Carbonic anhydrase activity in the red blood cells of sea
level and high altitude natives

JORGE GAMBOA, RICARDO CACEDA, ALFREDO GAMBOA, CARLOS MONGE-C.

Laboratorio de Transporte de Oxígeno, Departamento de Ciencias Fisiológicas, Universidad Peruana Cayetano Heredia.

ABSTRACT

Red blood cell carbonic anhydrase (CA) activity has not been studied in high altitude natives. Because CA is an intraerythocytic enzyme and high altitude natives are polycythemic, it is important to know if the activity of CA per red cell volume is different from that of their sea level counterparts. Blood was collected from healthy subjects living in Lima (150m) and from twelve subjects from Cerro de Pasco (4330m), and hematocrit and carbonic anhydrase activity were measured. As expected, the high altitude natives had significantly higher hematocrits than the sea level controls (p=0.0002). No difference in the CA activity per milliliter of red cells was found between the two populations. There was no correlation between the hematocrit and CA activity.

Key words: Carbonic anhydrase, hypoxia, polycythemia.

Carbonic anhydrase (CA) is a common enzyme, and it is contained in key regulatory organs. The carotid bodies respond to hypoxia with an increase in pulmonary ventilation; see Iturriaga (1993) for a review of the mechanisms of this response. In the kidney (Swenson, 1998), CA is involved in regulating acid-base equilibrium. Its action on the renal tubules produces a mild metabolic acidosis by increasing bicarbonate excretion, which increases pulmonary ventilation. The inhibition of this enzyme by acetazolamide is used as a prophylactic treatment for acute mountain sickness (Cain and Dunn, 1966; Hackett and Rennie, 1976; Johnson and Rock, 1998). It is known that this substance produces an elevation of the arterial PO₂ during acute exposure to hypoxia (Cain and Dunn, 1966). In the lungs, red cell CA is responsible for the dehydration of H₂CO₃ and its hydration in the tissues. Without a catalyst, the interconversion between CO₂ and H₂CO₃ requires more than 1 minute for completion, while the capillary transit of red cells takes 1 second. The acceleration of these reactions between 13000 and 25000 fold is achieved by red cell CA (Geers and Gros, 2000). Our concern was to evaluate the activity of this enzyme in high altitude and sea level natives because there was no information available with respect to the effects of chronic hypobaric hypoxia on the CA activity of red blood cells and its relation to polycythemia. Red cell levels in patients with secondary polycythemia, as opposed to hypobaric hypoxia, were similar to the normal subjects (Funakoshi and Deutsch, 1971). It is expected that an increased production of red cell CA occurs during hypobaric hypoxia as it does with hemoglobin. This information may be useful in future attempts to treat excessive polycythemia with acetazolamide.

Blood was collected from fifteen healthy native subjects of Cerro de Pasco (4300 m) and another twelve natives of Lima (130 m). The blood was centrifuged to separate the red cell fraction, and the red cells were diluted 1/400 (v/v). The carbonic anhydrase activity was measured using the changing pH technique with a barbital buffer, a continuous flow of CO₂ and the addition of increasing amounts of diluted red cells as described by Maren (1960). Units were expressed per milliliter of red cells (U/ml).

The hematocrit was measured by the microhematocrit method. Student's t test was used to compare the means between groups. Data were analyzed by SPSS for Windows v7.5, using p<0.05 for statistical significance.

Results are shown in Table I There was no correlation between hematocrit and CA activity when expressed by red cell volume. At high altitudes there is an increase of the blood volume, primarily due to an increase in the red cell fraction, otherwise the plasma volume is contracted (Sánchez et al, 1970). Blood flow at high altitude and sea level is similar (Winslow and Monge, 1987). This study showed no difference in the carbonic anhydrase activity per milliliter of red cells. However, due to the intraerythrocytic location of this enzyme, the circulating CA activity of red cells is increased, as occurs with the mass of circulating hemoglobin.

High altitude natives have a low CO₂ tension and a low arterial HCO₃^- level (Monge et al, 1964), which is the result of their greater ventilation. However, the role of CA in this acid base equilibrium at high altitudes is unknown. CA inhibitors such as acetazolamide have been used in the treatment of many diseases, including acute mountain sickness and sleep apnea syndrome (Swenson, 1998). Chronic Mountain Sickness (CMS) is a disabling disease suffered by high altitude natives and is characterized by an excessive hypoxemia and erythrocytosis and a variety of symptoms related to the nervous system (Winslow and Monge, 1987). An attempt to treat this disease with acetazolamide required the knowledge of CA activity in the blood of those patients and was a stimulus to undertake this research. Also, blood letting used in the treatment of CMS with significant acute reduction of polycythemia could be an important experimental and clinical tools for assessing the role of CA in excessive polycythemia.

Table I

Age, hematocrit, and carbonic anhydrase activity in healthy subjects in Cerro de Pasco
(4300m) and Lima (150 m)

x ± s.d.

Corresponding Author: Carlos Monge-C. Universidad Peruana Cayetano Heredia. Apartado 4314, Lima 100, Perú Email: cmonge@upch.edu.pe

Received: 14, 2000. Revised: uly 21, 2000 Accepted: July 27, 2000

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