Material and Methods: Fourty T1DM patients attending to the Diabetes and Endocrinology outpatient clinic in Haseki Hospital and 17 healthy volunteers as control group were included in this study. AGEs and AOPPs were measured spectrophotometrically and fibronectin was measured with nephelometric method.

Results: Fibronectin levels of diabetic patients were significantly higher than control group (p<0.05). There was not any significant difference in fibronectin levels between patients with and without diabetic complications. In addition, no statistically significant difference was observed in AGE and AOPP levels among diabetic patients and control group. No correlation was determined between plasma fibronectin and AGE and AOPP levels (r=0.4) either.

Conclusion: No significant correlation was determined between fibronectin levels and AGE and AOPP which play an important role in progression of diabetic complications in T1DM. The lack of significant difference between healthy controls and patients with T1DM suggests that treatment counteracts the increase in free radical production.

Advanced oxidation protein product (AOPP) was first described in 1996 by Witko-Sarsat et al. They occur during oxidative stress by the action of hypochlorous acid and chloramines which are produced by myeloperoxidase. They act like AGEs in induction of proinflammatory cytokins and adhesive molecules⁶. Reactive oxygen species (ROS) effects the proteins directly which leads to formation of oxydated amino acids. It oxydates thyrosine amino acid directly and generates dithyrosine structure causing agreggation and fragmantation in protein structure. The product produced by this configuration is called AOPP⁷. Chromatographic and electrophoretic techniques demonstrate that AOPP consists of heavy chain disulfide bonds or/and aggreagates ofalbuminwith thyrosine cross binding⁷.

Endothelial dysfunction is considered to be the first step in the development of arterial disease. It is possible to assess endothelial dysfunction by vascular function tests which use invasive or noninvasive techniques or by determining plasma concentrations of specific endothelial proteins. Fibronectin is also another marker of endothelial activation⁸.

Fibronectin is a large glycoprotein present in extracellular matrix and on cell surfaces which improves cell-cell and cell-matrix interactions and thus plays an important role in tissue construction and reconstruction⁹. It has two major forms of which one is predominantly soluble in body fluids like plasma, CSF, sinovial, amniotic and seminal fluid. The other form is found on cell surfaces and in extracellular matrix in an insoluble multimeric form. A fibronectin molecule has a length of 600 A, width of 25 A and its molecular weight is 550 KD. It consists of two subunits. The disulfide bonds which bind these subunits with each other stand near the carboxy terminal ends of each one¹⁰.

Fibronectin plays an important role in embryogenesis, oncogenic transformation, cell adhesion, wound healing, tissue reparation, platelet functions and cell migration Furthermore it may have a role in inflammation as an opsonin and chemotactic agent. While the majority amount of circulating fibronectin is synthesized by hepatocytes it is also synthesized and secreted by several types of cells like macrophages, thrombocytes, fibroblasts, amniotic cells, endothelial cells, melanocytes, mast cells, schwann cells, sinovial cells and chondrocytes. Plasma fibronectin has a half life of 24-72 hours. Average concentration of plasma fibronectin in normal people is between 250-400 pg/ml. It is reported that the plasma levels do not show any difference considering age and gender¹⁰.

Elevated levels of plasma fibronectin are reported in rheumatoid arthritis, pre-eclamptic pregnancies, collagenous vascular diseases, acute trauma, septic syndrome and thrombotic thrombocytopenic purpura^11-15. These results indicate that intravascular accumulation of fibronectin damages blood vessels.

Characteristic endothelial extracellular matrix alterations and damages in blood vessels are also present in diabetic patients. In diabetic patients the accumulation of proteins in subendothelial matrix is seen as PAS positive in light microscobe. The increased plasma levels of fibronectin reflect the vessel wall injury and matrix modifications in diabetic patients¹⁶.

Although oxidative stres is increased in both types of DM we could not find satisfactory number of studies concerning the patients with T1DM. With this study, we aimed to assess fibronectin, AOPP and AGE levels in T1DM diabetic patients as well as the interaction of these parameters with each other and diabetic complications.

Click Here to Zoom

Table I: The demography of patients and control group

Fifty of diabetic patients had diabetic complications. 11 had retinopathy, 7 had nephropathy and 9 had neuropathy. Biochemical parameters like glucose, creatinine, cholesterol, triglyceride, HDL and HbA1c levels were determined by Olympus AU 2700 (Japan) with original reactive and methods. Fibronectin levels were determined through nephelometric method with Dade Behring ProsPec analyser (Australia).

The results were expressed as mean ±SD (Standard deviation of mean). Data were evaluated by SPSS 2.0 for Windows (SPSS Inc., Chicago, II, USA). Statistical analysis was performed using Mann Whitney-U and Sperman correlation tests. A P value less than 0.05 was considered as statistically significant. Levene F test was used for assessing the compliance of the data to normal distribution. Nonparametric test was used because variance was not homogenous and the distribution was not normal.

AGEs assay. The sera were diluted 1:50 with phosphate buffered saline (PBS; pH 7.4). Fluorescence intensity was recorded at the emission maximum 350 nm (spectrofluorimeter Biorad). Maximum emission was recorded as 440 nm. Fluorescence intensity was expressed in arbitrary units (AU) and in AU/g protein⁶.

AOPP assay. Chloramine-T stock solution (10 mmol), 1.16mol/L of potassium iodure (KI), glaciel acetic acide (% 100 v/v ) and PBS (20 mmol/L) were used as reactives. Chloramine-T stock solution was diluted 100 times with PBS and main standart solution of 100µmol/L concentration was prepared. Chloramine-T standards for PBS calibration curve were prepared by diluting this solution with PBS to concentrations of 12.5, 25, 50, 75, 100 µmol/L. The stability of Chloramine-T stock solution is 3 to 4 months at 5° C, whereas stability of the diluted solution is 3 to 4 days at 5° C. The samples were processed at room temperature with a programme adapted to Olympus AU 2700 analyser. 160 µL of PBS reactive was added on 40µL plasma and incubated for 25 seconds. The absorbance of the mixture was measured at 340 nm. Subsequently 20µL of acetic acide was added and incubated for 25 seconds. the absorbance was re-measured. Finally 10µL of KI solution was added and absorbance was re-measured after another 25 sec. of incubation. Concentration of AOPP was determined in chloramines units (ìmol/L) and values adjusted for albumin levels were calculated (µmol/L/g albumin). All of the steps of the process were performed in a single measure cell at 37°C. Depending on the programme characteristics of the analyser used, time intervals might be arranged at 25 seconds or longer for each step¹⁷.

Click Here to Zoom

Table II: Data of patients and control group (mean ± SD).

Some matrix proteins are found in high concentrations in diabetes mellitus. Big vessels also involve laminin and fibronectin molecules in big amounts like small ones. Unfortunately polimerisation in intracellular matrix causes fibronectin to be released in the circulation in an insoluble form. The polarised secretion modifications induces an increase in fibronectin concentrations in diabetic patients⁸.

Simo and co-workers did not demonstrate any significant difference between fibronectin concentrations of diabetic patients with and without complications²³. We could not demonstrate any significant difference either. Zozulinska et al found fibronectin levels higher in type 1 diabetic patients than healthy people but when patients with and without complications were compared there was not any difference²⁴. The results of this study support our data. These studies point out that elevation of plasma concentration in diabetic patients is independent of microangiopathy. A study by Testa and co-workers also revealed elevated levels of fibronectin in diabetic patients and their findings supported our study²⁵. Lee and co-workers demonstrated that fibronectin levels were higher in diabetic patients compared with healthy subjects²⁶.

We compared AOPP and AGE levels in diabetic and healthy group and could not find any significant difference between two groups. Furthermore these two parameters were correlated with fibronectin, cholesterol, triglyceride, HDL and creatinine levels of the patients and no correlation was found (r=0.4). Wagner and co-workers found a positive correlation between AGE levels and creatinine levels of the diabetic patients with renal insufficiency. They did not define an increase in AGE levels of diabetic patients with normal renal functions²⁷. In our study, renal functions of our patients were within normal ranges. Kalousova and co-workers described a positive correlation in AGE and triglyceride levels in diabetic patients. They believe that these results are because of the fact that diabetes mellitus is a complex metabolic disorder⁶.

Witko and Sarsat found AOPP levels elevated in patients undergoing hemodialysis regardless of being diabetic or not⁴. However Kalousova and co-workers did not find AOPP levels elevated in diabetic patients with normal renal functions⁶. We also could not find AOPP levels significantly different in patient and control group. Both group of investigators found significant correlation between AOPP and triglyceride levels. Early on, the association between lipids and AOPP was not so clear but now it is known that AOPP is effective on oxidative modification of LDL and this effect causes atherosclerosis. Wagner and co-workers could not find an association between AGE levels and diabetic complications²⁷. Stitt and co-workers could not demonstrate detrimental effects and the accumulation AGE in damaged tissues²⁸. We also could not demonstrate a significant association in AGE and AOPP levels of patients with or without complication (p>0.05). Kalousova and co-workers found AGE and AOPP levels slightly elevated in patients with micro and macrovascular complications but it was not statistically significant¹⁰ while Gleisner and co-workers assessed AOPP levels in type 1 diabetic patients and failed to show any significant difference from the control group²⁹. Kalousova and co-workersl found these levels markedly increased in type 2 diabetic patients. Probably oxidative stress has a more important role in production of AGE in type 2 diabetic patients. It is supposed to be the compensation mechanism of the illness⁶. With another point of view, it may be suggested that antidiabetic treatment prevents the formation of free radicals caused by oxidative stress.

Xie XM and co-workers demonstrated by a study in 2006 that expression of fibronectin mRNA is induced by AGE when compared to control group. They suggested that elevation of fibronectin levels induced by AGEs contributed to progression of diabetic complications³⁰. We conclude that fibronectin levels were higher in diabetic group than control group yet this elevation was not related with complications. We could not demonstrate the effects of formation of AOPP and AGE on diabetic complications and fibronectin levels.

1) Vlassara H. Recent progress in advanced glycation end products and diabetic complications. Diabetes 1997;46 Suppl 2: S19-S25.

2) Miyata T, Van Ypersele de Strihou C, Kurokawa K, Baynes JW. Alterations in nonenzymatic biochemistry in uremia: origin and significance of “carbonyl stress” in long term uremic complications. Kidney Int 1999;55: 389-399.

3) Yenigün M. Her yönüyle Diabetes Mellitus. 2. baskı. İstanbul: Nobel Tıp Kitabevleri ltd. şti. 2001. s.165.

4) Witko-Sarsat V, Friedlander M, Capeillere-Blandin C, et al. Advanced oxidation protein products as a novel marker of oxidative stres in uremia. Kidney Int 1996;49: 1304-1313.

5) Bierhaus A, Hofmann MA, Ziegler R, Nawroth PP. AGEs and their interaction with AGE- receptors in vasculer disease and diabetes mellitus. I. The AGE concept. CardiovascRes. 1998;378: 586-600.

6) Kalousova M, Skrha J, Zima T. Advanced glycation end- products and advanced oxidation protein products in patients with diabetes mellitus. Physiol Res. 2002;51: 597-604.

7) Gil-del Valle L, de la C Milian L, Toledo A, Vilaró N, Tápanes R, Otero MA. Altered redox status in patients with diabetes mellitus type 1. Pharmacological Research 2005; 51: 375-380.

8) Kanters SD, Banga JD, Algra A, Frijns RC, Beutler JJ, Fijnheer R. Plasma levels of cellular fibronectin in diabetes. Diabetes Care. 2001;24: 323-327.

9) Hynes RO. Fibronectins. Sci Am 1986;254: 42-51.

10) Demirer S, Marakoğlu I. Periodontolojide fibronektinin önemi ve kullanımı. Cumhuriyet Üniversitesi Dişhekimliği Dergisi 2001;4: 1.

11) Friedman SA, de Groot C, Taylor R, Golditch B, Roberts JM. Plasma cellular fibronectin as a measure of endothelial involvement in preeclampsia and intrauterine growth retardation. Am J Obstet Gynecol 1994;170:838 -841.

12) Fijnheer R, Frijns CJ, Korteweg J, et al. Origin of P- selectin as a circulating plasma protein. Thromb Haemost 1997;77: 1081 -1085.

13) Peters JH, Maunder RJ, Woolf AD, Cochrane CG, Ginsberg MH. Elevated plasma levels of ED1+ ("cellular") fibronectin in patients with vascular injury. J Lab Clin Med 1989;113: 586-597.

14) Van der Plas RM, Schiphorst ME, Huizinga EG, et al. von Willebrand factor proteolysis is deficient in classic, but not in bone marrow transplantation-associated, thrombotic thrombocytopenic purpura. Blood 1999; 15;93: 3798-3802

15) Voskuyl AE, Emeis JJ, Hazes JMW, Van Hogezand RA, Biemond I, Breedveld FC. Voskuyl AE, Emeis JJ, Hazes JM, van Hogezand RA, Biemond I, Breedveld FC. Levels of circulating cellular fibronectin are increased in patients with rheumatoid vasculitis. Clin Exp Rheumatol 1998;16: 429-434.

16) Andresen JL, Rasmussen L, Ledet T. Diabetic macroangiopathy and atherosclerosis. Diabetes 1996;45: 91-94.

17) Selmeci L, Seres L, Antal M, Lukács J, Regöly-Mérei A, Acsády G. Advanced oxidation protein products (AOPP) for monitoring oxidative stress in critically ill patients: a simple, fast and inexpensive automated technique. Clin Chem Lab Med 2005;43: 294-297.

18) Dinneen SF, Gerstein HC. The association of microalbuminuria and mortality in non-insulin-dependent diabetes mellitus: a systematic overview of the literature. Arch Intern Med 1997;157: 1413-1418.

19) Johnstone MT, Creager SJ, Scales KM, Cusco JA, Lee BK, Creager MA. Impaired endothelium-dependent vasodilation in patients with insulin-dependent diabetes mellitus. Circulation 1993;88: 2510-2516

20) Van Mourik JA, Boertjes R, Huisveld IA, et al. von Willebrand factor propeptide in vascular disorders: A tool to distinguish between acute and chronic endothelial cell perturbation. Blood 1999;94: 179-185.

21) Vischer UM, Emeis JJ, Bilo HJG, at al. von Willebrand factor (vWf) as a plasma marker of endothelial activation in diabetes: improved reliability with parallel determination of the vWf propeptide (vWf:AgII). Thromb Haemost 1998;80: 1002 -1007.

22) Williams SB, Cusco JA, Roddy MA, Johnstone MT, Creager MA: Impaired nitric oxide—mediated vasodilation in patients with non-insulin-dependent diabetes mellitus. J Am Coll Cardiol 1996;27:567-574.

23) Simo R, Segura RM, Garcia-Pascual L, et al. Fibronectin and diabetes mellitus: the factors that influence its plasma concentrations and its usefulness as a marker of late complications (Spanish). Med Clin (Barc) 1999;112: 45-50.

24) Zozuliñska D, Derc K, Majchrzak A, Szczepanik A, Wierusz-Wysocka B. Assessment of plasma fibronectin concentration in type 1 diabetic patients (Polish). Pol Arch Med Wewn 1999 ;102:773-7777.

25) Testa R, Bonfigli AR, Sirolla C, et al. Fibronectin and lipoprotein(a) are inversely related to plasminogen activator inhibitor type-1 levels in Type 2 diabetic patients without complications. Diabetes Nutr Metab 2000;13: 269-275.

26) Lee IK, Park KY, Oh HK, Park RW, Jo JS. Plasma type IV collagen and fibronectin concentrations in diabetic patients with microangiopathy. Korean Med Sci 1994;9: 341-346.

27) Wagner Z, Wittman I, Mazak I, et al. N(epsilon)- (carboxymethyl)lysine levels in patients with type 2 diabetes: role of renal function. Am j Kidney Dis 2001;38: 785-791.

28) Stitt AW, HE C, Friedman S, et al. Elevated AGE- modified ApoB in sera of euglycemic, normolipidemic patients with atherosclerosis: relationship to tissue AGEs. Mol Med 1997;3: 617-627.

29) Gleisner A, Martinez L, Pino R, et al. Oxidative stress markers in plasma and urine of prepubertal patients with type 1 diabetes mellitus. J Pediatr Endocrinol Metab. 2006;19: 995-1000.

30) Xie XM, Yang ZW, Chen MF. Effects of advanced glycation endproducts on the activity of NF-kappaB and the expression of fibronectin mRNA in the endothelial cells in aged rats (Chinese). Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2006;31: 883-887.