- Email: [email protected]
Differential expression of carbonic anhydrase isoenzymes in various types of anemia
Clinica Chimica Acta 351 (2005) 79 – 86 www.elsevier.com/locate/clinchim
Differential expression of carbonic anhydrase isoenzymes in various types of anemia Wu-Hsien Kuoa,b, Shun-Fa Yangb, Yih-Shou Hsiehb, Chiou-Sheng Tsaib,c, Wen-Li Hwangc,d, Shu-Chen Chue,* a
Division of Gasroenterology, Department of Internal Medicine, Armed-Force Taichung General Hospital, Taiping City 411, Taichung, Taiwan b Institute of Biochemistry, Chung Shan Medical University, Taichung 402, Taiwan c Blood Bank, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung 403, Taiwan d Division of Hematology/Oncology, Taichung Veterans General Hospital, Taichung 403, Taiwan e Department of Food Science, Chungtai Institute of Health Sciences and Technology, No.11 Pu-tzu Lane, Pu-tzu Road, Taichung 406, Taiwan Received 8 July 2004; received in revised form 22 July 2004; accepted 22 July 2004
Abstract Background: The aim of the present study was to determine the concentrations of cytosolic carbonic anhydrase (CA) isoenzymes in erythrocytes of patients with aplastic, autoimmune hemolytic, iron deficiency or h-thalassemia anemia. Methods: Western blotting and CA esterase activity analysis were used to analyze cytosolic erythrocyte CA isoenzymes in 118 subjects with various types of anemia and 35 healthy controls. Results: Total CA activity and CAII concentration of anemia patients were significantly higher than that of the control subjects while CAI concentration was significantly lower in patients of autoimmune hemolytic anemia ( Pb0.01). Compared with controls, CAIII concentration was lower in iron deficiency anemia ( Pb0.01), but higher in h-thalassemia anemia ( Pb0.001). Conclusions: Carbonic anhydrase isoenzymes may contribute differently to various types of anemia. CAI may be an indicator to differentiate autoimmune hemolytic anemia from other types of anemia. CAII provides the CA activity necessary for maintaining ion transport in erythrocytes while CAIII may play an agent against oxidative damage in iron deficiency and hthalassemia anemia. D 2004 Elsevier B.V. All rights reserved. Keywords: Carbonic anhydrase isoenzymes; Aplastic anemia; Autoimmune hemolytic anemia; Iron deficiency anemia; h-Thalassemia anemia
1. Introduction * Corresponding author. Tel.: +886 4 22391647x7503; fax: +886 4 2239 6771. E-mail address: [email protected] (S.-C. Chu). 0009-8981/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2004.07.009
Anemia is one of the most frequently encountered and ignored medical problems [1]. Anemia
80
W.-H. Kuo et al. / Clinica Chimica Acta 351 (2005) 79–86
may be defined as any condition resulting from a significant decrease in the total body erythrocyte mass and anemia can be classified by cytometric schemes. For example, those that depend on cell size and hemoglobin-content parameters, such as mean cell volume (MCV) and mean cell hemoglobin concentration (MCHC), erythrokinetic schemes (those that take into account the rates of RBC production and destruction), and biochemical/molecular schemes (those that consider the etiology of the anemia at the molecular level) [2]. Using the MCV and MCHC from the hemogram, anemia can be classified as microcytic, macrocytic, or normocytic. Normocytic anemia is characterized with a decreased erythrocyte cell production and increased cell destruction and occurs in primary hematopoietic disorders, such as autoimmune hemolytic anemia and aplastic anemia. Microcytosis occurs when there is insufficient hemoglobin production in the developing erythrocyte. The most common causes of microcytic anemia include iron deficiency, anemia of chronic disease, and h-thalassemia [3]. Electrophoretic analysis of human red cell lysates reveals, in addition to the major and minor hemoglobin components, several non-hemoglobin proteins, the most prominent of which are carbonic anhydrases [4]. The carbonic anhydrase (EC 4.2.1.1) reversibly catalyzes the hydration of carbonic dioxide to bicarbonate and hydrogen ions. This enzyme has been detected in almost all types of tissues with 14 different isoforms and the distribution patterns of these isoenzymes are different from each other [5–10]. It also plays important roles in gas transport, acid– base regulation, and various secretory functions in tissues [6,11]. Two main types of CA have been isolated from erythrocytes: isoenzymes I and II, together with a minor component named CAIII. CAI has a lower enzymatic activity than CAII, but constitutes 89% of the total erythrocyte CA activity in adults [12–14]. In previous studies, it is known that the levels of erythrocyte carbonic anhydrase isoenzymes change under some pathologic conditions. The concentration of CAI was increased in uremic anemia, cancer anemia, megaloblastic anemia and chronic acidosis [15]. Furthermore, in our previous study, it was found that the total CA activity was significantly higher in G6PD deficiency induced hemolytic
anemia [16]. However, previous studies were focused on only a certain CA isoenzyme and only in a single type of anemia without revealing the overall interaction. The aim of the present study was to determine the concentrations of erythrocyte CAI, CAII and CAIII in various types of anemia patients to provide further information for the involvement of CAs in anemia.
2. Materials and methods 2.1. Subjects and specimen collection Human venous blood samples were obtained via routine venipuncture from anemia patients of Taichung Veteran General Hospital and ArmedForce Taichung General Hospital, Taichung, Taiwan. A total of 153 patients were voluntarily recruited into this study and subdivided into five groups, including control (n=35), aplastic anemia (n=28), autoimmune hemolytic anemia (n=28), iron deficiency anemia (n=50) and h-thalassemia anemia (n=12). The taken EDTA anticoagulated blood samples were centrifuged at 1000g for 10 min and then the pelleted red blood cells were washed three times with normal saline. Thereafter, red blood cells were frozen in 20 8C to break cell membranes to induce hemolysis. The hemolysates were centrifuged again at 11,600g for 5 min and then the supernatants were collected for CA activity assay and Western blotting analysis. Clinical characteristics and laboratory findings of patients in every group are summarized in Table 1. 2.2. Determination of erythrocyte CA activity Hemoglobin was removed from erythrocyte lysates as follows: 1 ml of lysate was extracted for 1 min with 0.2 ml 40% (v/v) toluene, and centrifuged at 3000g for 20 min at 4 8C. The toluene layer and membranous interface were discarded. At 4 8C, 0.2 ml lysate was mixed for exactly 1 min with 0.2 ml 40% (v/v) ethanol and 0.1 ml chloroform, both kept at 20 8C, and then centrifuged at 13,000g for 5 min. CA esterase activity was measured in the hemoglobin-free lysates by the conversion of p-nitrophenyl-acetate
W.-H. Kuo et al. / Clinica Chimica Acta 351 (2005) 79–86
81
Table 1 Clinical characteristics and laboratory findings of subjects Clinical characteristics
Normal subjects (n=35)
Aplastic anemia (n=28)
Autoimmune hemolytic anemia (n=28)
Iron deficiency anemia (n=50)
h-Thalassemia anemia (n=12)
Age (years) Sex Male Female WBC (103) Reticulocyte (%) Hb (g/dl) HCT (%) RBC (107) MCV MCHC
50.71F10.49
36.37F17.33
58.06F16.58
55.55F17.49
30.2F10.23
16 (45.7%) 19 (54.3%) 6.77F1.99 0.985F0.04 13.75F1.52 41.79F4.24 4.67F0.66 90.32F8.85 29.72F3.16
16(57.1%) 12(42.9%) 3.17F2.08 1.064F0.12 9.82F2.66 28.54F7.4 2.95F0.84 97.88F10.5*** 33.43F3.51***
13 (46.4%) 15 (53.6%) 7.26F3.92 7.15F0.98*** 11.52F2.42 33.26F8.78 3.72F1.12 89.17F12.48 32.33F10.56
22 (44%) 28 (56%) 6.34F2.12 1.153F0.06 11.42F2.08 35.28F5.18 4.35F0.74 81.8F9.8*** 26.23F4.48***
5 (41.7%) 7 (58.3%) 8.65F1.95 1.41F0.14*** 10.1F3.85 34.1F8.11 4.79F0.56 72.16F10*** 21.95F6.15***
*** P b 0.001.
to nitrophenol and quantitated by reading at 410 nm [17]. 2.3. Western blot analysis Protein contents of the hemolysates were measured by Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA) [18] with bovine serum albumin as a standard. Samples were prepared as described above. SDS-PAGE was carried out as described by Laemmli
[19] using 12.5% polyacrylamide gel. A total of 20 Ag proteins were electrophoresed at 140 V for 3.5 h; the gels were equilibrated for 15 min in 25 mmol/l Tris– HCl, pH 8.3, containing 192 mmol/l glycine and 20% (v/v) methanol [20]. Electrophoresed proteins were transferred to nitrocellulose paper (Whatman, BioBond-NC, 0.45 Am) using a Bio-Rad Trans-Blot apparatus (Bio-Rad Laboratories) at 100 V for 1 h. Nitrocellulose papers were incubated at room temperature for 1 h in blocking buffer containing 100 mmol/l
Fig. 1. Comparison of CA activity in various types of anemia and normal subjects. *Pb0.05, **Pb0.01, ***Pb0.001.
82
W.-H. Kuo et al. / Clinica Chimica Acta 351 (2005) 79–86
Tris–HCl, pH 7.5, 0.9% (w/v) NaCl, 0.05% (v/v) Tween-20 and 5% skim milk. Polyclonal antibodies to human CA isoforms were diluted 1:2000 in antibody binding buffer containing 100 mmol/l Tris–HCl, pH 7.5, 0.9% (w/v) NaCl, 0.05% (v/v) Tween-20 and 5% skim milk. Incubations were performed at room temperature for 3.5 h. The immunoblots were washed three times in 50 ml blotting buffer for 10 min and then immersed in the second antibody solution containing horseradish peroxidase goat anti-rabbit IgG (Promega) for 1 h and diluted 5000-fold in
binding buffer. The filters were then washed three times in blotting buffer for 10 min. Color development was presented in 20 ml mixture consisting of 15 mg 4-chloro-1-napthol, 3 mg 3,3V-diaminobenzidine, 0.9% (w/v) NaCl in Tris–HCl, pH 7.5. 2.4. Quantitative analysis of concentrations of cytosolic CA isoenzymes The relative concentrations of CA isoenzymes were quantitated by analyzing the developed bands
Fig. 2. (A) Immunoblot analysis of cytosolic expression of CAI and CAII in various types of anemia. Lane PC represents the positive control as CAI and CAII. (B) Densitometric analysis of the CAI and CAII concentrations in various types of anemia. *Pb0.05, **Pb0.01, ***Pb0.001.
W.-H. Kuo et al. / Clinica Chimica Acta 351 (2005) 79–86
83
Fig. 3. (A) Immunoblot analysis of cytosolic expression of CAIII in various types of anemia. Lane PC represents the positive control as CAIII. (B) Densitometric analysis of the CAIII concentration in various types of anemia. **Pb0.01, ***Pb0.001.
on the Western blot membrane with a gel documentation and analysis system (Alpha Imager 2000, Alpha Innotech) as previously described [16].
Whitney rank sum test. SigmaStat software (Jandel Scientific Software, USA) was used for all statistical analyses. A P value b0.05 was considered significant.
2.5. Statistical analysis 3. Results Values were expressed as meansFS.E. The statistical significance of the means for cytosolic CA isoenzyme between groups was determined by Mann–
The hematologic, demographic and clinical characteristics of controls and patients (patients with
84
W.-H. Kuo et al. / Clinica Chimica Acta 351 (2005) 79–86
various types of anemia including aplastic anemia, autoimmune hemolytic anemia, iron deficiency anemia and h-thalassemia anemia) are shown in Table 1. No significant difference was observed for any studied parameter between controls and studied groups. However, reticulocyte count was significantly increased in both groups of autoimmune hemolytic anemia and h-thalassemia anemia, compared to that of control subjects ( Pb0.001). MCV and MCHC were significantly increased in the group of aplastic anemia while, on the contrary, lower in iron deficiency and hthalassemia anemia, compared to the control subjects ( Pb0.001). The results of erythrocyte total CA activity for various types of anemia and statistical analysis showed that the activities of erythrocyte total CA activity were significantly increased in all patients with various types of anemia (Fig. 1). Furthermore, Western blotting analysis revealed that CAII concentration was higher in autoimmune hemolytic and iron deficiency, compared to that of controls ( Pb0.05 and Pb0.05, respectively) (Fig. 2A and B). In h-thalassemia anemia, CA II concentration was threefold higher than controls ( Pb0.001) while the density of CAI was significantly less in hemolytic anemia ( Pb0.01) (Fig. 2A and B). In addition, CAIII concentration in iron deficiency anemia was significantly less than controls ( Pb0.01). Nevertheless, in the h-thalassemia anemia, the concentration of CAIII was about 1.8-fold higher than control subjects ( Pb0.001) (Fig. 3A and B).
4. Discussion Hemolytic anemia is a state of hemolysis in which increased erythrocyte production is insufficient to keep up with accelerated RBC destruction, thus producing anemia. This anemia is characterized as normochromic/normocytic, except when sufficient outpouring of the larger reticulocytes produces a resulting elevation of the MCV (Table 1). Hemolytic anemia is a classification of disorders in which red cells are destroyed prematurely, which leads to anemia. In the classification, hemolytic anemia can be classified as autoimmune hemolytic anemia, mechanical hemolytic anemia, paroxysmal nocturnal hemoglobinuria and glucose-6-phosphate dehyrogenase (G6PD) deficiency, etc. [21]. In the present
study, patients with autoimmune hemolytic anemia have been identified as positive with the direct antiglobulin test and autoimmune anemia patients have fallen ill for at least 3 months. As in our previous report, patients with G6PD deficiency anemia had a lower CAI concentration than control subjects and a consistent result was observed for autoimmune hemolytic anemia. Thus, CAI may be an applicable indicator for hemolytic anemia. Previous studies have reported a statistically significant increase of human erythrocyte CAI in the erythrocytes of subjects suffering from uremic anemia, cancer anemia and megaloblastic anemia while in sideropenic anemia the content was normal [15]. In erythrocytes from patients with acute bleeding anemia, the content of erythrocyte CAI was significantly decreased, since there was a younger cell population with a lower content of CAI/g Hb present in the patients’ erythrocytes. In normal subjects, there was no difference in CAI content between young and old erythrocytes [11,15]. A quite similar phenomenon was also noticed in our study group. The lower content of CAI in patients with autoimmune hemolytic anemia might be caused by a slower synthesis rate of CAI compared with that of hemoglobin. Although the capability of CAI for CO2 hydration turnover is lower than that of CAII and CAII is more significant physically, the data clearly showed that the variation of CAI between patients with autoimmune hemolytic anemia and controls was greater than that of CAII. Furthermore, from the present data, we found that the concentration of CAII and total CA activity in various types of anemia were significantly higher than that of normal subjects. These results were consistent with our previous study [16]. Those individuals with a G6PD deficiency, a subtype of hemolytic anemia, have higher total CA activity and CAII concentration than that of control subjects [16]. The significantly increased total CA activity and CAII may be able to compensate for the function of CAI. On the other hand, CAII has been demonstrated to be capable of binding to the carboxyl terminus of band 3 protein on the erythrocyte membrane [22] and previous researches have shown that the abnormality of erythrocyte membrane in hemolytic anemia was due to an abnormality of band 3 protein and deficiency of band 4.2 protein [23]. Thus, increased CAII concentration in various types of anemia may serve to
W.-H. Kuo et al. / Clinica Chimica Acta 351 (2005) 79–86
compensate the decreased band 3 protein concentration. Besides, CA II may provide the amount of CA activity to maintain the ion transport in erythrocytes. In Fig. 3A and B, we found that CAIII concentration was decreased in the iron deficiency anemia, but increased in the h-thalassemia. CAIII, a CA isoenzyme abundant in the red skeletal muscle, has been demonstrated to function as an oxygen radical scavenger [24]. Previous studies have demonstrated that several intra-erythrocyte enzyme systems, including glutathione peroxidase and catalase, are protective against oxidative damage [25,26]. Further, the activities of erythrocyte glutathione peroxidase and catalase have been reported to be decrease in patients with iron deficiency anemia [27–29]. In iron deficiency anemia, decreased antioxidant defense may increase oxidant stress and may result in a tendency towards platelet aggregation [30]. Since erythrocyte CAIII concentration in iron deficiency anemia was decreased, this may play the same role as antioxidants [24]. However, increases in the levels of superoxide dismutase and glutathione peroxidase have been observed in h-thalassemia anemia [31]. These findings suggested that oxidative stress occurs in those red cells with increased CAIII and provided indirect evidence to support the suggestion that CAIII may play a role as an antioxidant in erythrocytes. In conclusion, our data suggested that different carbonic anhydrase isoenzymes might serve different roles in various types of anemia erythrocyte. CAI could be used as an indicator for autoimmune hemolytic anemia based on the great variation of CAI between autoimmune hemolytic anemia and other types of anemia. CAII may be able to compensate the functions of not only CAI but also band 3 protein while CAIII could act against oxidative damage in iron deficiency and h-thalassemia anemia. The precise function of these CAs in various types of anemia still remains uncertain and, therefore, warrants further studies.
Acknowledgment This study was supported by Taichung Veterans General Hospital (TCVGH 924301A) and the ArmedForce Taichung General Hospital, Taichung, Taiwan (grant 0930000526).
85
References [1] Handin RI, Lux SE, Stossel TP. Blood, principles and practice of hematology, 2nd ed. Lippincott Willliams & Wilkins., p. 1260 – 345. [2] Wintrobe MM. Clinical hematology. 8th ed. Philadelphia7 Lea & Febiger; 1981. [3] Lessels S, Davidson RJ. The low mean cell volume in routine haematology. Clin Lab Haematol 1979;1:291 – 8. [4] Rickli EE, Ghazanfar SA, Gibbons BH, Edsall JT. Carbonic anhydrases from human erythrocytes. Preparation and properties of two enzymes. J Biol Chem 1964;239:1065 – 78. [5] Sly WS, Hu PY. Human carbonic anhydrases and carbonic anhydrase deficiencies. Annu Rev Biochem 1995;64:375 – 401. [6] Tashian RE. The carbonic anhydrases: widening perspectives on their evolution, expression and function. Bioessays 1989;10:186 – 92. [7] Pastorek J, Pastorekova S, Callebaut I, Mornon JP, Zelnik V, Opavsky R, et al. Cloning and characterization of MN, a human tumor-associated protein with a domain homologous to carbonic anhydrase and a putative helix-loop-helix DNA binding segment. Oncogene 1994;9:2877 – 88. [8] Tureci O, Sahin U, Vollmar E, Siemer S, Gottert E, Seitz G, et al. Human carbonic anhydrase XII: cDNA cloning, expression, and chromosomal localization of a carbonic anhydrase gene that is overexpressed in some renal cell cancers. Proc Natl Acad Sci U S A 1998;95:7608 – 13. [9] Supuran CT, Scozzafava A, Casini A. Carbonic anhydrase inhibitors. Med Res Rev 2003;23:146 – 89. [10] Supuran CT, Scozzafava A. In: Conway J, editor. Carbonic Anhydrase, Its Inhibitors and Activators. Boca Raton, FL7 CRC Press, 2004. p. 1 – 363. [11] Ali Akbar SA, Brown PR. Measurement of human erythrocyte CAI and CAII in adult, newborn, and fetal blood. Clin Biochem 1996;29:157 – 64. [12] Nyman PO. Purification and properties of carbonic anhydrase from human erythrocytes. Biochim Biophys Acta 1961;52:1 – 12. [13] Wistrand PJ, Rao S. Immunologic and kinetic properties of carbonic anhydrases from various tissues. Biochim Biophys Acta 1968;154:130 – 44. [14] Funakoshi S, Deutsch HF. Human carbonic anhydrase III. Immunochemical studies. J Biol Chem 1970;245:2852 – 6. [15] Mondrup M, Anker N. Carbonic anhydrase isoenzyme B in erythrocytes of subjects with different types of anemia. Clin Chim Acta 1976;69:463 – 9. [16] Chiang WL, Chu SC, Lai JC, Yang SF, Chiou HL, Hsieh YS. Alternations in quantities and activities of erythrocyte cytosolic carbonic anhydrase isoenzymes in glucose-6phosphate dehydrogenase-deficient individuals. Clin Chim Acta 2001;314:195 – 201. [17] Goode HF, Kelle J, Walker BE. Relation between zinc status and hepatic functional reverse in patients with liver disease. Gut 1990;31:694 – 7. [18] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248 – 54.
86
W.-H. Kuo et al. / Clinica Chimica Acta 351 (2005) 79–86
[19] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680 – 5. [20] Brockenbrough JS, Meyer SA, Li C, Jirtle RL. Reversible and phorbol ester-specific defect of protein kinase C translation in hepatocytes isolated from phenobarbital-treated rats. Cancer Res 1991;51:130 – 6. [21] Costea N. The differential diagnosis of hemolytic anemias. Med Clin North Am 1973;57:289 – 302. [22] Vince JW, Reithmeier RA. Carbonic anhydrase II binds to the carboxyl terminus of human band 3, the erythrocyte C1 / HCO3 exchanger. J Biol Chem 1998;273:28430 – 7. [23] Kay MM, Bosman GJ, Shapiro SS, Bendich A, Bassel PS. Oxidation as a possible mechanism of cellular aging: vitamin E deficiency causes premature aging and IgG binding to erythrocytes. Proc Natl Acad Sci U S A 1986;83:2463 – 7. [24] Raisanen SR, Lehenkari P, Tasanen M, Rahkila P, Harkonen PL, Vaananen HK. Carbonic anhydrase III protects cells from hydrogen peroxide-induced apoptosis. FASEB J 1999; 13:513 – 22. [25] Mates JM, Perez-Gomez C, Blanca M. Chemical and biological activity of free radical dscavengersT in allergic diseases. Clin Chim Acta 2000;296:1 – 15.
[26] Reiter RJ, Acuna-Castroviejo D, Tan DX, Burkhardt S. Free radical-mediated molecular damage. Mechanisms for the protective actions of melatonin in the central nervous system. Ann N Y Acad Sci 2001;939:200 – 15. [27] Macdougall LG. Red cell metabolism in iron deficiency anemia: 3. The relationship between glutathione peroxidase, catalase, serum vitamin E, and susceptibility of irondeficient red cells to oxidative hemolysis. J Pediatr 1972;80:775 – 82. [28] Perona G, Cellerino R, Guidi GC, Moschini G, Stievano BM, Tregnaghi C. Erythrocytic glutathione peroxidase: its relationship to plasma selenium in man. Scand J Haematol 1977;19:116 – 20. [29] Kumerova A, Lece A, Skesters A, Silova A, Petuhovs V. Anaemia and antioxidant defence of the red blood cells. Mater Med Pol 1998;30:12 – 5. [30] Tekin D, Yavuzer S, Tekin M, Akar N, Cin S. Possible effects of antioxidant status on increased platelet aggregation in childhood iron-deficiency anemia. Pediatr Int 2001;43:74 – 7. [31] Chakraborty D, Bhattacharyya M. Antioxidant defense status of red blood cells of patients with beta-thalassemia and E betathalassemia. Clin Chim Acta 2001;305:123 – 9.
Differential expression of carbonic anhydrase isoenzymes in various types of anemia Wu-Hsien Kuoa,b, Shun-Fa Yangb, Yih-Shou Hsiehb, Chiou-Sheng Tsaib,c, Wen-Li Hwangc,d, Shu-Chen Chue,* a
Division of Gasroenterology, Department of Internal Medicine, Armed-Force Taichung General Hospital, Taiping City 411, Taichung, Taiwan b Institute of Biochemistry, Chung Shan Medical University, Taichung 402, Taiwan c Blood Bank, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung 403, Taiwan d Division of Hematology/Oncology, Taichung Veterans General Hospital, Taichung 403, Taiwan e Department of Food Science, Chungtai Institute of Health Sciences and Technology, No.11 Pu-tzu Lane, Pu-tzu Road, Taichung 406, Taiwan Received 8 July 2004; received in revised form 22 July 2004; accepted 22 July 2004
Abstract Background: The aim of the present study was to determine the concentrations of cytosolic carbonic anhydrase (CA) isoenzymes in erythrocytes of patients with aplastic, autoimmune hemolytic, iron deficiency or h-thalassemia anemia. Methods: Western blotting and CA esterase activity analysis were used to analyze cytosolic erythrocyte CA isoenzymes in 118 subjects with various types of anemia and 35 healthy controls. Results: Total CA activity and CAII concentration of anemia patients were significantly higher than that of the control subjects while CAI concentration was significantly lower in patients of autoimmune hemolytic anemia ( Pb0.01). Compared with controls, CAIII concentration was lower in iron deficiency anemia ( Pb0.01), but higher in h-thalassemia anemia ( Pb0.001). Conclusions: Carbonic anhydrase isoenzymes may contribute differently to various types of anemia. CAI may be an indicator to differentiate autoimmune hemolytic anemia from other types of anemia. CAII provides the CA activity necessary for maintaining ion transport in erythrocytes while CAIII may play an agent against oxidative damage in iron deficiency and hthalassemia anemia. D 2004 Elsevier B.V. All rights reserved. Keywords: Carbonic anhydrase isoenzymes; Aplastic anemia; Autoimmune hemolytic anemia; Iron deficiency anemia; h-Thalassemia anemia
1. Introduction * Corresponding author. Tel.: +886 4 22391647x7503; fax: +886 4 2239 6771. E-mail address: [email protected] (S.-C. Chu). 0009-8981/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2004.07.009
Anemia is one of the most frequently encountered and ignored medical problems [1]. Anemia
80
W.-H. Kuo et al. / Clinica Chimica Acta 351 (2005) 79–86
may be defined as any condition resulting from a significant decrease in the total body erythrocyte mass and anemia can be classified by cytometric schemes. For example, those that depend on cell size and hemoglobin-content parameters, such as mean cell volume (MCV) and mean cell hemoglobin concentration (MCHC), erythrokinetic schemes (those that take into account the rates of RBC production and destruction), and biochemical/molecular schemes (those that consider the etiology of the anemia at the molecular level) [2]. Using the MCV and MCHC from the hemogram, anemia can be classified as microcytic, macrocytic, or normocytic. Normocytic anemia is characterized with a decreased erythrocyte cell production and increased cell destruction and occurs in primary hematopoietic disorders, such as autoimmune hemolytic anemia and aplastic anemia. Microcytosis occurs when there is insufficient hemoglobin production in the developing erythrocyte. The most common causes of microcytic anemia include iron deficiency, anemia of chronic disease, and h-thalassemia [3]. Electrophoretic analysis of human red cell lysates reveals, in addition to the major and minor hemoglobin components, several non-hemoglobin proteins, the most prominent of which are carbonic anhydrases [4]. The carbonic anhydrase (EC 4.2.1.1) reversibly catalyzes the hydration of carbonic dioxide to bicarbonate and hydrogen ions. This enzyme has been detected in almost all types of tissues with 14 different isoforms and the distribution patterns of these isoenzymes are different from each other [5–10]. It also plays important roles in gas transport, acid– base regulation, and various secretory functions in tissues [6,11]. Two main types of CA have been isolated from erythrocytes: isoenzymes I and II, together with a minor component named CAIII. CAI has a lower enzymatic activity than CAII, but constitutes 89% of the total erythrocyte CA activity in adults [12–14]. In previous studies, it is known that the levels of erythrocyte carbonic anhydrase isoenzymes change under some pathologic conditions. The concentration of CAI was increased in uremic anemia, cancer anemia, megaloblastic anemia and chronic acidosis [15]. Furthermore, in our previous study, it was found that the total CA activity was significantly higher in G6PD deficiency induced hemolytic
anemia [16]. However, previous studies were focused on only a certain CA isoenzyme and only in a single type of anemia without revealing the overall interaction. The aim of the present study was to determine the concentrations of erythrocyte CAI, CAII and CAIII in various types of anemia patients to provide further information for the involvement of CAs in anemia.
2. Materials and methods 2.1. Subjects and specimen collection Human venous blood samples were obtained via routine venipuncture from anemia patients of Taichung Veteran General Hospital and ArmedForce Taichung General Hospital, Taichung, Taiwan. A total of 153 patients were voluntarily recruited into this study and subdivided into five groups, including control (n=35), aplastic anemia (n=28), autoimmune hemolytic anemia (n=28), iron deficiency anemia (n=50) and h-thalassemia anemia (n=12). The taken EDTA anticoagulated blood samples were centrifuged at 1000g for 10 min and then the pelleted red blood cells were washed three times with normal saline. Thereafter, red blood cells were frozen in 20 8C to break cell membranes to induce hemolysis. The hemolysates were centrifuged again at 11,600g for 5 min and then the supernatants were collected for CA activity assay and Western blotting analysis. Clinical characteristics and laboratory findings of patients in every group are summarized in Table 1. 2.2. Determination of erythrocyte CA activity Hemoglobin was removed from erythrocyte lysates as follows: 1 ml of lysate was extracted for 1 min with 0.2 ml 40% (v/v) toluene, and centrifuged at 3000g for 20 min at 4 8C. The toluene layer and membranous interface were discarded. At 4 8C, 0.2 ml lysate was mixed for exactly 1 min with 0.2 ml 40% (v/v) ethanol and 0.1 ml chloroform, both kept at 20 8C, and then centrifuged at 13,000g for 5 min. CA esterase activity was measured in the hemoglobin-free lysates by the conversion of p-nitrophenyl-acetate
W.-H. Kuo et al. / Clinica Chimica Acta 351 (2005) 79–86
81
Table 1 Clinical characteristics and laboratory findings of subjects Clinical characteristics
Normal subjects (n=35)
Aplastic anemia (n=28)
Autoimmune hemolytic anemia (n=28)
Iron deficiency anemia (n=50)
h-Thalassemia anemia (n=12)
Age (years) Sex Male Female WBC (103) Reticulocyte (%) Hb (g/dl) HCT (%) RBC (107) MCV MCHC
50.71F10.49
36.37F17.33
58.06F16.58
55.55F17.49
30.2F10.23
16 (45.7%) 19 (54.3%) 6.77F1.99 0.985F0.04 13.75F1.52 41.79F4.24 4.67F0.66 90.32F8.85 29.72F3.16
16(57.1%) 12(42.9%) 3.17F2.08 1.064F0.12 9.82F2.66 28.54F7.4 2.95F0.84 97.88F10.5*** 33.43F3.51***
13 (46.4%) 15 (53.6%) 7.26F3.92 7.15F0.98*** 11.52F2.42 33.26F8.78 3.72F1.12 89.17F12.48 32.33F10.56
22 (44%) 28 (56%) 6.34F2.12 1.153F0.06 11.42F2.08 35.28F5.18 4.35F0.74 81.8F9.8*** 26.23F4.48***
5 (41.7%) 7 (58.3%) 8.65F1.95 1.41F0.14*** 10.1F3.85 34.1F8.11 4.79F0.56 72.16F10*** 21.95F6.15***
*** P b 0.001.
to nitrophenol and quantitated by reading at 410 nm [17]. 2.3. Western blot analysis Protein contents of the hemolysates were measured by Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA) [18] with bovine serum albumin as a standard. Samples were prepared as described above. SDS-PAGE was carried out as described by Laemmli
[19] using 12.5% polyacrylamide gel. A total of 20 Ag proteins were electrophoresed at 140 V for 3.5 h; the gels were equilibrated for 15 min in 25 mmol/l Tris– HCl, pH 8.3, containing 192 mmol/l glycine and 20% (v/v) methanol [20]. Electrophoresed proteins were transferred to nitrocellulose paper (Whatman, BioBond-NC, 0.45 Am) using a Bio-Rad Trans-Blot apparatus (Bio-Rad Laboratories) at 100 V for 1 h. Nitrocellulose papers were incubated at room temperature for 1 h in blocking buffer containing 100 mmol/l
Fig. 1. Comparison of CA activity in various types of anemia and normal subjects. *Pb0.05, **Pb0.01, ***Pb0.001.
82
W.-H. Kuo et al. / Clinica Chimica Acta 351 (2005) 79–86
Tris–HCl, pH 7.5, 0.9% (w/v) NaCl, 0.05% (v/v) Tween-20 and 5% skim milk. Polyclonal antibodies to human CA isoforms were diluted 1:2000 in antibody binding buffer containing 100 mmol/l Tris–HCl, pH 7.5, 0.9% (w/v) NaCl, 0.05% (v/v) Tween-20 and 5% skim milk. Incubations were performed at room temperature for 3.5 h. The immunoblots were washed three times in 50 ml blotting buffer for 10 min and then immersed in the second antibody solution containing horseradish peroxidase goat anti-rabbit IgG (Promega) for 1 h and diluted 5000-fold in
binding buffer. The filters were then washed three times in blotting buffer for 10 min. Color development was presented in 20 ml mixture consisting of 15 mg 4-chloro-1-napthol, 3 mg 3,3V-diaminobenzidine, 0.9% (w/v) NaCl in Tris–HCl, pH 7.5. 2.4. Quantitative analysis of concentrations of cytosolic CA isoenzymes The relative concentrations of CA isoenzymes were quantitated by analyzing the developed bands
Fig. 2. (A) Immunoblot analysis of cytosolic expression of CAI and CAII in various types of anemia. Lane PC represents the positive control as CAI and CAII. (B) Densitometric analysis of the CAI and CAII concentrations in various types of anemia. *Pb0.05, **Pb0.01, ***Pb0.001.
W.-H. Kuo et al. / Clinica Chimica Acta 351 (2005) 79–86
83
Fig. 3. (A) Immunoblot analysis of cytosolic expression of CAIII in various types of anemia. Lane PC represents the positive control as CAIII. (B) Densitometric analysis of the CAIII concentration in various types of anemia. **Pb0.01, ***Pb0.001.
on the Western blot membrane with a gel documentation and analysis system (Alpha Imager 2000, Alpha Innotech) as previously described [16].
Whitney rank sum test. SigmaStat software (Jandel Scientific Software, USA) was used for all statistical analyses. A P value b0.05 was considered significant.
2.5. Statistical analysis 3. Results Values were expressed as meansFS.E. The statistical significance of the means for cytosolic CA isoenzyme between groups was determined by Mann–
The hematologic, demographic and clinical characteristics of controls and patients (patients with
84
W.-H. Kuo et al. / Clinica Chimica Acta 351 (2005) 79–86
various types of anemia including aplastic anemia, autoimmune hemolytic anemia, iron deficiency anemia and h-thalassemia anemia) are shown in Table 1. No significant difference was observed for any studied parameter between controls and studied groups. However, reticulocyte count was significantly increased in both groups of autoimmune hemolytic anemia and h-thalassemia anemia, compared to that of control subjects ( Pb0.001). MCV and MCHC were significantly increased in the group of aplastic anemia while, on the contrary, lower in iron deficiency and hthalassemia anemia, compared to the control subjects ( Pb0.001). The results of erythrocyte total CA activity for various types of anemia and statistical analysis showed that the activities of erythrocyte total CA activity were significantly increased in all patients with various types of anemia (Fig. 1). Furthermore, Western blotting analysis revealed that CAII concentration was higher in autoimmune hemolytic and iron deficiency, compared to that of controls ( Pb0.05 and Pb0.05, respectively) (Fig. 2A and B). In h-thalassemia anemia, CA II concentration was threefold higher than controls ( Pb0.001) while the density of CAI was significantly less in hemolytic anemia ( Pb0.01) (Fig. 2A and B). In addition, CAIII concentration in iron deficiency anemia was significantly less than controls ( Pb0.01). Nevertheless, in the h-thalassemia anemia, the concentration of CAIII was about 1.8-fold higher than control subjects ( Pb0.001) (Fig. 3A and B).
4. Discussion Hemolytic anemia is a state of hemolysis in which increased erythrocyte production is insufficient to keep up with accelerated RBC destruction, thus producing anemia. This anemia is characterized as normochromic/normocytic, except when sufficient outpouring of the larger reticulocytes produces a resulting elevation of the MCV (Table 1). Hemolytic anemia is a classification of disorders in which red cells are destroyed prematurely, which leads to anemia. In the classification, hemolytic anemia can be classified as autoimmune hemolytic anemia, mechanical hemolytic anemia, paroxysmal nocturnal hemoglobinuria and glucose-6-phosphate dehyrogenase (G6PD) deficiency, etc. [21]. In the present
study, patients with autoimmune hemolytic anemia have been identified as positive with the direct antiglobulin test and autoimmune anemia patients have fallen ill for at least 3 months. As in our previous report, patients with G6PD deficiency anemia had a lower CAI concentration than control subjects and a consistent result was observed for autoimmune hemolytic anemia. Thus, CAI may be an applicable indicator for hemolytic anemia. Previous studies have reported a statistically significant increase of human erythrocyte CAI in the erythrocytes of subjects suffering from uremic anemia, cancer anemia and megaloblastic anemia while in sideropenic anemia the content was normal [15]. In erythrocytes from patients with acute bleeding anemia, the content of erythrocyte CAI was significantly decreased, since there was a younger cell population with a lower content of CAI/g Hb present in the patients’ erythrocytes. In normal subjects, there was no difference in CAI content between young and old erythrocytes [11,15]. A quite similar phenomenon was also noticed in our study group. The lower content of CAI in patients with autoimmune hemolytic anemia might be caused by a slower synthesis rate of CAI compared with that of hemoglobin. Although the capability of CAI for CO2 hydration turnover is lower than that of CAII and CAII is more significant physically, the data clearly showed that the variation of CAI between patients with autoimmune hemolytic anemia and controls was greater than that of CAII. Furthermore, from the present data, we found that the concentration of CAII and total CA activity in various types of anemia were significantly higher than that of normal subjects. These results were consistent with our previous study [16]. Those individuals with a G6PD deficiency, a subtype of hemolytic anemia, have higher total CA activity and CAII concentration than that of control subjects [16]. The significantly increased total CA activity and CAII may be able to compensate for the function of CAI. On the other hand, CAII has been demonstrated to be capable of binding to the carboxyl terminus of band 3 protein on the erythrocyte membrane [22] and previous researches have shown that the abnormality of erythrocyte membrane in hemolytic anemia was due to an abnormality of band 3 protein and deficiency of band 4.2 protein [23]. Thus, increased CAII concentration in various types of anemia may serve to
W.-H. Kuo et al. / Clinica Chimica Acta 351 (2005) 79–86
compensate the decreased band 3 protein concentration. Besides, CA II may provide the amount of CA activity to maintain the ion transport in erythrocytes. In Fig. 3A and B, we found that CAIII concentration was decreased in the iron deficiency anemia, but increased in the h-thalassemia. CAIII, a CA isoenzyme abundant in the red skeletal muscle, has been demonstrated to function as an oxygen radical scavenger [24]. Previous studies have demonstrated that several intra-erythrocyte enzyme systems, including glutathione peroxidase and catalase, are protective against oxidative damage [25,26]. Further, the activities of erythrocyte glutathione peroxidase and catalase have been reported to be decrease in patients with iron deficiency anemia [27–29]. In iron deficiency anemia, decreased antioxidant defense may increase oxidant stress and may result in a tendency towards platelet aggregation [30]. Since erythrocyte CAIII concentration in iron deficiency anemia was decreased, this may play the same role as antioxidants [24]. However, increases in the levels of superoxide dismutase and glutathione peroxidase have been observed in h-thalassemia anemia [31]. These findings suggested that oxidative stress occurs in those red cells with increased CAIII and provided indirect evidence to support the suggestion that CAIII may play a role as an antioxidant in erythrocytes. In conclusion, our data suggested that different carbonic anhydrase isoenzymes might serve different roles in various types of anemia erythrocyte. CAI could be used as an indicator for autoimmune hemolytic anemia based on the great variation of CAI between autoimmune hemolytic anemia and other types of anemia. CAII may be able to compensate the functions of not only CAI but also band 3 protein while CAIII could act against oxidative damage in iron deficiency and h-thalassemia anemia. The precise function of these CAs in various types of anemia still remains uncertain and, therefore, warrants further studies.
Acknowledgment This study was supported by Taichung Veterans General Hospital (TCVGH 924301A) and the ArmedForce Taichung General Hospital, Taichung, Taiwan (grant 0930000526).
85
References [1] Handin RI, Lux SE, Stossel TP. Blood, principles and practice of hematology, 2nd ed. Lippincott Willliams & Wilkins., p. 1260 – 345. [2] Wintrobe MM. Clinical hematology. 8th ed. Philadelphia7 Lea & Febiger; 1981. [3] Lessels S, Davidson RJ. The low mean cell volume in routine haematology. Clin Lab Haematol 1979;1:291 – 8. [4] Rickli EE, Ghazanfar SA, Gibbons BH, Edsall JT. Carbonic anhydrases from human erythrocytes. Preparation and properties of two enzymes. J Biol Chem 1964;239:1065 – 78. [5] Sly WS, Hu PY. Human carbonic anhydrases and carbonic anhydrase deficiencies. Annu Rev Biochem 1995;64:375 – 401. [6] Tashian RE. The carbonic anhydrases: widening perspectives on their evolution, expression and function. Bioessays 1989;10:186 – 92. [7] Pastorek J, Pastorekova S, Callebaut I, Mornon JP, Zelnik V, Opavsky R, et al. Cloning and characterization of MN, a human tumor-associated protein with a domain homologous to carbonic anhydrase and a putative helix-loop-helix DNA binding segment. Oncogene 1994;9:2877 – 88. [8] Tureci O, Sahin U, Vollmar E, Siemer S, Gottert E, Seitz G, et al. Human carbonic anhydrase XII: cDNA cloning, expression, and chromosomal localization of a carbonic anhydrase gene that is overexpressed in some renal cell cancers. Proc Natl Acad Sci U S A 1998;95:7608 – 13. [9] Supuran CT, Scozzafava A, Casini A. Carbonic anhydrase inhibitors. Med Res Rev 2003;23:146 – 89. [10] Supuran CT, Scozzafava A. In: Conway J, editor. Carbonic Anhydrase, Its Inhibitors and Activators. Boca Raton, FL7 CRC Press, 2004. p. 1 – 363. [11] Ali Akbar SA, Brown PR. Measurement of human erythrocyte CAI and CAII in adult, newborn, and fetal blood. Clin Biochem 1996;29:157 – 64. [12] Nyman PO. Purification and properties of carbonic anhydrase from human erythrocytes. Biochim Biophys Acta 1961;52:1 – 12. [13] Wistrand PJ, Rao S. Immunologic and kinetic properties of carbonic anhydrases from various tissues. Biochim Biophys Acta 1968;154:130 – 44. [14] Funakoshi S, Deutsch HF. Human carbonic anhydrase III. Immunochemical studies. J Biol Chem 1970;245:2852 – 6. [15] Mondrup M, Anker N. Carbonic anhydrase isoenzyme B in erythrocytes of subjects with different types of anemia. Clin Chim Acta 1976;69:463 – 9. [16] Chiang WL, Chu SC, Lai JC, Yang SF, Chiou HL, Hsieh YS. Alternations in quantities and activities of erythrocyte cytosolic carbonic anhydrase isoenzymes in glucose-6phosphate dehydrogenase-deficient individuals. Clin Chim Acta 2001;314:195 – 201. [17] Goode HF, Kelle J, Walker BE. Relation between zinc status and hepatic functional reverse in patients with liver disease. Gut 1990;31:694 – 7. [18] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248 – 54.
86
W.-H. Kuo et al. / Clinica Chimica Acta 351 (2005) 79–86
[19] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680 – 5. [20] Brockenbrough JS, Meyer SA, Li C, Jirtle RL. Reversible and phorbol ester-specific defect of protein kinase C translation in hepatocytes isolated from phenobarbital-treated rats. Cancer Res 1991;51:130 – 6. [21] Costea N. The differential diagnosis of hemolytic anemias. Med Clin North Am 1973;57:289 – 302. [22] Vince JW, Reithmeier RA. Carbonic anhydrase II binds to the carboxyl terminus of human band 3, the erythrocyte C1 / HCO3 exchanger. J Biol Chem 1998;273:28430 – 7. [23] Kay MM, Bosman GJ, Shapiro SS, Bendich A, Bassel PS. Oxidation as a possible mechanism of cellular aging: vitamin E deficiency causes premature aging and IgG binding to erythrocytes. Proc Natl Acad Sci U S A 1986;83:2463 – 7. [24] Raisanen SR, Lehenkari P, Tasanen M, Rahkila P, Harkonen PL, Vaananen HK. Carbonic anhydrase III protects cells from hydrogen peroxide-induced apoptosis. FASEB J 1999; 13:513 – 22. [25] Mates JM, Perez-Gomez C, Blanca M. Chemical and biological activity of free radical dscavengersT in allergic diseases. Clin Chim Acta 2000;296:1 – 15.
[26] Reiter RJ, Acuna-Castroviejo D, Tan DX, Burkhardt S. Free radical-mediated molecular damage. Mechanisms for the protective actions of melatonin in the central nervous system. Ann N Y Acad Sci 2001;939:200 – 15. [27] Macdougall LG. Red cell metabolism in iron deficiency anemia: 3. The relationship between glutathione peroxidase, catalase, serum vitamin E, and susceptibility of irondeficient red cells to oxidative hemolysis. J Pediatr 1972;80:775 – 82. [28] Perona G, Cellerino R, Guidi GC, Moschini G, Stievano BM, Tregnaghi C. Erythrocytic glutathione peroxidase: its relationship to plasma selenium in man. Scand J Haematol 1977;19:116 – 20. [29] Kumerova A, Lece A, Skesters A, Silova A, Petuhovs V. Anaemia and antioxidant defence of the red blood cells. Mater Med Pol 1998;30:12 – 5. [30] Tekin D, Yavuzer S, Tekin M, Akar N, Cin S. Possible effects of antioxidant status on increased platelet aggregation in childhood iron-deficiency anemia. Pediatr Int 2001;43:74 – 7. [31] Chakraborty D, Bhattacharyya M. Antioxidant defense status of red blood cells of patients with beta-thalassemia and E betathalassemia. Clin Chim Acta 2001;305:123 – 9.