Serum hyaluronidase activity in chronic liver damage

47

CLINICA CHIMICA ACTA

SERUM HYALURONIDASE ACTIVITY IN CHRONIC L I V E R DAMAGE

N O B U T O NAKAMURA, NAMIO I W A B O R I A.~D T A K E O KOIZUMI

Department of Medicine, Osaka University Medical School, Osaka (lapan) (Received J u l y 3t, x969)

SUMMARY

t. Hyaluronidase activity in serum of rat with chronic hepatic damage induced by serial carbon tetrachloride inhalation was tound to be considerably elevated. In an advanced stage of the hepatic damage, however, the elevation ot the activity was not so marked. 2. Elevated serum enzyme activity was seen in patients with chronic hepatitis whereas no significant change was seen in cirrhotic patients. These results seem to show a similar tendency as seen in rats with experimental hepatic damage. 3. The change of the enzyme activity in rat serum was folmd to be closely related to the activity in liver homogenate. 4. The relationship between the changes in hepatic acid mucopolysaccharides and the serum enzyme activity in chronic hepatic damage is discussed.

INTRODUCTION

Hyaluronidase (E.C. 3.2.1.35) is an endomucopolysaccharidase which participates in the degradation of acid mucopolysaccharides in the connective tissue. Tiffs enzyme activity has been demonstrated in various mammalian tissues, serum and other body fluids t-8. In pathological conditions with connective tissue disorders or metabolic disturbance of acid mucopolysaccharides, it is postulated that hyaluronidase activity in the affected tissue as well as in the serum is elevated. We have reported changes in hepatic acid mucopolysaccharides in chronic hepatic damage previouslyL and Hutterer ~ observed changes in hepatic hyaluronidase activity in ethionine-induced liver damage. Since the liver is thought to be one of the organs which contain the highest hyaluronidase activity t°, it is reasonable to consider that liver injury brings about changes therein. In this paper, changes in serum hyaluronidase activity of rats with experimental liver damage and patients with chronic liver diseases are described. Activity of N-acetyl-/~-glucosaminidase--a lysosomal enzyme possessing similar properties to hyaluronidase as far as its metabolic role in acid mucopolysaccharides degradation la and its distribution TM are concerned--is also determined simultaneously and the relationship between these enzyme activities is studied. Clin. Chim. Acta, 27 (x97 o) 47-52

48

NAKAMURA et al.

MATERIALS AND METHODS

Male albino rats of Sprague-Dawley strain, weighing I5o--2oo g at the beginning of the experiment, were used. They were fed on a standard laboratory diet ad libitum. Chronic hepatic damage was induced by carbon tetrachloride inhalation twice a week for 14 weeks. At the periods of 4, 8, IO and 14 weeks, 4 to 8 animals were killed by exsanguination from the carotid artery, and the blood was collected, allowed to clot at 4 ° and the serum was separated by centrifugation after I h. The liver was perfused in situ through the portal vein with approximately 20 ml of ice-cold sucrose solution and removed. The liver homogenates were prepared as described elsewhere lz. For determination of human serum hyaluronidase activity, IO healthy adults and 34 patients with chronic liver diseases, including 19 with chronic hepatitis, IO with liver cirrhosis and 5 with hepatoma were selected. The diagnosis was established by history, clinical symptoms, laboratory findings and histological examination on biopsy specimens. Blood obtained from healthy subjects and patients was allowed to clot, centrifuged immediately, and the serum was stored at --20 ° . Hyaluronic acid, potassium salt from umbilical cord used as a substrate, was dissolved in 0.05 M acetate buffer (pH 3.9) in a concentration of I mg/ml before use. The serum was diluted I:IO with o.15 M NaCI. Routinely, the assay system for serum hyaluronidase activity contained 0.5 ml of substrate solution, 0.2 ml of o.15 M NaCI and 0.3 ml of the diluted serum and incubated at 37 ° for 18 h with constant shaking. Test tubes were covered hermetically with Parafilm to prevent the nonenzymatic degredation of hyaluronic acid. After the incubation period, the reaction was stopped by boiling for 3 min, and the liberated N-acetylglucosamine end-group was determined by the method of Reissig el al. 13 with N-acetylglucosamine-HCl as a standard. Enzyme activity was expressed as ttmoles of N-acetylglucosamine liberated/ml serum/I8 h. Assay of activities of liver hyaluronidase and serum N-acetyl-fl-glucosaminidase was performed as previously describedt~, 14. All determinations were done in duplicate. RESULTS

Between 6 and 21 h of incubation period, the amount of N-acetylglucosamine liberated by serum hyaluronidase was proportional to the time as shown in Fig. 1. From the results, 18 h incubation was employed for the advantage of obtaining a sufficient quantity of the product. Fig. z demonstrates that product formation was proportional to the amount of serum under o.o 4 ml per ml of the incubation mixture, o.o3 ml of serum (i.e., o. 3 ml of Io-fold diluted serum) was used for routine determination. A study on the substrate concentration showed that the amount of the reaction product was almost constant when more than 3o0 pg of hyaluronic acid per ml of the incubation mixture was used as a substrate. The pH optimum of serum hyaluronidase was found between 3.8 and 3.9 in acetate buffer. Changes in hyaluronidase activity in the liver and serum of rats with chronic hepatic damage were summarized in Table I. The hyaluronidase activity in the serum, as well as in the liver homogenate of rats with chronic hepatic damage increased Clin. Chim. Acta, 27 (197 o) 47-52

49

SERUM HYALURONIDASE IN LIVER DAMAGE

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Fig. I, T i m e course of t h e serum hy a l ur o ni da s e reaction. The reaction m i x t u r e consisted of 5oo p g of p o t a s s i u m salt of hyaluronic acid. o. 3 ml of d i l u t e d s e r u m ( i " I o ) a n d o,o 5 M a c e t a t e buffer, p H 3.9, was incubated at 37 ~. Fig. 2. E f fect of serum concentration on the e n z y m e reaction. The reaction mixture consisted of 5oo # g of potassium salt of hyaluronic acid. o.o 5 M a c e t a t e buffer, p H 3.9 a n d s e r u m as e n z y m e source, o . o l , o.o2, o.o3, o.o 4 a n d o.o 5 ml of t h e s e r u m was added, respectively. Incubation was p e r f o r m e d for t8 h at 37 ':~. TABLE

1

SERUM AND LIVER HYALURONIDASE AND SERUM N-ACETYL-/~-GLUCOSAMINIDASF ACTIVITIF-S OF RATS W I T H CHRONIC HEPATIC DAMAGE BY CARBON T E T R A C l t L O R I D E

D a t a are listed as m e a n ~: S.D. N u m b e r of rats is given in parentheses.

Hyaluromdase Healthy 4 weeks 8 weeks I o weeks x 4 weeks

damage damage damage damage

N-acetyl-lLglucosam inidase

l~it, e;* . . . . . . . . . . . . . . . . . . .

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8.15 Io.84 I t.94 13.24 5.80

2.3o 2.55 3.I7 4.93 3.27

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I4.2 17.6 ~9.7 20.7 23.2

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• t~moles of N - a c e t y l g l u c o s a m i n e / g wet liver/6 h. • * p m o l e s of N - a c e t y l g l u c o s a m i n e / m l s e r u m / 1 8 h. • ** m g of p - n i t r o p h e n o l / d l s e r u m / 3 o rain.

gradually. In the group after Io weeks of the damage, the serum enzyme activity was more than twice as high as in the healthy animals. In the more advanced stage, hyaluronidase activity in the liver homogenate was reduced markedly, and a similar tendency was seen in the serum, although the level remained higher than in the healthy rats. On the other hand, serum N-acetyl-/~-glucosaminidase activity of rats with chronic hepatic damage was elevated, even when the level of hyaluronidase activity in both liver and serum began to decrease. Table II shows the serum hyaluronidase and N-acetyl-/~-glucosaminidase activity of healthy adults and patients with chronic liver diseases. The serum hyaluronidase activity of Io healthy adults ranged from 1.72 to 2.3o pmoles N-acetylglucosamine/ml of serum/I8 h, and no obvious sex difference was seen. A significant increase of the enzyme activity was demonstrated in patients with chronic hepatitis. Clin. Chim. Aaa, 27 (x97 o) 4 7 - 5 2

NAKAMURA et al.

50 "I'ABLE I I

SERUM H Y A L U R O N I D A S E AND N - A C E T Y L - / ~ - G L U C O S A M I N I D A S E A C T I V I T I E S I N I I E A L T H Y S U B J E C T S AND IN P A T I E N T S W I T H C H R O N I C L I V E R D I S E A S E S

Number of subjects

Hyah~ronidase*

N-acetyl-[J-glucosaminidase* *

zo I9 IO 5

2.05 2.97 2.08 z.95

7.4 z2.9 t4.5 t9.9

Healthy Chronic hepatitis Liver cirrhosis Hepatoma

~ ± N :]:

o.z7 0.39 0.29 o.19

± z.8 ± 3.z ± 2.3 -& 3.8

* pmoles of N-acetylglucosamine/ml serum/z8 h. ** rng of p-nitrophenol/dl serum/3 o min.

However, in patients with liver cirrhosis, as well as with hepatoma, serum hyaluronidase activity was quite unchanged compared with that of healthy adults. On the other hand, a marked increase of the serum N-acetyl-fl-glucosaminidase activity was observed in patients with various chronic liver diseases, as previously reportedtL especially in hepatoma, when much higher values were found. Therefore, as shown in Fig.3, plotting the values of N-acetyl-fl-glucosaminidase and hyaluroni•.-,

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Fig, 3. Serum hyaluronidase and N-acetyl-~-glucosaminidase activities of h e a l t h y subjects and patients with chronic hepatitis, liver cirrhosis and hepatoma, The enzyme activities of N-acetyl-/Jglucosaminidase and hyaluronidase were plottefl on vertical and horizontal scales, respectively. A pair of dotted lines on each scale shows the normal range (mean + 2a) of each serum enzyme activity.

dase activity of each case in vertical and horizontal scales, tile areas showing values of healthy subjects, patients with chronic hepatitis, and patients with liver cirrhosis or hepatoma respectively, were fairly distinct. Namely, in the chronic hepatitis group, both serum enzyme activities were distinctly elevated, in cirrhosis or hepatoma, serum hyaluronidase activity was within normal range and serum N-acetyl-fl-glucosaminidase activity was obviously increased. Clin. Chim. ,4aa, 27 (I97O) 47-52

SERUM HYALURONIDASE IN LIVER DAMAGE

5I

DISCUSSION

The abnormalities of acid mucopolysaccharides in the liver during hepatic fibrogenesis have been reported from our laboratory. °. These changes have been suggested to be caused by the increase of acid mucopolysaccharides synthesis~L but it might be presumed that the disturbance in the degradation of these macromolecules is involved. Recently, we have reported the characteristic decrease of liver acid mucopolysaccharides in rats given a high dose of vitamin A ~4, and the decrease was remarkable in acid mucopolysaccharides sensitive to hyaluronidase, such as the nonsulfated type or chondroitin sulfate A and/or C, and both increased hepatic hyaluronidase activity and the release of the enzyme into the cell supernatant were found consistently. From this study, a similar phenomenon .was postulated in the liver with chronic damage. Data presented in this paper show the elevation of hyaluronidase activity in both liver homogenate and serum in chronic liver damage, except in a far advanced stage. The serum from patients with chronic hepatitis also shows elevated enzyme activity, but the activity of serum from cirrhotic patients was not increased. These results seem to suggest that the metabolism of acid nmcopolysaccharides becomes more inactive in the liver in the irreversible stage of hepatic damage. The reduction of serum N-acetyl-t/-glucosaminidase activity in the advanced stage of hepatic damage was not found either in rats or in human subjects. This disparity, although not yet elucidated, seems to be related with the difference in the properties of these two enzymes. Weissmann et al. ~T reported that N-acetyl-/5-glucosaminidase was tightly membrane-bound and could be hardly released from the lysosome particle with various disruptive techniques, such as freezing and thawing, or Triton X-Ioo treatment. In patients with hepatoma, serum N-acetyl-/~-glucosaminidase activity increased markedly, but unexpectedly, serum hyaluronidase activity showed the level seen in healthy controls. As several investigators demonstrated a large amount of acid nmcopolysaccharides in cancerous tissue Is-zi, it is not unreasonable to expect a high hyaluronidase activity in the serum of cancer patients. The presence of a specific hyaluronidase inhibitor in cancer patients' serum was reported2L and this unknown factor might have some relation to the enzyme activity in the serum. The simultaneous determination of serum hyaluronidase and N-acetyl-/~glucosaminidase activity seems to provide information for differentiating patients with cirrhosis or hepatoma from patients with chronic hepatitis. These phenomena might be related to the changes in the metabolism of acid nmcopolysaccharides in these liver diseases. REFERENCES t 2 3 4 5 6 7 8

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9 I0 It 12 13 14

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15 16 17 i8 19 zo zl zz

Ciin. Chim. zlcta, 27 (197 o) 47-52