A radioimmunosorbent technique for the assay of B- and C-types of carbonic anhydrase in human tissues

A radioimmunosorbent technique for the assay of the human carbonic anhydrase isoenzymes HCA B and HCA C in tissue fluids was developed. The sensitivity of the method was 0.2 ng/ml and the precision was 5 % in duplicate determinations for both enzymes. The presence in a tissue of up to 20 times higher concentrations of one isoenzyme will not interfere with the assay of the other. Haemolysates contained (mean ± SE, n = 11) 12.1±0.52 and 1.5 ±0.06 mg enzyme/g Hb, and serum 0.63 ±0.12 and 0.2 ±0.02 μg/ml of HCA B and HCA C, respectively. Pilot experiments indicated that the isoenzymes can be determined also in tissues, i.e. urine, saliva and cerebrospinal fluid, where catalytic methods previously have indicated absence of or only weak carbonic anhydrase activity. N-terminals of both enzymes were not antigenic.     

Carbonic anhydrase isoenzyme determination: An aid to the diagnosis of "small-for-date" infants

A specific and quantitative immunological method for the determination of human erythrocyte carbonic anhydrase isoenzymes B and C has been used to determine these enzymes in erythrocytes of umbilical cord blood. The investigations have shown a content of both isoenzymes less than a tenth of that in adults. The isoenzyme content correlated significantly with the estimated gestational age. The ratio between isoenzyme B and C rose with gestational age, reaching adult level at term. A group of 19 "small-for-date" infants had isoenzyme levels in the same range as normal full-term infants, significantly higher than the levels in the same range as normal full-term infants, significantly higher than the levels in truly premature infants. It is concluded that the determination of carbonic anhydrase isoenzymes is an aid in distinguishing "small-for-date" infants from the truly premature.     

Estimations of active and inactive carbonic anhydrase isozyme B in human red cells

Human erythrocyte carbonic anhydrase isozyme B was measured by a specific and immunological method. The levels of carbonic anhydrase B were determined in normal subjects, patients with hyperthyroidism, and patients with chronic obstructive lung disease and patients with epilepsy under treatment with acetazolamide, using the rapid assay method of single radial immunodiffusion. The levels of carbonic anhydrase B showed a decrease in hyperthyroidism, and increased in chronic obstructive lung diseases and epilepsies. Closely negative correlations were observed between carbonic anhydrase B and T₃ resin sponge uptake or protein bound iodine (PBI) levels in hyperthyroidism. Simultaneously, carbonic anhydrase B-dependent esterase activity (active carbonic anhydrase B enzyme) was determined kinetically using the immunoadsorbent method in the above-mentioned patients. The results were compared with the total enzyme protein (active and inactive carbonic anhydrase B enzyme), estimated by the single radial immunodiffusion technique. In patients treated with acetazolamide, the “true” specific activity of the carbonic anhydrase B (carbonic anhydrase B-dependent esterase activity/total carbonic anhydrase B protein) decreased remarkably. In chronic obstructive lung disease, no remarkable changes were observed. On the other hand the specific activity was elevated in hyperthyroidism to 2 times higher than that of normal subjects. The clinical significance of these active enzymes is discussed.     

The determination of osteopetrotic phenotypes by selective inactivation of red cell carbonic anhydrase isoenzymes

Red cell carbonic anhydrase isoenzyme activities (HCA-I and HCA-II) were quantitated in blood hemolysates of two female siblings affected with autosomal recessive osteopetrosis in addition to family members who were both obligate and potential heterozygotes as well as normal controls. Selective inactivation of red cell hemolysates with 20 mmol/l bromopyruvic acid (pH 7.5) for 50 min at 25 degrees C lead to a near quantitative removal of HCA-I activity without significant loss of HCA-II activity. Comparison of untreated hemolysate HCA activity with residual activity in bromopyruvate treated samples allowed determination of HCA-II/HCA-I activity ratios. Estimation of activity ratios was also attempted by use of the inhibitor I- which is known to discriminate between the two isoenzymes. Hemolysate HCA activity of the two affected individuals (homozygotes) was completely abolished by the procedure, consistent with an absence of the bromopyruvate resistant HCA-II isoenzyme in the red cells of these individuals. HCA-I activities were found to be elevated significantly above normal. Both parents (obligate heterozygotes) were found to have normal HCA-I activities and HCA-II activities 60-80% of normal. Computed HCA-II/HCA-I activity ratios fell into these ranges: 0 for homozygotes, 0.82-0.87 for obligate heterozygotes and 1.08-1.16 for normal controls. KI values for I- inhibition of hemolysate HCA activity gave 0.25-0.30 mmol/l, 0.9-1.5 mmol/l and 2.0 mmol/l for the three categories. These relations should prove useful in the screening of osteopetrotic phenotypes.     

The Influence of the Carbonic Anhydrase Inhibitor, Benzolamide (CL-11,366), on the Reabsorption of Chloride, Sodium, and Bicarbonate in the Proximal Tubule of the Rat

Benzolamide (CL-11,366), a potent carbonic anhydrase inhibitor, was used to examine the influence of carbonic anhydrase inhibition on the reabsorption of chloride, sodium, and bicarbonate in the rat proximal convoluted tubule. Administration of 2 mg/kg benzolamide was associated with a decrease in the tubular fluid/plasma (TF/P) chloride ratio from 1.19+/-0.10 (SEM) to 1.06+/-0.01, and an increase in the TF/P bicarbonate ratio from 0.181+/-0.02 to 0.584+/-0.02. This dose of benzolamide significantly reduced proximal fractional reabsorption of chloride by 29.14%, of sodium by 34.3%, and of bicarbonate by 35.64%. These results indicate that benzolamide administration inhibits the reabsorption of all three electrolytes in the proximal convoluted tubule. Although 20 mg/kg benzolamide accentuated the changes in fractional reabsorption, the differences between 2 and 20 mg/kg were not statistically significant.Inhibition of proximal tubular cytoplasmic and luminal carbonic anhydrase could well explain the diminished bicarbonate reabsorption and a fraction of the diminished sodium reabsorption noted in these studies. The fall in chloride reabsorption, and a portion of the fall in sodium reabsorption, however, may be a direct or indirect consequence of carbonic anhydrase inhibition or of an influence of benzolamide on a transport mechanism not dependent upon carbonic anhydrase.     

An evaluation of cytosolic erythrocyte carbonic anhydrase and catalase in carcinoma patients: an elevation of carbonic anhydrase activity

OBJECTIVES: The antioxidant enzyme catalase and the CO2/HCO3- exchange enzyme carbonic anhydrase are both present in significant amounts in the cytosol of erythrocytes. The aim of the present study was to investigate whether these erythrocyte enzyme activities are altered in patients who have carcinoma. DESIGN AND METHODS: Cytosolic erythrocyte enzyme activities were measured in 108 cancer patients presenting with carcinoma at one of variable sites, prior to clinical treatment. These were compared with an age- and sex-matched control group of 31 healthy volunteers. RESULTS: Mean (+/-SD) catalase activities did not differ significantly, i.e. 0.0035 (+/-0.0015) EU/ml in carcinoma patients vs. 0.0031 (+/-0.00075) EU/ml in controls. However, mean carbonic anhydrase activities of 204 (+/-91) EU/ml in the carcinoma patients were significantly higher than the 158 (+/-35) EU/ml in controls (P value of 0.0065). CONCLUSION: Cytosolic erythrocyte carbonic anhydrase levels may warrant further investigation as a potential peripheral marker in cancer.     

A simple enzyme-linked immunoassay of human carbonic anhydrase I for the study of developing erythroid cells

A simple and rapid enzyme-linked immunoassay for human erythrocyte carbonic anhydrase isozyme I was developed. The assay was found to be sensitive enough for the detection of nanogram amounts of the enzyme in incubation mixtures. The first incubation with anti-human carbonic anhydrase I IgG was carried out for 6 hours at room temperature. The second incubation of the enzyme was carried out in the presence of goat plasma C1q coupled with peroxidase for 1 h at room temperature. The enzymatic reaction was performed for 30 min using 2,2'-azino-di(3-ethyl-benzthiazoline sulfonate) as a substrate, and absorbance at 414 nm was recorded. Carbonic anhydrase I was assayed on the range of 1 to 200 ng/ml using this method. The levels of carbonic anhydrase I in K562 cells induced by erythropoietin and in other blood cells were determined. This enzyme-linked immunoassay has application for the study of developing erythroid cells.     

Human erythrocyte carbonic anhydrase B and C in chronic obstructive lung disease.

The levels of carbonic anhydrase B and C isozymes in human red cells were determined using a quantitative immunological technique in patients with chronic obstructive lung disease. A significant increase in the level of carbonic anhydrase B was observed in these patients, while the level of carbonic anhydrase C did not change substantially. Positive correlations were found between the level of carbonic anhydrase B and arterial CO2 tension and plasma HCO3 concentration. A negative correlation was observed between the levels of carbonic anhydrase B and blood pH. These findings suggest that the synthesis or degradation of carbonic anhydrase B isozyme is affected by arterial CO2 tension or plasma HC03 concentration. The clinical significance was also discussed in relation to these isozyme levels in red cell.     

Spontaneous luminal disequilibrium pH in S3 proximal tubules. Role in ammonia and bicarbonate transport.

We determined whether a spontaneous luminal disequilibrium pH, pHdq (pH measured - pH equilibrium), was present in isolated perfused rabbit S2 and S3 proximal tubules. Luminal pH was measured by perfusing with the fluorescent pH probe 1,4-DHPN, and the equilibrium pH was calculated from the measured collected total CO2 and dissolved CO2 concentrations. S2 tubules failed to generate a spontaneous pHdq. S3 tubules generated a spontaneous acidic pHdq of -0.46 +/- 0.15 (P less than 0.05), which was obliterated following the addition of carbonic anhydrase (0.1 mg/ml) to the perfusate. In S3 tubules perfused and bathed in 4 mM total ammonia, luminal total ammonia rose from 4.08 +/- 0.05 mM (perfusate) to 4.95 +/- 0.20 mM (collected fluid) (P less than 0.02). Carbonic anhydrase added to the perfusate prevented the rise in the collected total ammonia concentration. We conclude that the rabbit S3 proximal tubule lacks functional luminal carbonic anhydrase. The acidic pHdq in the S3 segment enhances the diffusion of NH3 into the lumen. In contrast, the S2 segment has functional luminal carbonic anhydrase.     

The measurement of human erythrocyte carbonic anhydrase I by the enzyme linked immunosorbent assay (ELISA)

A method is described for the assay of human erythrocyte (RBC) carbonic anhydrase I (HCAI) by the enzyme linked immunosorbent assay. The method was found to be a simple, reliable and precise technique and gave a mean C.V. for 40 samples, assayed in quadruplicate, of 3.32% and a range of 0.84-5.9. The mean erythrocyte HCAI value and standard deviation for 20 normal men and women were respectively 16.9 +/- 3.4 and 15.4 +/- 2.1 mg HCAI/g haemoglobin. The use of heparin as an anticoagulant interfered with the assay resulting in apparent HCAI concentrations as low as 60% of those obtained using EDTA.     

A single-step solid phase radioimmunoassay for quantifying human carbonic anhydrase I and II in cerebrospinal fluid

A single-step solid phase radioimmunoassay was developed to detect human carbonic anhydrase (CA) isoenzymes I (CA I) and II (CA II) in cerebrospinal fluid (CSF). The assay is capable of routinely detecting both isoenzymes at ng levels compared to the μg levels of the traditional catalytic methods, which failed to demonstrate any CA activity in CSF. When the values of immunoreactive CA II in CSF were corrected for blood contamination (the CA I/CA II ratio of blood was about 7.9), the amount of brain tissue originated CA II could be calculated. The CA II values in CSF samples from 13 patients with multiple sclerosis were higher than those in CSF samples from 11 patients with various peripheral neurological disorders. Since CA II has been specifically localized to oligodendrocytes and myelin, our preliminary results suggest the possibility of CA II leakage from oligodendrocytes and myelin into CSF in demyelinating diseases.     

Bone resorption induced by parathyroid hormone and dibutyryl cyclic AMP: role of carbonic anhydrase

The role of carbonic anhydrase in bone resorption induced by parathyroid hormone (PTH) and dibutyryl cyclic AMP (DBcAMP) was studied using an in vitro neonatal mouse half-calvarial culture system. Both PTH (16.7 nM) and DBcAMP (0.3 mM) were effective in stimulating bone resorption, as assessed by measuring changes in media calcium concentrations. Bones treated with PTH or DBcAMP for 96 hr contained significantly greater carbonic anhydrase activity than control bones [PTH Treated/Control (T/C) = 2.44; DBcAMP T/C = 2.34]. Both PTH and DBcAMP significantly enhanced calvarial acid phosphatase activity relative to control values [PTH T/C = 1.48; DBcAMP T/C = 1.30]. Neither PTH nor DBcAMP significantly altered calvarial alkaline phosphatase activity. Bone resorption induced by PTH and DBcAMP was inhibited by the carbonic anhydrase inhibitor acetazolamide, but not by the acetazolamide analog CL 13,850 (N-t-butylacetazolamide), which does not inhibit carbonic anhydrase. These results support the concepts that PTH-induced bone resorption requires the action of osteoclastic carbonic anhydrase and that the action of PTH on bone is mediated, in part, by the action of cyclic AMP.     

Inactive form of erythrocyte carbonic anhydrase B in patients with primary renal tubular acidosis.

Evidence was found for an inactive form of carbonic anhydrase type B in the erythrocytes of two children with primary renal tubular acidosis. The addition of zinc chloride to hemolysates from these patients resulted in a marked increase in the activity of this enzyme. No such effect was noted with hemolysates of control subjects. No significant differences were observed in the zinc levels of hemolysates of these patients and of normal individuals. However, the level of zinc in the carbonic anhydrase B isolated from one of these patients was low, suggesting a modified form of the enzyme. The restoration of activity upon the addition of zinc was reversed by ethylenediamine tetraacetate, but no such effects were noted for the carbonic anhydrase B of normal individuals. Thus the abnormal carbonic anhydrase B has decreased zinc binding. The ultraviolet difference spectrum of the carbonic anhydrase B of normal individuals and that of a patient showed a peak at 305 nm which decreased upon the addition of zinc. The abnormal form of carbonic anhydrase B was not distinguishable from that of normal individuals by either immunological or electrophoretic criteria.     

Inhibition of renal carbonic anhydrase as a respiratory stimulant-- an obsolete indication?

Inhibition of carbonic anhydrase in general or specific inhibition of one the different isoenzymes results in a significant metabolic acidosis due to renal bicarbonate loss. The increase of arterial pCO2 stimulates central and peripheral chemoreceptors and enhances ventilation. The inhibition of carbonic anhydrase as a respiratory stimulant is an accepted measure for the prevention of acute mountain sickness, has been used for a restricted number of subjects with sleep-disordered breathing or chronic hypoxaemic lung disease. The few indications and the narrow therapeutic index restrict the use of carbonic anhydrase blockers as stimulants for ventilation.     

Carbonic anhydrase isoenzyme B in the erythrocytes of uremic subjects

A specific and quantitative immunological method for determination of human erythrocyte carbonic anhydrase isoenzyme B has been used to determine the contents of enzyme in the erythrocytes from healthy persons and subjects with uremia.The investigations have shown a significant increase of carbonic anhydrase type B in the erythrocytes of patients with renal failure.Correlation was found between human erythrocyte carbonic anhydrase type B and Pt-creatinine-clearance but not between it and plasma standardbicarbonate.     

Analysis of marker enzymes in the K562 erythroleukaemia cell line: no coordinate expression of red cell enzymes on induction of haemoglobin synthesis

Six sublines of the K562 erythroleukaemic cell line were examined by starch gel electrophoresis for the red cell enzymes carbonic anhydrase, CA I and II, pyruvate kinase, PK1 (R), and lactic dehydrogenase, LDH (B), both uninduced and following induction of haemoglobin synthesis with sodium butyrate or haemin. Neither CA or PK1 (R) enzymes were present constitutively found to be inducible characteristics; the LDH isoenzyme pattern showed equal expression of A and B subunits and did not change to the red cell form (B greater than A) on induction. Subline heterogeneity was also examined by analysis of a further 11 polymorphic enzymes, and cell line contamination was found not to account for the previously reported variations in inducibility by sodium butyrate.     

Ethacrynic acid inhibits transcellular NaCl reabsorption in dog kidneys in doses of 1 to 10 mg.kg-1 and proximal bicarbonate-dependent reabsorption at higher doses

In anesthetized dogs, sodium reabsorption in the kidney tubule was continuously reduced with increasing dosage of ethacrynic acid until 59% of the filtered load was excreted with 25 mg.kg-1 at constant glomerular filtration rate. With 50 mg.kg-1, glomerular filtration rate fell, but fractional sodium reabsorption was further reduced by 7%. In the dosage range of 1 to 10 mg.kg-1, ethacrynic acid inhibited chloride but not bicarbonate reabsorption, and the ratio between reductions in sodium reabsorption and oxygen consumption (delta Na/delta O2) averaged 25.5 +/- 5.2. With doses of 25 and 50 mg.kg-1, ethacrynic acid further reduced chloride reabsorption, reduced fractional bicarbonate reabsorption by 17% and almost halved phosphate reabsorption, whereas delta Na/delta O2 rose significantly to 65.3 +/- 12.2. Renal cortical carbonic anhydrase activity was normal and the inhibitory effect of acetazolamide, a carbonic anhydrase inhibitor, on bicarbonate reabsorption was unimpaired. We conclude that ethacrynic acid in doses up to 10 mg.kg-1 exclusively inhibits transcellular NaCl reabsorption, but in higher doses interferes with bicarbonate reabsorption and bicarbonate-dependent passive reabsorption of NaCl in the proximal tubules without inhibiting carbonic anhydrase activity.     

Carbonic anhydrase function and the epithelial organization of H+ secretion in turtle urinary bladder

The function of carbonic anhydrase in H(+) secretion by the turtle bladder was studied in vitro. Dose response curves were obtained for the carbonic anhydrase inhibitors, acetazolamide and ethoxzolamide, with and without addition of CO(2) to the system. In addition, carbonic anhydrase was assayed in homogenates of mucosal cells. The activity in the homogenates was 155+/-16 U/g dry wt, of which only 11 U represented contamination from erythrocytes; after addition of 5 x 10(-6)m acetazolamide, no enzyme activity was detectable.In the intact preparation free of exogenous CO(2), the dose response curve for acetazolamide showed two plateaus of inhibition, one at 50% and one at more than 80% inhibition. At 50% inhibition (from 5 x 10(-6) to 5 x 10(-5)m acetazolamide), H(+) secretion was restored or enhanced by CO(2) addition to the same extent as bladders not exposed to acetazolamide. At concentrations of more than 1 x 10(-4)m, H(+) secretion was no longer restorable by CO(2). Unlike acetazolamide, ethoxzolamide caused progressive inhibition of H(+) secretion in the CO(2)-free system. The maximal extent of inhibition with ethoxzolamide and the behavior of inhibition in the presence of 2.5% CO(2) were the same as for acetazolamide. Evidence is presented that all surface epithelial cells secrete H(+) and generate OH(-) within the cell interior. The capacity of cells to dispose of OH(-) by CO(2) hydroxylation varies with the availability of cytoplasmic carbonic anhydrase. A small population of cells contains abundant carbonic anhydrase and secretes at high rates even when CO(2) is in short supply.On the basis of these results and histochemical data on the distribution of carbonic anhydrase within the mucosa, an analysis is presented of the epithelial organization of acidification by the turtle bladder.     

Carbonic anhydrase isoenzyme B in erythrocytes of anemic and non-anemic uremic subjects.

A specific and quantitative immunological method for determination of human erythrocyte carbonic anhydrase isoenzyme B has been used to determine the contents of this enzyme in the erythrocytes from healthy persons and anemic and non-anemic subjects with renal insufficiency. The investigations have shown significant increase of carbonic anhydrase type B in the erythrocytes of patients with renal failure. The erythrocytes of anemic uremic patients have a significantly higher content of erythrocyte carbonic anhydrase isoenzyme B than the erythrocytes of non-anemic uremic subjects. Significant correlations were found between human erythrocyte carbonic anhydrase type B and hemoglobin and between it and plasma bicarbonate. No correlation was found between human erythrocyte carbonic anhydrase and plasma creatinine or creatinine clearance and serum zinc.     

Effect of acetazolamide on rat gastric mucosal protection and stimulated bicarbonate secretion with 16,16-dimethyl PGE2

The effect of pretreatment with acetazolamide, a carbonic anhydrase inhibitor, was studied on the mucosal protection provided by and the gastric alkaline secretion stimulated by 16,16-dimethyl prostaglandin E2. Using a model employing a chamber of the rat whole stomach, 16,16-dimethyl prostaglandin E2 (1 microgram/ml) was found to significantly (p less than 0.05) increase the secretion of bicarbonate by 31.1 +/- 4.1 mu Eq/hr over basal values. This stimulated secretion was inhibited (to 18.0 +/- 2.2 mu Eq/hr) by pretreatment with acetazolamide (50 mg/kg body weight). In a separate series of experiments, the ability of this concentration of 16,16-dimethyl prostaglandin E2 to protect the rat stomach from necrosis caused by absolute ethanol was not impaired by prior exposure to the same dose of acetazolamide.