Klinische Wochenschrift
Klin Wochenschr (1990) 68:60-64
9 Springer-Verlag 1990
Originals Lipolytic Enzymes of the Human Pancreas III. Auxiliary Function of Lipase in the Cholesterol-Esterase-Dependent Oral Test on Exocrine Pancreatic Output J.G. Meyer, J.G. Renczes, and H. Kaffarnik Zentrallabor des Stadtkrankenhauses Hanau und Medizinische Poliklinik der Universitfit Marburg
Summary. In the oral exocrine pancreatic function test using fluorescein dilaurate, this synthetic substrate attaches primarily to the triglyceride surfaces of the neutral lipids administered as part of the breakfast: these fluorescein dilaurate molecules cannot be attacked by cholesterol esterase. In the course of triglyceride saponification by lipase and colipase, however, the fluorescein dilaurate is liberated and hydrolyzed by cholesterol esterase. The pancreatic function test, therefore, measures the lipolytic activities not merely of cholesterol esterase, but indirectly of lipase, as well. Key words: Cholesterol esterase - Lipase - Colipase - Fluorescein dilaurate - Oral pancreatic function test - Bile salts - Micelles
Exactly 20 years ago, in this journal [7] we presented the first noninvasive oral test of pancreatic function. That report, entitled" Methods and clinical significance of a new pancreas lipase test using fluorescein di-laurin acid ester," was based on results from experiments involving lipase from Rhizopus arrhizus, at that time the only lipase preparation available that was void of cross reactivities. Within 1 year, it had been proved that human pancreas lipase in the form of triacylglycerol hydrolase (EC 3.1.1.3) had a higher substrate specificity than Rhizopus lipase and that hydrolysis of fluorescein dilaurate (FDL) is catalysed by other human pancreas esterases (" arylesterases ") [8, 9, 15, 16]. Today, we know that this is due to cholesterol ester hydrolase (CEH, sterolester acylhydrolase; EC 3.1.1.13) in the pancreas secrete [13, 14]. Abbreviations: CEH=Cholesterol ester hydrolase; CMCCritical micellar concentration; FDL=Fluorescein dilaurate; PLT=Pancreolauryl test; TC=Taurocholate; TDC=Taurodeoxycholate
Only recently it has been shown that the oral pancreatic function test using F D L (Pancreolauryl test, PLT) actually measures - even though only indirectly - the lipase activity in pancreatic secretion, since the major part of the indicator reaction can only progress after lipase has transformed the substrate F D L on the triglyceride surface into a form accessible to CEH [17].
Objective Already, during the preparation of a standardized test method, it was noticed that hydrolysis of orally administered FDL resulted only when the test breakfast included neutral fats, even though pancreas secretion can be stimulated by a number of fat-free foods. Additionally, it remained unexplained why F D L hydrolysis dropped from 72% to 46% when the triglyceride dose was either below (10 g) or exceeded (50 g) the optimum amount (20 g butter). These findings warranted further quantitative and qualitative analysis of the role of triglycerides in the PLT [17].
Methods Standard Solution
A solution of 10 ml bicarbonate buffer (1000 ml sodium bicarbonate solution, 0.1mol/1, plus 150 ml disodium hydrogen phosphate, 0.066 tool/l, with approximately 15 ml hydrochloric acid, 0.01 mol/1, adjusted for pH=8.35) was mixed with 14mg F D L (cristalline; Temmler-Werke, Marburg). In the case of complete solution, dependent on further additives, the concentration of F D L would be 0.2 mmol/1.
J.G. Meyer et al. : Role of Lipase in the Pancreatic Function Test PLT
Mixing Procedures Closed Erlenmeyer flasks filled with the solutions and containing ten glass balls (6 mm in diameter) were shaken for 15 min at 37 ~ C (rotational movement). Immediately afterwards, the solutions were filtered (Selecta 595 1/2) and only the filtrates were further analysed (temperature 37 ~ C).
Quantification of the Dissolved FDL Portion In one aliquot of filtrate, the FDL (colorless) was saponified completely by the addition of the same volume of ethanolic potassium hydroxide solution (0.5 mol/1) and the fluorescein concentration was determined using fluorescence spectrophotometry (Perkin-Elmer 650-40) with respect to a calibration line (2ex-- 494 nm; hem = 520 nm).
6t
Results
1. Solubility of Fluorescein Dilaurate Lipids as Solubilizer FDL remains completely undissolved while being shaken in a primary solution without additives. Already small amounts of triglycerides (butter or triolein) bind FDL to the surfaces of the emulsion, as measurable by KOH hydrolysis of the filtrates; for example, 20 mg triglycerides solubilize 0.5 ~tmol/1 FDL. Analogous testing of 1,2-dilauryl lecithin, free lauric acid, and 1-monolauryl glyceride led by comparison to the following results: lecithin 0.008; lauric acid 0.38, and monoglyceride 0.9 gmol/1 FDL.
Enzymatic Hydrolysis of FDL
Contribution of Bile Salts
CEH derived from porcine pancreas (Calbiochem) was dissolved in phosphate buffer (0.066 mol/1, pH = 6.2) resulting in a primary solution of 2000 U/1 (calculated as cholesteryl oleate hydrolysis) and containing 5 mg CEH in 7 ml buffer; 20 gl/ 1000 gl test solution was used for the experiments, corresponding to an activity of 40 U/1. To measure the fluorescein concentration liberated from FDL by CEH, the photometer was set for the accepted optimum at pH=8.35: 2ex= 483 rim, 2 e m = 512 nm. Measurements in the concentration range of 10 -8 mol/1 fluorescein were possible by comparison with an analog calibration line. The results from CEH of 48% acetone precipitate [12] isolated from human pancreas were used as a control for porcine CEH.
By adding sodium salts of various bile salts to the primary solutions, varying dissolution of FDL resulted dependent on the type of salt employed. At a bile salt concentration of 1 mmol/1 the following FDL concentrations (gmol/1) in the filtrates were measured (KOH hydrolysis): both taurocholate and taurodeoxycholate 0.4, taurochenodeoxycholate 1.7, glycocholate 0.3 and glycochenodeoxycholate 4.2.
Additives Tested bile salts (Calbiochem) were added to the standard mixtures as dry substance. Lipase derived from porcine pancreas was used in a stock solution of 50000 U/1 (phosphate buffer, pH 7.8, 0.066 mol/ 1); colipase (porcine pancreas, Boehringer Mannheim) was used in a stock solution of 250 pg/ml with the same buffer. Testing entailed diluting 20 gl to 1000 ~tl which corresponds to an activity of 1000 U/1 lipase and to a colipase concentration of 5 gg/ml in the stock solutions. Lipase activity (turbidimetric reduction in opaqueness) was measured on an Abbott-VP, Hitachi 705. Sigma Chemical supplied triolein (99% glyceryl-trioleate), lauric acid, monolaurin (1-monolauryl-rac-glycerol), and trilaurin (1,2,3-propantriol-tridodecanoate). L-e-Lecithin (1,2-dilauroyl-glycero-3-phosphatidyl choline) was supplied by Calbiochem.
Combination of Triglycerides and Bile Salts Since an increasing amount of triglycerides led to increasing cloudiness of the emulsion, which then disturbs measurements, 20 mg butter per 10 ml buffer was examined in all subsequent analyses. The FDL solubility did not increase with the addition of taurocholate or taurodeoxycholate; after KOH hydrolysis of the filtrates, 0.5 gmol/1 fluorescein was invariably measured. Thus, an additive effect of both solubilizers was not observed.
Enzymatic Hydrolysis of FDL by CEH Effect of Bile Salts CEH hydrolyses cholesterol ester only in the presence of relatively high concentrations of bile salts, e.g., 16 to 44 mmol/1 taurocholate [18]. By comparison, should be synthetic substrate FDL be employed after it has been partially dissolved into micelles by ethyl-glycol-monomethyl ether, the bile salts would interfere by forming mixed micelles [12]. With increasing concentration and micelle size, FDL evades attack by CEH. In Fig. 1, two bile salts demonstrate this relation. After exceeding
62
J.G. Meyer et al. : Role of Lipase in the Pancreatic Function Test PLT
Ft
t[min]
ColipQse U/L Lipase
15-
,700
2~0"
1400 U/L
1,5 84
~00 U/L
+CEH
0,5
1
2
3
4
5
Fig. 1. Hydrolysis of fluorescein dilaudate by CEH (40 U/l) in bile salt-triglyceride emulsion (ordinate: Fl, fluorescein liberation in 10 8 mmol/1); o - - o , Na-taurodeoxycholate, critical micelle concentration ( C M C ) = I . 5 mmol/1 [5]; x - - x , Nataurocholyte, CMC = 4.5 mmol/1 [5]
+Lipase* Co[ipase
..
t [mini
15-
their critical micelle concentrations (CMCs), only a minimal FDL turnover is measured. Effect of Lipase and Colipase in Triglyceride-FDL Emulsions
10-
/
/ ....
/
/
9 § , Dpase Co[rpase
~s
§
@9 -5
IF J]
100 200 300 Fig. 3. Dependence of enzymatic FDL hydrolysis (CEH) on the activity of the lipase additives employed for the initial preincubation period of 5 min. Without colipase, the lipase remains inefficacious. Analyses did not include bile salts
I(}0
200
,
300
9
[FI]
Fig. 2. Filtrate from triglyceride emulsion of FDL without (1) and with (2) taurocholate (1 retool/l). Abscissa: relative fluorescence intensity (FDL hydrolysis). Ordinate. time in minutes. (1) : unaccompanied CEH results only in minimal FDL hydrolysis which increases sharply only after addition of lipase (700 U/ 1) and colipase (5 rag/l). Taurocholate (2) mediates the immediate FDL hydrolysis by CEH; the effect of lipase/colipase is less pronounced
In a comparative study, the FDL turnover in the natural triglyceride mixture butter was compared with trilaurin and triolein, and, during the (minimal) CEH hydrolysis of FDL embodied in the emulsion, the triglyceride phase was attacked by the addition of lipase and colipase. In all three analyses the result was an increase of the FDL turnover. A lag phase dependent on the lipase activity was also noted which, in the case of lipase, lasts 5 to 7 min before an increase in fluorescein release occurs (Fig. 2). In view of the noted lag phase, we examined whether an increase in CEH activity could be achieved by initially preincubating the triglycerideFDL emulsion with lipase/colipase before adding CEH. In fact, a very nearly perfect linear correlation was found under these conditions between lipase activity and FDL turnover (Fig. 3). Even in the absence of bile salts, the reaction is dependent on colipase and, indeed, without it a lipase effect on FDL hydrolysis is not to be achieved.
J.G. Meyer et al. : Role of Lipase in the Pancreatic Function Test PLT Table 1. FDL turnover using various combinations of the standard mixture a with TDC and TC and CEH or lipase/colipase + CEH Analysis
FDL turnover (10- s mol/min) using CEH
Lipase/colipase + CEH
Standard mixture Std. + T D C 0.5 mmol/1 TDC 1.0 mmol/1 TDC 2.0 retool/1
0.01 1.84 0.9 0.04
0.43 4.40 1.35 0.04
Std.+TC0.5 mmol/1 TC1.0 mmol/1 TC2.0 retool/1
0.90 0.80 0.53
2.0 (factor 2.2) 1.5 (factor 1.9) 0.66 (factor 1.2)
(factor (factor (factor (factor
43) 2.4) 1.5) 1.0)
a Standard mixture (Std)=filtrate from 14 mg FDL and 20 mg butter in 10 ml buffer Enzyme activities tested: 50 U/I CEH; 1100 U/I lipase. Colipase concentration was 5 mg/1. TC=taurocholate; T D C = t a u r o deoxycholate
If commercially available porcine pancreatic CEH is substituted by a CEH preparation from h u m a n pancreas, then an analog increase in F D L turnover is measurable, dependent on the activity of the lipase/colipase supplement. Combination of Triglycerides, Bile Salts and Lipase/Colipase The results reported above were derived from analyses without bile salts. If emulsions with taurocholate and taurodeoxycholate are examined, again by adding lipase and colipase to the already initiated F D L hydrolytic reaction, the increase in turnover appears less pronounced than in the standard mixture free of bile salts, but the absolute F D L turnover was consistently greater, i.e., by a factor of 10 for 0.5 mmol/l taurodeoxycholate (turnover 4.4 instead of 0.43 in the standard mixture). In comparison with the basic value of 0.01 (CEH effect in the standard mixture), an increase of 184% results with the addition of taurodeoxycholate alone and the combined effect of lipase/ colipase leads to an increase of 440% (see Table 1). Discussion
The results establish the combined effects of cholesterol ester hydrolase, lipase, and colipase, physiologically in conjunction with bile salts, for the high pancreatic specificity of the Pancreolauryl test. The presence of bile salts is no longer deemed to be an essential prerequisite [10]. Our analyses performed on cholecystectomized patients without any evidence of diminished exocrine pancreatic
63
function delivered, without exception, normal results with the PLT. The initial hypothesis [14] that the orientation of bipolar F D L on the lipid-water interface seems to be an essential step of its solubilization is apparently very similar to the actual physicochemical conditions. For enzymatic hydrolysis of the triglyceride globules covered by F D L the presence of colipase has been experimentally proven to be a requirement. This leads to the conclusion that the function of colipase, in analogy to the cancellation of the inhibitive effect of bile salts on lipase activity [2], is to displace F D L from the triglyceride surface - whereby lipase is then provided access to its substrate. Monoglycerides are produced as the final product of triglyceride hydrolysis; these are also effective F D L solubilizers and, contrary to the triglyceride-FDL emulsion, facilitate a high turnover by CEH [17]. Thus, the increase in F D L hydrolysis by a factor of 43 after preincubation with lipase/ colipase is explained (Table 1, standard mixture). Apparently, CEH has no immediate access to the F D L components attached to the triglyceride spheres. Providing bile salts are present, in low concentrations mixed micelles are produced from bile salts and FDL, as has been described previously [12]. The synthetic substrate is in a similar state like physiological cholesterol esters which are only saponified in mixed micelles with bile salts [18]. F D L hydrolysis is remarkable inasmuch as accessibility for CEH apparently decreases as the bile salt concentration increases and activity eventually ceases after the C M C has been reached. This is not only true for lipase-free analysis (Fig. 1) but also after preincubation with lipase/colipase (Table 1; 2.0 mmol/1 TDC). Here is a noteworthy analogy to previous experiments with colipase which revealed that, without the addition of colipase, lipase activity is inhibited above the C M C of the bile salts present [2, 3]. These new findings in the biochemistry of PLT are an important contribution towards the interpretation of the sensitivity (81%-100%) and specificity (84%-98%) of accurate clinical studies [1, 4, 6, 11]. The clinical valency, i.e., the predictive value of the tests, should only be discussed with respect to the prevalence. Assuming a sensitivity of 81% and a specificity of 92% [6], the probability of a pathological test result in the case of actual pancreatic insufficiency increases from the predictive value of 50% (in a group with 10% prevalence, based solely on the clinical history) to over 90% (if through careful preselection the prevalence in
64
J.G. Meyer et al. : Role of Lipase in the Pancreatic Function Test PLT
the group being considered can be increased to 50%). On the other hand, the probability that a negative PLT designates no pancreatic insufficiency is exactly 99.998% for a prevalence of 10 cases of chronic pancreatitis per 100000 population, i.e, for 100000 screening tests the results are only uncertain in two instances: a normal Pancreolauryl test, therefore, can be taken as proof of a normal exocrine pancreatic function [1, 4].
9. Kaffarnik H, Meyer-Bertenrath JG (1971) Zur Pankreasspezifit/it des oralen Funktionstests mit Fluorescein-Di bzw. Monolaurat. Verh Dtsch Ges Inn Med 77:524 10. Kay G, Hine P, Braganza J (1982) The Pancreolauryl Test. A method of assessing the combined efficacy of pancreatic esterase and bile salts in vivo? Digestion 24:241 11. Lankisch PG, Schreiber A, Otto J (1983) Pancreolauryl Test. Evaluation of a tubeless pancreatic function test in comparisons with other indirect and direct tests for exocrine pancreatic function. Dig Des Sci 28:490 12. Meyer Jfirgen G (1989) Lipolytic enzymes of the human pancreas. II. Purification and properties of cholesterol ester hydrolase. Biochim Biophys Acta 1002:89 13. Meyer-Bertenrath JG (1981) Cholesterol ester hydrolase splits fluorescein dilaurate. III. International congress of clinical enzymology, Salzburg 1981. Berichte Osterr Ges Klin Chem 4: P-A26 14. Meyer-Bertenrath JG (1982) Lipolytic enzymes of the human pancreas. I. A highly sensitive, specific, and simple measurement of cholesterol ester hydrolase activity. Enzyme 28 : 336 15. Meyer-Bertenrath JG, Heckmann H, Kaffarnik H (1970) Uber Esterasen des Pankreas mit hydrolyticher Aktivit/it gegenfiber Fluorescein - Estern. Herbsttagung der Dtsch. Ges f. Biolog. Chem in Bad Neuenahr 1969. Hoppe Seyler's Z Physiol Chem 351:1316 16. Meyer-Bertenrath JG, Heckmann G, Kaffarnik H (1978) Zur Biochemie und klinischen Bedeutung des oralen Pankreasfunktionstests mit Fluoresceindilaurinsfiureester. Klin Wochenschr 56:917 17. Renczes JG (1985) In vitro Untersuchungen zum Verhalten des Fluoresceindilaurats im Pancreolauryl-Test. Inaugural dissertation, Universit/it Frankfurt 18. Schaller G (1987) Zur Kinetik der Hydrolyse von Cholesteryloleat durch Cholesterol-Ester-Hydrolase. Lab Med 11:311
Acknowledgement. The authors thank Mrs. D. Miiller and Mr. G. Asche for their excellent technical assistance.
References 1. Barry RE, Erie MD, Barry R, Parker G (1982) Fluorescein dilaurate - tubeless test for pancreatic exocrine failure. Lancet (October 2) : 742 2. Borgstr6m B (1975) On the interactions between pancreatic lipase and colipase and the substrate, and the importance of bile salts. J Lipid Res 16:411 3. Borgstr6m B, Erlanson-Albertsson Ch, Wieloch T (1979) Pancreatic colipase: chemistry and physiology. J Lipid Res 20: 805 4. Boyd EJS, Cumming JGR, Cuschieri A, Wood RAB, Wormsley K G (1982) Prospective comparison of the fluorescein-dilaurate test with the secretin-cholecystokinin test for pancreatic exocrine function. J Clin Pathol 35:1240 5. Carey MC, Small DM (1969) Micellar properties of dihydroxy and trihydroxy bile salts: effects of counterion and temperature. J Colloid Interface Sci 31:381 6. Freise J, Ranft U, Fricke K, Schmidt FW (1984) Chronische Pankreatitis: Sensitivit/it, Spezifitfit und prfidiktiver Wert des Pancreolauryltests. Z Gastroenterol 22:705 7. Kaffarnik H, Meyer-Bertenrath JG (1969) Zur Methodik und klinischen Bedeutung eines neuen Pankreaslipase-Tests mit Fluorescein-Dilaurinsfiureester. Klin Wochenschr 47 : 221 8. Kaffarnik H, Meyer-Bertenrath JG (1970) Bestimmung von Esteraseaktivitfit in vivo mit dem neuen Substrat Fluorescein - Dilaurinsfiureester. Arzneimittelforschung Drug Res 20:754
Received: July 20, 1989 Accepted: September 26, 1989
-
Prof. Dr. Dr. Jfirgen G. Meyer Stadtkrankenhaus Hanau Akademisches Lehrkrankenhaus der Universitfit Frankfurt Leimenstr. 20 D-6450 Hanau
Klin Wochenschr (1990) 68:60-64
9 Springer-Verlag 1990
Originals Lipolytic Enzymes of the Human Pancreas III. Auxiliary Function of Lipase in the Cholesterol-Esterase-Dependent Oral Test on Exocrine Pancreatic Output J.G. Meyer, J.G. Renczes, and H. Kaffarnik Zentrallabor des Stadtkrankenhauses Hanau und Medizinische Poliklinik der Universitfit Marburg
Summary. In the oral exocrine pancreatic function test using fluorescein dilaurate, this synthetic substrate attaches primarily to the triglyceride surfaces of the neutral lipids administered as part of the breakfast: these fluorescein dilaurate molecules cannot be attacked by cholesterol esterase. In the course of triglyceride saponification by lipase and colipase, however, the fluorescein dilaurate is liberated and hydrolyzed by cholesterol esterase. The pancreatic function test, therefore, measures the lipolytic activities not merely of cholesterol esterase, but indirectly of lipase, as well. Key words: Cholesterol esterase - Lipase - Colipase - Fluorescein dilaurate - Oral pancreatic function test - Bile salts - Micelles
Exactly 20 years ago, in this journal [7] we presented the first noninvasive oral test of pancreatic function. That report, entitled" Methods and clinical significance of a new pancreas lipase test using fluorescein di-laurin acid ester," was based on results from experiments involving lipase from Rhizopus arrhizus, at that time the only lipase preparation available that was void of cross reactivities. Within 1 year, it had been proved that human pancreas lipase in the form of triacylglycerol hydrolase (EC 3.1.1.3) had a higher substrate specificity than Rhizopus lipase and that hydrolysis of fluorescein dilaurate (FDL) is catalysed by other human pancreas esterases (" arylesterases ") [8, 9, 15, 16]. Today, we know that this is due to cholesterol ester hydrolase (CEH, sterolester acylhydrolase; EC 3.1.1.13) in the pancreas secrete [13, 14]. Abbreviations: CEH=Cholesterol ester hydrolase; CMCCritical micellar concentration; FDL=Fluorescein dilaurate; PLT=Pancreolauryl test; TC=Taurocholate; TDC=Taurodeoxycholate
Only recently it has been shown that the oral pancreatic function test using F D L (Pancreolauryl test, PLT) actually measures - even though only indirectly - the lipase activity in pancreatic secretion, since the major part of the indicator reaction can only progress after lipase has transformed the substrate F D L on the triglyceride surface into a form accessible to CEH [17].
Objective Already, during the preparation of a standardized test method, it was noticed that hydrolysis of orally administered FDL resulted only when the test breakfast included neutral fats, even though pancreas secretion can be stimulated by a number of fat-free foods. Additionally, it remained unexplained why F D L hydrolysis dropped from 72% to 46% when the triglyceride dose was either below (10 g) or exceeded (50 g) the optimum amount (20 g butter). These findings warranted further quantitative and qualitative analysis of the role of triglycerides in the PLT [17].
Methods Standard Solution
A solution of 10 ml bicarbonate buffer (1000 ml sodium bicarbonate solution, 0.1mol/1, plus 150 ml disodium hydrogen phosphate, 0.066 tool/l, with approximately 15 ml hydrochloric acid, 0.01 mol/1, adjusted for pH=8.35) was mixed with 14mg F D L (cristalline; Temmler-Werke, Marburg). In the case of complete solution, dependent on further additives, the concentration of F D L would be 0.2 mmol/1.
J.G. Meyer et al. : Role of Lipase in the Pancreatic Function Test PLT
Mixing Procedures Closed Erlenmeyer flasks filled with the solutions and containing ten glass balls (6 mm in diameter) were shaken for 15 min at 37 ~ C (rotational movement). Immediately afterwards, the solutions were filtered (Selecta 595 1/2) and only the filtrates were further analysed (temperature 37 ~ C).
Quantification of the Dissolved FDL Portion In one aliquot of filtrate, the FDL (colorless) was saponified completely by the addition of the same volume of ethanolic potassium hydroxide solution (0.5 mol/1) and the fluorescein concentration was determined using fluorescence spectrophotometry (Perkin-Elmer 650-40) with respect to a calibration line (2ex-- 494 nm; hem = 520 nm).
6t
Results
1. Solubility of Fluorescein Dilaurate Lipids as Solubilizer FDL remains completely undissolved while being shaken in a primary solution without additives. Already small amounts of triglycerides (butter or triolein) bind FDL to the surfaces of the emulsion, as measurable by KOH hydrolysis of the filtrates; for example, 20 mg triglycerides solubilize 0.5 ~tmol/1 FDL. Analogous testing of 1,2-dilauryl lecithin, free lauric acid, and 1-monolauryl glyceride led by comparison to the following results: lecithin 0.008; lauric acid 0.38, and monoglyceride 0.9 gmol/1 FDL.
Enzymatic Hydrolysis of FDL
Contribution of Bile Salts
CEH derived from porcine pancreas (Calbiochem) was dissolved in phosphate buffer (0.066 mol/1, pH = 6.2) resulting in a primary solution of 2000 U/1 (calculated as cholesteryl oleate hydrolysis) and containing 5 mg CEH in 7 ml buffer; 20 gl/ 1000 gl test solution was used for the experiments, corresponding to an activity of 40 U/1. To measure the fluorescein concentration liberated from FDL by CEH, the photometer was set for the accepted optimum at pH=8.35: 2ex= 483 rim, 2 e m = 512 nm. Measurements in the concentration range of 10 -8 mol/1 fluorescein were possible by comparison with an analog calibration line. The results from CEH of 48% acetone precipitate [12] isolated from human pancreas were used as a control for porcine CEH.
By adding sodium salts of various bile salts to the primary solutions, varying dissolution of FDL resulted dependent on the type of salt employed. At a bile salt concentration of 1 mmol/1 the following FDL concentrations (gmol/1) in the filtrates were measured (KOH hydrolysis): both taurocholate and taurodeoxycholate 0.4, taurochenodeoxycholate 1.7, glycocholate 0.3 and glycochenodeoxycholate 4.2.
Additives Tested bile salts (Calbiochem) were added to the standard mixtures as dry substance. Lipase derived from porcine pancreas was used in a stock solution of 50000 U/1 (phosphate buffer, pH 7.8, 0.066 mol/ 1); colipase (porcine pancreas, Boehringer Mannheim) was used in a stock solution of 250 pg/ml with the same buffer. Testing entailed diluting 20 gl to 1000 ~tl which corresponds to an activity of 1000 U/1 lipase and to a colipase concentration of 5 gg/ml in the stock solutions. Lipase activity (turbidimetric reduction in opaqueness) was measured on an Abbott-VP, Hitachi 705. Sigma Chemical supplied triolein (99% glyceryl-trioleate), lauric acid, monolaurin (1-monolauryl-rac-glycerol), and trilaurin (1,2,3-propantriol-tridodecanoate). L-e-Lecithin (1,2-dilauroyl-glycero-3-phosphatidyl choline) was supplied by Calbiochem.
Combination of Triglycerides and Bile Salts Since an increasing amount of triglycerides led to increasing cloudiness of the emulsion, which then disturbs measurements, 20 mg butter per 10 ml buffer was examined in all subsequent analyses. The FDL solubility did not increase with the addition of taurocholate or taurodeoxycholate; after KOH hydrolysis of the filtrates, 0.5 gmol/1 fluorescein was invariably measured. Thus, an additive effect of both solubilizers was not observed.
Enzymatic Hydrolysis of FDL by CEH Effect of Bile Salts CEH hydrolyses cholesterol ester only in the presence of relatively high concentrations of bile salts, e.g., 16 to 44 mmol/1 taurocholate [18]. By comparison, should be synthetic substrate FDL be employed after it has been partially dissolved into micelles by ethyl-glycol-monomethyl ether, the bile salts would interfere by forming mixed micelles [12]. With increasing concentration and micelle size, FDL evades attack by CEH. In Fig. 1, two bile salts demonstrate this relation. After exceeding
62
J.G. Meyer et al. : Role of Lipase in the Pancreatic Function Test PLT
Ft
t[min]
ColipQse U/L Lipase
15-
,700
2~0"
1400 U/L
1,5 84
~00 U/L
+CEH
0,5
1
2
3
4
5
Fig. 1. Hydrolysis of fluorescein dilaudate by CEH (40 U/l) in bile salt-triglyceride emulsion (ordinate: Fl, fluorescein liberation in 10 8 mmol/1); o - - o , Na-taurodeoxycholate, critical micelle concentration ( C M C ) = I . 5 mmol/1 [5]; x - - x , Nataurocholyte, CMC = 4.5 mmol/1 [5]
+Lipase* Co[ipase
..
t [mini
15-
their critical micelle concentrations (CMCs), only a minimal FDL turnover is measured. Effect of Lipase and Colipase in Triglyceride-FDL Emulsions
10-
/
/ ....
/
/
9 § , Dpase Co[rpase
~s
§
@9 -5
IF J]
100 200 300 Fig. 3. Dependence of enzymatic FDL hydrolysis (CEH) on the activity of the lipase additives employed for the initial preincubation period of 5 min. Without colipase, the lipase remains inefficacious. Analyses did not include bile salts
I(}0
200
,
300
9
[FI]
Fig. 2. Filtrate from triglyceride emulsion of FDL without (1) and with (2) taurocholate (1 retool/l). Abscissa: relative fluorescence intensity (FDL hydrolysis). Ordinate. time in minutes. (1) : unaccompanied CEH results only in minimal FDL hydrolysis which increases sharply only after addition of lipase (700 U/ 1) and colipase (5 rag/l). Taurocholate (2) mediates the immediate FDL hydrolysis by CEH; the effect of lipase/colipase is less pronounced
In a comparative study, the FDL turnover in the natural triglyceride mixture butter was compared with trilaurin and triolein, and, during the (minimal) CEH hydrolysis of FDL embodied in the emulsion, the triglyceride phase was attacked by the addition of lipase and colipase. In all three analyses the result was an increase of the FDL turnover. A lag phase dependent on the lipase activity was also noted which, in the case of lipase, lasts 5 to 7 min before an increase in fluorescein release occurs (Fig. 2). In view of the noted lag phase, we examined whether an increase in CEH activity could be achieved by initially preincubating the triglycerideFDL emulsion with lipase/colipase before adding CEH. In fact, a very nearly perfect linear correlation was found under these conditions between lipase activity and FDL turnover (Fig. 3). Even in the absence of bile salts, the reaction is dependent on colipase and, indeed, without it a lipase effect on FDL hydrolysis is not to be achieved.
J.G. Meyer et al. : Role of Lipase in the Pancreatic Function Test PLT Table 1. FDL turnover using various combinations of the standard mixture a with TDC and TC and CEH or lipase/colipase + CEH Analysis
FDL turnover (10- s mol/min) using CEH
Lipase/colipase + CEH
Standard mixture Std. + T D C 0.5 mmol/1 TDC 1.0 mmol/1 TDC 2.0 retool/1
0.01 1.84 0.9 0.04
0.43 4.40 1.35 0.04
Std.+TC0.5 mmol/1 TC1.0 mmol/1 TC2.0 retool/1
0.90 0.80 0.53
2.0 (factor 2.2) 1.5 (factor 1.9) 0.66 (factor 1.2)
(factor (factor (factor (factor
43) 2.4) 1.5) 1.0)
a Standard mixture (Std)=filtrate from 14 mg FDL and 20 mg butter in 10 ml buffer Enzyme activities tested: 50 U/I CEH; 1100 U/I lipase. Colipase concentration was 5 mg/1. TC=taurocholate; T D C = t a u r o deoxycholate
If commercially available porcine pancreatic CEH is substituted by a CEH preparation from h u m a n pancreas, then an analog increase in F D L turnover is measurable, dependent on the activity of the lipase/colipase supplement. Combination of Triglycerides, Bile Salts and Lipase/Colipase The results reported above were derived from analyses without bile salts. If emulsions with taurocholate and taurodeoxycholate are examined, again by adding lipase and colipase to the already initiated F D L hydrolytic reaction, the increase in turnover appears less pronounced than in the standard mixture free of bile salts, but the absolute F D L turnover was consistently greater, i.e., by a factor of 10 for 0.5 mmol/l taurodeoxycholate (turnover 4.4 instead of 0.43 in the standard mixture). In comparison with the basic value of 0.01 (CEH effect in the standard mixture), an increase of 184% results with the addition of taurodeoxycholate alone and the combined effect of lipase/ colipase leads to an increase of 440% (see Table 1). Discussion
The results establish the combined effects of cholesterol ester hydrolase, lipase, and colipase, physiologically in conjunction with bile salts, for the high pancreatic specificity of the Pancreolauryl test. The presence of bile salts is no longer deemed to be an essential prerequisite [10]. Our analyses performed on cholecystectomized patients without any evidence of diminished exocrine pancreatic
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function delivered, without exception, normal results with the PLT. The initial hypothesis [14] that the orientation of bipolar F D L on the lipid-water interface seems to be an essential step of its solubilization is apparently very similar to the actual physicochemical conditions. For enzymatic hydrolysis of the triglyceride globules covered by F D L the presence of colipase has been experimentally proven to be a requirement. This leads to the conclusion that the function of colipase, in analogy to the cancellation of the inhibitive effect of bile salts on lipase activity [2], is to displace F D L from the triglyceride surface - whereby lipase is then provided access to its substrate. Monoglycerides are produced as the final product of triglyceride hydrolysis; these are also effective F D L solubilizers and, contrary to the triglyceride-FDL emulsion, facilitate a high turnover by CEH [17]. Thus, the increase in F D L hydrolysis by a factor of 43 after preincubation with lipase/ colipase is explained (Table 1, standard mixture). Apparently, CEH has no immediate access to the F D L components attached to the triglyceride spheres. Providing bile salts are present, in low concentrations mixed micelles are produced from bile salts and FDL, as has been described previously [12]. The synthetic substrate is in a similar state like physiological cholesterol esters which are only saponified in mixed micelles with bile salts [18]. F D L hydrolysis is remarkable inasmuch as accessibility for CEH apparently decreases as the bile salt concentration increases and activity eventually ceases after the C M C has been reached. This is not only true for lipase-free analysis (Fig. 1) but also after preincubation with lipase/colipase (Table 1; 2.0 mmol/1 TDC). Here is a noteworthy analogy to previous experiments with colipase which revealed that, without the addition of colipase, lipase activity is inhibited above the C M C of the bile salts present [2, 3]. These new findings in the biochemistry of PLT are an important contribution towards the interpretation of the sensitivity (81%-100%) and specificity (84%-98%) of accurate clinical studies [1, 4, 6, 11]. The clinical valency, i.e., the predictive value of the tests, should only be discussed with respect to the prevalence. Assuming a sensitivity of 81% and a specificity of 92% [6], the probability of a pathological test result in the case of actual pancreatic insufficiency increases from the predictive value of 50% (in a group with 10% prevalence, based solely on the clinical history) to over 90% (if through careful preselection the prevalence in
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J.G. Meyer et al. : Role of Lipase in the Pancreatic Function Test PLT
the group being considered can be increased to 50%). On the other hand, the probability that a negative PLT designates no pancreatic insufficiency is exactly 99.998% for a prevalence of 10 cases of chronic pancreatitis per 100000 population, i.e, for 100000 screening tests the results are only uncertain in two instances: a normal Pancreolauryl test, therefore, can be taken as proof of a normal exocrine pancreatic function [1, 4].
9. Kaffarnik H, Meyer-Bertenrath JG (1971) Zur Pankreasspezifit/it des oralen Funktionstests mit Fluorescein-Di bzw. Monolaurat. Verh Dtsch Ges Inn Med 77:524 10. Kay G, Hine P, Braganza J (1982) The Pancreolauryl Test. A method of assessing the combined efficacy of pancreatic esterase and bile salts in vivo? Digestion 24:241 11. Lankisch PG, Schreiber A, Otto J (1983) Pancreolauryl Test. Evaluation of a tubeless pancreatic function test in comparisons with other indirect and direct tests for exocrine pancreatic function. Dig Des Sci 28:490 12. Meyer Jfirgen G (1989) Lipolytic enzymes of the human pancreas. II. Purification and properties of cholesterol ester hydrolase. Biochim Biophys Acta 1002:89 13. Meyer-Bertenrath JG (1981) Cholesterol ester hydrolase splits fluorescein dilaurate. III. International congress of clinical enzymology, Salzburg 1981. Berichte Osterr Ges Klin Chem 4: P-A26 14. Meyer-Bertenrath JG (1982) Lipolytic enzymes of the human pancreas. I. A highly sensitive, specific, and simple measurement of cholesterol ester hydrolase activity. Enzyme 28 : 336 15. Meyer-Bertenrath JG, Heckmann H, Kaffarnik H (1970) Uber Esterasen des Pankreas mit hydrolyticher Aktivit/it gegenfiber Fluorescein - Estern. Herbsttagung der Dtsch. Ges f. Biolog. Chem in Bad Neuenahr 1969. Hoppe Seyler's Z Physiol Chem 351:1316 16. Meyer-Bertenrath JG, Heckmann G, Kaffarnik H (1978) Zur Biochemie und klinischen Bedeutung des oralen Pankreasfunktionstests mit Fluoresceindilaurinsfiureester. Klin Wochenschr 56:917 17. Renczes JG (1985) In vitro Untersuchungen zum Verhalten des Fluoresceindilaurats im Pancreolauryl-Test. Inaugural dissertation, Universit/it Frankfurt 18. Schaller G (1987) Zur Kinetik der Hydrolyse von Cholesteryloleat durch Cholesterol-Ester-Hydrolase. Lab Med 11:311
Acknowledgement. The authors thank Mrs. D. Miiller and Mr. G. Asche for their excellent technical assistance.
References 1. Barry RE, Erie MD, Barry R, Parker G (1982) Fluorescein dilaurate - tubeless test for pancreatic exocrine failure. Lancet (October 2) : 742 2. Borgstr6m B (1975) On the interactions between pancreatic lipase and colipase and the substrate, and the importance of bile salts. J Lipid Res 16:411 3. Borgstr6m B, Erlanson-Albertsson Ch, Wieloch T (1979) Pancreatic colipase: chemistry and physiology. J Lipid Res 20: 805 4. Boyd EJS, Cumming JGR, Cuschieri A, Wood RAB, Wormsley K G (1982) Prospective comparison of the fluorescein-dilaurate test with the secretin-cholecystokinin test for pancreatic exocrine function. J Clin Pathol 35:1240 5. Carey MC, Small DM (1969) Micellar properties of dihydroxy and trihydroxy bile salts: effects of counterion and temperature. J Colloid Interface Sci 31:381 6. Freise J, Ranft U, Fricke K, Schmidt FW (1984) Chronische Pankreatitis: Sensitivit/it, Spezifitfit und prfidiktiver Wert des Pancreolauryltests. Z Gastroenterol 22:705 7. Kaffarnik H, Meyer-Bertenrath JG (1969) Zur Methodik und klinischen Bedeutung eines neuen Pankreaslipase-Tests mit Fluorescein-Dilaurinsfiureester. Klin Wochenschr 47 : 221 8. Kaffarnik H, Meyer-Bertenrath JG (1970) Bestimmung von Esteraseaktivitfit in vivo mit dem neuen Substrat Fluorescein - Dilaurinsfiureester. Arzneimittelforschung Drug Res 20:754
Received: July 20, 1989 Accepted: September 26, 1989
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Prof. Dr. Dr. Jfirgen G. Meyer Stadtkrankenhaus Hanau Akademisches Lehrkrankenhaus der Universitfit Frankfurt Leimenstr. 20 D-6450 Hanau
