Tissue and organ expression of catalase in acatalasemic beagle dogs.

Acatalasemic Beagle dogs which were maintained in our laboratories showed no sign of catalase activity at all in the erythrocytes, and glutathione peroxidase and superoxide dismutase were at normal levels. Immunoblotting analysis demonstrated that no catalase protein is detectable in their erythrocytes. On the other hand, catalase activity was detected in other tissues and organs, albeit at varying, lower levels than in normal dogs. Quantitative immunoblotting analysis consistently demonstrated that the catalase protein is expressed in the liver and kidneys of acatalasemic dogs in proportion to the activity in these organs. The catalase mRNA expressions in the blood, liver and kidneys in acatalasemic dogs were almost the same as those in normal dogs. These results suggested that catalytically normal catalase protein is translated from mRNA in the tissues and organs including erythrocytes, but in erythrocytes this enzyme protein is disposed of by an unknown mechanism.     

Immunotitration of the catalase in the blood of Japanese subjects and mice suffering from acatalasemia and hypocatalasemia.

Catalase in hemolysates of normal, heterozygous hypocatalasemic and acatalasemic Japanese was immunotitrated with an anti-human blood catalase rabbit serum. Equivalence points were calculated from the regression lines between catalase activity added and catalase activity remaining in the supernatant. Catalase activities at the equivalence points of Japanese normal, hypocatalasemia and acatalasemia were similar. The results indicate that the specific activities of catalase in the normal and of the variant bloods are identical. Catalase in hemolysates of normal and variant mice was immunotitrated with an anti-mouse liver catalase rabbit serum. In contrast to Japanese acatalasemic subject, the equivalence points of catalase in heterozygous hypocatalasemic, homozygous hypocatalasemic, acatalasemic and normal hemolysates were different, and the ratios of specific activity in these variant mice to that in normal were 0.72, 0.46 and 0.21, respectively. The differences in catalase activities at equivalence points were also supported by the statistical analysis on parameters of regression lines of catalase activities remaining in the supernatant on catalase activities added in the immunotitration. These findings suggest that the molecular properties of residual catalase of Japanese acatalasemia and those of mouse acatalasemia are entirely different.     

Increased erythrocyte catalase activity in patients with hyperthyroidism

Levels of human erythrocyte catalase activity were determined in 38 patients with thyroidal dysfunction. In patients with hyperthyroidism, erythrocyte catalase activities were found to be higher than the levels of normal subjects (P less than 0.001). In hypothyroidism, erythrocyte catalase activities were of the same order as those of normal subjects. Significantly high positive correlation was found between erythrocytes catalase activity and the levels of thyroxine (r = 0.5794, n = 36, P less than 0.001), and slight positive correlation was detected between catalase activity and the levels of triiodothyronine (r = 0.3978, n = 33, P less than 0.05). A decreased erythrocyte catalase activity was observed when erythrocytes lysate was incubated with thyroid hormones. It was suggested that erythrocyte catalase activity had close relationship with thyroid state, however, direct effect of thyroid hormones were not observed on erythrocyte catalase assay system in vitro.     

Human aging brain disorders: Role of antioxidant enzymes

In order to investigate the role of two free radical detoxificant enzymes in patients with aging brain disorders, superoxide dismutase (SOD) and catalase (CAT) activities have been measured in blood from male and female human patients of different ages with several types of aging brain disorders. When compared with activities in the normal population, we have detected: 1) SOD and CAT activities are decreased in patients with Parkinson disease. 2) SOD activity seems to be normal and CAT activity is decreased in patients with dementia. 3) In the patients with stroke, SOD activity is normal, while CAT activity is decreased. SOD activity was measured in red blood cells using the Minami and Yoshikawa method. CAT activity was measured in hemolysates by the method of Aebi. We can conclude that SOD and CAT activities in patients with Parkinson disease are decreased.     

Properties of erythrocyte catalase from homozygotes and heterozygotes for Swiss-type acatalasemia

The unstable catalase variant found in the blood of individuals homozygous for Swiss-type acatalasemia and the enzyme species present in heterozygous carriers of this rare defect have been further characterized. The mutant enzyme isolated from acatalasemic red cells is considerably more heat labile and differs in electrophoretic mobility from the normal enzyme. Catalase preparations obtained from heterozygotes consist of an apparently uniform enzyme species, probably representing a molecular hybrid, with properties intermediate to those of the normal and the variant enzyme. However, antigenic identity of catalase from all three sources is observed. Model experiments indicate that hybrid catalase molecules can be produced by recombining normal and variant dimer subunits. Fractionation of erythrocytes according to density and age shows that most of the residual catalase activity is localized in juvenile acatalasemic cells, whereas in normal and heterozygous individuals the catalase activity level does not alter significantly during the life span of the red cells. These findings agree with the observation that there is no gene dosage in heterozygotes, their catalase activity values falling within the normal range.     

Catalase activity measured with a micro oxygen electrode in a pressurized reaction vessel

The assembly and the use of a simple airtight pressurized reaction vessel are described for the measurement of catalase activity with a micro oxygen electrode in an optically heterogenous medium. The oxygen concentration is expressed as the ratio of observed current to the current in an air-saturated solution. Thus, an individual standard can be obtained for each measurement and the calibration is less affected by changes in the amplification factor. Different procedures for calibration of the oxygen electrode were compared. Specific activities of crystalline catalase, of red blood cells from humans and from normal or acatalasemic mice, and of liver homogenates from normal or amino-triazole-pretreated rats were determined. The specific catalase activity of human erythrocytes, as found by this method, agrees with that obtained photometrically.     

Effect of C111T polymorphism in exon 9 of the catalase gene on blood catalase activity in different types of diabetes mellitus

Hydrogen peroxide plays a major role in the pathomechanism of diabetes mellitus and its main regulator is enzyme catalase. The blood catalase and the C111T polymorphism in exon 9 was examined in type 1, type 2 and gestational diabetes mellitus. Compared to the control group (104.7 +/- 18.5 MU/l) significantly decreased (p < 0.001) blood catalase activities were detected in type 2 (71.2 +/- 14.6 MU/l), gestational (68.5 +/- 12.2 MU/l) diabetes mellitus and without change in type 1 (102.5 +/- 26.9 MU/l). The blood catalase decreased (p = 0.043) with age for type 2 diabetics and did not change (p>0.063) for type 1, gestational diabetic patients and controls. Blood catalase showed a weak association with hemoglobin A1c for type 1 diabetic patients (r = 0.181, increasing). The mutant T allele was increased in type 1 and gestational diabetes mellitus, and CT+TT genotypes showed decreased blood catalase activity for type 1 and increased activities for type 2 diabetic patients. The C111T polymorphism may implicate a very weak effect on blood catalase activity in different types of diabetes mellitus.     

Identification of two highly divergent catalase genes in the fungal tomato pathogen, Cladosporium fulvum.

Catalases of pathogenic micro-organisms have attracted attention as potential virulence factors. Homology-based screens were performed to identify catalase genes in the fungal tomato pathogen Cladosporium fulvum. Two highly divergent genes, Cat1 and Cat2, were isolated and characterized. Cat1 codes for a putative 566-amino-acid catalase subunit and belongs to the gene family that also encodes the mainly peroxisome-localized catalases of animal and yeast species. Cat2 codes for a putative catalase subunit of 745 amino acids and belongs to a different gene family coding for the large-subunit catalases similar to ones found in bacteria and filamentous fungi. Neither catalase had an obvious secretory signal sequence. A search for an extracellular catalase was unproductive. The Cat1 and Cat2 genes showed differential expression, with the Cat1 mRNA preferentially accumulating in spores and the Cat2 mRNA preferentially accumulating in response to external H(2)O(2). With Cat2-deleted strains, activity of the Cat2 gene product (CAT2) was identified among four proteins with catalase activity separated on non-denaturing gels. The CAT2 activity represented a minor fraction of the catalase activity in spores and H(2)O(2)-stressed mycelium, and no phenotype was observed for Cat2-deleted strains, which showed a normal response to H(2)O(2) treatment. These results indicate the existence of a complex catalase system in C. fulvum, with regard to both the structure and regulation of the genes involved. In addition, efficient C. fulvum gene-replacement technology has been established.     

A quantitative genetic analysis of tissue-specific catalase activity inMus musculus

Tissue-specific catalase activity in 3-week-old animals from inbred mouse strains 129/ReJ, BALB/c, C3H/HeAnl/Cas-1^b, C3H/HeSnJ, C3H/S, C57BL/6J, and Swiss-Webster was found to be highly variable by analysis of variance (P=0.01). Appropriate crosses were made among strains which were classified as normal (BALB/c, C3H/HeSnJ, C3H/S), hypocatalasemic (129/ReJ, C57BL/6J), and acatalasemic (C3H/HeAnl/Cas-1^b) with respect to blood catalase activity to study the inheritance of the blood, kidney, liver, and lung catalase activity levels in a number of generations (reciprocal F₁'s, F₂, two backcrosses —BC₁ and BC₂— and some RI lines). Segregation analysis and statistical methods which tested different models of inheritance as well as calculations of heritability were used in an effort to assess and evaluate genetic parameters that affect catalase activity. Results indicate that the inheritance of blood catalase activity in the cross involving acatalasemic and normal (BALB/c, C3H/HeSnJ) strains is compatible with the single-locus difference between the parental strains; however, the difference between the acatalasemic and the hypocatalasemic strain (C57BL/6J) would require additional genetic interaction for a satisfactory explanation. A similar pattern of generalization also applies to the inheritance of kidney catalase activity. The segregation pattern for the liver and lung catalase activity in most crosses is significantly different from the expectations of the single locus model. These results are compatible with the concept that a number of genes must affect tissue-specific catalase activity in mice. These may include previously described (e.g., Ce-1 and Ce-2) or novel genetic regulators/modifiers which interact with a single structural gene (Cas-1) or its product to produce the catalase phenotype characteristic of specific tissues in each strain.This investigation was supported by a Natural Sciences and Engineering Research Council of Canada operating grant to S.M.S.     

Properties of residual catalase in the erythrocytes of Japanese-type acatalasemia

Summary In Japanese-type acatalasemia erythrocytes, the presence and properties of residual catalase were determined and compared with those of normal erythrocyte catalase. Residual catalase activity was proved by titration, active staining after polyacrylamide gel electrophoresis, and measurement of oxygen evolution. Residual catalase protein, demonstrated by double immunodiffusion, was similar to that of normal catalase. The properties of residual catalase activity were identical with those of normal catalase activity. It occurred as three fractions of equal specific activity by DEAE column chromatography. These observations suggest that Japanese-type acatalasemia contains residual catalase with properties similar to those of normal catalase.     

Effect of C111T polymorphism in exon 9 of the catalase gene on blood catalase activity in different types of diabetes mellitus

Hydrogen peroxide plays a major role in the pathomechanism of diabetes mellitus and its main regulator is enzyme catalase.

The blood catalase and the C111T polymorphism in exon 9 was examined in type 1, type 2 and gestational diabetes mellitus.

Compared to the control group (104.7 ± 18.5 MU/l) significantly decreased (p < 0.001) blood catalase activities were detected in type 2 (71.2 ± 14.6 MU/l), gestational (68.5 ± 12.2 MU/l) diabetes mellitus and without change in type 1 (102.5 ± 26.9 MU/l). The blood catalase decreased (p = 0.043) with age for type 2 diabetics and did not change (p>0.063) for type 1, gestational diabetic patients and controls. Blood catalase showed a weak association with hemoglobin A1c for type 1 diabetic patients (r = 0.181, increasing).

The mutant T allele was increased in type 1 and gestational diabetes mellitus, and CT+TT genotypes showed decreased blood catalase activity for type 1 and increased activities for type 2 diabetic patients.

The C111T polymorphism may implicate a very weak effect on blood catalase activity in different types of diabetes mellitus.     

Effect of age, sex, and smoking on serum catalase activity

In selected hospitalized patients (9294) with normal serum catalase activity, 7.58 per cent higher (p less than 0.001) serum catalase activity was detected in males than in females. With age, serum catalase activity decreased (slope-0.0683) in males, increased (slope 0.101) in females and did not change in all patients (slope 0.049). Cigarette smoking did not influence serum catalase activity (p greater than 0.05).     

Catalase −262C>T polymorphisms in Hungarian vitiligo patients and in controls: further acatalasemia mutations in Hungary

Catalase is the main regulator of hydrogen peroxide metabolism. In vitiligo patients there are conflicting data on its activity and no data on the effect of −262C>T polymorphism in the catalase gene. Blood catalase activity, −262C>T polymorphism and acatalasemia mutations were examined in 75 vitiligo patients and in 162 controls, in Hungary. We measured blood catalase activity and conducted analyses with PCR-SSCP, polyacrylamide gel electrophoresis and silver staining in combination with RFLP and nucleotide sequencing. Comparison of the wild (CC) genotype and the mutant (TT) genotype in the vitiligo patients revealed a non significant (P > 0.19) increase in blood catalase. Male controls with the CT genotype had significantly (P < 0.04) lower blood catalase activity than CC genotype controls. Female vitiligo patients with CC genotype had lower (P < 0.04) blood catalase than female controls. The frequency of wild genotype (CC) and C alleles is significantly (P < 0.04) decreased in Hungarian controls when compared to controls in Slovenia, Morocco, UK, Greece, Turkey, USA, China. The detection of a novel acatalasemia mutation (37C>T in exon 9) and the 113G>A (exon 9) mutation in Hungary are further proofs of genetic heterogeneity origin of acatalasemia mutations. In conclusion, the −262 C>T polymorphism has a reverse effect on blood catalase in vitiligo patients and in controls. In controls the mutant genotypes and alleles are more frequent in Hungary than in several other populations. The new acatalasemia mutations are further examples of heterogeneity of acatalasemia.     

The inherited enzymatic defect in porphyria variegata

Summary Protoporphyrinogen oxidase activity and ferrochelatase activity have been measured in blood lymphocytes from patients with porphyria variegata, and from some members of the family of one patient; the mean activity of protoporphyrinogen oxidase from patients was about 50% of that in lymphocytes from normal subjects; similar results were obtained from asymptomatic carriers in two generations of the patient's family. This finding confirms that a protoporphyrinogen oxidase decreased activity reflects the primary genetic defect in Porphyria Variegata. Data of ferrochelatase activity have been found usually in the normal range and these results are discussed.     

Catalase levels in patients with aniridia and/or Wilms' tumor: utility and limitations

The gene for red blood cell (RBC) catalase has recently been mapped to 11p13, and a gene dosage effect has been demonstrated for individuals with triplication or deletion of that region. Deletion of the 11p13 band has also been associated with aniridia, with and without Wilm's tumor. We studied the RBC catalase levels in individuals without detectable chromosomal abnormalities but with aniridia, Wilm's tumor, and the combination of aniridia and Wilms' tumor, to determine whether catalase levels might provide evidence for a submicroscopic chromosomal deletion in the 11p13 region. All karyotypically normal patients were found to have normal catalase levels.     

The influence of progressive growth on the specific catalase activity of human diploid cell strains. I. Effect of cellular genotype: Homozygous strains

The specific catalase activity of human diploid cell strains increases with progressive growth of the culture, and falls again following subculture. Although the increase is small, it is readily demonstrable, and is exponential with time. The response of catalase activity to proggressive growth of the culture was studied in three abnormal human cell lines. A diploid cell strain, developed from a patient homozygous for the gene causing acatalasia I, had no detectable catalase activity throughout the life cycle of the culture. Another diploid cell strain, developed from a patient homozygous for the gene causing acatalasia II, had about 5% normal catalase activity, but the proportionate increase in specific activity as the culture grew was the same as for normal cells. Thus the mutation causing acatalasia II does not change the responsiveness of the cell in terms of catalase activity to progressive growth of the culture. The behavior of a heteroploid line was similar to that of the normal diploid strains, but when the growth of the heteroploid cultures reached a plateau, their population densities were four times higher than those of the diploid strains and they had about twice the specific catalase activity.     

The effects of hydrogen peroxide promoted by homocysteine and inherited catalase deficiency on human hypocatalasemic patients

Elevated plasma homocysteine can generate oxygen free radicals and hydrogen peroxide. The enzyme catalase is involved in the protection against hydrogen peroxide. We examined the effect of oxidative stress promoted by homocysteine on erythrocyte metabolism (blood hemoglobin, MCV, folate, B12, serum LDH, LDH isoenzymes, haptoglobin) in the oxidative stress sensitive Hungarian patients with inherited catalase deficiency. The plasma homocysteine (HPLC method, Bio-Rad), folate, B12 (capture binding assay, Abbott), blood hemoglobin concentrations, blood catalase activity (spectrophotometric assay of hydrogen peroxide), and MCV values were determined in 7 hypocatalasemic families including hypocatalasemic (male:12, female:18) patients and their results were compared to those of the normocatalasemic (male:17 female: 12) family members. We found decreased (p <.036) folate (ng/ml) concentrations (male hypocatalasemic 5.44 +/- 2.81 vs. normocatalasemic 7.56 +/- 1.97, female 5.01 +/- 1.93 vs. 6.61 +/- 1.91), blood hemoglobin (p <.010, male:140.2 +/- 11.0 vs. 153.6 +/- 11.6 g/l, female: 128.4 +/- 10.9 vs. 139.6 +/- 9.2 g/l). Increased levels of MCV (p <.001) were detected in hypocatalasemic patients (male: 98.6 +/- 3.4 vs. 90.1 +/- 7.5 fl, female: 95.9 +/- 3.9 vs. 90.1 +/- 2.5 fl), plasma homocysteine (p <.049, male: 9.72 +/- 3.61 vs. 7.36 +/- 2.10 umol/l, female: 9.06 +/- 3.10 vs. 6.84 +/- 2.50 umol/l) and not significant (p >.401) plasma B12 (male: 336 +/- 108 vs. 307 +/- 76 pg/ml, female: 373 +/- 180 vs. 342 +/- 75 pg/ml). The serum markers of hemolysis (LDH, LDH isoenzymes, haptoglobin) did not show significant (p >.228) signs of oxidative erythrocyte damage. We report firstly on increased plasma homocysteine concentrations in inherited catalase deficiency. The increased plasma homocysteine and inherited catalase deficiency together could promote oxidative stress via hydrogen peroxide. The patients with inherited catalase deficiency are more sensitive to oxidative stress of hydrogen peroxide than the normocatalasemic family members. This oxidative stress might be responsible for the decreased concentration of the blood hemoglobin via the oxidation sensitive folate and may contribute to the early development of arteriosclerosis and diabetes in these patients.     

Relationship of hydrogen peroxide production by Mycoplasma pulmonis to virulence for catalase-deficient mice

Mycoplasma pulmonis, an etiological agent of murine pneumonia, produced about 0.065 mumoles of hydrogen peroxide (H(2)O(2)) per hr per 10(10) colony-forming units. When glucose was present at a concentration of 0.01 m, H(2)O(2) production was increased by 50%. To determine if H(2)O(2) production by M. pulmonis could be correlated with virulence, normal, acatalasemic, and acatalatic mice were infected with the organism. Three days after infection with M. pulmonis significantly more acatalatic mice had pneumonia than did normal or acatalasemic mice. The pneumonia in acatalatic mice was also more severe than in the other two groups. Five days after infection, pneumonia in the acatalatic mice was resolved, whereas normal mice were severely affected. The presence of pneumonia and the severity were correlated with the recovery of M. pulmonis from the lesions. In vitro studies of the effect of catalase on M. pulmonis showed that exogenously supplied catalase stimulated the growth of M. pulmonis at 37 C and prolonged its survival at 25 C. Hemolysis of sheep blood, guinea pig blood, rabbit blood, and normal and acatalasemic mouse blood by M. pulmonis was inversely related to the catalase activity of the erythrocytes. These findings suggest that H(2)O(2) secretion contributes to the virulence of M. pulmonis and to the death of the microorganism in the absence of host catalase.     

Curcumin and its synthetic analogue dimethoxycurcumin differentially modulates antioxidant status of normal human peripheral blood mononuclear cells

Curcumin is a polyphenol derived from the herb Curcuma longa, which has been extensively studied in terms of its antitumour, antioxidant, and chemopreventive activity as well as various other effects. In the present work we compared curcumin with its synthetic analogue dimethoxycurcumin (dimc) in terms of its antioxidant enzyme-modulating effects in human peripheral blood mononuclear cells (PBMC). We found that these compounds modulate antioxidant enzymes differentially. Both curcumin and dimethoxycurcumin effected a decrease in lipid peroxidation status in PBMC, however, curcumin had better activity in this regard. An increase in the activity of catalase was seen in the case of curcumin-treated PBMC, whereas dimc increased catalase activity significantly to almost twofold level. Real time-polymerase chain reaction (RT-PCR) analysis revealed significant up-regulation of catalase at mRNA level post treatment with curcumin as well as dimc, however, dimc had better activity in this regard. Glutathione reductase (GR) activity and reduced glutathione levels increased in the case of peripheral blood mononuclear cells (PBMC) treated with curcumin, however, the trend was reversed with dimethoxycurcumin where, both glutathione reductase activity and reduced glutathione levels were significantly reduced. RT-PCR analysis of glutathione reductase mRNA levels showed decrease in mRNA levels post treatment with dimethoxycurcumin (dimc) further corroborating GR enzyme assay results, however, we could not obtain significant result post curcumin treatment. NFkB reporter assay and western blot analysis of nuclear as well as cytosolic fractions of NFkB revealed that curcumin inhibits NFkB activation whereas inhibition was much less with dimc. It has been reported that curcumin and dimc exerts differential cytotoxicity in normal and tumour cells and the reason for this had been attributed to the differential uptake of these compounds by normal cells and tumour cells. Based on our results we propose that differential modulation of antioxidant enzymes via NFkB pathway could be the reason behind differential cytotoxicity of dimc as well as curcumin in normal cells and tumour cells in addition to differential uptake of these compounds as reported previously.