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Red meat linked to diabetes risk: The study, which included around 210,000 men and women, found that after adjustment for other risk factors, the pooled HRs for a one serving/d increase of unprocessed, processed and total red meat consumption was 1.12, 1.32 and 1.14 respectively.

Association between erythrocyte Na+K+-ATPase activity and some blood lipids in type 1 diabetic patients from Lagos, Nigeria

Bamidele A Iwalokun and Senapon O Iwalokun

Diabetes mellitus, a metabolic disease characterized by altered carbohydrate, lipid and protein metabolism and requiring strict glycemic control for delaying or preventing cardiovascular, nephropathic and neurological complications in humans remains a public health problem in developing and developed countries of the world [1]. Several studies in animals and humans have shown that pathogenic course of both Type 1 and Type 2 diabetes mellitus involves alterations in the structures, organization and protein functions of membranes of cells and tissues (e.g. retina, glomerulli, erythrocyte, nerve), culminating in diabetic complications such as retinopathy, nephropathy and peripheral neuropathy [2-4].

Na+K+-ATPase is a soluble conserved trimeric pump (α, 133 kDa, β, 35 kDa, γ, 10 kDa) involved in transmembrane cation regulation via ATP – dependent efflux and influx of sodium and potassium ions in various cells [5]. The pump is isoenzymic in nature and has its α1 catalytic subunit encoded by the ATP1A1 gene present in both erythrocyte and nerve cells [5,6]. Na+K+-ATPase has also been reported as one of the membrane proteins affected structurally and functionally in diabetes mellitus [5].

Numerous experimental studies conducted on streptozotocin-induced diabetic rats show that for instance, the delayed motor nerve conduction velocity, decreased nerve blood flow, degeneration of peripheral nerves, persistent hyperglycemia and other metabolic aberrations involve a common denominator of down-regulated Na+K+-ATPase [4,7,8]. Reduced activity of Na+K+-ATPase has also been implicated in streptozotocin-induced diabetic rats with nephropathy [9]. De Leo et al [10] and Kowluru [11] also reported impairment in the level of this enzyme in diabetic rats and mice with retinopathy. However, in most of these studies, the observed Na+K+-ATPase reduction was found with other metabolic derangements which include myoinositol depletion, aldolase reduction, sorbitol accumulation and protein kinase C activity [7,12]. Subsequent associations of Na+K+-ATPase with these parameters have been unfolded [8,12]. Also in humans, several studies have consistently reported a declined Na+K+-ATPase activity from the membranes of nerve cells and erythrocytes preparations of Type 1 and Type 2 diabetic patients coupled with alterations in membrane protein composition [13-15]. Among diabetic Nigerian patients, defectiveness in Ca2+ homeostasis and qualitative and quantitative changes in erythrocyte membrane proteins have also been reported [16,17]. The work of Okegbile et al [18] also revealed a reduction in erythrocyte Na+K+-ATPase activity in Nigerian patients with Type 2 diabetes mellitus similar to the finding of DeLuise et al [19] in obese patients. The latter is characterized by dyslipidemia caused by an alteration in the regulations of cholesterol, triglycerides, high density and low-density lipoproteins [20]. Abnormal levels of these lipids in the blood have subsequently been observed in diabetics with or susceptible to cardiovascular complications and microangiopathy [2].

However, research is still needed to enhance our present understanding about factors responsible for the decreased Na+K+-ATPase observed in diabetes mellitus especially in Type 1 diabetes mellitus, which occurs early in life and requires life-long insulin therapy [1]. Furthermore, this group of diabetics is also susceptible to cardiovascular complications of diabetes mellitus [1-4] and in our recent study we observed microalbuminuria of comparable prevalence rates in Type 1 and Type 2 diabetic cohorts and in whom elevated blood pressure was also found [21].

Understanding the pattern and magnitude of association between Na+K+-ATPase activity and cardiovascular risk factors of lipid metabolism would provide a leap forward in our understanding of how diabetes mellitus progresses to cardiovascular complications in patients. Clues to this possibility have emanated from few studies. They include the observation by Rabini et al [22] that elevated plasma lysophosphate choline (LPC) due to decreased lecithin-cholesterol acteyltransferase (LCAT) associate strongly with reduced Na+K+-ATPase activity in diabetic patients. Kiziltunc et al [23] also reported cholesterol as an inhibitor of erythrocyte membrane Na+K+-ATPase in vitro. Inverse correlation between erythrocyte membrane Na+K+-ATPase activity and polyunsaturated fatty acid levels has also been reported by Djemli-Shipkolye et al, [8]. Taken together, there is currently a paucity data on the relationship of Na+K+-ATPase with cardiovascular risk factors such as total cholesterol, triglycerides and low density lipoprotein cholesterol as well as high density lipoprotein cholesterol. These lipid metabolites are more synonymous with cardiovascular complications than LPC in diabetic patients from Nigeria and other parts of the world 21, 24, 25]. Therefore, knowledge of the metabolic effects of these lipid metabolites on Na+K+-ATPase activity is essential for a better understanding of the pathogenesis of diabetes mellitus and evolution of strategies that would improve the management and prognosis of the disease.

In the present study, erythrocyte ghost membrane preparations from Nigerian patients with Type 1 diabetes mellitus were evaluated for protein content and Na+K+-ATPase activity in association with duration of diabetes and their plasma levels of total cholesterol, triglycerides, LDL-cholesterol and HDL-C. The kinetics of the enzyme based on reaction with ATP in vitro was also investigated.

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All the reagents used in this study were of analytical grade and highest purity. Fatty acid free bovine serum albumin (BSA), sodium dodecyl sulfate (SDS), Tris base and disodium salts of adenosine triphosphate (vanadate free), ethylene diamine tetraacetic acid (EDTA), and Ethyleneglycol – bis-2-aminoethyl ether) N, N, N1, N1 – tetracetic acid (EGTA) were purchased from Sigma Chemical Co., St. Louis, MO, U.S.A. Hydrochloric acid (HCl), trichloroacetic acid (TCA), sulphuric acid (H2SO4), magnesium chloride (MgCl2), Deoxycholate, potassium chloride (KCl) and sodium chloride (NaCl) were procured from British Drug House (BDH), UK. All buffers were prepared to their respective pH values measured with the aid of a pH meter (Mettler, Inc. Germany).

Study design

This study enrolled 34 (male/female, 23/11) Type 1 diabetic out-patients aged 18 – 37 years attending diabetic clinics at General Hospital, Lagos, Nigeria. The patients, diagnosed clinically based on proneness to polydipsia, polyuria, thirst, weight loss and biochemically based on sustained hyperglycemia were placed absolutely on a continuous insulin therapy by attending endocrinologists. A total of twenty-seven apparently healthy non-diabetic volunteers (male/female, 17/10) were also recruited as control. Members in the Type 1 diabetes mellitus group were further subdivided into two subgroups: 'poor glycemic control group' (fasting glucose > 5.6 mmole/L) (group A1) and 'good glycemic control group' (fasting glucose ≤ 5.6 mmole/L) (group A2). The classification was based on WHO treatment guidelines [1]. Fasting blood glucose of members in the control group was also determined twice at three days interval to ascertain their normoglycemic status. Meanwhile, to participate in the study, each member of the study groups had haematocrit and hemoglobin levels greater than or equal to 33% and 11 g/dl respectively, no clinical evidence of pancreatitis and gave written informed consent. Approval to conduct the study was granted by the ethics review committee of the Hospital Management Board. Demographic data including age and duration of diabetics were collected using a questionnaire.

Sample collection and clinical chemistry

Fasting venous blood samples (5 – 7 mL each) were collected between 8.00 and 9.00 h on each clinic day from the patients and control into labeled heparinized test tubes. The blood samples were centrifuged at 1,500 g at 25°C for 10 min and the resulting plasma transferred into plain test tubes for glucose and lipid analysis.

Fasting plasma glucose was determined using a glucometer (ACCU-CHEK Advantage®. Roche Diagnostics Corporation, IND, USA). Plasma cholesterol and triglycerides were determined by the enzymatic method of Siedel et al [26] and McGowan et al [27] respectively. HCL-cholesterol in plasma was determined following the precipitation of chylomicrons, very low-density lipoproteins (VLDL), intermediate density lipoproteins (IDL) cholesterol and low-density lipoprotein (LDL) cholesterol by MgCl2 – phosphotungstic acid mixture as described by Lopez-Virrela et al [28]. LDL-cholesterol was estimated according to Friedewald et al [29]. Hypercholesterolemia (HCL) was defined as plasma total cholesterol of 200 mg/dl according to the European Artherosclerosis Society guidelines [30]. Hypertriglyceridaemia was put at plasma TAG > 160 mg/dl according to Isselbacher et al, [31]. Elevated LDL-C and reduced HDL-C were defined by values > 150 mg/dl and < 35 mg/dl respectively [32].

Erythrocyte ghost membrane preparation

A simplified procedure of DeLuise and Flier [33] was used for erythrocyte ghost membrane preparation. Briefly, 10 volumes of ice cold 5 mM Tris/0.1 mM Na2EDTA, pH 7.6 were added to each of the tubes containing buffy coat free – packed erythrocytes of diabetics and non-diabetic samples to achieve osmotic lysis. The resulting membranes were centrifuged at 20,000 g for 20 min at 4°C. They were then washed three times in 0.017 M NaCl/5 mM Tris-HCl, pH 7.6 and three times with 10 mM Tris-HCl (pH 7.5). The haemoglobin-free membrane suspension was finally stored at -20°C in the 10 mM washing Tris-HCl buffer (pH 7.5) but used within three days of preparation for Na+K+-ATPase assays and membrane protein determination.

Na+K+-ATPase assays

The erythrocyte total ATPase activity was determined by incubating 50 μL of ghost membrane suspension (~200 μg of membrane protein) of type 1 diabetic or healthy subject with 5 mM Tris-ATP, 25 mM KCl, 75 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 25 mM Tris-HCl, pH 7.5 in a total volume of 500 μL for 90 min at 37°C in a shaking water bath (150 rpm). The reaction was stopped by adding TCA to a final concentration of 5% (wt/vol). After centrifugation for 20 min at 1,500 g, an aliquot of the supernatant was used to measure total inorganic phosphate liberated by the reaction of Fiske and Subbarow [34]. This assay was repeated in the presence of 200 μM methyldigoxin, an inhibitor of Na+K+-ATPase activity. Total ATPase activity was expressed as micromole of inorganic phosphate liberated per milligram membrane protein per hour. The activity of Na+K+-ATPase was subsequently determined by subtracting total ATPase activity in the presence of digoxin from enzyme activity in the absence of the inhibitor drug.

For the enzymes kinetics experiment, Na+K+-ATPase activity was measured in assays containing 0.02 – 0.1 mM Tris-ATP concentration range. A negative control assay devoid of ghost membrane suspension was run in parallel to the test assays and used as blank during absorbance measurement at 660 nm. Enzyme activity was then determined as described previously

The kinetic constants: Vmax and Km were determined by extrapolation from Lineweaver-Burke plot of reciprocally transformed activity (V) and substrate (ATP) data.

Ghost erythrocyte membrane protein determination

This was determined according to the method of Lowry et al [35] after solubilizing aliquots of ghost membrane suspension with 0.2% SDS. Bovine serum albumin (BSA) (50 – 300 μg) was used as standard. Absorbance was measured in a Beckmann D700 spectrophotometer (Beckmann, USA) at 720 nm.

Statistical analysis

Data obtained was entered into SPSS statistical software version 11 for descriptive and deductive statistics and multivariate regression analyses. All data were expressed as mean ± SD and analyzed by Student's t-test. The metabolic and demographic parameters obtained were subjected to correlation analysis to test their association with erythrocyte membrane (Na+K+-ATPase activity. P value less than 0.05 was regarded as significant.

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Demographic and haematological characteristics of the diabetic and non diabetic subjects are presented in Table ​Table1.1. The duration of diabetes ranged from 2 to 8 years with a mean duration of 4.6 years in the type 1 diabetic patients investigated. No significant (P > 0.05) differences were noted between the diabetic patients and non-diabetic control with respect to their age (28.2 ± 3.9 vs. 28.4 ± 4.3 years), haematocrit (39.1 ± 1.4 vs. 39.7 ± 1.2%) and haemoglobin levels (13.1 ± 0.3 vs. 13.2 ± 0.2 g/dL).