Enzyme Substitution in Pancreatic Disease: Is Colipase Activity Sufficient? C. ERLANSON-ALBERTSSON & 0. WISEN Dept. of Medical and Physiological Chemistry, University of Lund, Lund, and Gastrointestinal Unit, Dept. of Medicine, Karolinska Hospital, Stockholm, Sweden
Scand J Gastroenterol Downloaded from informahealthcare.com by Nyu Medical Center on 05/13/15 For personal use only.
Erlanson-Albertsson C, W i s h 0. Enzyme substitution in pancreatic disease: is colipase activity sufficient? Scand J Gastroenterol 1992, 27. 108-110 The aim of the present study was to determine colipase activity in various pancreatic enzyme tablets to ascertain whether these contained sufficient amounts of colipase to activate lipase during fat digestion. Colipase activity in all preparations tested exceeded that of lipase activity by a factor of 1.4-1.9 on a molar activity basis. Since optimal activity of lipase is obtained with colipase being present in a colipase to lipase molar activity ratio of 1.0, it is concluded that these preparations contain a sufficient amount of colipase to activate lipase. K e y words: Colipase; pancreatic enzyme substitution; pancreatic insufficiency; pancreatic lipase; steatorrhea Charlotte Erlanson-Albertsson, Dept. of Medical and Physiological Chemistry, University of Lund, P. 0. Box 94, S-221 00 Lund, Sweden
Pancreatic enzyme substitution is widely used to alleviate the maldigestion that accompanies severe exocrine insufficiency (1). Besides sufficient contents of pancreatic enzymes in preparations developed for this purpose, other factors such as the galenic preparation and the influence of gastric acid secretion have to be considered. Especially lipase is rapidly inactivated by hydrochloric acid. Another aspect of importance for full lipase activity under physiologic conditions is the concomitant presence of colipase (2, 3). As the colipase content of even the most widely used pancreatic enzyme preparations is unknown, we have now determined the colipase activity in a number of such preparations. MATERIALS AND METHODS Materials The following pancreatic substitution tablets were tested: Combizym forte (no. 118851, Luitpold-Werk, Munich/ Selena Lakemedel AB, Stockholm), Pancreon comp. forte (no. 054023, Meda AB, Gothenburg), Pankreon pancreatin enterocapsules (no. 081083, Kali-Chemie, Pharma GmbH, Hannover/Meda AB), Combizym compositum (no. 149997, Luitpold-Werk, Munich/Selena Lakemedel AB), Combizym (no. 190934, Luitpold-Werk, Munich/Selena Lakemedel AB), Pankreon pankreatin (no. 431262, 5-g dose granulate) (Kali-Chemie, Pharma GmbHlMeda AB), Pancrease enterocapsules (no. 013961, McNeil Pharmaceutical, Canada, Slouffville, Ontario/Cilag AB, Sollentuna, Sweden). Combizym (Luitpold-Werk/Selena) is an acid-resistant capsule consisting of 220 mg pancreatin containing pancreatic enzymes with specified activities of lipase, amylase,
and trypsin. The protective film around the capsule contains acid-resistant plant enzymes (cellulase, amylase, and protease). Combizym forte (Luitpold- Werk/Selena) is an acid-resistant capsule of pancreatin at a higher dose, 700 mg, in which the protective film contains plant enzymes. Combizym compositum (Luitpold-Werk/Selena) contains, in addition to pancreatin, 400 mg constituents of bile, 60 mg in an acid-resistant coat containing plant enzymes. Pancrease (McNeil Pharmaceutical/Cilag) contains 170 mg pig pancreatic enzymes as acid-resistant granules in a gelatinized capsule. Pankreon (Kali-Chemie/Meda) enterocapsules contain 300 mg pig pancreatic enzymes in acid-resistant granules surrounded by a gelatinized capsule. Pankreon comp-forte (Kali-Chemie/Meda) contains 700 mg pancreatin from pig pancreas and 75 mg bile in acidresistant tablets releasing the enzymes mainly in duodenum. Pankreon Dosgranulat (Kali-Chemie/Meda) contains pancreatin at a high dose of 1.6 g in granules that are mainly released in the duodenum.
Methods Lipase and colipase activities were determined by titration, using a pH-stat (Mettler components DK 10, DK 11, and DV 11) with tnbutyrin as substrate (4). Reactions were carried out at 23°C in 15 ml buffer on 500 pi of tributyrin, with emulsification obtained by the use of a magnetic stirrer. For lipase assays the buffer contained 2 m M Tris maleate, pH 7.0, 1 mM CaCI2, and 150 mM NaCI. For colipase assays, lipase buffer plus 4 mM taurodeoxycholate and a saturating amount of purified porcine lipase (40 units) were
Enzyme Substitution
109
Table I. Preparation of enzyme solutions from the pancreatic capsules. One tablet was dissolved in the volume indicated in a glass beaker and stirred until the coat disappeared completely or until a homogeneous solution was obtained (around 20 min). Aliquots for lipase and colipase activities were taken as indicated below Dissolved in NaCI, mi
Lipase, WI
Colipase*,
10 30 20 10 10 30 50
5 5 5 5 5 5 5
5 10 5 10 5 10 5
Scand J Gastroenterol Downloaded from informahealthcare.com by Nyu Medical Center on 05/13/15 For personal use only.
Combizym Combizym forte Combizym compositum Pancrease Pancreon enterocapsule Pancreon comp. forte Pancreon dose granulate
PI
* Diluted 1:lO
used. A lipase or colipase unit of activity expresses the number of micromoles of butyric acid released per minute in the assay system used at 23°C. For determination of enzyme activity in the various pancreatin preparations the capsules were dissolved in 0.15 M NaCl with stirring until complete dissolution had occurred. The volumes for each capsule and the amount taken for lipase/colipase determination are indicated in Table I. RESULTS In Table I1 are shown the activities of lipase and colipase in the various pancreatic capsules used for enzyme substitution. The activities of lipase determined by the various manufacturers are also listed. As can be seen, the activity of colipase in all preparations is in a 1-2 molar excess of lipase. The activity measurements of lipase used by us agrees fairly well with those of the manufacturers. DISCUSSION All the tested enzyme preparations contained large amounts of colipase. Lipase acts at a hydrophobic-hydrophilic interface. such as is found in an emulsified fat solution. In
the presence of bile salts, all lipase activity is lost and is only and specifically restored by the addition of colipase (2). The optimal conditions for lipid digestion are obtained when the colipase to lipase ratio is 1.0. In all of the preparations tested the colipase activity on average exceeded that of lipase by a factor of 1.5, with minor differences between the various enzyme tablets. This means that colipase is present in sufficient amounts for optimal fat digestion provided the content of the capsules is released in the intestine. Further studies are needed to verify this. The reason for the relatively high colipase content of the enzyme preparations is that the pancreatic enzymes are of porcine origin in all cases. Previous determinations of colipase activity in the pig have shown a colipase to lipase ratio of 2-4 (C. Erlanson-Albertsson, unpublished data), whereas the ratio is 1.0 in man ( 5 ) and 0.8 in the rat (6). TOconclude, the results of this study show that pancreatic enzyme substitution, using preparations of porcine origin, contain a sufficient amount of colipase activity to ensure effective lipid digestion. ACKNOWLEDGEMENTS
Ms. Ulla Johannesson is thanked for skillful technical assist-
Table 11. Lipase and colipase activity in different pancreatic granule capsules, determined with tributyrin as substrate. The procedure was repeated three times on three different occasions. Each sample was analyzed twice. The values are means t SD. Lipase activity as determined by the commercial manufacturer is also given
Pancreatic enzyme capsules Combizym Combizym forte Combizym compositum Pancrease Pancreon (enterocapsules) Pancreon (comp. forte) Pancreon (dose granulate)
Lipase activity (units) according to manufacturer
Lipase activity (units)
7400 30,000 13,500 6200 8000 28,000 60,000
8220 t 2100 38,100 t 3400 17,800 t 2000 7280 t 95 7450 t 500 30,200 t 3400 57,500 t 1800
Colipase activity (units) 12,800 5 850 55,000 5 2900 24,800 2 5000 10,200 5 600 14,500 5 900 43,300 5 5000 98,800 2 10,500
Colipase to lipase ratio 1.8 1.4 1.4 1.4 1.9 1.4 1.7
Scand J Gastroenterol Downloaded from informahealthcare.com by Nyu Medical Center on 05/13/15 For personal use only.
110
C. Erlanson-Albertsson & 0.WisCn
ance and Ms. Ruth LovCn for typing the manuscript. This work was supported by grants from the Swedish Medical Research Council (B91-03X-07904-05C).
REFERENCES 1. Saunders, JHB, Wormsley KG. Pancreatic extracts in the treatment of pancreatic exocrine insufficiency. Gut 1975,16, 157-162 2. Borgstrom B, Erlanson-Albertsson C . Pancreatic colipase. In: Borgstrom B, Brockman HL, editors. Lipases. Elsevier NorthHolland, Amsterdam, 1984, 151-184 Received 28 May 1990 Accepted 29 August 1991
3. Hildebrand H, Borgstrom B, BCkBssy A, Erlanson-Albertsson C, Helin I. Isolated colipase deficiency in two brothers. Gut 1982, 23, 243-246 4. Patton J , Albertsson P-A, Erlanson C, Borgstrom B. Binding of porcine pancreatic lipase and colipase in the absence of substrate studied by two-phase partition and affinity chromatography. J Biol Chem 1978, 253, 4195-4202 5. Borgstrom B, Hildebrand H. Lipase and colipase activities of human small intestinal contents after a liquid test meal. Scand J Gastroenterol 1975, 10, 585-591 6 . Erlanson-Albertsson C, Larsson A , Duan, R-D. Secretion of pancreatic lipase and colipase from rat pancreas. Pancreas 1987, 2, 531-535
Scand J Gastroenterol Downloaded from informahealthcare.com by Nyu Medical Center on 05/13/15 For personal use only.
Erlanson-Albertsson C, W i s h 0. Enzyme substitution in pancreatic disease: is colipase activity sufficient? Scand J Gastroenterol 1992, 27. 108-110 The aim of the present study was to determine colipase activity in various pancreatic enzyme tablets to ascertain whether these contained sufficient amounts of colipase to activate lipase during fat digestion. Colipase activity in all preparations tested exceeded that of lipase activity by a factor of 1.4-1.9 on a molar activity basis. Since optimal activity of lipase is obtained with colipase being present in a colipase to lipase molar activity ratio of 1.0, it is concluded that these preparations contain a sufficient amount of colipase to activate lipase. K e y words: Colipase; pancreatic enzyme substitution; pancreatic insufficiency; pancreatic lipase; steatorrhea Charlotte Erlanson-Albertsson, Dept. of Medical and Physiological Chemistry, University of Lund, P. 0. Box 94, S-221 00 Lund, Sweden
Pancreatic enzyme substitution is widely used to alleviate the maldigestion that accompanies severe exocrine insufficiency (1). Besides sufficient contents of pancreatic enzymes in preparations developed for this purpose, other factors such as the galenic preparation and the influence of gastric acid secretion have to be considered. Especially lipase is rapidly inactivated by hydrochloric acid. Another aspect of importance for full lipase activity under physiologic conditions is the concomitant presence of colipase (2, 3). As the colipase content of even the most widely used pancreatic enzyme preparations is unknown, we have now determined the colipase activity in a number of such preparations. MATERIALS AND METHODS Materials The following pancreatic substitution tablets were tested: Combizym forte (no. 118851, Luitpold-Werk, Munich/ Selena Lakemedel AB, Stockholm), Pancreon comp. forte (no. 054023, Meda AB, Gothenburg), Pankreon pancreatin enterocapsules (no. 081083, Kali-Chemie, Pharma GmbH, Hannover/Meda AB), Combizym compositum (no. 149997, Luitpold-Werk, Munich/Selena Lakemedel AB), Combizym (no. 190934, Luitpold-Werk, Munich/Selena Lakemedel AB), Pankreon pankreatin (no. 431262, 5-g dose granulate) (Kali-Chemie, Pharma GmbHlMeda AB), Pancrease enterocapsules (no. 013961, McNeil Pharmaceutical, Canada, Slouffville, Ontario/Cilag AB, Sollentuna, Sweden). Combizym (Luitpold-Werk/Selena) is an acid-resistant capsule consisting of 220 mg pancreatin containing pancreatic enzymes with specified activities of lipase, amylase,
and trypsin. The protective film around the capsule contains acid-resistant plant enzymes (cellulase, amylase, and protease). Combizym forte (Luitpold- Werk/Selena) is an acid-resistant capsule of pancreatin at a higher dose, 700 mg, in which the protective film contains plant enzymes. Combizym compositum (Luitpold-Werk/Selena) contains, in addition to pancreatin, 400 mg constituents of bile, 60 mg in an acid-resistant coat containing plant enzymes. Pancrease (McNeil Pharmaceutical/Cilag) contains 170 mg pig pancreatic enzymes as acid-resistant granules in a gelatinized capsule. Pankreon (Kali-Chemie/Meda) enterocapsules contain 300 mg pig pancreatic enzymes in acid-resistant granules surrounded by a gelatinized capsule. Pankreon comp-forte (Kali-Chemie/Meda) contains 700 mg pancreatin from pig pancreas and 75 mg bile in acidresistant tablets releasing the enzymes mainly in duodenum. Pankreon Dosgranulat (Kali-Chemie/Meda) contains pancreatin at a high dose of 1.6 g in granules that are mainly released in the duodenum.
Methods Lipase and colipase activities were determined by titration, using a pH-stat (Mettler components DK 10, DK 11, and DV 11) with tnbutyrin as substrate (4). Reactions were carried out at 23°C in 15 ml buffer on 500 pi of tributyrin, with emulsification obtained by the use of a magnetic stirrer. For lipase assays the buffer contained 2 m M Tris maleate, pH 7.0, 1 mM CaCI2, and 150 mM NaCI. For colipase assays, lipase buffer plus 4 mM taurodeoxycholate and a saturating amount of purified porcine lipase (40 units) were
Enzyme Substitution
109
Table I. Preparation of enzyme solutions from the pancreatic capsules. One tablet was dissolved in the volume indicated in a glass beaker and stirred until the coat disappeared completely or until a homogeneous solution was obtained (around 20 min). Aliquots for lipase and colipase activities were taken as indicated below Dissolved in NaCI, mi
Lipase, WI
Colipase*,
10 30 20 10 10 30 50
5 5 5 5 5 5 5
5 10 5 10 5 10 5
Scand J Gastroenterol Downloaded from informahealthcare.com by Nyu Medical Center on 05/13/15 For personal use only.
Combizym Combizym forte Combizym compositum Pancrease Pancreon enterocapsule Pancreon comp. forte Pancreon dose granulate
PI
* Diluted 1:lO
used. A lipase or colipase unit of activity expresses the number of micromoles of butyric acid released per minute in the assay system used at 23°C. For determination of enzyme activity in the various pancreatin preparations the capsules were dissolved in 0.15 M NaCl with stirring until complete dissolution had occurred. The volumes for each capsule and the amount taken for lipase/colipase determination are indicated in Table I. RESULTS In Table I1 are shown the activities of lipase and colipase in the various pancreatic capsules used for enzyme substitution. The activities of lipase determined by the various manufacturers are also listed. As can be seen, the activity of colipase in all preparations is in a 1-2 molar excess of lipase. The activity measurements of lipase used by us agrees fairly well with those of the manufacturers. DISCUSSION All the tested enzyme preparations contained large amounts of colipase. Lipase acts at a hydrophobic-hydrophilic interface. such as is found in an emulsified fat solution. In
the presence of bile salts, all lipase activity is lost and is only and specifically restored by the addition of colipase (2). The optimal conditions for lipid digestion are obtained when the colipase to lipase ratio is 1.0. In all of the preparations tested the colipase activity on average exceeded that of lipase by a factor of 1.5, with minor differences between the various enzyme tablets. This means that colipase is present in sufficient amounts for optimal fat digestion provided the content of the capsules is released in the intestine. Further studies are needed to verify this. The reason for the relatively high colipase content of the enzyme preparations is that the pancreatic enzymes are of porcine origin in all cases. Previous determinations of colipase activity in the pig have shown a colipase to lipase ratio of 2-4 (C. Erlanson-Albertsson, unpublished data), whereas the ratio is 1.0 in man ( 5 ) and 0.8 in the rat (6). TOconclude, the results of this study show that pancreatic enzyme substitution, using preparations of porcine origin, contain a sufficient amount of colipase activity to ensure effective lipid digestion. ACKNOWLEDGEMENTS
Ms. Ulla Johannesson is thanked for skillful technical assist-
Table 11. Lipase and colipase activity in different pancreatic granule capsules, determined with tributyrin as substrate. The procedure was repeated three times on three different occasions. Each sample was analyzed twice. The values are means t SD. Lipase activity as determined by the commercial manufacturer is also given
Pancreatic enzyme capsules Combizym Combizym forte Combizym compositum Pancrease Pancreon (enterocapsules) Pancreon (comp. forte) Pancreon (dose granulate)
Lipase activity (units) according to manufacturer
Lipase activity (units)
7400 30,000 13,500 6200 8000 28,000 60,000
8220 t 2100 38,100 t 3400 17,800 t 2000 7280 t 95 7450 t 500 30,200 t 3400 57,500 t 1800
Colipase activity (units) 12,800 5 850 55,000 5 2900 24,800 2 5000 10,200 5 600 14,500 5 900 43,300 5 5000 98,800 2 10,500
Colipase to lipase ratio 1.8 1.4 1.4 1.4 1.9 1.4 1.7
Scand J Gastroenterol Downloaded from informahealthcare.com by Nyu Medical Center on 05/13/15 For personal use only.
110
C. Erlanson-Albertsson & 0.WisCn
ance and Ms. Ruth LovCn for typing the manuscript. This work was supported by grants from the Swedish Medical Research Council (B91-03X-07904-05C).
REFERENCES 1. Saunders, JHB, Wormsley KG. Pancreatic extracts in the treatment of pancreatic exocrine insufficiency. Gut 1975,16, 157-162 2. Borgstrom B, Erlanson-Albertsson C . Pancreatic colipase. In: Borgstrom B, Brockman HL, editors. Lipases. Elsevier NorthHolland, Amsterdam, 1984, 151-184 Received 28 May 1990 Accepted 29 August 1991
3. Hildebrand H, Borgstrom B, BCkBssy A, Erlanson-Albertsson C, Helin I. Isolated colipase deficiency in two brothers. Gut 1982, 23, 243-246 4. Patton J , Albertsson P-A, Erlanson C, Borgstrom B. Binding of porcine pancreatic lipase and colipase in the absence of substrate studied by two-phase partition and affinity chromatography. J Biol Chem 1978, 253, 4195-4202 5. Borgstrom B, Hildebrand H. Lipase and colipase activities of human small intestinal contents after a liquid test meal. Scand J Gastroenterol 1975, 10, 585-591 6 . Erlanson-Albertsson C, Larsson A , Duan, R-D. Secretion of pancreatic lipase and colipase from rat pancreas. Pancreas 1987, 2, 531-535
