|Year : 2015 | Volume
| Issue : 2 | Page : 63-68
Paraoxonase-1 activity in type 2 diabetes mellitus with and without nephropathy
Nehal H El-said1, Mohammed M Nasr-Allah1, Noha A Sadik1, Saher A Sharaf2
1 Department of Internal Medicine, Kasr Al Ainy Hospital, Faculty of Medicine, Cairo University, Giza, Egypt
2 Department of Clinical and Chemical Pathology, Kasr Al Ainy Hospital, Faculty of Medicine, Cairo University, Giza, Egypt
|Date of Submission||24-Mar-2015|
|Date of Acceptance||22-Apr-2015|
|Date of Web Publication||29-Jun-2015|
Noha A Sadik
7 Al Mahrossa Street, Ahmed Orabi, Al Mohandeseen, app 3, Giza, 694
Source of Support: None, Conflict of Interest: None
Paraoxonase-1 (PON-1) is an enzyme synthesized in the liver that has antioxidant functions as it binds to the HDL particles and prevents the oxidation of LDL, which possibly plays a role in the prevention of atherosclerosis and coronary artery disease.
To determine PON-1 activity in type 2 diabetic patients with and without diabetic nephropathy and its correlation with the lipid profile, disease duration, and glycemic status.
Patients and methods
This study was carried out on 30 patients with type 2 diabetes mellitus who attended the diabetes and endocrine clinic at Kasr Al Ainy Hospital, Cairo University, including 20 patients with evidence of diabetic nephropathy and 10 patients without diabetic nephropathy as well as 15 healthy age-matched control participants. Fasting blood sugar, 2 h postprandial blood sugar, total cholesterol, HDL, LDL, triglycerides, and serum creatinine were measured. PON-1 activity was detected using a colorimetric method.
PON-1 activity was reduced significantly in diabetic patients with and without nephropathy, with mean 226.1 135.4 and 221.7 119.6 nmol/ml/min, respectively, versus 758.5 353.9 nmol/ml/min in the control group (P < 0.001). PON-1 activity was not significantly different between diabetic patients with and without nephropathy. PON-1 activity was correlated negatively with HDL (r = −0.496, P = 0.026) in diabetic patients with nephropathy. PON-1 activity was not correlated significantly with disease duration and glycemic status.
PON-1 activity was significantly reduced in type 2 diabetes, but did not differ between diabetics with or without nephropathy.
Keywords: Diabetic nephropathy, oxidative stress, paraoxonase-1, type 2 diabetes
|How to cite this article:|
El-said NH, Nasr-Allah MM, Sadik NA, Sharaf SA. Paraoxonase-1 activity in type 2 diabetes mellitus with and without nephropathy. Egypt J Intern Med 2015;27:63-8
|How to cite this URL:|
El-said NH, Nasr-Allah MM, Sadik NA, Sharaf SA. Paraoxonase-1 activity in type 2 diabetes mellitus with and without nephropathy. Egypt J Intern Med [serial online] 2015 [cited 2019 Sep 19];27:63-8. Available from: http://www.esim.eg.net/text.asp?2015/27/2/63/159451
| Introduction|| |
Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia because of defects in insulin secretion, insulin action, or both . Chronic hyperglycemia can induce reactive oxygen species and increase the oxidative stress, which may be a result of glycation and glucoxidation. Increased oxidative stress induces lipid oxidation and formation of lipid peroxides, which have been implicated in the pathogenesis of diabetic complications .
Diabetic nephropathy is one of the most leading causes of end-stage renal disease in Egypt after hypertensive nephropathy . The pathogenesis of diabetic nephropathy is still unclear and complex. Previous studies have documented the important role of oxidative stress in the development of diabetic nephropathy ,.
Human serum paraoxonase-1 (PON-1) is an enzyme synthesized in the liver and released in blood binding to HDL . It is an antioxidant enzyme that hydrolyzes the toxic peroxides in the oxidized lipids in both LDL and HDL; thus, it has been implicated in the prevention of atherosclerosis and cardiovascular diseases . Serum PON-1 activity and concentrations vary widely in the population, PON-1 can vary by more than 40-fold, and the PON-1 protein levels by more than 13-fold in a single PON-1 genotype . Decreased PON-1 activity, independent of genotype, has been documented in diabetes, hypercholesterolemia, and renal failure . It was noted that patients with low PON-1 activity are more vulnerable to diseases involving increased oxidative damage and lipid peroxidation compared with patients with high PON-1 activity .
Diabetic complications are a major cause of morbidity and early mortality; therefore, early detection and treatment of lipoprotein-related disorders may facilitate early recognition and treatment of diabetic patients .
This study was carried out to determine the PON-1 activity in type 2 diabetic patients with and without nephropathy and its correlation with the lipid profile, disease duration, and glycemic status.
| Patients and methods|| |
This study was carried out on 30 patients with type 2 diabetes mellitus who had attended the diabetes and endocrine clinic at Kasr Al Ainy Hospital, Cairo University, and 15 healthy age-matched control participants. The study was carried out from January 2014 to June 2014. Diabetes and diabetic complications were defined according to WHO 1999 . All patients had been diagnosed with diabetes mellitus for at least 5 years. Diabetic patients were subdivided into 20 with diabetic nephropathy and 10 with no evidence of nephropathy. All patients were subjected to an assessment of history, clinical examination, retinal examination, ECG, urine analysis, serum creatinine, fasting blood sugar, 2 h postprandial blood glucose, total cholesterol (TC), triglycerides (TAG), LDL-cholesterol (LDL-C), HDL-cholesterol (HDL-C), and determination of estimated glomerular filtration rate. Determination of serum PON-1 activity was performed using the calorimetric method.
Patients receiving renal replacement therapy, kidney transplant recipients as well as those with liver disease, congestive heart failure, and cerebrovascular disorders were excluded.
Research protocols were approved by the medical ethics committee of Kasr Al Ainy Medical School, Cairo University. All participants provided a written informed consent after the research protocols were carefully explained to them. Informed consent was obtained from all the study participants and their approval was obtained by a signature.
| Methods|| |
All laboratory investigations were performed on a Roche Diagnostics Hitachi 917 automated analyzer. LDL-C was calculated using the method of Friedewald et al. . Urinary albumin excretion was calculated as 24 h urine collection. Levels of albuminuria lower than 30 mg/24 h were reported to be negative, levels of 30-300 mg/24 h were reported as microalbuminuria, and levels above 300 mg/24 h were reported as macroalbuminuria. Estimated glomerular filtration rate was determined on the basis of the MDRD 4-variable equation .
Paraoxoase activity in enzymatic assay was assessed on the basis of the method described by Sampson et al. . As recommended by Abbott et al.  before the analysis of PON-1 activity, serum from controls and diabetics was preincubated with 5 μmol/l serine for 10 min at room temperature to inhibit serum butyrylcholinesterease activity, which is elevated in diabetes and would otherwise interfere with the determination of PON-1 activity in the serum from individuals with diabetes.
Paraoxon (O,O-diethyl P-nitrophenyl phosphate; 1.0 mmol/l; Sigma chemical Co., St Louis, Missouri, USA) was used as a substrate, and enzyme activity was measured at 37°C in 50 mmol/l Tris/HCL buffer (pH 6.8) containing 1.0 mmol/l CaCl 2 . The rate of P-nitrophenol generation was determined on a spectrophotometer RA50 (Bayer, Pittsburgh, Pennsylvania, USA) by recording the increase in A405 at 37°C.
Data were analyzed using the statistical package of social science software program (SPSS Inc., Chicago, Illinois, USA), version 21.
Data were summarized as range, mean, SD, and median for quantitative variables or frequency and percentage for qualitative variables.
Comparison between groups was performed using one-way analysis of variance (if parametric) or Mann-Whitney and Kruskal-Wallis tests (if nonparametric) for quantitative variables and χ2 with Fisher's exact test for qualitative variables.
Spearman correlation coefficients were calculated to signify the association between different parametric quantitative variables and Pearson's test for nonparametric variables.
Receiver operating characteristics curve analyses were carried out to explore the discriminant ability of PON-1 activity to differentiate patients with diabetes mellitus.
P values less than 0.05 were considered statistically significant.
| Results|| |
The demographic and laboratory data of the studied groups are shown in [Table 1]. Using the analysis of variance test, paroxonase-1 activity was significantly lower in both diabetic patients groups compared with nondiabetic controls (P < 0.001) [Table 1] and [Figure 1]. There was no significant difference between paroxonase activity levels among those with and without nephropathy. TC and TAG were elevated significantly in the diabetic nephropathy group compared with the other groups (P < 0.001) but HDL-C was not significantly different between the three groups (P = 0.4) [Table 1]. Paroxonase activity was not correlated to any of the parameters measured apart from the HDL levels in patients with diabetic nephropathy [Table 2]. [Table 3] and [Figure 2] show the cut-off point level of PON-1 activity to discriminate between diabetic patients and nondiabetic controls.
|Figure 1: Box plot showing the distribution of paraoxonase-1 activity among the studied gro ups.|
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|Figure 2: ROC curve analysis to explore the ability of paraoxonase-1 activity to discriminate diabetic cases from controls. ROC, receiver operating characteris tic.|
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|Table 2: Correlation of PON-1 activity with different parameters within each group|
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| Discussion|| |
In our study, the mean PON-1 activity was significantly decreased in the diabetic patients with nephropathy and the diabetic patients without nephropathy compared with the control participants. We found that the cut-off point up to 500 nmol/ml/min was the level of PON-1 activity to discriminate the diabetic patients from the control group using receiver operating characteristic curve analysis. These results were in agreement with previous studies that reported low PON-1 activity in type 2 diabetes ,,.
In contrast to these results, Valabhji et al.  did not report any difference in PON-1 activity in type 2 diabetes and controls and this may have been because of the technique used to detect PON-1 activity in that study, which used substrates such as phenyl acetate, which may not be the most appropriate substrate to assess PON-1 activity. Also, the study carried out by Kopporasch et al.  did not find a significant difference in PON-1 activity in impaired glucose-tolerant patients and early diabetes compared with the controls, and they suggested that the decrease in PON-1 activity may occur later in the course of diabetes mellitus .
There may be several explanations for the decreased PON-1 activity in type 2 diabetes. This may have been because of the conformational changes of the enzyme as a result of glucoxidation or lipoxidation . Ferretti et al.  found that in-vitro incubation of HDL with glucose decreased the activity of HDL-associated paraoxanase; thus, glycated HDL leads to physiochemical modification in the properties of HDL. Also, Hedrick et al.  found that in-vitro glycation of purified paraoxanase protein might cause a 40% reduction in its enzymatic activity and Deakin et al.  found that the decrease in the size of HDL particles and the accumulation of unesterified cholesterol in the HDL particles that occur in diabetes may affect the ability of HDL to release PON-1 from cells and may also affect its stabilization. These decreases in PON-1 activity prevent the antioxidant function of HDL and accelerate atherogenesis and related complications .
In our study, there was no significant difference in PON-1 activity between diabetics with or without nephropathy. This was in agreement with Ikeda et al. , who did not report differences in PON-1 activity in type 2 diabetes with and without nephropathy, although there was a significant difference between patients with and without retinopathy. This was in contrast to the previous two studies ,, which found a significant decrease in diabetic patients with nephropathy compared with diabetics without nephropathy. In the study by Khodeir and colleagues, the patients with diabetic nephropathy were older and had a longer duration of diabetes compared with diabetic patients without nephropathy. In the other study, the duration of diabetes was not reported and this may have been a drawback that could have affected the results of that study. In our study, the mean duration of diabetes in patients with and without nephropathy was longer, 9.7 ± 4.2 years, and was not significantly different between both groups, which may explain the similarity in PON-1 activity among patients in the two groups.
Seres et al.  reported a progressive decrease in PON-1 activity in elderly patients and this decrease was related to the occurrence of oxidative stress that develops with aging. In our study, there was no significant difference in PON-1 activity in terms of age; this may be related to our selected participants, who were in the middle age group 50 ± 3 years, and this was in agreement with Khodeir et al. .
In our study, we found a negative correlation between duration of diabetes in patients with nephropathy and PON-1 activity, but this was not significant. Also, we found a nonsignificant negative correlation between fasting blood sugar and 2 h postprandial blood glucose and PON-1 activity in diabetic patients without nephropathy, and this was in agreement with Elattar et al. . In contrast to these results, Kordonouri et al.  reported a negative correlation between blood glucose levels and PON-1 activity.
In our study, there were no significant correlations between TC, TAG, LDL-C, and PON-1 activity in any of the groups and this was in agreement with Elattar et al. , who also did not find any significant correlations in the lipid profile and PON-1 activity in both the control and the diabetic group.
However, we found a negative correlation between HDL and PON-1 activity in the diabetic patients with nephropathy. The relationship between PON-1 and lipids has shown conflicting results in different studies. Some studies have shown a positive correlation ,,, whereas others have found no correlation . This may be explained by the association of PON-1 with HDL particles  and the consequent impact of HDL levels  and drug intake, for example statins on the levels of PON-1 . Moreover, the PON-1 genotype is a major determinant of serum lipids and lipoprotein concentrations . Nonetheless, Mackness et al.  have suggested that PON-1 is associated exclusively with a discrete subpopulation of HDL particles, namely, apo-A1 and clusterin, which explains the poor correlation between PON-1 and HDL found in the population studies.
| Conclusion|| |
Our study supports previous observations that type 2 diabetes mellitus patients have significantly decreased PON-1 activity, which did not differ between diabetic patients with and without nephropathy.
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
| References|| |
American diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 2009; 32:S26-S67.
Baynes JW. Role of oxidative stress in development of complications in diabetes. Diabetes 1991; 40:405-412.
Dihazi H, Müller GA, Lindner S, Meyer M, Asif AR, Oellerich M, Strutz F. Characterization of diabetic nephropathy by urinary proteomic analysis: identification of a processed ubiquitin form as a differentially excreted protein in diabetic nephropathy patients. Clin Chem 2007; 53:1636-1645.
Prabhakar S, Starnes J, Shi S, Lonis B, Tran R. Diabetic nephropathy is associated with oxidative stress and decreased renal nitric oxide production. J Am Soc Nephrol 2007; 18:2945-2952.
Ha H, Kim KH. Pathogenesis of diabetic nephropathy: the role of oxidative stress and protein kinase C. Diabetes Res Clin Pract 1999; 45:147-151.
Li HL, Liu DP, Liang CC. Paraoxonase gene polymorphisms, oxidative stress, and diseases. J Mol Med (Berl) 2003; 81:766-779.
Rani JA, Mythili SV. Paraoxonase 1 in type 2 diabetes mellitus - a review. Int J Pharm Sci Rev Res 2014; 28:175-179.
Costa LG, Cole TB, Jarvik GP, Furlong CE. Functional genomic of the paraoxonase (PON1) polymorphisms: effects on pesticide sensitivity, cardiovascular disease, and drug metabolism. Annu Rev Med 2003; 54:371-392.
Mackness B, Davies GK, Turkie W, Lee E, Roberts DH, Hill E, et al
. Paraoxonase status in coronary heart disease: are activity and concentration more important than genotype?. Arterioscler Thromb Vasc Biol 2001; 21:1451-1457.
Seres I, Paragh G, Deschene E, Fulop T Jr, Khalil A. Study of factors influencing the decreased HDL associated PON1 activity with aging. Exp Gerontol 2004; 39:59-66.
Jenkins AJ, Best JD, Klein RL, Lyons TJ. Lipoproteins, glycoxidation and diabetic angiopathy. Diabetes Metab Res Rev 2004; 20:349-368
World Health Organization. Definition, diagnosis and classification of diabetes mellitus and its complications, 1999
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; 18:499-502.
National Kidney Foundation. K/DOQI clinical practice guidelines for chronic kidney disease: evaluation, classification, and stratification. Am J Kidney Dis 2002; 39(Suppl 1): S1-S266.
Sampson MJ, Braschi S, Willis G, Astley SB. Paraoxonase-1 (PON1) genotype and activity and in vivo oxidized plasma low-density lipoprotein in type II diabetes. Clin Sci (Lond) 2005; 109:189-189.
Abbott CA, Mackness MI, Kumar S, Boulton AJ, Durrington PN. Serum paraoxonase activity, concentration, and phenotype distribution in diabetes mellitus and its relationship to serum lipids and lipoproteins. Arterioscler Thromb Vasc Biol 1995; 15:1812-1818.
Mackness B, Durrington PN, Abuashia B, Boulton AJ, Mackness MI. Low paraoxonase activity in type II diabetes mellitus complicated by retinopathy. Clin Sci (Lond) 2000; 98:355-363.
Karabina SA, Lehner AN, Frank E, Parthasarathy S, Santanam N. Oxidative inactivation of paraoxonase - implications in diabetes mellitus and atherosclerosis. Biochim Biophys Acta 2005; 1725:213-221.
Valabhji J, McColl AJ, Schachter M, Dhanjil S, Richmond W, Elkeles RS. High-density lipoprotein composition and paraoxonase activity in type I diabetes. Clin Sci (Lond) 2001; 101:659-670.
Kopprasch S, Pietzsch J, Kuhlisch E, Graessler J. Lack of association between serum paraoxonase 1 activities and increased oxidized low-density lipoprotein levels in impaired glucose tolerance and newly diagnosed diabetes mellitus. J Clin Endocrinol Metab 2003; 88: 1711-1716.
Baynes JW, Thorpe SR. Glycoxidation and lipoxidation in atherogenesis. Free Radic Biol Med 2000; 28:1708-1716.
Ferretti G, Bacchetti T, Marchionni C, Caldarelli L, Curatola G. Effect of glycation of high density lipoproteins on their physicochemical properties and on paraoxonase activity. Acta Diabetol 2001; 38:163-169.
Hedrick CC, Thorpe SR, Fu MX, Harper CM, Yoo J, Kim SM, et al
. Glycation impairs high-density lipoprotein function. Diabetologia 2000; 43:312-320.
Deakin S, Leviev I, Gomaraschi M, Calabresi L, Franceschini G, James RW. Enzymatically active paraoxonase-1 is located at the external membrane of producing cells and released by a high affinity, saturable, desorption mechanism. J Biol Chem 2002; 277:4301-4308.
Ikeda Y, Suehiro T, Inoue M, Nakauchi Y, Morita T, Arii K, et al.
Serum paraoxonase activity and its relationship to diabetic complications in patients with non-insulin-dependent diabetes mellitus. Metabolism 1998; 47:598-602.
Khodeir SA, Abd El Raouf YM, Amer AE, El Fadaly NH, Abd El Latif EA. Paraoxonase gene polymorphism and activity in type 2 diabetes mellitus with microvascular complications. J Am Sci 2012; 8 :27-34.
Vanitha Gowda MN, Kusuma KS, Vasudha KC. Serum paraoxonase (Arylesterase) activity in type 2 diabetes mellitus and diabetic nephropathy. Indian J Appl Res 2013; 3 :351-353.
Elattar N, Swelam EE, Hamed E, Elnahal A, Mostafa E. Paraoxonase 1 gene polymorphism relationship with type 2 diabetes mellitus. Life Sci J 2012; 9:1742-1751.
Kordonouri O, James RW, Bennetts B, Chan A, Kao YL, Danne T, et al
. Modulation by blood glucose levels of activity and concentration of paraoxonase in young patients with type 1 diabetes mellitus. Metabolism 2001; 50:657-660.
Saha N, Roy AC, Teo SH, Tay JS, Ratnam SS. Influence of serum paraoxonase polymorphism on serum lipids and apolipoproteins. Clin Genet 1991; 40:277-282.
Deepthi SK, G, Amar Raghu Narayan. Paraoxonase activity and its concentration in type 2 diabetes mellitus. Int J Pharm Bio Sci 2012; 3:969-973.
Patil Asmita B, Ganu Jayashree V. Paraoxonase 1, total cholesterol and HDL cholesterol in diabetes mellitus. Indian J Basic Appl Med Res 2013; 2:998-1001.
Tomas M, Latorre G, Senti M, Marrugat J. The antioxidant function of high density lipoproteins: a new paradigm in atherosclerosis. Rev Esp Cardiol 2004; 57:557-569.
Kural BV, Orem C, Uydu HA, Alver A, Orem A. The effects of lipid-lowering therapy on paraoxonase activities and their relationships with the oxidant-antioxidant system in patients with dyslipidemia. Coron Artery Dis 2004; 15:277-283.
Hegele RA, Brunt JH, Connelly PW. A polymorphism of the paraoxonase gene associated with variation in plasma lipoproteins in a genetic isolate. Arterioscler Thromb Vasc Biol 1995; 15:89-95.
Mackness B, Durrington PN, Boulton AJ, Hine D, Mackness MI. Serum paraoxonase activity in patients with type 1 diabetes compared to healthy controls. Eur J Clin Invest 2002; 32:259-264.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]