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Title:
DETERMINATION OF CATALASE ACTIVITY
Document Type and Number:
WIPO Patent Application WO/2001/034831
Kind Code:
A2
Abstract:
An assay for catalase in a test sample comprising contacting the test sample with enzymatically produced H¿2?O¿2?, a peroxidase and a chromogenic dye; and measuring the change in color intensity.

Inventors:
O'brien, Peter James (SmithKline Beecham Pharmaceuticals The Frythe Welwyn Hertfordshire AL6 9AR, GB)
Slaughter, Mark Raymond (SmithKline Beecham Pharmaceuticals The Frythe Welwyn Hertfordshire AL6 9AR, GB)
Application Number:
PCT/GB2000/004305
Publication Date:
May 17, 2001
Filing Date:
November 10, 2000
Export Citation:
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Assignee:
Smithkline, Beecham P. L. C. (New Horizons Court Brentford Middlesex TW8 9EP, GB)
SMITHKLINE BEECHAM CORPORATION (One Franklin Plaza Philadelphia, PA, 19103, US)
O'brien, Peter James (SmithKline Beecham Pharmaceuticals The Frythe Welwyn Hertfordshire AL6 9AR, GB)
Slaughter, Mark Raymond (SmithKline Beecham Pharmaceuticals The Frythe Welwyn Hertfordshire AL6 9AR, GB)
International Classes:
C12Q1/28; C12Q1/30; (IPC1-7): C12Q1/00
Attorney, Agent or Firm:
Blakey, Alison (SmithKline Beecham Two New Horizons Court Brentford Middlesex TW8 9EP, GB)
Giddings, John Peter (SmithKline Beecham . Corporate Intellectual Property Two New Horizons Court Brentford, Middlesex TW8 9EP, GB)
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Claims:
Claims:
1. An assay for catalase in a test sample comprising contacting the test sample with enzymatically produced H202, a peroxidase and a chromogenic dye; and measuring the change in color intensity.
2. An assay according to claim 1 wherein the peroxidase is horseradish peroxidase.
3. An assay according to claim 1 or 2 wherein the source of H202 is an oxidative enzyme and it's substrate selected from uricase and uric acid; cholesterol oxidase and cholesterol; glucose oxidase and glucose; alphaglycerol phosphate oxidase and glycerol and glycerol kinase; creatine amidinohydrolase and sarcosine oxidase and creatine; sarcosine and sarcosine oxidase; oxalate oxidase and oxalate; choline and choline oxidase; and xanthine oxidase and xanthine.
4. An assay according to claim 3 wherein the source of H202 is uricase and uric acid.
5. An assay according to claim 3 or 4 wherein the substrate is used in excess.
6. An assay according to any one of the preceding claims wherein the chromogenic dye is Trinder reagent (4aminophenazone and 3,5dichloro2hydroxybenzenesulfonate) or 2,2'azinobis (3ethyl benzthiazoline6sulfonic acid).
7. A kit for assaying catalase in a test sample which comprises an enzymatic source of H202, a peroxidase and a chromogenic dye as defined in any one of the preceding claims.
8. A method for assessing the oxidative stress, pathological effect, peroxisomal proliferation or peroxidatic activity inducing effect of a selected environmental or genetic factor on a biological system comprising the steps of : (a) exposing the biological system to an environmental or genetic factor for a sufficient time to affect genetic expression or activity of catalase in said system; (b) extracting a test sample from the system of step (a); (c) assaying the test sample for catalase by a process according to any one of claims 1 to 6; and (d) comparing the concentration of catalase assayed in step (c) with the amount of catalase present in a control test sample from a biological system which has not been exposed to the environmental or genetic factor, to identify any change in catalase expression or activity, indicative of an oxidative stress, pathological effect, peroxisomal proliferation or peroxidatic activity inducing effect of the environmental or genetic factor on the biological system.
9. A method according to claim 8 wherein the biological system is a mammal.
10. A method according to claim 8 or 9 wherein the environmental factor is an exogenous synthetic compound.
Description:
NOVEL PROCESS The present invention relates to an assay for the enzyme catalase, in particular for the antioxidant catalatic activity of catalase.

Catalase is a primary component of the antioxidant system, which defends against oxidative stress which is ubiquitously associated with pathological conditions, for example cancer and diabetes.

Oxidative stress is also critically and mechanistically involved in mediating many drug toxicities. Catalase decomposes hydrogen peroxide (H202), a major reactive oxygen species that in the presence of iron or other metal ions oxidizes cellular lipid, protein, and nucleic acid and causes cytotoxicity as described in Van Lente F et al, Clin. Chem., 1990, 36,1339-1343 andWheelerCR etal, Anal. Bioch., 1990,184,193-199. Genetic overexpression of catalase and exogenous catalase or catalase mimetics protects against oxidative injury whereas catalase deficiency increases susceptibility to oxidative stress- induced disease.

Serum catalase is increased in a wide variety of diseases, including pancreatic, liver, hemolytic, renal, skin, respiratory, graft-vs-host, acquired immunodeficiency, and oxidant-mediated vascular diseases. Conversely, serum catalase is decreased in acatalasemia, alcoholism, certain cancers and psychiatric diseases. Finally, catalase's location in peroxisomes also makes it an important tissue biomarker of peroxisome proliferation, a toxic effect in rodents of a wide range of drugs and chemicals, such as fibrates, thiazolidinediones, certain non-steroidal anti-inflammatory drugs, and fatty acid- derived molecules.

Historically, oxidative stress assays have been used investigatively as described in Góth L et al, Hungarian Scientific Instruments., 1984,57,7-12, Beers RF et al, J. Biol.

Chem., 1952,95,133-40 and Leighton F et al, J. Cell Biol., 1968,37,482-513, but interest in their diagnostic and prognostic use is developing. Clinical chemistry laboratories are increasingly measuring plasma and tissue biomarkers of oxidative stress and activity of the antioxidant system. There are fully-automated assays for superoxide dismutase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and total antioxidant status, see Woolliams JA et al, Res. Vet. Sci., 1983, 34,253-6; Paglia DE et al, J. Lab. Clin. Med., 1967,70,158-69; Goldberg DM et al, in: Bergmeyer HU ed. Methods of enzymatic analysis, 3rd ed, Vol 3. Verlag Chemie: Deerfield Beach, 1983: 258-265, Kornberg A, in: Colowick SP, Kaplan NO, eds. Methods in enzymology, Vol I. Pp. 323. New York: Academic Press, 1955; and Miller NJ et al, Clin. Sci. (Colch)., 1993,84,407-12.

A fully-automated, direct assay for the antioxidant, H20z-metabolizing activity of catalase (H2O2 : HzOz oxidoreductase ; EC 1.11.1.6) is not currently available, although assay of its alcohol-metabolizing activity has been automated, see Van Lente F et al, ibid, Wheeler CR et al, ibid, and Yasmineh WG et al, Clin. Biochem., 1992,26,21-27. In the former, so-called"catalatic"activity, H202 is decomposed to water and oxygen, whereas in the latter"peroxidatic"activity, hydrogen donor molecules such as methanol and ethanol are oxidized by H202 to their respective aldehydes, see Oshino N et al, Biochem.

J., 1973,131,555-567 and Sichak SP et al, Arch. Biochem. Biophys., 1986,249,286-295.

Both reactions proceed by formation of a catalase-H202 complex, but catalatic decomposition occurs faster than the peroxidatic reaction by several orders of magnitude.

Increasing the concentration of alcohol compared to H202 favours the peroxidatic reaction but does not exclude the catalatic reaction, although the peroxidatic reaction is excluded by the absence of alcohol. Accordingly, catalase assays based on peroxidatic activity underestimate catalatic activity.

Although numerous methods of assaying catalatic activity of catalase have been reported, none are amenable to the standard, clinical chemistry autoanalyser. Catalatic activity has been measured from O2 production (Góth L et al, ibid), absorbance of H202 at 240 nm (Beers RF et al, ibid), permanganate (Cohen G et al, Anal. Biochem., 1970,34, 30-38), ferrithiocyanate (Hosono M et al, Rinsho Byori, 1996,44,444-448), indamine (Goth L et al, Acta Biol. Hung., 1987,38,287-290), ammonium molybdate (Goth L et al, Clin. Chem., 1988,29,741-743) or luminol and sodium hypochlorite (Goth L et al., Clin.

Chim. Acta, 1989,186,39-44). These methods are inappropriate for standard clinical chemistry autoanalysers because of their need for oxygen or luminescence detectors, non- standard wavelength or temperature settings, capacity for automated addition of more than two reagents, or because of their use of undesirably hazardous chemicals.

Automation of assays for peroxidatic activity of catalase has been reported, see Van Lente, F. et al, ibid and Oshino, N. et al, ibid, but this activity is only indirectly and inexactly related to catalatic activity, and the relationship between these activities has not been defined for catalases of different species. Peroxidatic activity has been measured from formaldehyde production via its reaction with chromogenic dye (Purpald; 4-amino- 3-hydrazino-5-mercapto-1, 2,4-triazole; Wheeler CR et al, ibid, and LeffJA et al, J. Appl.

Physiol., 1991,71,1903-1906) or with nicotinamide adenine dinucleotide and aldehyde dehydrogenase (Van Lente F et al, ibid, and Yasmineh WG et al, ibid). Full automation of the former assay requires more steps than can be performed on the standard clinical analyzer. Furthermore, peroxidatic activity is sensitive to assay conditions, and cannot be fully dissociated from catalatic activity. Accurate prediction of catalatic activity from peroxidatic activity requires knowledge of the relative proportions catalatic and peroxidatic activities.

In contrast to traditional enzyme assays, in which substrate is added in excess to achieve zero-order kinetics and optimal activity, catalase must be assayed at sub- saturating concentrations of H202. This requirement reflects the high H202 concentration required to saturate catalase (above 5 M) and the much lower H202 concentration that inhibits catalase (0.1 M,), see Aebi HE, in: Bergmeyer HU ed.

Methods of enzymatic analysis, 3rd ed, Vol 3. Verlag Chemie: Deerfield Beach, 1983: 273-86. Consequently, international units of activity cannot be defined for catalase.

The requirement to use subsaturating concentrations of H202 in catalase assays has advantages. It allows minimization of potential hazardous effects of its corrosive, oxidizing, and decomposing properties. Furthermore, it allows the alternative, slow, steady-state production of H202 by endogenous enzymes such as glucose oxidase. This obviates the hazard concern and additionally produces more consistent results.

Endogenous, enzymatic provision of H202 has been used to assay peroxidatic activity of catalase, see Van Lente F et al, ibid and Sichak SP et al, ibid. H202 concentration may be measured as described in Barham D et al, Analyst, 1972,97,142-145, van Gent CM et al, Clin. Chim. Acta., 1977,75,243-251; Fossati P et al, Clin. Chem., 1980,26,227-31, Fossati P et al, Clin. Chem., 1982,28,2077-2080; Fossati P et al, Clin. Chem., 1983,29, 1494-1496; and Petrarulo M et al, Clin. Chem., 1994,40,2030-2034.

A fully-automated catalase assay would be a desirable addition to the panel of parameters available to assess the activity of the antioxidant system.

The present invention provides an assay for the antioxidant catalatic activity of catalase using enzymatic production of H202 and monitoring H202 concentration, which is susceptible to automation. The assay is simple, precise, relatively inexpensive, and rapid, e. g. 600 tests may be performed per hour on a Hitachi 717 analyzer.

According to the invention there is provided an assay for catalase in a test sample comprising contacting the test sample with enzymatically produced H202, a peroxidase and a chromogenic dye; and measuring the change in color intensity.

Any peroxidases which are capable of catalyzing oxidation of a chromogenic dye by H202 may be used in the assay. A preferred peroxidase is horseradish peroxidase (HRP). Other peroxidases include soybean peroxidase, fungal peroxidase, lignin peroxidase, lactoperoxidase (with substrate), myeloperoxidase (with substrate) and glutathione peroxidase (with substrate).

Suitable enzymatic sources ou for use in the assay include oxidative enzymes and their substrates for example uricase and uric acid; cholesterol oxidase and cholesterol; glucose oxidase and glucose; alpha-glycerol phosphate oxidase and glycerol and glycerol kinase; creatine amidinohydrolase and sarcosine oxidase and creatine; sarcosine and sarcosine oxidase; oxalate oxidase and oxalate; choline oxidase and choline; and xanthine

oxidase and xanthine. The substrate is preferably added in excess to maximize H202 production. The preferred enzymatic source of H202 is uricase and uric acid. The uric acid may be added as the free acid or in the form of a salt for example sodium urate.

Chromogenic dyes for use in the assay of the invention are dyes which undergo a color change with H202. Suitable dyes include Trinder reagent (4-aminophenazone (AP) and 3,5-dichloro-2-hydroxybenzenesulfonate (DHBS)), 2,2'-azino-bis (3-ethyl benzthiazoline-6-sulfonic acid) (ABTS), 4-aminoantipyrine plus phenol, 4-chlorophenol or 2,4,6-tribromophenol, 4-aminophenazone plus phenol, 3-methyl-2-benzothiazolone hydrazone plus N, N-diethylaniline, 3,3'-diaminobenzidine, and o-tolidine. Preferred dyes for use in the assay are Trinder reagent and ABTS, especially Trinder reagent.

Measurement of the change in color intensity may be performed using techniques known to those skilled in the art, e. g. absorbance measurement at a wavelength appropriate for the chromogenic dye.

Since peroxidases consume H202 to effect oxidation of the chromogenic dye, to produce in the case of Trinder reagent the quinoneimine, the assay is effectively based on competition between catalase and peroxidase. The activity of the peroxidase is preferably adjusted to provide sufficient sensitivity towards catalase such that inhibition of color formation shows a linear relationship over a wide range of catalase activities. In the examples that follow conditions were derived specifically for liver homogenates, erythrocytes and plasma, however, sensitivity can be adjusted for test samples with lower catalase activity, by reduction of the enzymatic source of H202, e. g. by reducing the uricase, and peroxidase activity in the reaction medium and using standards with lower catalase activity.

The chemical reactions of the catalase assay according to the invention are illustrated in Scheme 1 using uricase/uric acid as the source of HzOz. Abbreviations: AP, 4-aminophenazone ; DHBS, 3,5-dichloro-2-hydroxybenzenesulphonate (sodium salt). Uricase Uric acid Allantoin H202 CATALASE PEROXIDASE H20DHBS + 4AP Quinoneimine + (color at 505 nm) °2 (reduced color at 505 nm) Scheme 1

When performing the assay of the invention it may also be necessary to consider known interferents with the peroxidase catalyzed oxidation of the chromogenic dye for specific biological test samples, for the Trinder reaction such interferents include bilirubin, haemoglobin, and ascorbate, see Fossati P et al, ibid and Mimic-Oka J et al, Clin. Nephrol., 1999,51,233-241. In these cases the reagent may also contain potassium hexacyanoferrate (II) to eliminate bilirubin interference and to enhance color production.

If necessary, interference from ascorbate can be eliminated by addition of ascorbate oxidase. Haemoglobin is unlikely to interfere with the measurement of erythrocyte catalase since its high specific activity in this cell enables dilutions that reduce haemoglobin concentration to below levels that cause interference with the Trinder reaction. Other potential interferents and methods for their avoidance will be apparent to those skilled in the art.

The assay is preferably a quantitative assay. Because of the unique aspects of the assay of the invention catalase activity is conveniently expressed in a manner similar to that described for superoxide dismutase (Woolliams JA et al, Res. Vet. Sci., 1983,34, 253-6) rather than as previously described for catalase. Firstly, the assay is based on inhibition of color development rather than on color formation, secondly, H202 decomposition is not directly measured. Accordingly, expression of activity as the rate constant for the exponential decomposition H202 by catalase, as is frequently done in other assays, is not appropriate. Thirdly, because catalase activity cannot be assayed under conditions of substrate saturation, and therefore its activity is proportional to H202 concentration, it is considered inappropriate to use international units. Finally, the steady state concentration of H202 in this assay is unknown.

Inability to assay catalase at saturating substrate concentrations, as is conventionally done in standardized enzyme assays, has resulted in there being no official reference method and a wide variation in the reaction conditions and methods that are used. Consequently, estimates of catalase activity for different tissues vary widely, depending on the method of assay, concentration of H202 and units of measurement. For example, human plasma activity of catalase is reported to be 70 29 U/L using a peroxidatic method, 1 mM H202 and catalase standards, but approximately 200-fold or 700-fold higher in catalatic methods based on measurement of the decrease in absorbance of 10 and 54 mM H202, respectively. Rat liver catalase activity is reported to be 0.002 IU/mg protein using the peroxidatic method and 2 mM H202, but approximately 700-fold higher in a catalatic assay based on measurement of the decrease in absorbance of 10 mM H202. Human erythrocyte catalase activity is reported to be 15.6 2.2 units/g hemoglobin using a peroxidatic method, endogenous production of peroxidase by glucose oxidase, and catalase standards, but varied from 313 48 k/g hemoglobin at 10 mM H202

to 42 6.0 k/g hemoglobin at 70 FM H202. The latter was shown to be equivalent to 70 llmol H202 removed/min/g hemoglobin. Rat erythrocyte catalase activity is reported as 52 k/g hemoglobin in a catalatic assay based on measurement with 10 mM H202.

Measurements of catalase activity from the assay of the invention cannot be readily compared to those previously reported because it is the first to use steady-state production of H202 in a catalatic assay and the H202 concentration is unknown. The steady state concentration of H202 will reflect the balance of its production, e. g. by uricase, and its decomposition by peroxidase in competition with catalase.

In the assay of the invention test samples are preferably assayed together with standard samples of known catalase activity. In the assay the units of activity are independent of the absolute activity of the standards and only require of the standard that there is sufficient activity to inhibit the peroxidase and color development. The units are similar to those for superoxide dismutase, and are not international units nor equivalent units of standard enzyme (Sigma-Aldrich Co. Ltd, Poole, UK). Unit activity is defined as that half-maximally inhibiting 400 U/L peroxidase (under the specified reaction conditions) and not as the equivalent amount of standard. The standards allow definition of the log-linear relationship between inhibition of color formation and linear increase in catalase activity. The standards also assist in quality control of the reagents. For example, reagent deterioration causes a decrease in the amount of standard to produce 50% inhibition of color development.

For purposes of convenience and reagent quality control the catalase assay may be adapted to a kit format.

Therefore, according to a further aspect of the invention, there is provided a kit for assaying catalase in a test sample which comprises an enzymatic source of H202, a peroxidase and a chromogenic dye.

The assay of the invention may be used to measure catalase as a marker of oxidative stress in biological systems and in particular to measure the potential of environmental factors to cause oxidative stress in such systems. Additionally, it may be used as a biomarker in a wide variety of diseases and as a biomarker of peroxisome proliferation.

Therefore, according to a further aspect of the invention, there is provided a method for assessing the oxidative stress, pathological effect, peroxisomal proliferation or peroxidatic activity inducing effect of a selected environmental or genetic factor on a biological system comprising the steps of : (a) exposing the biological system to an environmental or genetic factor for a sufficient time to affect genetic expression or activity of catalase in said system; (b) extracting a test sample from the system of step (a);

(c) assaying the test sample for catalase by a process comprising contacting the test sample with enzymatically produced Ho02, a peroxidase and a chromogenic dye; and measuring the change in color intensity; and (d) comparing the concentration of catalase assayed in step (c) with the amount of catalase present in a control test sample from a biological system which has not been exposed to the environmental or genetic factor, to identify any change in catalase expression or activity, indicative of an oxidative stress, pathological effect, peroxisomal proliferation or peroxidatic activity inducing effect of the environmental or genetic factor on the biological system.

The determination of environmental factors and genetic factors having an oxidative stress, pathological effect, peroxisomal proliferation, or peroxidatic activity inducing potential is useful, e. g. in the screening and development of new pharmaceutical agents and therapies.

In this aspect of the invention the biological system is preferably an animal, plant, or bacteria, more preferably a mammal.

Test samples from a mammal which may be assayed for catalase activity include liver muscle, heart, blood cells, lens, and kidney samples.

Environmental factors include a wide variety of physical, chemical or biological factors which have the potential to cause oxidative stress in biological systems. Physical environmental stimuli include the diet of a living organism e. g. a mammal, increases or decreases in temperature, and increases or decreases in exposure to ionizing or ultraviolet radiation. Biological and chemical stimuli include administering a transgene to a biological system, eliminating a gene from a biological system, and administering an exogenous synthetic compound or exogenous agent or an endogenous compound, agent or analog thereof to the biological system.

Exogenous synthetic compounds can be, for example, pharmaceutical compounds, toxic compounds, proteins, peptides or chemical compositions.

Exogenous agents can be e. g. natural pathogens, such as microbial agents, which can alter gene transcription. Examples of pathogens include bacteria, viruses, and lower eukaryotic cells such as fungi, yeast, molds and simple multicellular organisms, which are capable of infecting a biological system, e. g. a mammal, and replicating its nucleic acid sequences in the cells or tissue of that system.

An endogenous compound is a compound which occurs naturally in the body, e. g. a hormone, enzyme, receptor or ligand. An analogue is an endogenous compound which is preferably produced by recombinant techniques and which differs from said naturally occurring endogenous compound in some way.

All publications cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.

The invention is illustrated by the following example which uses the catalase assay of the invention to study variables that affect tissue catalase activity, including tissue type, species, and age.

Example Materials 4-Aminophenazone (AP), 3,5-dichloro-2-hydroxybenzenesulphonate (sodium salt, DHBS), horseradish peroxidase (HRP ; hydrogen peroxide oxidoreductase; EC 1.11.1.7) type XII, uric acid (sodium salt), uricase (urate: oxygen oxidoreductase; EC 1.7.3.3 from Candida utilis, type IV), potassium hexacyanoferrate (II), tris (hydroxymethyl) aminomethane (Tris), and catalase (H202 oxidoreductase ; EC 1.11.1.6; bovine liver) from Sigma-Aldrich Co. Ltd, Poole, UK. Potassium dihydrogen phosphate and dipotassium hydrogen phosphate from BDH Chemicals Ltd, Poole, UK.

Reagent Preparation Color-developing reagent (reagent 1) was prepared from DHBS, AP, uricase and HRP. Separate stock solutions of DHBS (4mmol/L) and of AP (1 mM) with potassium hexacyanoferrate (II) (100 pM) were prepared in phosphate buffer (50 mmol/L, pH 7.0) containing Triton X-100 (1.5 g/L) and stored for up to 1 month at 4°C. Reagent 1 was prepared on the day of assay by first mixing equal volumes of DHBS and AP solutions.

Stock solution of uricase (1.0 x 103 U/L, prepared fresh) and HRP (1.0 x 105 U/L, stable 1 month) in phosphate buffer were then added to give final concentrations of 2 mmol/L DHBS, 0.5 mmol/L AP, 50 pM potassium hexacyanoferrate II, 57 units/L uricase and 400 units/L HRP (unless stated otherwise).

Reaction-initiating reagent (reagent 2) was prepared by dissolving uric acid (0.5 mM) in phosphate buffer/Triton X-100 and stored at 4°C (stable for at least one month).

Unless otherwise stated, reagent 2 contained 500} je uric acid.

A concentrated solution of freshly prepared catalase (218,000 Sigma units/L) in Tris buffer (20 mmol/L, pH 7.4) was used to produce a series of standards. Tris buffer was used as a blank. A unit (U, international unit) of catalase activity used to prepare standards is specifically defined by the vendor (Sigma-Aldrich Co. Ltd, Poole, UK) as decomposing 1.0 Rmole of H202 per min at pH 7.0 at 25°C, while the H202 concentration falls from 10.3 to 9.2 mM

Sample Preparation Twenty male Sprague-Dawley rats (Charles River Ltd, UK), 10 aged 8 to 9 weeks and 10 aged 6 months were killed by exanguination under deep halothane anaesthesia.

Livers were excised, blotted clean of blood, and immediately frozen in liquid nitrogen and stored at-80°C until the day of assay (within one month). Liver samples from the 6 month old rats were also assayed fresh in order to determine the effects of freezing and storage. For catalase analysis of liver, approximately 180 to 200 mg tissue was homogenized on ice for 20 sec at 9,500 rpm (IKA T25 homogenizer; Janke and Junkel, Germany) in 3.2 mL of ice-cold, Tris buffer, centrifuged at 3000 g for 30 min at 4°C and the supernatants further diluted with two volumes of ice-cold Tris buffer immediately before assay.

Heparinised blood was obtained from 10 human volunteers, 7 male beagle dogs, and from the 10 male rats aged 6 months described above. All subjects were healthy and without clinical signs of disease. Plasma was separated and samples without any visible signs of hemolysis were used to assay catalase. Samples of human plasma were immediately frozen in liquid nitrogen and stored at-80°C until the day of assay (within one week) in order to determine the effects of freezing and storage. All rat samples showed visible hemolysis and so were discarded; human and canine samples were free of hemolysis. Red cells were washed in saline three times, lysed and assayed for hemoglobin concentration as described in Van Lente F et a/, ibid.

Catalase Assay Assays were conducted on a Hitachi 717 automated chemistry analyzer (Roche Diagnostics Ltd, Lewes, U. K.). Reagents and samples were placed on the instrument and assayed using the settings in Table 1.

Standards and blanks were assayed in duplicate and treated as samples (i. e. not placed in the calibration positions). Analytical conditions were set so that sample was first added to reagent 1, and after a 5 min blank incubation period, reagent 2 was added to initiate H202 generation and color development. The Hitachi 717 analyzer automatically records absorbance readings every 12 sec for 10 min; however, the analyzer settings determine the specific readings used for the assay. In this case, the reading times were selected to allow for a 12-sec lag phase after addition of uric acid, followed by the absorbance change after an 84-sec incubation. This end point reading was used to calculate the percentage inhibition of color development for all samples and standards, using the Tris blank as the uninhibited reaction. One unit of catalase activity was defined as the amount of enzyme activity that causes 50% inhibition of color development.

Standards were plotted on a semi-log graph of percentage inhibition against log enzyme

activity and the line of best fit was calculated by least squares linear regression. This was used to determine activity in liver homogenates.

Hitachi 717 Analyzer Settings for Measurement of Catalase Activity Temperature 37°C Assay code 2 point: 26-33 Sample volume (uL) 20 Reagent 1 (uL) 300 Reagent 2 (uL) 50 Wavelength (nm) 700-505 Calibration K factor: 0-0 STD1 0-1 SD limit 0.1 Duplicate limit 100 ABS limit inc/dec 0-increase Prozone limit 32000-upper Instrument factor 1.0 Table 1 Data Analysis Data were analyzed and graphed with commercially available software (Graphpad Prism version 3.00, Graphpad Software, San Diego, CA). Differences between mean values for different species or tissues were tested for using the unpaired, two-tailed, Student's t-test when comparing two groups. For comparing three or more groups analysis of variance was used, followed by the Newman-Keuls post test when the F ratio was statistically significantly greater than 1. The line of best fit for calibration curves was calculated by least squares regression. Goodness of fit was determined from the square of the correlation coefficient, and defined as the proportion of total variance in'Y'values accounted for by the linear regression model (Snedecor GW et al, Statistical Methods, Iowa State University Press, 1991, Ames).

Method imprecision for within-run and between run replicate values was determined by calculating the coefficients of variation (CV; 100 % SD/mean). Since catalase standards were assayed in duplicate, the SD for the difference between duplicates (d) of n standards was calculated from ( /2n) (Gowenlock AH, Varley's Practical Clinical Biochemistry, Heinemann Medical Books, 1988, London). Statistical significance was considered to occur for P < 0.05.

Results Figure 1 shows the effect of uric acid concentration on absorbance change in the catalase assay. Samples contained 1000 (), 12500 (), or 50000 () U/mL catalase.

A U of catalase activity used to prepare standards is specifically defined by the vendor (Sigma-Aldrich Co. Ltd, Poole, UK) as decomposing 1.0 umole of H202 per min at pH 7.0 at 25 C, while the H202 concentration falls from 10.3 to 9.2 mM. However, the activity of a U of catalase activity is likely to substantially lower in this assay because of substantially lower H202 concentration and because its activity is counteracted by HRP.

The concentration of uric acid causing maximal color development in the catalase assay is demonstrated to be approximately 500 pM in reagent 2 for a wide range of catalase activities. At 250 jj. M uric acid in reagent 2, there was almost 50% lower color development.

Figure 2 shows the time course of color formation in the catalase assay (a) in the absence of catalase; and (b) after addition of catalase solution containing 25000 Sigma units/mL. Absorbance was measured every 12 sec. UA: addition of reaction-initiating reagent (reagent 2) containing 500 pM uric acid; dA: measurement point for absorbance change used in the catalase assay. Absorbance increased linearly from initiation of the reaction with uric acid for the 84 sec over which the reaction was measured by the analyzer, and hyperbolically from approximately this time on. At the end of the 84-sec reaction, absorbance was approximately 0.5 in the absence of catalase and 0.1 with samples containing 50,000 Sigma units/mL of catalase.

Figure 3 shows: (a) a typical standard curve used for determining catalase activity in samples, using inhibitory units. One unit represents the amount of enzyme activity that causes 50% inhibition of color development and was equivalent to approximately 6,000 Sigma units.

(b) relationship between Sigma units and inhibitory units for catalase activity. In order to determine catalase activity in liver, tissue was diluted approximately 51-fold, to cause 30 to 60% inhibition of color development.

Figure 4 shows the effect of HRP activity (in color developing reagent) on color development in the catalase assay for a wide range of activities of catalase in the sample.

Start reagent contained 500 uM uric acid. (a) Absorbance change after 84 sec assay incubation period after addition of catalase solutions each (), 12500 (), or 50000 () U/mL. (b) Goodness of fit for linearity of standard curve (standard catalase solutions ranged 1000 to 50000 Sigma units/mL). r: correlation coefficient. Standard curves were most linear when the catalase assay used an HRP activity of 500 units/L.

Figure 5 shows the inhibitory effect of catalase on color development in the optimized catalase assay:

(a) shows the final absorbance change. The final absorbance decreases exponentially as sample catalase activity is increased.

(b) shows percentage inhibition of color formed in the absence of catalase. Color- developing reagent (reagent 1) contained 400 units/L HRP ; reaction initiating reagent (reagent 2) contained 500 tM uric acid. The log catalase activity is plotted against the amount of inhibition of color development in the catalase assay. Color development was half-maximally inhibited at approximately 6.0 x 10'U/mL.

Figure 6 shows the effect of dilution and freezing of samples in the catalase assay.

(a) Dilutional parallelism for liver catalase activity. (b) Dilutional parallelism for erythrocyte catalase activity. (c) Effect of freezing human plasma on its catalase activity.

(d) Effect of freezing rat liver on its catalase activity. In Figure 6, dilutional parallelism is demonstrated for rat liver homogenates and human erythrocyte lysates in the catalase assay. The linear regression line for activity versus concentration shows that dilutions of up to 8-fold over the range 0.1 to 1.1 units/mL produced 107% of the predicted activity for rat liver homogenates and 104% of the predicted activity for human erythrocyte lysates. Rapid freezing of human plasma and rat liver in liquid nitrogen followed by storage (1 week) at-80 °C had no discernible effect on catalase activity.

Figure 7 shows the plasma, liver, and erythrocyte activity of catalase in man, rats and dogs. (a) Rats aged 8 to 9 weeks compared to rats aged 6 months. (b) Plasma activity in man and dogs. (c) Erythrocyte activity in man, dogs, and rats. (d) Recovery of catalase activity added to rat liver homogenates. In Figure 7, plasma, erythrocyte and liver activities are compared for man, dog, and rats. Erythrocyte catalase activity in man was 18% lower than for rats but was 3.3-fold that for dogs (61.2 7.5 [47.6-70.4], 74.3 8.7 [60.6-89.4], 18.4 0.7 [17.4-19.5] units/g hemoglobin, respectively). Assuming average blood hemoglobin concentrations of 154,138,157 g/L and average hematocrits of 0.44,0.44,0.48 L/L, these activities are equivalent to 21.4,23.3, and 6.0 units/mL erythrocytes, respectively, for man, rats, and dogs. In contrast to erythrocytes, plasma activity of catalase was not significantly different between man and dogs (0.36 0.02 [0.33-0.40] and 0.34 0.04 [0.29-0.41] units/mL, respectively). Mean catalase activity for rat livers was 20.2 2.9 [17.3-24.2] and 31.9 4.9 [19.4-37.1] units/g wet weight tissue at ages 8 to 9 weeks and 6 months, respectively, indicating a maturation effect on hepatic catalase activity, increasing approximately 50% from 2 months of age to maturity. Recovery of catalase added to liver homogenates was 107%.

For estimates of analytical precision, within-run coefficients of variation (cv) were 2.1 % for the standard curve (based on the SD of differences between duplicates), and 2.2 % for replicates of a pool of rat liver homogenates (n=20). Between-run cv values were 3.1 % for standard curves (n=7) and 7.3% for a homogenate pool.