Postprandial hypertriglyceridemia and oxidative stress in patients of type 2 diabetes mellitus with macrovascular complications

Clinica Chimica Acta 359 (2005) 101 – 108 www.elsevier.com/locate/clinchim

Postprandial hypertriglyceridemia and oxidative stress in patients of type 2 diabetes mellitus with macrovascular complicationsB Ritu Saxenaa, Sri Venkata Madhub, Rimi Shuklaa, Keshav M. Prabhua, Jasvinder K. Gambhir a,* a

Department of Biochemistry, University College of Medical Sciences and Guru Teg Bahadur Hospital, Shahdara, Delhi-110095, India b Department of Medicine, University College of Medical Sciences and Guru Teg Bahadur Hospital, Shahdara, Delhi-110095, India Received 7 February 2005; received in revised form 11 March 2005; accepted 11 March 2005 Available online 12 May 2005

Abstract Background: Oxidative stress has been implicated in vascular complications of diabetes mellitus (DM). This study aims to evaluate the relationship between postprandial hypertriglyceridemia (PP-HTG) and oxidative stress in Indian patients of type 2 DM with macrovascular complications. Methods: Plasma triglycerides (TG), thiobarbituric acid reactive substances (TBARS), erythrocyte reduced glutathione (GSH) and superoxide dismutase (SOD) were measured in fasting and postprandial (PP) state at 2, 4, 6 and 8 h after a high fat meal challenge in controls (Group I) and patients of type 2 DM without (Group II) and with macrovascular complications (Group III). Results: Postprandial TGs increased significantly in patients with type 2 DM, which showed an exaggerated response to high fat meal challenge in Group III as compared to Group II. Highest PP-TBARS were also observed in Group III which correlated positively with TG. However, GSH and SOD were lower in both groups of diabetics as compared to controls. Conclusions: The magnitude of PP-HTG appears to be the major determinant of oxidative stress in type 2 DM, which along with a compromised antioxidant status may lead to endothelial dysfunction and macrovascular complications. D 2005 Elsevier B.V. All rights reserved. Keywords: Oxidative stress; Type 2 diabetes mellitus; Macrovascular complications; Postprandial hypertriglyceridemia; High fat meal challenge

1. Introduction Abbreviations: AUC, area under curve; DM, diabetes mellitus; GSH, reduced glutathione; HTG, hypertriglyceridemia; PP, postprandial; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances; TRL’s, triglyceride-rich lipoproteins. B All the authors have seen the final manuscript and there is no conflict of interest. * Corresponding author. Fax: +91 11 22590495. E-mail address: [email protected] (J.K. Gambhir). 0009-8981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2005.03.036

Macrovascular complications, which manifest in about 80 percent of patients with type 2 diabetes mellitus (type 2 DM), are a leading cause of morbidity and mortality worldwide [1,2]. Hyperglycemia, hyperinsulinemia and dyslipidemia in diabetic patients have been implicated in the development of macroangiopathies which possibly act by their ability to induce

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oxidative stress, leading to endothelial dysfunction and atherosclerosis. The typical secondary dyslipidemia of DM is characterized by increased concentration of total triglycerides (TG), very low density lipoproteins (VLDL) and decreased levels of high density lipoprotein (HDL) [3,4]. After a high fat meal, the increased influx of exogenous triglyceride-rich lipoproteins (TRL’s) into plasma inhibits the clearance of such lipoproteins of endogenous origin. Further, an increase in the secretion of TRL’s of endogenous origin inhibits the removal of newly absorbed exogenous TRL’s from plasma. The net effect of all these processes would be to accentuate the magnitude of postprandial lipemia (PPL) [5–8]. Thus, PPL represents the post absorptive state during which the capacity to metabolize TG is under challenge [9]. Further, the postmeal TG enrichment of VLDL (VLDL-TG) may lead to increased production of lipid derived free radicals and reactive oxygen species (ROS). When the generation of reactive oxygen species exceeds the availability of antioxidant defense mechanisms, as seen in DM, oxidative stress ensues. Accordingly, some markers of oxidative stress like plasma malondialdehyde (MDA) have been found to be increased in diabetic patients in the postmeal state [10]. A number of studies have evaluated the role of oxidative stress in microvascular and macrovascular complications of diabetes in the fasting state. However, there is paucity of such data in the postprandial state, especially in Indian diabetic patients with evidence of macroangiopathy. Hence, the present study has been carried out to address the relationship between postprandial TG levels and selected parameters of oxidative stress in Indian patients of type 2 DM with macrovascular complications.

2. Methods The study was conducted in 13 patients of type 2 DM with macrovascular complications (Group III) and 13 patients without macrovascular complications (Group II), recruited from the Medical OPD and Diabetic Clinic of Guru Teg Bahadur Hospital along with 13 healthy controls (Group I). All patients were N30 years with diagnosed diabetes of at least 1 year duration. Diagnosis of diabetes was made according to revised American Diabetes Association Criteria [11].

The group with macrovascular complications included patients with coronary artery disease (CAD), peripheral vascular disease (PVD) and cerebrovascular disease (CVD). Coronary artery disease was defined on the basis of history, clinical examination and ECG findings or positive treadmill test or positive coronary angiography. Peripheral vascular disease was defined on the basis of history, clinical examination and ankle brachial index (ABI) determined by handheld vascular Doppler (ABI V 0.90 was taken as evidence of PVD). Cerebrovascular disease was defined by the history of proven stroke. The diabetic patients had no evidence of acute infection, hepatic or renal disease and hypercholesterolemia (fasting cholesterol N250 mg/ dl). Subjects taking drugs affecting lipid metabolism, antioxidants, insulin and supplemental vitamins were also excluded. The protocol of the study was approved by the Institutional Ethical Committee and all participants gave informed written consent before being tested. Studies began at 9 AM, after a 12–14 h overnight fast. Fasting blood samples were drawn in EDTA vials (1 mg/ ml) and plasma was separated immediately by centrifugation and stored at 20 8C until analysis. Whole blood and washed erythrocytes were used for estimation of erythrocyte GSH content and SOD activity, respectively. Following this, the study subjects were given a standardized high fat meal in the form of whipped cream with sugar and fruit. The total energy content of the meal was 729 kcal/m2 body surface area, with 5.3 gm protein, 24.75 gm carbohydrate, 240 mg cholesterol and 65.2 gm fat/m2 body surface area with a polyunsaturated to saturated fat ratio of 0.06. All parameters evaluated at baseline were repeated serially at 2, 4, 6 and 8 h after the test meal. Plasma glucose, TG, total and high density lipoprotein cholesterol (HDL-C) were estimated on Synchron CX-4 autoanalyzer by enzymatic methods using kits from Accurex Biomedical Private Limited, Mumbai. Low density lipoprotein cholesterol (LDL-C) was calculated by Friedwald and Fredrickson’s formula. Glycosylated hemoglobin (HbA1c) was determined by ionexchange chromatography as described by Goldstein et al. [12] using kits from ERBA Test. Serum thiobarbituric acid reactive substances (TBARS) were measured as an index of lipid peroxidation using the colorimetric method as described by Satoh [13]. Reduced glutathione was estimated by the method

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of Beutler et al. [14]. The activity of erythrocyte SOD was measured by the method of Marklund and Marklund [15] as modified by Nandi and Chatterjee [16].

3. Statistical analysis Results are given as mean F S.D. For a desired p value of 0.05 and 80% power to detect an actual difference, a sample size of 10 subjects per group has been considered satisfactory as reported by Dawson-Saunders et al. [17]. The significance of difference was determined using analysis of variance (ANOVA-F) test followed by post hoc Tukey’s test for multiple comparisons. Correlation between variables was tested using Pearson’s correlation analysis. A p b 0.05 was considered statistically significant.

4. Results 4.1. Nondiabetic controls Age and gender matched healthy controls (Group I) showed BMI, WHR, fasting and 2 h postprandial Table 1 Baseline characteristics of the study groups Parameter

Group I

Group II

Group III

Age (years) Gender (M/F) BMI (kg/m2) WHR Duration of DM (years) Fasting glucose (mg/dl) 2 h Postprandial glucose (mg/dl) HbA1c(g%) Total cholesterol (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl)

50.6 F 8.9 3/10 23.4 F 2.0 0.83 F 0.03 –

54.6 F 7.4 3/10 22.9 F 3.3 0.94 F 0.08a 4.62 F 3.64

54.5 F 9.5 3/10 23.5 F 2.3 0.95 F 0.08a 7.31 F 5.38

85.2 F 10.1

145.9 F 46.2a

157.1 F 45.3a

227 F 74.9a

250.0 F 41.2a

113.8 F 9.0 6.2 F 0.8 151.5 F 11.6

8.5 F 1.6a 158.2 F 15.9

8.6 F 1.9a 188.6 F 39.9

46.9 F 5.5 85.4 F 10.8

39.0 F 7.2 101.7 F 17.2

34.0 F 3.5a 125.5 F 38.0a

Data are presented as mean F S.D.; n = 13. Group I: controls; Group II and Group III: diabetes without and with macroangiopathy, respectively. BMI = body mass index; HbA1c = glycosylated hemoglobin; WHR = waist–hip ratio; HDL-C = high density lipoprotein cholesterol; LDL-C = low density lipoprotein cholesterol. a p b 0.05 vs. Group I.

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Table 2 Area under the curve (AUC) for plasma glucose and lipid profile parameters in different study groups Parameter

Group I

Group II

Group III

Glucose TC HDL-C LDL-C TG

466.0 F 42.4 1318.5 F 99.3 395.8 F 36.2 743.6 F 90.8 901.3 F 152.5

817.0 F 267.0 1453 F 159.1 313.9 F 56.6a 874.6 F 148.8 1409.3 F 364.4

789.0 F 245.0a 1694 F 352.2a 284.4 F 27.8a 988.8 F 308.7a 2114.2 F 1392.8a

Values are mean F S.D. for AUC between 0 and 8 h. a p b 0.05 vs. Group I.

plasma glucose, glycosylated hemoglobin and lipid profile parameters were within the normal range (Table 1). Following high fat meal challenge total cholesterol at 4 and 6 h, and HDL-C at all time points showed a small but significant increase as a function of baseline level ( p b 0.05). No significant change in LDL-C was seen in postmeal state. Overall changes in different parameters from 0 to 8 h (AUC) are shown in Table 2. There was a slight and insignificant increase in serum triglyceride levels which peaked at 4 h (Table 3). Plasma TBARS increased significantly ( p b 0.05) from fasting to postmeal state which peaked at 4–6 h and declined at 8 h (Table 3), suggesting that there is a slight meal generated oxidative stress in healthy subjects. Similar trend with significant postprandial increase in GSH as a function of baseline was also seen ( p b 0.05), whereas there was no significant change in the activity of SOD post-meal (Table 4). 4.2. Diabetic subjects The two groups of diabetics, Group II and Group III, were matched for age, sex and BMI (Table 1). Mean duration of DM was higher in Group III as compared to Group II; however, the difference was not significant statistically. WHR in both groups of diabetics were significantly higher as compared to healthy controls ( p b 0.001), suggesting that diabetics had truncal obesity. Similarly, glycosylated hemoglobin was also significantly higher in both groups of diabetics ( p b 0.05) as compared to controls. The baseline glucose and lipid profile parameters in study groups and AUC (0–8 h) are shown in Tables 1 and 2, respectively. The fasting as well as postprandial glucose concentration in diabetics was significantly higher as compared to controls ( p b 0.05). The percent increase in blood glucose from basal to

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Table 3 Baseline and postprandial plasma triglyceride and TBARS levels in different study groups Hours

0 2 4 6 8 Increase from basal to peak (%)

Group I

Group II

Group III

TG (mg/dl)

TBARS (nmol/ml)

TG (mg/dl)

TBARS (nmol/ml)

TG (mg/dl)

TBARS (nmol/ml)

95.8 F 22.4 106.5 F 16.3 121.5 F 20.2 121.4 F 25.4 106.4 F 17.4 26.8

2.86 F 0.41 3.18 F 0.48c 3.36 F 0.52c 3.36 F 0.59c 3.19 F 0.52c 17.5

96.2 F 27.6 150.7 F 39.7a,c 204.3 F 60.2a,c 206.0 F 66.4a,c 190.0 F 70.6a,c 114.1

3.03 F 0.96 3.89 F 1.37c 4.10 F 1.46c 3.85 F 1.14c 3.76 F 1.11 35.3

145.0 F 85.3 208.6 F 138.6c 282.3 F 166.6 c 346.5 F 248.0c 294.1 F 249.2c 139.0

3.97 F 0.96a,b 5.16 F 1.08a,b,c 5.48 F 1.38a,b,c 5.42 F 1.38a,b,c 4.95 F 1.40a,b,c 38.1

Data are presented as mean F S.D.; n = 13; p b 0.05, a vs. Group I, b Group II vs. Group III, and c baseline vs. postmeal. Group I: controls; Group II and Group III: diabetics without and with macroangiopathy. TBARS = thiobarbituric acid reactive substances; TG = triglycerides.

peak level (4 h) was 23% in controls as compared to 41% and 39% in Group II and Group III, respectively. Total cholesterol rose significantly in both diabetic groups with peak at 4 h ( p b 0.05 as compared to baseline). Similar trend was seen in HDL-C and LDL-C with marginal increases in the postprandial phase (total change shown as AUC in Table 2). However, fasting as well as postmeal HDL-C levels were significantly lower in Group III as compared to healthy controls ( p b 0.05). Plasma triglycerides showed a peak at 4–6 h in Group II and at 6 h in Group III with a mean percent increase of 114% and 139% in Group II and III, respectively, which remained elevated until 8 h in contrast to healthy subjects in which triglycerides returned to near normal levels by this time (Table 3; Fig. 1). Postprandial increase in plasma TG is determined by fasting levels; however, there was a variable response in Group III subjects, since 4 patients from this group with normal fasting TG also developed postprandial hypertriglyceridemia. These results suggest that fasting TG levels do not always predict the derangements during postprandial phase. Baseline TBARS levels were significantly higher in Group III as compared to Group II and Group I (Table 3).

Plasma TBARS peaked at 4 h postmeal in all the groups, the increase being significant at all time points ( p b 0.05) though the percent rise among diabetics was higher at peak (38.03% in Group III and 35.3% in Group II) as compared to controls (17.48%). When Group III patients were divided according to fasting TG, 4 out of 13 patients, which showed fasting plasma TG level N 200 mg/dl (251.0 F 60.8 mg/dl), also had higher fasting MDA levels (4.6 F 0.74 nmol/ml) as compared to the group with fasting TG b200 mg/dl (3.68 F 0.84 nmol/ml). The former group also showed more pronounced rise in MDA in the postprandial state with a higher peak (6.68 F 1.18 nmol/ml) as compared to the latter group (4.94 F 0.98 nmol/ml) (Fig. 1). Moreover, a positive correlation was also found between MDA and TG levels in all study groups at all time points (Fig. 2; p b 0.05; r = 0.337). Erythrocyte GSH levels were significantly lower ( p b 0.01) in diabetics in the fasting state as compared to controls (Table 4). Though a statistically significant rise in GSH was seen in postprandial state as compared to baseline ( p b 0.05) in all three groups, the levels remained lower in diabetics than the control group at all time points. Red Cell SOD activity was lower in diabetics in the fasting state as compared to

Table 4 Levels of antioxidants in different study groups Parameter Erythrocyte GSH (mg/g Hb)

SOD (U/g Hb)

Hours 0 4 6 0

Group I 3.44 F 0.52 3.94 F 0.43c 3.72 F 0.44c 1127.0 F 193.0

Group II

Group III a

2.54 F 0.67 3.11 F 0.59c 3.13 F 0.62c 941.5 F 152.3

2.33 F 0.69a 2.87 F 0.77c 3.00 F 0.76c 846.3 F 147.6a

Data are presented as mean F S.D.; n = 13, a p b 0.05, vs. Group I, c p b 0.05, baseline vs. postmeal. Group I: controls; Group II and Group III: diabetics without and with macroangiopathy, respectively. GSH = reduced glutathione; SOD = superoxide dismutase.

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800

8

700

7

600

6

500

5

400

4

300

3

200

2

100

1

0

TBARS (nmol/ml)

TG (mg/dl)

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0 0

2

4

6

8

Time (hrs) Fasting TG (<200)

Fasting TG (>200)

TBARS

TBARS

Fig. 1. Plasma triglycerides (mg/dl) and TBARS (nmol/ml) at different time points (h) in type 2 diabetics with macroangiopathy (Group III) with fasting TG b and N 200 mg/dl (mean F S.E.) [ p b 0.05].

controls (Table 3) which did not change significantly in the postprandial phase (data now shown).

5. Discussion There is growing evidence suggesting the role of increased oxidative stress in the etiology of macrovascular complications in diabetes mellitus [18,19]. According to the NCEP ATP-III guidelines, diabetes

has been classified as a coronary heart disease (CHD) risk equivalent [20]. Hyperglycemia and characteristic dyslipidemia of DM along with increased oxidative stress leading to endothelial dysfunction have been implicated as early events in the pathogenesis of atherothrombotic macrovascular disease [9]. Most of this information is available in fasting individuals, therefore, the present study has evaluated the relationship between postprandial abnormalities in the lipid metabolism, especially hypertriglyceridemia and

9 Y=0.0058+2.9454 R2=0.3368

8

TBARS (nmol/ml)

7 6 5 4 3 Group 3 Group 2 Group 1 line

2 1 0 0

200

400

600

800

1000

TG (mg/dl) Fig. 2. Correlation between plasma triglycerides and TBARS in three study groups (n = 13 each) at all time points ( y = 0.0058x + 2.945, r = 0.336, p b 0.05).

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increased oxidative stress vis-a-vis development of diabetic macrovascular complications in Indian ethnic population. Our results suggest that there is increased oxidative stress in the postprandial state, which is of higher magnitude and longer lasting in diabetics, more so in the group with macrovascular complications. Moreover, there are unfavorable changes in lipid profile especially lower HDL-C and persistent postprandial hypertriglyceridemia in diabetics which correlated with postmeal oxidative stress. Secondary dyslipidemia frequently observed in diabetics is characterized by triglyceride enrichment of VLDL in the postprandial phase, which along with low HDL-C and coexisting hyperglycemia is strongly associated with the risk of development of atherosclerotic lesions [21]. In addition TG-rich lipoproteins can influence endothelial function indirectly by changing the phenotype of the LDL towards small and dense LDL particles which have higher atherogenicity [22]. An acute increase in PPL along with increased influx of free fatty acids and co-existing oxidative stress often seen in diabetics causes damage to the endothelium. This along with lowered antioxidant defenses (GSH, SOD, vitamin E) and HDL-C levels in diabetics may play an important role in the development of diabetic macrovascular complications. The higher concentration of TBARS in diabetics even in the fasting state in our study is consistent with previous reports [23,24] and gives an assessment of existing oxidative stress with end product accumulation. In healthy controls, as well as in Group II diabetics, there was a gradual increase in TBARS postmeal which was significant as compared to baseline values, while in Group III, the exaggerated postprandial rise in TBARS seemed to be a consequence of a rapid and steep increase in TG which may have resulted in an acute oxidative milieu. In addition more pronounced depletion of antioxidants led to very high TBARS, more so in individuals with high fasting TG levels (Fig. 1). There is in vitro data showing that oxidative damage caused by lipid components in remnant rich lipoproteins leads to deterioration of cell surface membranes and may be partly responsible for remnant induced impairment of endothelium dependent relaxation [25]. The extracellularly generated O2S and H2O2 have been shown to traverse erythrocyte membranes [26]

and circulating human erythrocytes possess the ability to scavenge these extracellularly generated ROS [26,27]. In general, oxidative injury occurs when endogenous antioxidant mechanisms are unable to balance the rate of production of free radicals [28]. Increased availability of lipid substrates especially in PPL of diabetes leads to increased lipid peroxidation as evidenced by the higher levels of TBARS in DM groups in our study. In addition, we have also observed lower activity of SOD in diabetics. Both increased [29] and decreased [30–32] activities of SOD have been reported in diabetics. Decreased activity of SOD and other related antioxidant systems has been linked to progressive glycation of enzyme proteins in diabetes [33]. In addition, the lower baseline levels of GSH observed in diabetics further reflect the enhanced proxidant milieu. Lower intracellular GSH is a common finding in DM [34]; important causes being the shunting of glucose through polyol pathway leading to depletion of NADPH which reduces glutathione-redox cycle [35]. GSH is the major intracellular antioxidant and vitamin E is the major lipid soluble antioxidant; GSH also helps in the regeneration of other antioxidants like ascorbate and vitamin E [36]. Thus depletion of GSH further impairs the activity of antioxidant defense mechanisms like vitamins C and E. Vitamin E markedly reduces the susceptibility of isolated LDL to oxidation and inhibits the secretion of pro inflammatory cytokines [37]. Infusion of GSH has also been shown to prevent the increase in the plasma intercellular adhesion molecule-1 (ICAM-1) levels in the postprandial state in the humans [38]. Thus an oxidative mechanism seems to mediate the atherogenic effects of meals in general and high fat meals in particular. This ensuing oxidative stress further adds on to the already existing endothelial injury among diabetics which may play an important role in the development of atherosclerotic macrovascular complications.

6. Conclusions The present study has demonstrated a significant increase in postprandial oxidative stress in type 2 DM patients after a high fat challenge particularly in those with macroangiopathy. This appears to be related to increased production of lipid derived free radicals that

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follow postprandial hypertriglyceridemia. Thus, postprandial oxidative stress seems to play an important role in development of vascular disease in general, and in diabetics in particular. Therefore, the screening of diabetic patients for postprandial abnormalities in lipid metabolism and oxidative stress will be beneficial in the prevention of diabetic vascular complications.

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