¹ Department of Biochemistry, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt;

² Department of Internal Medicine, Specialized Medical Hospital, Faculty of Medicine, Mansoura University, Mansoura, Egypt

Corresponding Author: Mohamed N. Amin, Department of Biochemistry, Faculty of pharmacy, Mansoura University,Mansoura 35516, Egypt. E-mail: dr.mohamednasr@hotmail.com.

	ABSTRACT
	INTRODUCTION
	SUBJECTS AND METHODS
	RESULTS
	DISCUSSION
	CONCLUSION
	ACKNOWLEDGEMENT
	REFERENCES

	ABSTRACT

Advanced glycation end products (AGEs) are complex, heterogenous molecules generated by glycation and oxidation of proteins in vivo, which are thought to markedly increase in diabetic patients. One of the recently identified AGEs is carboxy methyl lysine (CML), which is the main ligand of receptors for advanced glycation end products (RAGE). The present study aimed to assess the effect of obesity on such pathways in presence and absence of Type 2 diabetes mellitus. CML, soluble receptors for advanced glycation end products (sRAGE), Hb_A1C, lipid profile, liver function tests and kidney function tests were determined in 29 diabetic obese, 29 diabetic non-obese, 15 non-diabetic obese and 15 non-diabetic non-obese subjects. The study compared obese and non-obese subjects in presence and absence of type 2 diabetes. The results showed a significant increase in CML and a significant decrease in sRAGEineach of the diabetic obese group when compared with the diabetic non-obese group and the non-diabetic obese group when compared with the non-diabetic non-obese group. A significant positive correlation was found between CML and markers of obesity (body mass index and waist/hip ratio). These results suggest that obesity can increase CML independent of diabetes and support the reports that CML could be generated from both sugars and lipids. The present study suggests that treatment using glycation inhibitors like aminoguanidine or recombinant sRAGEwill not only retard the diabetic complications, but may also have a prophylactic effect.

	INTRODUCTION

   AGEs are heterogeneous compounds including compounds derived from protein glycation and their precursors.Protein glycation (Maillard reaction) occurs between reducing sugars and free amino-groups of a protein via nucleophilic addition that forms a Schiff base (1). The formation of reactive α-dicarbonyls occurs due to several rearrangements of Schiff base.The α-dicarbonyls can react with lysine and arginine functional groups on proteins, leading to the formation of stable AGE compounds, such as N-ε-(carboxymethyl)lysineadducts (CML).

   CMLcan originate from both sugar and lipid oxidation products and were called either advanced glycation or lipoxidation end products (2). Consequently, CML is thought to be a general marker of oxidative stress (3). One of the main downstream pathways of CML is activation of the receptors of advanced glycation end products (RAGE). RAGE is a multiligand member of the immunoglobulin super-family of cell-surface molecules.

   RAGE was first described as receptor for adducts modified by non-enzymatic glycosylation occurring on proteins and lipids in a wide variety of settings, mainly in diabetes mellitus (4).The best documented consequences of RAGE activation isthe generation of reactive oxygen species, activation and translocation of pro-inflammatory kinases and transcription factors like nuclear factor kappa B (NFκB)(5).

   AGEs are able to alter physiological processes in vivo bydifferent mechanisms. AGE-mediated cross-linked proteins have decreased solubility and high resistance to proteolytic digestion, with the consequence of altered physicochemical and mechanical properties of tissue components (e.g. increased stiffness of collagen) and the generation of new immunogenic epitopes. Also, the binding of AGEs on the RAGE induces activation of NFκB of the RAGE bearing cells, resulting in increased expression of cytokines, growth factors and adhesion molecules. In addition, the accumulation of AGEs intensifies the expression of RAGE. RAGE is not only a receptor for AGEs but also for other pro-inflammatory molecules such as high-mobility group protein B1 and the S 100/calgranulin family, possibly increasing inflammatory reactions by this indirect way (6). On the other hand, blockade of AGE-mediated effects was observed in the presence sRAGE (7). sRAGE has been suggested to act as a decoy that captures the circulating AGEs preventing activation of RAGE signaling pathway (1).

   The present study aimed to evaluate the effect of obesity on markers of the non-enzymatic glycation pathway (CML, sRAGE and Hb_A1C) and the contribution of type 2 diabetes. In addition, we investigated their diagnostic value for type 2 diabetes and obesity. In vivo studies of CML in obesity are scarce.Advanced glycation has been well studied in diabetes but studies have determined CML in vivo in obesity are still scarce. We tried to explore such unrevealed area to find out CML levels in obesity and type 2 diabetes. The study also aimed to investigate the origin of CML andthe decoy function of sRAGE.

	SUBJECTS AND METHODS

   Subjects

   The present study was carried out on 73 patientsselected from the outpatients of the Diabetes Clinics and the Obesity Clinics of the Specialized Internal Medicine Hospital, Mansoura University, Egypt,between May 2009 and November 2009.Patients were divided into 29 Diabetic Obese group (D.O), 29 Diabetic Non-Obese group (D.N.O) and 15 Non-Diabetic Obese group (N.D.O). 15 Non-Diabetic Non-Obese subjects of Mansoura City inhabitants were selected as control group (N.D.N.O).

   All patients and controls were subjected to history taking,thorough clinical examination, and blood pressure measurement. The exclusion criteria included: altered hepatic function (either by clinical examination or liver function tests),chronic inflammatory diseases, malignancy, and smoking.Body weight (to the nearest kg) and body height (to the nearest cm) were measured to calculate the body mass index (BMI).Obesity was defined by BMI more than 30 kg/m²(8).The waist/hip ratio (WHR) was measured as a marker for abdominal obesity (9).

   Blood sampling

   The selected patients and control subjects were fasted for 12 hours.Blood samples were collected byclean venipuncture and divided into two portions.1.5 mlwas collected into EDTA powder-containing wassermann tube, stored at 2-8ºC and used for determination of blood glycated hemoglobin % (Hb_A1C) using ion-exchange resin separation method (10).3.5 ml was collected into clean dry wassermann tube, allowed to clot for 15 min at room temperature and centrifuged for 10 min at 600 g. The clear non-hemolyzed serum was used for the determination of the levels of fasting blood glucose (FBG) (11), total cholesterol (Total-C) (12), triglycerides (TAG) (13), high density lipoprotein-cholesterol(HDL-C) (14), alanine aminotransferase activity(ALT) (15),aspartate aminotransferase activity(AST) (16), albumin (ALB) (17) and creatinine(CRT) (18),immediately. Low density lipoprotein-cholesterol (LDL-C) was calculated from HDL-C, TAG and Total-C (19).The rest of serum was kept frozen at -20ºC for the determination of CMLusing commercially available ELISA kit (UscnLifescience& Technology Co., Ltd, U.S.A) (20)and sRAGE concentrationusingcommercially available ELISA kit (R&D Systems, Inc., U.S.A) (21). The ELx800 Absorbance Microplate ELISA Reader from BioTek Instruments, U.S.A with BioTek's Gen5™ Data Analysis Software was used for ELISA measurements.

   Statistical analysis

   Datawereexpressed as mean ± standard deviation (S.D).One way ANOVAtest was used to compare between groups, followed by post hoc test (least significant difference) for inter-group comparisons. For correlation study, Pearson correlation was applied. Statistical calculations were done using the computer software SPSS version 10 (Chicago, IL, USA) andMicrosoft Excel (Microsoft^®OfficeExcel^®2007). Statistical significance was set at P<0.05.

	RESULTS

   Clinical Characteristics

   The clinical characteristics of the study population (Mean ± SD)including: sex, age, diabetic duration, body mass index,waist/hip ratio, systolic blood pressureand diastolic blood pressureare shown in Table 1.

   The sex percentages were kept constant within the groups with a male percentage ranged between (13.3-13.8%) and a female percentage ranged between (86.2-86.7%). No significant differenceswere found between the groups for all clinical characteristics shown in Table 1.

   Biochemical Changes

   The different biochemical markers of the study population (Mean ± SD) including: FBG, Hb_A1C, Total-C, TAG, HDL-C, ALB, CRT, ALT, AST, CML and sRAGEare shown in Table 2.

   FBG and Hb_A1C. The D.O group showed a significant increase in both FBGand Hb_A1C when compared with those of D.N.Ogroup.The N.D.O group showed a significant increase in Hb_A1C, but insignificant increase in FBG when compared with those of the N.D.N.O group.

   Lipid Profile. The D.O group showed a significant increase in serum Total-C, TAG and LDL-Cwhen compared withthose of the D.N.O group. Also, the N.D.O group showed a significant increase in Total-C and LDL-C, but an insignificant increase in TAG, when compared withthose of the N.D.N.O group.A significant decrease in HDL-Cwas found inthe D.O group compared with the D.N.O groupand inthe N.D.O compared with N.D.N.O group.

   Albumin. The D.O group showeda significant decrease in serum ALB when compared with that of the D.N.Ogroup, while no significant relationship was found between ALB of the N.D.O and that of the N.D.N.O group. However, serum albumin in the four studied groups was within the normal range.

   Creatinine. The D.O group showed a significant increase in serum CRT when compared with that of the D.N.O group. On the other hand,the N.D.O group showed insignificant increase in CRT when compared with that of the N.D.N.O group.

   ALT and AST. The D.O group showed a significant increase in serum ALT and AST when compared with those of the D.N.O group, while the N.D.O group showed insignificant change in both ALT and AST when compared withthose of the N.D.N.O group.

   CML and sRAGE. The D.O group showed a significant increase in CML and a significant decrease in sRAGE when compared with those of the D.N.O group. Also, the N.D.O group showed a significant increase in CML and a significant decrease in sRAGE when compared with those of the N.D.N.O group.

   Regression Analysis

   The whole study samples were divided into two groups: Diabetic and Non-Diabetic to determine the independent predictors of type 2 diabetes. CML (P=0.01), sRAGE (P=0.00), Hb_A1C (P=0.00) and FBG (P=0.04) were found to be predictors for diabetes (Table 3).The whole study samples were divided into two groups: Obese and Non-Obese to determine the independent predictors of obesity. CML (P=0.00), sRAGE (P=0.04), Hb_A1C (P=0.00) were found to be predictors for obesity as shown in Table 3.

   Correlation study

   Pearson correlation coefficient of sRAGE, CML and HbA1C with different clinical characteristics and biochemical parameters measured in the measured in the four groups are shown in Tables 4-7. sRAGE showed a significant negative correlation with BMI in the four groups with r values of -0.73, -0.67, -0.71 and -0.79 in D.O, D.N.O, N.D.O and N.D.N.O groups, respectively as shown in Figure 1. sRAGE showed a significant negative correlation with WHR in the four groups with r values of -0.81, -0.73, -0.52 and -0.63 in D.O, D.N.O, N.D.O and N.D.N.O groups, respectively as shown in Figure 2. Also, sRAGE showed a significant negative correlation with CML in the four groups with r values of -0.74, -0.57, -0.53 and -0.61 in D.O, D.N.O, N.D.O and N.D.N.O groups, respectively. CML showed a significant positive correlation with BMI in the four groups with r values of 0.52, 0.63, 0.59 and 0.56 in D.O, D.N.O, N.D.O and N.D.N.O groups, respectively.

 

View larger version : [in a new window] Figure 1.Significant negative correlation between soluble receptors of advanced glycation end products (sRAGE) (pg/ml) and body mass index (BMI) (kg/m2) in:(A)Diabetic Obese group (r=-0.73, p<0.01); (B)Diabetic Non-Obese group (r=-0.67, p<0.05); (C)Non-Diabetic Obese group (r=-0.71, p<0.01); (D)Non-Diabetic Non-Obese group (r=-0.79, p<0.01).

 

View larger version : [in a new window] Figure 2. Significant negative correlation between soluble receptors of advanced glycation end products (sRAGE) (pg/ml) and waist/hip ratio (WHR) in: (A) Diabetic Obese group (r=-0.81, p<0.01); (B)Diabetic Non-Obese group (r=-0.73, p<0.01); (C)Non-Diabetic Obese group (r=-0.52, p<0.05); (D)Non-Diabetic Non-Obese group (r=-0.63, p<0.05).

	DISCUSSION

   Obesity has been recognized as an important determinant of insulin sensitivity (22).Abdominal obesity plays a central role in the metabolic syndrome and considered a major risk factor for chronic diseases, such as type 2 diabetes and cardiovascular disease. This suggestion is further stressed by the fact that body fat distribution and weight gainthroughout adulthood are important predictors of diabetes (23).

   In the present study, D.O group showed significant increases inFBG, Total-C, TAG, LDL-C andCRT and significant decreases inHDL-C and ALB when compared with D.N.O group. On the other hand, N.D.O group showed a significant increase in Total-C and LDL-C and a significant decrease in HDL-C when compared with N.D.N.O group.These results are consistent with reports showing that obesity would aggravate type 2 diabetes effects on FBG (24, 25), Total-C (26,27), TAG (27,28), LDL-C (25,27), HDL-C (27, 28), CRT (29, 30) and ALB (29, 31). In addition, our results are consistent with reports indicating that obesity per se would affect Total-C, LDL-C and HDL-C (32).

   In the present study, there were significant increases ofHb_A1Cin D.O group and N.D.O group when compared with those of D.N.O group and N.D.N.O group, respectively.

   These results are consistent with reports demonstrating an increase in Hb_A1C associated with diabetes (33) and obesity (34)suggesting that both diabetes and obesity contribute to the elevation in Hb_A1C. In diabetes mellitus the extent of the non-enzymatic glycation of proteins increases, compared with non-diabetic subjects, which may comprise at least a part of diabetic complications. Hb_A1C provides an integrated measurement of blood glucose during previous 2-3 months, reflecting 120-day life span of erythrocytes (35).

   This may explain the significant positive correlation found between Hb_A1C and FBG in D.Oand D.N.O groups.

   Hb_A1C elevation associated with obesity may be also attributed to the disordered glucose metabolism.The role of glucose metabolism in Hb_A1Celevation may be illustrated by the significant positive correlation between Hb_A1C and FBG in N.D.O and N.D.N.O groups and the significant positive correlation between Hb_A1C and both BMI and WHR. Hb_A1C and BMI correlation is consistent with the findings of Koga and his colleagues (2007),who showed a significant positive correlation between Hb_A1C and BMI in 212 non-diabetic normal, overweight and obese subjects (35).Although Hb_A1C was significantly increased in the N.D.O group versus N.D.N.O group, it remained within the normal range. Moreover, our findings also revealed a significant positive correlation between Hb_A1C and age, which are consistent with the previous study of Koga et al (2007) (35).

   In the present study, there wasa significant increase of CMLand a significant decrease of sRAGEin D.O and N.D.O groups when compared with those of D.N.O and N.D.N.O groups, respectively. Furthermore, our results showed significant positive correlations between CML and each of BMI, LDL-C and FBG and a significant negative correlation with HDL-C.In addition, sRAGE showed significant negative correlations with CML, BMI and WHR.

   Several reports have demonstrated that AGEs are markedly elevated within type 2 diabetic patients and/or illustrated a strong positive correlation between AGEs and BMI. These reports mostly used ELISA kits with polyclonal antibodies showing cross reactivity with many other AGEs or monoclonal ones but specific for AGEs other than CML (36-38).Few reports specifically illustrated CML (using ELISA kits with monoclonal antibodies) and its association with diabetes and BMI (39-41).However, the data still scarce and needs further confirmation. On the other hand, several studies have demonstrated a decline in sRAGE associated with either type 2 diabetes (38, 39)or obesity (21).CML may beconsidered as a general biomarker for oxidative stress. This could be reasoned by the fact that CML, not only originates from sugars, but also generated from lipids.It is generated in vitro from LDL incubated with copper ions and glucose and therefore,is believed to be both lipid and protein adducts. The pathways of CML formation suggest that a direct link of inflammation to AGE formation exists (1).

   RAGEwas initially identified as a receptor for CML. RAGE–ligand interactions lead to prolonged inflammation, mainly as a result of RAGE-dependent expression of pro-inflammatory cytokines and chemokines (1).

   The decrease of sRAGE in type 2 diabetes and obesity has been explained by the beneficial role of sRAGE and its function as decoy to capture the circulating AGEs preventing activation of RAGE signaling pathway as it still possesses the V-ligand essential for ligand binding (1). The decoy function theory is supported by an in vitro study, which demonstrated that the treatment with sRAGEnormalized the increases in most of the inflammatory markers in diabetic mice to those seen in age matched, non-diabetic controls (40).

   Importantly, ligand stimulated RAGE activation results in the generation of reactive oxygen species (ROS), at least in part via the activation of NADPH oxidase. A plethora of evidence suggests that AGEs are involved in a vicious cycle of inflammation, generation of ROS, amplified production of AGEs, enhanced inflammation, and so on. In addition to the ligation of RAGE, AGEs may be linked to increased generation of ROS by multiple mechanisms, such as decreasing activities of superoxide dismutase and catalase, diminishing glutathione stores, and activation of protein kinase C (4).It has been demonstrated that elevated AGEs and advanced lipoxidation end products may participate in the development of vascular disease complications (42). The association between low levels of sRAGE and high levels of CML-protein in patients with microvascular complications suggests that the production of sRAGE was probably insufficient to clear the excess CML-protein. This unbalanced ratio allowed circulating AGE to bind to cell RAGE and activates its signaling pathway (43).

   Diet is a major source of exogenous AGEs, as well as, AGEs are especially high in Western diets where foods are processed under elevated temperatures such as by broiling, roasting, deep frying, oven frying, or grilling. In addition, cola drinks, which contain large amounts of caramel additives, are rich sources of AGEs (44). The AGEs content of the same food item can be increased 10-200fold by increasing the temperature and conditions used in cooking. About 10% of dietary AGEs are absorbed, of which about one third is excreted and two thirds are deposited in tissues (44). Dietary AGEs are implicated in insulin resistance, visceral obesity, and the development of diabetes (36, 45). This may explains the significant positive correlations found in the present work between CML and either BMI or WHR.

   The significant positive correlations between CML and either age or diabetic duration may be explained depending upon its role as a general biomarker for oxidative stress. The links between normal physiological ageing and degenerative age-related disease are obvious and there are firm associations between diminished capacity for ageing cells to respond to stress and susceptibility to degenerative disease (46).sRAGE showed a significant negative correlation with CML. The correlation may be explained on the basis of a proposed negative feedback mechanism between CML and serum endogenous soluble receptors of advanced glycation end products (esRAGE) which in turn affect the whole pool of sRAGE. The relationship may include also oxidized LDL whose formation is favored by increased levels of CML and is known to be one of the RAGE ligands (37).In the present work, sRAGE showed a significant negative correlation with systolic and diastolic blood pressure. These results are coincident with some reports which indicated that low sRAGE is associated with hypertension in diabetics and non-diabetics (38, 47, 48).

	CONCLUSION

   The present study suggests that non-enzymatic glycation is the pathway connecting diabetes, obesity and diabetic complications including: Hb_A1C, CML and sRAGE. It indicates that obesity would affect components of the glycation pathway independent of type 2 diabetes. The increase in CML and the decrease in sRAGE associated with obesity found in the present study may support CML role as a general marker of oxidative stress. Also, it supports the decoy function of sRAGEand its beneficial effect in capturing CML. The results may represent a start for more future studies upon the clinical use of sRAGE as an effective medication for various conditions including type 2 diabetes and vascular diseases associated with either type 2 diabetes or obesity. However, the regulation of CML and sRAGE needs further investigation.

	ACKNOWLEDGEMENT

   We would like to express our sincere gratitude and deep thanks to Prof. Dr. Ahmad M. Rabie (God mercy him), Professor of Biochemistry, Biochemistry Department, Faculty of Pharmacy, Mansoura University, for his intellectual guidance, valuable advice and objective criticism.

	REFERENCES

1. Basta G. Receptor for advanced glycationendproducts and atherosclerosis: From basic mechanisms to clinical implications. Atherosclerosis. 2008; 196 (1): 9-21.
2. Ahmed N. Advanced glycationendproducts: role in pathology of diabetic complications. Diabetes. Res. Clin. Pract. 2005; 67 (1): 3-21.
3. DrindaS, FrankeS, CanetCC, PetrowP, et al. Identification of the advanced glycation end products N (epsilon)-carboxymethyllysine in the synovial tissue of patients with rheumatoid arthritis. Ann. Rheum. Dis. 2002; 61 (6):488-492.
4. Ramasamy R, Vannucci SJ, Yan SS, Herold K, et al. Advanced glycation end products and RAGE: a common thread in aging, diabetes, neurodegeneration, and inflammation. Glycobiology. 2005; 15 (7): 16R-28R.
5. Vincent AM, Perrone L, Sullivan KA, Backus C, et al. Receptor for advanced glycation end products activation injures primary sensory neurons via oxidative stress. Endocrinology. 2007; 148 (2):548-558.
6. Hein GE. Glycation end products in osteoporosis - Is there a pathophysiologic importance? Clinica.Chimica.Acta. 2006; 371: 32-36.
7. Schmidt AM, Stern DM. RAGE: a new target for the prevention and treatment of the vascular and inflammatory complications of diabetes. Trends Endocrinol.Metab. 2000; 11 (9):368-375.
8. World Health Organisation. Physical status: the use and interpretation of anthropometry: report of a WHO Expert Comittee.World Health Organ. Tech. Rep. Ser. 1995; 854:1-452.
9. Coutinho T, Goel K, Corrêa de Sá D, Kragelund C, et al. Central obesity and survival in subjects with coronary artery disease a systematic review of the literature and collaborative analysis with individual subject data. J. Am. Coll. Cardiol. 2011; 57 (19):1877-1886.
10. James TM, Davis JE, McDonald JM, Santiago JV, et al. Comparison of hemoglobin A1c and hemoglobin A1 in diabetic patients. Clin. Biochem. 1981; 14 (1):25-27.
11. Trinder P. Determination of blood glucose using 4-amino phenazone as oxygen acceptor.J. Clin. Pathol. 1969; 22 (2): 246.
12. Richmond W. Preparation and properties of a cholesterol oxidase from Nocardia sp. and its application to the enzymatic assay of total cholesterol in serum. Clin. Chem. 1973; 19 (12):1350-1356.
13. Trinder P. Triglycerides estimation by GPO-PAP method. Ann. Clin. Biochem. 1969; 6 (2): 24-27.
14. Izawa S, Okada M, Matsui H,Horita Y. A new direct method for measuring HDL cholesterol which does not produce any biased values. J. Med. Pharm. Sci. 1997; 37:1385-1388.
15. SchumannG, BonoraR, CeriottiF, FérardG, et al. IFCC primary reference procedure for the measurement of catalytic activity concentrations of enzymes at 37°C. Part 4. Reference procedure for the measurement of catalytic concentration of alanine aminotransferase. Clin. Chem. Lab. Med. 2002; 40 (7):718-724.
16. SchumannG, BonoraR, CeriottiF, FérardG, et al. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. International Federation of Clinical Chemistry and Laboratory Medicine. Part 5. Reference procedure for the measurement of catalytic concentration of aspartate aminotransferase. Clin. Chem. Lab. Med. 2002; 40 (7):725-733.
17. Doumas BT, Watson WA, Biggs HG. Albumin standards and measurement of serum albumin with bromcresol green. Clin. Chim. Acta. 1971; 31 (1): 87-96.
18. Larsen K. Creatinine assay by a reaction-kinetic principle. Clin. Chim. Acta. 1972; 41:209-217.
19. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge.Clin. Chem. 1972; 18 (6): 499-502.
20. Schleicher ED, Wagner E,Nerlich AG. Increased accumulation of the glycoxidation product Nε- (carboxymethyl) lysine in human tissues in diabetes and aging. J. Clin. Invest. 1997; 99 (3): 457-468.
21. NorataGD, GarlaschelliK, GrigoreL, TibollaG, et al. Circulating soluble receptor for advanced glycation end products is inversely associated with body mass index and waist/hip ratio in the general population. Nutr. Metab. Cardiovasc. Dis. 2009; 19 (2):129-134.
22. AkehiY, AnzaiK,KatsutaH,YoshidaR,et al. Adverse effects of obesity on β-cell function in Japanese subjects with normal glucose tolerance. Obes. Res. Clin. Pract. 2008; 2 (3): 195-202.
23. Goossens GH. The role of adipose tissue dysfunction in the pathogenesis of obesity-related insulin resistance. Physiol. Behav. 2008; 94 (2): 206-218.
24. Swiatkowska-Stodulska R, BabinskaA,Skibowska-BielinskaA,SworczakK. Assessment of α1-antitrypsin and α2-macroglobulin levels in obese patients. Pol. Arch. Med. Wewn. 2008; 118(12): 713-717.
25. GokalpD, BahceciM, OzmenbS,ArikanaS,et al. Adipocyte volumes and levels of adipokines in diabetes and obesity. DiabMetabSyn: Clin. Res. Rev. 2008; 2 (4): 253-258.
26. AiM,OtokozawaS,SchaeferEJ,AsztalosBF,et al. Glycated albumin and direct low density lipoprotein cholesterol levels in type 2 diabetes mellitus. Clin. Chim. Acta. 2009; 406 (1-2): 71-74.
27. YounBS,KlötingN,KratzschJ,LeeN,et al. Serum vaspin concentrations in human obesity and type 2 diabetes. Diabetes. 2008; 57 (2): 372-373.
28. KogaM, OtsukiM, MatsumotoS, SaitoH,et al. Negative association of obesity and its related chronic inflammation with serum glycated albumin but not glycated hemoglobin levels. Clin. Chim. Acta. 2007; 378 (1-2): 48-52.
29. Alberti KG, ZimmetP, ShawJ, IDF Epidemiology Task Force Consensus Group. The metabolic syndrome--a new worldwide definition. Lancet. 2005; 366 (9491):1059-1062.
30. Betteridge DJ. Diabetic dyslipidaemia. Eur. J. Clin. Invest. 1999; 29(2):12-16.
31. ValerioG,LicenziatiMR,IannuzziA,FranzeseA,et al. Insulin resistance and impaired glucose tolerance in obese children and adolescents from Southern Italy. Nutr. Metab. Cardiovasc. Dis. 2006; 16 (4): 279-284.
32. Sikaris KA. The clinical biochemistry of obesity-more than skin deep.Heart Lung Circ. 2007; 16 (3): S45-S50.
33. Quilliot D, Zannad F,Ziegler O. Impaired response of cardiac autonomic nervous system to glucose load in severe obesity. Metabolism. 2005; 54 (7): 966- 974.
34. Nakatsuji H,KishidaK,KitamuraT,NakajimaC,et al. Dysregulation of glucose, insulin, triglyceride, blood pressure, and oxidative stress after an oral glucose tolerance test in men with abdominal obesity. Metabolism. 2010; 59 (4): 520-526.
35. ChrysohoouC,PanagiotakosDB,PitsavosC,SkoumasI,et al. The implication of obesity on total antioxidant capacity in apparently healthy men and women: The ATTICA study. Nutr. Metab. Cardiovasc. Dis. 2007; 17 (8): 590-597.
36. Yamagishi S,Adachi H,Nakamura K,Matsui T,et al. Positive association between serum levels of advanced glycation end products and the soluble form of receptor for advanced glycation end products in non-diabetic subjects. Metabolism.2006; 55 (9): 1227-1231.
37. Saisho Y, Maruyama T, Hirose H,Saruta T. Relationship between proinsulin-to-insulin ratio and advanced glycation end products in Japanese type 2 diabetic subjects. Diabetes Res. Clin. Pract. 2007; 78 (2): 182-188.
38. Nakamura K,Yamagishi S,Adachi H,Matsui T,et al. Serum levels of soluble form of receptor for advanced glycation end products (sRAGE) are positively associated with circulating AGEs and soluble form of VCAM-1 in patients with type 2 diabetes. Microvasc. Res. 2008; 76 (1): 52-56.
39. Grossin N, Wautier MP, Meas T, Guillausseau PJ, et al. Severity of diabetic microvascular complications is associated with a low soluble RAGE level. Diabetes Metab. 2008; 34 (4 Pt 1): 392-395.
40. Boor P,Celec P,Behuliak M,Grancic P,et al. Regular moderate exercise reduces advanced glycation and ameliorates early diabetic nephropathy in obese Zucker rats. Metabolism.2009; 58 (11): 1669-1677.
41. Semba RD, Najjar SS, Sun K,Lakatta EG, et al. Serum carboxymethyl–lysine, an advanced glycation end product, is associated with increased aortic pulse wave velocity in adults. Am. J. Hypertens. 2009; 22 (1): 74-79.
42. Miyashita Y, Nishimura R, Morimoto A, Matsudaira T, et al. Glycated albumin is low in obese, type 2 diabetic patients. Diabetes Res. Clin. Pract. 2007; 78 (1): 51-55.
43. Folsom AR, Ma J, Eckfeldt JH, Nieto FJ, et al. Low serum albumin association with diabetes mellitus and other cardiovascular risk factors but not with prevalent cardiovascular disease or carotid artery intima-media thickness. Ann. Epidemiol. 1995; 5 (3):186-191.
44. Rossi MC,Nicolucci A,Pellegrini F,Comaschi M,et al. Obesity and changes in urine albumin/Creatinine ratio in patients with type 2 diabetes: The DEMAND Study. Nutr. Metab. Cardiovasc. Dis. 2010; 20 (2): 110-116.
45. Jerums G, Premaratne E,Panagiotopoulos S,Clarke S,et al. New and old markers of progression of diabetic nephropathy. Diabetes Res. Clin. Pract. 2008; 82 (1): s30-s37.
46. Lee IT, Huang CN, Lee WJ, Lee HS, et al. Aggravation of albuminuria by metabolic syndrome in type 2 diabetic Asian subjects. Diabetes Res. Clin. Pract. 2008; 81 (3): 345-350.
47. Falcone C, Emanuele E, D'Angelo A, Buzzi MP, et al. Plasma levels of soluble receptor for advanced glycation end products and coronary artery disease in nondiabetic men. Arterioscler.Thromb.Vasc. Biol. 2005; 25 (5):1032-1037.
48. Geroldi D, Falcone C, Emanuele E, D'Angelo A, et al. Decreased plasma levels of soluble receptor for advanced glycation end-products in patients with essential hypertension. J. Hypertens. 2005; 23 (9):1725-1729.

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