Data Sheet - Pancreatin

Pancreatin is the name of an extract from porcine pancreas.

CAS-No.     8049-47-6

The pancreas is a gland with a small endocrine part (Islets of Langerhans), and a large exocrine part. The Islets of Langerhans secrete insulin and glucagon, whilst the cells of the exocrine part produce various hydrolytic enzymes most of which have digestive functions. Upon synthesis, the proteins are deposited in granules, either as active enzymes or as inactive precursors (zymogens). When the secretion is induced, the contents of the granules are released into a system of ducts and finally reach the duodenum where the zymogens are being transformed into active enzymes.

The pancreatic enzymes can be classified in four groups:

Pancreatin contains many of these enzymes. The quality of any Pancreatin preparation is defined by three enzyme activities, Protease, Amylase and Lipase. The pharmacopoeias specify minimum activity levels (in units per mg) for each of these three enzymes in Pancreatin. In both absolute and relative terms, the activities of these enzymes vary with, and depend upon, the origin of the pancreas and the treatment of the glands. Porcine pancreas is preferred for the industrial manufacture of Pancreatin for the following reasons:

The following sections will deal with some characteristics of the Pancreatin enzymes which are relevant for their biological function and for their assay.

Pancreas proteases

The pancreas contains a mixture of endo- and exopeptidases. Trypsin (EC 3.4.21.4), Chymotrypsin (EC 3.4.21.1) and Elastase (EC 3.4.21.36) are specific endopeptidases which cleave peptide bonds preferentially at the carboxyl end of basic, aromatic and aliphatic L-amino acids, respectively. Carboxypeptidase A (EC 3.4.17.1) and Carboxypeptidase B (EC 3.4.17.2) are exopeptidases which cleave L-amino acids from the free carboxyl terminus of peptides with a preference for C-terminal hydrophobic and basic amino acids, respectively.

This broad spectrum of specificities has two important implications. Physiologically, it provides an efficient tool for the degradation of proteinaceous food components into resorbable fragments. Biochemically, in any assay for Protease activity in Pancreatin only a protein substrate, e.g. casein, can supply a sufficient range of susceptible bonds for all Proteases present. In the assay, the protein substrate should be denatured prior to use because Trypsin and Chymotrypsin which together contribute the most abundant part of the endopeptidases in Pancreatin both display a preference for unfolded proteins.

In Pancreatin the major amount of each proteolytic enzyme is present as the corresponding Zymogen, i.e. as Trypsinogen, Chymotrypsinogen, procarboxypeptidase A, procarboxypeptidase B, and proelastase. Therefore an assay performed with Pancreatin as such will simply determine any free Protease activity. The total Protease activity can only be measured after preincubation of a Pancreatin solution with an activating compound. Enterokinase is routinely used for this purpose. This enzyme renders Trypsin active and thus triggers the activation of the other zymogens.

The catalytic optima of the pancreas proteases are in good agreement with the physiological requirements, i.e. about 37 °C and between pH 7 and 9. Due to the nature of the enzymes a Pancreatin solution is not very stable under these conditions because autolytic and proteolytic degradation of the enzymes takes place. Calcium ions increase the activity and stabilize Trypsin against the autolytic attack. The same holds true for Chymotrypsin though the stabilizing effect of calcium is less pronounced than with Trypsin (1).

Pancreas Lipase

Pancreatic Lipase (EC 3.1.1.3) can be characterized both by its ability to liberate fatty acids from emulsified long-chain triglycerides and by its action at the oil-water interface (2, 3). Like other esterases, pancreatic Lipase has a low substrate specificity. The Enzyme hydrolyzes not only tri-, di- and monoglycerides but also esters of n-alcohols and fatty acids with short to long carbon chains. The rate of hydrolysis decreases in the order tri-, di- and monoglyceride (1, 3, 4). The reaction velocity also decreases with increasing fatty acid chain lengths of triglycerides, that is e.g. from tributyrin to triolein.

On the other hand, in the same order triglycerides become more specific substrates (2). Therefore triolein (or olive oil as its natural substitute) is used as a substrate in most of the assays on pancreatic Lipase.

Several factors are known to affect the activity of pancreatic Lipase. Since pancreatic Lipase acts at the oil-water interface, the reaction rate depends on the surface area of the substrate and thus on the degree of emulsification (3). Bile salts promote emulsification and they are therefore used - together with gum arabic - for the preparation of stable substrate emulsions. Colipase, a small protein (molecular weight approx. 10,000), is a specific cofactor of Pancreatin Lipase. It is synthesized and secreted by the Pancreas and serves to anchor pancreatic Lipase on the substrate surface in the presence of bile acids or other amphipaths like proteins and fatty acids (2). Sodium chloride is absolutely required for and increases the activity of pancreatic Lipase (1). Calcium ions improve the thermal stability of pancreatic Lipase and also enhance the activity of the enzyme (3). Pancreatic Lipase is active in the range of pH 6.5 to 9.0 (1).

Pancreas Amylase

α-Amylase (EC 3.2.1.1; 1,4-α-D-glucan Glucanohydrolase) hydrolyzes 1,4-α-D-glucosidic linkages in polysaccharides containing 3 or more 1,4-α-linked D-glucose units (5). The enzyme from porcine pancreas is composed of 2 subunits and has a molecular weight of 50,000 (6).

Three types of amylases can be distinguished:
a-Amylase is an endoamylase which is found in all living organisms. a-Amylase acts in a random fashion by a multiple-attack mechanism on starch, glycogen and related polysaccharides and oligosaccharides ultimately yielding glucose and maltose as well as larger oligosaccharides. Reducing groups are in the α-configuration. The Enzyme cannot hydrolyze 1,6-α-bonds in glycogen and amylopectin, but it is able to bypass these branch points (1, 5).
β-Amylase and g-Amylase are exoamylases which are exclusively found in plants and microorganisms.

β-Amylase (EC 3.2.1.2; 1,4-α-D-glucan maltohydrolase) can neither hydrolyze 1,6-α-bonds nor bypass these branch points. This enzyme acts on the same substrates as a-Amylase but it removes successive maltose units from the non-reducing end; by inversion the maltose units released are in the β-configuration (5).

γ-Amylase (EC 3.2.1.3; 1,4-α-D-glucan Glucohydrolase) releases β-D-glucose successively from the non-reducing end of the polysaccharide chains (5).

Calcium and chloride ions are essential for a-Amylase. One Ca2+ is tightly bound by each enzyme molecule thereby keeping it in the correct conformation for activity. Excess calcium stabilizes a-Amylase towards heat (1). Thus, chelating agents are inhibitory. Based on their strong effect on α-Amylase chloride ions have been regarded as natural activators of the enzyme (6). NaCl activates the enzyme when added to the assay in a concentration of 10-100 mM. At higher concentrations NaCl is inhibitory (1, 7). The catalytic optima are a temperature between 40 °C and 45 °C and a pH about neutral (1).

Assays

The methods described here are those given in the monography on Pancreas powder in the Ph.Eur. (Pancreatis pulvis). They are fairly identical to the methods given by the FIP (see: Lauwers & Scharpé (1)) which originally developed these assay methods.

Note: The biological reference preparations Pancreas powder (Protease) BRP and Pancreas powder (Amylase and Lipase) BRP are issued by: Technical Secretariat, European Pharmacopoeia Commission, Council of Europe, P.O. Box 907, 67029 Strasbourg CEDEX 1, France.

Assay for proteolytic activity

Principle
Pancreatin proteases hydrolyze casein into peptides. The amount of peptides which cannot be precipitated with trichloroacetic acid is determined spectrophotometrically at 275 nm.

The total proteolytic activity of Pancreas powder is determined by comparing the quantity of non-precipitable peptides released per minute from a casein substrate solution with the quantity of such peptides released by Pancreas powder (Protease) BRP from the same substrate in the same conditions.

Reagents

  1. 0.1 N Sodium hydroxide.
  2. 0.1 N Hydrochloric acid.
  3. 0.02 M Calcium chloride: Dissolve 2.94 g of CaCl2 x 2 H2O in 900 ml of water, adjust the pH to 6.0-6.2 and add water to a final volume of 1000.0 ml. Store at 5 ± 3 °C.
  4. Borate buffer solution pH 7.5: Dissolve 2.5 g of NaCl, 2.85 g of disodiumtetraborate (Na2B4O7 x 10 H 2O) and 10.5 g of boric acid (H3BO3) in water and add water to 1000.0 ml. Adjust the pH if necessary. Store at 5 ± 3 °C.
  5. 5 % (m/V) Trichloroacetic acid.
  6. Enterokinase solution: Dissolve 50 mg of Enterokinase BRP in 0.02 M calcium chloride (C) and dilute to 50.0 ml with the same solvent. Use on the day of preparation.
  7. Casein solution: Suspend a quantity of casein BRP equivalent to 1.25 g of dried substance (determine the water content of casein BRP prior to the test by heating at 60 °C in vacuo for 4 h) in 5 ml of water, add 10 ml of 0.1 N sodium hydroxide (A) and stir for 1 min. Add 60 ml of water and stir with a magnetic stirrer until the solution is practically clear. Adjust to pH 8.0 with 0.1 N sodium hydroxide (A) or 0.1 N hydrochloric acid (B). Dilute to 100.0 ml with water. Use on the day of preparation.
  8. Test suspension: Prepare the suspension and carry out the dilution at 0 °C to 4 °C. Triturate 0.100 g of the substance to be examined for 5 min adding gradually 25 ml of 0.02 M calcium chloride (C). Transfer completely to a volumetric flask and dilute to 100.0 ml with 0.02 M calciumchloride (C). To 10.0 ml of this suspension add 10.0 ml of Enterokinase solution (F) and heat in a waterbath at 35 ± 0.5 °C for 15 min. Cool and dilute with borate buffer solution pH 7.5 (D) at 5 ± 3 °C to a final concentration of about 0.065 Ph. Eur. units of total proteolytic activity per ml calculated on the basis of the stated activity.
  9. Reference suspension: Prepare a suspension of Pancreas powder (Protease) BRP as described for the test suspension but without addition of Enterokinase so as to obtain a known final concentration of about 0.065 Ph. Eur. units per ml calculated on the basis of the stated activity.

Preparations

Filter paper control: A suitable filter paper complies with the following test: filter 5 ml of 5 % (m/V) trichloroacetic acid (E) on a 7 cm disc of white filter paper. The absorbance of the filtrate measured at 275 nm against an unfiltered portion as blank is less than 0.04.

Procedure

Label test tubes in duplicate T, Tb, S1, S1b, S2, S2b, S3, S3b and 1 tube B. Add to the test tubes in the order given:

Mix by shaking. Place a glass rod in each tube and place all tubes together with the casein solution (G) in a waterbath at 35 ± 0.5 °C. When temperature equilibrium is reached, add 2.0 ml of casein solution (G) to tubes B, S1b, S2b, S3b and Tb. Mix. At t = 0 and at intervals of 30 s, add 2.0 ml of casein solution (G) successively to tubes S1, S2, S3 and T. Mix immediately after each addition. Exactly 30 min after addition of casein solution, taking into account the regular interval adopted, add 5.0 ml of 5 % trichloroacetic acid (E) to tubes S1, S2, S3 and T. Mix. Withdraw the tubes from the waterbath and allow to stand at room temperature for 20 min. Filter the contents of each tube twice through the same suitable filter paper previously washed with 5 % trichloroacetic acid (E), then with water and dried. Measure the absorbance of the clear filtrates at 275 nm against the filtrate from tube B. See the flow chart below.

Label S1 S1b S2 S2b S3 S3b T Tb B
ml buffer (D) 2,0 2,0 1,0 1,0 ---- ---- 1,0 1,0 3,0
ml standard (I) 1,0 1,0 2,0 2,0 3,0 3,0 ---- ---- ----
ml sample (H) ---- ---- ---- ---- --- --- 2,0 2,0 ---
ml 5% TCA (E) --- 5,0 --- 5,0 --- 5,0 --- 5,0 5,0
Mix X X X X X X X X X
equilibrate to 35 °C in a waterbath X X X X X X X X X
ml Casein (G) 35 °C --- 2,0 --- 2,0 --- 2,0 --- 2,0 2,0
mix X X X X X X X X X
ml Casein (G) 35 °C 2,0 --- 2,0 --- 2,0 --- 2,0 --- ---
Mix and incubate at 35 °C
in a waterbath for exactly 30 min
X --- X --- X --- X --- ---
ml 5% TCA (E) 5,0 --- 5,0 --- 5,0 --- 5,0 --- ---
Mix X X X X X X X X X

Remove from waterbath after 30 minutes and allow to stand at room temperature for 20 min, filter twice through the same suitable filter paper, measure the absorbance at 275 nm in 1 cm cells.

Correct the average absorbance values for the filtrates obtained from tubes S1, S2 and S3 by subtracting the average values obtained for the filtrates from tubes S1b, S2b and S3b, respectively. The test is not valid unless the corrected absorbance values are between 0.15 and 0.60. Draw a calibration curve of the corrected values against the volume of the reference suspension used. Determine the activity of the substance to be examined using the corrected absorbance for the test suspension (T-Tb) and the calibration curve and taking into account the dilution factors.

Assay for lipolytic activity

Principle

The concentration of fatty acids which are liberated by Lipase from an olive oil emulsion is determined titrimetrically with sodium hydroxide.

The lipolytic activity is determined by comparing the rate at which a suspension of Pancreas powder hydrolyzes an olive oil substrate emulsion with the rate at which a suspension of Pancreas powder (Amylase and Lipase) BRP hydrolyzes the same substrate under the same conditions.

Reagents

  1. Acacia solution: Dissolve 100 g of acacia (Acaciae Gummi Ph.Eur.; gum arabic) in 1000 ml of water. Stir with a mechanical stirrer for 2 h. Centrifuge at about 2000 x g for 30 min to obtain a clear solution. Store in polyethylene containers of about 250 ml capacity at 0 °C to -20 ° C.
  2. Sodium taurocholate solution: 8 % (w/v). Dissolve 2 g of sodium taurocholate in water to a final volume of 25 ml. Prepare freshly every day.
  3. Tris(hydroxymethyl)aminomethane solution: Dissolve 60.6 mg of tris(hydr-oxymethyl)aminomethane and 234 mg of sodium chloride in water to a final volume of 100 ml. Store at 2 °C to 8 °C and use within 3 days.
  4. 0.1 N Sodium hydroxide.
  5. Olive oil emulsion: To a 800 ml beaker (9 cm diameter) transfer 40 ml of olive oil (Olivae Oleum Ph.Eur.), 330 ml of acacia solution (A) and 30 ml of water. Place an electric mixer at the bottom of the beaker. Place the beaker in a vessel containing ethanol and a sufficient quantity of ice as a cooling mixture. Emulsify using the mixer at a speed of 1000 rpm to 2000 rpm. Cool to 5 °C to 10 °C. Increase the mixing speed to 8000 rpm.

    Mix for 30 min, keeping the temperature below 25 °C by continuous addition of crushed ice into the cooling mixture. (A mixture of calcium chloride and crushed ice is also suitable.)
    Store the emulsion in the refrigerator and use within 14 days. The emulsion must not separate into 2 distinct layers. Check the diameter of the globules of the emulsion under the microscope: At least 90 % of the droplets have a diameter below 3 µm and none has a diameter greater than 10 µm. Shake thoroughly before preparing the substrate emulsion.
  6. Substrate emulsion: For 10 determinations, mix the following solutions in the order indicated: 100 ml of olive oil emulsion (E), 80 ml of tris(hydroxy-methyl)aminomethane solution (C), 20 ml of sodium taurocholate solution (B) and 95 ml of water. Use on the day of preparation.
  7. Lipase solvent: Maleate buffer solution pH 7.0. Dissolve 10.0 g sodiumchloride, 6.06 g tris(hydroxymethyl)aminomethane and 4.90 g maleic anhydride in 900 ml of water. Adjust with 4 N sodium hydroxide to pH 7.0 and add water to a final volume of 1000 ml. Store at 2 °C to 8 °C and use within 3 days.
  8. Test suspension: In a small mortar cooled to 0 °C to 4 °C, triturate carefully a quantity of the substance to be examined equivalent to about 2500 Ph. Eur. units of lipolytic activity with 1 ml of cooled Lipase solvent (G) until a very fine suspension is obtained. Dilute with cold Lipase solvent (G), transfer quantitatively to a volumetric flask and add cold Lipase solvent (G) to a final volume of 100.0 ml..
  9. Reference suspension: Equilibrate the bottle with standard to room temperature before opening to prevent moistening by condensing water. Prepare a suspension of standard (Pancreas powder (Amylase and Lipase) BRP) as described for the test suspension using a quantity equivalent to about 2500 Ph. Eur. units.

Keep the test and reference suspensions in iced water during the test!

Apparatus

Use a reaction vessel of about 50 ml capacity provided with:

An automatic or manual titration apparatus may be used. In the latter case, the burette is graduated in 0.005 ml and the pH-meter is provided with a wide reading scale and glass-calomel electrodes. After each test the reaction vessel is evacuated by suction and washed several times with water, the washings being removed each time by suction.

Procedure

Carry out the titrations immediately after the preparation of the test suspension and the reference suspension.

Place 29.5 ml of substrate emulsion (F) in the reaction vessel equilibrated at 37 ± 0.5 °C. Fit the vessel with the electrodes, a stirrer and a burette (the tip being immersed in the olive oil suspension), put the lid in place and switch on the apparatus. Carefully add 0.1 N sodium hydroxide (D) with stirring to adjust to pH 9.2. Using a rapid-flow graduated pipette transfer about 0.5 ml of the previously homogenized reference suspension (I), start the chronometer and add continuously 0.1 N sodium hydroxide (D) to maintain the pH at 9.0. Read the volume of 0.1 N sodium hydroxide (D) used after exactly 1 min and repeat this reading four times. Determine the average of the last four readings (S1). Make 2 further determinations (S2 and S3) and calculate the average n1 of the values S1, S2 and S3. The average volume of 0.1 N sodium hydroxide used should be about 0.12 ml/min with limits of 0.08 and 0.16 ml/min.

Carry out 3 determinations in the same manner for the test suspension (T1, T2 and T3) and calculate the average n1 of the values T1, T2 and T3. If the quantity of 0.1 N sodium hydroxide used is outside the limits of 0.08 to 0.16 ml/min, the assay should be started again with a quantity of test suspension which is more suitable but situated between 0.4 and 0.6 ml. Otherwise the quantity of the substance to be examined should be adjusted to comply with the conditions of the test.

Calculate the activity in Ph. Eur. units/mg from the expression:

n  *  m1
x A
n1  *  m

n     =   average volume of 0.1 N sodium hydroxide used per min during the titration of the test suspension,
n1   =   average volume of 0.1 N sodium hydroxide used per min during the titration of the reference suspension,
m    =   mass in mg of the substance to be examined,
m1  =   mass in mg of the reference preparation,
A    =   activity of the Pancreas powder (Amylase and Lipase) BRP in Ph. Eur. units/mg.

Assay for amylolytic activity

Principle
α-Amylase liberates from starch reducing groups which in alkaline solution react with iodine which is added in excess. The amount of iodine not used by the reaction is determined titrimetrically with thiosulfate.

The amylolytic activity is determined by comparing the rate at which a suspension of Pancreas powder hydrolyzes a starch substrate solution with the rate at which a suspension of Pancreas powder (Amylase and Lipase) BRP hydrolyzes the same substrate under the same conditions.

Reagents

  1. Phosphate buffer solution pH 6.8 (0.2 M): To 51 ml of 0.2 M KH2PO4 (2.72 % m/V) add about 49 ml of 0.2 M Na2HPO4 x 12 H2O (7.16 % m/V) to give a pH of 6.8. Store at 5 ± 3 °C.
  2. Starch solution: To a quantity of starch BRP equivalent to 2.0 g of dried substance add 10 ml of water and mix (determine the water content of starch BRP prior to the test by heating at 120 °C for 4 h). Add this suspension with continuous stirring to 160 ml of boiling water. Rinse the starch container several times with water, 10 ml each, and add the washings to the hot starch solution. Heat to boiling, stirring continuously. Cool to room temperature and dilute to 200 ml with water. Use on the day of preparation.
  3. 0.2 M Sodium chloride (1.17 % m/V).
  4. 1 N Hydrochloric acid.
  5. 0.1 N Iodine.
  6. 0.1 N Sodium hydroxide.
  7. Sulfuric acid (96 %).
  8. 0.1 N Sodium thiosulfate.
  9. Test suspension: Triturate an amount of the substance to be examined equivalent to about 1500 Ph. Eur. units of amylolytic activity with 60 ml of phosphate buffer solution pH 6.8 (A) for 15 min. Transfer quantitatively to a volumetric flask and dilute to 100.0 ml with phosphate buffer solution pH 6.8 (4 °C) (A).
  10. Reference suspension: Prepare a suspension of standard (Pancreas powder (Amylase and Lipase) BRP) as described for the test suspension, using a quantity equivalent to about 1500 Ph. Eur. units.

Keep all suspensions in iced water during the test!

Procedure

In a test tube (200 mm long, 22 mm diameter; with ground-glass stopper) place 25.0 ml of starch solution (B), 10.0 ml of phosphate buffer solution pH 6.8 (A) and 1.0 ml of 0.2 M sodium chloride (C). Stopper the tube, mix the contents and place in a waterbath at 25.0 ± 0.1 °C. When the temperature equilibrium has been reached, add 1.0 ml of the test suspension (I) and start the chronometer. Mix thoroughly and place the tube in the waterbath. After exactly 10 min, add 2 ml of 1 N hydrochloric acid (D) to stop the reaction. Transfer the mixture quantitatively to a 300 ml conical flask fitted with a ground-glass stopper. While shaking continuously, add 10.0 ml of 0.1 N iodine (E) and immediately 45 ml of 0.1 N sodium hydroxide (F).

Allow to stand in the dark at 15 °C to 25 °C for 15 min. Add 4 ml of a mixture of 4 volumes of water and 1 volume of sulfuric acid (G). Titrate the excess of iodine with 0.1 N sodium thiosulfate (H) using a microburette.

Carry out a blank titration adding 2 ml of 1 N hydrochloric acid (D) before introducing the test suspension (I).

Carry out the titration of the reference suspension (J) in the same manner.

Calculate the amylolytic activity in Ph. Eur. units/mg from the expression:

(n'  -  n ) * m1
x A
(n'1 -  n1) * m

n     =   number of ml of 0.1 N sodium thiosulfate used in the titration of the test suspension,
n1   =   number of ml of 0.1 N sodium thiosulfate used in the titration of the reference suspension,
n'     =   number of ml of 0.1 N sodium thiosulfate used in the blank titration of the test suspension,
n'1   =   number of ml of 0.1 N sodium thiosulfate used in the blank titration of the reference suspension,
m    =   mass in mg of the substance to be examined,
m1  =   mass in mg of the reference preparation,
A    =   activity of Pancreas powder (Amylase and Lipase) BRP in Ph. Eur. units/mg.

Availability

Standard qualities
1 x USP (powder)
2 x USP (powder)
3 x USP (powder)
4 x USP (powder or granules)
5 x USP (powder or granules)
6 x USP (powder or granules)
7 x USP (powder or granules)
8 x USP (powder or granules)
8 x USP (powder or granules) „high Lipase“
8 x USP (powder or granules) „high Protease“
1 x Ph. Eur. / FIP (powder or granules)
2 x Ph. Eur. / FIP (powder or granules)
3 x Ph. Eur. / FIP (powder or granules)
Concentrate (powder or granules) very high activities

Customized qualities are available upon request.

References

  1. Lauwers, A., Scharpé, S.: Pharmaceutical Enzymes, Drugs and Pharmaceutical Sciences, Volume 84, Marcel Dekker, Inc., New York-Basel-Hong Kong, 1997.
  2. Junge, W. in: Methods of Enzymatic Analysis (Bergmeyer, J., Graál, M., eds.) 3rd ed., Vol. IV, p.15. Verlag Chemie, Weinheim, 1984.
  3. Neumann, U., Kaspar, P., Ziegenhorn, J. in: Methods of Enzymatic Analysis (Bergmeyer, J., Graál, M., eds.) 3rd ed., Vol. IV, p.26. Verlag Chemie, Weinheim, 1984.
  4. Barman, T.E.: Enzyme Handbook, Vol. II. Springer, New York - Heidelberg-Berlin-Tokyo, 1985.
  5. International Union of Biochemistry. Nomenclature Committee: Enzyme Nomenclature. Academic Press, Inc., London, 1984.
  6. Dixon, M., Webb, E.C.: Enzymes. 3rd ed. Academic Press, New York, San Francisco, 1979.
  7. Rauscher, E. in: Methods of Enzymatic Analysis (Bergmeyer, J., Graál, M., eds.) 3rd., Vol. IV, p.152. Verlag Chemie, Weinheim, 1984.

 

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