Fasting and Postprandial Glycoxidative and Lipoxidative Stress Are Increased in Women With Type 2 Diabetes

The present study was a substudy of a cross-sectional study to assess the effect and relative contributions of two consecutive fat-rich and carbohydrate-rich meals on markers of CVD risk in women with NGM (fasting glucose <6.1 mmol/l and 2-h postload glucose <7.8 mmol/l) (n = 76) and type 2 diabetes (n = 79). Women with type 2 diabetes (n = 522), recruited from the registry of the Diabetes Care System in Hoorn, the Netherlands, and women who were randomly selected from the municipal registry of Hoorn (n = 541), aged 50–65 years at the beginning of the study, were invited to participate in the study. Of these 1,063 women, 431 were complete nonresponders, 258 were not willing to participate, and 220 did not meet the inclusion criteria. Inclusion criteria were as follows: postmenopausal status, nonsmoking, no untreated endocrine disorder other than type 2 diabetes, no use of short-acting insulin analogs, no use of peroxisome proliferator–activated receptor α and γ agonists, no use of oral corticosteroids, no use of hormone replacement therapy, no statin use (for NGM subjects only), A1C <9.0%, fasting cholesterol <8.0 mmol/l, fasting triglycerides <4.0 mmol/l, systolic blood pressure <190 mmHg, and no liver impairment or renal impairment. For the present study, a subsample of 26 women with type 2 diabetes (no use of statins or insulin analogs) and 27 women with NGM, matched for age, were studied. All participants gave written informed consent, and the study protocol was approved by the ethics committee of the Vrije University Medical Center in Amsterdam, the Netherlands. The study consisted of a screening visit and two visits for the test meals. On the screening visit, fasting blood samples were drawn after a 12-h overnight fast. Blood pressure was measured thrice with an oscillometric blood pressure measuring device (Colin Press-Mate BP-8800; Colin, Komaki-City, Japan) after a 15-min supine rest. Weight and height were measured in participants wearing only light clothing. Smoking and alcohol consumption were assessed by a questionnaire (14).

Postprandial meal responses were examined following two standardized consecutive test meals (breakfast and lunch). On 1 day, both meals were high in fat content, and, on the other occasion, both meals were high in carbohydrate content. The test meal occasions were performed in random order. The fat-rich meals consisted of two croissants, 10 g butter, 40 g high-fat cheese, and 300 ml high-fat milk (3,349 kJ; 50 g fat; and 56 g carbohydrates). The carbohydrate-rich meals consisted of two slices of bread, 25 g marmalade, 30 g cooked chicken breast, 50 g ginger bread, and 300 ml drinking yogurt fortified with 45 g soluble carbohydrates (3,261 kJ; 4 g fat; and 162 g carbohydrates). Both meals were eaten within 10 min. Apart from the test meals and water (ad libitum), participants were not allowed to eat.

Plasma glucose and serum total cholesterol, HDL cholesterol, and triglycerides were measured by enzymatic colorimetric assays (Roche, Mannheim, Germany). Fasting and postprandial LDL cholesterol was calculated according to the Friedewald formula if triglycerides were <4.5 mmol/l (15). A1C was measured with cation-exchange chromatography (Menarini Diagnostics, Florence, Italy).

Oxidized LDL was measured in EDTA plasma in duplicate with a competitive enzyme-linked immunoabsorbant assay kit (Mercodia, Uppsala, Sweden) with interassay and intra-assay coefficients of variation (CVs) of 7.8 and 4.8%, respectively. Because a fairly strong correlation between oxidized LDL and LDL cholesterol is a consistent finding in several studies (16,17), we expressed oxidized LDL as a ratio per LDL cholesterol (oxidized LDL–to–LDL cholesterol ratio), as proposed by Holvoet et al. (18).

3DG was measured with a newly developed liquid chromatography tandem mass spectrometry method after precolumn derivatization, according to a published procedure (19). In brief, 0.5 ml whole blood was mixed with 1.0 ml of 1.2 mol/l perchloric acid and immediately stored at −80°C until assayed. After thawing, the samples were centrifuged (5 min at 20,000g), and 80 μl of the supernatant was mixed with 100 μl of the internal standard solution (1 μmol/l 2,3-pentanedione dissolved in ethanol). After the addition of 20 μl of the derivatization reagent (10 mmol/l 2,4-dinitrophenylhydrazine dissolved in 1.2 mol/l perchloric acid), the samples were incubated for 16 h at room temperature. Chromatographic separation was performed on a Xterra MS C18 column (4.6 × 50 mm, 3.5-μm particle size) (Waters, Milford, MA) using a linear gradient from 40 to 95% acetonitrile in water over 9 min at a flow rate of 1 ml/min. Mass transitions of 521.1→ 431.0 and 521.1→182.1 for 3DG and 459.1→182.1 for the internal standard were monitored in negative ion mode. Intra-assay and interassay CVs for 3DG were 8 and 12%, respectively. Two subjects with NGM had missing 3DG samples because of missing blood samples. In a pilot study applying the same study protocol, we assessed the time-course of 3DG in nine type 2 diabetic subjects and eight NGM subjects and measured 3DG with a high-performance liquid chromatography method with fluorescence detection, as previously described (20). The highest 3DG concentration was observed at 8 h after the first meal (21).

Unbound CML and CEL were measured in EDTA plasma by high-performance liquid chromatography with stable isotope dilution tandem mass spectrometric detection using a procedure developed for the analysis of protein-bound CML and CEL (22), with a modified sample preparation procedure. Briefly, after centrifugation of the plasma samples (5 min at 20,000g), 50 μl of the supernatant was mixed with 150 μl of a combined internal standard solution (containing deuterated CML and CEL), and proteins were removed by centrifugation (20 min at 15,000g) in a 10-kDa cutoff ultrafiltration device (Vivaspin 500 PES; Vivascience, Hannover, Germany). A 90-μl aliquot of the ultrafiltrate was mixed with 10 μl of a 50 mmol/l nonafluoropentanoic acid solution, and 25 μl of this mixture was subjected to liquid chromatography tandem mass spectrometry analysis, as described before (22). Intra-assay and interassay CVs for CML were 3 and 4%, respectively, and for CEL, 5 and 9%, respectively.

To assess the cumulative effect of both consecutive meals, we measured oxidized LDL, 3DG, CML, and CEL at baseline and at 8 h.

Analyses were performed with SPSS for Windows version 11.01 (SPSS, Chicago, IL). Data are presented as means ± SD, and, in case of a skewed distribution, as median (interquartile range). Data were ln-transformed before testing, in case of a skewed distribution. Differences between women with NGM and type 2 diabetes were tested with Student's t test for continuous variables and χ² test for dichotomous variables. Differences between meals were tested with paired-samples t tests. Postprandial changes in glucose and triglycerides were calculated as incremental areas under the curve (iAUCs) by the trapezoid method. Correlations between postprandial responses were calculated by Spearman's correlation coefficients. We considered two-sided P < 0.05 to indicate statistical significance.

The clinical and biochemical characteristics of the participants at the screening visit are presented in Table 1. Figure 1 illustrates the 8-h time courses of triglycerides and glucose after two consecutive fat-rich and two consecutive carbohydrate-rich meals. At both meal visits, fasting triglyceride and glucose levels were higher in women with type 2 diabetes compared with the women with NGM (both P < 0.001). The iAUC for triglycerides was higher following the fat-rich meals than after the carbohydrate-rich meals in both women with type 2 diabetes and NGM (both P < 0.001) but was similar in both groups. Following the carbohydrate-rich meals, the iAUC for glucose was higher in women with type 2 diabetes compared with NGM (P < 0.001), whereas this difference was borderline significant after the fat-rich meals (P = 0.06).

LDL cholesterol significantly decreased from baseline to 8 h in both groups and after both meal types (from 3.6 ± 0.8 to 3.2 ± 0.7 mmol/l and 3.7 ± 0.8 to 3.3 ± 0.7 mmol/l for fat-rich and carbohydrate-rich meals, respectively, in NGM; 3.3 ± 0.7 to 2.9 ± 0.7 mmol/l and 3.3 ± 0.7 to 3.0 ± 0.7 mmol/l, respectively, in type 2 diabetes; all P < 0.001).

The oxidized LDL–to–LDL cholesterol ratio, 3DG, CML, and CEL values in women with NGM and type 2 diabetes at baseline and at 8 h are listed in Table 2. Baseline oxidized LDL–to–LDL cholesterol ratio, 3DG, and CML were significantly higher in type 2 diabetes compared with NGM. The increase of the oxidized LDL–to–LDL cholesterol ratio during the fat-rich meals was higher than the increase during the carbohydrate-rich meals (17 ± 10 vs. 10 ± 7% in NGM and 15 ± 13 vs. 7 ± 9% in type 2 diabetes, both P < 0.05). Of interest, the fasting values of the oxidized LDL–to–LDL cholesterol ratio, 3DG, and CML in women with type 2 diabetes were similar to the postprandial values at 8 h in women with NGM (all P > 0.1).

The baseline oxidized LDL–to–LDL cholesterol ratio was correlated with fasting triglycerides in women with type 2 diabetes (r = 0.39, P < 0.05) but not in women with NGM. After the fat-rich meals, changes in the oxidized LDL–to–LDL cholesterol ratio correlated with changes in triglycerides, either expressed as triglyceride iAUC (r = 0.32, P < 0.05) (Fig. 2A) or as the increase over baseline at 8 h (r = 0.50, P < 0.001).

The mean increase of 3DG after the carbohydrate-rich meals was higher than after the fat-rich meals (29 ± 21 vs. 13 ± 24%, P < 0.05 in NGM and 36 ± 17 vs. 15 ± 20%, P < 0.001 in type 2 diabetes). Fasting plasma glucose concentration was correlated with baseline 3DG in women with type 2 diabetes (r = 0.50, P < 0.01) but not in women with NGM. Postprandial changes in 3DG correlated with the glucose iAUC after the fat-rich meals (r = 0.37, P < 0.01) (Fig. 2B) but not with the glucose iAUC after the carbohydrate-rich meals. The changes of CML and CEL neither correlated with the triglycerides iAUC nor with the glucose iAUC, irrespective of meal composition.

In the present cross-sectional study in postmenopausal women with NGM and type 2 diabetes, we studied the postprandial changes of the oxidized LDL–to–LCL cholesterol ratio, 3DG, CML, and CEL following two consecutive fat-rich or carbohydrate-rich meals. The main findings are that the fasting values of the oxidized LDL–to–LDL cholesterol ratio, 3DG, and CML are higher in women with type 2 diabetes compared with women with NGM. In women with type 2 diabetes, the fasting values of the oxidized LDL–to–LDL cholesterol ratio, 3DG, and CML were similar to the postprandial values at 8 h in women with NGM. Overall, the postprandial rises of the oxidized LDL–to–LDL cholesterol ratio and 3DG were of similar magnitude in type 2 diabetes and NGM. The oxidized LDL–to–LDL cholesterol ratio showed a greater increase after the fat-rich meals, whereas CML and 3DG increased more after the carbohydrate-rich meals. The increase in the oxidized LDL–to–LDL cholesterol ratio was correlated with the rise in postprandial triglycerides, whereas the increase in 3DG was correlated with the postprandial rise of glucose.

There is increasing evidence that postprandial glucose and triglycerides may play an important role in macro- and microvascular complications (23). However, their relative contributions and the mechanisms leading to the increased CVD risk are not yet fully understood (5). Prospective studies have shown that postprandial or postload glucose levels are more strongly associated with increased CVD than fasting glucose (24,25). Oxidative stress is hypothesized to be an important pathway linking postprandial glucose and triglyceride responses to CVD (26,27). Indeed, in a recent study, it was found that postprandial oxidized LDL was a determinant of the extent of coronary atherosclerosis in patients with CAD (13). A limitation of most previous studies is that responses after a single meal were studied, which might not reflect the “real-life” burden of the postprandial state, possibly leading to an underestimation of the associations between postprandial responses and CVD risk. A recent study from our group that applied two consecutive meals (breakfast and lunch) in healthy young men demonstrated that even slightly increased levels of triglycerides and glucose were associated with endothelial dysfunction and increased markers of oxidative stress, including oxidized LDL (12). We concluded that these findings might be relevant for insulin-resistant subjects and type 2 diabetic individuals who have a more pronounced (exaggerated and prolonged) postprandial elevation of glucose and triglycerides, possibly leading to more oxidative stress. Our present findings support this conjecture, as we found that baseline oxidized LDL was higher in type 2 diabetes than in NGM, with a greater increase of oxidized LDL after the fat-rich meals compared with the carbohydrate-rich meals. Interestingly, although the fasting oxidized LDL–to–LDL cholesterol ratio was higher in the type 2 diabetic group, we found no difference in the postprandial increase in the oxidized LDL–to–LDL cholesterol ratio between NGM and type 2 diabetes.

There are multiple sources and mechanisms in the formation of AGEs in vivo, involving both oxidative and nonoxidative reactions of reducing carbohydrates and other metabolic intermediates with amino acid residues (28). The AGE precursor 3DG, which is increased in patients with diabetes (29), is formed by nonoxidative modifications of Amadori adducts and from fructose-3-phosphate (30). In line with these observations, we found that 3DG increased more after the carbohydrate-rich meals than after the fat-rich meals. In addition, postprandial changes in 3DG were significantly correlated to changes in glucose but, surprisingly, only after the fat-rich meals. This suggests involvement of additional oxidative mechanisms in its formation. In patients with type 1 diabetes, Beisswenger et al. (31) observed an increase in 3DG within 2 h, whereas in our pilot study in patients with type 2 diabetes we did not observe a rise in 3DG after the first meal (21).

Some studies have shown that total serum CML is higher in type 2 diabetes than in healthy individuals (32,33), although in other studies, no difference was observed (22). We chose not to measure total CML and CEL but rather the unbound forms of these compounds that are continuously released during degradation of intracellular proteins with a rapid turnover. Because oxidative stress not only enhances formation of protein-bound CML and CEL but also triggers proteasomal degradation of damaged proteins (34,35), the unbound fraction of CML and CEL may better reflect formation of AGEs by oxidative stress. We observed that CML was affected by both meal types. However, possibly due to a relatively large biological variation, as reflected by differences in the baseline CML and CEL values at the two meal visits, these changes were less consistent than for oxidized LDL and 3DG. Future studies should assess whether changes in the protein-bound forms of CML and CEL are more informative.

The major precursor of CML is glyoxal, and oxidative stress seems to enhance the formation of glyoxal from glucose (36), which is consistent with our finding of a greater increase in CML after the carbohydrate-rich meals compared with the fat-rich meals.

This study has few limitations. First, both groups were not matched for BMI, lipids, blood pressure, and alcohol intake, all of which may have independent effects on the parameters measured. However, we chose not to match for these variables to avoid selection of relatively healthy type 2 diabetic patients. Second, our study was performed in elderly postmenopausal women, and the changes observed might be different in younger women as well as in men.

In summary, we found significant elevations of postprandial oxidized LDL–to–LDL cholesterol ratios, 3DG, and CML in postmenopausal women with type 2 diabetes and NGM. Because postprandial increases in α-dicarbonyls and associated AGEs are related to cellular damage, therapies aimed at reducing postprandial glycemia and triglyceridemia may contribute to the reduction in micro- and macrovascular complications.

This study was supported by Novartis Switzerland and a grant from Dutch Diabetes Research Foundation (no. 2001.00.052).

We thank Danielle van Assema for her assistance in organization and Rick Vermue for his excellent technical assistance.