Life Sciences, Vol. 47, pp. PL-145-PL-150 Printed in the U.S.A.
Pergamon Press
PHARMACOLOGY LETTERS Accelerated Communication
THE DETERMINATION OF MYELOPEROXIDASE ACTIVITY IN LIVER D.L. Duval, D. Howard, T.A. McCalden and R.E. Billings, Departments o f Surgery and Pharmacology, University of Nevada School of Medicine, Reno, NV 89557-0046. (Submitted August 20, 1990; accepted August 30, 1990; received in final form October I0, 1990) Abstract. Myeloperoxidase (MPO) is an enzyme found in granulocytes of neutrophils, but not in mammalian tissues. Previous studies have directly correlated MPO activity with neutrophil accumulation in tissues. This study presents a method for determining MPO activity in liver. Neutrophil accumulation in rat liver was provoked by creating partial ischemia followed by reperfusion. Liver homogenates prepared b y a standard procedure showed no MPO activity. The homogenate was applied to Sephadex GI00 and DEAE Sepharose CL6B columns which separated ~ P O activity from inhibitory activity. The inhibitor was identified as catalase based upon its elution from the columns and removal with 3-amino- 1,2,4-triazole (AT), a catalase inhibitor. Based upon these findings, it was determined that full MPO a c t i v i t y c a n be assayed in unfractionated liver homogenates by first inactivatinq catalase with AT. Introduction Myeloperoxidase (MPO) (donor: H202 oxidoreductase, ECI.II.I.7) is a l e u k o c y t e - s p e c i f i c enzyme found primarily in the granules of neutrophils and to a lesser extent in macrophages. MPO in neutrophils is released from granules in response to NADPH-oxidase activation [1-3]. MPO catalyses the reaction of H202 with halide ions to form h y p o h a l o u s acids. These acids serve as the main antimicrobial compounds released by the neutrophils as well as causing considerable damage to surrounding tissues when released extracellularly [1-5]. Neutrophils are drawn to areas of injury by a wide variety of chemotactic factors [2]. Their presence has been used to quantify inflammation, myocardial infarct and other types of tissue injury. Although, histological examination of the tissues has been used to determine neutrophil accumulation, the timeconsuming and semi-quantitative nature of this method led to the measurement of MPO activity as a marker of neutrophil accumulation [5-7]. Unfortunately, the recovery of MPO activity is often negligible, possibly b e c a u s e of incomplete degranulation of the enzyme and the presence of a number of endogenous inhibitors [5]. In vitro studies have shown that MPO activity can be blocked by a variety of inhibitors including azide, cyanide, and catalase [3]. This study examines the inhibitors of MPO in liver samples and introduces a method to increase MPO activity recovery. Corresponding Author: Dr. Ruth Billings, Depts. of Pharmacology and Surgery, University of Nevada School of Medicine, Reno, NV 895570046. 0024-3205/90 $3.00 + .00 Copyright (c) 1990 Pergamon Press plc
PL-146
M y e l o p e r o x i d a sActivity e
Vol. 47, No. 24, 1990
Materials and Methods Rat Liver Ischemia-Reperfusion Model. Male Sprague-Dawley rats (275 - 3 0 0 g m ) were anesthetized by injection of Nembutal (pentobarbital) at 50 mg/ kg (i.p.) for the surgery. A previously described partial ischemia model was used [8]. Briefly, ischemia was provoked in the left lateral and median lobes of the liver by the application of a microaneurysm clip (Bruno Vet Supply, San Leandro, CA ) to the portal vessels supplying those lobes. After one hour of ischemia, the clips were removed to restore blood flow to the ischemic lobes. The animals were maintained in a stable temperature environment. Reperfusion was allowed for varying durations. Under Nembutal anesthesia non-ischemic and ischemic liver lobe samples were collected from the rats. The tissue samples were immediately frozen in liquid nitrogen and stored at -70 ° C until assayed. Myeloperoxidase SpectroDhotometric Assay. The samples were prepared by an established method using cetyltrimethylammonium bromide as a detergent [6,7]. The assay contained 50 ~ potassium phosphate buffer, pH 6.0, 0.167 mg/ml a-dianisidine dihydrochloride, and 0.0005% H202. The change in absorbance at 460 nm was measured for 5 minutes. Protein was determined by the method of Lowry et al [9]. Chromatography of Myeloperoxidase. Liver homogenate from a 24 hour reperfused tissue was applied to a 26 mm x 30 cm column of Superfine (SF) Sephadex G-100 and eluted with 50 mM potassium phosphate buffer, pH 6.0 at 4 ° C. The fractions (2.25 ml) were assayed for MPO activity. Inhibitory activity was monitored by combining fractions with neutrophil exudate of known MPO activity. Peritoneal neutrophils were collected as described by Krawisz et al [7]. A second liver sample was dialyzed in 3500 molecular weight cut off (MWCO) tubing with 25 mM potassium phosphate buffer, pH 8.0 and applied to a 16mm x 24 cm DEAE Sepharose CL6B column charged with phosphate ions and equilibrated with 25mM potassium phosphate buffer, pH 8.0. The column was eluted with Ii0 ml of equilibration buffer followed by a linear gradient of 0.5 M KCI (i00 ml). Fractions (1-23: 4.5 ml, 24-103: 2.3 ml) were assayed for MPO activity and inhibition as described previously. Catalase activity was determined by measuring the absorbance change at 240 nm [i0]. Inhibition of catalase with 3-Amino-l,2,4-Triazole CAT). Catalase activity was inhibited by incubating I:i0 dilutions of the prepared samples with 200 mM AT at 25 ° C with .i, i, and i0 mM H202 for 2 hours [ii]. Later tests used 0.i mM H202. Results Initial assays for MPO in techniques resulted in low values failed to correlate histological examinations
liver samples using standard preparation and inconsistent activity values. T h e s e with the neutrophil accumulation seen in of the samples.
Application of dialyzed liver homogenate to a resulted in separation of an MPO activity inhibitory activity. MPO activity was eluted whereas MPO inhibition was observed with the 76 - 97.
Sephadex GIOOSF column peak from a peak of in fractions ii0 - 148 void volume fractions,
Vol. 47, No. 24, 1990
Myeloperoxidase Activity
PL-147
A p p r o x i m a t e l y , 90% of M P O a c t i v i t y was e l u t e d f r o m a D E A E S e p h a r o s e CL6B column with the equilibration buffer (Figure i). Two a d d i t i o n a l a c t i v i t y p e a k s w e r e a t t r i b u t e d to M P O - l i k e a c t i v i t y such as h e m o g l o b i n , r a t h e r t h a n MPO, due to the n o n - s p e c i f i c n a t u r e of od i a n i s i d i n e oxidation. M P O i n h i b i t o r y a c t i v i t y w a s r e t a i n e d on the D E A E c o l u m n and e l u t e d in f r a c t i o n s 50 - 62 at a p p r o x i m a t e l y 0.2 M KCl. T h i s p e a k d i r e c t l y c o r r e l a t e d to a p e a k of c a t a l a s e activity.
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Pergamon Press
PHARMACOLOGY LETTERS Accelerated Communication
THE DETERMINATION OF MYELOPEROXIDASE ACTIVITY IN LIVER D.L. Duval, D. Howard, T.A. McCalden and R.E. Billings, Departments o f Surgery and Pharmacology, University of Nevada School of Medicine, Reno, NV 89557-0046. (Submitted August 20, 1990; accepted August 30, 1990; received in final form October I0, 1990) Abstract. Myeloperoxidase (MPO) is an enzyme found in granulocytes of neutrophils, but not in mammalian tissues. Previous studies have directly correlated MPO activity with neutrophil accumulation in tissues. This study presents a method for determining MPO activity in liver. Neutrophil accumulation in rat liver was provoked by creating partial ischemia followed by reperfusion. Liver homogenates prepared b y a standard procedure showed no MPO activity. The homogenate was applied to Sephadex GI00 and DEAE Sepharose CL6B columns which separated ~ P O activity from inhibitory activity. The inhibitor was identified as catalase based upon its elution from the columns and removal with 3-amino- 1,2,4-triazole (AT), a catalase inhibitor. Based upon these findings, it was determined that full MPO a c t i v i t y c a n be assayed in unfractionated liver homogenates by first inactivatinq catalase with AT. Introduction Myeloperoxidase (MPO) (donor: H202 oxidoreductase, ECI.II.I.7) is a l e u k o c y t e - s p e c i f i c enzyme found primarily in the granules of neutrophils and to a lesser extent in macrophages. MPO in neutrophils is released from granules in response to NADPH-oxidase activation [1-3]. MPO catalyses the reaction of H202 with halide ions to form h y p o h a l o u s acids. These acids serve as the main antimicrobial compounds released by the neutrophils as well as causing considerable damage to surrounding tissues when released extracellularly [1-5]. Neutrophils are drawn to areas of injury by a wide variety of chemotactic factors [2]. Their presence has been used to quantify inflammation, myocardial infarct and other types of tissue injury. Although, histological examination of the tissues has been used to determine neutrophil accumulation, the timeconsuming and semi-quantitative nature of this method led to the measurement of MPO activity as a marker of neutrophil accumulation [5-7]. Unfortunately, the recovery of MPO activity is often negligible, possibly b e c a u s e of incomplete degranulation of the enzyme and the presence of a number of endogenous inhibitors [5]. In vitro studies have shown that MPO activity can be blocked by a variety of inhibitors including azide, cyanide, and catalase [3]. This study examines the inhibitors of MPO in liver samples and introduces a method to increase MPO activity recovery. Corresponding Author: Dr. Ruth Billings, Depts. of Pharmacology and Surgery, University of Nevada School of Medicine, Reno, NV 895570046. 0024-3205/90 $3.00 + .00 Copyright (c) 1990 Pergamon Press plc
PL-146
M y e l o p e r o x i d a sActivity e
Vol. 47, No. 24, 1990
Materials and Methods Rat Liver Ischemia-Reperfusion Model. Male Sprague-Dawley rats (275 - 3 0 0 g m ) were anesthetized by injection of Nembutal (pentobarbital) at 50 mg/ kg (i.p.) for the surgery. A previously described partial ischemia model was used [8]. Briefly, ischemia was provoked in the left lateral and median lobes of the liver by the application of a microaneurysm clip (Bruno Vet Supply, San Leandro, CA ) to the portal vessels supplying those lobes. After one hour of ischemia, the clips were removed to restore blood flow to the ischemic lobes. The animals were maintained in a stable temperature environment. Reperfusion was allowed for varying durations. Under Nembutal anesthesia non-ischemic and ischemic liver lobe samples were collected from the rats. The tissue samples were immediately frozen in liquid nitrogen and stored at -70 ° C until assayed. Myeloperoxidase SpectroDhotometric Assay. The samples were prepared by an established method using cetyltrimethylammonium bromide as a detergent [6,7]. The assay contained 50 ~ potassium phosphate buffer, pH 6.0, 0.167 mg/ml a-dianisidine dihydrochloride, and 0.0005% H202. The change in absorbance at 460 nm was measured for 5 minutes. Protein was determined by the method of Lowry et al [9]. Chromatography of Myeloperoxidase. Liver homogenate from a 24 hour reperfused tissue was applied to a 26 mm x 30 cm column of Superfine (SF) Sephadex G-100 and eluted with 50 mM potassium phosphate buffer, pH 6.0 at 4 ° C. The fractions (2.25 ml) were assayed for MPO activity. Inhibitory activity was monitored by combining fractions with neutrophil exudate of known MPO activity. Peritoneal neutrophils were collected as described by Krawisz et al [7]. A second liver sample was dialyzed in 3500 molecular weight cut off (MWCO) tubing with 25 mM potassium phosphate buffer, pH 8.0 and applied to a 16mm x 24 cm DEAE Sepharose CL6B column charged with phosphate ions and equilibrated with 25mM potassium phosphate buffer, pH 8.0. The column was eluted with Ii0 ml of equilibration buffer followed by a linear gradient of 0.5 M KCI (i00 ml). Fractions (1-23: 4.5 ml, 24-103: 2.3 ml) were assayed for MPO activity and inhibition as described previously. Catalase activity was determined by measuring the absorbance change at 240 nm [i0]. Inhibition of catalase with 3-Amino-l,2,4-Triazole CAT). Catalase activity was inhibited by incubating I:i0 dilutions of the prepared samples with 200 mM AT at 25 ° C with .i, i, and i0 mM H202 for 2 hours [ii]. Later tests used 0.i mM H202. Results Initial assays for MPO in techniques resulted in low values failed to correlate histological examinations
liver samples using standard preparation and inconsistent activity values. T h e s e with the neutrophil accumulation seen in of the samples.
Application of dialyzed liver homogenate to a resulted in separation of an MPO activity inhibitory activity. MPO activity was eluted whereas MPO inhibition was observed with the 76 - 97.
Sephadex GIOOSF column peak from a peak of in fractions ii0 - 148 void volume fractions,
Vol. 47, No. 24, 1990
Myeloperoxidase Activity
PL-147
A p p r o x i m a t e l y , 90% of M P O a c t i v i t y was e l u t e d f r o m a D E A E S e p h a r o s e CL6B column with the equilibration buffer (Figure i). Two a d d i t i o n a l a c t i v i t y p e a k s w e r e a t t r i b u t e d to M P O - l i k e a c t i v i t y such as h e m o g l o b i n , r a t h e r t h a n MPO, due to the n o n - s p e c i f i c n a t u r e of od i a n i s i d i n e oxidation. M P O i n h i b i t o r y a c t i v i t y w a s r e t a i n e d on the D E A E c o l u m n and e l u t e d in f r a c t i o n s 50 - 62 at a p p r o x i m a t e l y 0.2 M KCl. T h i s p e a k d i r e c t l y c o r r e l a t e d to a p e a k of c a t a l a s e activity.
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