- Research article
- Open Access
Activities of three erythrocyte enzymes of hyperglycemic rats (Rattus norvegicus) treated with Allium sativa extract
© Chikezie and Uwakwe; licensee BioMed Central Ltd. 2014
- Received: 5 December 2013
- Accepted: 14 April 2014
- Published: 22 April 2014
The present study sought to investigate erythrocyte glutathione S-transferases (GST), NADH-Methaemoglobin reductase (NADH-MR) and Na+/K+-ATPase activities of hypoglycemic rats treated with ethanol/water (1:2 v/v) extract of A. sativa as agent of glycemic control.
Hyperglycemia was induced by a single intra-peritoneal injection of 0.1 mol/L alloxan monohydrate in phosphate buffer saline (PBS) solution (pH = 7.4); dosage = 140 mg/kg. At the end of the experimental time (t = 76 h), erythrocyte GST, NADH-MR and Na+/K+-ATPase activities as well as serum fasting blood sugar (FBS) levels were measured by spectrophotometric methods.
Serum FBS levels of control/normal (C/N) rats ranged between 72.93 ± 0.82–95.12 ± 0.92 mg/dL, whereas experimental rats without glycemic control gave: 249.41 ± 1.03–256.11 ± 1.23 mg/dL. Hyperglycemic rats treated with ethanol/water (1:2 v/v) extract of A. sativa exhibited comparative reduced serum levels of FBS alongside with erythrocyte GST, NADH-MR and Na+/K+-ATPase activities. The average relative activities of the three enzymes and corresponding order of enzyme activity in hyperglycemic rats treated with ethanol/water (1:2 v/v) extract of A. sativa was: NADH-MR = 60.99% > GST = 47.81% > Na+/K+-ATPase = 46.81%. In the same order, relative activities of the three enzymes in rats without glycemic control were: NADH-MR = 49.65% > GST = 23.69% > Na+/K+-ATPase = 17.02%.
Erythrocyte GST, NADH-MR and Na+/K+-ATPase activities gave insights into the pathophysiology of diabetic state and served as biomarkers for ascertaining therapeutic control in Type 1 diabetes mellitus.
- Glutathione S-transferases
- NADH-Methaemoglobin reductase
- Allium sativa
- Diabetes mellitus
Diabetic mellitus is an endocrine disorder characterized by insufficiency in circulating plasma level of insulin (Type 1, or Insulin-Dependent Diabetes Mellitus; IDDM) and peripheral resistance and insensitivity to insulin (Type 2, or Non-Insulin-Dependent Diabetes Mellitus; NIDDM). Unlike Type 1 diabetes mellitus, Type 2 is associated with hyperinsulinism. Primarily, overall physiologic distortions prompted by poor control of metabolism in absence or insufficiency of insulin engender hyperglycemia and associated metabolic disorders [1, 2]. Striking consequential effects of prolong hyperglycemia are changes in structure and function of macromolecules [3, 4], auto-oxidation of glycated proteins, increased production of reactive oxygen species (ROS), decreased antioxidant defense, increased lipid peroxidation, and associated apoptosis or necrosis occasioned by membrane degeneration [4, 5]. Notably, alterations/adjustments in most glycolytic, tricarboxylic acid cycle (TCA) enzymes activities are associated with diabetic states [5, 6]. Activities of these enzymes (pyruvate kinase, pyruvate dehydrogenase, glycogen synthase, pyruvate carboxylase, fructose 1, 6-bisphosphate etc.) are regulated by insulin and have been observed to be phosphoenzymes. Activation of enzyme activity in response to insulin stimulus is prompted by cyclic adenosine monophosphate (cAMP) phosphodiesterase mediated pathway  or through secondary metabolic events connected to insulin action.
Glutathione S-transferases (GSTs) are multi-gene and multifunctional antioxidant enzymes that comprise several classes of GST isozymes. These enzymes by virtue of their activities act as subset of numerous cellular antioxidants defense systems against ROS species that are associated with many disease-causing electrophiles [2, 7, 8]. NADH-Methaemoglobin reductase (NADH-MR) (EC: 188.8.131.52) transfers electrons from NADH + H+ to cytochrome b5 via its flavin adenine dinucleotide (FAD) prosthetic group . This erythrocyte enzyme maintains hemoglobin in its ferrous (Fe2+) state . Na+/K+-ATPase, also called the sodium pump, is a soluble conserved trimeric pump (α-133 kDa; β-35 kDa; γ-10 kDa) involved in transmembrane cation regulation via ATP–dependent dual efflux/influx of sodium (Na+) and potassium (K+) ions in various cells [11, 12]. The regulation of this pump activity is dependent on the phosphorylation of the α-subunit of Na+/K+-ATPase [11, 13].
Allium sativa has been widely reported to exhibit therapeutic benefits to numerous pathologic states whose etiology is linked to oxidative stressors and electrophiles  such as diabetes mellitus [15–18], atherosclerosis [19, 20], hyperlipidemia [20, 21] thrombosis , hypertension . Phytochemical and biochemical profile of A. sativa has been reported elsewhere . The present study was based on the premise that hyperglycemia is one of the various indicators and promoters of distortional haemostasis associated with diabetes mellitus. Therefore, we sought to investigate level of alterations in erythrocyte GST, NADH-MR and Na+/K+-ATPase activities of hypoglycemic rats treated with ethanol/water (1:2 v/v) extract of A. sativa as agent of glycemic control.
Collection of plant specimen
Fresh samples of A. sativa were obtained in July, 2012 from local market at Umoziri-Inyishi, Imo State, Nigeria. The plant specimen was identified and authenticated by Dr. F.N. Mbagwu at the Herbarium of the Department of Plant Science and Biotechnology, Imo State University, Owerri, Nigeria. A voucher specimen was deposited at the Herbarium for reference purposes.
Preparation of extract
Fresh bulbs of A. sativa were washed under a continuous stream of distilled water for 15 min and air-dried at room temperature for 5 h. The bulbs were chopped and further dried for 5 h in an oven at 60°C and subsequently ground with a ceramic mortar and pestle. Twenty-five grams (25 g) of pulverized specimen was suspended in 250 mL of ethanol/water mixture (1:2 v/v) in stoppered flasks and allowed to stand at −4°C for 24 h. The suspensions were filtered with Whatman No. 24 filter papers. The filtrate was concentrated in a rotary evaporator at 50°C and dried in vacuum desiccator. The yield was calculated to be 3.4% (w/w). The extract was finally suspended in phosphate buffered saline (PBS) solution (extract vehicle), osmotically equivalent to 100 g/L PBS (90.0 g NaCI, 17.0 Na2HPO4.2H2O and 2.43 g NaH2PO4.2H2O), and used in all the studies with doses expressed in mg/kg of body weight of the animals.
Male rats Rattus norvegicus (8–10 weeks old) weighing 150–200 g were generous gift from Professor A.A. Uwakwe (Department of Biochemistry, University of Port Harcourt, Nigeria). The rats were maintained at room temperatures of 25 ± 5°C, 30–55% of relative humidity on a 12-h light/12-h dark cycle, with access to water and food ad libitum for 2 weeks acclimatization period. The handling of the animals was in accordance with the standard principles of laboratory animal care of the United States National Institutes of Health (NIH, 1978).
Induction of hyperglycemia and study design
Hyperglycemia was induced by a single intra-peritoneal injection of 0.1 mol/L alloxan monohydrate in PBS solution (pH = 7.4) at a dosage of 140 mg/kg. The animals were considered hyperglycemic when their blood glucose concentrations exceeded 250 mg/dL 72 h after alloxan treatment, which was in conformity with our previous study . The animals were deprived of food and water for additional 16 h before commencement of treatment (control and test experiments) as described elsewhere .
A total of twenty four (24) rats were divided into six (6) groups of four (n = 4) each as follows:
Group C1; Control-Normal (C/N): Normal rats received only PBS (Vehicle; 1.0 mL/kg/16 h, i. p.) for 64 h.
Group C2; Control-Hyperglycemic (C/H): Hyperglycemic rats received PBS (Vehicle; 1.0 mL/kg/16 h, i. p.) for 64 h.
Group T1; H[A. sativa] = 1.0 mg/kg: Hyperglycemic rats received A. sativa (1.0 mg/kg/16 h, i. p.) for 64 h.
Group T2; H[A. sativa] = 2.0 mg/kg: Hyperglycemic rats received A. sativa (2.0 mg/kg/16 h, i. p.) for 64 h.
Group T3; H[A. sativa] = 4.0 mg/kg: Hyperglycemic rats received A. sativa (4.0 mg/kg/16 h, i. p.) for 64 h.
Group T5; H[Glibenclamide] = 5.0 mg/kg: Hyperglycemic rats received glibenclamide (5.0 mg/kg/16 h, i. p.) for 64 h.
Measurement of fasting blood sugar
After alloxan treatment, blood samples were drawn from apical region of the tails of the rats i.e., at experimental t = 0 h and by carotid artery puncture at experimental t = 76 h for measurement of fasting blood sugar (FBS). Determination of serum level of FBS was by glucose oxidase method according to the Randox® kit manufacturer’s procedure (Randox® Laboratories Ltd. Ardmore, United Kingdom). Glibenclamide, a standard anti-diabetic agent is a product of Aventis Pharma. Ltd. Goa, India.
Collection of blood and preparation of erythrocyte haemolysate
At the end of treatment, the animals were fasted for 12 h  and subsequently sacrificed according to United States National Institutes of Health approved protocols (NIH, 1978). Blood volume of 4.0 mL was obtained by carotid artery puncture using hypodermic syringe. The erythrocytes were separated from plasma by bench centrifugation for 10 min. The harvested erythrocytes were washed by methods of Tsakiris et al., as described by Chikezie et al.,. Within 2 h of collection of blood specimen, 1.0 mL of harvested erythrocyte was introduced into centrifuge test tubes containing 3.0 mL of buffer solution pH = 7.4: 250 mM tris (hydroxyl methyl) amino ethane–HCl (Tris–HCl)/140 mM NaCl/1.0 mM MgCl2/10 mM glucose). The erythrocytes suspension was further centrifuged at 1200 g for 10 min and repeated 3 times. According to Chikezie , to remove platelets and leucocytes, the pellet was re-suspended in 3.0 mL of phosphate-buffered saline (PBS) solution (pH = 7.4) and passed through a column (3.5 cm in a 30 mL syringe) of cellulose-microcrystalline cellulose (ratio w/w 1:1) . The eluted fraction was passed twice through a new column of cellulose-microcrystalline cellulose (ratio 1:1 w/w) to obtain erythrocyte suspension sufficiently devoid of leucocytes and platelets. Finally, erythrocytes were re-suspended in 1.0 mL of this buffer and stored at 4°C. The washed erythrocytes were lysed by freezing/thawing as described by Galbraith and Watts,  and Kamber et al.,. The erythrocyte haemolysate was used for the determination of erythrocyte glutathione S-transferase (GST) and NADH-Methaemoglobin reductase (NADH-MR) activity.
Erythrocyte haemolysate haemoglobin concentration
The cyanomethaemoglobin reaction modified method of Baure,  as described by Chikezie et al.,  was used for measurement of haemolysate haemoglobin concentration. A 0.05 mL portion of erythrocyte haemolysate was added to 4.95 mL of Drabkins reagent (100 mg NaCN and 300 mg K4Fe(CN)6 per liter). The mixture was left to stand for 10 min at 25 ± 5°C and absorbance read at λmax = 540 nm against a blank. The absorbance was used to evaluate for haemolysate haemoglobin concentration by comparing the values with the standard.
Erythrocyte glutathione S-transferase
GST activity was measured by the method of Habig,  as described by Pasupathi et al., with minor modifications according to Chikezie et al.,. The reaction mixture contained 1.0 mL of 0.3 mM phosphate buffer (pH = 6.5), 0.1 mL of 30 mM 1-chloro-2, 4-dinitrobenzene (CDNB) and 1.7 mL of distilled water. After pre-incubating the reaction mixture at 37°C for 5 min, the reaction was started by the addition of 0.1 mL of erythrocyte haemolysate and 0.1 mL of glutathione (GSH) as substrate. The absorbance was followed for 5 min at λmax = 340 nm. The enzyme activity was expressed as erythrocyte GST activity in international unit per gram haemoglobin (IU/gHb) using an extinction coefficient (∑) of 9.6 mM−1 cm−1 in reaction in which 1 mole of GSH is oxidized (Eq. 1).
Erythrocyte NADH-Methaemoglobin reductase
NADH-MR activity was assayed according to the method of Board, . A mixture of 0.2 mL Tris–HCl/EDTA buffer pH = 8.0, 0.2 mL NADH and 4.35 mL of distilled water was introduced into a test tube and incubated for 10 min at 30°C. The content was transferred into a cuvette and the reaction started by adding 0.2 mL of K3Fe(CN)6/0.05 mL erythrocyte haemolysate. The increase in absorbance of the medium was measured at λmax = 340 nm per min for 10 min at 30°C against a blank solution. NADH-MR activity was expressed in international unit per gram haemoglobin (IU/gHb) using an extinction coefficient (∑) of 6.22 mM−1 cm−1 in reaction in which 1 mole of NADH + H+ is oxidized (Eq. 1).
Calculation of GST and NADH-MR activities
EA = Enzyme activity in IU/gHb
D/min = Change per min in absorbance at 340 nm.
VC = Cuvette volume (total assay volume) = 1.0 mL.
VH = Volume of haemolysate in the reaction system (0.05 mL).
Erythrocyte ghost membrane preparation
A simplified procedure of DeLuise and Flier,  as reported by Iwalokun and Iwalokun,  was used for erythrocyte ghost membrane preparation. Briefly, 10 mL of ice cold 5 mM Tris/0.1 mM Na2EDTA (pH = 7.6) were added to test tubes containing buffy coat free–packed erythrocytes of test and control rats to achieve osmotic lysis. The resulting membranes were centrifuged at 20,000 g for 20 min at 4°C. The membrane suspensions were washed 3 times in 0.017 M NaCl/5 mM Tris–HCl, pH = 7.6 and 3 times with 10 mM Tris–HCl (pH = 7.5). The haemoglobin-free membrane suspension was finally stored at −20°C in 10 mM Tris–HCl buffer (pH = 7.5).
The erythrocyte total ATPase activity was determined by incubating 50 μL of ghost membrane suspension (~200 μg of membrane protein) of test and control rats with 5 mM Tris-ATP, 25 mM KCl, 75 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, 25 mM Tris–HCl (pH = 7.5) in 500 μL for 90 min at 37°C in a shaking water bath. The reaction was stopped by adding tricarboxylic acid (TCA) to a final concentration of 5% (w/v). After centrifugation for 20 min at 1,500 g, an aliquot of the supernatant was used to measure total inorganic phosphate liberated according to Fiske and Subbarow,  reaction. 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 (μM pi/mg protein/h). 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.
Ghost erythrocyte membrane protein
Membrane protein was measured according to the method of Lowry et al., after solubilizing aliquots of ghost membrane suspension with 0.2% sodium dodecyl sulfate (SDS). Bovine serum albumin (BSA) (50–300 μg), product of Sigma Chemical Company, Saint Louis, Missouri, USA, was used as standard. Absorbance was measured with Beckmann D700 spectrophotometer (Beckmann, USA) at λmax = 720 nm.
The data collected were analyzed by the analysis of variance procedure while treatment means were separated by the least significance difference (LSD) incorporated in the statistical analysis system (SAS) package of 9.1 version (2006). The correlation coefficients between the results were determined with Microsoft Office Excel, 2010 version.
Serum FBS levels of hyperglycemic rats with/without glycemic control
t = 0 h
t = 76 h
95.12 ± 0.92a
72.93 ± 0.82a
256.11 ± 1.23b
249.41 ± 1.03b
H[A. sativa] = 1.0 mg/kg
255.64 ± 1.09b,c
125.11 ± 0.91c
H[A. sativa] = 2.0 mg/kg
261.13 ± 2.00b,c,d
129.32 ± 1.50c,d
H[A. sativa] = 4.0 mg/kg
267.94 ± 0.92c,d,e
132.61 ± 0.81d,e
H[Glibenclamide] = 5.0 mg/kg
265.49 ± 49c,d,e,f
101.12 ± 0.80f
However, these values represented marginal variations in serum FBS levels amongst the three categories of A. sativa treated hyperglycemic rats (Group T1, Group T2 and Group T3) within the experimental time: 0 h ≤ t ≤ 76 h. Specifically, at t = 76 h, serum FBS[A. sativa] = 1.0 mg/kg = 125 ± 0.91 mg/dL; FBS[A. sativa] = 2.0 mg/kg = 129.32 ± 1.50 mg/dL and FBS[A. sativa] = 4.0 mg/kg = 132.61 ± 0.81 mg/dL.; p < 0.05 compared to C/N rats. Furthermore, t = 76 h, the three groups of A. sativa treated hyperglycemic rats exhibited: H[A. sativa] = 1.0 mg/kg = 51.06%, H[A. sativa] = 2.0 mg/kg = 50.48% and H[A. sativa] = 4.0 mg/kg = 50.41% reduction in serum FBS levels compared to their corresponding FBS levels at t = 0 h. Similarly, compared to serum FBS levels at t = 0 h, H[Glibenclamide] = 5.0 mg/kg rats showed reduced serum FBS level by 61.91% at t = 76 h, representing a ratio of 1: 1.4 decrease in serum FBS levels compared to C/N rats; p < 0.05. At the end of the experiment, serum FBS levels of H[A. sativa] = 1.0 mg/kg was not significantly different (p > 0.05) from H[A. sativa] = 2.0 mg/kg rats. Likewise, FBS levels of H[A. sativa] = 2.0 mg/kg showed no significantly difference (p > 0.05) compared to H[A. sativa] = 4.0 mg/kg rats.
The use of experimental animal model for study of Type 1 diabetes mellitus has been widely reported [38–41]. The cytotoxic action of diabetogenic agents is mediated by formation of superoxide radicals and other related ROS, causing massive destruction of the β-cells [39, 42, 43]. From the present study, experimental rats treated with the widely used diabetogenic agent–alloxan, in conformity with previous reports elsewhere [24, 39, 42, 43], showed evidence of hyperglycemia (Table 1). Hyperglycemia is the earliest and primary clinical presentation in diabetic states [3, 44]. Studies on the application of nutraceuticals, sourced from spices and other edible plants and their products, for the treatment and management of diabetes mellitus have received the attention of several research endeavours . The present study showed evidence of the capacity of ethanol/water extract of A. sativa to reduce serum level of FBS in hyperglycemic rats, which compared fairly with the standard anti-diabetic drug-glibenclamide (Table 1). The anti-diabetic properties of A. sativa extract have been previously reported [15, 24]. The therapeutic action of A. sativa as it applies to its role in the treatment and management of diabetes mellitus is identical to the mode of action of other numerous anti-diabetic agents of plant origin such as Coriandrum sativum; Gongronema latifolium; Allium cepa Linn . However, other mechanism of therapeutic action, which involves increase peripheral glucose consumption induced by Eugenia Floccosa, Berberis lyceum and Tinospora cordifolia roots  have been documented. The active principles of these plant extracts exhibited insulin-like effect by mimicry. However, within the experimental time, administration of the three experimental doses of ethanol/water (1:2 v/v) extract of A. sativa as an instrument of glycemic control did not restore normal serum level of FBS (72.93 ± 0.82–95.12 ± 0.92 mg/dL) in hyperglycemic rats with [FBS] > 250 mg/dL.
According to Raza et al., oxidative stress is an important factor in the etiology and pathogenesis of diabetes mellitus. Furthermore, Pasupathi et al., had observed significant (p < 0.001) decrease in reduced glutathione (GSH) concentration in diabetic erythrocytes compared to control participants. They further averred that decreased level of GSH was an aftereffect of increased utilization of the coenzyme for scavenging ROS due to elevated oxidative stress associated with diabetes. Consequently, we observed decreased levels of erythrocyte GST activity in hyperglycemic rats, which was in conformity with previous studies [3, 44, 45, 50–52], since the co-substrate (GSH) required for GST antioxidant protective activity [1, 53] may have been utilized for other non-enzymatic reductive pathways. Judging from erythrocyte GST activity of rats without glycemic control, the relatively higher levels of erythrocyte GST activity of hyperglycemic rats treated with A. sativa extract in dose dependent pattern (Figure 1) was an obvious indication of the capability of ethanolic extract of plant extract to serve as anti-diabetic agent, fairly comparable to the standard anti-diabetic drug-glibenclamide. Erythrocyte GST activity has been proven to be a reliable biochemical index and basis for diagnosis and monitoring of therapeutic events in the course of treatment and management of other pathologic/metabolic disorders whose etiologies and manifestations are linked to oxidative stress. Notable among which are: parasitic infections [26, 54], gout and rheumatoid arthritis [55, 56], haemoglobinopathies , malignancy , hypertension , stroke  and atherosclerosis . In a related perspective, Moasser et al., had previously given account of the use of GST activity as a reliable biomarker in depicting the etiology of diabetes mellitus. They posited that two isoforms of GST (GSTM1 and GSTT1) might be involved in the pathogenesis of Type 2 diabetes mellitus in South Iranian population. In addition, investigations by Yalin et al., showed that the GSTM1 gene may play a significant role in the aetiopathogeneses of diabetes mellitus and could serve as a useful biomarker in the prediction of diabetes mellitus susceptibility of the Turkish population.
According to Coleman,  poor glycemic control in diabetes and combination of oxidative, metabolic, and carbonyl stresses caused restriction in supply but excessive demand for reducing equivalents. Therefore, repressed NADH-MR activity in hyperglycemic rats could be linked to the substantial diversion and utilization of reducing equivalents to other reductive pathways in efforts to minimize oxidative stress, prompted by erythrocyte high ROS content. Thus, the decreased level of erythrocyte NADH-MR activity of hyperglycemic rats (Figure 2) is a reflection of a compromised erythrocyte antioxidant status associated with hyperglycemia [61, 62]. Furthermore, in concordance with the present reports, Zerez et al., had stated that conditions that engender decreased erythrocyte NADH content resulted to decreased rate of methaemoglobin reduction in connection to impaired NADH-MR activity. This condition is responsible, in part, for relatively high methaemoglobin content in sickle erythrocytes and susceptibility to oxidative damage . Based on the present observations, it is presumed that adjustments in diabetic erythrocyte methaemoglobin levels might provide early indication of diabetic antioxidant and oxidative stress status.
Studies suggest that insulin plays a stimulatory role in Na+/K+-ATPase activity through tyrosine phosphorylation process . The relatively reduced levels of erythrocyte Na+/K+-ATPase activity in hyperglycemic rats (Figure 3) was consistent with the findings of previous authors. Soulis-Liparota et al., reported reduced Na+/K+-ATPase activity streptozotocin-induced diabetic rats with nephropathy, whereas, Di Leo et al., and Kowluru,  reported impairment in the enzyme activity in diabetic rats and mice with retinopathy. In a different study, using human participants, Iwalokun and Iwalokun,  noted compromised erythrocyte Na+/K+-ATPase activity in Type 1 diabetic patients from Lagos, Nigeria. This finding was corroborated by Mimura et al., study, in which they noted reduction of erythrocyte Na+/K+-ATPase activity in Type 2 diabetic patients with hyperkalemia. Raccah et al., suggested that diabetes-induced Na+/K+-ATPase activity dysfunction could be implicated in the pathogenesis of human diabetic neuropathy and the electrophysiological abnormalities.
The findings reported here was in concordance with those of Konukoglu et al.,. They noted that hypercholesterolemia and free radical-induced mechanisms may be responsible for the inhibition of erythrocyte Na+/K+-ATPase activity in patients with Type 2 diabetes mellitus. According to the present study, decreased erythrocyte Na+/K+-ATPase activity of hyperglycemic rats was analogous to altered enzyme activity in peripheral neurons of individuals with diabetic neuropathy. According to Greene et al.,, impaired Na+/K+-ATPase activity is induced by hyperglycemia with characteristic distortions in myo-inositol and phosphoinositol metabolism, which normalizes with intensive insulin therapy that controls hyperglycemia . Thus, decreased erythrocyte Na+/K+-ATPase activity was an obvious confirmation of a connection between the capacity of erythrocyte to actively transport Na+/K+ ions (antiport) and obligatory utilization of ATP for α-subunit of Na+/K+-ATPase phosphorylation required for enzyme activity [11, 13, 72]. Hyperglycemia with associated depressed glucose utilization in diabetic states results in low intracellular ATP concentration, insufficient for the required obligatory phosphorylation of the enzyme. The dose dependent increase in erythrocyte Na+/K+-ATPase activity in hyperglycemic rat treated with extract of A. sativa as instrument of glycemic control was an indication of improve glucose utilization exemplified in hyperglycemic rats treated with the standard anti-diabetic drug. The role and mechanism of insulin in regulation of Na+/K+-ATPase activity has been described elsewhere . In another study, Konukoglu et al., reported that hypercholesterolemia and free radical-induced mechanisms may be responsible for the inhibition of erythrocyte Na+/K+-ATPase activity patients with type 2 diabetes mellitus.
The present study showed that erythrocyte GST, NADH-MR and Na+/K+-ATPase activities gave insights into the pathophysiology of diabetic state and could serve as a biomarker for ascertaining therapeutic control in Type 1 diabetes mellitus.
- Nowier SR, Kashmiry NK, Rasool HAA, Morad H, Ismail S: Association of Type 2 diabetes mellitus and glutathione S-transferase (GSTM1 and GSTT1) genetic polymorphism. Res J Med Medical Sci 2009, 4(2):181–188.Google Scholar
- Moasser E, Kazemi-Nezhad SR, Saadat M, Azarpira N: Study of the association between glutathione S-transferase (GSTM1, GSTT1, GSTP1) polymorphisms with type II diabetes mellitus in southern of Iran. Mol Biol Rep 2012, 39(12):10187–10192. 10.1007/s11033-012-1893-4PubMedView ArticleGoogle Scholar
- Pasupathi P, Chandrasekar V, Kumar US: Evaluation of oxidative stress, antioxidant and thyroid hormone status in patients with diabetes mellitus. J Med 2009, 10: 60–66.View ArticleGoogle Scholar
- Velladath SU, Das A, Kumar RKN: Erythrocyte glutathione S-transferase activity in diabetics and its association with HbA1c. Webmed Central Clin Biochem 2011., 2(7): WMC002004Google Scholar
- Raza H, Prabu SK, Robin MA, Avadhani NG: Elevated mitochondrial cytochrome P450 2E1 and glutathione S-transferase A4–4 in streptozotocin-induced diabetic rats: tissue-specific variations and roles in oxidative stress. Diabetes 2004, 53: 185–194. 10.2337/diabetes.53.1.185PubMedView ArticleGoogle Scholar
- Grodsky GM: Chemistry and functions of hormones: III. Pancreas and gastrointestinal tract. In Harper’s Review of Biochemistry. Los Altos: Lange Medical Publications; 1983:511–522.Google Scholar
- Yalin S, Hatungil R, Tamer L, Ates NA, Dogruer N, Yildirim H, Karakas S, Atik U: Glutathione S-transferase gene polymorphisms in Turkish patients with diabetes mellitus. Cell Biochem Function 2007, 25(5):509–513. 10.1002/cbf.1339View ArticleGoogle Scholar
- Bid HK, Konwar R, Saxena M, Chaudhari P, Agrawal CG, Banerjee M: Association of glutathione S-transferase (GSTM1, T1 and P1) gene polymorphisms with type 2 diabetes mellitus in north Indian population. J Postgrad Med 2010, 56: 176–181. 10.4103/0022-3859.68633PubMedView ArticleGoogle Scholar
- Yubisui T, Takeshita M: Reduction of methaemoglobin through flavin at the physiological concentration by NADPH-flavin reductase of human erythrocytes. J Biochem 1980, 87(6):1715–1720.PubMedGoogle Scholar
- Rockwood GA, Armstrong KR, Baskin SI: Species comparison of methaemoglobin reductase. Exp Biol Med 2003, 228: 79–83.Google Scholar
- Feraille E, Carranza ML, Gonin S, Beguin P, Pedemonte C, Rousselot M, Caverzasio J, Geering K, Martin PY, Favre H: Insulin-induced stimulation of Na+/K+-ATPase activity in kidney proximal tubule cells depends on phosphorylation of the α-subunit at Tyr-10. Mol Biol Cell 1999, 10: 2847–2859. 10.1091/mbc.10.9.2847PubMedPubMed CentralView ArticleGoogle Scholar
- Kaplan JH: Biochemistry of Na+/K+-ATPase. Annu Rev Biochem 2002, 71: 511–535. 10.1146/annurev.biochem.71.102201.141218PubMedView ArticleGoogle Scholar
- Carranza ML, Féraille E, Favre H: Protein kinase C-dependent phosphorylation of the Na+/K+-ATPase α-subunit in rat kidney cortical tubules. Am J Physiol 1996, 271: C136-C143.PubMedGoogle Scholar
- Banerjee SK, Maulik SK: Effect of garlic on cardiovascular disorders: a review. Nutr J 2002, 1: 4. 10.1186/1475-2891-1-4PubMedPubMed CentralView ArticleGoogle Scholar
- El-Demerdash FM, Yousef MI, Abou El-Naga NI: Biochemical study on the hypoglycemic effects of onion and garlic in alloxan-induced diabetic rats. Food Chem Toxicol 2005, 43: 57–63. 10.1016/j.fct.2004.08.012PubMedView ArticleGoogle Scholar
- Chauhan A, Sharma PK, Srivastava P, Kumar N, Duehe R: Plants having potential antidiabetic activity: a review. Der Pharm Lett 2010, 2(3):369–387.Google Scholar
- Ayodhya S, Kusum S, Anjali S: Hypoglycemic activity of different extracts of various herbal plants Singh . Int J Res Ayurveda Pharm 2010, 1(1):212–224.Google Scholar
- Patel DK, Prasad SK, Kumar R, Hemalatha S: An overview on antidiabetic medicinal plants having insulin mimetic property. Asian Pac J Trop Biomed 2012, 2012: 320–330.View ArticleGoogle Scholar
- Lau BHS, Adetumbia MA, Sanchez A: Allium sativum (garlic) and atherosclerosis: a review. Nutr Res 1983., 3(1): (83)80128 http://dx.doi.org/10.1016/S0271–5317Google Scholar
- Choudhary R: Beneficial effect of Allium sativum and Allium tuberosum on experimental hyperlipidemia and atherosclerosis. Pak J Physiol 2008, 4(2):7–9.Google Scholar
- Mahmoodi M, Islami MR, Karam AGR, Khaksari M, Sahebghadam LA, Hajizadeh MR, Mirzaee MR: Study of the effects of raw garlic consumption on the level of lipids and other blood biochemical factors in hyperlipidemic individuals. Pak J Pharmacol Sci 2006, 19: 295–298.Google Scholar
- Fukao H, Yoshida H, Tazawa YI, Hada T: Antithrombotic effects of odorless garlic powder Bothin vitroandin vi vo . Biosci Biotechnol Biochem 2007, 71: 84–90. 10.1271/bbb.60380PubMedView ArticleGoogle Scholar
- Benavides GA, Squadrito GL, Mills RW, Patel HD, Isbell TS, Patel RP, Darley-Usmar VM, Doeller JE, Kraus DW: Hydrogen sulfide mediates the vasoactivity of garlic. PNAS 2007, 104: 17977–17982. 10.1073/pnas.0705710104PubMedPubMed CentralView ArticleGoogle Scholar
- Ibegbulem CO, Chikezie PC: Hypoglycemic properties of ethanolic extracts of Gongronema latifolium, Aloe perryi, Viscum album and Allium sativum administered to alloxan-induced diabetic albino rats (Rattus norvegicus ). Pharmacog Commun 2012, 3(2):12–16.Google Scholar
- Tsakiris S, Giannoulia-Karantana A, Simintzi I, Schulpis KH: The effect of aspartame metabolites on human erythrocyte membrane acetylcholinesterase activity. Pharmacol Res 2005, 53: 1–5.PubMedView ArticleGoogle Scholar
- Chikezie PC, Uwakwe AA, Monago CC: Glutathione S-transferase activity of three erythrocyte genotypes (HbAA, HbAS and HbSS) of male subjects/volunteers administered with Fansidar and Quinine. Afr J Biochem Res 2009, 3(5):210–214.Google Scholar
- Chikezie PC: Methaemoglobin concentration and NADH-methaemoglobin reductase activity of three human erythrocyte genotypes. Asian J Biochem 2011, 6(1):98–103.View ArticleGoogle Scholar
- Kalra VK, Sikka SC, Sethi GS: Transport of amino acids in gamma-glutamyl transpeptidase-implanted human erythrocytes. J Biol Chem 1981, 256: 5567.PubMedGoogle Scholar
- Galbraith DA, Watts DC: Changes in some cytoplasmic enzymes from red cells fractionated into age groups by centrifugation in Ficoll™/Triosil™ gradients: comparison of normal human and patients with Duchenne muscular dystrophy. Biochem J 1980, 191: 63–70.PubMedPubMed CentralView ArticleGoogle Scholar
- Kamber K, Poyiagi A, Delikonstantinos G: Modifications in the activities of membrane-bound enzymes during in vivo ageing of human and rabbit erythrocytes. Comp Biochem Physiol 1984, B.77B: 95–99.Google Scholar
- Baure JD: Laboratory investigation of hemoglobin. In Gradwohl’s Clinical Laboratory Methods and Diagnosis. Edited by: Sonnenwirth AC, Jarett L. St. Louis: Mosby; 1980.Google Scholar
- Habig WH, Pabst MJ, William BJ: Glutathione S-transferases; the first enzymatic step in mecapturic acid formation. J Biol Chem 1974, 249(6):130–137.Google Scholar
- Board P, Coggan M, Johnston P, Ross V, Suzuki T, Webb G: Genetic heterogeneity of the human glutathione transferases; a complex of gene families. Pharmacol Ther 1990, 48: 357–69. 10.1016/0163-7258(90)90054-6PubMedView ArticleGoogle Scholar
- DeLuise M, Flier JS: Functionally abnormal Na+/K+-ATPase pump in erythrocytes of a morbidly obese patient. J Clin Invest 1982, 69: 38–44. 10.1172/JCI110439PubMedPubMed CentralView ArticleGoogle Scholar
- Iwalokun BA, Iwalokun SO: Association between erythrocyte Na+/K+-ATPase activity and some blood lipids in type 1 diabetic patients from Lagos, Nigeria. BMC Endocrine Disorders 2007, 7: 7. 10.1186/1472-6823-7-7PubMedPubMed CentralView ArticleGoogle Scholar
- Fiske CH, Subbarrow Y: The colorimetric determination of phosphorous. J Biol Chem 1925, 66: 375–400.Google Scholar
- Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the folin phenol reagent. J Biol Chem 1951, 193: 265–275.PubMedGoogle Scholar
- El-Missiry MA, El Gindy AM: Amelioration of alloxan induced diabetes mellitus and oxidative stress in rats by oil of Eruca sativa seeds. Ann Nutr Metab 2000, 44: 97–100. 10.1159/000012829PubMedView ArticleGoogle Scholar
- Szkudelski T: The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas. Physiol Res 2001, 50(6):537–546.PubMedGoogle Scholar
- Gwarzo MY, Nwachuku VA, Lateef AO: Prevention of alloxan induced diabetes mellitus in rats by vitamin a dietary supplementation. Asian J Ani Sci 2010, 4: 190–196.View ArticleGoogle Scholar
- Shahaboddin ME, Pouramir M, Moghadamnia AA, Lakzaei M, Mirhashemi SM, Motallebi M: Antihyperglycemic and antioxidant activity of Viscum album extract. Afr J Pharm Pharmacol 2011, 5(3):432–436.View ArticleGoogle Scholar
- Lankin VZ, Korchin VI, Konovalova GG, Lisina MO, Tikhaze AK, Akmaev IG: Role of antioxidant enzymes and antioxidant compound probucol in antiradical protection of pancreatic beta-cells during alloxan-induced diabetes. Bull Exp Biol Med 2004, 137: 20–23.PubMedView ArticleGoogle Scholar
- Sharma US, Kumar A: Anti-diabetic effect of Rubus ellipticus fruits extracts in alloxan-induced diabetic rats. J Diabetol 2011, 2: 4.Google Scholar
- Choudhuri S, Dutta D, Chowdhury IH, Mitra B, Sen A, Mandal LK, Mukhopadhyay S, Bhattacharya B: Association of hyperglycemia mediated increased advanced glycation and erythrocyte antioxidant enzyme activity in different stages of diabetic retinopathy. Diabetes Res Clin Pract 2013, 100(3):376–384. 10.1016/j.diabres.2013.03.031PubMedView ArticleGoogle Scholar
- Rajeshwari CU, Andallu B: Oxidative stress in NIDDM patients: influence of coriander ( Coriandrum sativum ) seeds. Res J Pharmaceut, Biol Chem Sci 2011, 2(1):31–41.Google Scholar
- Ugochukwu NH, Babady NE: Antihyperglycaemic of effect aqueous and ethanolic extracts of Gongronema latifolium leaves on glucose and glycogen metabolism in livers of normal and streptozotocin induced diabetic rats. Life Sci 2003, 73(15):1925–1938. 10.1016/S0024-3205(03)00543-5PubMedView ArticleGoogle Scholar
- Kala SMJ, Tresina PS, Mohan VR: Antioxidant, anti-hyperlipidemic and antidiabetic activity of Eugenia floccosa Bedd leaves in alloxan induced diabetic rats. J Basic Clin Pharm 2012, 3(001):235–240. 10.4103/0976-0105.103831View ArticleGoogle Scholar
- Gulfraz M, Qadir G, Nosheen F, Parveen Z: Antihyperglycemic effects of Berberis lyceum Royle in alloxan induced diabetic rats. Diabetologia Croatica 2007, 36–3: 49–54.Google Scholar
- Stanely P, Prince M, Menon VP: Hypoglycaemic and other related actions of Tinospora cordifolia roots in alloxan-induced diabetic rats. J Ethnopharmacol 2000, 70(1):9–15. 10.1016/S0378-8741(99)00136-1PubMedView ArticleGoogle Scholar
- Mcrobie DJ, Glover DD, Tracy TS: Effects of gestational and overt diabetes on human placental cytochromes P450 and glutathione S-transferase. Drug Metab Disposition 1997, 26(4):367–371.Google Scholar
- Rathore N, Kale M, John S, Bhatnagar D: Lipid peroxidation and antioxidant enzymes in isoproterenol induced oxidative stress in rat erythrocytes. Indian J Physiol Pharmacol 2000, 44: 161–166.PubMedGoogle Scholar
- Surapanenin KM: Oxidant–antioxidant status in gestational diabetes patients. J Clin Diagnostic Res 2007, 1(4):235–238.Google Scholar
- Bekris LM, Shephard C, Peterson M, Hoehna J, Van Yserloo B, Rutledge E, Farin F, Kavanagh TJ, Lernmark A: Glutathione S-transferase M1 and T1 polymorphisms and associations with Type 1 diabetes age-at-onset. Autoimmunity 2005, 38(8):567–575. 10.1080/08916930500407238PubMedView ArticleGoogle Scholar
- Sohail M, Kaul A, Raziuddin M, Adak T: Decreased glutathione S-transferase activity: diagnostic and protective role in vivax malaria. Clin Biochem 2007, 40(5–6):377–382.PubMedView ArticleGoogle Scholar
- Hassan MQ, Hadi RA, Al-Rawi ZS, Padron VA, Stohs SJ: The glutathione defense system in the pathogenesis of rheumatoid arthritis. J Appl Toxicol 2001, 21: 69–73. 10.1002/jat.736PubMedView ArticleGoogle Scholar
- Bohanec GP, Logar D, Tomsic M, Rozman B, Dolzan V: Genetic polymorphisms of glutathione S-transferases and disease activity of rheumatoid arthritis. Clin Exp Rheumatol 2009, 27(2):229–236.Google Scholar
- Zafereo ME, Sturgis EM, Aleem S, Chaung K, Wei Q, Li G: Glutathione S-transferase polymorphisms and risk of second primary malignancy after index squamous cell carcinoma of the head and neck. Cancer Prevention Res (Phila) 2009, 2(5):432–439. 10.1158/1940-6207.CAPR-08-0222View ArticleGoogle Scholar
- Lee BK, Lee SJ, Joo SJ, Cho KS, Kim NS, Kim HJ: Association of glutathione S-transferase genes (GSTM1 and GSTT1) polymorphisms with hypertension in lead-exposed workers. Mol Cell Toxicol 2012, 8: 203–208. 10.1007/s13273-012-0025-5View ArticleGoogle Scholar
- Turck N, Robin X, Walter N, Fouda C, Hainard A, Sztajzel R, Wagner G, Hochstrasser DF, Montaner J, Burkhard PR, Sanchez JC: Blood Glutathione S-transferase-p as a time indicator of stroke onset. Plos One 2012, 7(9):e43830. 10.1371/journal.pone.0043830PubMedPubMed CentralView ArticleGoogle Scholar
- Yang Y, Yang Y, Xu Y, Lick SD, Awasthi YC, Boor PJ: Endothelial glutathione- S -transferase A4–4 protects against oxidative stress and modulates iNOS expression through NF-κB translocation. Toxicol Appl Pharmacol 2008, 230(2):187–196. 10.1016/j.taap.2008.03.018PubMedPubMed CentralView ArticleGoogle Scholar
- Coleman MD: Use of in vitro methaemoglobin generation to study antioxidant status in the diabetic erythrocyte. Biochem Pharmacol 2000, 60(10):1409–1416. 10.1016/S0006-2952(00)00333-6PubMedView ArticleGoogle Scholar
- Memişoğullari R, Türkeli M, Bakan E, Akçay F: Effect of Metformin or Gliclazide on lipid peroxidation and antioxidant levels in patients with diabetes mellitus. Turk J Med Sci 2008, 38(6):545–548.Google Scholar
- Zerez CR, Lachant NA, Tanaka KR: Impaired erythrocyte methaemoglobin reduction in sickle cell disease: dependence of methaemoglobin reduction on reduced nicotinamide adenine dinucleotide content. Blood 1990, 76: 1008–1014.PubMedGoogle Scholar
- Soulis-Liparota T, Cooper ME, Dunlop M, Jerums G: The relative roles of advanced glycation, oxidation and aldose reductase inhibition in the development of experimental diabetic nephropathy in the Sprague–Dawley rat. Diabetologia 1995, 38: 1492–1493. 10.1007/BF00400619View ArticleGoogle Scholar
- Di Leo MA, Santini SA, Cercone S, Lepore D, Gentiloni Silveri N, Caputo S, Greco AV, Giardina B, Franconi F, Ghirlanda G: Chronic taurine supplementation ameliorates oxidative stress and Na+/K+-ATPase impairment in the retina of diabetic rats. Amino Acids 2002, 23: 401–406. 10.1007/s00726-002-0202-2PubMedView ArticleGoogle Scholar
- Kowluru RA: Retinal metabolic abnormalities in diabetic mouse: comparison with diabetic rat. Curr Eye Res 2002, 24: 123–128. 10.1076/ceyr.184.108.40.20658PubMedView ArticleGoogle Scholar
- Mimura M, Makino H, Kanatsuka A, Yoshida S: Reduction of erythrocyte (Na+-K+) ATPase activities in non-insulin dependent diabetic patients with hyperkalemia. Metab 1992, 41(4):426–430. 10.1016/0026-0495(92)90079-PView ArticleGoogle Scholar
- Raccah D, Fabreguettes C, Azulay JP, Vague P: Erythrocyte Na+-K+-ATPase activity, metabolic control, and neuropathy in IDDM patients. Diabetes Care 1996, 19(6):564–568. 10.2337/diacare.19.6.564PubMedView ArticleGoogle Scholar
- Konukoglu D, Kemerli GD, Sabuncu T, Hatemi H: Relation of erythrocyte Na+-K+- ATPase activity and cholesterol and oxidative stress in patients with Type 2 diabetes mellitus. Clin Invest Med 2003, 26(6):279–284.PubMedGoogle Scholar
- Greene DG, Lattimer SA, Sima AAF: Are disturbances of sorbitol, phosphoinositide, and Na+/K+-ATPase regulation involved in pathogenesis of diabetic neuropathy? Diabetes 1988, 37: 688–693. 10.2337/diab.37.6.688PubMedView ArticleGoogle Scholar
- Greene DA, DeJesus PV, Winegrad AL: Effects of insulin and dietary myo-inositol on impaired peripheral motor nerve conduction velocity in acute streptozotocln diabetes. J Clin Invest 1975, 55: 1326–1336. 10.1172/JCI108052PubMedPubMed CentralView ArticleGoogle Scholar
- Mishra G, Routray R, Das SR, Behera HN: Alloxan diabetes in Swiss mice: activity of Na+-K+-ATPase and succinic dehydrogenase. Indian J Physiol Pharmacol 1995, 39(3):271–274.PubMedGoogle Scholar
- Hatou S, Yamada M, Akune Y, Mochizuki H, Shiraishi A, Joko T, Nishida T, Tsubota K: Role of insulin in regulation of Na+/K+-dependent ATPase activity and pump function in corneal endothelial cells. Invest Ophthalmol Visual Sci 2010, 51(8):3935–3942. 10.1167/iovs.09-4027View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.