERIFIED
CHOLES-
IN RAT TISSUE EXTRACTS wet weight of tissue. Esterified cholesterol
Enzymatic
GC
Enzymatic
1.51 1.27 1.24 1.48 1.28 1.36 0.13
1.36 1.13 1.30 1.45 1.20 1.32 0.09
2.62 3.89 3.06 3.46 3.12 3.23 0.47
2.86 3.64 2.81 3.20 2.79 3.06 0.36
2.15 2.00 2.03 2.03 2.25 2.05 2.10 0.10
1.81 2.08 1.92 1.92 2.07 1.72 1.92 0.16
5.06 4.85 4.85 5.51 5.07 4.93 5.08 0.26
4.93 4.91 4.91 5.22 5.07 4.65 4.96 0.21
2.32 2.62 2.46 2.76 2.55 2.54 0.17
2.33 2.52 2.46 2.71 2.36 2.48 0.15
1.21 1.28 1.38 1.38 1.27 1.30 0.07
1.17 1.24 1.30 1.33 1.20 1.24 0.07
2.45 1.91 2.23 2.26 1.99 2.17 0.22 32.2 34.7 36.6 34.3 27.6 33.1 3.4
GC 2.33 1.80 2.08 2.19 1.92 2.06 0.21 26.2 32.2 33.1 30.8 28.8 30.2 2.8
manually, or with an automated instrument as in the present study. The gaschromatographic method utilized for comparison purposes in this study is also a highly accurate and specific method, and if employed with an automatic sample injecting system and automatic digital integration of peak areas can also be a rapid means of routine determination of free and esterified cholesterol, with many advantages over the digitonin precipitation-calorimetric type methods. Reference8 1 Tonvik, H.. Gjone, E. and Norum, K.R. (1968) Acta, Med. Stand. 183.387 2 Jones, D.P.. Sosa, F.R., Shartsis. J.. Shah, P.T.. Skromak, E. and Beher, W.T. (1971) 3 4 5 6 7
50.259 CaIandra, S.. Martin. M.J. and McIntyre, N. (1971) J. Clin. Invest., 352 Schoenheimer. R. and Sperry, W.M. (1934) J. Biol. Chem. 106.745 Clarke, D.H. and Mamey. A.F. (1945) J. Lab. Clin. Med. 30. 615 Sperry, W.M. and Webb, M. (1950) J. Biol. Chem. 187.97 B~OWII, H.H., Zlatkis. A.. Zak, B. and Boyle. A.J. (1954) Anal. Chem. 26. 379
J. CIin. Invest.
80
8
Crawford,
9
Webster,
N. (1958)
Clin.
D. (1962)
10
Creech,
11
Cham,
B.G.
12
Zeitman,
and
B.E..
Clin. SeweU,
Hurwood,
B.B.
13
Beukers, Bondjers,
G. and
15
Ikekawa,
N.,
16
Waxman,
S. and
AlIain,
18
Witte,
C.C.,
19
Blomhoff,
20
Driscoll,
J.L.,
21
MacGee,
J.,
Med.
656
J.P.
82,
22
Morin.
R.J.
23
Abell,
L.L.,
L.S.,
Ishikawa.
Levy,
Elms. B.B.,
Res.
Ghan, Clin. T.,
N.J.
K.
Jap. Chem.
Richmond,
Wycoff,
Chim.
Acta
Descoteaux,
(1975)
Biochem.
(1971) Clin.
Miller,
G.J.M.
Anal.
C.S.G..
Brodie,
Powell,
3,
119
L.W.
(1973)
Clin.
Chim.
Acta
49.
109
6, 578
and
D.,
Biochem. and
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