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Oxidative enzymes of polymorphonuclear leucocytes and plasma fibrinogen, ceruloplasmin, and copper levels in Behcet's disease
Clinical Biochemi~y, Vol. 27, No. 5, pp. 413--418, 1994 Copyright© 1994 The Canadian Societyof Clinical Chemists Printed in the USA. All fightsre~rv~ 0009-9120/94 $6.00 + .00
Pergamon 0009-9120(94)~m~1-4
Oxidative Enzymes of Polymorphonuclear Leucocytes and Plasma Fibrinogen, Ceruloplasmin, and Copper Levels in Behc;et's Disease PAKIZE DO,AN, 1 GURSEL TANRIKULU, 1 0MIT SOYUER,2 and KADER KOSE1 Departments of 1Biochemistry and 2 Dermatology, Faculty of Medicine, Erciyes University, 38039 Kayseri, Turkey This study was performed to investigate the antioxident mechanisms of polymorphonuclear leucocytes (PMN) in active stage of Behget's Disease. PMN activities of myeloperoxidase (p < 0.02), superoxide dlsmutase (p < 0.001), catalase (p < 0.005), and glutathlone peroxidsee (p < 0.005) were significantly lower in the patients: the NADPH oxidase activity was significantly hlgher (p < 0.001) than those in controls. The plasma levels of ceruloplasmln (Cp), flbrinogen, and copper (Cu) were also signiflcantly higher in the patients group (p < 0.001). Slgnlflcant and poslUve correlations were found between the glutathione peroxldsee and catslase activities (p < 0.001) and also between the plasma Cp and Cu levels (p < 0.001) in the patients group. However, no correlation was observed among the other enzyme activities. In the control group, a significantly positive correlatlon was present only between the plasma ceruloplasmin and Cu levels (p < 0.001). It was concluded that (impaired PMN functions) decreased enzyme activities in the antioxidant system and Increased levels of oxygen free radicals may play a role in tissue damage in Sehget's disease.
Introduction
reported that functional abnormalities were present in the neutrophils of patients with Behget's Disease, such as chemot~Tis and phagocytosis (6). Furthermore, although they are not specific to neutrophils, some lysosemal enzymes, ~-glucuronidase and acid phosphatase, were reported to be higher during the attack than in the remission phase or control (7). A decrease in the activity of myeloperoxidase (MPO), an enzyme specific to the neutrophils, was shown by various authors (8-10). It has been suggested that stimulated neutrophils from patients with Behget's disease generate high levels of oxygen intermediates (OI), resulting in endothelial tissue damage .(11). Because of the relationship between OI and oxidative enzymes, and t h e possible correlation between Beh~et's disease and functions of polymorphonuclear (PMN) leucocytes, we attempted to clarify the significance and correlation of superoxide dismutase (SOD), and MPO, glutathione peroxidase (GSH-Px), catalase, NADPH-oxidase in PMN leucocytes, and serum ceruloplasmin (Cp), fibrinogen, and copper (Cu) in Behget's disease.
B
Patients, materials, and methods
KEY WORDS: superoxide dismutase activity; myeloperoxidase activity; catalase activity; NADPH oxidase activity; glutathione peroxidase activity; ceru]opln~min activity; Cu; fibrinogen; polymorphonuclear leucocytes; Behqet's disease.
ehget syndrome is a multisystem disorder characterized by recurrent oral and genital ulcers, arthritis, thrombophlebitis, uveitis. Its basic underlying pathological process is that of a vasculitis (1). Genetic and environmental factors probably play a role in pathogenesis of this disease. Some studies show an increased prevalence of HI~-B5 (2), HLA135-1, and HLA-DQw3 (3) in Behget's disease. Recent studies have demonstrated an increase in circulation activated T lymphocytes (4). In addition high levels of circulating immune complexes have been detected in patients with this disease (5). It has been Correspondence: Prof. Dr. PRkiTe Do,an. Manuscript received April 15, 1994; revised June 24, 1994; accepted June 27, 1994. CLINICAL BIOCHEMISTRY, VOLUME 27, OCTOBER 1994
Forty patients (14 males and 26 femRles) with the complete type of Behget's disease were studied. Their ages ranged from 14 to 65 years (mean +- SD, 33.5 -+ 1.7). Thirty-six healthy subjects (16 males and 20 females, 29.3 -+ 1.3 years old) that were 1947 years-old were evaluated as the control group. ISOLATION OF P M N
Blood was taken from the antecubital vein with a heparinized disposable syringe, and mi~ed with 6% (w/v) d e x ~ m in saline in a ratio of 4:1 and allowed to stand for 60 rain at room temperature. The supernatant was removed and centrifuged at 275 x g for 10 rain. The resulting cell pellet was resuspended in 413
D O O A N ET AL.
8 mL of Krebs Ringer Phosphate Buffer (KRPB), layered over 3 mL of Histopaque-1077 (Sigma Chemical Co., St. Louis, MO) spun at 450 × g for 30 rain at 37 °(3. The cell pellet contained the PMN. Residual erythrocytes were lysed by adding 9 volumes of distilled water to this pellet, followed by vigorous sbMdng for 30 s; osmolality was restored by adding i volume of 9% (w/v) NaC1. The cell suspension was centrifuged at 275 × g for 5 rain, and the s u p e r n a t a n t was discarded. The pellet was washed three times with K R P B and resusponded in 2 m L of KRPB. Cell numbers in the finalsuspension were counted and the suspension stored at -20 °C. The purity of P M N population was 95%.
added to the reaction mixture to inhibitM P O activity (15). MEASUREMENT OF CATALASEACTIVITY
Activity was determined at 25 °C according to the modified technique of Beers and Sizer (16) by following the absorbance of hydrogen peroxide at 240 nm. One unit of catalase activity was defined as the amount of enzyme that decomposes 1 ~mol H202/ rain at an initial H202 concentration of 30 mM/L at pH 7.0 and 25 °C. A standard curve was constructed by using purified bovine liver catalase (Sigma). Catalase activity of the sample was expressed as U/mg protein.
PREPARATION OF PMN HOMOGENATES MEASUREMENT OF N A D P H OXIDASE ACTIVITY
The PMN suspension was frozen at - 2 0 °C and thawed six times, then homogenized using a motor driven Teflon-glass homogenizer (900 rpm for 5 rain at 0 °C). The protein content of the homogenate was measured using Lowry et al.'s method (12). MZASUR~.MENT OF MPO ACTIVITY
MPO activity was determined by a modification of the o-disnlsidine method (13). The assay mixture, in a cuvette of 1 cm path length, contained: 0.3 mL 100 mM/L phosphate buffer (pH 6.0; 0.3 mL 10 mM/L HsO2, 0.5 mL 20 mM/L o-dianisidine (freshly prepared without contaminating surroundings, with extreme caution, using disposable gloves because it is carcinogenic) in deionized water; and 0.010 mL PMN homogenate in a final volume of 3.0 mL. The PMN homogenate was added last and the change in absorbance at 460 n m was followed for 10 rain. All measurements were carried out in duplicate. One unit of MPO was defined as that degrading 1 tLmol HsO2/min at 25 °C; specific activity was given as U/rag of protein. A molar extinction coefficient (e) of 11.300 for oxidized o-dianisidine was used for the calculation. MgAS~MENTS OF SOD ACTIVITY
S O D activitywas detected by its abilityto inhibit the autoxidation of adrenalin at p H 10.2, using a modification of the method described by Misra and Fridowich (14). The assay mixture contained: 0.5 ml, 1.8 m M / L adrenalin (freshly prepared, pH 2.0); 0.4 mL 0.75 mM/L EDTA; 0.5 mL 300 mM/L sodium carbonate (pH 10.2); and 0.1 mL 60 mM/L sodium azide and PMN homogenate (0.75 protein/lnL) in a final volume of 3.0 mL. The reaction was started by the addition of the adrenalin and the increase in absorbance at 480 n m was followed in a P e r k i n Elmer spectrophotometer. A standard containing 0.75 ~g SOD inhibited the conversion of adrenalin to adrenochrome by 73%. Control experiments were run with boiled enzyme and with bovine serum albumin. Sodium aside was 414
NADPH oxidase catalyzes the reduction of 3-(4,5 dimethyl-2-thiazolyl-2,5-diphenyl-2H-diphenyl-2H tetrazolium bromide (MTT) (Sigma), which acts as hydrogen accepter. NADPH oxidase activity was determined by measuring the increase in absorbance at the absorption maximum of MTT formazan, at 560 nm, resulting from the reduction of MTT by NADPH (17). The assay mixture contained the following: 2.3 mL of 130 mM/L potassium phosphate buffer, pH 7.6; 0.5 mL 1 mg/mL MTT; 0.1 mL 10 mg/mL NADPH. The assay temperature was 30 °C, and reaction was initiated by adding 0.1 mL leucocyte homogenate. We measured absorbance change per minute (A/min) from the linear portion of the recorder tracing with a spectrophotemeter. One unit of activity is the activity necessary to reduce 1 ttmol MTT to MTT-formazan per minute under specified conditions. Purified diaphorase from Clostridium kluyveri (Sigma) was used to obtain the standard curve. Specific activity was given as U/g protein. MEASUREMENT OF G S H - P x ACTIVITY
Glutathione peroxidase activity was determined by the method of Paglia and Valentine (18). The reaction mixture contained 2.48 m L of 50 m M / L phosphate buffer,p H 7, containing: 5 m M / L EDTA; 0.1 m L 8.4 m M / L N A D P H ; 0.1 m L 150 m M / L reduced glutathione (Sigma); 0.01 m L 112.5 m M / L sodium azide; and 4.6 U glutathione reductase (Type HI, Sigma). The reaction was initiated by the addition of 0.1 mL 2.2 mM H2O 2 to the reaction mixture containing 500-1000 ~g protein. The change in the optical density was read at 340 n m for 4 rain. The data were expressed as nmol NADPH oxidized to NADP/mg protein/min by using the molar extinction coefficient (e) of 6.200 at 340 nm. All data were corrected for the linear NADPH oxidation by HsO 2 alone in the absence of enzyme protein that was always less than 5% of the total reaction (19). CLINICAL BIOCHEMISTRY, VOLUME 27, OCTOBER 1994
OXIDATIVE ENZYMES IN BEHCET~ DISEASE MEASURBMENT OF PLASMA Cp ACTIVITY
Discussion
Plasma Cp levels were determined by measurement of p-phenylene diamine oxidase activity according to the method of Sunderman and Nomoto (2O).
Previous studies have demonstrated important disorders of neutrophil functions in Beh~et's disease such as an increase in the chemot~TiR and in the activity of NADPH oxid"~e, increased lysosomal enzyme activities, shortened neutrophil life, and increased levels of oxygen r a s c a l s (6-8,10,11). Yamada et al. (7) studied neutrophils from patients with Beh~et's disease and found out that the levels of lysosomal enzymes, ~-glucuronidase, and collage nase were increased in the active phase of the disease, whereas MPO activity was found to be lower during the active stage when compared to the rest phase. However, they reported that the difference between MPO levels was statistically insignificant. On the other hand, Namba (9) reported that acid phosphatase and ~-glucuronida~e levels were no different from those in controls but MPO activity was significantly lower in Beh;et's patients compared with that in the controls. In a previous preliminary study with a few patients with Beh~et's disease and controls, we examined the alterations in the activities of SOD, MPO, and catalase in P M N s a n d serum Cp and Cu content. Although, there were numerical decreases in MPO and catalase activities in the patient group compared to controls, no statistically significant difference was observed (23). However in this study, because the n u m b e r of patients and controls were greater then our previous study, we could be able to detect the significant decrease in those enzyme levels. Our results dealing with MPO are in good agreement with those obtained by Namba (9) and Yamada et al. (7). MPO is present in the azurophile granules of neutrophils and is discharged into the phagoseme during phagocytosis, where it interacts with H202 generated by the respiratory burst and a halide. This microbicidal system has been shown to be effective in the killing of bacteria, yeast, mycoplasma, viruses, and tumor cells (24). The finding of diminished MPO levels in these PMNs could be due to the partial exhaustion of the PMN granule (azurophil and a-granules) contents due to a greater systemic state of PMN activation in these patients as a result of their underlying a u t o i m m u n e disease. Thus, it would be suggested that there might be an impair-
MEASUREMENT OF TOTAL PLASMA Cu LEVELS
Plasma Cu levels were measured in an atomic absorption spectrophotometer (Hitachi Z-8000) at 324.7 n m (21). Method was according to the manufacturer's instructions. MEASUREMENT OF FIBRINOGEN
The blood was collected in tubes containing sodium citrate 3.8% (4.5/0.5 v/v); and then the plasma was promptly separated by low speed centrifugation. Fibrinogen was measured after precipitation with calcium chloride in plasma samples (22). STATISTICAL ANALYSIS
All values are expressed as mean ± SD. The resuits were analysed using Student's t-test, and linear regression analysis. Differences ofp < 0.05 were considered significant. Results Mean MPO, SOD, catalase, and GSH-Px activities were significantly lower in Beh@et's patients than in controls (Table 1). However, NADPH oxidase activity was found to be significantly higher in patients compared with controls (p < 0.001). Plasma Cp, fibrinogen, and Cu levels are shown in Table 2. It was observed that all of t h e m were significantly higher in Beh~et's patients than in controls (p < 0.001). Regression analysis was carried out to test for a correlation between enzyme activities. The only statistically significant correlation found was a significant positive correlation between catalase and GSHPx in the patients group (r = 0.525, p < 0.001). In addition, there was a significant positive correlation between plasma Cp and Cu levels in both Beh~et's patients and controls (r = 0.803, p < 0.001, and r = 0.810, p < 0.001, respectively).
TABL~ 1 Comparison of Enzyme Activities in PMN Leucocytes Obtained From 36 Control and 40 Subjects With Beh~et's Disease Enzymes
Controls
Patients
p
MPO (U/mg protein) Catalase (U/rag protein) GSH-Px (U/mg protein)
24.59 _+ 1.41 67.42 + 3.38 29.94 -+ 1.38
20.30 -+ 1.07 54.52 -+ 2.84 24.31 - 0.98
<0.02 <0.005 <0.005
1.86 _+0.01
0.77 - 0.07
<0.001
0.77 -+ 0.05
1.05 --_0.05
<0.005
SOD (U/L) NADPH oxidase (U/g protein)
Values are mean + SD. Statistical analysis is compared to control. CLINICAL BIOCHEMISTRY, VOLUME 27, OCTOBER 1994
415
D O ' A N ET AL.
TABI~ 2 Plasma Ceruloplasmin, Copper, and Fibrinogen Levels in PatientsWith Behget's Disease and Controls
Copper (ttmol/L) Ceruloplasmin (pJmol/L) Fibrinogen (mg/dL)
n
Controls
n
Patients
p
36
17.47 _+ 0.54
40
21.71-+ 0 . 9 2
<0.001
36
362.21-+ 10.48
40
634.70-+ 20.60
<0.001
15
299.46+- 21.13
15
475.27-+ 96.81
<0.001
Values are mean -+ SD. n, Number of subjects.Statisticalanalysisis compared to control. ment in MPO-depondent microbicidal activity in P M N s with Beh~et's disease. Niwa et al. (11) and Mizushima (25) reported that phagocytosis is enhanced in Beh~et's patients. Furthermore, it has been demonstrated that when the normal T-lymphocyte population were stimulated with T-cell mitogens or with streptococcal preparations, the supernatants from these cultures potentiated neutrophil chemotaxis, phagocytosis, and 0 2 generation not only in Beh~et's patients but also in healthy controls (4). N A D P H oxidase is an enzyme known to be activated during phagocytosis and is responsible for the respiratory burst (24). In this study, a significant elevation of NADPH-oxidase activity in P M N s of Beh~et's patients was detected compared to controls. The present report shows the first correlation between P M N NADPH-oxidase activity and Beh~et's disease. The increased NADPH-oxidase activity in patient's group could be partly responsible for the elevation in the 0 2 production. It has been suggested that NADPH-oxidase activityand duration of 0 2 generation during the respiratory burst are regulated by M P O . Indeed, termination of the respiratory burst depends on M P O activity (26).Therefore, less M P O activity m a y extend the duration of NADPH-oxidase resulting in the formation of large amounts of 0 2 that m a y cause tissue damage in Beh~et's disease. Catalase, SOD, and glutathione reductase are the primary intracellular, antioxidant defense mechanisms to cope with increased oxidant stress. They eliminate 0 2 and hydroperexides that may oxidize cellular substrates, and they prevent free radical chain reactions. These antioxidants have specific requirements for different metals at their active catalytic sites (27). Catalase is a tetrameric hemoprotein that undergoes alternate divalent oxidation and reduction at its active site in the presence of H202 and catalyzes the dismutation of H20s to water and molecular oxygen. This important cytoplasmic hemoprotein can be inhibited by 0 2 . 0 2 converts catalase to the ferroxy and ferryl states that are inactive forms of the enzyme (28). GSH-Px is the key enzyme in the glutathione redox cycle responsible for the reduction of hydroperoxides and contains four atoms of selenium (Se) bound as selenocystein moieties that confer the cat416
alytic activity.GSH-Px has an absolute requirement for reduced glutathione as a cosubstrate that undergoes inactivation by hydroxyl radicals (OH') and 0 2 (29). Catalase and GSH-Px both have capacities for the elimination of intracellular H202. It has been stated that GSH-Px is the preferential pathway for the elimination of low concentrations of H202 (30). O n the other hand, catalase has a greater activity toward H202 at higher H2Os concentrations (31). In our study, we observed that both catalase and glutathione peroxidase activitieswere significantly lower in Beh~et's patients. Because both of these enzymes can be inhibited by 02, then itcan be thought that increased O 2 during enhanced respiratory burst m a y be the responsible factor for decreased activities of catalase and GSH-Px. The integrity of GSH-Px requires adequate intake of Se. Se has been shown to have antinflammatory, antiploriporative, antiviral, and immune altering effects (32).In a previous study, plasma Se levels in patients with Beh~et's disease have been reported to be significantlylower and suggest that Se m a y effect the prognosis (33). Decreased levels determined in our patients group m a y be attributed to redistribution from the plasma pool into the tissues as a defense mechanism against oxidative processes so that it modulates the effectof infections.The decrease in the activity of GSH-Px m a y also be related to decreased Se levels in those patients (33). S O D has been identified as an enzyme system, dealing with O 2 as a substrate, and provided the second layer of defense after GSH-Px and catalase against free radical injury (34). S O D catalyze the dismutation of 0 2 to H202. Previously, Niwa and collaborators (35) reported increased neutrophil active oxygen generation in Beh~et's patients at the active stage leading to tissue injury (11).In another study, they applied liposomal SOD, a clinical scavenger of 0 2 radicals for patients showing an increased neutrophil active oxygen generation and reported that liposomal SOD in effective and is highly applicable to this disease. In our study, SOD activity in PMNs in patients with Beh~et's disease was found to be statistically lower than in controls. This finding is in agreement with our previous preliminary study (23). It has been demonstrated by Rister and Baehner (36) that increased H202 concentrations inactivate SOD thus impairing the defense mechanism against CLINICALBIOCHEMISTRY,VOLUME27, OCTOBER1994
OXIDATIVEENZYMESIN BEH~ETS DISEASE 0 2 . In fact H202, the dismutation product of superoxide, can inhibit CuZn-SOD by reducing enzymebound Cu 2+ to Cu 1+ and then reacting with Cu 1+ to give a potent oxidant, probably OH'. Then OH" attacks an adjacent active site necessary for catalytic activity (37). Decreases in the SOD level could be dependent on three factors: a suppression in SOD synthesis due to a genetic defect; leakage of SOD out of the cell due to increase in the production of oxygen radicals causing cell membrane damage; or inactivation of SOD by increased H20 2 production in the cell. According to our observations less catalase and GSH-Px may increase H20 2 to concentrations that inactivate SOD, because H20 2 can also be formed spontaneously in a considerable degree if 0 2 production increases (38). In our study, w e observed that there was an approximate increase of 30% in mean NADPH oxidize levels and a greater than 60% decrease in PMN SOD activity in patients with Beh~et's syndrome. These results were more important than the other enzyme differences in MPO, catalase, and GSH-Px activity that although statistically significant were relatively minor compared to control patients. The results of our present study thus seem to suggest that the fn,st cause of 0 2 generation is the enhanced activity of NADPH oxidase, a membrane enzyme of neutrophils. Second, the decreased activity of MPO, catalase, and GSH-Px that are responsible from the degradation of H20 2. Third, the decrease in the activity of SOD is inhibited primarily by the increased levels of H20 2. It has been suggested that Cp may be the major antioxidant in plasma as a scavenger of 0 2 radicals (39). Cp is also as predominant an acute phase protein as fibrinogen. In our study we observed that Cp, Cu, and fibrinogen levels were significantly higher in patient's sera than in controls. Our fndings about Cp and Cu are in good agreement with our previous preliminary study (23). Increases in Cp, Cu, and fibrinogen may be attributable to inflammation associated with the disease. Fibrinogen has a distinct place between these parameters. Our findings about fibrinogen is in agreement with the previous reports (40,41). Fibrinogen is the major determinant of plasma viscosity and induces reversible red cell aggregation. Both phenomena limit the fluidity of blood and affect it by reducing flow, and by predisposing to thrombosis. Obstruction of both the superior and inferior vena cava seen in Beh~et's disease patients supports our findings (42). Therefore, the examination of fibrinogen levels may be helpful in treatment and following the prognosis of the disease. In conclusion, all of these phenomena might provide some insight into the pathophysiological mechanisms involved in Beh~et's Disease. Acknowledgement The authors are grateful to the Erciyes University Research Foundation for supporting this study. CLINICALBIOCHEMISTRY,VOLUME27, OCTOBER1994
References
1. O'Duffy JD. Beh~et's syndrome. N Engl J Med 1990; 322: 326-7. 2. Seylu M, Ereoz TR, Erken E, The association between HLA B5 and ocular involvement in Beh~t's disease in southern Turkey. Acta Ophthalmol (Copenh) 1992; 70: 786-9. 3. Mizoguchi M, Matsuki K, Mochizu]fi M, et al. Human leukocyte antigen in Sweet's syndrome and its relationship to Beh~et's disease. Arch Dermatol 1988; 124: 1069-73. 4. Niwa Y, Mizuahima Y. Neutrophil-potentiating factors released from stimulated lymphocytes; special reference to the increase in neutrophil-petsntiating factors from streptococcus-stimulated lymphocytes of patients with Beh~et's disease. Clin Exp Immunol 1990; 79: 353-90. 5. Sunakawa M, Ohshio G. Serum secretory IgA levels in patients with Behget disease. MeCab Pediatr Syst Ophthalmol 1989; 12: 110-2. 6. James DW, Walker JR, Smith JD. AbhorreR!polymorphonuclear leucocyte chemotaxis in Beh~et's syndrome. Ann Rheum Dis 1979; 38: 219-21. 7. Yamada M, Mimura Y, Wa~nAbe K, Yuasa T, Mural Y. Neutrophil lysosome enzyme activities in patients witth Beh~et's disease. In: Inaba G, Ed. Behcet's dis. ease. Jap Med Res Found Pub No 18, pp. 279-89. Japan, 1981, 8. Yamada M, Namba K. Lysesome] enzymes from patients with Beh~t's disease. In: Inaba G, Ed. Beh~et's disease. Jap Med Res Found Pub No 18, pp. 259-67. Japan, 1981. 9. Namba K. Leucocytes and lysosomal enzymes in the patients with Behf~t's disease. Jpn J Ophthalmol 1981; 25: 83-9. 10. Namba K. Types of ocular attacks and lysosomal enzymes in Beh~et's disease. Jpn J Ophthalmo11984; 28: 80-8. 11. Niwa Y, Miyake S, Sakane T, Sbingu M, YokoysmA M. Auto-oxidative damage in Beh~et's diseaseendothelial cell damage following the elevated oxygen radicals generated by stimulated neutrophils. Clin Exp Immunol 1982; 49: 247-55. 12. Lowry OH, Rosobrongh NJ, Farr AI, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 265-75. 13. Klebanoff SJ. Inactivation of estrogen by rat uterine preparations. Endocrinology 1965; 76: 301-11. 14. Misra HP, Fridowich I. Superexide dismutaso. A photochemical augmentation assay. Arch Biochem Biophys 1977; 181: 308-12. 15. Do,an P, Seyuer U, Tanrikulu G. Superoxide dismutaso and myloperoxidase activity in polymorphonuclear leucecytes and serum cerulop|RAminand copper levels, in psoriasis. Br J Dermatol 1989; 120: 239-44. 16. Torii KK, Hayashi S, Nakamoto H, Nakamura S. Properties of Aspergillus niger catalase. J Biochem 1982; 92: 1449-56. 17. Boetling RS, Weawer TL. A new assay for diaphorase activity in reagent formulations, based on the reduction of thiazolyl blue. Clin Chem 1979; 25: 2040-2. 18. Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte 417
DOGAN~T ~.L.
19.
20. 21. 22. 23.
24. 25. 26.
27. 28. 29. 30.
418
glutathione peroxidase. J Lab Clin Med 1967; 70: 158-69. Thomson CD, Rea HM, Dosburg VM, Robinson MF. Selenium concentrations and glutathione peroxidase activities in whole blood of New Zealand residents. Br J Nutr 1977; 37: 457-60. Sunderman FW, Nomoto S. Measurement of human serum ceruloplasmin by its p-phenylene-dismiue oxidase activity. Clin Chem 1970; 16: 903-10. Tietz NW. Textbook of Clinical Chemistry. Pp. 595-6, 787, 981-5, 1499. Philadelphia: WB Saunders, 1986. Lockland H. Quantitative fibrinogen. Lab Digest 1965; 28: 3-7. Do,an P, Soyuer U, Tanrikulu G, Utas S. Changes in serum and polymorphonuclear antioxidant defense systems in Behget's Disease. Invest Dermatol 1989; 93: 297. Badwey JA, Karnovsky ML. Active oxygen species and the functions of phagocytic leucocytes. Annu R e v Biochem 1980; 49: 695-726. MizuRhimAY. Recent research into Beh~et's disease in Japan. Int J Tissue React 1988; 10: 59-65. Edwards SW, Swan TF. Regulation of superexide generation by myeloperoxidase during the respiratory burst of human neutrophils. Biochem J 1986; 237: 601-4. Sies H. Antioxidant activity in cells and organs. Am Rev Respir Dis 1987; 136: 478-80. Kono Y, Fridovich I. Superoxide radical inhibits catalase. J Biol Chem 1982; 257: 5751-5. Blum J, Fridovich I. Inactivation of glutathione peroxidase by superoxide radical. Arch Biochem Biophys 1985; 280: 500-8. Jacob HS, Jandl JH. Effects of sulphhydryl inhibition on red blood cells. II. Glutathione in the regulation of the hexose monophosphate pathway. J Biol Chem 1966; 241: 4243-50.
31. Fridovich I, Freeman B. Antioxidant defences in the lung. Annu Rev Physiol 1986; 48: 693-702. 32. Lockitch G. Selenium: Clinical significance and analytical concepts. Crit Rev Clin Lab Sci 1989; 27: 483541. 33. Do,an P, Do,an M, Klockenk~mper R. Determination of trace elements in blood serum of patients with Beh~et disease by total reflection X-ray fluorescence analysis. Clin Chem 1993; 39: 1037-41. 34. Fridovich I. Superexide radical: An endogeneous toxicant. Annu Rev Pharmacol Toxicol 1983: 23: 239-57. 35. Niwa Y, Somiya K, Michelsen AM, Puget K. Effect of liposomal-encapsulated superoxide dismutese on active oxygen related human disorders. A preliminary study. Free Rad Res Commun 1985; 1: 137-53. 36. Rister M, Baehner R. The alteration of superoxide dismutase, catalase, glutathione peroxidase and NADPH (H) cytochrome C reductase in guinea pig polymorphonuclear leucocytes and alveolar macrophagse during hyperoxia. J Clin Invest 1976; 58: 1174-84. 37. Freeman BA, Crapo JD. Biology of disease, free radicals and tissue injury. Lab Invest 1982; 47: 412-26. 38. Fridovich I. Superoxide dismutases. Annu Rev Biochem 1975; 44: I47-51. 39. Halliwel B, Gutteridge JMC. The antioxidants of human extracellular fluids. Arch Biochem Biophys 1990; 280: 1-8. 40. Kluft C, Michiels JJ, Wijngoards G. Factural or artificial inhibition of fibrinolysis and the occurrence of venous thrombosis in 3 cases of Beh~et's disease. Scand J Haematol 1980; 25: 423-30. 41. Hampton KK, Chamberlein MA, Menon DK, Davies JA. Coagulation and fibrinolytic activity in Beh~et's disease. Thromb Haemost 1991; 66: 292-4. 42. Ernst E. The rele of fibrinogen as a cardiovascular risk factor. Atherosclerosis 1993; 100: 1"12.
CLINICALBIOCHEMISTRY,VOLUME 27, OCTOBER1994
Pergamon 0009-9120(94)~m~1-4
Oxidative Enzymes of Polymorphonuclear Leucocytes and Plasma Fibrinogen, Ceruloplasmin, and Copper Levels in Behc;et's Disease PAKIZE DO,AN, 1 GURSEL TANRIKULU, 1 0MIT SOYUER,2 and KADER KOSE1 Departments of 1Biochemistry and 2 Dermatology, Faculty of Medicine, Erciyes University, 38039 Kayseri, Turkey This study was performed to investigate the antioxident mechanisms of polymorphonuclear leucocytes (PMN) in active stage of Behget's Disease. PMN activities of myeloperoxidase (p < 0.02), superoxide dlsmutase (p < 0.001), catalase (p < 0.005), and glutathlone peroxidsee (p < 0.005) were significantly lower in the patients: the NADPH oxidase activity was significantly hlgher (p < 0.001) than those in controls. The plasma levels of ceruloplasmln (Cp), flbrinogen, and copper (Cu) were also signiflcantly higher in the patients group (p < 0.001). Slgnlflcant and poslUve correlations were found between the glutathione peroxldsee and catslase activities (p < 0.001) and also between the plasma Cp and Cu levels (p < 0.001) in the patients group. However, no correlation was observed among the other enzyme activities. In the control group, a significantly positive correlatlon was present only between the plasma ceruloplasmin and Cu levels (p < 0.001). It was concluded that (impaired PMN functions) decreased enzyme activities in the antioxidant system and Increased levels of oxygen free radicals may play a role in tissue damage in Sehget's disease.
Introduction
reported that functional abnormalities were present in the neutrophils of patients with Behget's Disease, such as chemot~Tis and phagocytosis (6). Furthermore, although they are not specific to neutrophils, some lysosemal enzymes, ~-glucuronidase and acid phosphatase, were reported to be higher during the attack than in the remission phase or control (7). A decrease in the activity of myeloperoxidase (MPO), an enzyme specific to the neutrophils, was shown by various authors (8-10). It has been suggested that stimulated neutrophils from patients with Behget's disease generate high levels of oxygen intermediates (OI), resulting in endothelial tissue damage .(11). Because of the relationship between OI and oxidative enzymes, and t h e possible correlation between Beh~et's disease and functions of polymorphonuclear (PMN) leucocytes, we attempted to clarify the significance and correlation of superoxide dismutase (SOD), and MPO, glutathione peroxidase (GSH-Px), catalase, NADPH-oxidase in PMN leucocytes, and serum ceruloplasmin (Cp), fibrinogen, and copper (Cu) in Behget's disease.
B
Patients, materials, and methods
KEY WORDS: superoxide dismutase activity; myeloperoxidase activity; catalase activity; NADPH oxidase activity; glutathione peroxidase activity; ceru]opln~min activity; Cu; fibrinogen; polymorphonuclear leucocytes; Behqet's disease.
ehget syndrome is a multisystem disorder characterized by recurrent oral and genital ulcers, arthritis, thrombophlebitis, uveitis. Its basic underlying pathological process is that of a vasculitis (1). Genetic and environmental factors probably play a role in pathogenesis of this disease. Some studies show an increased prevalence of HI~-B5 (2), HLA135-1, and HLA-DQw3 (3) in Behget's disease. Recent studies have demonstrated an increase in circulation activated T lymphocytes (4). In addition high levels of circulating immune complexes have been detected in patients with this disease (5). It has been Correspondence: Prof. Dr. PRkiTe Do,an. Manuscript received April 15, 1994; revised June 24, 1994; accepted June 27, 1994. CLINICAL BIOCHEMISTRY, VOLUME 27, OCTOBER 1994
Forty patients (14 males and 26 femRles) with the complete type of Behget's disease were studied. Their ages ranged from 14 to 65 years (mean +- SD, 33.5 -+ 1.7). Thirty-six healthy subjects (16 males and 20 females, 29.3 -+ 1.3 years old) that were 1947 years-old were evaluated as the control group. ISOLATION OF P M N
Blood was taken from the antecubital vein with a heparinized disposable syringe, and mi~ed with 6% (w/v) d e x ~ m in saline in a ratio of 4:1 and allowed to stand for 60 rain at room temperature. The supernatant was removed and centrifuged at 275 x g for 10 rain. The resulting cell pellet was resuspended in 413
D O O A N ET AL.
8 mL of Krebs Ringer Phosphate Buffer (KRPB), layered over 3 mL of Histopaque-1077 (Sigma Chemical Co., St. Louis, MO) spun at 450 × g for 30 rain at 37 °(3. The cell pellet contained the PMN. Residual erythrocytes were lysed by adding 9 volumes of distilled water to this pellet, followed by vigorous sbMdng for 30 s; osmolality was restored by adding i volume of 9% (w/v) NaC1. The cell suspension was centrifuged at 275 × g for 5 rain, and the s u p e r n a t a n t was discarded. The pellet was washed three times with K R P B and resusponded in 2 m L of KRPB. Cell numbers in the finalsuspension were counted and the suspension stored at -20 °C. The purity of P M N population was 95%.
added to the reaction mixture to inhibitM P O activity (15). MEASUREMENT OF CATALASEACTIVITY
Activity was determined at 25 °C according to the modified technique of Beers and Sizer (16) by following the absorbance of hydrogen peroxide at 240 nm. One unit of catalase activity was defined as the amount of enzyme that decomposes 1 ~mol H202/ rain at an initial H202 concentration of 30 mM/L at pH 7.0 and 25 °C. A standard curve was constructed by using purified bovine liver catalase (Sigma). Catalase activity of the sample was expressed as U/mg protein.
PREPARATION OF PMN HOMOGENATES MEASUREMENT OF N A D P H OXIDASE ACTIVITY
The PMN suspension was frozen at - 2 0 °C and thawed six times, then homogenized using a motor driven Teflon-glass homogenizer (900 rpm for 5 rain at 0 °C). The protein content of the homogenate was measured using Lowry et al.'s method (12). MZASUR~.MENT OF MPO ACTIVITY
MPO activity was determined by a modification of the o-disnlsidine method (13). The assay mixture, in a cuvette of 1 cm path length, contained: 0.3 mL 100 mM/L phosphate buffer (pH 6.0; 0.3 mL 10 mM/L HsO2, 0.5 mL 20 mM/L o-dianisidine (freshly prepared without contaminating surroundings, with extreme caution, using disposable gloves because it is carcinogenic) in deionized water; and 0.010 mL PMN homogenate in a final volume of 3.0 mL. The PMN homogenate was added last and the change in absorbance at 460 n m was followed for 10 rain. All measurements were carried out in duplicate. One unit of MPO was defined as that degrading 1 tLmol HsO2/min at 25 °C; specific activity was given as U/rag of protein. A molar extinction coefficient (e) of 11.300 for oxidized o-dianisidine was used for the calculation. MgAS~MENTS OF SOD ACTIVITY
S O D activitywas detected by its abilityto inhibit the autoxidation of adrenalin at p H 10.2, using a modification of the method described by Misra and Fridowich (14). The assay mixture contained: 0.5 ml, 1.8 m M / L adrenalin (freshly prepared, pH 2.0); 0.4 mL 0.75 mM/L EDTA; 0.5 mL 300 mM/L sodium carbonate (pH 10.2); and 0.1 mL 60 mM/L sodium azide and PMN homogenate (0.75 protein/lnL) in a final volume of 3.0 mL. The reaction was started by the addition of the adrenalin and the increase in absorbance at 480 n m was followed in a P e r k i n Elmer spectrophotometer. A standard containing 0.75 ~g SOD inhibited the conversion of adrenalin to adrenochrome by 73%. Control experiments were run with boiled enzyme and with bovine serum albumin. Sodium aside was 414
NADPH oxidase catalyzes the reduction of 3-(4,5 dimethyl-2-thiazolyl-2,5-diphenyl-2H-diphenyl-2H tetrazolium bromide (MTT) (Sigma), which acts as hydrogen accepter. NADPH oxidase activity was determined by measuring the increase in absorbance at the absorption maximum of MTT formazan, at 560 nm, resulting from the reduction of MTT by NADPH (17). The assay mixture contained the following: 2.3 mL of 130 mM/L potassium phosphate buffer, pH 7.6; 0.5 mL 1 mg/mL MTT; 0.1 mL 10 mg/mL NADPH. The assay temperature was 30 °C, and reaction was initiated by adding 0.1 mL leucocyte homogenate. We measured absorbance change per minute (A/min) from the linear portion of the recorder tracing with a spectrophotemeter. One unit of activity is the activity necessary to reduce 1 ttmol MTT to MTT-formazan per minute under specified conditions. Purified diaphorase from Clostridium kluyveri (Sigma) was used to obtain the standard curve. Specific activity was given as U/g protein. MEASUREMENT OF G S H - P x ACTIVITY
Glutathione peroxidase activity was determined by the method of Paglia and Valentine (18). The reaction mixture contained 2.48 m L of 50 m M / L phosphate buffer,p H 7, containing: 5 m M / L EDTA; 0.1 m L 8.4 m M / L N A D P H ; 0.1 m L 150 m M / L reduced glutathione (Sigma); 0.01 m L 112.5 m M / L sodium azide; and 4.6 U glutathione reductase (Type HI, Sigma). The reaction was initiated by the addition of 0.1 mL 2.2 mM H2O 2 to the reaction mixture containing 500-1000 ~g protein. The change in the optical density was read at 340 n m for 4 rain. The data were expressed as nmol NADPH oxidized to NADP/mg protein/min by using the molar extinction coefficient (e) of 6.200 at 340 nm. All data were corrected for the linear NADPH oxidation by HsO 2 alone in the absence of enzyme protein that was always less than 5% of the total reaction (19). CLINICAL BIOCHEMISTRY, VOLUME 27, OCTOBER 1994
OXIDATIVE ENZYMES IN BEHCET~ DISEASE MEASURBMENT OF PLASMA Cp ACTIVITY
Discussion
Plasma Cp levels were determined by measurement of p-phenylene diamine oxidase activity according to the method of Sunderman and Nomoto (2O).
Previous studies have demonstrated important disorders of neutrophil functions in Beh~et's disease such as an increase in the chemot~TiR and in the activity of NADPH oxid"~e, increased lysosomal enzyme activities, shortened neutrophil life, and increased levels of oxygen r a s c a l s (6-8,10,11). Yamada et al. (7) studied neutrophils from patients with Beh~et's disease and found out that the levels of lysosomal enzymes, ~-glucuronidase, and collage nase were increased in the active phase of the disease, whereas MPO activity was found to be lower during the active stage when compared to the rest phase. However, they reported that the difference between MPO levels was statistically insignificant. On the other hand, Namba (9) reported that acid phosphatase and ~-glucuronida~e levels were no different from those in controls but MPO activity was significantly lower in Beh;et's patients compared with that in the controls. In a previous preliminary study with a few patients with Beh~et's disease and controls, we examined the alterations in the activities of SOD, MPO, and catalase in P M N s a n d serum Cp and Cu content. Although, there were numerical decreases in MPO and catalase activities in the patient group compared to controls, no statistically significant difference was observed (23). However in this study, because the n u m b e r of patients and controls were greater then our previous study, we could be able to detect the significant decrease in those enzyme levels. Our results dealing with MPO are in good agreement with those obtained by Namba (9) and Yamada et al. (7). MPO is present in the azurophile granules of neutrophils and is discharged into the phagoseme during phagocytosis, where it interacts with H202 generated by the respiratory burst and a halide. This microbicidal system has been shown to be effective in the killing of bacteria, yeast, mycoplasma, viruses, and tumor cells (24). The finding of diminished MPO levels in these PMNs could be due to the partial exhaustion of the PMN granule (azurophil and a-granules) contents due to a greater systemic state of PMN activation in these patients as a result of their underlying a u t o i m m u n e disease. Thus, it would be suggested that there might be an impair-
MEASUREMENT OF TOTAL PLASMA Cu LEVELS
Plasma Cu levels were measured in an atomic absorption spectrophotometer (Hitachi Z-8000) at 324.7 n m (21). Method was according to the manufacturer's instructions. MEASUREMENT OF FIBRINOGEN
The blood was collected in tubes containing sodium citrate 3.8% (4.5/0.5 v/v); and then the plasma was promptly separated by low speed centrifugation. Fibrinogen was measured after precipitation with calcium chloride in plasma samples (22). STATISTICAL ANALYSIS
All values are expressed as mean ± SD. The resuits were analysed using Student's t-test, and linear regression analysis. Differences ofp < 0.05 were considered significant. Results Mean MPO, SOD, catalase, and GSH-Px activities were significantly lower in Beh@et's patients than in controls (Table 1). However, NADPH oxidase activity was found to be significantly higher in patients compared with controls (p < 0.001). Plasma Cp, fibrinogen, and Cu levels are shown in Table 2. It was observed that all of t h e m were significantly higher in Beh~et's patients than in controls (p < 0.001). Regression analysis was carried out to test for a correlation between enzyme activities. The only statistically significant correlation found was a significant positive correlation between catalase and GSHPx in the patients group (r = 0.525, p < 0.001). In addition, there was a significant positive correlation between plasma Cp and Cu levels in both Beh~et's patients and controls (r = 0.803, p < 0.001, and r = 0.810, p < 0.001, respectively).
TABL~ 1 Comparison of Enzyme Activities in PMN Leucocytes Obtained From 36 Control and 40 Subjects With Beh~et's Disease Enzymes
Controls
Patients
p
MPO (U/mg protein) Catalase (U/rag protein) GSH-Px (U/mg protein)
24.59 _+ 1.41 67.42 + 3.38 29.94 -+ 1.38
20.30 -+ 1.07 54.52 -+ 2.84 24.31 - 0.98
<0.02 <0.005 <0.005
1.86 _+0.01
0.77 - 0.07
<0.001
0.77 -+ 0.05
1.05 --_0.05
<0.005
SOD (U/L) NADPH oxidase (U/g protein)
Values are mean + SD. Statistical analysis is compared to control. CLINICAL BIOCHEMISTRY, VOLUME 27, OCTOBER 1994
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D O ' A N ET AL.
TABI~ 2 Plasma Ceruloplasmin, Copper, and Fibrinogen Levels in PatientsWith Behget's Disease and Controls
Copper (ttmol/L) Ceruloplasmin (pJmol/L) Fibrinogen (mg/dL)
n
Controls
n
Patients
p
36
17.47 _+ 0.54
40
21.71-+ 0 . 9 2
<0.001
36
362.21-+ 10.48
40
634.70-+ 20.60
<0.001
15
299.46+- 21.13
15
475.27-+ 96.81
<0.001
Values are mean -+ SD. n, Number of subjects.Statisticalanalysisis compared to control. ment in MPO-depondent microbicidal activity in P M N s with Beh~et's disease. Niwa et al. (11) and Mizushima (25) reported that phagocytosis is enhanced in Beh~et's patients. Furthermore, it has been demonstrated that when the normal T-lymphocyte population were stimulated with T-cell mitogens or with streptococcal preparations, the supernatants from these cultures potentiated neutrophil chemotaxis, phagocytosis, and 0 2 generation not only in Beh~et's patients but also in healthy controls (4). N A D P H oxidase is an enzyme known to be activated during phagocytosis and is responsible for the respiratory burst (24). In this study, a significant elevation of NADPH-oxidase activity in P M N s of Beh~et's patients was detected compared to controls. The present report shows the first correlation between P M N NADPH-oxidase activity and Beh~et's disease. The increased NADPH-oxidase activity in patient's group could be partly responsible for the elevation in the 0 2 production. It has been suggested that NADPH-oxidase activityand duration of 0 2 generation during the respiratory burst are regulated by M P O . Indeed, termination of the respiratory burst depends on M P O activity (26).Therefore, less M P O activity m a y extend the duration of NADPH-oxidase resulting in the formation of large amounts of 0 2 that m a y cause tissue damage in Beh~et's disease. Catalase, SOD, and glutathione reductase are the primary intracellular, antioxidant defense mechanisms to cope with increased oxidant stress. They eliminate 0 2 and hydroperexides that may oxidize cellular substrates, and they prevent free radical chain reactions. These antioxidants have specific requirements for different metals at their active catalytic sites (27). Catalase is a tetrameric hemoprotein that undergoes alternate divalent oxidation and reduction at its active site in the presence of H202 and catalyzes the dismutation of H20s to water and molecular oxygen. This important cytoplasmic hemoprotein can be inhibited by 0 2 . 0 2 converts catalase to the ferroxy and ferryl states that are inactive forms of the enzyme (28). GSH-Px is the key enzyme in the glutathione redox cycle responsible for the reduction of hydroperoxides and contains four atoms of selenium (Se) bound as selenocystein moieties that confer the cat416
alytic activity.GSH-Px has an absolute requirement for reduced glutathione as a cosubstrate that undergoes inactivation by hydroxyl radicals (OH') and 0 2 (29). Catalase and GSH-Px both have capacities for the elimination of intracellular H202. It has been stated that GSH-Px is the preferential pathway for the elimination of low concentrations of H202 (30). O n the other hand, catalase has a greater activity toward H202 at higher H2Os concentrations (31). In our study, we observed that both catalase and glutathione peroxidase activitieswere significantly lower in Beh~et's patients. Because both of these enzymes can be inhibited by 02, then itcan be thought that increased O 2 during enhanced respiratory burst m a y be the responsible factor for decreased activities of catalase and GSH-Px. The integrity of GSH-Px requires adequate intake of Se. Se has been shown to have antinflammatory, antiploriporative, antiviral, and immune altering effects (32).In a previous study, plasma Se levels in patients with Beh~et's disease have been reported to be significantlylower and suggest that Se m a y effect the prognosis (33). Decreased levels determined in our patients group m a y be attributed to redistribution from the plasma pool into the tissues as a defense mechanism against oxidative processes so that it modulates the effectof infections.The decrease in the activity of GSH-Px m a y also be related to decreased Se levels in those patients (33). S O D has been identified as an enzyme system, dealing with O 2 as a substrate, and provided the second layer of defense after GSH-Px and catalase against free radical injury (34). S O D catalyze the dismutation of 0 2 to H202. Previously, Niwa and collaborators (35) reported increased neutrophil active oxygen generation in Beh~et's patients at the active stage leading to tissue injury (11).In another study, they applied liposomal SOD, a clinical scavenger of 0 2 radicals for patients showing an increased neutrophil active oxygen generation and reported that liposomal SOD in effective and is highly applicable to this disease. In our study, SOD activity in PMNs in patients with Beh~et's disease was found to be statistically lower than in controls. This finding is in agreement with our previous preliminary study (23). It has been demonstrated by Rister and Baehner (36) that increased H202 concentrations inactivate SOD thus impairing the defense mechanism against CLINICALBIOCHEMISTRY,VOLUME27, OCTOBER1994
OXIDATIVEENZYMESIN BEH~ETS DISEASE 0 2 . In fact H202, the dismutation product of superoxide, can inhibit CuZn-SOD by reducing enzymebound Cu 2+ to Cu 1+ and then reacting with Cu 1+ to give a potent oxidant, probably OH'. Then OH" attacks an adjacent active site necessary for catalytic activity (37). Decreases in the SOD level could be dependent on three factors: a suppression in SOD synthesis due to a genetic defect; leakage of SOD out of the cell due to increase in the production of oxygen radicals causing cell membrane damage; or inactivation of SOD by increased H20 2 production in the cell. According to our observations less catalase and GSH-Px may increase H20 2 to concentrations that inactivate SOD, because H20 2 can also be formed spontaneously in a considerable degree if 0 2 production increases (38). In our study, w e observed that there was an approximate increase of 30% in mean NADPH oxidize levels and a greater than 60% decrease in PMN SOD activity in patients with Beh~et's syndrome. These results were more important than the other enzyme differences in MPO, catalase, and GSH-Px activity that although statistically significant were relatively minor compared to control patients. The results of our present study thus seem to suggest that the fn,st cause of 0 2 generation is the enhanced activity of NADPH oxidase, a membrane enzyme of neutrophils. Second, the decreased activity of MPO, catalase, and GSH-Px that are responsible from the degradation of H20 2. Third, the decrease in the activity of SOD is inhibited primarily by the increased levels of H20 2. It has been suggested that Cp may be the major antioxidant in plasma as a scavenger of 0 2 radicals (39). Cp is also as predominant an acute phase protein as fibrinogen. In our study we observed that Cp, Cu, and fibrinogen levels were significantly higher in patient's sera than in controls. Our fndings about Cp and Cu are in good agreement with our previous preliminary study (23). Increases in Cp, Cu, and fibrinogen may be attributable to inflammation associated with the disease. Fibrinogen has a distinct place between these parameters. Our findings about fibrinogen is in agreement with the previous reports (40,41). Fibrinogen is the major determinant of plasma viscosity and induces reversible red cell aggregation. Both phenomena limit the fluidity of blood and affect it by reducing flow, and by predisposing to thrombosis. Obstruction of both the superior and inferior vena cava seen in Beh~et's disease patients supports our findings (42). Therefore, the examination of fibrinogen levels may be helpful in treatment and following the prognosis of the disease. In conclusion, all of these phenomena might provide some insight into the pathophysiological mechanisms involved in Beh~et's Disease. Acknowledgement The authors are grateful to the Erciyes University Research Foundation for supporting this study. CLINICALBIOCHEMISTRY,VOLUME27, OCTOBER1994
References
1. O'Duffy JD. Beh~et's syndrome. N Engl J Med 1990; 322: 326-7. 2. Seylu M, Ereoz TR, Erken E, The association between HLA B5 and ocular involvement in Beh~t's disease in southern Turkey. Acta Ophthalmol (Copenh) 1992; 70: 786-9. 3. Mizoguchi M, Matsuki K, Mochizu]fi M, et al. Human leukocyte antigen in Sweet's syndrome and its relationship to Beh~et's disease. Arch Dermatol 1988; 124: 1069-73. 4. Niwa Y, Mizuahima Y. Neutrophil-potentiating factors released from stimulated lymphocytes; special reference to the increase in neutrophil-petsntiating factors from streptococcus-stimulated lymphocytes of patients with Beh~et's disease. Clin Exp Immunol 1990; 79: 353-90. 5. Sunakawa M, Ohshio G. Serum secretory IgA levels in patients with Behget disease. MeCab Pediatr Syst Ophthalmol 1989; 12: 110-2. 6. James DW, Walker JR, Smith JD. AbhorreR!polymorphonuclear leucocyte chemotaxis in Beh~et's syndrome. Ann Rheum Dis 1979; 38: 219-21. 7. Yamada M, Mimura Y, Wa~nAbe K, Yuasa T, Mural Y. Neutrophil lysosome enzyme activities in patients witth Beh~et's disease. In: Inaba G, Ed. Behcet's dis. ease. Jap Med Res Found Pub No 18, pp. 279-89. Japan, 1981, 8. Yamada M, Namba K. Lysesome] enzymes from patients with Beh~t's disease. In: Inaba G, Ed. Beh~et's disease. Jap Med Res Found Pub No 18, pp. 259-67. Japan, 1981. 9. Namba K. Leucocytes and lysosomal enzymes in the patients with Behf~t's disease. Jpn J Ophthalmol 1981; 25: 83-9. 10. Namba K. Types of ocular attacks and lysosomal enzymes in Beh~et's disease. Jpn J Ophthalmo11984; 28: 80-8. 11. Niwa Y, Miyake S, Sakane T, Sbingu M, YokoysmA M. Auto-oxidative damage in Beh~et's diseaseendothelial cell damage following the elevated oxygen radicals generated by stimulated neutrophils. Clin Exp Immunol 1982; 49: 247-55. 12. Lowry OH, Rosobrongh NJ, Farr AI, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 265-75. 13. Klebanoff SJ. Inactivation of estrogen by rat uterine preparations. Endocrinology 1965; 76: 301-11. 14. Misra HP, Fridowich I. Superexide dismutaso. A photochemical augmentation assay. Arch Biochem Biophys 1977; 181: 308-12. 15. Do,an P, Seyuer U, Tanrikulu G. Superoxide dismutaso and myloperoxidase activity in polymorphonuclear leucecytes and serum cerulop|RAminand copper levels, in psoriasis. Br J Dermatol 1989; 120: 239-44. 16. Torii KK, Hayashi S, Nakamoto H, Nakamura S. Properties of Aspergillus niger catalase. J Biochem 1982; 92: 1449-56. 17. Boetling RS, Weawer TL. A new assay for diaphorase activity in reagent formulations, based on the reduction of thiazolyl blue. Clin Chem 1979; 25: 2040-2. 18. Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte 417
DOGAN~T ~.L.
19.
20. 21. 22. 23.
24. 25. 26.
27. 28. 29. 30.
418
glutathione peroxidase. J Lab Clin Med 1967; 70: 158-69. Thomson CD, Rea HM, Dosburg VM, Robinson MF. Selenium concentrations and glutathione peroxidase activities in whole blood of New Zealand residents. Br J Nutr 1977; 37: 457-60. Sunderman FW, Nomoto S. Measurement of human serum ceruloplasmin by its p-phenylene-dismiue oxidase activity. Clin Chem 1970; 16: 903-10. Tietz NW. Textbook of Clinical Chemistry. Pp. 595-6, 787, 981-5, 1499. Philadelphia: WB Saunders, 1986. Lockland H. Quantitative fibrinogen. Lab Digest 1965; 28: 3-7. Do,an P, Soyuer U, Tanrikulu G, Utas S. Changes in serum and polymorphonuclear antioxidant defense systems in Behget's Disease. Invest Dermatol 1989; 93: 297. Badwey JA, Karnovsky ML. Active oxygen species and the functions of phagocytic leucocytes. Annu R e v Biochem 1980; 49: 695-726. MizuRhimAY. Recent research into Beh~et's disease in Japan. Int J Tissue React 1988; 10: 59-65. Edwards SW, Swan TF. Regulation of superexide generation by myeloperoxidase during the respiratory burst of human neutrophils. Biochem J 1986; 237: 601-4. Sies H. Antioxidant activity in cells and organs. Am Rev Respir Dis 1987; 136: 478-80. Kono Y, Fridovich I. Superoxide radical inhibits catalase. J Biol Chem 1982; 257: 5751-5. Blum J, Fridovich I. Inactivation of glutathione peroxidase by superoxide radical. Arch Biochem Biophys 1985; 280: 500-8. Jacob HS, Jandl JH. Effects of sulphhydryl inhibition on red blood cells. II. Glutathione in the regulation of the hexose monophosphate pathway. J Biol Chem 1966; 241: 4243-50.
31. Fridovich I, Freeman B. Antioxidant defences in the lung. Annu Rev Physiol 1986; 48: 693-702. 32. Lockitch G. Selenium: Clinical significance and analytical concepts. Crit Rev Clin Lab Sci 1989; 27: 483541. 33. Do,an P, Do,an M, Klockenk~mper R. Determination of trace elements in blood serum of patients with Beh~et disease by total reflection X-ray fluorescence analysis. Clin Chem 1993; 39: 1037-41. 34. Fridovich I. Superexide radical: An endogeneous toxicant. Annu Rev Pharmacol Toxicol 1983: 23: 239-57. 35. Niwa Y, Somiya K, Michelsen AM, Puget K. Effect of liposomal-encapsulated superoxide dismutese on active oxygen related human disorders. A preliminary study. Free Rad Res Commun 1985; 1: 137-53. 36. Rister M, Baehner R. The alteration of superoxide dismutase, catalase, glutathione peroxidase and NADPH (H) cytochrome C reductase in guinea pig polymorphonuclear leucocytes and alveolar macrophagse during hyperoxia. J Clin Invest 1976; 58: 1174-84. 37. Freeman BA, Crapo JD. Biology of disease, free radicals and tissue injury. Lab Invest 1982; 47: 412-26. 38. Fridovich I. Superoxide dismutases. Annu Rev Biochem 1975; 44: I47-51. 39. Halliwel B, Gutteridge JMC. The antioxidants of human extracellular fluids. Arch Biochem Biophys 1990; 280: 1-8. 40. Kluft C, Michiels JJ, Wijngoards G. Factural or artificial inhibition of fibrinolysis and the occurrence of venous thrombosis in 3 cases of Beh~et's disease. Scand J Haematol 1980; 25: 423-30. 41. Hampton KK, Chamberlein MA, Menon DK, Davies JA. Coagulation and fibrinolytic activity in Beh~et's disease. Thromb Haemost 1991; 66: 292-4. 42. Ernst E. The rele of fibrinogen as a cardiovascular risk factor. Atherosclerosis 1993; 100: 1"12.
CLINICALBIOCHEMISTRY,VOLUME 27, OCTOBER1994