Clin Biochem, Vol. 23, pp. 167-171, 1990 Printed in Canada. All rights reserved.

0000-9120/90 $3.00 + .00 Copyright © 1990 The Canadian Society of Clinical Chemists.

Automated Measurement of Trypsin Inhibitor in Urine with a Centrifugal Analyzer: Comparison with Other Acute Phase Reactants SHIRO KUWAJIMA, 1 TADASHI MATSUI, 2 SHIGERU KITAHASHI, 2 TAKUYA KISHIDA, 1 TADAHUMI NODA, ~ YOSHIHITO IZUMI, ~ KEIICHI NAKA, ~ and KIYOSHI OKUDA 1'2 1Department of Laboratory Medicine, and 2Central Clinical Laboratory, Osaka City University Medical School, 1-5-7, Asahi-Machi, Abeno-Ku, Osaka, 545 Japan Automated measurement of trypsin inhibitor in urine was performed with good precision using the COBAS FARA. Elevated levels of both trypsin inhibitor in urine and acute phase proteins in serum were shown in most cases of major abdominal surgery. We suggest that the automated assay of urinary trypsin inhibitor might be useful for the clinical diagnosis of acute phase response.

KEY WORDS: acute phase protein; urinary trypsin inhibitor; mingin; centrifugal analyzer; haptoglobin; ~l-antitrypsin; C-reactive protein. Introduction

rinary trypsin inhibitor (UTI) is a glycoprotein (1) which inhibits enzyme activity of trypsin U and ~-chymotrypsin, hyaluronidase, and, creatine phosphokinase (2). UTI has the activity of an endothelial cell growth factor (3), and is useful for therapy of shock and acute pancreatitis (4-7). By 1965 (8), a dramatic increase of UTI excretion in urine had already been noted in the acute phase of various conditions: bacterial infections (9-13), surgery (11), cancer (12), myocardial thrombosis (8), and normal pregnancy (13). However, it has been neglected as an acute phase protein (APP) in more recent publications (14,15). To re-evaluate UTI from the view point of APP, levels were determined by using the COBAS FARA automated centrifugal analyzer and compared with other acute phase reactants of serum in postoperational cases. Methods PATIENTS

T w e n t y - f o u r - h - u r i n e s taken before and after major abdominal surgery for gall stone, stomach and

Correspondence: S. Kuwajima, Department of Laboratory Medicine, Osaka City University Medical School, 1-5-7, Asahi-Machi, Abeno-Ku, Osaka, 545 Japan. Manuscript received December 14, 1988; revised August 29, 1989; accepted September 19, 1989. CLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990

colon cancer were examined. The healthy staff of our hospital served as controls. ASSAYS

Automated determinations of UTI activity were performed using the COBAS FARA centrifugal analyzer (Roche, Switzerland) at 405 nm, 25 °C, for 5 min. The unit of UTI (U) to inhibit trypsin was equivalent to that of urinastatin (=Miraclid, Mochida Pharmaceutical, Japan, MW; 67,000, pI; 2.8), which was used as standard: *2.5 mL of the buffer solution of triethanolamine which was adjusted to 0.2 mol/L, pH 7.8, containing 20 mM CaC12, was mixed with 100 ~L of test urine. Five min later, it was centrifuged for 5 min, 1,000 x g at room temperature. Then, using the COBAS FARA, 91 ~L of the above supernatant or standard solution was mixed with 70 ~L of trypsin solution (16 ~g/mL water, bovine pancreas, type IH, Sigma, USA), and incubated for 180 s at 25 °C. Thereafter, 70 ~L of substrate, N-a-benzoyl-Da-arginine-p-nitroanilide hydrochloride (Sigma, 4.0 mmol/L dimethyl sulfoxide) was added. The final volume of the reaction mixture was 250 ~L. Alpha 1-antitrypsin, and haptoglobin of serum were measured by single radial immunodiffusion (SRID, Behring, FRG). Serum sialic acid was determined by colorimetric assay using neuraminidase and N-acetyl neuraminic acid aldolase (Kyokuto Seiyaku, Japan) (16). Serum C-reactive protein (CRP) was measured quantitatively by using the LA-system (latex agglutination turbidimetry, Eiken, Japan). Results

With standard UTI from 0-500 U, linear time course of reaction was obtained for at least 15 min (Figure 1). Dilution up to tenfold of patient's urine

*One unit inhibits 50% of 2 }xg trypsin, specific activity > 2,500 U/mg from the same supplier. 167

KUWAJIMA, MATSUI, KITAHASHI, ~T AL.

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Figure 1--Time course of inhibitory activity using standard urinary trypsin inhibitor. which showed a high concentration of UTI, resulted in linear decrease of UTI activity (Figure 2). Precision using patient material was good (Table 1). Diurnal variation was observed in healthy subjects who showed higher levels in the morning (Figure 3). The mean value of healthy males in the afternoon was 31.6 _ 28.6 U/mL (mean_+2SD, n = 110), and t h a t of females was 27.6 _+29.0 U/mL (n = 149) (Figure 4). Elevated excretion of UTI was observed in 76.9% (20 cases out of 26) of post-surgical cases in whom increased levels of serum APP were seen as well (Table 2). CRP was increased in all cases (100%); ~l-antitrypsin in 91.7% (22 cases out of 24); and haptoglobin in 83.3% (20 cases out of 24). Figure 5 shows a typical case. UTI levels remained high for approximately 3 weeks in some cases (Figure 6).

Discussion In this study, inhibitory activity of UTI was successfully measured with an automatic anlayzer. In most cases (76.9%), elevated levels of UTI were observed as well as those of the other serum APP after operation, confirming the study by Dillard (11). While the UTI concentration of six cases did not exceed the normal value as shown in Table 2, all of these showed an acute phase response with respect to CRP; in four of these ~ - a n t i t r y p s i n and haptoglobin were also increased. In 1960, Mayehiro (17) recognized t h a t UTI was parallel with erythrocyte sedimentation rate (ESR).

168

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Figure 2--Dilution of patient's urine which showed a high level of UTI activity (TI as U/mL). Rudman et al. (18,19) showed t h a t 30-60% of patients with disseminated neoplastic disease excreted 100-500 times more UTI in their urine t h a n normal. Some of the normal cases in our study showed a relatively higher excretion of UTI in the morning, as shown by Dillard (11). Exceptions were observed by Mayehiro (17). Three patients with myelogeneous leukemia, in whom massive tissue destruction was easily detected, did not show elevated UTI. Viral infection might also be exceptional (10), in t h a t patients with upper respiratory infection with high fever showed normal levels of UTI.

TABLE 1

Precision of UTI Assay Within-run Sample N £ (u/mL) SD CV(%)

A 10 458.4 5.9 1.3

B

Day to Day C

A

B

C

10 10 6 6 6 157.7 6 9 . 5 3 1 1 . 7 1 2 6 . 8 49.6 6.9 3.2 22.8 8.6 4.4 4.4 4.7 7.3 6.8 8.9

CLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990

TRYPSIN INHIBITOR AND ACUTE PHASE REACTANTS UTI

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Figure 3--Diurnal variation of UTI activity in six normal persons. Another aspect of the study of UTI is that preparations of UTI from various laboratories have different molecular sizes: 70,000 (20), 67,000 (21), 34,000 (22), 27,000 (18), and 16,700 (23). Enzymatic degradation of UTI leads to the production of UTIs with different molecular weights and pI without losing inhibitory activity(22,24,25). HI-14(25) which formed a 1:1 complex with trypsin was also one of these digestive products of UTIs with the smaller molecular weight of 14,000. These findings may explain why UTIs from different institutes have many names: mingin (26), UTI (20), T.I.(17), Kunitz-type proteinase inhibitor (22), HI-30 (1), urinastatin( = Miraclid) (6), ASTI (= acid stable trypsin inhibitor) (27), EDC1 (18), ECGF-2b (= endothelial cell growth factor-2b) (3), etc.

As mentioned by Faarvang (8), increased excretion of UTI in urine has been recognized in response to inflammatory conditions; this was confirmed by automated assay in our study with post-surgical cases as a model of inflammation. In addition, renal disease and normal pregnancy have caused the augmented excretion of UTI (8). Consequently, increased levels of UTI will reflect the acute phase response when these conditions are absent. References 1. Hochstrasser K, Schbnberger OL, Rossmanith I, Wachter

E. Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-alpha-trypsin inhibitor V. Attachments of carbohydrates in the human urinary

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Figure 4--Frequency distribution of UTI activity of healthy controls during 1:00-4:00 p.m.

CLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990

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KUWAJIMA, MATSUI, KITAHASHI, ET AL. TABLE 2 Comparison of UTI and Other Serum APP in Postoperative Cases Postoperative Days to Case No.

Name

Sex

Age

Diagnosis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

M.N. H.G. K.H. K.K. S.K. H.T. M.K. T.W. S.T. M.H. H.T. M.K. N.N. S.F. K.T. N.S. Y.I. Y.K. S.Y. S.E. M.T.

M F F M F F M F M F M F M M M F M F F M F

40 69 56 59 54 51 66 56 55 82 63 65 70 46 51 62 61 52 41 65 54

22 23 24 25 26

S.K. N.K. T.M. T.K. I.M.

M F F M M

58 52 58 51 55

Gall Stone Gall Stone Gall Stone Gall Stone Gall Stone Colon Ca Gall Stone Stomach Ca Stomach Ca Stomach Ca Colon Ca Stomach Ca Stomach Ca Gall Stone Stomach Ca Rectal Ca Gall Stone Gall Stone Liver Cyst Stomach Ca Intrahepatic Lithiasis Colon Ca Rectum Ca Gall Stone Stomach Ca Gall Stone

*Peak UTI (U/mL)

Peak UTI

185.4 356.5 117.2 294.0 100.3 44.7 168.2 256.0 177.7 443.4 71.9 248.2 229.1 97.4 36.8 216.0 95.5 53.8 57.2 279.6 0

2 6 4 2 6 3 3 11 2 9 5 4 3 2 2 3 3 3 2 1 (- )

0 283.7 155.0 462.7 246.0

(- ) 3 1 2 8

Return to Normal

*Peak Values (g/L)

al-AT

CRP

Haptoglobin

>10 20 >7 16 7 (- ) 14 17 11 21 7 21 21 4 (- ) 7 4 (- ) (- ) 3 (- )

2.96 3.34 3.34 **N.D. N.D. 4.71 4.34 6.70 6.03 5.78 5.02 6.38 7.13 3.86 5.59 5.40 4.76 3.88 7.09 5.24 4.87

0.098 0.399 0.104 0.267 0.061 0.092 0.145 0.280 0.135 0.255 0.091 0.255 0.187 0.078 0.231 0.106 0.126 0.179 0.179 0.256 0.109

1.81 5.40 1.50 N.D. N.D. 3.45 4.59 5.03 3.00 1.05 3.12 5.18 5.32 2.59 3.04 3.97 3.52 2.80 3.43 5.08 4.70

(- ) >16 4 20 9

5.43 12.00 6.13 4.16 2.84

0.105 0.282 0.113 0.322 0.214

5.65 9.92 2.85 5.10 3.42

*Normal Values; (a) UTI (Urinary Trypsin Inhibitor) M;31.6-+ 28.6, F;27.6--29.0 (U/mL); (b) ~l-AT(~l-Antitrypsin) M; 2.60---0.45, F;2.20--0.50 (g/L); (c) CRP (C-Reactive Protein) less than 0.004 (g/L); (d) Haptoglobin 2.20_+0.40 (g/L). **N.D. = Not Determined; Ca = Cancer; ( - ) Peak value of UTI normal.

2.

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trypsin inhibitor isolated by affinity chromatography. Hoppe-Seyler's Z Physiol Chem 1981; 362: 1357-62. Kosuzume H, Ashida Y, T a k e m u r a M, Kato K, Ohnishi H. Mechanisms of enzyme inhibition by trypsin inhibitor in h u m a n urine. Igaku-no-Ayumi 1983; 125: 18790. McKeehan WL, Sakagami Y, Hoshi H, McKeehan KA. Two apparent h u m a n endothelial cell growth factors from h u m a n hepatoma cells are tumor-associated proteinase inhibitors. J Biol Chem 1986; 261: 5378-83. Ohnishi H, Kosuzume H, Ashida Y, Kato K, Suzuki Y, Honjo I. Therapeutic effects of h u m a n urinary trypsin inhibitor on acute experimental pancreatitis. Folia Pharmacol Japon 1983; 81: 235-44. T a m a k u m a S, Ozeki K, Ohtsuka T, et al. Clinical study of MR-20 on patients in shock. Kyukyu Igaku 1984; 8: 619-24. Sato M, Ishikawa H, Niho T, Mizota M. Effects of urinastatin on energy metabolism disorder during shock. Folia Pharmacol Japon 1986; 88: 195-203. Inaba H, Kato K, Ohzawa N, N a k a y a m a K. Effects of urinastatin on disseminated intravascular coagnla-

tion. Folia Pharmacol Japon 1986; 88: 239-44. 8. F a a r v a n g HJ. U r i n a r y tryspin inhibitor in man ("Mingin"). Scand J Clin Lab Invest 1965; 17 Suppl. 83: 1-78. 9. Bauer J, Reich Z. Ueber die antitryptische W i r k u n g des H a m s . Med Klin 1909; 5 : 1 7 4 4 47. 10. DSblin A. Ueber den Nachweis von Antitrypsin im Urin. Z Immun-Forsch 1910; 4: 224-28. 11. Dillard GHL. The trypsin inhibitor of the urine in health and disease. J Lab Clin Med 1950; 36: 266-82. 12. Schippers JC. Uber die antitryptische W i r k u n g Pathologischer Harne. Dtsch Arch Klin Med 1911; 101: 543-56. 13. F a a r v a n g HJ. The excretion of trypsin inhibitor in urine during normal pregnancy. Acta Endocrino11959; 31: 117-22. 14. K u s h n e r I, Volanakis JE, Gewurtz H. C-reactive protein and the plasma protein response to tissue injury. Ann N Y Acad Sci 1982; 389: 1-482. 15. Endo T. (ed.). Tissue injury and host response. Nihon Rinsho 1987; 45(7): 927-1158. 16. Taniuchi K, Chifu K, Hayashi N, et al. A new enzymatic method for the determination of sialic acid in CLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990

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Figure 5 - - U T I and serum APP in a typical case (S.T.), male, 55 y.o., stomach cancer. CRP, C-reactive protein; HP, haptoglobin; AT, ~l-antitrypsin; OPE, operation. serum and its application for a marker of acute phase reactants. Kobe J Med Sci 1981; 27: 91-102. 17. Mayehiro A. Studies of trypsin inhibitor in urine. Report II. The trypsin inhibitor of the urine in various pediatric patients. Yokohama Med Bull 1960; 11: 111-124. 18. Chawla RK, Wadsworth AD, Rudman D. Relation of

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CLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990

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the urinary cancer-related glycoprotein EDC 1 to plasma inter-alpha-trypsin inhibitor. J Immunol 1978; 1 2 1 : 1636-39. Rudman D, Chawla RK, Wadsworth AD, Nixon DW, Schwartz M. A system of cancer-related urinary glycoproteins: biochemical properties and clinical applications. Trans Assoc A m Phys 1977; 90: 286-99. Proksch GJ, Routh JI. The purification of the trypsin inhibitor from human pregnancy urine. J Lab Clin Med. 1972; 79: 491-99. Sumi H, Takada Y, Takada A. Studies on human urinary trypsin inhibitor 1. Its modification on treatment of urine with acid. Thrombos Res 1977; 11: 747-54. Hochstrasser K, Bretzel G, Feuth H, Hilla W, Lempart K. The inter-alpha-trypsin inhibitor as precursor of the acid-stable proteinase inhibitors in human serum and urine. Hoppe-Seyler's Z Physiol Chem 1976; 357: 153-62. Shulman NR. A proteolytic inhibitor with anticoagulant activity separated from human urine and plasma. J Biol Chem 1955; 213: 655-71. Sumi H, Toki N, Takasugi S, et al. Low molecular weight trypsin-plasmin inhibitors isolated from papain treated urinary trypsin inhibitor. Haemostasis 1982; 47: 14-18. Wachter E, Hochstrasser K, Bretzel G, Heindl S. Kunitz-type proteinase inhibiters derived by limited proteolysis of the inter-alpha-trypsin inhibitor II. Characterization of a second inhibitory inactive domain by amino acid sequence determination. Hoppe-Seyler's Z Physiol Chem 1979; 360: 1297-1303. Astrup T, Alkajaer K, Soardi F. Partial purification of the trypsin inhibitor in urine. Scand J Clin Lab Invest 1959; 11: 181--84. Yamamoto Y, Sumi H, Maruyama M, et al. Acid stable trypsin inhibitor in bile. Clin Chim Acta 1986; 158: 91--8.

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