[Bicentennial of catalase research, 1818-2018]. / A katalázkutatás kétszáz éve, 1818­2018.

L. J. Thénard and J. L. Gay-Lussac discovered hydrogen peroxide in 1818. Later, Thénard noticed that animal and plant tissues decompose hydrogen peroxide. The substance which is responsible for this reaction was named as catalase by O. Loew in 1900. The catalase enzyme was regarded as a diagnostic and a tumour marker in the late years of the 19th century and in the early years of the 20th century. Acatalasemia, an inherited deficiency of enzyme catalase, was studied in Japan, Switzerland and Hungary. The recent findings on catalase are focusing on the effects of reactive oxygen species and on the association of acatalasemia and diabetes mellitus. Orv Hetil. 2018; 159(24): 959-964.

[Acatalasemia and type 2 diabetes mellitus]. / A veleszületett katalázhiány (acatalasaemia) és a 2-es típusú diabetes mellitus.

The catalase enzyme decomposes the toxic concentrations of hydrogen peroxide into oxygen and water. Hydrogen peroxide is a highly reactive small molecule and its excessive concentration may cause significant damages to proteins, deoxyribonucleic acid, ribonucleic acid and lipids. Acatalasemia refers to inherited deficiency of the catalase enzyme. In this review the authors discuss the possible role of the human catalase enzyme, the metabolism of hydrogen peroxide, and the phenomenon of hydrogen peroxide paradox. In addition, they review data obtained from Hungarian acatalasemic patients indicating an increased frequency of type 2 diabetes mellitus, especially in female patients, and an early onset of type 2 diabetes in these patients. There are 10 catalase gene variants which appear to be responsible for decreased blood catalase activity in acatalasemic patients with type 2 diabetes. It is assumed that low levels of blood catalase may cause an increased concentration of hydrogen peroxide which may contribute to the pathogenesis of type 2 diabetes mellitus.

Further acatalasemia mutations in human patients from Hungary with diabetes and microcytic anemia.

In blood, the hydrogen peroxide concentration is regulated by catalase. Decreased activity of catalase may lead to increased hydrogen peroxide concentration, which may contribute to the manifestation of age-related disease. The aim of this study is to examine association of decreased blood catalase activity and catalase exon mutations in patients (n=617) with diabetes (n=380), microcytic anemia (n=58), beta-thalassemia (n=43) and presbycusis (n=136) and in controls (n=295). Overall, 51 patients (8.3%) had less than half of normal blood catalase activity. Their genomic DNA was used for mutation screening of all exons and exon/intron boundaries with polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and PCR-heteroduplex analyses, and mutations were verified with nucleotide sequencing. Seven patients (type 2 diabetes (n=3), gestational diabetes (n=1), microcytic anemia (n=2)) had four novel catalase exon mutations namely, c.106_107insC, p.G36Afs*5(n=3, Hungarian type G1), c.379C>T, p.R127Y (n=2, Hungarian type H1), c.390T>C, p.R129L, (n=1, Hungarian type H2) and c.431A>T, p.N143V (n=1, Hungarian type H3). In patients with decreased blood catalase, the incidence of acatalasemia mutations was significantly high (P<0.0002) in microcytic anemia, type 2 and gestational diabetes. The four novel mutations were probably responsible for low blood catalase activity in 7/51 patients. In the remainder of the cases, other polymorphisms and epigenetic/regulatory factors may be involved.

Inherited catalase deficiency: is it benign or a factor in various age related disorders?

Hydrogen peroxide was - and is still - considered toxic for a wide range of living organisms. Oxidative stress occurs when there is an excess of pro-oxidants over antioxidants and it has been implicated in several diseases. Catalase is involved in hydrogen peroxide catabolism and is important in defense against oxidative stress. Acatalasemia means the inherited near-total deficiency of catalase activity, usually in reference to red cell catalase. Acatalasemia was thought at first to be an asymptotic disorder. In the absence of catalase, neither the Japanese, or Hungarian acatalasemics nor acatalasemic mice had significantly increased blood glutathione peroxidase activity. In animal models, catalase deficient tissues show much slower rates of removal of extracellular hydrogen peroxide. In catalase knock-out mice, a decreased hydrogen peroxide removing capacity and increased reactive oxygen species formation were reported. Hydrogen peroxide may cause methemoglobinemia in patients with catalase deficiency. During anesthesia for a Japanese acatalasemic patient the disinfection with hydrogen peroxide solution caused severe methemoglobinemia. Patients with inherited catalase deficiency, who are treated with uric acid oxidase (rasburicase) may experience very high concentrations of hydrogen peroxide and may suffer from methemoglobinemia and hemolysis. The high (18.5%) prevalence of diabetes mellitus in inherited catalase deficient individuals and the earlier (10 years) manifestation of the disease may be attributed to the oxidative damage of oxidant sensitive, insulin producing pancreatic beta-cells. Ninety-seven of 114 acatalasemics had diseases related to oxidative stress and aging. The oxidative stress due to catalase deficiency could contribute to the manifestation of diabetes while for the other diseases it may be one of the factors in their causations. In summary, inherited catalase deficiency is associated with clinical features, pathologic laboratory test results, age and oxidative stress related disorders. Rather than considering it a benign condition, it should be considered as a complicating condition for aging and oxidative stress.

Effects of rs769217 and rs1001179 polymorphisms of catalase gene on blood catalase, carbohydrate and lipid biomarkers in diabetes mellitus.

Oxidative stress and deficiency of the enzyme catalase, which is the primary scavenger of the oxidant H(2)O(2), may contribute to diabetes. The current study examined two polymorphisms in the catalase gene, -262C>nT in the promoter and 111C>T in exon 9, and their effects on blood catalase activity as well as on concentrations of blood glucose, haemoglobin A1c, triglyceride, cholesterol, HDL, LDL, ApoA-I and ApoB. Subjects were type-1 and type-2 diabetics. We evaluated PCR-single strand conformational polymorphism for 111C>T and PCR-restriction fragment length polymorphism for - 262C>T. TT genotype frequency of 111C>T polymorphism was increased in type-1 diabetes. Type-2 diabetics with the CC or CT genotypes had decreased catalase and increased glucose, hemoglobinA1c and ApoB. Type-2 diabetics who have TT genotype in -262C>T may have elevated risk for diabetes complications; these patients had the lowest mean catalase and HDL, as well as the highest glucose, haemoglobin A1c, cholesterol and ApoB.

Acatalasemia and diabetes mellitus.

The enzyme catalase catalyzes the breakdown of hydrogen peroxide into oxygen and water. It is the main regulator of hydrogen peroxide metabolism. Hydrogen peroxide is a highly reactive small molecule formed as a natural byproducts of energy metabolism. Excessive concentrations may cause significant damages to protein, DNA, RNA and lipids. Low levels in muscle cells, facilitate insulin signaling. Acatalasemia is a result of the homozygous mutations in the catalase gene, has a worldwide distribution with 12 known mutations. Increased hydrogen peroxide, due to catalase deficiency, plays a role in the pathogenesis of several diseases such as diabetes mellitus. Diabetes mellitus is a disorder caused by multiple genetic and environmental factors. Examination of Hungarian diabetic and acatalasemic patients showed that an increased frequency of catalase gene mutations exists among diabetes patients. Inherited catalase deficiency may increase the risk of type 2 diabetes mellitus, especially for females. Early onset of type 2 diabetes occurs with inherited catalase deficiency. Low levels of SOD and glutathione peroxidase could contribute to complications caused by increased oxidative stress.

A simple method for examination of polymorphisms of catalase exon 9: rs769217 in Hungarian microcytic anemia and beta-thalassemia patients.

Catalase decreases the high, toxic concentrations of hydrogen peroxide but it lets the physiological, low concentrations in the cells mainly for signaling purposes. Its decreased activity may contribute to development of several pathological conditions. Catalase mutations occur frequently in exon 9, these were examined with different, complicated and costly methods. The aim of the current study was to evaluate a method for screening of polymorphisms in catalase exon 9. We used the slab gel electrophoresis of PCR amplicons without denaturation and silver staining for visualization of the DNA bands. We detected extra DNA bands in the 400-800 bp region of the catalase exon 9. Their single stranded nature was proved with nucleotide sequence analyses, comparison with the standard SSCP, staining with Sybr Green II and Sybr Green I, ethidium bromide, no digestion with RFLP (BstX I), and digestion with plant nuclease. We used this method for examination of polymorphisms of catalase exon 9 in microcytic anemia and beta-thalassemia patients. The lowest blood catalase activities were detected in microcytic anemia and beta-thalassemia patients with the TT genotypes of the C111T polymorphism. This method was sensitive for detection of G113A acatalasemia mutation, but poorly detected C37T and G5A acatalasemia mutations.

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.

[Analytical problems in determination of hemoglobin A1c and the different ways of its interpretation]. / A hemoglobin-A1c-meghatározás analitikai problémái és az eredményközlés gondjai.

Glycated proteins are formed during the nonenzymatic reaction of glucose and amino groups of proteins. Hemoglobin A1c is formed by the condensation of glucose with the N-terminal valine residue of each beta-chain of hemoglobin A. The amount of glycated hemoglobin in blood depends on both life-span of red blood cells and blood glucose concentration. As the rate of formation of hemoglobin A1c is directly proportional to the concentration of glucose in the blood, it represent the integrated values for glucose over the preceding 6 to 8 weeks. Hemoglobin A1c determination is widely used for monitoring long-term glycemic control, and it is a risk factor for complications of diabetes. The concentration of blood hemoglobin A1c depends on further factors such as half-life of hemoglobin, blood carbohydrates, blood analytes, methods of determination and calibration. Committees were established under the auspices of the American Association of Clinical Chemistry, American Diabetes Association, International Federation of Clinical Chemistry (IFCC) to standardize HbA1c assays (DCCT: Diabetes Control and Complications Trial, NGSP: National Glycohemoglobin Standardization Program, IFCC reference method for measurement of HbA1c). The NGSP recommends to report HbA1c result in % (g HbA1c/g hemoglobin) while IFCC suggests mmol HbA1c/mol hemoglobin A. Reports are presenting mathematical relationship between HbA1c and average glucose concentration in blood, however, the clinical usefulness of estimating average serum glucose from HbA1c level is under discussion.

[Rasburicase therapy may cause hydrogen peroxide shock]. / Az uratoxidaz- (rasburicas-) terápia lehetséges szövôdménye a hidrogen-peroxid okozta sokk.

Hyperuricemia contributes to the pathomechanism of diseases such as renal failure, gout, tumor lysis syndrome and metabolic syndrome. Tumor lysis syndrome is a complication of malignancies caused by massive tumor cell lysis due to either spontaneous tumor cell lysis or to different therapies and it may cause hyperuricemia. Recently, for treatment of hyperuricemia the recombinant urate oxidase (rasburicase) therapy has been used. This enzyme converts uric acid with high affinity into soluble allantoin which is eliminated by the kidneys. In this reaction high concentration of hydrogen peroxide is generated. This hydrogen peroxide could cause hemolysis and especially methemoglobin formation, in case of glucose-6-phosphate-dehydrogenase and catalase deficiencies. Therefore it is recommended that these enzymes are determined before therapy. For monitoring of rasburicase therapy the determination of serum uric acid concentration is used. More than 95 per cent of Hungarian clinical laboratories are using the uricate oxidase/peroxidase reactions and hydrogen peroxide measurements in the uric acid assays. These assays may be interfered by ascorbic acid and hydrogen peroxide which is generated by rasburicase either in vivo or in vitro.

Catalase deficiency may complicate urate oxidase (rasburicase) therapy.

Patients with low (inherited and acquired) catalase activities who are treated with infusion of uric acid oxidase because they are at risk of tumour lysis syndrome may experience very high concentrations of hydrogen peroxide. They may suffer from methemoglobinaemia and haemolytic anaemia which may be attributed either to deficiency of glucose-6-phosphate dehydrogenase or to other unknown circumstances. Data have not been reported from catalase deficient patients who were treated with uric acid oxidase. It may be hypothesized that their decreased blood catalase could lead to the increased concentration of hydrogen peroxide which may cause haemolysis and formation of methemoglobin. Blood catalase activity should be measured for patients at risk of tumour lysis syndrome prior to uric acid oxidase treatment.

Detection of a novel familial catalase mutation (Hungarian type D) and the possible risk of inherited catalase deficiency for diabetes mellitus.

The enzyme catalase is the main regulator of hydrogen peroxide metabolism. Recent findings suggest that a low concentration of hydrogen peroxide may act as a messenger in some signalling pathways whereas high concentrations are toxic for many cells and cell components. Acatalasemia is a genetically heterogeneous condition with a worldwide distribution. Yet only two Japanese and three Hungarian syndrome-causing mutations have been reported. A large-scale (23 130 subjects) catalase screening program in Hungary yielded 12 hypocatalasemic families. The V family with four hypocatalasemics (60.6 +/- 7.6 MU/L) and six normocatalasemic (103.6 +/- 23.5 MU/L) members was examined to define the mutation causing the syndrome. Mutation screening yielded four novel polymorphisms. Of these, three intron sequence variations, namely G-->A at the nucleotide 60 position in intron 1, T-->A at position 11 in intron 2, and G-->T at position 31 in intron 12, are unlikely to be responsible for the decreased blood catalase activity. However, the novel G-->A mutation in exon 9 changes the essential amino acid Arg 354 to Cys 354 and may indeed be responsible for the decreased catalase activity. This inherited catalase deficiency, by inducing an increased hydrogen peroxide steady-state concentration in vivo, may be involved in the early manifestation of type 2 diabetes mellitus for the 35-year old proband.

Catalase enzyme mutations and their association with diseases.

Enzyme catalase seems to be the main regulator of hydrogen peroxide metabolism. Hydrogen peroxide at high concentrations is a toxic agent, while at low concentrations it appears to modulate some physiological processes such as signaling in cell proliferation, apoptosis, carbohydrate metabolism, and platelet activation. Benign catalase gene mutations of 5' noncoding region (15) and intron 1 (4) have no effect on catalase activity and are not associated with disease. Catalase gene mutations have been detected in association with diabetes mellitus, hypertension, and vitiligo. Decreases in catalase activity in patients with tumors is more likely to be due to decreased enzyme synthesis rather than to catalase mutations.Acatalasemia, the inherited deficiency of catalase has been detected in 11 countries. Its clinical features might be oral gangrene, altered lipid, carbohydrate, homocysteine metabolism and the increased risk of diabetes mellitus. The Japanese, Swiss, and Hungarian types of acatalasemia display differences in biochemical and genetic aspects. However, there are only limited reports on the syndrome causing these mutations. These data show that acatalasemia may be a syndrome with clinical, biochemical, genetic characteristics rather than just a simple enzyme deficiency.

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.

[Proteomics in clinical enzymology: polymorphism of creatine kinase and alkaline phosphatase]. / A fehérjekutatás a klinikai enzimológiai gyakorlatban: a kreatinkináz és alkalikus foszfatáz enzimek polimorfizmusa.

INTRODUCTION: The clinical proteomics is dedicated to the use of the proteomics in the clinical laboratory science. AIMS: Examples for the importance of clinical proteomics are provided by studies on the polymorphisms of creatine kinase and alkaline phosphatase enzymes. METHODS: The different (immunoinhibition, electrophoretic, immunochemistry) assays for determination of a cardiac marker (creatine kinase 2) are compared in hospital patients. Isoform analysis of alkaline phosphatase is performed in benign, transient hyperphosphatasemia. RESULTS: The author reviews recent knowledge on polymorphism of creatine kinase (isoenzymes, isoforms, macro types), and alkaline phosphatase (isoenzymes, their cancer related variants, isoforms). The origin of falsely high cardiac marker (creatine kinase 2) determined by the immunoinhibition assay could be related to the presence of macro creatine kinase (both types), creatine kinase isoenzyme 1, and their mixtures. After reviewing the recent findings on the syndrome of benign, transient hyperphosphatasemia, a case of a 1-year-old Hungarian boy with this syndrome is presented. CONCLUSIONS: The author points out that the appropriate use of clinical proteomics (the polymorphisms of creatine kinase and alkaline phosphatase enzymes) may improve the diagnostic efficiency of the clinical laboratory tests.