Effects of Diaceto-Dipropyl-Disulphide on Plasma Sialic Acid and Renal Tissue Thiol Levels in Alloxan Diabetic Rats

DN is major life threatening microvascular complication of Diabetes Mellitus (DM) and is the commonest cause of end stage renal disease, in India [1,2]. Prevalence of DN among DM patients varies from 13% to 70.8% in India [3].

SA, also known as N-acetyl neuraminic acid, is an essential component of glycoproteins and glycolipids present on cell membrane [4]. SA is a nine-carbon sugar derived from mannosamine and pyruvate [5]. It acts as a cofactor of many cell surface receptors (e.g., insulin receptor) [6]. The unique structural feature of SA, which includes a negative charge owing to a carboxyl group, enable it to play an important role in cellular functions, such as cell-to-cell repulsion, recognition, transportation of positively charged compounds and tumour cell metastasis. These sialic acid molecules apparently enter the circulation by either shedding or cell lysis [7].

SA contributes to the maintenance of the negative charge of the renal Glomerular Basement Membrane (GBM) [8]. Plasma SA level is elevated in DM patients with microalbuminuria and clinical proteinuria i.e., DN, which is a well-established diabetic complication accompanied by an increased risk of coronary artery disease [9].

Cellular functional variations as well as cellular protein alterations are commonly observed in DM which are due to increased production of Reactive Oxygen Species (ROS). This diabetes induced ROS elevation may result in decreased total cellular / tissue thiol levels [10].

Disulphides of garlic, (Allium sativum Linn) are known for their anti-diabetic, anti-hyperlipidaemic, anti-atherogenic, antioxidant as well as anti-carcinogenic actions [11]. Diallyl Disulphide (DADS), the principle disulphide compound of garlic oil is responsible for the above-mentioned beneficial properties of garlic. In our previous studies we have documented the anti-glycation effect of DADS on GBM and its beneficial effect in reducing de-sialation of GBM [12,13]. But DADS is very pungent oil and often it is difficult to feed rats. Also, few studies have shown toxic effects of DADS on kidneys [14,15]. But its structural analogue, Diaceto- Dipropyl Disulphide (DADPDS) is pleasant smelling, more palatable, less toxic and possesses similar anti-diabetic properties [15].

This study was undertaken to assess the effects of DADPDS on GBM sialic acid and renal tissue antioxidant levels in alloxan diabetic rats.

Materials and Methods

Materials

Alloxan and thiopropanol were procured from Sigma Chemicals. All other chemicals used in the present study were of analytical grade. DADPDS was synthesized from thiopropanol as explained by Veena GR [15].

Maintenance of Animals

Male albino rats, weighing 200-250g were randomly selected and used for the present study. The animals were maintained on a standard rat feed supplied from Amrut rat feeds, Bengaluru. The rats were maintained at room temperature of 25°C with 24 hours water and food supply.

Ethics

The experiments were conducted according to the norms approved by Ministry of Social Justice and Empowerment, Government of India and Institutional Animal Ethics Committee (IAEC) guidelines.

Induction of Diabetes

The animals were fasted overnight and diabetes was induced by a single IP injection of freshly prepared alloxan (150mg/kg body wt.) [11,13] in sterile normal saline. The animals were considered diabetic if their blood glucose were above 250mg/dl and urine showed consistent glucosuria. The treatment was started on 5^th day after alloxan injection and was considered as first day of treatment.

Grouping of Animals

The rats were divided into three groups comprising six rats in each group as follows:

Group I: Normal rats – which were fed on 30 ml of normal saline per kg body weight, through gastric intubation, for a period of 60 days.

Group II: Diabetic Control rats – which were fed on normal saline 30ml/kg body weight, through gastric intubation, for a period of 60 days.

Group III: Diaceto- Dipropyl Disulphide (DADPDS) treated Diabetic rats – which were fed on DADPDS (100mg/ kg body weight) prepared in normal saline, given as 30ml/kg body weight suspension, through gastric intubation, for a period of 60 days.

Procedure

On completion of 60 days, the rats were anaesthetised and then sacrificed. Blood was collected in heparinised test tubes. Kidneys were dissected and their net weight was noted. Immediately the kidneys were processed as follows. One part of kidney was homogenised with 9 parts of cold Phosphate buffer (pH 7.4) and the extract was used for estimation of tissue total thiols [16] and total proteins [17]. A second part of the kidney was homogenised with 9 parts of trichloro acetic acid (10%) and the extract was used for the estimation of thiobarbituric acid reactive substances (TBARS) levels by Nitroprusside reaction method [18]. A part of whole blood was centrifuged at 3500 rpm for 6-8mins and the plasma was used for the estimation of glucose by glucose oxidase-peroxidase method [19] and sialic acid levels by Ehrlich’s method [20].

Statistical Analysis

The obtained results were statistically analysed by Student’s t-test.

Results

Results obtained in the present study have been presented in [Table/Fig-1]. The plasma glucose levels, plasma creatinine levels, plasma sialic acid levels along with renal glycated proteins, renal thiol levels and renal TBARS levels of normal rats (group I), diabetic control rats (group II) and DADPDS treated diabetic rats (group III) have been incorporated.

[Table/Fig-1]:

Plasma glucose, sialic acid, renal tissue thiols and TBARS levels in all the rat groups.

	Plasma glucose(mg/dl)	PlasmaSialic acid(mg/dl)	PlasmaCreatinine(mg/dl)	Renal thiols(mg of Cysteine equivalent/g)	RenalTBARS(μ mol/g)
Normal rats (n=6)	122.61 ± 20.1	71.08 ± 8.35	0.67 ± 0.08	1.77 ± 0.44	7.96 ± 1.81
Diabetic control rats(n=6)	421.66*** ± 102.08(p< 0.0001)	98.81*** ± 17.33(p= 0.0011)	0.72 ± 0.04(p= 0.1362)	0.88*** ± 0.16(p <0.0001)	21.01*** ± 4.23(p <0.0001)
DADPDS treated rats(n=6)	264.18*** ± 87.41(p= 0.0051)	73.97*** ± 11.49(p= 0.0045)	0.70 ± 0.03(p= 0.2769)	1.08*** ± 0.06(p= 0.0052)	10.68*** ± 2.29(p< 0.0001)

1. Number in parentheses indicate the number of animals in each group.

2. The values are expressed as their mean ± SD

3. Significance level * p < 0.05; ** p < 0.01; *** p < 0.001.

There was a significant increase in plasma glucose (p<0.001), plasma sialic acid levels (p<0.001) and renal tissue TBARS levels (p<0.001); significant decrease (p<0.001) in renal tissue thiol levels in group II rats as compared to group I rats. However, there was no significant change in plasma creatinine levels between these two groups. Further, it was observed that there was a significant decrease in plasma sialic acid levels (p<0.001) and renal tissue TBARS levels (p<0.001) as well as significant increase (p<0.001) in renal tissue thiol levels in group III rats as compared to group II rats. However, there was no appreciable observed in plasma glucose and plasma creatinine levels between these two groups.

Discussion

SA residues are important constituents of the carbohydrate moiety of the GBM. Along with glycosaminoglycans, SA contributes to the polyanionic components and the charge-selective permeability / barrier of the GBM. Sialic acid content of GBM has been found to be decreased in diabetic subjects where the decrease seems to correlate with duration of diabetes. Similar reductions in sialo-glycoproteins were found in alloxan diabetic rats. These findings were due in part to the raised activity of the degradative enzyme, sialidase, in the diabetic kidney cortex [21]. Studies have suggested possible role of hyperglycation of GBM proteins in DM, altering its structural orientation and exposing sialic acid to sialidase. This may lead to removal of sialic acid which in part may account for the increased plasma sialic acid levels [Table/Fig-1]. The observed elevated sialic acid levels in alloxan diabetic rats is in agreement with earlier studies in diabetic nephropathy [8,11,13,22,23]. In our previous study we have documented that this de-sialation of GBM proteins may alter their ionic nature; a decrease in negative charges leading to percolation of little albumin which probably results in microalbuminuria, a predisposing factor observed prior to frank nephropathy [12,13]. A significant decrease in plasma sialic acid levels (p<0.001) as well as a significant increase in tissue thiol levels (p<0.001) in DADPDS treated alloxan diabetic rats (group II rats) decisively indicates that DADS has a significant role in inhibiting de-sialation of GBM in alloxan diabetic rats.

DADPDS a disulphide, may be involved in sulphydryl exchange reactions with proteins or enzymes [24,25] similar to any other disulphide as follows:

Sialidase being a sulphydryl enzyme [26], may be involved in sulphydryl exchange reaction with DADPDS. Such a sulphydryl exchange reaction with sialidase, may alter the activity of the enzyme resulting in retention of sialic acid residues on the GBM and thus maintaining its shape as well as negative nature of GBM proteins. Such an action of DADPDS should result in lower activity of sialidase enzyme and hence the decreased plasma sialic acid levels observed in the present study [Table/Fig-1,2].

[Table/Fig-2]:

Possible actions of DADPDS in preventing diabetic nephropathy in alloxan diabetic rats. (* Advanced glycated end products/advanced lipid glycation products).

Total thiol pool contributes to the majority of antioxidant activity of the body. Disturbances of thiol-related mechanisms have been observed in diabetes. Major contribution to total thiol pool is from reduced glutathione and free thiol group present in the amino acid cysteine, which can be found in many cellular peptides or proteins [27]. For instance, plasma levels of protein-bound thiols were found to be lower in type 2 diabetics compared with their controls [28]. Thiol groups can be oxidised to form disulphide. The oxidation of the thiol groups in several peptides and proteins including GBM proteins are specifically coupled with the reduction of other molecules. These thiol antioxidative peptides or proteins, such as glutathione, thioredoxin etc., play an important role in the cellular antioxidative defence as well as the regulation of cellular functions involving the thiol-disulphide exchange [25,29]. Such a thiol-disulphide exchange reaction with DADPDS should result in increased thiol content in the renal tissue and decreased TBARS levels which has been observed in the present study [Table/Fig-1].

Limitation

The study was conducted in short term (2 months) diabetic rats. Histopathological changes of the renal tissue in these rats were not done.

Conclusion

Hence, it may be concluded that DADPDS helps in preventing de-sialation of GBM in alloxan diabetic rats and improves renal tissue antioxidant defence mechanisms, possibly through thioldisulphide exchange reaction.

1. Number in parentheses indicate the number of animals in each group.2. The values are expressed as their mean ± SD3. Significance level * p < 0.05; ** p < 0.01; *** p < 0.001.

[1]. Ashok Kumar EA, Jijiya Bai P, Development of nephropathy in Type II Diabetes Mellitus with normalbuminuria, micro albuminuria, and macro proteinuria IAIM 2016 3(3):106-17.  [Google Scholar]

[2]. Joydeep G, Subinay D, Mrinal P, Role of sialic acid in prediction of diabetic nephropathy Al Ameen J Med Sci 2016 9(1):58-64.  [Google Scholar]

[3]. Mohan V, Nadia RP, Agnelo F, Frederick V, Manoj K, Metabolic syndrome and hypertension in diabetic nephropathy patients in rural Goa, India International Journal of Contemporary Medical Research 2016 3(4):1174-76.  [Google Scholar]

[4]. Kokoglu E, Sonmez H, Uslu E, Uslu I, Sialic acid levels in various types of cancer Cancer Biochem Biophys 1992 13:57-64.  [Google Scholar]

[5]. Kulkarni SP, Mallikurjana CR, Jayaprakash Murthy DS, Cerebrospinal fluid free sialic acid and aspartate transaminase levels in meningitis Indian Journal of Clinical Biochemistry 2006 21(1):185-88.  [Google Scholar]

[6]. Salhanide AI, Amatruda JM, Role of sialic acid in insulin activity and the insulin resistance of diabetes mellitus Am J Physiol 1988 255:E173-79.  [Google Scholar]

[7]. Surangkul D, Pothacharoen P, Suttajit M, Kongtawelert P, A periodate-resorcinol microassay for the quantitation of total sialic acid in human serum Chiang Mai Med Bull 2001 40(3):111-18.  [Google Scholar]

[8]. Melidonis A, Tournis S, Hraklioti S, Hraklianou S, Serum sialic acid concentration and diabetic nephropathy in type 2 diabetes mellitus Diabetica Croatica 1998 27:4-8.  [Google Scholar]

[9]. Crook M, Tutt P, Pickup JC, Serum sialic acid in non insulin dependent diabetes mellitus and its relationship to blood pressure and retinopathy Diabetes Care 1993 16:57-60.  [Google Scholar]

[10]. Mendez JD, Xie J, Agular HM, Mendez VV, Molecular susceptibility to glycation and its implication in diabetes mellitus and related diseases Mol Cell Biochem 2010 344(1-2):185-93.  [Google Scholar]

[11]. Augusti KT, Therapeutic values of onion (Allium cepa Linn) and garlic (Allium sativum Linn) Ind J Exp Biol 1996 34:634-40.  [Google Scholar]

[12]. Vijay V, Vickram Kashinath RT, Effects of diallyl disulphide on renal glycated proteins and plasma sialic acid levels in alloxan diabetic rats Global Journal of Medical Research 2011 11(3/1):31-35.  [Google Scholar]

[13]. Vijay V, Vickram Kashinath RT, Effect of DADS in protein and lipid glycation, and lipid peroxidation in brain of alloxan diabetic rats Global Journal of Medical Research 2014 13(7/1):7-11.  [Google Scholar]

[14]. Kashinath RT, Joseph PK, A study on garlic toxicity Journal of Advanced Research in Biological Sciences 2009 1(2):39-45.  [Google Scholar]

[15]. Veena GR, Vickram Vijay V, Kashinath RT, Hypolipidemic effects of diaceto dipropyl disulphide in alloxan diabetic rats Global Journal of Medical Research 2012 12(5/1):1-8.  [Google Scholar]

[16]. Colowinck SP, Kaplan NO, Enzymes in lipid metabolism in Methods in Enzymology 1957 1New YorkAcademic Press:623-24.  [Google Scholar]

[17]. Nadigar HA, Marcus SR, Chandrakala MV, Kulkarani DD, Malonyl-dialdehyde levels in different organs of rats subjected to acute alcohol toxicity Ind J Clin Biochem 1986 1:133-36.  [Google Scholar]

[18]. Varley H, Gowenloch AH, Bell M, Determination of Blood glucose (O’Toluidine method) Practical Clinical Biochemistry 1991 Vol 15th editionLondonHeimann Professional publishing Ltd:395-97.  [Google Scholar]

[19]. Varley H, Gowenloch AH, Bell M, The plasma proteins Practical Clinical Biochemistry 1991 Vol 15th editionLondonHeimann Professional publishing Ltd:368-79.  [Google Scholar]

[20]. Aminoff D, Methods for quantitative estimation of N-acetyl Neuraminic acid and their applications to hydrolysate of sialomucoids Biochem J 1961 81:384-92.  [Google Scholar]

[21]. Faud NZ, Stanley G, Elizabeth FO, Diabetic nephropathy: Metabolic and biochemical mechanisms in The kidney in diabetes mellitus by Benner BM, Stein JH 1989 Vol 20Churchill Livingstone publishers. University of Michigan:87-113.  [Google Scholar]

[22]. Powie JK, Watts GF, Crook MA, Ingham JN, Shaw KM, Serum sialic acid and the long term complications of insulin dependent diabetes mellitus Diabetic Med 1996 13:238-42.  [Google Scholar]

[23]. Nayak SB, Duncan H, Lalloo S, Maraj K, Matmungal V, Matthews F, Correlation of microalbumin and sialic acid with anthropometric variables in type 2 diabetic patients with and without nephropathy Vasc Health Risk Manag 2008 4(1):243-47.  [Google Scholar]

[24]. Amadu I, Joseph PK, Augusti KT, Hypolipidemic action of onion and garlic unsaturated oils in sucrose fed rats over a two month period Experentia 1982 38:899-901.  [Google Scholar]

[25]. Adoga GI, Effect of garlic oil extract on glutathione reductase levels in rats fed on high sucrose and alcohol diets - A Possible Mechanism of the Activity of the Oil Bioscience Reports 1986 6(10):909-12.  [Google Scholar]

[26]. Hata K, Wada T, Hasegawa A, Kiso M, Miyagi T, Purification and Characterization of a Membrane-Associated Ganglioside Sialidase from Bovine Brain J Biochem 1998 123(5):899-905.  [Google Scholar]

[27]. Sarkar A, Dash S, Barik BK, Muttigi MS, Kedage V, Shetty JK, Copper and ceruloplasmin levels in relation to total thiols and GST in type 2 diabetes mellitus patients Indian J of Clin Biochem 2010 25(1):74-76.  [Google Scholar]

[28]. Osama MA, Adel AM, Ibrahim AY, Ayman MM, Antihyperglycemic, antihyperlipidemic and antioxidant effects & probable mechanisms of action of Ruta graveolens infusion and Rutin in nicotinamide-STZ induced diabetic rats Diabetologica Croatica 2010 39(1):15-31.  [Google Scholar]

[29]. Liang M, Jennifer LP, Thiol related genes in diabetic complications Arterioscler Thromb Vascl Biol 2007 27:77-83.  [Google Scholar]