Glycation inhibition activity of 5-Sulfosalicyclic acid in diabetic condition.
Objective: To study the glycation inhibition effect of Sulfosalicyclic acid.
Methodology: The study was conducted by using plasma from apparently healthy non diabetic and diabetic persons. Samples of different concentrations of glucose and inhibitor were incubated for 5 weeks at 37oC temperature. Glucose was estimated by glucose oxidase kit method before and after dialysis. Glycation level was assessed and measured by TBA and periodate assays.
Results: Increase in glycation was observed from 1st to 3rd week of incubation while it was decreased after 5th week due to formation of advanced glycation end products. Three different concentrations of inhibitor showed variable responses. It was also noted that 10 mM concentration of Sulfosalicyclic acid exhibited fairly good response to decrease glycation. Conclusions: Highest concentration of Sulfosalicyclic acid produced overall high level of inhibition. Periodate borohydride proved to be more reliable and sensitive glycation assay when compared to TBA test.
KEY WORDS: Glycation, Diabetes, Maillard reaction, Sulfosalicyclic acid, TBA, Periodate.
How to cite this article:
Kousar S, Asghar M, Rashid R. Glycation inhibition activity of 5-Sulfosalicyclic acid in diabetic condition. Pak J Med Sci 2011;27(3):626-629
Diabetes and its complications are rapidly becoming the world's most significant epidemic disease.1 The morbidity caused by diabetes has traditionally been classified into macroand micro vascular complications. Although in diabetes and hyperglycaemia, there have been more focus on macro vascular complications, however micro vascular complications also have a pivotal role in the development of diabetes and hyperglycaemia.2 Hyperglycemia is known to be play a major role in the pathophysiology of microand macrovascular complications associated with both type 1 and type 2 diabetes.3
The adverse effects of hyperglycaemia depend upon the type of the cells. Cells which express a high level of the glucose transporter 1 (GLUT1), are unable to regulate intracellular glucose concentrations and thus are very susceptible to hyperglycemia-induced damage.4 Its deleterious or detrimental effects are attributable, to the formation of sugar-derived substances called Maillard products. The Maillard reaction is the interaction between the carbonyl group (aldehyde or ketone) of the reducing sugar with the amino group of the biomolecule to form a reversible Schiff base which can undergo an intramolecular rearrangement to form the Amadori products.5 These can undergo further rearrangements by dehydration, and condensation to form irreversible end products.
These products may be fluorescent and yellowbrown in color; some can form stable intermolecular and intramolecular cross-links called advanced glycation end products (AGEs).6 Nonenzymatic glycation of proteins have now been implicated in the pathogenesis of different diseases like diabetes, renal failure and aging.7
Living system has devised various defense mechanisms to protect the tissues against deleterious effects of advanced glycation end products. These include glyoxylase system (I and II) having oxaldehyde reductase and aldose reductase that catalyze the deglycation and detoxification of methylglyoxal, the most common reactive intermediates of AGEs to D-lactate.8
To explore or isolate new compounds either from plants or synthetically to control diabetes and other age accelerating diseases is the need of the day.
Aminoguanidine (AG) is the first compound which has been extensively studied in vitro and in vivo as a powerful inhibitor of glycation and AGE formation.9
AG is a nucleophilic compound that traps reactive carbonyl intermediates partially inhibiting carboxy methyl lysine and carbxy ethyl lysine.10 A derivative of vitamin B6, (pyridoxamine) has a similar mechanism of action, like AG and inhibits the post Amadori steps of the Maillard reaction.11
The major aim of this study was to investigate glycation inhibition by 5-Sulfoslicyclic acid in normal and diabetic human plasma and its comparison with sensitivities of Thiobarbituric Acid (TBA) and Periodate methods.
Effect on glycation was monitored by synthetic/ chemical inhibitor 5-Sulfoslicyclic acid (5-SSA). Selection of conditions and concentrations: Four concentrations of glucose (G1=500 mM, G2=250 mM, G3=50 mM and G4=5.5 mM) were used. Plasma fractions were collected from diabetic patient's blood (Type II) and from normal/ healthy male and female volunteer. Samples were stored at -20oC until used. At the time of use, all plasma were polled together to (Normal and diabetic separately). Normal and diabetic plasma samples were diluted to the range having protein concentration up to 20 mg/mL. Two glycation assays were performed to measure glycation level. Protein estimation was carried out before and after dialysis by Biuret method.12 Three different concentrations of inhibitor (I1= 10 mM, I2= 5 mM and I3 = 1 mM) were used is in this study.
Selection of Combinations: To study the effect of 5-
Sulfoslicyclic acid with normal (PN) and diabetic (PD) plasma sixteen combinations were made and all were placed at 37oC at same time for five weeks (Table-I).
Glycation of Plasma: All plasma combinations were incubated with four concentrations of glucose (in PBS) at 37oC for 1-5 weeks. Plasma samples were dialyzed to remove free glucose after incubation as free glucose is the major hindrance in estimation of glycation level and AGE production. Glucose was again estimated after dialysis to confirm that concentration of glucose is decreased or not. Glycation inhibition of plasma: Different concentrations of glucose and inhibitor were incubated at 37oC for 1-5 weeks.
Thiobarbituric Acid (TBA) Colorimetric Technique: Enzymatic and non-enzymatic glycation was determined by TBA technique. This method is based on the reaction between fructose, amino acids and week acid, yielding 5hydroxymethyl furfural (HMF) compound.13
Non enzymatic glycation was determined as follows. NE Glycation = (C Glycation + E Glycation) E Glycation
NE =Non Enzymatic, E= Enzymatic, C =Collective Periodate Borohydride Assay: This test is based on the production of formaldehyde by periodate oxidation of cis-diol, aminol, ketol or ketoamine structures. Two moles of formaldehyde are formed from hexose sugar. The amount of formaldehyde produced was quantified as the fluorescent adduct formed by condensation of formaldehyde with acetyl acetone and ammonia. Fructose was used as standard, being the analogous to the ketoamine form of the glycogroups.14,15
Table-I: Different combinations for Plasma glycation inhibition.
S# Combinations for###Combinations for
Effect of 5-sulfosalicyclic acid (5-SSA) on glycation level with normal human plasma: The results ob tained from 5-SSA as an inhibitor of AGEs and glycation in normal plasma indicated 250 mM (G2 ) concentration of glucose exhibited maximum glycation after 3rd week of incubation as compared to 1st and 5th week (Figure.1). TBA measured maximum glycation level was 5.015 mole/mole that reduced to 2.019 mole/mole by G4 (5.5 mM) concentration of glucose after 1st week incubation at 37oC.
Glycation was maximally decreased by I1 (10 mM) of 5SSA: When Periodate borohydride assay was used to measure glycation level, maximum glycation was observed after 3rd week with G (5.312 mole/mole) and minimum with G4 glucose concentration (3.068 mole/mole) after 1st week of incubation (Figure.2).
The maximum inhibition of 5-SSA was seen by I1 (10 mM) concentration as compare to other two. Effect of 5-sulfosalicyclic acid (5-SSA) on glycation level with diabetic human plasma: In Figure.3, results obtained with diabetic human plasma indicated that glucose concentration G2 (250 mM) produced maximum glycation (7.159 mole/mole) after 3rd week of incubation with TBA test. Minimum level of glycation was given by G4 (5.5 mM) with value 3.458 mole/mole after 5th week incubation. Maximum glycation inhibition response was given by I1 (10 mM) concentration of 5-SSA.
With periodate borohydride assay, maximum glycation was observed after 3rd week with G (7.718 mole/mole) and minimum with G4 mole) after 1st week of incubation 1 (3.153 mole/ maximum inhibition of 5-SSA was seen by I (10 mM) as compare to I (5 mM) and I (1mM) 1 concentration.
The results indicated that overall 250 mM(G ) concentration of glucose exhibited maximum 2 glycation both in normal and diabetic condition. (Figures 1-4). Maximum glycation inhibition was observed in I1 (10 mM) concentration of 5SSA. Our results are supported by Rahbar and Figarola16, who studied PostAmadori AGEbreaking by novel compounds and observed after 3rd week with G (5.312 mole/mole) maximum inhibition was seen by aminosalicylic acid and minimum with G4 glucose concentration (3.068 mole/mole) after 1st week of incubation (Figure.2).
The maximum inhibition of 5-SSA was seen by I1 (10 mM) concentration as compare to other two. Effect of 5-sulfosalicyclic acid (5-SSA) on glycation level with diabetic human plasma: In Figure.3, results obtained with diabetic human plasma indicated that glucose concentration G2 (250 mM) produced maximum glycation (7.159 mole/mole) after 3rd week of incubation with TBA test. Minimum level of glycation was given by G4 (5.5 mM) with value 3.458 mole/mole after 5th week incubation. Maximum glycation inhibition response was given by I1 (10 mM) concentration of 5-SSA. (10 mM concentration). This is also corroborated with previous findings17 where effects of DL-penicillamine showed effect on early and advanced glycation inhibition. Our results are also in accordance with a previous report18 claiming that glycation can be inhibited by acetylsalicylic acid (aspirin), thiamine, and pyridoxine.
Glycation assays; TBA and Periodate showed same trend of glycation measurement but periodate proved to be more sensitive and affective method to measure glycation level. Our findings are also in conformity with Jakus et al results,17 where glycation level of membrane proteins from diabetics was found to be elevated as compared to normoglycemic
Normal plasma protein (PN H" 20 mg/mL) was incubated with all glucose concentrations (G1= 500 mM, G2= 250 mM, G3= 50 mM and G4= 5.5 mM) and inhibitor with three concentrations (I1= 10 mM, I2= 5 mM and I3 =1mM) in 0.075 M PBS. Reaction mixtures were incubated at 37oC for 5 weeks at the same time. Samples were analysed after 1st, 3rd and 5th week and glycation level was measured in mole\ mole (glucose\ protein). Values were the average of experiments carried out at n= 3
Periodate borohydride assay in normal human plasma. Normal plasma protein (PN H" 20 mg/mL) was incubated with all glucose concentrations (G1= 500 mM, G2= 250 mM, G3= 50 mM and G4= 5.5 mM) and inhibitor with three concentrations (I1= 10 mM, I2= 5 mM and I3 =1mM) in 0.075 M PBS. Reaction mixtures were incubated at 37oC for 5 weeks at the same time. Samples were analysed after 1st, 3rd and 5th week and glycation level was measured in mole\ mole (glucose\ protein). Values were the average of experiments carried out at n= 3
Diabetic plasma protein (PD H" 20 mg/mL) was incubated G3= 50 mM and G4= 5.5 mM) and inhibitor with three concentrations (I1= 10 mM, I2= 5 mM and I3 =1mM) in 0.075 M PBS. Reaction mixtures were incubated at 37oC for 5 weeks at the same time. Samples were analysed after 1st, 3rd and 5th week and glycation level was measured in mole\ mole (glucose\ protein). Values were the average of experiments carried out at n= 3
control tissue. It was concluded that periodate glycation assay for the measurement of Amadori products, was the most convenient. It was observed from the present study that glycation was increased from 1st to 3rd week of incubation and slight decrease in glycation level was seen after 5th week of incubation (due to formation of advanced glycation end products). It was also proved from present findings that glucose concentrations G1 (500 mM) and G2 (250 mM) can produce maximum glycation.
It was concluded from above findings, that 10 mM concentration of 5-SSA produced overall good response to decrease glycation. Periodate borohydride proved more reliable and sensitive glycation assay than TBA test.
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Samina Kousar, Muhammad Asghar, Robina Rashid , Samina Kousar, PhD, Dept. of Biochemistry, University College of Medicine, University of Lahore, Pakistan. Prof. Muhammad Asghar, PhD, Dept. of Chemistry and Biochemistry, University of Agriculture, Faisalabad, Pakistan. Robina Rashid, M.Phil, Dept. of Biochemistry, Nawaz Sharif Medical College (NSMC), University of Gujrat, Pakistan. Correspondence: Samina Kousar, Assistant Professor, Department of Biochemistry, University College of Medicine and Dentistry, University of Lahore, 1-KM Defense Road, OFF Raiwaind Road, Lahore, Pakistan. Email: email@example.com firstname.lastname@example.org Received for Publication: December 30, 2010 1st Revision Received: January 18, 2011 2nd Revision Received: February 26, 2011 3rd Revision Received: April 8, 2011 4th Revision Received: April 18, 2011 Final Revision Accepted: April 26, 2011
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|Author:||Kousar, Samina; Asghar, Muhammad; Rashid, Robina|
|Publication:||Pakistan Journal of Medical Sciences|
|Date:||Sep 30, 2011|
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