Biomedical Research 2 (3) 334-337, 1981

Adaptation to : Changes in the level of erythrocyte 2.3-diphosphoglycerate and liver adenylates

MASATAKA YOSHINO1, RYOICHI HAYASHI2, YOSHINAO KATSUMATA3, KOHEI SUGANO4, KOZO HIRATA4 and TETSUO NAGASAKA4 ‘Department of Biochemistry, Yokohama City University School of Medicine, Yokohama 232, Zlnstitute of Equilibrium Research, Gifu University School of Medicine, Gifu 500, 3Department of Legal Medicine, Nagoya University School of Medicine, Nagoya 466, and 4Department of Physiology, Kanazawa University School of Medicine, Kanazawa 920, Japan

ABSTRACT The effects of acute hypoxia (9% O2) on the levels of erythroycte 2,3-diphospho- glycerate (2,3-DPG) and hepatic adenine nucleotides were investigated. Measure- ments were made from both fed and starved mice. 1) Exposure of fed mice to 9% O2 rapidly increased erythrocyte 2,3-DPG, which is considered to be a reflection of metabolic adaptation to hypoxia. The liver adenylate pool remained unchanged for one day under hypoxic states, and a marked reduction in the hepatic adenylates occurred after 3 days. 2) When mice were starved, little or no increase in erythrocyte 2,3-DPG was observed, and hepatic adenylates decreased rapidly within 1 day. Thus, a decrease in the liver adenylate pool and an increase in erythrocyte 2,3-DPG, the most important factor favoring oxygen release at tissue levels, were demonstrated, and this suggests the importance of the adenylate level in liver for metabolic adaptation to hypoxia.

KEY WORDS diphosphoglycerate / hypoxia / adaptation / liver adenylate pool

Hypoxia results in a series of physiological the mechanism of rapid adaptation to hypoxia and biochemical changes that can maintain for facilitating the supply of oxygen to tissues normal oxygen delivery and also induce a is not operative above an altitude of 6,000 m metabolic adaptation to an anaerobic environ- (20). This paper concerns metabolic adapta- ment (11). Some metabolic changes to a tion to hypoxia, that is, the changes in eryth- deficient oxygen supply may act in most rocyte 2,3-DPG and hepatic adenylate contents tissues: an elevation of erythrocyte 2,3-di- which occurred in mice exposed to 9% O2 phosphoglycerate (2,3-DPG) (10), an increased (6,000m simulated altitude). An increase in rate of glycolysis (13), and adenine nucleotide erythrocyte 2,3-DPG and a decrease in hepatic degradation (7, 14~16) under the conditions adenylates were demonstrated: the importance causing tissue hypoxia. The increase in eryth- of the hepatic adenylate level is discussed in rocyte 2,3-DPG during hypoxia, which is relation to liver , which can considered to be the most important factor supply , a precursor of 2,3-DPG in favoring oxygen release at tissue level (10), erythrocytes. is explained by the activation of erythrocyte Male ddy-mice, aged 4 weeks, were separated phosphofructokinase (EC 2.7.1.11) due to into control and fasted groups. The control respiratory alkalosis in hypoxia (8). Our groups were fed standard laboratory chow and recent studies on the relationship between the water ad libftuur. Five mice per group were increase in 2,3-DPG and the oxygen saturation exposed to a gas mixture of 9% O2 and 91% N2 in arterial blood at high altitude suggest that in chambers through which the gas mixture was ADAPTATION TO HYPOXIA 335 passed at 700 ml per min. The composition of Fig. 1 shows the increase in the concentra- the gas mixture was controlled and monitored tions of erythrocyte 2,3-DPG in mice occur- by regulating the flow of each component into ring with exposure to a gas mixture of 9% O2 a mixing vessel with calibrated flowmeters. (corresponding to an altitude of 6,000 rn). The compositions of all gas mixtures were A remarkable and rapid increase in 2,3-DPG expressed on a v/v basis. In the fasted group was observed. During the following 7 days the mice were fasted for 24 hr prior to exposure of O2 deficiency, 2,3-DPG concentrations still to hypoxia, and starvation was continued under remained elevated (Fig. 1). When the animals the hypoxic conditions. were exposed to normal air, 2,3-DPG levels Blood samples were collected by decapita- returned to normal value within one day. It tion, and erythrocytes were separated by cen- should be noted that 2,3-DPG did not tend to trifugation of heparinized blood. The livers increase when the animals were starved: the were separated and immediately frozen in concentration of 2,3-DPG remained at normal liquid nitrogen. The frozen livers were then levels (Fig. 1). broken, rapidly weighed, and ground in frozen We also determined the concentrations of form with a mortar and pestle. The powder was liver adenine nucleotides during exposure to homogenized in 5 parts (w/v) of 6% perchloric hypoxic conditions. The adenylate pool in acid. The precipitate was removed by cen- liver remained unchanged for a day after the trifugation and the supernatant fluid was exposure to hypoxia. The concentration dras- neutralized with 0.6M KZCOB. The super- tically decreased on the third day, and the natants were used for the determination of decreased level of liver adenylates was further adenylates. Adenylates were measured after maintained under the hypoxic conditions. enzymatic conversion to ATP, which was The level returned to normal value when the analyzed by luciferase reaction (5). 2,3-DPG mice were returned to normal air (Fig. 2). was determined according to the method of Animals which had been starved for 24 hr Keitt (9). Hemoglobin was measured as cya- showed a significant decrease in liver adenylates nomethemoglobin. Data are given as means on the first day after exposure to hypoxia iSD. Standard statistical methods were em- (Fig. 2). ployed. Exposure to hypoxia initiates a series of

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Fig. 1 Changes in erythrocyte 2,3-diphosphoglycerate (2,3-DPG) concen- trations in mice during exposure to 9% O2 (corresponding to 6,000m simulated altitude). Blood was collected by , and erythrocytes were separated by centrifugation of the heparinized blood. 2,3-DPG was determined by the method of Keitt (9). O, fed; A, starved for 24 hr prior to exposure to 9% O2 and further fasted for 24 hr during exposure to hypoxia 336 M. YOSHINO er al.

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"'(,umoAdeny/gEH25<3wetghtwe ,< --- -9<% Q[------>eAn+* Tot caO i. 10 i H 2'4-1r i st Hours Days Exposure Fig. 2 Changes in mouse liver adenylate pool during exposure to 9% O2. Conditions and symbols are the same as in Fig. 1. Sample preparation and determination of adenylates are described in the text. Data are given as means :1; SD. physiological and biochemical changes that hypoxia (2, 6) could be responsible for the tend to maintain oxygen delivery and also degradation of adenine nucleotides. Second, induce a metabolic adaptation to lower oxygen liver adenylate content decreased more rapidly delivery (11). Our previous paper has shown under starved-hypoxic conditions than under that a rapid increase in the plasma oxypurines fed-hypoxic conditions. Stimulation of gluco- and erythrocyte 2,3-DPG occurs during hypoxia neogenesis, which is an energy-requiring reac- (21). Erythrocyte 2,3-DPG, which is a power- tion, can result. in a slight decrease in the liver ful modifier of the oxygen afiinity of hemoglobin adenylate pool under starved conditions (18). (1, 4), is considered to be the most important Hypoxia-induced increase in catecholamines factor favoring oxygen release at tissue levels and cortisol would favor an increased glyco- (10). A close correlation of oxypurines and genolysis and hepatic gluconeogenesis (2, 3, 6, 17), 2,3-DPG with the oxygen pressure in which can provide blood glucose, a good sub- suggests that the levels of these metabolites strate for anaerobic metabolism in many may be indicative of tissue hypoxia (21). Fur- tissues under hypoxic conditions, and for the thermore, a relation between the increase in synthesis of erythrocyte 2,3-DPG. When fed erythrocyte 2,3-DPG and the recovery of ad lfbffruiz before and during hypoxia, little oxygen saturation in arterial blood during a or no depletion of liver glycogen and no stay at high altitude suggests that the mechanism stimulation of hepatic gluconeogenesis may whereby oxygen is more efiiectively transported occur at the first stage; thus, the liver adenylate to the tissues has a central role in the rapid pool remains unchanged for 3 days. Prolonged acclimatization toward hypoxia below 6,000m exposure to hypoxia can result in the exhaus- altitude (20). tion of liver glycogen (12, 17); liver gluco- We have demonstrated a decrease in liver neogenesis has a central role in the production adenylate pool and an increase in erythrocyte of blood glucose, and a decrease in the adenylate 2,3-DPG under hypoxic conditions. Our data pool can occur. On the other hand, if the do not provide a detailed explanation of the organism is starved prior to hypoxic exposure, mechanism, but the following conclusions blood glucose must be supplied mainly by can be drawn. First, hypoxic results hepatic gluconeogenesis because of depletion in marked increase in plasma glucose (2), of the glycogen stores in liver; thus, a rapid lactate (2), fatty acids (3), cortisol (2), and decrease in liver adenylates can be observed. catecholamines (2), which are known to induce A decrease in the hepatic adenylate pool, the degradation of adenine nucleotides (19). which may be the result of both the direct The increased level of catecholamines during effect of catecholamines and the stimula- ADAPTATION TO HYPOXIA 337 tion of gluconeogenesis, and an increase in diphosphoglyceric acid: Spectrophotometric erythrocyte 2,3-DPG were clearly demonstrat- and fluorometric procedures. J. Lab. Clix. Med. 77, 470-475 ed during hypoxia: the liver adenylate level LENFANT C., TORRANCE J., Ewousn E., Fmcn and energy states seem to be important factors C. A., REYNAFARJE C., RAMOS J. and FAURA in metabolic adaptation to hypoxia. J. (1968) Effect of altitude on oxygen binding by hemoglobin and on organic phosphate levels. We would like to express our gratitude to Professor J. Clfn. Invest. 47, 2652-2656 K. Tsushima in the Department of Biochemistry, LENFANT C., TORRANCE J. D., WOODSON R. Yokohama City University School of Medicine for and Fmcn C. A. (1970) Adaptation to hypoxia. his valuable discussions and criticisms concerning In Red Cell M6f0b0lfSl?l and Function (ed. this work. BREWER G. J.) Plenum Press, New York, pp. 203-212 Receivedfor publication 17 December 1980 PURSHOTTAM T., KAvEEsnwAR U. and BRAHMACHARI H. D. (1977) Changes in tissue glycogen stores of rats under acute and chronic REFERENCES hypoxia and their relationship to hypoxia toler- 1. Bawsscn R. and Bawsscn R. E. 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