Limits...
Role of N-(carboxymethyl)lysine in the development of ischemic heart disease in type 2 diabetes mellitus.

A Ahmed K, Muniandy S, S Ismail I - J Clin Biochem Nutr (2007)

Bottom Line: Serum CML levels were measured by enzyme-linked immunosorbent assay using polyclonal anti-CML antibodies.In conclusion, this study demonstrates the effect of both diabetes and oxidative stress on the higher levels of circulating CML.These results showed that increased serum levels of CML are associated with the development of IHD in Type 2 diabetes mellitus.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.

ABSTRACT
This study aims to determine the levels of N(epsilon)-(carboxymethyl)lysine (CML) in patients with Type 2 diabetic patients with and without ischemic heart disease (IHD) and to find for a possible association between circulating CML and a number of clinical parameters including lipids, hemoglobin A1c (HbA1c) and malondialdehyde (MDA) in Type 2 diabetic IHD patients. Serum CML levels were measured by enzyme-linked immunosorbent assay using polyclonal anti-CML antibodies. Serum levels of CML and MDA were assessed in 60 IHD patients with Type 2 diabetes, 43 IHD patients without Type 2 diabetes, 64 Type 2 diabetics without IHD, and 80 sex- and age-matched healthy subjects. Correlations studies between CML levels and lipids, HbA1c, and lipid peroxidation were performed in Type 2 diabetes patients with and without IHD. A statistical significance was observed in the levels of serum glucose, lipids (triglyceride, total cholesterol, HDL-cholesterol), MDA, HbA1c, CML and LDL-cholesterol (p<0.05) between the groups of the study. CML levels were significantly increased in diabetic IHD patients compared with Type 2 diabetes patients but without IHD (537.1 +/- 86.1 vs 449.7 +/- 54.9, p<0.001). A positive correlation was observed between serum levels of CML and MDA, r = 0.338 (p = 0.008) in Type 2 diabetes patients with IHD. However, age, HbA1c and lipids had no significant influence on CML levels among diabetics (p>0.05). In conclusion, this study demonstrates the effect of both diabetes and oxidative stress on the higher levels of circulating CML. These results showed that increased serum levels of CML are associated with the development of IHD in Type 2 diabetes mellitus.

No MeSH data available.


Related in: MedlinePlus

Immunoreactivity of the polyclonal anti-CML antibody to CML-proteins. The immunoreactivity of the polyclonal anti-CML antiserum was determined by competitive ELISA. The microplate wells were coated with 100 µl of CML-BSA (1.0 µg/ml) in 50 mM carbonate buffer (pH 9.6) at room temperature for 1 h. Wells were washed three times with buffer A and blocked with 6% skimmed milk for 1 h followed by triple washing. Each protein or competitor to be tested with different concentrations (60 µl) was mixed with equal volume of antiserum (final dilution 1:2000) for 1 h and a portion (100 µl) of the mixture was then added to the wells followed by incubation for 1 h. After triple washing, HRP-conjugated anti-rabbit IgG antibody (0.1 µg/ml) was used to detect the antibodies bound to wells as described in the methods section.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2170948&req=5

Figure 2: Immunoreactivity of the polyclonal anti-CML antibody to CML-proteins. The immunoreactivity of the polyclonal anti-CML antiserum was determined by competitive ELISA. The microplate wells were coated with 100 µl of CML-BSA (1.0 µg/ml) in 50 mM carbonate buffer (pH 9.6) at room temperature for 1 h. Wells were washed three times with buffer A and blocked with 6% skimmed milk for 1 h followed by triple washing. Each protein or competitor to be tested with different concentrations (60 µl) was mixed with equal volume of antiserum (final dilution 1:2000) for 1 h and a portion (100 µl) of the mixture was then added to the wells followed by incubation for 1 h. After triple washing, HRP-conjugated anti-rabbit IgG antibody (0.1 µg/ml) was used to detect the antibodies bound to wells as described in the methods section.

Mentions: To develop an antiserum that would selectively and specifically recognize CML adducts, but not epitopes of native proteins, the rabbit was immunized with CML-BSA. The anti-CML-BSA titer was determined to be greater than 36000 in a non-competitive ELISA. The specificity of the antiserum was tested by a competitive ELISA and results are shown in Fig. 2. CML-BSA and CML-Hb were highly effective competitors and significantly reacted with anti-CML antibodies, whereas the native (BSA, Hb) and CEL-modified proteins (CEL-BSA, CEL-Hb) were not recognized by the antibody. Thus, this monospecific antiserum is specifically directed to the CML epitope, able to recognize it on any of a variety of different proteins.


Role of N-(carboxymethyl)lysine in the development of ischemic heart disease in type 2 diabetes mellitus.

A Ahmed K, Muniandy S, S Ismail I - J Clin Biochem Nutr (2007)

Immunoreactivity of the polyclonal anti-CML antibody to CML-proteins. The immunoreactivity of the polyclonal anti-CML antiserum was determined by competitive ELISA. The microplate wells were coated with 100 µl of CML-BSA (1.0 µg/ml) in 50 mM carbonate buffer (pH 9.6) at room temperature for 1 h. Wells were washed three times with buffer A and blocked with 6% skimmed milk for 1 h followed by triple washing. Each protein or competitor to be tested with different concentrations (60 µl) was mixed with equal volume of antiserum (final dilution 1:2000) for 1 h and a portion (100 µl) of the mixture was then added to the wells followed by incubation for 1 h. After triple washing, HRP-conjugated anti-rabbit IgG antibody (0.1 µg/ml) was used to detect the antibodies bound to wells as described in the methods section.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2170948&req=5

Figure 2: Immunoreactivity of the polyclonal anti-CML antibody to CML-proteins. The immunoreactivity of the polyclonal anti-CML antiserum was determined by competitive ELISA. The microplate wells were coated with 100 µl of CML-BSA (1.0 µg/ml) in 50 mM carbonate buffer (pH 9.6) at room temperature for 1 h. Wells were washed three times with buffer A and blocked with 6% skimmed milk for 1 h followed by triple washing. Each protein or competitor to be tested with different concentrations (60 µl) was mixed with equal volume of antiserum (final dilution 1:2000) for 1 h and a portion (100 µl) of the mixture was then added to the wells followed by incubation for 1 h. After triple washing, HRP-conjugated anti-rabbit IgG antibody (0.1 µg/ml) was used to detect the antibodies bound to wells as described in the methods section.
Mentions: To develop an antiserum that would selectively and specifically recognize CML adducts, but not epitopes of native proteins, the rabbit was immunized with CML-BSA. The anti-CML-BSA titer was determined to be greater than 36000 in a non-competitive ELISA. The specificity of the antiserum was tested by a competitive ELISA and results are shown in Fig. 2. CML-BSA and CML-Hb were highly effective competitors and significantly reacted with anti-CML antibodies, whereas the native (BSA, Hb) and CEL-modified proteins (CEL-BSA, CEL-Hb) were not recognized by the antibody. Thus, this monospecific antiserum is specifically directed to the CML epitope, able to recognize it on any of a variety of different proteins.

Bottom Line: Serum CML levels were measured by enzyme-linked immunosorbent assay using polyclonal anti-CML antibodies.In conclusion, this study demonstrates the effect of both diabetes and oxidative stress on the higher levels of circulating CML.These results showed that increased serum levels of CML are associated with the development of IHD in Type 2 diabetes mellitus.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.

ABSTRACT
This study aims to determine the levels of N(epsilon)-(carboxymethyl)lysine (CML) in patients with Type 2 diabetic patients with and without ischemic heart disease (IHD) and to find for a possible association between circulating CML and a number of clinical parameters including lipids, hemoglobin A1c (HbA1c) and malondialdehyde (MDA) in Type 2 diabetic IHD patients. Serum CML levels were measured by enzyme-linked immunosorbent assay using polyclonal anti-CML antibodies. Serum levels of CML and MDA were assessed in 60 IHD patients with Type 2 diabetes, 43 IHD patients without Type 2 diabetes, 64 Type 2 diabetics without IHD, and 80 sex- and age-matched healthy subjects. Correlations studies between CML levels and lipids, HbA1c, and lipid peroxidation were performed in Type 2 diabetes patients with and without IHD. A statistical significance was observed in the levels of serum glucose, lipids (triglyceride, total cholesterol, HDL-cholesterol), MDA, HbA1c, CML and LDL-cholesterol (p<0.05) between the groups of the study. CML levels were significantly increased in diabetic IHD patients compared with Type 2 diabetes patients but without IHD (537.1 +/- 86.1 vs 449.7 +/- 54.9, p<0.001). A positive correlation was observed between serum levels of CML and MDA, r = 0.338 (p = 0.008) in Type 2 diabetes patients with IHD. However, age, HbA1c and lipids had no significant influence on CML levels among diabetics (p>0.05). In conclusion, this study demonstrates the effect of both diabetes and oxidative stress on the higher levels of circulating CML. These results showed that increased serum levels of CML are associated with the development of IHD in Type 2 diabetes mellitus.

No MeSH data available.


Related in: MedlinePlus