169
Clinica Chimica Acta, 89 (1978) 169-171 0 Elsevier/North-Holland Biomedical Press
BRIEF TECHNICAL
NOTE
CGA 9728
INORGANIC ASPIRATES
PHOSPHATE
IN BILE AND ‘BILE-RICH’
DUODENAL
JEAN WIEGARD and G.M. MURPHY * Gastroenterology
Unit, Guy’s Hospital Medical School,
London
(U.K.)
(Received May 18th, 1978)
Measurements of the phospholipid content of bile and ‘bile-rich’ duodenal aspirates (samples of duodenal fluid obtained following gallbladder contraction) have played an important role in studies of cholesterol gallstone formation and dissolution. Recently the accuracy of the methods used to determine biliary phospholipid has been questioned [ 11. Most methods for phospholipid determination involve strong acid digestion of the phospholipids to inorganic phosphorus followed by the estimation of the total resultant phosphorus with the Fiske and SubbaRow [Z] colour reaction. Some workers have suggested that the contribution of inorganic phosphate to the total phosphorus content of bile and duodenal aspirates is minimal compared with that made by phospholipid and that therefore no preliminary extraction of phospholipid is required [ 3,4]. Other workers have advocated the extraction of phospholipids before their digestion [l]. To date, however, no direct measurements of inorganic phosphate concentrations in such samples have been reported. We therefore have assayed inorganic phosphate in biles and duodenal aspirates and calculated its contribution to the corresponding total phosphorus concentration. Inorganic phosphate concentration was measured using the enzymatic method of Hwang and Cha [5]. No increase in absorbance was observed when either lecithin or lysolecithin was added to the assay system. No difference was found in the results if sample blanks were prepared without enzymes or if they included as the only enzyme xanthine oxidase, indicating that any hypoxanthine which may have been originally present in the samples did not generate detectable amounts of uric acid. Total phosphorus concentrations were determined as described by Bartlett [ 61, a method which uses the Fiske-SubbaRow reagent without previous lipid extraction.
* To whom correspondenceshouldbe addressed.
170 6-
0 0
4Inorganic phosphate mmol/l
00 0
0
l
0 +-78
2-
0 00 000
0
*
10
20 Total phosphorus,
30
40
mmol /I
Fig. 1. Inorganic phosphate concfmtrations measured enzymatica& [Sl vs. total phosphorus concentrations measured by the method of Bartlett 161 in duodenal aspirates (open circles) and gallbladder biles (closed circles).
Inorganic phosphate was detected in all samples of duodenal aspirates and g~lbladder biles studied (Fig. 1). No si~ific~t relationship was found between the total phosphorus concentration and the inorganic phosphate concentration. In general, however, the higher the total phosphorus concentration the smaller the contribution made to it by the inorganic fraction. The major component of biliary phospholipid is phosphatidyl choline (lecithin), minor fractions being lysophosphatidyl choline (lysolecithin) and phosphatidyl eth~olamine [7-91, As recently demonstrated, inorganic phospha~ is the major fraction of non-lipid phosphorus in bile [lo]. Lysoleeithin is more polar than lecithin and is water soluble, it is, therefore, possible to lose some lysolecithin whilst separating biliary phospholipid from biliary inorganic phosphate. For example, losses of up to lo%, as lysolecithin, have been reported during Folch extractions [ll] of bile. In addition to bile, duodenal aspirates may contain intestinal, pancreatic and gastric secretions. Pancreatic secretions contain phospholipases and thus lysolecithin may form a larger proportion of the phospholipids found in duodenal aspirates compared to that in gallbladder biles and losses, therefore, are greater if lipid extraction is performed on duodenal aspirates. If no extraction procedure is used then phospholipid determinations include inorganic phosphate and as the present study shows the errors are considerable at low concentrations. Previous workers [1] using lipid extraction, selected a total phosphorus concentration of 5 mmol/l as an arbitrary lower limit for concentrated samples; they found there was a >20% difference between total phosphorus and lipid phosphorus in 10 of 17 samples with phospholipid concentration >5 mmol/l. In only 5 of the 32 samples in the present study with total phosphorus concentrations >5 mmol was the contribution made by the inorganic phosphate fraction >20%. From an analytical viewpoint the methods commonly used for the determination of total phospholipid concentrations in duodenal aspirates are unsatisfactory, particularly with dilute samples. Correction for inorganic phosphate is relatively simple to make compared to the difficulties of estimating lysolecithin.
171
References 1 2 3 4 5 6 7 8 9 10 11
Murison, J., Festi, D.. Ross, P.E. and Bouchier, I.A.D. (1976) CIin. Chim. Acta 68.159-166 Fiske, C.H. and SubbaRow, Y. (1925) J. Biol. Chem. 66.376-400 Mackay, C., Crook, J.N., Smith, D.C. and McAIIister, R.A. (1972) Gut 13,769-762 Bolton. C.H., Low-Beer, T.S., Pomare, E.W.. Wicks, A.C.B.. Yeates. J. and Heaton. K.W. (1978) Clin. Chhn. Acta 83.177-181 Hwang, WI. and Cha, S. (1973) Anal. Biochem. 55.379-387 Bartlett, G.R. (1959) J. Biol. Chem. 234,466-468 PhiIIips. G.D. (1960) Biochim. Biophys. Acta 41.361-369 Spitzer, H.L.. Kyriakides, E.S. and Bahnt, T.A. (1964) Nature 204. 288 Gottfries, A., Nilsson. S.. Sammuelsson, B. and Schersten, T. (1968) Stand. J. Clin. Lab. Invest. 21, 168-173 Sutor, D.J. and WiIkie. L.G. (1977) Clin. Chim. Acta 77,31-36 Folch, J., Lees, M. and Sloane-Stanley. G.H. (1957) J. Biol. Chem. 226,497-509
Clinica Chimica Acta, 89 (1978) 169-171 0 Elsevier/North-Holland Biomedical Press
BRIEF TECHNICAL
NOTE
CGA 9728
INORGANIC ASPIRATES
PHOSPHATE
IN BILE AND ‘BILE-RICH’
DUODENAL
JEAN WIEGARD and G.M. MURPHY * Gastroenterology
Unit, Guy’s Hospital Medical School,
London
(U.K.)
(Received May 18th, 1978)
Measurements of the phospholipid content of bile and ‘bile-rich’ duodenal aspirates (samples of duodenal fluid obtained following gallbladder contraction) have played an important role in studies of cholesterol gallstone formation and dissolution. Recently the accuracy of the methods used to determine biliary phospholipid has been questioned [ 11. Most methods for phospholipid determination involve strong acid digestion of the phospholipids to inorganic phosphorus followed by the estimation of the total resultant phosphorus with the Fiske and SubbaRow [Z] colour reaction. Some workers have suggested that the contribution of inorganic phosphate to the total phosphorus content of bile and duodenal aspirates is minimal compared with that made by phospholipid and that therefore no preliminary extraction of phospholipid is required [ 3,4]. Other workers have advocated the extraction of phospholipids before their digestion [l]. To date, however, no direct measurements of inorganic phosphate concentrations in such samples have been reported. We therefore have assayed inorganic phosphate in biles and duodenal aspirates and calculated its contribution to the corresponding total phosphorus concentration. Inorganic phosphate concentration was measured using the enzymatic method of Hwang and Cha [5]. No increase in absorbance was observed when either lecithin or lysolecithin was added to the assay system. No difference was found in the results if sample blanks were prepared without enzymes or if they included as the only enzyme xanthine oxidase, indicating that any hypoxanthine which may have been originally present in the samples did not generate detectable amounts of uric acid. Total phosphorus concentrations were determined as described by Bartlett [ 61, a method which uses the Fiske-SubbaRow reagent without previous lipid extraction.
* To whom correspondenceshouldbe addressed.
170 6-
0 0
4Inorganic phosphate mmol/l
00 0
0
l
0 +-78
2-
0 00 000
0
*
10
20 Total phosphorus,
30
40
mmol /I
Fig. 1. Inorganic phosphate concfmtrations measured enzymatica& [Sl vs. total phosphorus concentrations measured by the method of Bartlett 161 in duodenal aspirates (open circles) and gallbladder biles (closed circles).
Inorganic phosphate was detected in all samples of duodenal aspirates and g~lbladder biles studied (Fig. 1). No si~ific~t relationship was found between the total phosphorus concentration and the inorganic phosphate concentration. In general, however, the higher the total phosphorus concentration the smaller the contribution made to it by the inorganic fraction. The major component of biliary phospholipid is phosphatidyl choline (lecithin), minor fractions being lysophosphatidyl choline (lysolecithin) and phosphatidyl eth~olamine [7-91, As recently demonstrated, inorganic phospha~ is the major fraction of non-lipid phosphorus in bile [lo]. Lysoleeithin is more polar than lecithin and is water soluble, it is, therefore, possible to lose some lysolecithin whilst separating biliary phospholipid from biliary inorganic phosphate. For example, losses of up to lo%, as lysolecithin, have been reported during Folch extractions [ll] of bile. In addition to bile, duodenal aspirates may contain intestinal, pancreatic and gastric secretions. Pancreatic secretions contain phospholipases and thus lysolecithin may form a larger proportion of the phospholipids found in duodenal aspirates compared to that in gallbladder biles and losses, therefore, are greater if lipid extraction is performed on duodenal aspirates. If no extraction procedure is used then phospholipid determinations include inorganic phosphate and as the present study shows the errors are considerable at low concentrations. Previous workers [1] using lipid extraction, selected a total phosphorus concentration of 5 mmol/l as an arbitrary lower limit for concentrated samples; they found there was a >20% difference between total phosphorus and lipid phosphorus in 10 of 17 samples with phospholipid concentration >5 mmol/l. In only 5 of the 32 samples in the present study with total phosphorus concentrations >5 mmol was the contribution made by the inorganic phosphate fraction >20%. From an analytical viewpoint the methods commonly used for the determination of total phospholipid concentrations in duodenal aspirates are unsatisfactory, particularly with dilute samples. Correction for inorganic phosphate is relatively simple to make compared to the difficulties of estimating lysolecithin.
171
References 1 2 3 4 5 6 7 8 9 10 11
Murison, J., Festi, D.. Ross, P.E. and Bouchier, I.A.D. (1976) CIin. Chim. Acta 68.159-166 Fiske, C.H. and SubbaRow, Y. (1925) J. Biol. Chem. 66.376-400 Mackay, C., Crook, J.N., Smith, D.C. and McAIIister, R.A. (1972) Gut 13,769-762 Bolton. C.H., Low-Beer, T.S., Pomare, E.W.. Wicks, A.C.B.. Yeates. J. and Heaton. K.W. (1978) Clin. Chhn. Acta 83.177-181 Hwang, WI. and Cha, S. (1973) Anal. Biochem. 55.379-387 Bartlett, G.R. (1959) J. Biol. Chem. 234,466-468 PhiIIips. G.D. (1960) Biochim. Biophys. Acta 41.361-369 Spitzer, H.L.. Kyriakides, E.S. and Bahnt, T.A. (1964) Nature 204. 288 Gottfries, A., Nilsson. S.. Sammuelsson, B. and Schersten, T. (1968) Stand. J. Clin. Lab. Invest. 21, 168-173 Sutor, D.J. and WiIkie. L.G. (1977) Clin. Chim. Acta 77,31-36 Folch, J., Lees, M. and Sloane-Stanley. G.H. (1957) J. Biol. Chem. 226,497-509
