Carbonic anhydrase I and II autoantibody levels in primary hypertension: our preliminary results.

OBJECTIVE: The pathogenesis of primary hypertension (HT) is still not completely clear, although autoimmunity has been implicated in recent years. Carbonic anhydrase (CA) is an enzyme involved in a number of important metabolic processes. CA I and II autoantibodies have been linked to various autoimmune diseases. However, CA I and II autoantibody levels in primary HT have not been previously investigated. The purpose of this study was, therefore, to investigate levels of CA I and II autoantibodies in primary HT. PATIENTS AND METHODS: Fifty-six patients newly diagnosed with primary HT and 33 healthy individuals were included in the study. Twenty-four-hour ambulatory blood pressure monitoring was performed following office controls. Blood specimens were collected under appropriate conditions for CA I and II autoantibody level investigation and biochemical tests. Urine sodium and protein excretion were measured after 24 h. Demographic and biochemical parameters and CA I and II autoantibody levels were then compared between the patient and healthy groups. RESULTS: CA II autoantibody and uric acid levels were significantly higher in the hypertensive group than in the control group (p=0.005, and p<0.001, respectively). CA II autoantibody (exp ß: 79.06 CI: 4.44-1407.02) (p=0.003) and uric acid elevation (exp ß: 2.10 CI: 1.31- 3.34) (p=0.002) were identified as independent predictors of HT development at logistic regression analysis. CONCLUSIONS: CA II autoantibody levels were higher in hypertensive patients, and this elevation is an independent predictor of HT development.

Carbonic anhydrase I and II autoantibodies in Behçet's disease.

BACKGROUND: Behçet's disease is a vasculitis, seen more frequently around the Mediterranean and the Far East, and evinces with oral and genital ulcerations, skin lesions and uveitis. Carbonic anhydrase (CA) is a metalloenzyme which is widely distributed in the living world, and it is essential for the regulation of acid-base balance. Anti-CA antibodies have been reported in many disorders, such as systemic lupus erythematosus, Sjögren's syndrome, rheumatoid arthritis, endometriosis, idiopathic chronic pancreatitis, type 1 diabetes and Graves' disease. The goal of this study was to investigate CA I and II autoantibodies in Behçet's disease (BD). METHODS: 35 patients with BD and 29 healthy controls were included in the study and CA I and II autoantibody levels were investigated by ELISA. RESULTS: The CA I and II autoantibody levels of BD group were significantly higher than the healthy group (p=0.013, p inf 0.0001, respectively). A cut-off value of 0.250 ABSU for anti-CA I was associated with 34 % sensitivity and 100 % specificity and a cut-off value of 0.171 ABSU for anti-CA II was associated with 54 % sensitivity and 100 % specificity for predicting BD. CONCLUSION: The CA I and II autoantibody levels in patients with BD were found higher compared to control group and the results suggest that CA I and II autoantibodies may be involved in the pathogenesis of BD.

Specific alterations in plasma proteins during depressed, manic, and euthymic states of bipolar disorder

Bipolar disorder (BD) is a common psychiatric mood disorder affecting more than 1-2% of the general population of different European countries. Unfortunately, there is no objective laboratory-based test to aid BD diagnosis or monitor its progression, and little is known about the molecular basis of BD. Here, we performed a comparative proteomic study to identify differentially expressed plasma proteins in various BD mood states (depressed BD, manic BD, and euthymic BD) relative to healthy controls. A total of 10 euthymic BD, 20 depressed BD, 15 manic BD, and 20 demographically matched healthy control subjects were recruited. Seven high-abundance proteins were immunodepleted in plasma samples from the 4 experimental groups, which were then subjected to proteome-wide expression profiling by two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight tandem mass spectrometry. Proteomic results were validated by immunoblotting and bioinformatically analyzed using MetaCore. From a total of 32 proteins identified with 1.5-fold changes in expression compared with healthy controls, 16 proteins were perturbed in BD independent of mood state, while 16 proteins were specifically associated with particular BD mood states. Two mood-independent differential proteins, apolipoprotein (Apo) A1 and Apo L1, suggest that BD pathophysiology may be associated with early perturbations in lipid metabolism. Moreover, down-regulation of one mood-dependent protein, carbonic anhydrase 1 (CA-1), suggests it may be involved in the pathophysiology of depressive episodes in BD. Thus, BD pathophysiology may be associated with early perturbations in lipid metabolism that are independent of mood state, while CA-1 may be involved in the pathophysiology of depressive episodes.

CA1 contributes to microcalcification and tumourigenesis in breast cancer.

BACKGROUND: Although mammary microcalcification is frequently observed and has been associated with poor survival in patients with breast cancer, the genesis of calcification remains unclear. Carbonic anhydrase I (CA1) has been shown to promote calcification by catalysing the hydration of CO2. This study aimed to determine whether CA1 was correlated with microcalcification and with other processes that are involved in breast cancer tumourigenesis. METHODS: CA1 expression in breast cancer tissues and blood samples was detected using western blotting, real-time PCR, immunohistochemistry and ELISA. Calcification was induced in the cultured 4T1 cell line originating from mouse breast tumours, using ascorbic acid and ß-glycerophosphate. Acetazolamide, a chemical inhibitor of CA1, was also added to the culture to determine the role of CA1 in calcification. The MCF-7 human breast cancer cell line was treated with anti-CA1 siRNA and was assessed using a CCK-8 cell proliferation assay, an annexin V cell apoptosis assay, transwell migration assay and a human breast cancer PCR array. The tag SNP rs725605, which is located in the CA1 locus, was genotyped using TaqMan® genotyping. RESULTS: Increased CA1 expression was detected in samples of breast carcinoma tissues and blood obtained from patients with breast cancer. A total of 15.3 % of these blood samples exhibited a 2.1-fold or higher level of CA1 expression, compared to the average level of CA1 expression in samples from healthy controls. Following the induction of calcification of 4T1 cells, both the number of calcium-rich deposits and the expression of CA1 increased, whereas the calcification and CA1 expression were significantly supressed in the presence of acetazolamide. Increased migration and apoptosis were observed in MCF-7 cells that were treated with anti-CA1 siRNA. The PCR array detected up-regulation of the androgen receptor (AR) and down-regulation of X-box binding protein 1 (XBP1) in the treated MCF-7 cells. Significant differences in the allele and genotype frequencies of rs725605 were detected in the cohort of patients with breast cancer but not in other tumours. CONCLUSION: The results of this study suggested that CA1 is a potential oncogene and that it contributes to abnormal cell calcification, apoptosis and migration in breast cancer.

Specific alterations in plasma proteins during depressed, manic, and euthymic states of bipolar disorder.

Bipolar disorder (BD) is a common psychiatric mood disorder affecting more than 1-2% of the general population of different European countries. Unfortunately, there is no objective laboratory-based test to aid BD diagnosis or monitor its progression, and little is known about the molecular basis of BD. Here, we performed a comparative proteomic study to identify differentially expressed plasma proteins in various BD mood states (depressed BD, manic BD, and euthymic BD) relative to healthy controls. A total of 10 euthymic BD, 20 depressed BD, 15 manic BD, and 20 demographically matched healthy control subjects were recruited. Seven high-abundance proteins were immunodepleted in plasma samples from the 4 experimental groups, which were then subjected to proteome-wide expression profiling by two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight tandem mass spectrometry. Proteomic results were validated by immunoblotting and bioinformatically analyzed using MetaCore. From a total of 32 proteins identified with 1.5-fold changes in expression compared with healthy controls, 16 proteins were perturbed in BD independent of mood state, while 16 proteins were specifically associated with particular BD mood states. Two mood-independent differential proteins, apolipoprotein (Apo) A1 and Apo L1, suggest that BD pathophysiology may be associated with early perturbations in lipid metabolism. Moreover, down-regulation of one mood-dependent protein, carbonic anhydrase 1 (CA-1), suggests it may be involved in the pathophysiology of depressive episodes in BD. Thus, BD pathophysiology may be associated with early perturbations in lipid metabolism that are independent of mood state, while CA-1 may be involved in the pathophysiology of depressive episodes.

The effects of bronchodilator drugs and antibiotics used for respiratory infection on human erythrocyte carbonic anhydrase I and II isozymes.

Carbonic anhydrase (CA) is an enzyme which plays role/roles in various homeostatic mechanisms, such as the acid-base balance and electrolyte secretion in various tissues. This study aimed to determine and to compare possible alterations in activity of this enzyme caused by use of bronchodilator drugs and respiratory infection antibiotics. CA I and II were purified from human erythrocytes by a simple one step procedure using Sepharose 4B-L-tyrosine-sulfonamide affinity column. The iso-enzymes were purified 259.16-fold with a yield of 31.74%. CAI and II isozymes were treated with several drugs, then the inhibition or activation of the enzymes were determined. The results of this study show that itrapropium bromide is the most effective inhibitor for human erythrocytes carbonic anhydrase compared with the other bronchodilator drugs.

Autoantigenicity of carbonic anhydrase 1 in patients with abdominal aortic aneurysm, revealed by proteomic surveillance.

Abdominal aortic aneurysm (AAA) is sometimes detected in patients with atherosclerosis. One of the histological characteristics of AAA walls is infiltration of inflammatory cells, in which autoimmunity may be involved. Thereby, we here surveyed autoantigens in AAA walls by proteomics. Specifically, we separated proteins extracted from AAA wall samples by 2-dimensional electrophoresis and detected candidate autoantigens by western blotting. One of the detected candidates was carbonic anhydrase 1 (CA1). ELISA confirmed that the autoantibodies to CA1 were detected more frequently in AAA patients (n=13) than in healthy donors (n=25) (p=0.03). Interestingly, some serum samples from the AAA patients reacted to CA1 of the AAA walls stronger than to CA1 of peripheral blood mononuclear cells from healthy donors. Our data indicate that CA1 in the AAA walls would be modified to express neo-epitope(s) and that the autoimmunity to CA1 may be involved in the pathogenesis of AAA.

Molecular interactions of re-released proteins in electrophoresis of human erythrocytes.

Recently, we found that hemoglobin (Hb) could be re-released from live erythrocytes during electrophoresis release test (ERT). The re-released Hb displays single-band and multiple-band re-release types, but its exact mechanism is not well understood. In this article, the protein components of the single-band re-released Hb were examined. First, the re-released band of erythrocytes and the corresponding band of hemolysate, which was used as control, were cut out from starch-agarose mixed gel. Next, proteins were recovered from the starch-agarose mixed gel by freeze-thaw method. After condensing in a vacuum freeze drier, the samples were loaded onto a 5-12% SDS-PAGE. After electrophoresis, three protein bands (16, 28.9, and 29.3 kDa) emerged from the erythrocytes re-released Hb single-band (R-R), but only one band (29.3 kDa) emerged from the corresponding hemolysate control band (H-R). Finally, these bands were analyzed by MALDI-TOF MS. The results showed that these proteins were beta-globin (16 kDa), carbonic anhydrase 1 (CA1, 28.9 kDa), and carbonic anhydrase 2 (CA2, 29.3 kDa). Because CA2 exists in both erythrocytes re-released band and hemolysate control band, we conclude that the single-band re-released Hb is mainly composed of HbA and CA1. Studying the possible interaction between HbA and CA1 will help us further understand the in vivo function of Hb.

Effects of dopaminergic compounds on carbonic anhydrase isozymes I, II, and VI.

Studies on carbonic anhydrase (CA, EC 4.2.1.1) inhibitors have increased due to several therapeutic applications while there are few investigations on activators. Here we investigated CA inhibitory and activatory capacities of a series of dopaminergic compounds on human carbonic anhydrase (hCA) isozymes I, II, and VI. 2-Amino-1,2,3,4-tetrahydronaphthalene-6,7-diol hydrobromide and 2-amino-1,2,3,4-tetrahydronaphthalene-5,6-diol hydrobromide were found to show effective inhibitory action on hCA I and II whereas 2-amino-5,6-dibromoindan hydrobromide and 2-amino-5-bromoindan hydrobromide exhibited only moderate inhibition against both isoforms, being more effective inhibitors of hCA VI. K(i) values of the molecules 3-6 were in the range of 41.12-363 µM against hCA I, of 0.381-470 µM against hCA II and of 0.578-1.152 µM against hCA VI, respectively. Compound 7 behaved as a CA activator with K(A) values of 27.3 µM against hCA I, of 18.4 µM against hCA II and of 8.73 µM against hCA VI, respectively.

Genetic structure and variation of Van cats.

To determine the genetic structure and variation of Van cats and some other cats, seven enzyme loci were examined using horizontal starch gel electrophoresis. ME bands were observed for the first time in cats. For the enzyme loci CA ( 1 ), SOD, GPI, and GOT, neither the individual Van cats nor the specimens of other cat species exhibited any variation. These enzymes presented identical bands, all of which were homozygous. With respect to the PGD, ME, and ESD loci, however, genetic variation was observed in all of the cats. Hence, three of the seven gene-enzyme systems (43%) were polymorphic with two alleles, contributing to an estimate of average heterozygosity of 0.33-0.49 for the Van cats. PGD was the most discriminatory among the three polymorphic loci. The phylogenetic tree indicated that the Van, Persian, Turkish Angora, and Turkish Tekir cats are distinct from Siamese and Bombay cats.

Carbonic anhydrase inhibitors. Inhibition of human erythrocyte isozymes I and II with a series of antioxidant phenols.

The inhibition of two human cytosolic carbonic anhydrase (hCA, EC 4.2.1.1) isozymes I and II, with a series of phenol derivatives was investigated by using the esterase assay, with 4-nitrophenyl acetate as substrate. 2,6-Dimethylphenol, 2,6-diisopropylphenol (propofol), 2,6-di-t-butylphenol, butylated hydroxytoluene, butylated hydroxyanisole, vanillin, guaiacol, di(2,6-dimethylphenol), di(2,6-diisopropylphenol), di(2,6-di-t-butylphenol), and acetazolamide showed K(I) values in the range of 37.5-274.5 microM for hCA I and of 0.29-113.5 microM against hCA II, respectively. All these phenols were non-competitive inhibitors with 4-nitrophenylacetate as substrate. Some antioxidant phenol derivatives investigated here showed effective hCA II inhibitory effects, in the same range as the clinically used sulfonamide acetazolamide, and might be used as leads for generating enzyme inhibitors possibly targeting other CA isoforms which have not been yet assayed for their interactions with such agents.

Highly efficient depletion strategy for the two most abundant erythrocyte soluble proteins improves proteome coverage dramatically.

In-depth human erythrocyte proteome studies are severely hampered by the presence of hemoglobin and carbonic anhydrase-1, which account for more than 98% of the total erythrocyte soluble protein content. We developed a specific depletion approach that resulted in a drastic increase in the number of identified proteins. This depletion technique is valuable for proteome studies of human erythrocyte disorders with unknown etiology and of tissue samples that contain blood.

Morphine inhibits erythrocyte carbonic anhydrase in vitro and in vivo.

Morphine is implicated in diverse functions, from development to immune modulation in the central and peripheral nervous systems. At the present time, morphine is one of the most effective antinociceptive agents used to manage pain. It has been used extensively in the clinical management of pain due to its potent analgesic effect. In this study, the in vitro and in vivo inhibitory effects of morphine on erythrocyte carbonic anhydrase (CA) were investigated. Human erythrocyte isoenzymes, HCA-I and HCA-II, were purified by Sepharose-4B affinity chromatography column with a yield of 66.95 and 62.82%, a specific activity of 3892.3 and 11663.2 EU/mg proteins with 745.1 and 2232.6-fold purification of each isoenzyme, respectively. To determine enzyme purity, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. In vitro inhibition of erythrocyte HCA-I and HCA-II by morphine using the CO(2)-hydratase enzyme gave IC(50) values 4.50 x 10(-5) M (r(2): 0.954) and 9.23 x 10(-5) M (r(2): 0.996), respectively. CA activity was significantly attenuated in vivo in Spraque-Dawley rats for up to 3 h (p<0.001) following intraperitoneal administration of morphine. In conclusion, morphine inhibited CA activity both in vitro and in vivo.

[Erythrocyte carbonic anhydrase I and zinc concentrations in thyrotoxicosis reflect integrated thyroid hormone levels over the previous few months].

In the present review, the clinical utility of determining red blood cell (RBC) carbonic anhydrase I isozyme (CA1) and zinc(Zn) concentrations in patients with various forms of thyroid disease is discussed. RBC CAI and Zn concentrations were both decreased in patients with hyperthyroid Graves' disease. After treatment, the normalization of RBC CA1 and Zn lagged two months behind the normalization of plasma thyroxine (T4) and triiodothyronine (T3) levels. Furthermore, the highest correlation coefficients were observed between RBC CA1 and Zn levels, and plasma thyroid hormone levels measured eight weeks earlier. These results indicate that both RBC CAI and zinc levels reflect integrated plasma thyroid hormone levels over the previous few months. Transient thyrotoxicosis due to destructive thyroiditis did not cause significant changes in RBC CA1 and Zn concentrations. T3 at a physiological free concentration significantly decreased the level of CAl mRNA and the concentration of CA1 in burst forming unit erythroid-derived cells. These results indicate that the measurement of RBC CA1 and Zn concentrations may be useful as follows: (1) to obtain an accurate estimate of the extent of elevated thyroid hormone levels in hyperthyroid patients in whom serial measurements were not obtained over time; (2) to differentiate patients with hyperthyroid Graves' disease from those with transient thyrotoxicosis.

Measurement of erythrocyte carbonic anhydrase isozymes (CA-I and CA-II) in racehorses and riding horses.

Equine carbonic anhydrase isozymes (CA-I and CA-II) were purified from erythrocytes by several column chromatography. Polyclonal anti-CA-I and anti-CA-II sera were produced in rabbits. Sensitive competitive enzyme-linked immunosorbent assays (ELISA) were established to determine the developmental changes in CA-I and CA-II levels in equine erythrocytes. Concentrations of CA-I and CA-II in erythrocytes from 150 clinically normal thoroughbreds (123 racehorses and 27 riding horses) were determined by ELISA. Mean (+/- SD) concentrations of CA-I and CA-II in racehorses were 1.70 +/- 0.48 and 0.94 +/- 0.13 mg/g hemoglobin (Hb), respectively. Mean concentrations of CA-I and CA-II in riding horses were 2.34 +/- 0.52 and 0.76 +/- 0.08 mg/g Hb, respectively. When the CA levels in racehorses and riding horses were compared, the CA-I level in riding horses was higher than that in racehorses (p=0.01). The CA-II level in racehorses was higher than that in riding horses (p=0.02). These data suggest that the levels of CA isozymes in erythrocytes of racehorses were influenced by chronic physical stress. The CA-I concentration in erythrocytes of 2-month-old horses was approximately 0.25 mg/g Hb. The CA-I level noticeably increased during the first year of life and approached normal adult levels by 2 years. The CA-II level decreased slightly with age, indicating different regulation of CA-I and CA-II expression during development.

Investigation of red blood cell carbonic anhydrase, glucose 6-phosphate dehydrogenase, hexokinase enzyme activities, and zinc concentration in patients with hyperthyroid diseases.

We have recently reported on the activities of glucose-6-phosphate dehydrogenase (G6PD), hexokinase (HK) and carbonic anhydrase I (CA-I) and II (CA-II) isoenzymes obtained from erythrocytes of healthy subjects and untreated patients with hyperthyroid diseases. Also, erythrocyte zinc concentrations were measured. Red blood cell (RBC) zinc (Zn) concentration was measured by atomic absorption spectrophotometry. Activities of carbonic anhydrase II and I isoenzymes were determined with CO2-hydratase activity method by using selective inactivation with bromopyruvate. G6PD and HK enzyme activities were measured spectrophotometrically via absorbance change (at 340 nm) in NADPH formed as a result of the reactions catalysed by these enzymes. In statistical analysis of all these parameters, activity of CA-I was 4388 +/- 207 (EU/gHb) and 2881 +/- 869 (EU/gHb) in healthy and untreated hyperthyroid subjects, respectively. The activity values for CA-II were 5391 +/- 257 (EU/gHb) and 4688 +/- 12.6 (EU/gHb) in healthy and untreated hyperthyroid subjects. Glucose 6-phosphate dehydrogenase (G6PD) activity was 10.19 +/- 1.87 (EU/gHb) in healthy group and 4.92 +/- 2.49 (EU/gHb) in patient group. While hexokinase enzyme activity was 1.575 +/- 0.898 in healthy subjects, it was 0.651 +/- 0.418 (EU/gHb) in the patient group. While erythrocyte zinc concentration in the healthy subjects was 49.32 +/- 23.5 (mg/gHb), this concentration for patients with uncontrolled hyperthyroid diseases was significantly decreased to 29.62 +/- 4.26 (mg/gHb). As a conclusion, CA-I isoenzyme, G6PD, hexokinase activities and erythrocyte zinc concentration had decreased in untreated patients carrying hyperthyroid diseases as compared to those of the healthy subjects.