glycylprolyl β-naphthylamidase activity in human serum

Clinica Chimica Acta, 62 (1975) 5-l 1 0 Elsevier Scientific Publishing Company,

Amsterdam

-Printed

in The Netherlands

CCA 6935

GLYCYLPROLYL SERUM

M. HINOa,

P-NAPHTHYLAMIDASE

T. NAGATSUa,

S. KAKUMUb,

ACTIVITY

S. OKUYAMAb,

IN HUMAN

Y. YOSHIIC

and I. NAGATSUd

‘Department of Biochemistry, School of Dentistry, AichCGakuin University, Nagoya (Japan), bThird Department of Internal Medicine, Faculty of Medicine, Nagoya University, Nagoya (Japan), ‘Department of internal Medicine, Aichi Cancer Center Hospital, Sciences, (Received

Nagoya (Japan) and dLaboratory of Molecular Fujita-Gakuen University, Toyoake (Japan) November

Biology,

Institute

for Medical

4, 1974)

Summary Glycylprolyl /3-naphthylamidase activities in sera from 40 normal subjects (18-81 years) were: 22.6 f 0.9 (S.E.) (11.8-38.2) I.U./l serum at 37°C. The enzyme activities did not differ significantly with age between the younger group under 40-years-old and the older group over 40-years-old. Males, especially under 40-years-old, had slight but significantly higher activities than females. The levels were decreased in patients with gastric cancer. The levels were elevated in patients with hepatobiliary diseases, and had significant correlations with the results of the serum tests in hepatic diseases such as glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, alkaline phosphatase and total bilirubin, but had no correlation with serum lactate dehydrogenase. In cellulose acetate electrophoresis, normal sera had a single peak at the P-globulin region, but the sera in hepatitis or liver cirrhosis showed not only an increase in the normal peak at the P-globulin region but also the appearance of the other one or two new peaks in the a, and 01~-globulin regions.

Introduction In 1966, Hopsu-Havu and Glenner [l] discovered in rat liver and kidney a new diaminopeptidase, glycylprolyl /3-naphthylamidase, which liberates N-terminal glycylproline from glycyl-L-prolyl /3-naphthylamide. This enzyme was purified from rat liver [2] and hog kidney [3]. We found the enzyme activity in human saliva and serum [4] , in human salivary gland [ 5,6] and in bovine dental pulp [7], and purified the enzyme to a homogeneous form from human submaxillary gland [6]. This enzyme has been shown to hydrolyze the peptide bond between N-terminal glycylproline and adjacent amino acids and peptides

6

[S] . The physiological significance of this enzyme is not yet clear, but since the structure of glycylprolyl-X is present in the collagen molecule, the enzyme might have some role in the degradation of peptides derived from collagen. We are interested in the presence of this enzyme in human serum [4] and have examined the enzyme activity in serum from normal subjects in relation to age and sex. The enzyme activity in serum from patients has also been studied. Materials and Methods Glycyl-L-prolyl /3-naphthylamide was kindly synthesized by the Protein Research Foundation, Osaka. Fast Garnet GBC was obtained from Sigma Chem. Co. Human sera from healthy subjects were obtained from the members of Aichi-Gakuin University, School of Dentistry. The serum samples from patients were obtained from the clinics of Aichi Cancer Center Hospital and of the 3rd Department of Internal Medicine, Nagoya University Hospital. Diagnosis of chronic hepatitis and liver cirrhosis was made based on the findings of laparoscopy and liver biopsy. Diagnosis of gastric cancer was confirmed by gastric biopsy before the operation. The blood samples were collected by venipuncture and after clotting, the serum was separated by centrifuging at 3000 rpm for 15 min. Glycylprolyl /3-naphthylamidase activity did not change for at least 3 months when stored frozen at -2O”C, and for at least 24 hours at 4°C. The enzyme activity for hydrolysis of glycylprolyl /3-naphthylamide was measured by calorimetry as described previously [6] (Fig. 1). The incubation mixture (total volume 0.90 ml) contained 90 pmoles of Tris/maleate buffer, pH 7.0; 0.45 pmole of glycylprolyl /3-naphthylamide; and 20 (~1of human serum diluted to 0.30 ml with water. Diluted serum heated at 95°C for 5 min was used for the blank incubation. Incubation was carried out at 37°C for 30 min, and terminated by adding 0.30 ml of 10% Tween 20 in 1 M acetate buffer, pH 4.2, containing 0.45 mg of stabilized diazonium salt Fast Garnet GBC. After 30 min, absorbance at 530 nm was measured. The enzyme reaction was linear for at least 6 hours, and with 5 to 50 1.11of human serum. One unit of enzyme activity was defined as the amount of enzyme catalyzing the formation of 1 pmole of /3-naphthylamine per min (International Unit, I.U.) per 1 serum at 37°C. Electrophoretic behaviour of the enzyme was determined on cellulose acetate strips. 10 ~1 of the serum were placed along a center line of a 2.5 cm X 10 cm strip of cellulose acetate, and the samples were electrophoresed for 3.5 hours in Verona1 buffer, pH 8.6, ionic strength 0.075 at a constant current of

t&N-CH,-CO I

&?“a

/

Gly-Pro-NAose,

Gly-Pro-NA H~-cH2-~o fiCOOH

+

HzNm f-naphfhylamine

Fig. 1. Principle

of the assay of glycylprolyl

P-naphthylamidase

activity.

0.5 mA/cm width of a strip. One strip was stained for the identification of serum proteins, and the other strip was cut into pieces at 2.5-mm intervals, they were extracted with 0.3 ml water, and the enzyme activity was determined as described above. Glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase activities were assayed by the method of Karmen [9], and the activities were expressed in Karmen units at 25” C. The activities in some sera were assayed by the method of Reitman and Frankel [lo]. Alkaline phosphatase activity was assayed by the modified phenylphosphate method of Kind and King [ 111, and the activity was expressed as mg phenol/l5 min/lOO ml serum (King-Armstrong units) at 37°C. Lactate dehydrogenase activity was assayed by the method of Cabaud and Wroblewski [12], and the activity was expressed in the unit as a decrease of 0.001 in absorbance per min at 340 nm per ml of serum. A thymol turbidity test was carried out using the method of Shank and Hoagland [13] and the result expressed in Maclagan units. Total bilirubin and total cholesterol in serum were measured by the methods of Michaelson [14] and Zak [15], respectively, and the results expressed as mg per 100 ml serum. Results Glycylprolyl/3-naphthylamidase

activities

in normal

human

sera

The enzyme activities in sera from 40 healthy human subjects from 18 to 81 years-of-age are shown in Table I. The mean value was 22.6 * 0.9 (S.E.) (range, 11.8-38.2) I.U./l serum. To find out whether or not the enzyme activity changes with age, the values from subjects under 40-years-old (the younger group) were compared with those from subjects over 40-years-old (the older group). The older group had a slightly higher mean value than the younger group, but the difference is not statistically significant. Males had a slightly higher mean enzyme activity than females, and the difference is statistically significant (P < 0.05). The sex differences were clearly observed in the younger (P < 0.05) but not in the older group.

TABLE

I

GLYCYLPROLYL

Number

@NAPHTHYLAMIDASE

of cases

Age mean

Total Male Female

ACTIVITY

HUMAN

Glycylprolyl

(seam.) * S.E.

IN NORMAL

(range)

(I.U./I

SERUM

P-naphthylamidase

serum)

mean

40

37.7

f 3.5

(19-81)

22.6

+ 0.9

(11.8-38.2)

24

37.7

* 4.4

(19-81)

23.6

* 1.2

(15.8-38.2) (11.8-30.8)

16

36.8

f 5.6

(20-74)

20.4

t 1.3

29

24.6

+ 0.9

(19-39)

21.8

? 1.1

(11.8-38.2)

Male

18

25.8

i 1.2

(19-39)

24.1

f 1.4

(15.8-38.2)

F.?ttlale

11

22.5

t 1.4

(20-35)

18.2

* 1.1

(11.8-22.8)

11

70.9

f 1.8

(60-81)

24.5

+ 1.5

(17.2-31.8)

Male

6

73.3

+ 2.4

(67-81)

23.8

f 2.4

(17.2-31.8)

Female

5

68.0

+ 2.5

(60-74)

25.4

* 2.0

(18.6-30.8)

Younger

Older

group

group

f S.E.

(range)

activity

8 TABLE

II

GLYCYLPROLYL

P-NAPHTHYLAMIDASE

ACTIVITY

IN HUMAN

SERUM

Diagnosis

Number of cases

Glycylprolyl P-naphthylamidase (I.U./l serum) mean + S.E.

Normal (control) Gastric cancer Pancreatic cancer Gastric ulcer Pancreatitis Bile duct cancer Acute hepatitis Chronic hepatitis active type inactive type Liver cirrhosis

40 27 2 3 2 2 8

22.6 15.1 11.9 17.4 21.0 42.6 37.6

k 0.9 * 1.1* ? 2.8* i 2.3 * 2.5 + 10.5* i 6.4*

3 5 11

33.4 29.5 35.5

+ + f

Glycylprolyl

@uzphthylamidase

FROM activity

PATIENTS *Difference from control

p < 0.001 p < 0.001 n. s. n.s. p < 0.001 p < 0.05

1.8* 4.2 3.6+

< 0.001 n.s. p < 0.01

I-,

activities in sera from patients

Since the enzyme activity is high in the liver [ 1,2], any change in the serum activity would be expected in hepatic diseases. Therefore, the enzyme activities have been determined in sera from patients with hepatobiliary diseases and, for comparison, sera from patients with diseases of digestive organs other than the liver have been also examined (Table II). Patients with gastric cancer had significantly lower enzyme activities than the normal subjects. The patients with pancreatic cancer without any sign of obstructive jaundice also had lower activities. On the contrary, the enzyme activities in sera from patients with gastric ulcer or pancreatitis did not differ significantly from control values. Patients with hepatobiliary diseases had significantly elevated enzyme activities. The levels in acute hepatitis, chronic active hepatitis and liver cirrhosis were significantly high. The activity with chronic inactive hepatitis was also higher than the control value, but the difference was not statistically significant. In contrast to gastric or pancreatic cancer, the patients with cancer of the bile duct had significantly higher serum enzyme activities. These patients also had signs of obstructive jaundice and liver damage as indicated in the results of serum biochemical tests for liver diseases. The correlations between the serum glycylprolyl fl-naphthylamidase activity and the results of serum biochemical tests for liver diseases have been examined. Glutamic-oxaloacetic transaminase activity (r = 0.67) (Fig. 2), glutamic-pyruvic transaminase activity (r = 0.65), alkaline phosphatase activity (r = 0.61) and thymol turbidity test (r = 0.52), total bilirubin (r = 0.54) and total cholesterol (r = 0.33) had significant correlations with glycylprolyl P-naphthylamidase activity, while lactate dehydrogenase activity (r = -0.16) had no correlation. Isoenzymes

of glycylprolyl

fl-naph thylamidase

in human serum

The electrophoretic behaviour of glycylprolyl fl-naphthylamidase was examined on cellulose acetate strips (Fig. 3). Normal human sera showed a single

I

r= 0.67 Alb.

03

d, 62

B

t

T I

O:u 543210123 SERUM

IN

05-ACUTE

0

20

GlyFig. 2. Correlation glutamic-oxaloacetic

40

60

80

Pro-NAase

between glycylprolyl transaminase (GOT)

j%naphthylamidase (Gly-Pro-NAase) activity (Karmen units).

0

’

I

’

,

I

543210123 DISTANCE

activity

(I.U./l

I

(Cml

serum)

and

Fig. 3. Separation of glycylprolyl /.%naphthylamidase (Gly-Pro-NAase) by cellulose acetate electrophoresis. Typical examples of serum from a normal subject (15.9 I.U./l) and from a patient with acute hepatitis (73.8 I.U./l) are shown. Electrophoresis was carried out as described in Materials and Methods. GUY-ProNAase activity is expressed in terms of absorbance at 530 nm due to fl-naphthylamine.

peak of enzyme activity in the &globulin region. However, the human sera from patients with hepatitis and liver cirrhosis showed not only an elevated normal peak in the /3-globulin region, but also one or two new peaks in the regions of (Y1-globulin and (1~~ -globulin. Discussion We had previously observed the presence of glycylprolyl /3-naphthylamidase activity in human serum [4]. In the present investigation, the activity in normaI human serum has been examined in relation to age and sex and the normal values determined (Table I), The enzyme activity in normal human serum was about 20 I.U./l serum and the variations were relatively small. Therefore, if the enzyme activity changes significantly in some disease conditions it can be used as a diagnostic tool. A small but significant sex difference was found in the serum enzyme activity; the activity in male serum was slightly higher than that in female serum. This sex difference appears to be distinct only in ages under 40 years, the older group did not show the sex difference. The cause of sex difference remains for further investigation. Another factor which must be considered is age. The physiological function of this enzyme is not yet clear, but since the substrate specificity, i.e. the requirement for N-terminal glycylprolyl sequence, suggests some implication of

10

the enzyme on the degradation of peptides derived from collagen, changes in collagen metabolism during the process of development and aging may influence the enzyme activity. However, when the enzyme activities were compared between the younger and older groups, the mean activities were similar. Therefore, it is concluded that difference with age is not present, at least with adults (18-81 years) examined in this study. A highly significant elevation of the enzyme activity was found in sera from patients with hepatobiliary diseases (Table II). These results suggest that the serum glycylprolyl /3-naphthylamidase activity may be useful in the differential diagnosis of hepatobiliary diseases. The serum enzyme activity showed a good correlation (r = 0.61) with the serum alkaline phosphatase activity, which is classified by Zimmerman and Seeff [16] as the enzyme of group 1 whose levels are high in patients with obstructive jaundice and infiltrative disease of the liver, Total bilirulin also showed a close correlation (r = 0.54) with the enzyme activity. These results suggest that glycylprolyl /3-naphthylamidase belongs to the enzymes of group 1 along with alkaline phosphatase and leucyl /3-naphthylamidase, and that therefore the enzyme test may be of diagnostic value for obstructive jaundice and infiltrative disease of the liver. On the other hand, the enzyme activity also showed a close correlation with glutamic-oxaloacetic transaminase (r = 0.67) (Fig. 2) or glutamic pyruvic transaminase (r = 0.65), which are classified as enzymes of group 2 by Zimmerman and Seeff [ 161, and whose levels are markedly elevated in acute hepatitis and therefore indicate hepatobiliary injury. In support of the possibility of the release of the enzyme in hepatobiliary injury, we have recently observed that liver damage by carbon tetrachloride in rats caused a decrease of enzyme activity in the liver and an increase in serum (unpublished results). Obviously, the serum enzyme must be determined in more cases of hepatobiliary diseases and assay of the enzyme level in liver biopsy specimens is necessary to establish the diagnostic significance of the elevated serum enzyme activity in hepatobiliary diseases. When the possibility of multiple forms of glycylprolyl P-naphthylamidase was examined by electrophoresis, normal human sera contained only one peak in the P-globulin region. However, electrophoresis of sera from patients with hepatitis and cirrhosis has revealed the presence of one or two new activity peaks in the (Ye-globulin and (Ye-globulin regions, besides an increase in the activity peak in the /3-globulin observed in normal human serum (Fig. 3). The results indicate the presence of the multiple forms of the enzyme in human serum at least in pathological conditions and the usefulness of the isoenzymes in clinical diagnosis is under investigation. Patients with gastric cancer had significantly lower enzyme activity than the normal subjects and patients with gastric ulcer. These low values do not appear to be due to the older age in the group of gastric cancer patients, because no significant difference in the activity was observed between the younger and older groups of normal controls. The question arises whether these low values occur only in gastric cancer or in malignant disease generally. Two cases of pancreatic cancer also showed lower activities, while cases of pancreatitis had normal levels. Significant elevation of the level in cancer of the bile duct appears to be due to obstructive jaundice. The serum enzyme levels in patients with various cancers, and the changes in the level of patients with

gastric cancer after the complete removal of the cancer are currently under investigation in order to assess the significance of the observed decrease in gastric cancer. In summary, the present clinical studies suggest that glycylprolyl @-naphthylamidase activity in serum may be useful for the diagnosis of hepatobiliary diseases and some type of cancers such as gastric cancer. Acknowledgements We are grateful to Drs S. Sakakibara and K. Takada (Protein Research Foundation, Osaka) for the synthesis of glycyl-L-prolyl ~-naphthyl~ide. The technical assistance from Miss N. Yamada and Miss M. Suzuki is gratefully acknowledged. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

V.K. Hopsu-Havu and G.G. Glenner, Histochemie. 7 (1966) 197-201 V.K. Hopsu-Havu and S.R. Sarimo, Hoppe-Seyler’s Z. Physiol. Chem., 348 (1967) 1540-1550 V.K. Hopsu, P. Rintola and G.G. Glenner, Aeta Chem. &and., 22 (1968) 299-308 1. Nagatsu, T. Nagatsu and T. Yamamoto, Experientia, 24 (1968) 347-348 H. Oya, I. Nagatsu, M. Harada and T. Nagatsu, Experientia, 26 (1970) 252-253 H. Oya, I. Nagatsu and T. Nagatsu, Biochim. Biophys. Acta. 258 (1972) 591-599 Ii. Oya, T. K&o, I. Nagatsu and T. Nag&u, Arch. Oral Biol., 17 (1972) 1245-1248 H. Oya, M. Harada and T. Nagatsu, Arch. Oral BioI., 19 (1974) 489-491 A, Karmen, 3. Ciin. Invest., 34 (1955) 131-133 S. Reitman and S. Frankel, Am. J. Clin. Pathol.. 28 (1957) 56-63 P.R.N. Kind and E.J. King, J. Clin. Pathol.. 7 (1954) 322-326 P.G. Cabaud and F. Wrbblewski. Am. J. Clin. Pathol., 30 (1958) 234-236 R.E. Shank and C.L. Hoagtand, J. BioL Chem., 162 (1946) 133-138 M. Mieha&lsson, &and. J. Clin. Invest. Suppl., 56 (1961) l-80 B. Zak, Am. J. Clin. Pathol., 27 (1957) 583-588 H.J. Zimmerman and L.B. Seeff, in L. Coodley (ed.) Diagnostic Enzymology, Lea and Febiger, Philadelphia, 1970, pp. l-38