177

Clinica Chimica Acta, 83 (1978) 177-181 @ Elsevier/North-Holland Biomedical Press

CGA 9238

A SIMPLIFIED PROCEDURE FOR THE ANALYSIS OF CHOLESTEROL, PHOSPHOLIPIDS AND BILE SALTS IN HUMAN BILE

C.H. BOLTON, T.S. LOW-BEER *, E.W. POMARE, A.C.B. WICKS ***, J. YEATES and K.W. HEATON University Department

of Medicine, Bristol Royal Infirmary, Bristol BS2 8HW (U.K.)

(Received October lst, 1977)

Summary Cholesterol, total bile salts and phospholipids have been measured on alcoholic extracts from human bile-rich duodenal contents. Folch extraction before phospholipid assay was found to be unnecessary and, unless fresh samples are used, it is liable to give misleadingly low values. Removal of bile pigment before measuring cholesterol was unnecessary because this estimation was done by gasliquid chromatography. The methods described provide a simple, reproducible analysis of bile lipids.

Introduction The accurate measurement of cholesterol and phospholipids in human bile is an important step in studies of the pathogenesis of gallstones. Many different methods are at present in use for these analyses, making inter-laboratory comparisons difficult. There is a need for a simple, universally applicable method for the analysis of bile. We have encountered two serious problems in bile lipid analysis. The assay of phospholipids after a Folch extraction [ 11, is subject to some error if watersoluble phospholipids e.g. lysolecithin, are present since they are removed by the aqueous washes. Similarly, cholesterol assays performed spectrophotometritally have been shown to be influenced to a marked extent by pigmentation in the bile sample [ 21. In overcoming these problems, we have found it is possible to simplify the technique of bile analysis. * Present address: Department of Gastroenterology. Selly Oak Hospital. Bhmingham. U.K. ** Present address: Department of Medicine, Wellington Hospital, Wellington, New Zealand. *** Present address: Department of Medicine, Harani Hospital, Salisbury. Rhodesia.

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Materials and methods Bile-rich duodenal juice (hereafter called bile) was aspirated from fasting human subjects by duodenal intubation following the intravenous injection of pancreozymin (Boots, Nottingham, England 110 CHR units). Alcoholic extraction of biliary lipids 0.2 ml of whole bile was mixed with 1.8 ml isopropanol.

After standing at room temperature for 5 min, the mixture was centrifuged for 10 min to sediment the protein. The clear supernatant was decanted and used for the assay of total biliary cholesterol. For assay of total bile salts and phospholipids, bile was treated in exactly the same way but using methanol for the extraction. Folch extraction of biliary lipids 0.1-0.5 ml of bile was diluted to 0.6 ml with water and 2.4 ml of chloroform/methanol (2 : 1, v/v) was added to bring the final ratio of H,O/MeOH/ CHC13 to 3 : 4 : 8, v/v. After mixing and centrifugation the upper pigmented

layer was discarded. The lower layer was concentrated to dryness, dissolved in isopropanol or methanol respectively for cholesterol and phospholipid assay. If the lower layer remained coloured after this extraction, it was washed with 1.4 ml of the upper phase of an H,0/MeOH/CHC13 (3 : 4 : 8, v/v) mixture. Analyses (a) Cholesterol.

4 ~1 of the biliary lipid extract was analysed by GLC on a Pye 104 dual flame ionisation detector model fitted with 5 foot X 5 inch glass columns containing 1.5% E30 on Diatomite CQ. The column temperature was 25O”C, detector 300°C and the carrier gas (nitrogen) flow-rate 45 ml/min. Cholesterol concentration was estimated by comparing the peak height with that of a standard cholestane peak obtained under identical conditions. Detector response was linear over the range of concentrations required (0.65-5.2 mmol/l equivalent to 1-8 pg). After a number of injections (at least 30) into the same column, the support in the immediate vicinity of the injection often became contaminated and this affected the reproducibility of the method. When this point was reached the top 2 inch or so were removed and replaced with fresh packing. This invariably restored the efficiency of the column. Cholesterol was also estimated spectrophotometrically by Watson’s modification [ 21 of the standard Liebermann-Burchard assay. (b) Phospholipids. Total phospholipids were estimated by the method of King [3]. (c) Bile salts. Total bile salts were measured by &he method of Iwata and Yamasaki [ 41 using 3a-hydroxy-steroid dehydrogenase (Sigma, Kingston-uponThames, Surrey).

Results Using isopropanolic and methanolic extracts, total bile salts, phospholipids and cholesterol were measured on individual bile samples. Within assay and between assay precision was high for all three compounds (Table I).

179 TABLE I PRECISION OF ASSAYS

FOR BILE SALTS (BS), PHOSPHOLIPIDS

Within-assay * precision (96) Between-assay * * precision (%)

(PL) AND CHOLESTEROL

BS

PL

Chol

2.2 3.9

1.0 5.6

2.3 4.0

(ChoU

* Coefficient of variation of duplicates in same assay. ** Coefficient of variation of repeatedly assayed samples.

Effect of pigment

Cholesterol content of nine bile samples was measured using both the Liebermann-Burchard reaction and GLC analysis (Table II). The results suggest that the spectrophotometric (Liebermann/Burchard) method of cholesterol assay tends to overestimate the cholesterol level particularly in heavily pigmented bile samples. In confirmation of this, when bilirubin was added in increasing amounts to a standard solution of cholesterol, a linear relationship was found between the amounts of bilirubin present and the absorbance of the solution as measured in the Liebermann-Burchard reaction (Fig.1). Effect of extraction

When steps were taken to remove the pigmentation by repeated aqueous washing of the extract, for example during Folch extractions, the apparent phosphorus content of the sample fell progressively, indicating loss of some water-soluble phospholipids. Lysolecithin and glyceryl phosphoryl choline are both produced on degradation of lecithin [5,6]. This was confirmed in the following experiment: In 9 subjects, the lipid composition of fresh bile was compared before and after Folch extraction. There was no significant difference in the phospholipid content: 19.9 + 5.9 pmol/ml vs. 19.0 f 6.1 pmol/ml (mean f S.D.) respectively, but the cholesterol content showed a highly significant difference: 15.5 + 7.4 pmol/ml vs. 11.2 f 3.9 pmol/ml (p < 0.01) respectively accounted for by pig-

TABLE II COMPARISON

OF LIEBERMANN/BURCHARD

Cholesterol concentration (pmol/mI)

as determined by

Liebermann/Burchard

GLC

1.77 2.66 2.76 3.05 3.83 4.99 6.16 * 6.60 * 14.54 *

1.81 2.51 2.29 3.05 4.40 3.17 4.33 4.30 11.47

* Heavily pigmented samples.

AND GLC ASSAYS

FOR BILIARY

CHOLESTEROL

180

E’ 600 2 In 500 s 01 LOO :! ;

300

$ 200 100

20

LO

60

80

100

pg Bilirubin Fig. 1. The effect on absorbance amuunt of cholesterol (200 pg).

of increasing

amounts

of bilirubin.

alone and in the presence

of a fixed

mentation. In two further samples of bile which had been allowed to stand at room temperature for several hours, there was a large decrease in phospholipid content as measured after Folch extraction. Values before extraction were 20.6 and 25.3 I.rmol/ml, and after extraction 8.1 and 6.4 pmol/ml, representing reductions of 61% and 75%, respectively. Discussion We have confirmed previous work [2] showing that pigmentation of bile samples produces a falsely high reading for cholesterol if a spectrophotometric method is used for its determination. This can be overcome to a large xtent if the cholesterol extract is washed repeatedly with water. However, this process is tedious and final traces of pigments have proved difficult to remove [ 51. Gasliquid chromatographic analysis of cholesterol is not affected by the presence of pigments. This allows the measurement of cholesterol in simple isopropanol extracts of bile. Our comparison of GLC with the spectrophotometric assay of Liebermann-Burchard as modified by Watson [2] showed that in slightly pigmented samples agreement is close, but in more heavily pigmented samples, the spectrophotometric method gives high readings (Table II). The determination of total phospholipids in bile is usually performed on Folch extracts from bile. However, water-soluble phospholipids, e.g. lysolecithin, are selectively lost in this procedure during the aqueous washing process. Such partially hydrolysed products often arise from the parent lipid on storage of samples and may arise if bile samples are frozen and thawed (Bolton, C.H. and Pomare, E.W., unpublished). This can be avoided by processing bile immediately it is aspirated, but this is not always convenient. It is simpler to dispense with the lipid extraction step. The presence of lecithin breakdown products does not affect the estimation of phospholipid since this is based on the specific measurement of elemental phosphorus, which remains one mole per mole of phospholipid, regardless of its source. The use of a simple methanol extract for phospholipid assay has the theoreti-

181

cal objection that it will include non-lipid phosphorus present in bile or other duodenal secretions. However, any overestimate of phosphorus content arising from inorganic phosphate in the bile must be small [7] since as we have confirmed, differences between the phosphorus content of extracted and nonextracted bile are minimal. A recent report [8] suggested that the inorganic phosphorus content of bilerich duodenal juice, particularly in dilute samples, is significant and that, because of this, an extraction step is necessary. We have found no evidence, in a wide range of concentrations of bile, which supports this. It must be stressed, however, that our samples were obtained by cholecystokinin stimulation after an overnight fast. The situation may be different in bile obtained after meals. This analysis was therefore, carried out on a simple methanolic extract from bile with no aqueous washing. The method used for analysis of bile salts is essentially the same as that reported by Iwata and Yamasaki [ 41 using 3o-hydroxy-steroid dehydrogenase. There was no significant difference in results from extracted and non-extracted bile samples. We have shown therefore that it is possible to measure accurately and quickly total cholesterol, phospholipid and bile salts, and their relative proportions on alcoholic extracts from bile. This procedure, with the use of GLC for cholesterol assay, circumvents the need to perform a Folch extraction, which has previously been required to remove bile pigments, and which has been shown often not to give a true picture of phospholipid composition. Acknowledgments We are grateful to Kellogg Co. of Great Britain Ltd. for financial assistance. Miss C.S. White gave technical assistance in the early stages of this investigation. Dr. Mitropoulos of Hammersmith Hospital, London, kindly gave advice on setting up of the GLC method. References 1 2 3 4 5 6

Folch, J., Lees, M. and Sloane-Stanley, G.H. (1957) J. Biol. Chem. 226.497-509 Watson, D. (1960) Clin. Chim. Acta 5.637-643 King, E.J. (1932) Biochem. J. 26. 292-297 Iwata. T. and Yamasaki. K. (1964) J. Biocheh. 56,424-431 Pomare. E.W. (1974) M.D. Thesis, University of Bristol Ansell, G.B. and Hawthorne. J.N. (1964) in Phospholipids: Chemistry, Metabolism and Function, vier, Amsterdam 7 Sobotka, H. (1937) in Physiological Chemistry of the Bile. BaiIliere. TindaII and Cox. London 8 Murison. J.. Festi. D., Ross, P.E. and Bouchier, I.A.D. (1976) CIin. Chim. Acta 68.159-166

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A simplified procedure for the analysis of cholesterol, phospholipids and bile salts in human bile.

177 Clinica Chimica Acta, 83 (1978) 177-181 @ Elsevier/North-Holland Biomedical Press CGA 9238 A SIMPLIFIED PROCEDURE FOR THE ANALYSIS OF CHOLESTER...
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