Syed Muhammad Shahid  ( Department of Biochemistry, Fatima Jinnah Dental College )
Muhammad Jawed  ( Department of Biochemistry, Fatima Jinnah Dental College )
Tabassum Mahboob  ( Department of Biochemistry, University of Karachi )

Objective: To investigate the disturbances of serum and red cell electrolytes in association with membrane Na+-K+- ATPase activity as well as the status of serum Urea, Creatinine and osmolality in normotensive diabetic and hypertensive diabetic patients.

Methods: Thirty normotensive and thirty hypertensive patients (age and sex matched) were selected along with thirty control subjects. Erythrocytes were isolated from freshly drawn blood samples, washed and used for the estimation of sodium and potassium concentrations using flame photometer (Corning 410). Erythrocyte membranes were prepared for the estimation of Na+-K+-ATPase activity in terms of inorganic phosphate released/mg protein/hour. Serum glucose, creatinine and urea were determined by well-documented ortho toulidine, Jaffe's and diacetyl monoxime methods respectively. Osmomat 030 was used to estimate the plasma osmolality.

Results: The intra-erythrocyte sodium, serum glucose, urea, creatinine and osmolality were increased significantly in hypertensive diabetic patients as compared to normotensive diabetic patients whereas Na+-K+-ATPase activity, serum sodium, potassium, magnesium and calcium were decreased significantly in hypertensive diabetic patients as compared to normotensive diabetic patients.

Conclusion: Results confirmed that there is a significant difference between normotensive and hypertensive diabetic patients with respect to their electrolyte metabolism and associated pathways. These results will notably help the physicians to treat diabetic patients with associated morbidity like hypertension (JPMA 55: 153;2005).

Diabetes mellitus is a chronic metabolic disorder that can lead to serious cardiovascular, renal, neurologic and retinal complications.1 Observational studies suggested that cardiovascular morbidity is ~2 times higher in diabetic patients than in the general population, and that cardiovascular disease accounts for ~70% of all deaths in people with diabetes mellitus.2 In contrast with type I diabetes, hypertension in type II diabetes develops even without renal involvement. The constellation of abnormalities that include obesity, hypertension, diabetes and dyslipidemia is called metabolic syndrome, and this condition has been associated with high cardiovascular risk, morbidity and mortality.3

The rise in plasma insulin levels may elevate blood pressure levels by a variety of mechanisms, including increased sympathetic activity and sodium retention. This association could be explained on the basis of three different pathophysiological mechanisms: 1) abnormal electrolyte transport across cell membranes, a defect that alters sodium-potassium exchange and also sodium-calcium exchanges, increasing the concentration of intracellular calcium ions that heightens vessel wall tension and the smooth muscle process, 2) increased sympathetic nervous system activity and 3) altered cellular concentration that induces water logging in the peripheral arteriolar walls. These mechanisms increase peripheral resistance and arterial pressure.4

It is generally accepted that hypertension and other vascular pathologies increase in diabetic patients as a result of renin-angiotensin-aldosterone system5, which plays a key role in the regulation of fluid and electrolyte balance. The angiotensin converting enzyme inhibitors have been shown to be effective in many cardiovascular diseases particularly hypertension associated diabetes.6

Sodium retention occurs as a characteristic alteration in type I or type II diabetes. Possible sodium retaining mechanisms include increased glomerular filtration of glucose leading to enhanced proximal tubular sodium-glucose cotransport, hyperinsulinemia, an extravascular shift of fluid with sodium, and once it occurs, renal failure. The pathogenetic role of sodium retention in diabetes-associated hypertension is supported by positive correlation between systolic or mean blood pressure and exchangeable sodium and by normalization of blood pressure after removal of excess sodium by diuretic treatment in hypertensive diabetic subjects.7 Endothelium-derived nitric oxide is an important determinant of renal natriuresis and of peripheral vascular tone. Although the available data are conflicting, a number of studies have demonstrated impaired vascular response to endothelial dependent vasodilators or reactive hyperemia in diabetic patients. This suggests impaired production or enhanced degradation of nitric oxide or depressed smooth muscle responses to nitric oxide. The mechanisms underlying this abnormality in vascular function are not well established.8 Abnormal sodium metabolism has a critical role in the etiology of essential hypertension. This has lead to the hypothesis that can increase in the circulating concentration of Na+-K+-ATPase inhibitor, which is responsible for the cause of essential hypertension. Potassium depletion is a common feature of essential hypertension and type II diabetes. Treatment of hypertension at least partially restores potassium levels towards normal and fasting steady state potassium levels are closely linked to calcium and magnesium homeostasis.9 Because of the importance of magnesium and calcium in metabolic enzyme regulation and their interaction, several studies have demonstrated the disturbed concentrations of erythrocyte sodium, magnesium, calcium, potassium in diabetic and hypertensive patients as well as in normotensive patients.10 Streptozotocin-induced diabetic rats with hyperinsulinemia, hyperlipidemia and hypertension also showed increase in serum urea, creatinine, triglyceride, and cholesterol levels.11 Significant correlation has been found between fasting plasma glucose and plasma osmolality as well as concentrations of sodium and potassium in diabetic and hypertensive patients.12 These studies highlighted the high prevalence of vascular complications in diabetic and hypertensive patients, especially macrovascular complications.13

Due to the similar and coordinate nature of ionic defects, the present study was designed to investigate the disturbances of serum and red cell electrolytes in association with membrane Na+-K+- ATPase activity as well as the status of serum Urea, Creatinine and osmolality in normotensive diabetic and hypertensive diabetic patients.

Thirty hypertensive and thirty normotensive diabetic patients without intercurrent illness or severe diabetic complications were studied on a regular follow-up after obtaining verbal informed consent. Their mean age was 38 ± 2.5 years. None were taking any medication known to influence Na+-K+-ATPase, such as drugs like calcium channel blockers, thyroxin, glucocorticoid, mineralocorticoid or digitalis. Thirty age and sex matched healthy normotensive subjects with no known history of hyperglycemia were selected as controls.
Fasting blood samples were collected from control and diabetic subjects in lithium heparin coated tubes followed by measuring their blood pressures using sphygmomanometer. A small portion of blood was separated to obtain serum.

Heparinized blood was centrifuged and plasma was separated. Buffy coat was aspirated and discarded. Erythrocytes were washed three times at room temperature by suspension in magnesium chloride solution (112 mmol L-1), centrifugation at 450x g at 4oC for 5 minutes and aspiration of the supernatant as described earlier.14 Final supernatant was retained for the estimation of intra-erythrocyte sodium and potassium concentration. No electrolyte was detectable in the final wash. Washed erythrocytes were then lysed and used for the estimation of intra-erythrocyte sodium and potassium.

The red cell pack extracted by centrifugation at 4oC were resuspended and diluted in 25 volumes of Tris buffer at pH 7.4. The hemolyzed cells were then centrifuged at 12,000 rpm at 4oC and the membrane pellet was suspended in 30 ml of 0.11 mol L-1 Tris-HCl buffer. This centrifugation step was repeated three times. The final concentration of the membrane suspension was ~4 mg protein ml-1 of Tris buffer. The membrane suspension was stored at -80oC until the assay was performed.

Na+-K+-ATPase activity was measured in a final volume of 1 ml as follows: Membrane (400mg) were preincubated for 10 minutes at 37oC in a mixture containing 92mmol L-1 Tris-HCl (pH=7.4), 100mmol L-1 NaCl, 20 mmol L-1 KCl, 5 mmol L-1 MgSO4. H2O and 1 mmol L-1 EDTA.15 Assays were performed with and without 1mmol L-1 Ouabain, a specific inhibitor of Na+-K+-ATPase. After incubation with 4 mmol L-1 ATP (Vanadate free, Sigma) at 37oC for 10 minutes, the reaction was stopped by adding ice-cold trichloroacetic acid to a final concentration of 5%. After centrifugation at 4oC, 5500g for 10 minutes. The amount of inorganic phosphate in the supernatant was determined.16 Na+-K+-ATPase activity was calculated as the difference between inorganic phosphate released during the 10-minute incubation with and without Ouabain. Activity was corrected to a nanomolar concentration of inorganic phosphate released milligram-1 protein hour-1. The concentration of protein was estimated by Biuret method.

All assays were performed in duplicate, and blanks for substrate, membrane and incubation time were included to compensate for endogenous phosphate and non-enzyme related breakdown of ATP. Under these experimental conditions, the coefficient of variation was 7.5%.

Serum sodium, potassium and calcium were estimated by flame photometer (Corning 410). Serum magnesium was estimated by the method described earlier.17

Serum glucose was estimated by Ortho toulidine method, serum urea was estimated by Diacetyl monoxime method and serum creatinine was estimated by Jaffe's method. All the above discussed methods are very common and well documented. The plasma osmolality was estimated by using automatic Osmolality meter (Osmomat 030).

Results are presented as mean and standard deviation. Statistical significance and difference from control and test values were evaluated by Student's t-test.

Comparison of control subjects with normotensive diabetic patients

The intraerythrocyte sodium, serum potassium (Table 1) and serum glucose (Table 2) concentrations were increased significantly (P<0.01) in normotensive diabetic patients as compared to control subjects. The membrane Na+-K+-ATPase activity, serum sodium and serum magnesium concentrations decreased significantly (P<0.01) in normotensive diabetic patients as compard to control subjects (Table 1) whereas intra-erythrocyte potassium and serum calcium were decreased and increased significantly (P<0.05) in normotensive diabetic patients as compared to control subjects respectively (Table 1). The serum urea concentration was decreased and serum creatinine and plasma osmolality were increased non significantly in normotensive diabetic patients as compared to control subjects (Table 2). No significant increase or decrease was observed in systolic and diastolic blood pressures in normotensive diabetic patients as compared to control subjects (Table 3).

Comparison of Control subjects with Hypertensive diabetic patients

The intraerythrocyte sodium (Table 1), serum glucose, serum creatinine and plasma osmolality (Table 2) were increased significantly (p<0.01) in hypertensive diabetic patients as compared to control subjects. Serum urea was also increased but non significantly. The intraerythrocyte potassium, serum sodium, potassium, magnesium, calcium and membrane Na+-K+-ATPase activity were decreased significantly (p<0.01) in hypertensive diabetic patients as compared to control subjects (Table 1). A significant rise in systolic as well as diastolic blood pressures was observed in hypertensive diabetic patients as compared to control subjects (Table 3).

Table 1. Intraerythrocyte and serum electrolytes in control, normotensive diabetic and hypertensive diabetic patients.
	Controls	Normotensive Diabetics	Hypertensive Diabetics
RBC Sodium	11.55+4.83	14.96+3.95**	7.99+3.35++§§
(mmol L-1)
RBC Potassium	106.61+21.41	97.09+10.72*	97.55+9.52++
(mmol L-1)
Na+-K+-ATPase	443.37+282.04	100.66+43.8**	58.86+24.63++§§
(nmol mg-1 hr-1)
Serum Sodium	138.1+13.79	107.33+16.07**	92.67+13.1++§§
(mmol L-1)
Serum Potassium	5.03+1.51	7.41+1.8**	4.59+1.71++§§
(mmol L-1)
Serum Magnesium	1.25+0.68	0.84+0.32**	0.72+0.25++
(mmol L-1)
Serum Calcium	2.11+0.41	2.46+0.73*	1.57+0.49++§§
(mmol L-1)

Values are Mean + Standard deviation.
* P < 0.05, ** P < 0.01 Control vs Normotensive diabetic subjects.
++ P < 0.01 Control vs Hypertensive diabetic subjects.
§§ P < 0.01 Normotensive diabetics vs Hypertensive diabetics.

Table 2. Serum glucose, urea, creatinine and osmolality in control, normotensive diabetic and hypertensive diabetic patients.
	Controls	Normotensive diabetics	Hypertensive diabetics
Glucose (mmol L-1)	5.4 + 0.45 9.0 +	3.9** 9.16 +	2.52++
Urea (mmol/L)	10.58 + 2.63	9.69 + 1.93	11.26 + 2.46
Creatinine (mmol	L-1) 107.67 + 48.81	112.83 + 38.43	156.83 + 42.83++§§
Osmolality (mOsm Kg-1)	206.23 + 30.46	212.2 + 34.27	231.93 + 30.45++§

Values are Mean + Standard deviation.
** P < 0.01 Control vs Normotensive diabetic subjects.
++ P < 0.01 Control vs Hypertensive diabetic subjects.
§ P < 0.05, §§ P < 0.01 Normotensive diabetics vs Hypertensive diabetics.

Comparison of Normotensive diabetic patients with Hypertensive diabetic patients
The intraerythrocyte sodium (Table 1), serum creatinine and plasma osmolality (Table 2) were found increased significantly (p<0.01) in hypertensive diabetic patients as compared to normotensive diabetic patients. Serum glucose and serum urea concentrations were also found increased but non-significantly. The membrane Na+-K+-ATPase activity, serum sodium, serum potassium and serum calcium concentrations were found decreased significantly (p<0.01) in hypertensive diabetic patients as compared to normotensive diabetic patients (Table 1). The intraerythrocyte potassium and serum magnesium concentrations were also found decreased but non significantly (Table 1). A significant rise in systolic and diastolic blood pressures was observed in hypertensive diabetic patients as compared to normotensive diabetic patients (Table 3).

Table 3. Systolic and Diastolic blood pressures in control, normotensive diabetic and hypertensive diabetic patients.
	Controls	Normotensive diabetics	Hypertensive diabetics
Systolic BP (mm Hg)	123.77 + 8.13	125.33 + 6.3	143.87 + 13.55++§§
Diastolic BP (mm Hg)	79.37 + 6.79	78.73 + 4.11	+ 7.0++§§

Values are Mean + Standard deviation.
++ P < 0.01 Control vs Hypertensive diabetic subjects.
§§ P < 0.01 Normotensive diabetics vs Hypertensive diabetics.

The results of the present study reveal the significant variations in electrolyte balance along with the glucose and blood pressure rise; as discussed before.The dysregulation of chief electrolytes especially sodium, potassium, calcium and magnesium have a characteristic role in diabetic complications such as cardiovascular disturbances, primarily hypertension.18

The role of sodium, potassium, calcium and magnesium in blood pressure regulation, particularly in diabetes mellitus, is well established.19 Increased intra-erythrocyte sodium with decreased intra-erythrocyte potassium, serum sodium, calcium and magnesium is seen in diabetic subjects. Several evidences suggested that increased Na/H exchanger activity might be involved in this functional impairment. In Streptozotocin-induced Hypertensive Rats strain, increased functional sodium reabsorption and blunted pressure natriuresis have been reported.20 Sodium potassium exchange pump decreases the transport of sodium ions out of the cell and potassium ions into the cell causing a rise in intracellular sodium concentration and lowers the intracellular potassium concentration, as shown in the present study. In diabetes, the direction and activity of Na+-Ca+ exchanger is changed, which may be one of the mechanisms for the decreased extracellular calcium concentration and prolonged hyperglycemia is one of the stimulating factor for this alteration in Na+-Ca+ exchanger activity. These steady state kinetics are closely linked to calcium and magnesium homeostasis.21 Magnesium is a strong co factor for the Na+-K+-ATPase pump22 and its lower concentration in diabetes is justified by the reduced activity of Na+-K+-ATPase pump.

Long standing hyperglycemia is directly related to the raised blood pressure in hypertensive diabetic subjects. The increased intra-erythrocyte sodium with decreased intra-erythrocyte potassium, serum sodium, calcium and magnesium in diabetic subjects is a consequence of decreased Na+-K+-ATPase activity due to the increased systolic and diastolic blood pressures as observed in the present study. Na+-K+-ATPase is a ubiquitous enzyme that ensures that the transmembrane gradients of sodium and potassium concentrations are maintained. Alterations of this transport enzyme are thought to be linked to several complications of diabetes mellitus like hypertension.22 The present study revealed a decreased serum calcium and magnesium levels in association with increased blood pressure. The constellation of suppressed calcium and magnesium levels is the characteristic of hypertension associated with diabetes.9 Elevated cytosolic free calcium and reciprocally reduced, extracellular ionized calcium levels are observed in both hypertension and diabetes as observed in present study. These alterations of calcium metabolism underlie the predisposition to the alterations of blood pressure and insulin sensitivity.23 Magnesium appears to be a special kind of calcium antagonist in vascular smooth muscles. At vascular membrane, it can lower peripheral and cerebral vascular resistance.24 Decreased level of magnesium also results in the inhibition of Na+-K+-ATPase enzyme because magnesium is a co-factor for this enzyme.

The hyperosmolar polyuria is the characteristic sign of diabetes with hypertension resulting from increased urea, creatinine and plasma osmolality in these conditions and same results were obtained in the present study. Plasma osmolality and the concentrations of chief solutes responsible for hyperosmolality are directly linked with the increased plasma glucose concentrations and the arterial pressures.12 In various previous diabetic nephropathy trials, the risk of combined end point of doubling the base line serum creatinine level is strongly related with onset of end-stage renal disease.25 The increased levels of urea and creatinine resulted from the alterations in the renin-angiotensin-aldosterone system5, which is the main aberration during diabetes associated with hypertension leading to diabetic nephropathy.

These results emphasize the similar and coordinate nature of imbalances in ionic environment and their defective transportation in normotensive diabetics as compared to hypertensive diabetic patients and supports the role of extracellular glucose levels in regulating electrolyte metabolism. It may be concluded that in long standing diabetes with hypertension, the measurement and interpretation of these cellular markers may be useful to identify patients who are at risk of developing nephropathy.

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