GastroenterologiaJaponica Copyright 9 1978 by TheJapanese Society of Gastroenterology

Vol. 13, No. 6 Printed inJapan

- - O r i g i n a l Article--

ANALYSIS OF H U M A N P U R E P A N C R E A T I C JUICE I N CHRONIC PANCREATITIS AND CANCER OF T H E PANCREAS Hideo H A R A D A , M.D., T e n m e i H A Y A S H I , M.D., Akinori O N O , M.D., Nihei Y A M A M O T O , M.D., Itoko I K U B O , M . D . , T e t s u m a s a S H U N D O , M . D . , A r m a n d o N E G R O N , M.D., Kuniki M I S H I M A , M.D. a n d Ikuro K I M U R A , M.D.

Second Department of Internal Medicine, Okayama University Medical School 2-5-1, Shikada-cho, Okayama City, Japan

Summary Pure pancreatic juice was collected from 8 control subjects, 12 patients with chronic pancreatitis and 4 patients with cancer of the pancreas by endoscopic retrograde cannulation of the papilla. Samples were collected at 1 minute intervals for 20 minutes after rapid intravenous injection of secretin (Eisai, 1 U/kg) and for 10 minutes after rapid intravenous injection of CCK-PZ (Boots, 1 U/kg). Determinations of volume, bicarbonate concentration and three hydrolases (amylase, chymotrypsinogen and lipase) were made. Our tentative conclusions are (1) pancreatic enzymes are likely to be affected one after another, not in parallel fashion, in chronic pancreatitis and in cancer of the pancreas, (2) bicarbonate concentration and chymotrypsinogen or lipase are most susceptible in chronic pancreatitis and lipase secretion seems to be more susceptible than other parameters in cancer of the pancreas. Amylase is the least affected enzyme in both pancreatic diseases, and (3) determinations of chymotrypslnogen and/or lipase should be preferably performed among hydrolytic enzymes in the evaluation of exocrine pancreatic function in chronic pancreatitis and cancer of the pancreas.

Key Words: analysis of pure pancreatic juice, pure pancreatic juice, endoscopic catheterization of the papilla Secretin test or pancreozymin-secretin test has been used as one o f the most reliable m e t h o d s currently availabel for the estimation of exocrine p a n c r e a t i c function. This test is b a s e d on m e a s u r e m e n t s of volume, bicarb o n a t e concentration a n d enzymes in d u o d e n a l aspirates after s t i m u l a t i o n of the pancreas with secretin, with or without a d d e d pancreozymin ReceivedJuly 20, 1978. Accepted August 14, 1978. Address requests for reprints to: Dr. Hideo Harada, M.D., 2nd Department of Internal Medicine, Okayama University Medical School, 2-5-1, Shikada-cho, Okayama City, 700, Japan.

(CCK-PZ). Analysis of enzymes is confined to amylase for clinical purposes in most facilities because of stability a n d ease of m e a s u r e m e n t of the enzyme. As f r e q u e n t l y p o i n t e d out, howerver, this test does n o t reliably s e p a r a t e patients with m i l d d i s t u r b a n c e of p a n c r e a t i c function from n o r m a l individuals, although it is possible to recognize m o d e r a t e to severe p a n c r e a t i c dysfunction by the test. Therefore, m a n y efforts have been m a d e to improve a sensitivity or diagnositic value of the test, such as a re-evaluation o f m e t h o d s o f h o r m o n a l

462

H. H A R A D A

stimulation, appraisal system of the o b t a i n e d

Vol. 13, No. 6

ET AL.

results a n d c o m b i n a t i o n s of parameters to be

creatic a n d hepato-biliary tract diseases on the basis of sequential analysis of serum a n d u r i n e

evaluated. Confined ourself to the last probl e m , there is still a lot of controversy ml) about

tolerance

the type a n d n u m b e r of parameters (volume, b i c a r b o n a t e concentration, pancreatic enzymes

amylase, liver f u n c t i o n test, 50 g r a m glucose test,

p a n c r e o z y m i n secretin

test,

provocation test by p a n c r e o z y m i n a n d secretin, P F T using N-benzoyt-L-tyrosyl-p-aminoben-

etc.) which must be estimated in order to provide a satisfactory criterion of the secretory capacity of the exocrine pancreas. T h e purpose

pancreatography

of the present study is to evaluate the diagnostic

with chronic pancreatitis and bases for the

value of the parameters a n d to find better

diagnosis were as shown in T a b l e 1. All of the

zoic acid, scintigraphy with 75Se-methionine and

endoscopic

retrograde (ERCP).

cholangio-

Twelve

patients

c o m b i n a t i o n s of p a r a m e t e r s for the detection

12 patients had f r e q u e n t episodes of clinical

of pancreatic dysfunction in chronic p a n -

symptoms

creatitis a n d in cancer of the pancreas, based

suggestive of chronic pancreatitis and laboratory findings ( a b n o r m a l u r i n e amylase, serum amylase a n d / o r provocation test).

o n the analysis of h u m a n p u r e pancreatic juice obtained

by

endoscopic

retrograde

catheterization of the papilla.

(abdominal

pain)

and

findings

Diagnosis of chronic pancreatitis was m a d e o n the basis of clinical a n d histological findings in 6 patients, a b n o r m a l ERCP a n d PS test as

Patients and Methods Eight non-alcoholic control subjects were subjected to p a n c r e a t i c duct c a n n u l a t i o n

well as clinical findings in 5 patients a n d ab-

because of vague a b d o m i n a l symptoms b u t were subsequently f o u n d to be free of p a n -

Six patients were heavy alcoholic a n d 6 others were nonalcoholic. Findings of ERCP

n o r m a l ERCP a n d scintigraphy in one patient.

Table 1. Backgroundsof patients with chronic pancreatitis Case

Age

Sex

Diagnosis

Bases for diagnosis

S.T. n K.F.n K.Ko.* T.F.n O.M.n

65 33 29 52 78

F M M F F

F.I."

63

F

Y.H.**

48

M

Ki.Ka**

57

M

G.A. ** Ke.Ka. **

37 58

M M

K.N. **

57

M

M.K.**

70

M

Chronic pancreatitis Chronic pancreatitis Chronic pancreatitis Chronic pancreatitis Chronic pancreatitis & parapapillary diverticuli Chronic pancreatitis Cholecystectomy5 ys. ago Chronic pancreatitis & chemical diabetes Chronic pancreatitis & liver cirrhosis Chronic pancreatitis Chronic pancreatitis & liver cirrhosis Chronic pancreatitis & liver cirrhosis Chronic pancreatitis

ERCP and histology ERCP and scintography ERCP and PS test Histological findings ERCP, PFT and PS test Upper GI and endoscopy Histologicalfindings Operation for cholelithiasis ERCP, PFT and PS test 50 gram GTT & IRI Histological findings Histological findings ERCP and histology Histology Histology ERCP and PS test Histology ERCP and PS test

**: Alcoholicpatients who had enjoyedmore than 5.4 dl ofJapanese SAKEper day for more than 10 years. *: Lessthan 6.3 dl of beer/day n: No alcoholicbevarage at all

December 1978

463

Biochemical Analysis of Pure Pancreatic Juice

were i n t e r p r e t e d by the criteria (criteria II) d e s c r i b e d in our previous reporOZ). 4 patients with p a n c r e a t i c cancer were diagnosed as such on histological e x a m i n a t i o n b u t all were nonresectable. Cancer was f o u n d in the h e a d of the p a n c r e a s e in 3 cases a n d in the b o d y in one case (Case S.M.). P r e m e d i c a t i o n consisted o f subcutaneous injection of d i a z e p a m ( 5 m g ) a n d a t r o p i n ( 0 . 2 5 m g ) . T h e m a i n p a n c r e a t i c duct was c a n n u l a t e d to a d e p t h of 2.0 to 2.5 cm utilizing the duodenofiberscope O l y m p u s JF-B2. Pure p a n c r e a t i c juice was collected by siphoning at 1 m i n u t e intervals for 20 m i n u t e s after intravenous injection of secretin (Eisai secretin, 1 U p e r kg of body weight) a n d then for 10 m i n u t e s after intravenous injection of CCK-PZ (Boots, 1 U p e r kg of b o d y weight). All samples were collected in iced tubes a n d biochemical analysis was p e r f o r m e d as soon as the 30 m i n u t e s ' collection was c o m p l e t e d . Bicarb o n a t e c o n c e n t r a t i o n was m e a s u r e d by the use of ETS 600 titration system (Copenhagen). P r o t e i n concentration was d e t e r m i n e d by the

m e t h o d of Lowry et al.aS) using bovine serum a l b u m i n as s t a n d a r d . Before enzymatic assays, all samples were d i l u t e d b y physiological saline solution to a p p r o x i m a t e l y the same conc e n t r a t i o n (about 1.3 to 1.6 m g p r o t e i n per dl). This p r o c e d u r e allows all of the samples to b e in the same conditions for enzymatic assays. Amylase activity was assayed by t h e amyloclastic m e t h o d (Amylase-Test Wako)14). Lipase activity was m e a s u r e d by Lipase Kit " m a r u p i " using B A L t r i b u t y r a t e as the substratO~,16). Activation o f chymotrypsinogen to chymotrypsin was c o m p l e t e d in 2 hours at 4~ by m i x i n g 100/21 o f each s a m p l e with 200/21 o f a bovine pancreas trypsin (P-L Biochemicals, Inc.) solution (25/2g p e r ml) in 7 5 m M Tris, 20mM NaC1, 50mM CaC1 (pH 8.0). Chymotrypsin activity was e s t i m a t e d a c c o r d i n g to Kinugasa et al. 17) using N-benzoyl-L-tyrosylp - a m i n o b e n z o i c acid ( B T P A B A ) as the substrate. T h e activity was expressed as milli-moles o f p a r a a m i n o b e n z o i c acid l i b e r a t e d d u r i n g 15 m i n u t e s i n c u b a t i o n at 37~

Table 2. Secretion of pure pancreatic juice by secretin and CCK-PZ stimulation in control subjects Mean Maximal flowrate (ml/min) Maximal biarbonate concent. (mEq/L) Peak protein concentration (mg/ml) Peak concentration of enzymes Amylase (u/ml) CHTG (m mol 10Z/ml) Lipase (I.U./ml) Specific activity of enzymes Amylase (u/mg protein/ml) CHTG (m mol 102/mg protein/ml) Lipase (I.U./mg protein/ml) Maximal output of enzymes Amylase (u/min) CHTG (m mol 102/min) Lipase (I.U./min) CHTG: Chymotrypsinogen

S.D.

Coefficient of variation (%)

Low limits of controls (M-1.5 S.D.)

2.75 140.8 6.49

0.46 3.4 1.55

16.7 2.4 23.9

2.06 135.7 4.17

2789 2.300 284.0

787 0.614 87.1

28.2 26.7 30.7

1608 1.379 153.4

426 0.358 43.5

59 0.064 7.31

13.9 17.9 16.8

338 0.262 32.54

5854 4.886 585.3

1628 1.381 114.9

27.8 28.5 19.6

3412 2.815 413.0

464

Vol, 13, No. 6

H. HAR.dD~'I E T A L .

Results Results obtained in control subjects were as listed in T a b l e 2. Coefficients of variation was least in maximal bicarbonate concentration. Coefficients of variation in enzyme concentration and maximal output of enzymes were quite similar and ranged from 25 to 30%, except in maximal output of lipase in which it was somewhat low (19.6%). Calculation of maximal flow rate and maximal enzyme output per kg of body weight did not improve the coefficients of variation. Lowest limit of controls in each parameter was tentatively set at the level of Mean-1.5 S.D.. Results obtained in patients with chronic pancreatitis and in patients with cancer of the pancreas were listed in T a b l e 3 and 4. I n chronic pancreatitis, all parameters but flow rate and amylase specific activity showed significantly lower values than low limits of controls by Student t-test. Maximal bicarbonate concentration and maximal output of chymotrypsinogen were most frequently affected (in all of 12 cases). Maximal output of lipase was the next to be affected (in 11 of 12 cases), followed by peak chymotrypsinogen concentration (in 10 of 12 cases) and maximal output of amylase (in 8 of 12 cases). However, no statistically significant difference was recognized in the degree of decrement from low limits of controls among maximal amylase output, maximal chymotrypsinogen output and maximal lipase output, when only those cases with abnormally low values were compared. In cancer of the pancreas, in contrast, maximal flow rate and enzyme secretion were most frequently affected (in all of 4 cases). Maximal bicarbonate concentration was normal in a case'with cancer of the body of the pancreas. Among enzymes, peak concentration of lipase, maximal output of lipase and specific

activity of chymotrypsinogen and lipase were most frequently affected.

Discussion A review of literatures reveals that there is still a lot of controversy ~11) about the type and numbers of parameters which should be preferably measured in order to evaluate precisely the exocrine function of the pancreas. On the basis of analysis of duodenal aspirates, some investigators 1,~) advocate the measurement of secretory volume and bicarbonate as most sensitive means and others n-a0) consider the measurement of enzymes to be more sensitive means to detect exocrine pancreatic dysfunction. Furthermore, there have also been controversies about the type and number of enzymes to be l~referably estimated. Lagerl6fS) noted that amylase output was most susceptible and somewhat similar results were also reported by Diamond et al. 4) and Burton et al.2). Importance of chymotrypsin measurement was stressed by Barbezat et al.~>, Fitippini et al.6) and Hadorn et al.S). Choi et al.9) and Goldberg et al. as) reported taht trypsin was more sensitive than chymotrypsin in d e t e c t i n g pancreatic dysfunction. Lipase was regarded as the most susceptible enzyme by Diamond et al.~9), Rick et al.2~ Hadorn et al.S), Ribet et al.2a), Capitane et al.22), Gullo et al.~S), Mott et al.10), and Minaire et al.24). Zieve et al. 25) reported that there was little to choose among the specific enzymes and that multiple enzyme determinations seem unneccessary for purposes of detection of ordinary chronic pancreatitis and carcinoma of the pancreas. A thorough review of literatures suggests that these differences in opinions were derived not only from different methods of collecting samples and estimating enzymatic activities but also from different patients with

December

Biochemical Analysis of Pure Pancreatic Juice

1978

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