ISRAEL JOURNAL OF
VETERINARY MEDICINE
THE EFFECT OF HYPERTHYROIDISM ON THE LEVELS
OF Na+ K ATP+ ASE, GLUCOSE 6 PHOSPHATE
DEHYDROGENASE AND GLUTATHIONE
Vol. 57 (2) 2002
A. Bildik1, F. Belge2, F. Yur3, M. Alkan3 and D. Kili•alp2
1. Department of Biochemistry, Faculty of Veterinary Medicine, University
of Adnan Menderes, Ayd‫ץ‬n, Turkey.
2. Department of Physiology, Faculty of Veterinary Medicine, University of
YŸzŸncŸ Y‫ץ‬l, Van, Turkey.
3 . Department of Biochemistry, Faculty of Veterinary Medicine, University
of YŸzŸncŸ Y‫ץ‬l, Van, Turkey.
Abstract
This study was carried out to investigate the effect of hyperthyroidism on the activity of Na +
K+ ATP ase, glutathione and glucose 6 P dehydrogenase. Rabbits (n=16) were divided into
two groups of 8: control and treatment. Treatment group was given daily i.p. an injection of
L-tyrosine (MERCK) (100 µg/kg, for 21 days). After treatment, blood samples were collected
into vacutaine tubes containing sodium heparin. Concentrations of plasma T3 and T4 were
measured using DPC-Immulite kit. Na+ K+ ATP ase activity in erythrocyte was determined
colorimetrically by the release of inorganic phosphate from ATP in the presence and
absence of oubain. The activities of GSH and glucose 6 P dehydrogenase were assayed
spectrophotometrically. In the treatment group, Na + K+ ATP ase levels were decreased
(p<0.05) while GSH and glucose 6 P dehydrogenase levels (p<0.05) were elevated
compared to control values.
Introduction
Hyperthyroidism occurs as a result of excessive production of thyroid hormones. In most
hyperthyroid patients, the thyroid gland is two to three times larger than normal (1).
The stimulatory effect of thyroid hormones on the basal energy expenditure of the body is
well known. However, the mechanism at the cellular level is still unclear. The activity of
membrane bound Na+ /K+ ATPase has been proposed to be regulated by thyroid hormones
and accounts for a major proportion of the increased energy demand in hyperthyroidism (1,2).
Thyroid hormones include the iodinated amino acid derivates T3 (3,3’,5-triiodo-L-thyronine)
and T4 (3,3’,5,5-tetraiodo-L-thyronine), the only iodinated hormones produced endogenously.
T3 is the biologically active hormone, and is mostly produced from T 4 in extra thyroidal
tissues. T4 lacks significant bioactivity and is a hormone precursor. With the possible
exception of the adult brain, anterior pituitary, spleen, and testes, thyroid hormone exerts a
thermogenic (calorigenic) effect and increases oxygen consumption and energy expenditure
through its effect on ATP formation and breakdown (3).
In animal experiments, the activity of Na K ATP ase in different tissues was found to be
stimulated by thyroid hormones (4). On the other hand, in erythrocytes of hyperthyroid
patients the activity of this enzyme is reported to decrease (5,6) or increase (7). The role of
Na+ /K++ ATP ase for thyroid hormone dependent energy expenditure in cells remains
unclear.
Required energy for Na+ , K+ transport in erythrocyte is supplied by phosphoglyserate in
glycolysis (8). Glycolysis in erythrocytes, even under aerobic conditions, always end with
lactate because of the absence of mitochondria(9).
In the erythrocyte, glycolysis by the pentose phosphate pathway is the predominant pathway of
carbohydrate metabolism. Glycolysis supplies ATP for membrane ion pumps and NADH for
reoxidation of methemoglobin. The pentose phosphate pathway supplies NADPH to reduce glutathione
for protection against oxidant injury. Glucose 6 phosphate dehydrogenase (G6PD), the first enzyme of
the pentose phosphate pathway, can usually make an adequate supply of NADPH under normal
conditions (9,10). The carrier-mediated transport of thyroid hormones across the red cell membrane as
well as their biological effects has been described. There exists a good correlation between the binding
of thyroid hormone to membrane and its biological effects. In the rat erythrocyte membrane, many of
the enzyme reactions are known to be influenced by thyroid hormones (11,12,13,14).
The present study was aimed to examine Na+ /K+ ATPase, G-6-P and Glutathione (GSH)
in T3-induced hyperthyroidism.
Materials and Methods
Animals:
Sixteen male rabbits (Lepus europus) weighing 1500-1600g were fed ad libitum and
received daily i.p. injections of L tyrosine (MERCK) (100µg/kg, during 21 days, treatment
group) while the controls received an equivalent volume of 0.9% NaCl (15). After 21 days,
blood samples were collected into vacutainer tubes containing sodium heparin.
Hormone and enzyme activities measurements:
Concentrations of plasma T 3 and T4 using DPC-Immulite kit were assayed by DPC
IMMULITE Automated Analyzer (16).
Na+ /K+ ATPase activity in the erythrocytes was measured as the release of inorganic
phosphate from hydrolysis of ATP in the presence and absence of oubain.
Erythrocytes were incubated at 370C for 60 min in 1 ml of a solution containing 3 mM ATP
(pH=7.0), 50mM NaCl, 20 mM KCl, 3 mM MgCl2, 100 mM tris-HCl (pH=7.4). To inhibit Na+ /K+
ATPase activity, 1mM oubain was added. The reaction was stopped by the addition of
trichloracetic acid. ATPase activity was expressed as nanomoles of phosphorus released per
mg protein /hr (17,18).
The amount of reduced glutathione (GSH) in whole blood was determined by the method of
Beutler (19) and the activities of G-6-P dehydrogenase were measured using the Randox kit
(20).
Statistical analysis and significance of differences between control and T 3-treated animals
were determined by Student’s t test (21).
Results
After 21 days of treatment, the respective mean serum T3 and T4 values were 98.4± 6.6
ng/dl, 6.09±0.76 ug/dl in the controls and 266.1±19ng/dl, 32.75±3.2 ug/dl in the hyperthyroid
rabbits (p<0.001).
In the rabbits with hyperthyroidism, mean Na+ /K+ ATPase erythrocyte activity was 0.85±
0.2 µmol Pi/h.mg protein, whole blood mean GSH value was 38.1±1.5 mg/dl, mean G-6-P D
activity in the erythrocyte was 10.1± 0.8 U/g Hb. In the controls, the respective values were
1.77±0.25 µmol Pi/h.mg protein, 24.17±1.9 mg/dl, and 7.3±0.5 U/g Hb.
Na+ /K++ ATPase activity in hyperthyroid rabbits was significantly lower than that in
controls (p<0.05) (Figure 1), GSH level increased by 57% (p<0.001) (Figure 2). The mean G6-PD activity was also elevated in the hyperthyroid group (p<0.05) (Figure 3).
Figure1: Na+ /K+ ATPase activity of controls and rabbits with hyperthyroidism
Figure 2: GSH levels of controls and rabbits with hyperthyroidism
Figure 3: Glucose 6 P dehydrogenase activity of controls and rabbits with hyperthyroidism
Discussion
Our studies on erythrocytes in hyperthyroidism confirm that the Na +/K+ ATPase activity
decreased and that GSH and G-6 P dehydrogenase levels were increased.
It has been demonstrated that thyroid hormones increase the Na +/K+ ATPase activity in rat
liver, kidney and skeletal muscle (4,22,23). In contrast, enzyme activity was diminished in
RBCs (18,24). The exact mechanism responsible for the reduction in the number of RBC Na +
/K+ ATPase pumps in hyperthyroidism is still unknown.
Various possible mechanisms have been proposed to explain the decrease in Na +/K+
ATPase in erythrocytes. Rubython et al. (6) have suggested that thyroid hormones probably
inhibit the synthesis of the sodium pump during the maturation in the bone marrow. However,
this is unlikely as Aramunayam et al. have recently shown in a preliminary study that Na+ K+
ATPase activity of an erythroid cell line was stimulated by T 3 (25). Therefore, DeLuise and
Flier supported the hypothesis that the resultant change could be due to the degradation of
Na+ /K+ ATPase units in erythrocytes as thyroid hormones are known to accelerate
catabolism of cell protein (5). They have suggested that this degradation may occur in the
circulation during aging of the RBCs or during formation of the reticulocytes. According to the
results of this study, we think it is likely that the decreased erythrocyte Na+ /K+ ATPase
activity in hyperthyroidism may be due to an accelerated degradation of membrane proteins
Thyroid hormone exerts a thermogenic effect and increases oxygen consumption. Elevation
of the metabolic rate of rats made hyperthyroid by treatment with T3 is accompanied by
increased rates of hepatic O2 consumption, together with an enhanced microsomal oxidative
capacity and free radical activity. In addition, key antioxidant mechanisms including the
activity of superoxide dismutase and catalase as well as the total content of liver glutathione,
are depressed by T3 treatment (14).
Seven et al. (13) found that hyperthyroid patients were observed to have significantly higher
lipid peroxidation end product (TBARS) and antioxidant status parameters: glutathione,
glutathione peroxidase (GSH-Px) and CuZn superoxide dismutase (CuZnSOD) levels than
controls (13). They have suggested a selective modification of the antioxidative profile in
hyperthyroidism.
Previous studies have shown that GSH content of RBC’s are elevated (13,15) and liver
GSH levels are decreased (12,14) in subjects with hyperthyroidism, T 3 induces a marked
depletion of hepatic GSH, which can be ascribed mainly to a loss of GSH from the liver into
the sinusoidal space (14). In this study, GSH concentration in the blood was higher by 57% in
hyperthyroid rabbits than in controls.
Erythrocytes derive all their energy from the oxidation of glucose. Since RBC’s are nonnucleated and without mitochondria, the major route of glucose consumption is through the
anaerobic Embden-Meyerhoff pathway. Hexose monophosphate pathway metabolizes only
about 10% of total glucose uptake (3,11). G-6-P dehydrogenase, which is an enzyme of the
pentose phosphate pathway plays an important role. For this to occur, NADPH is also needed
for maintenance of reducing atmosphere in cells exposed to high concentrations of oxygen
radicals (9,10).
Our data agree with those by Nehal and Baquer (11) that both G-6-P dehydrogenase and
hexokinase increase in hyperthyroidism. However, the precise mechanism for the changes in
the activities of red cell enzymes stimulated by T 3 is yet to be understood, it is obvious that
thyroid hormones do have an overall effect on metabolism of responsive tissues and that their
effect is an indirect one.
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