331
Clinica Chimica Acta, 64 (1975) 337-341 0 Elsevier Scientific Publishing Company,
BRIEF TECHNICAL
Amsterdam
- Printed
in The Netherlands
NOTE
CCA 7218
ENZYMATIC
DETERMINATION
A. RODAa’*, D. FESTI, L. BARBARA
OF CHOLESTEROL
C. SAMA, G. MAZZELLA,
Istituto di Clinica Medica e Gastroenterologia a Cattedra di Chimica Generale e Inorganica,
R. ALDINI,
IN BILE
E. RODA and
dell’llniversitb di Bologna and Facolth di Farmacia, Universith di Bologna
(Italy)
(Received
May 1, 1975)
Summary A simple, sensitive and rapid method for measuring the cholesterol concentration in bile is described. The method is based on the combination of an enzymatic technique and spectrophotometry. In the present study the results obtained with this method are compared with those obtained by the standard Liebermann-Burchard reaction on the same samples and are found to be in close agreement.
Introduction The amount of free cholesterol in bile is usually determined by a chemical method based on the Liebermann-Burchard [ 1,2] reaction employing the reagents acetic acid, acetic anhydride and sulphuric acid. The compound obtained has a maximum absorption at 630 nm. However, the bile salts, which are present in bile in a concentration approximately l/lOth that of cholesterol, also react with sulphuric acid to form compounds which absorb at the same wavelength. Therefore, a preliminary separation of the cholesterol is necessary which can be carried out either by liquid-liquid extraction method of Folch et al. [3] or by precipitation with digitonin [4] . In the present study the possibility of direct measurement on the sample has been examined, thus avoiding the long extraction procedure and the use of corrosive reagents. Rtischlau et al. [5] introduced a new method of determining serum cholesterol based on the use of cholesterol oxidase. We have applied this tech* Address for reprint requests: Dr. Aldo Roda, Cattedra di Chimica Generale Inorganica, Facolti di Farmacia. Universit‘a di Bologna c/o 1st. Chimico G. Ciamician. Via Selmi, 2 - Bologna, Italy.
338
nique to bile and compared used previously. Principle
the results with those obtained
from the method
of the method
Cholesterol oxidase [6-91 oxidizes cholesterol in the presence of oxygen to give A4cholestenone and HzOz. Any cholesterol esters are first hydrolysed by preliminary treatment with cholesterol esterase. The hydrogen peroxide thus formed reacts with methanol in the presence of catalase to form formaldehyde. The fo~~dehyde then reacts with ammonium ions and acetylacetone to form 3,5-diacetyl-1,4-dihydrolutidine which can then be determined by light absorption measurements. Cholesterol
ti202+
Cholesterol
+ 02
CHjOH
H&O
Catclose
+ ‘2CH,COCH,COCH,
oxidase
t
HCHO
c_
+
44-CholestenonetH20z
2H20
+ NH3--+CH3
+3H>O
Method Reagents NaCl (Merck), CHCla (Merck), CHsOH (Merck), CiH,OH (Fluka). All reagents and solvents were of analytical grade and were used without further purification. Enzymatic color test Boehringer Mannheim. The cholesterol concentration was determined in 40 samples of bile, obtained by duodenal intubation after CCK-PZ (1 U/kg) stimulation, using both the enzymatic method and the method of Sperry and Webb ]4] (extraction for the latter Folch’s method [3] ). Each sample of bile was diluted 1 : 5 with saline solution (8% NaCl): 0.1 or 0.2 ml of this solution (depending on the bile concentration) was analysed by the method originally used by Rijschlau et al. [5] which is now (commercially) available (Boehringer). Finally, the absorption of the sample was measured at 410 nm (tungs~n lamp, Philips UNICAM spectrophotometer). AI1 measurements were carried out in duplicate. Calculation
The results were calculated in two different ways: cuZculation using the known absorption
(a) Direct
coefficient
of 3,5-
339
Fig. 1. Calibration curve.
diacetyl dihydrolutidine. centration of cholesterol C(mmol/lOO
For a sample of 0.1 ml of bile diluted 1 : 5, the conis given by :
ml) = 1.9 X (EA - EB)410 nm
(b) Calibration A standard solution of cholesterol (2 mg/ml) in ethanol was used in this method. 5-70 ~1, corresponding to 2.58 X 10P2-36.19 X 10P2 pmoles of cholesterol per sample. These sample were analysed as above obtaming the calibration curve shown in Fig. 1. Results The results obtained from the 40 samples of bile analysed in dublicate by Roschlau’s method and by the method of Sperry and Webb were correlated by polynomial regression and the correlation between the two sets of measurements are shown in Fig. 2. Analysis of regression shows close agreement of values, demonstrating that other lipids do not affect the method.
1
Cholesterol
- method
B bn~oles/~00ml)
Fig. 2. Correlation between Rlischlau’s method (A) and the method of Sperry and Webb (B).
340
0.2
Fig.
3. Effect
The effect
0.4 T.C.A. of bile
0.8 1.0 OJmoles)
0.6 added
salt. T.C.A.,
sodium
taurocholate.
of bile salts
Increasing quantities of sodium taurocholate (0.2-0.8 pmol) were added to 2 standard samples of cholesterol (containing 5.17 X 10P2 and 15.51 X lo-” pmol of cholesterol) and 5 measurements were carried out for each sample. Fig. 3 shows the mean values and indicates that bile salts have no significant effect on the absorption values. The effect
of bilirubin
Since bilirubin also has an absorption at 410 nm we thought it best to check any effect it may have on this method. We therefore added increasing amounts (0.05, 0.1, 0.3, 0.5 mmol) of bilirubin to 10 standard samples of cholesterol (containing 7.75 X 10P2 pmol cholesterol). No significant change was found in the measurements. Reproducibility
The data shown in Table I demonstrates the accuracy and reproducibility of the method used and this is confirmed by statistical analysis of the standard deviation. Recovery
test
The degree of recovery in 4 samples of bile to which an amount of cholesterol equal to 0.1, 0.2, 0.3 mmol 100 ml was added is from 96 to 102% (mean 100.3%) as shown in Table II.
TABLE
I
Cholesterol
analyzed
(/an01
X 10’)
Mean
of
5 measurements
Relative
S.D.
S.D.
2.40
2.40
0.41
0.010
4.00
4.02
0.64
0.026
3.00
3.02
1.12
0.034
6.00
5.99
0.50
0.030
5.50
5.48
0.45
0.025
341
TABLE II
Initial cholesterol
Cholesterol added
Cholesterol
in sample (mmo1/100
to the sample
measured
(mmol/lOO
(mmol/lOO
ml)
0.23 0.23 0.23 0.70 0.70 0.70
0.10 0.20 0.30 0.10 0.20 0.30
ml) (A)
0.32 0.43 0.525 0.8 0.88 0.99
M-A
Recovery (%)
0.22 0.23 0.225 0.7 0.68 0.69
96 100 98 100 97 98.5
ml) (M)
Discussion Since this method has proved to be more sensitive than that of Sperry and Webb and reproducibility is good, we consider it to be a valid alternative to the former method as it has the added advantages of simple, rapid operation eliminating most of the manual error and is especially suitable for such a complex liquid as bile. References Liebermann, C. (1885) Ber. 18, 1803 Lies?. L.A.. Levy, B.B., Brodie, B.B. and F.E. Kendall (1962) J. Biol. Chem. 195,357 Folch, J., Lees, M. and Sloane Stanley. G.H. (1957) J. Biol. Chem. 26,497 Sperry, W.M. and Webb, M. (1950) J. Biol. Chem. 187.96 Rb;scblau P. et al. (1974) Z. Klin. Biochem. 12, 226 Kageyama, N. (1971) Clin. Chim. Acta 31. 421426 Richmond, W. (1972) Stand. J. Clin. Lab. Invest., 29, Suppl. 126 Richmond, W. (1973) Clin. Chem. 19.1350 Flegg. H.M. (1973) Ann. Clin. Biochem. 10, 79
Clinica Chimica Acta, 64 (1975) 337-341 0 Elsevier Scientific Publishing Company,
BRIEF TECHNICAL
Amsterdam
- Printed
in The Netherlands
NOTE
CCA 7218
ENZYMATIC
DETERMINATION
A. RODAa’*, D. FESTI, L. BARBARA
OF CHOLESTEROL
C. SAMA, G. MAZZELLA,
Istituto di Clinica Medica e Gastroenterologia a Cattedra di Chimica Generale e Inorganica,
R. ALDINI,
IN BILE
E. RODA and
dell’llniversitb di Bologna and Facolth di Farmacia, Universith di Bologna
(Italy)
(Received
May 1, 1975)
Summary A simple, sensitive and rapid method for measuring the cholesterol concentration in bile is described. The method is based on the combination of an enzymatic technique and spectrophotometry. In the present study the results obtained with this method are compared with those obtained by the standard Liebermann-Burchard reaction on the same samples and are found to be in close agreement.
Introduction The amount of free cholesterol in bile is usually determined by a chemical method based on the Liebermann-Burchard [ 1,2] reaction employing the reagents acetic acid, acetic anhydride and sulphuric acid. The compound obtained has a maximum absorption at 630 nm. However, the bile salts, which are present in bile in a concentration approximately l/lOth that of cholesterol, also react with sulphuric acid to form compounds which absorb at the same wavelength. Therefore, a preliminary separation of the cholesterol is necessary which can be carried out either by liquid-liquid extraction method of Folch et al. [3] or by precipitation with digitonin [4] . In the present study the possibility of direct measurement on the sample has been examined, thus avoiding the long extraction procedure and the use of corrosive reagents. Rtischlau et al. [5] introduced a new method of determining serum cholesterol based on the use of cholesterol oxidase. We have applied this tech* Address for reprint requests: Dr. Aldo Roda, Cattedra di Chimica Generale Inorganica, Facolti di Farmacia. Universit‘a di Bologna c/o 1st. Chimico G. Ciamician. Via Selmi, 2 - Bologna, Italy.
338
nique to bile and compared used previously. Principle
the results with those obtained
from the method
of the method
Cholesterol oxidase [6-91 oxidizes cholesterol in the presence of oxygen to give A4cholestenone and HzOz. Any cholesterol esters are first hydrolysed by preliminary treatment with cholesterol esterase. The hydrogen peroxide thus formed reacts with methanol in the presence of catalase to form formaldehyde. The fo~~dehyde then reacts with ammonium ions and acetylacetone to form 3,5-diacetyl-1,4-dihydrolutidine which can then be determined by light absorption measurements. Cholesterol
ti202+
Cholesterol
+ 02
CHjOH
H&O
Catclose
+ ‘2CH,COCH,COCH,
oxidase
t
HCHO
c_
+
44-CholestenonetH20z
2H20
+ NH3--+CH3
+3H>O
Method Reagents NaCl (Merck), CHCla (Merck), CHsOH (Merck), CiH,OH (Fluka). All reagents and solvents were of analytical grade and were used without further purification. Enzymatic color test Boehringer Mannheim. The cholesterol concentration was determined in 40 samples of bile, obtained by duodenal intubation after CCK-PZ (1 U/kg) stimulation, using both the enzymatic method and the method of Sperry and Webb ]4] (extraction for the latter Folch’s method [3] ). Each sample of bile was diluted 1 : 5 with saline solution (8% NaCl): 0.1 or 0.2 ml of this solution (depending on the bile concentration) was analysed by the method originally used by Rijschlau et al. [5] which is now (commercially) available (Boehringer). Finally, the absorption of the sample was measured at 410 nm (tungs~n lamp, Philips UNICAM spectrophotometer). AI1 measurements were carried out in duplicate. Calculation
The results were calculated in two different ways: cuZculation using the known absorption
(a) Direct
coefficient
of 3,5-
339
Fig. 1. Calibration curve.
diacetyl dihydrolutidine. centration of cholesterol C(mmol/lOO
For a sample of 0.1 ml of bile diluted 1 : 5, the conis given by :
ml) = 1.9 X (EA - EB)410 nm
(b) Calibration A standard solution of cholesterol (2 mg/ml) in ethanol was used in this method. 5-70 ~1, corresponding to 2.58 X 10P2-36.19 X 10P2 pmoles of cholesterol per sample. These sample were analysed as above obtaming the calibration curve shown in Fig. 1. Results The results obtained from the 40 samples of bile analysed in dublicate by Roschlau’s method and by the method of Sperry and Webb were correlated by polynomial regression and the correlation between the two sets of measurements are shown in Fig. 2. Analysis of regression shows close agreement of values, demonstrating that other lipids do not affect the method.
1
Cholesterol
- method
B bn~oles/~00ml)
Fig. 2. Correlation between Rlischlau’s method (A) and the method of Sperry and Webb (B).
340
0.2
Fig.
3. Effect
The effect
0.4 T.C.A. of bile
0.8 1.0 OJmoles)
0.6 added
salt. T.C.A.,
sodium
taurocholate.
of bile salts
Increasing quantities of sodium taurocholate (0.2-0.8 pmol) were added to 2 standard samples of cholesterol (containing 5.17 X 10P2 and 15.51 X lo-” pmol of cholesterol) and 5 measurements were carried out for each sample. Fig. 3 shows the mean values and indicates that bile salts have no significant effect on the absorption values. The effect
of bilirubin
Since bilirubin also has an absorption at 410 nm we thought it best to check any effect it may have on this method. We therefore added increasing amounts (0.05, 0.1, 0.3, 0.5 mmol) of bilirubin to 10 standard samples of cholesterol (containing 7.75 X 10P2 pmol cholesterol). No significant change was found in the measurements. Reproducibility
The data shown in Table I demonstrates the accuracy and reproducibility of the method used and this is confirmed by statistical analysis of the standard deviation. Recovery
test
The degree of recovery in 4 samples of bile to which an amount of cholesterol equal to 0.1, 0.2, 0.3 mmol 100 ml was added is from 96 to 102% (mean 100.3%) as shown in Table II.
TABLE
I
Cholesterol
analyzed
(/an01
X 10’)
Mean
of
5 measurements
Relative
S.D.
S.D.
2.40
2.40
0.41
0.010
4.00
4.02
0.64
0.026
3.00
3.02
1.12
0.034
6.00
5.99
0.50
0.030
5.50
5.48
0.45
0.025
341
TABLE II
Initial cholesterol
Cholesterol added
Cholesterol
in sample (mmo1/100
to the sample
measured
(mmol/lOO
(mmol/lOO
ml)
0.23 0.23 0.23 0.70 0.70 0.70
0.10 0.20 0.30 0.10 0.20 0.30
ml) (A)
0.32 0.43 0.525 0.8 0.88 0.99
M-A
Recovery (%)
0.22 0.23 0.225 0.7 0.68 0.69
96 100 98 100 97 98.5
ml) (M)
Discussion Since this method has proved to be more sensitive than that of Sperry and Webb and reproducibility is good, we consider it to be a valid alternative to the former method as it has the added advantages of simple, rapid operation eliminating most of the manual error and is especially suitable for such a complex liquid as bile. References Liebermann, C. (1885) Ber. 18, 1803 Lies?. L.A.. Levy, B.B., Brodie, B.B. and F.E. Kendall (1962) J. Biol. Chem. 195,357 Folch, J., Lees, M. and Sloane Stanley. G.H. (1957) J. Biol. Chem. 26,497 Sperry, W.M. and Webb, M. (1950) J. Biol. Chem. 187.96 Rb;scblau P. et al. (1974) Z. Klin. Biochem. 12, 226 Kageyama, N. (1971) Clin. Chim. Acta 31. 421426 Richmond, W. (1972) Stand. J. Clin. Lab. Invest., 29, Suppl. 126 Richmond, W. (1973) Clin. Chem. 19.1350 Flegg. H.M. (1973) Ann. Clin. Biochem. 10, 79
