225
Clinica Chimica Acta, 62 (1975) 225-229 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7091
A SIMPLE METHOD FOR THE QUANTITATIVE STOOL TRYPSIN AND CHYMOTRYPSIN
PETER
G. ROBINSON,
Department of Paediatrics, (New Zealand)
(Received
January
PATRICIA School
DETERMINATION
A. SMITH and ROBERT of Medicine,
University
OF
B. ELLIOTT
of Auckland,
Auckland,
29, 1975)
Summary A simple method for the determination of the activity of trypsin and chymotrypsin in stool and duodenal aspirations is described. The method is based on that described by Barber0 et al. (Am. J. Dis. Child.: 112 (1966) 536) [l] but utilizes the time for a drop of 0.1 pH unit in the solution, instead of a constant pH titration, to determine enzyme activity. Results for a number of cystic fibrosis patients and normals compare favourably with the literature.
Introduction The earliest reported methods [2,3] for the determination of tryptic activity in stools used the non-specific digestion of gelatine coated X-ray film by serially diluted stool homogenate. A number of modifications to these have been suggested 14-71 in an attempt to improve the specificity. The most recent methods for assay of tryptic and chymotryptic activity have involved the use of specific substrates [1,8-121. The method of Barber0 et al. [l] uses specific substrates, together with a constant pH titration, to obtain the rate of substrate hydrolysis and hence the enzyme concentration. The assay described in this paper is a simplification of this latter method. Experimental Materials Trizma-HCl (tris(hydroxymethyl)aminomethane, Tris-HCl, T 3253), Trizma Base (Tris, T 1503), ATEE (N-acetyl-L-tyrosine ethyl ester, A 6751), TAME (p-tosyl-L-argininemethyl ester, T 4626), trypsin (T 0134) and chymotrypsin (C 4129) were obtained from Sigma Chemical Company, U.S.A. Other chemicals were LR or AR grade.
226
Reagent solutions 1. Trypsin Buffer (pH 8.2, 0.005 M) was prepared by dissolving Tris-HCl (0.354g), Tris (0.334 g), sodium chloride (2.34 g, 0.04 M) and calcium choride dihydrate (2.94 g, 0.02 M) in a liter of distilled water. Standard enzyme solution was made by dissolving trypsin (1 mg/ml) in buffer. Substrate solution was prepared by dissolving TAME (2.075 g) in 50 ml buffer. 2. Chymo trypsin Buffer (pH 7.8, 0.005 M) was made by dissolving Tris-HCl (0.532 g), Tris (0.198 g), sodium chloride (2.925 g, 0.05 M) and calcium chloride dihydrate (0.735 g, 0.005 M) in a liter of distilled water. Standard enzyme solution was prepared by dissolving chymotrypsin (1 mg/ml) in buffer. Substrate solution was made by dissolving ATEE (0.454 g) in 25 ml methanol and making up to 50 ml with buffer. Buffer solutions were stored at 37°C and standard enzyme solutions at 0-4°C. Calibration standards were prepared daily by diluting stock standard 1 : 1 (500 pg/ml), 1 : 10 (100 pg/ml) and 1 : 100 (10 pg/ml) with the appropriate buffer. Methods Stool samples were homogenized in 0.9% saline (1 g stool/l0 denal aspirates were diluted 1 : 10 with saline.
ml). Duo-
1. Trypsin assay Sample solution (2 ml) or working standard (2 ml), buffer (pH 8.2, 0.005 M, 5.5 ml) and TAME solution (2.5 ml) were placed in a 25 ml beaker on a magnetic stirrer. A small paper clip was used as a disposable stirring bar. The pH of the solution was adjusted to just above 8.2 (Radiometer Expanded Scale pH Meter Model 26, Combined Glass Electrode GK 2301B) with 0.2 M HCl or NaOH and the time for the pH to drop from 8.2 to 8.1 was noted (stopwatch). The pH was readjusted and a duplicate timing made. 2. Chymotrypsin
assay
As for trypsin but buffer (pH 7.8, 0.005 M, 3 ml) and ATEE (5 ml) used instead of pH 8.2 buffer and TAME. The pH drop was timed from 7.8 to 7.7.
Quantitation A calibration plot on log-log paper was constructed as shown in Fig. 1. Enzyme concentration in pg/g (or E.cg/ml duodenal aspirate) is read directly from the graph. Results are expressed as concentration of enzyme in pg/g for convenience although this is not strictly correct. It is assumed that the molecular activity
227
1
10 pH drop
hme
100 lsecl
Fig. 1. Calibration plots for activity of trypsin and chymotrypsin as a function (0.1 PH units). Average standard deviation
Clinica Chimica Acta, 62 (1975) 225-229 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7091
A SIMPLE METHOD FOR THE QUANTITATIVE STOOL TRYPSIN AND CHYMOTRYPSIN
PETER
G. ROBINSON,
Department of Paediatrics, (New Zealand)
(Received
January
PATRICIA School
DETERMINATION
A. SMITH and ROBERT of Medicine,
University
OF
B. ELLIOTT
of Auckland,
Auckland,
29, 1975)
Summary A simple method for the determination of the activity of trypsin and chymotrypsin in stool and duodenal aspirations is described. The method is based on that described by Barber0 et al. (Am. J. Dis. Child.: 112 (1966) 536) [l] but utilizes the time for a drop of 0.1 pH unit in the solution, instead of a constant pH titration, to determine enzyme activity. Results for a number of cystic fibrosis patients and normals compare favourably with the literature.
Introduction The earliest reported methods [2,3] for the determination of tryptic activity in stools used the non-specific digestion of gelatine coated X-ray film by serially diluted stool homogenate. A number of modifications to these have been suggested 14-71 in an attempt to improve the specificity. The most recent methods for assay of tryptic and chymotryptic activity have involved the use of specific substrates [1,8-121. The method of Barber0 et al. [l] uses specific substrates, together with a constant pH titration, to obtain the rate of substrate hydrolysis and hence the enzyme concentration. The assay described in this paper is a simplification of this latter method. Experimental Materials Trizma-HCl (tris(hydroxymethyl)aminomethane, Tris-HCl, T 3253), Trizma Base (Tris, T 1503), ATEE (N-acetyl-L-tyrosine ethyl ester, A 6751), TAME (p-tosyl-L-argininemethyl ester, T 4626), trypsin (T 0134) and chymotrypsin (C 4129) were obtained from Sigma Chemical Company, U.S.A. Other chemicals were LR or AR grade.
226
Reagent solutions 1. Trypsin Buffer (pH 8.2, 0.005 M) was prepared by dissolving Tris-HCl (0.354g), Tris (0.334 g), sodium chloride (2.34 g, 0.04 M) and calcium choride dihydrate (2.94 g, 0.02 M) in a liter of distilled water. Standard enzyme solution was made by dissolving trypsin (1 mg/ml) in buffer. Substrate solution was prepared by dissolving TAME (2.075 g) in 50 ml buffer. 2. Chymo trypsin Buffer (pH 7.8, 0.005 M) was made by dissolving Tris-HCl (0.532 g), Tris (0.198 g), sodium chloride (2.925 g, 0.05 M) and calcium chloride dihydrate (0.735 g, 0.005 M) in a liter of distilled water. Standard enzyme solution was prepared by dissolving chymotrypsin (1 mg/ml) in buffer. Substrate solution was made by dissolving ATEE (0.454 g) in 25 ml methanol and making up to 50 ml with buffer. Buffer solutions were stored at 37°C and standard enzyme solutions at 0-4°C. Calibration standards were prepared daily by diluting stock standard 1 : 1 (500 pg/ml), 1 : 10 (100 pg/ml) and 1 : 100 (10 pg/ml) with the appropriate buffer. Methods Stool samples were homogenized in 0.9% saline (1 g stool/l0 denal aspirates were diluted 1 : 10 with saline.
ml). Duo-
1. Trypsin assay Sample solution (2 ml) or working standard (2 ml), buffer (pH 8.2, 0.005 M, 5.5 ml) and TAME solution (2.5 ml) were placed in a 25 ml beaker on a magnetic stirrer. A small paper clip was used as a disposable stirring bar. The pH of the solution was adjusted to just above 8.2 (Radiometer Expanded Scale pH Meter Model 26, Combined Glass Electrode GK 2301B) with 0.2 M HCl or NaOH and the time for the pH to drop from 8.2 to 8.1 was noted (stopwatch). The pH was readjusted and a duplicate timing made. 2. Chymotrypsin
assay
As for trypsin but buffer (pH 7.8, 0.005 M, 3 ml) and ATEE (5 ml) used instead of pH 8.2 buffer and TAME. The pH drop was timed from 7.8 to 7.7.
Quantitation A calibration plot on log-log paper was constructed as shown in Fig. 1. Enzyme concentration in pg/g (or E.cg/ml duodenal aspirate) is read directly from the graph. Results are expressed as concentration of enzyme in pg/g for convenience although this is not strictly correct. It is assumed that the molecular activity
227
1
10 pH drop
hme
100 lsecl
Fig. 1. Calibration plots for activity of trypsin and chymotrypsin as a function (0.1 PH units). Average standard deviation
