Sodium Potassium ATPase and Glucose 6 Phosphate Dehydrogenase Activity in Patients with Hyperthyroidism: A Comparison with Euthyroid Patients

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Article Information:


Group: 2009
Subgroup: Volume 7, Issue 1, Winter
Date: March 2009
Type: Original Article
Start Page: 5
End Page: 11

Authors:

  • A Prasad
  • Department of Biochemistry, Kasturba Medical College, Madhavanagar, 576104, Manipal, India
  • S Nayak
  • Department of Biochemistry, Kasturba Medical College, Madhavanagar, 576104, Manipal, India

      Correspondence:

      Affiliation: Department of Biochemistry, Kasturba Medical College, Madhavanagar, 576104
      City, Province: Manipal,
      Country: India
      Tel:
      Fax:
      E-mail: anushreprasad@gmail.com

Abstract:


Background: Sodium potassium ATPase (Na+-K+ ATPase) and Glucose 6 phosphate dehydrogenase (G 6 PD) activities in different tissues have been found to be stimulated by thyroid hormones. In erythrocytes, the activities of these enzymes were reported to vary. The aim of our study was to determine the sodium potassium AT-Pase and glucose 6 phosphate dehydrogenase ac-tivities in patients with hyperthyroidism and compare them with those of patients with euthy-roid goiter.

Materials and Methods: After approval from the Institutional Ethics Committee and obtaining in-formed consent from all patients, 40 hyperthyro-id patients (17 men, 23 women; mean age 44.75±2.6 years) and 50 patients with euthyroid goiter (13 men, 37 women; mean age 37.2±1.6 years) were included in the study. They were classified based on T3, T4, TSH measurements. Erythrocyte Na+-K+ ATPase and G 6 PD activity were measured using spectrophotometry.

Results: In hyperthyroid patients, Na+-K+ AT-Pase activity was significantly lower compared to euthyroid controls (134.98±3.78 vs. 164.34±3.85 nmol pi/hr.mg protein, p<0.001) and G 6 PD le-vels were significantly elevated when compared to euthyroid controls (19.19±0.438 U/g Hb vs. 11.505±0.385 U/g Hb, p<0.001).

Conclusion: Hyperthyroidism is associated with decrease in Na+-K+ ATPase activity and increase in G 6 PD levels when compared to patients with euthyroid goiter. The measurement of Na+-K+ ATPase activity could be used as an early marker for diagnosis of hyperthyroidism.

Keywords: Sodium potassium ATPase; Glucose 6 phosphate dehydrogenase; Hyperthyroidism

Manuscript Body:


Introduction
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 pro-duced endogenously. T3 is a biologically ac-tive hormone, and is mostly produced from T4 in extra thyroidal tissues, while T4 lacks significant bioactivity and is a hormone pre-cursor. With the possible exception of the adult brain, anterior pituitary, spleen, and testes, these thyroid hormones exert a ther-mogenic (calorigenic) effect and increase oxygen consumption and energy expenditurethrough their effect on ATP formation and breakdown.
In animal experiments, the activity of so-dium potassium ATPase (Na+-K+ATPase) in different tissues was found to be stimulated by thyroid hormones. On the other hand, in erythrocytes of hyperthyroid patients the ac-tivity of this enzyme was reported to vary.1,2 The role of Na+-K+ ATPase for thyroid hor-mone dependent energy expenditure in cells remains unclear. It has been proposed that the activity of thyroid hormones at the cellular lev-el maybe due to their influence on the Na+-K+ ATPase, which accounts for a major proportion of the energy requirements.3 The energy re-quired for Na+-K+ ATPase transport in the eryt-hrocyte is supplied by phosphoglycerate in gly-colysis. Glycolysis in RBCs, under aerobic conditions, always ends with lactate due to the absence of mitochondria.4

Glucose 6 phosphate dehydrogenase (G6PD), a dimer of identical subunits, each with mole-cular weight of 60000 daltons, is the first en-zyme in the pentose phosphate pathway. Since, the erythrocyte lacks the TCA cycle, it depends on the pentose phosphate pathway, for supply of NADPH, which is required to maintain intracellular concentration of reduced gluta-thione; G6PD catalyzes the dehydrogenation of glucose 6 phosphate to 6 phospho gluco-nate via the formation of 6 phospho glucono-lactone. In RBCs, glycolysis and the pentose phosphate pathways are the predominant pathways of energy metabolism, which supply ATP for the membrane ion pumps and NADPH for the reduction of glutathione, which protects against oxidative injury;5 G6PD can usually make an adequate supply of NADPH under normal conditions.6,7 Earli-er studies have shown that thyroid hormones do influence the levels of G6PD in the eryt-hrocytes.8,9 In RBCs, the pentose phosphate pathway provides NADPH for the reduction of oxidized glutathione catalyzed by gluta-thione reductase, a flavoprotein containing FAD; reduced glutathione removes H2O2 in a reaction catalyzed by glutathione peroxidase, an enzyme containing selenium analogue of cysteine at the active site. Accumulation of H2O2 may decrease the lifespan of erythro-cytes by causing oxidative damage to the cell membrane, leading to hemolysis.10

The aim of our study was to estimate the levels of erythrocyte sodium potassium AT-Pase and glucose-6-phosphate dehydrogenase in patients with hyperthyroidism and com-pare these with those of euthyroid patients.

Materials and Methods
In this cross-sectional study, a total of 90 patients, aged between 18 to 60 years (40 hyperthyroid and 50 euthyroid) were in-cluded in the study. The research protocol was approved by the Institutional Ethics Committee and informed consent was ob-tained from all patients. Subjects in the hyperthyroid group were sampled from pa-tients seen in medical/surgical units in our hospital clinics. Patients were diagnosed as having hyperthyroidism if they matched one or more of the following criteria: T3>2 ng/dL, T4>14 µg/dL, TSH<0.05 ?U/mL. In-dividuals in the control group were selected from those who had goiter/thyroid nodules and normal thyroid function tests, and were not on any thyroid hormone medications; pa-tients with any other co-morbid illness and those on any medication that alters the thyro-id profile (e.g. amiodarone, lithium and digi-talis) were excluded from in the study. Five ml of blood with anticoagulant (EDTA) was collected from the patients for estimation of erythrocyte Na+-K+ATPase and G 6 PD le-vels.

Measurements:
Erythrocyte sodium potassium ATPase: Na+-K+ ATPase activity in the erythrocytes was measured as a release of inorganic phos-phate from hydrolysis of ATP in the presence and absence of ouabain.4,11
Sodium potassium ATPase activity was es-timated by the Fiske and Subbaraw method12 and the membrane protein was from the red blood cell ghosts estimated using the method adapted by Lowry et al.13,14

Glucose-6-phosphate dehydrogenate (G 6 PD).10 G6PD catalyzes the oxidation of glucose 6 phosphate (G6P) to 6 phospho glu-conate with a concurrent conversion of NADP+ to NADPH. The activity of G 6 PD was determined by measurement of the rate in increase in NADPH concentration. Whe-reas NADP+ is transparent to ultraviolet (UV) light, NADPH strongly absorbs UV light. Therefore, the rate of increase in absor-bance at 340 nm was used as a measure of enzyme activity. The absorbance of the test solution at 540 nm was used to estimate the hemoglobin concentration. A reference range value of 8 – 16 U / gm Hb was considered.

GPDactivityUgHb=14.67×ΔΑ340/ΔΑ540

 T3,15 T416 and TSH17 were estimated by the Electro-chemiluminescence immunoassay “ECLIA” of Roche Elecsys and Modular Analytics. Statistical analysis All values of analyzed parameters were ex-pressed as mean ± SEM. Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS/PC; SPSS-13, Chicago, USA). Independent sample t- test was used to compare the mean values in the two groups followed by the non-parametric test Man-Whitney test;p<0.05 was consi-dered statistically significant.

The study included 90 patients, 50 of whom were euthyroid, and 40 hyperthyroid. All demographic parameters, except for age were comparable between the groups (Table 1). The gender distribution between the two groups and T3, T4 and TSH levels in euthy-roid and hyperthyroid patients are shown in Table 1. Na+-K+ ATPase activity and glucose 6 phosphate dehydrogenase levels in euthyro-id and hyperthyroid patients are depicted in figures 1 and 2. In hyperthyroid patients, Na+-K+ ATP ase activity was significantly low when compared to euthyroid controls (134.98±3.78 vs 164.34±3.85 nmol pi/hr.mg protein, p<0.001). G6PD levels in hyperthy-roid patients however was significantly ele-vated when compared to euthyroid controls (19.19±0.43 U/g Hb vs 11.50±0.38 U/g Hb, p<0.001).

Table 1. Demographic parameters, T3, T4 and TSH levels (expressed as mean ± SEM) in the study groups * p<0.05

 

Euthyroid(n= 50)

Hyperthyro-id(n= 40)

Age (yr)

37.2±1.6*

44.75±2.6

Height (cm)

156.36±0.9

156.53±1.5

Weight (kg)

57.66±1.19

54.93±1.4

BMI (kg/m2)

23.59±0.4

22.57±0.6

Sex distribution (M/ F)

13/37

17/23

T3 (ng/dL)

1.23±0.32

2.69±1.55

T4 (µg/dL)

8.49±1.96

17.07±3.86

TSH (μU/mL)

1.68±0.87

0.03±0.05

 

 

fig1  

Fig.1. Mean Na+-K+ ATPase activity in euthyroid and hyperthyroid patients. The darker line depicts standard deviation

Fig.2. G6 PD levels in euthyroid and hyperthyroid patients

 Fig.2. G6 PD levels in euthyroid and hyperthyroid patients

Discussion

Various studies investigating the activity of Na+-K+ ATPase in tissues have consistently shown that the activity is increased in hyper-thyroidism.24-26 However, levels and activity of the enzyme in RBCs of patients with thy-roid dysfunction have not shown a consistent trend. While Sato et al18 found that the activi-ty of the enzyme decreased in both hyperthy-roid and hypothyroid patients, DeLuise M et al 2, Dasmahapatra et al 4, and others 6,19,20 found that the levels and activity of the en-zyme decreased in hyperthyroid and in-creased in hypothyroid patients.

Sato et al18 found that the Na+-K+ ATP ase activity was significantly reduced (11±4.6 vs 17.3±4.1 µg Pi/hr/mg protein, p<0.01) in hy-pothyroid and also in hyperthyroid patients (p<0.01) which normalized after treatment; they suggested that Na+-K+ ATPase activity can be used as a sensitive index of peripheral thyroid status.

In our study, the levels of Na+-K+ ATPase were significantly decreased in hyperthyroid patients (134.98±3.78 nmol Pi/mg.h vs 164.34± 3.85 nmol Pi/mg.h). The results of our study were similar to those documented by Dasma-hapatra et al4, who also found levels of the en-zyme to be decreased in hyperthyroidism (307±30 nmol Pi/mg.h vs 380±24 nmol Pi/mg.h, p<0.05). The exact mechanism re-sponsible for the reduction in the activity of RBC Na+-K+ ATPase pumps in hyperthyroid-ism is still uncertain. Various possible me-chanisms have been proposed to explain the decrease in Na+-K+ ATPase in erythrocytes; Rubython et al3 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 al21 have shown in a prelim-inary study that Na+-K+ ATPase activity of an erythroid cell line was stimulated by T3. De-Luise and Flier et al2 supported the hypothe-sis 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 proteins, sug-gesting that this degradation may occur in the circulation during aging of the RBCs or dur-ing formation of the reticulocytes. Dasmaha-patra et al4 have also postulated that the di-minished degradation of erythrocyte pump units in hypothyroidism may be responsible for the increased enzyme activity in this group of patients. Since our study correlated well with their study, we think it is likely that the decreased erythrocyte Na+-K+ ATPase ac-tivity in hyperthyroidism may be due to an accelerated degradation of membrane pro-teins.

Many of the abovementioned studies have shown normalization of the enzyme activity following treatment of thyroid dysfunc-tion.20,27,28 De Riva C et al20 suggested that the decrease in enzyme levels after cessation of treatment could be used as an early indica-tor of recurrence of hypothyroidism. Since we lost a number of patients on follow up, we could not assess the enzyme levels after treatment.

In view of the aforementioned data, we suggest that the measurement of activity of sodium potassium ATPase could be used as an early marker for the diagnosis of hyper-thyroidism.

The intracellular redox potential is deter-mined by the concentrations of oxidants and reductants. A critical modulator of the redox potential is NADPH, the principal intracellu-lar reductant in all cell types. Glucose- 6-phosphate dehydrogenase (G6PD), the rate limiting enzyme of the Pentose Phosphate Pathway (PPP), determines the amount of NADPH by controlling the metabolism of glucose via the PPP.23 It has been traditional-ly thought that G6PD was a typical ‘house-keeping’ enzyme that was regulated solely by the ratio of NADPH to NADP.29 But research suggests that this enzyme is highly regulated and plays important roles in a variety of cel-lular processes. G6PD is under close tran-scriptional, translational, and posttranslation-al control.29-31 Research has demonstrated that growth factors like the thyroid hormone can rapidly activate G6PD and stimulate translocation of G6PD.30,31 In addition, G6PD activity plays a critical role in cell growth via its role in intracellular redox regulation.23

In our study, G6PD activity was signifi-cantly elevated in hyperthyroid patients (p< 0.001), findings which correlated well with those of Nehal et al.8 Bildik et al1 also found a significant increase in the erythrocyte G6PD levels in hyperthyroid rabbits (P< 0.05). However, Odcikin E et al22 found that the levels of G6PD was reduced in hyperthy-roid patients when compared to euthyroid ones (10.19±1.87 Ug/Hb in the healthy group and 4.92±2.49 Ug/Hb in the patients).

The precise mechanism for the changes in the activities of red cell enzymes stimulated by T3 is not fully understood. It is obvious that thyroid hormones do have an overall ef-fect on metabolism of responsive tissues.1

Tian Wang-Ni et al23 have shown that intracellular levels of G6PD are crucial in the balance of oxidant- antioxidant activity, over-expression of G6PD increased resistance to H2O2-induced cell death and that reduced le-vels predisposed the cell to oxidant induced cell death.24 In view of the above findings, we postulate that increased levels of the en-zyme activity in hyperthyroidism can help prevent RBCs from oxidant induced destruc-tion.

To conclude, our findings show that eryt-hrocyte Na+-K+ ATPase activity is decreased in patients with hyperthyroidism which may be due to an accelerated degradation of membrane proteins. Measurement of the ac-tivity of sodium potassium ATPase could be used as an early marker for the diagnosis of hyperthyroidism. Increased levels of the G6PD enzyme activity in hyperthyroidism can help prevent RBCs from oxidant induced cell destruction.

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