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Journal of the American College of Nutrition, Vol. 24, No. 6, 474-485 (2005)
Published by the American College of Nutrition

Time Course of Oxidative Stress Status in the Postprandial and Postabsorptive States in Type 1 Diabetes Mellitus: Relationship to Glucose and Lipid Changes

Begoña Manuel-y-Keenoy, MD, PhD, Ann Van Campenhout, Petra Aerts, Jan Vertommen, Pascale Abrams, MD, Luc F. Van Gaal, MD, PhD, FACN, Carolien Van Gils and Ivo H. De Leeuw, MD, PhD, FACN

Metabolic Research Unit (B.M.-y-K., A.V.C., P.A., J.V.), University of Antwerp, Belgium
University Hospital (P.A., L.V.G., C.V.G., I.D.L.), University of Antwerp, Belgium

Address reprint requests to: B. Manuel-y-Keenoy, MD, PhD, University of Antwerp (UA) campus Drie Eiken, Metabolic Research Unit (AMRU) T4.37, Universiteitsplein 1, B-2610 Wilrijk-Antwerp, BELGIUM. E-mail: begona.manuelykeenoy{at}ua.ac.be


   ABSTRACT
TOP
 FOOTNOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Objective: The aim of this study was to compare oxidative stress status (OSS) with blood glucose and lipid changes during the fasting, postprandial and postabsorptive phases in type 1 diabetes mellitus.

Methods: Twenty-three patients on intensive insulin treatment received a standard fat-rich breakfast and lunch. OSS was monitored at fasting (F), just after the post-breakfast glycemia peak (BP) (identified by continuous subcutaneous glucose monitoring), 3.5-h post-breakfast (B3.5), just after the post-lunch peak (LP), just after the post-lunch dale (LD) and 5 hours after lunch (L5).

Results: Whereas whole blood glutathione and plasma protein thiols increased in the postprandial period (from 6.52 ± 1.20 (F) to 7.08 ± 1.45 µmol/g Hb (BP), p = 0.005), ascorbate decreased gradually from 44 ± 17 (F) to 39 ± 19 µmol/L (LD), p = 0.015. Retinol and {alpha}-tocopherol also decreased from 27.1 ± 7.0 (F) to 25.3 ± 5.2 µmol/L (BP), p = 0.005. Uric acid decreased later, from 213 ± 77 (BP) to 204 ± 68 µmol/L (B3.5), p = 0.01, but then increased in LP (231 ± 70 µmol/L) and LD to values higher than F (215 ± 64, µmol/L, p = 0.01). Malondialdehyde increased gradually from 1.02 ± 0.36 (F) to a maximum of 1.14 ± 0.40 µmol/L (LP). In the postabsorptive phase (L5) all parameters except for thiols reverted to fasting concentrations.

Conclusions: In type 1 diabetes lipid peroxidation increases during the postprandial phase in parallel to glucose and triglyceride changes. Blood antioxidants, however, followed diverse patterns of change.

Key words: type 1 diabetes mellitus, oxidative stress, postprandial, postabsorptive, lipid peroxidation, antioxidants

Abbreviations: B3.5 = 3.5-hour post-breakfast • BP = post-breakfast glycemia peak • F = fasting • GSH = reduced glutathione • L5 = 5-hour post-lunch • LD = post-lunch glycemia dale • LP = post-lunch glycemia peak • MAPK = mitogen activated protein kinase • MDA = malondialdehyde • NFkB = nuclear facto k B • OGTT = oral glucose tolerance test • OS = oxidative stress • T1DM = type 1 diabetes mellitus • T2DM = type 2 diabetes mellitus • TAC-PI = total antioxidant capacity as per cent inhibition of chemiluminescence • TACTE = total antioxidant capacity as Trolox equivalents


   INTRODUCTION
TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The two- to-fourfold higher risk and mortality from cardiovascular disease in diabetes mellitus has been independently associated with abnormalities of plasma lipid and lipoprotein metabolism [1] as well as with several pathologic effects of hyperglycemia which result in cellular dysfunction [2,3], glycation [4] and oxidative stress [5]. These all lead to increased susceptibility to lipid peroxidation [6,7] and to the accumulation of products of oxidative damage to lipids [8], proteins [9] and DNA [10]. However, it is not clear if these effects are directly caused by acute increases in blood glucose or are solely the result of an accumulation of an excess of glucose over long periods of time. To answer this question, interest on identifying and recording diurnal glucose and lipid patterns is increasing. For example, there is convincing evidence that postprandial changes of both lipids and glucose can predict cardiovascular risk more strongly than fasting values or even long-term integration parameters such as glycated hemoglobin. Several studies have identified significant associations with intima media thickness [11,12] and with clinical end-points [13–15] reviewed in [16].

More recently it has been suggested that the link between acute postprandial increases in blood glucose and cardiovascular risk is oxidative stress (OS) [17,18]. Indeed, several markers of oxidative damage such as dienes [19], TBARS [20], nitrotyrosine [21], F2 isoprostanes [22] and protein {alpha}-dicarbonyls [23] have been found to increase in the 2–3 hours after an oral glucose load (OGTT). In both healthy and diabetic subjects these are sometimes accompanied by a decrease in antioxidant defenses [20].

Most of these studies have investigated the effects of an OGTT in the more preponderant type of diabetes, namely Type 2DM. Much less information is available on the post-meal changes occurring during the course of a typical day in T1DM patients and how these might relate to the increased cardiovascular risk in these patients. There is evidence that these patients also suffer from increased oxidative stress, even at a young age [24,25], but these analyses have always been carried out in fasting blood. Moreover, the patterns of postprandial glycemia in T1 patients are more labile and intricately determined by the kinetics of exogenously administered insulin and its effect on glucose and lipid metabolism. Therefore, we considered it of interest to investigate the relationship between the acute changes in glucose and oxidative stress by monitoring subcutaneous glucose continuously and by taking blood samples in the course of the 8–9 hours after breakfast and lunch. The data on glucose changes has been published elsewhere [26]. An infrared connection for online monitoring allowed instantaneous identification of changes such as maximal and minimal glycemia (peaks and dales respectively) so that blood for analysis of oxidative stress could be extracted of at these crucial time points.


   MATERIALS AND METHODS
TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Study Subjects
Type 1 diabetic patients on intensive insulin treatment were asked to take part in the study. Exclusion criteria were < 18 and > 69 years of age, BMI < 20 and > 30 kg/m2, detectable C-peptide, pregnancy, alcohol or drug abuse, abdominal, renal or hepatic disease, anemia, low platelet count, clotting disorders, glycated hemoglobin > 9% and complication profile involving history of myocardial infarction, pre-proliferative retinopathy, neuropathy and microalbuminuria > 200 µg/min. After signing the informed consent form, each patient was hospitalised in the metabolic ward. On the evening before the test, the continuous glucose monitoring device (GlucoDay®, A. Menarini Diagnostics) was inserted subcutaneously in the periumbilical region to allow overnight monitoring for about 14 hours before the start of the test. The GlucoDay® device measures glucose concentration in the dialysate derived from the interstitial fluid and stores a value every 3 minutes until the device is removed.

Patients were asked not to modify their self-glucose monitoring and insulin dosages. During the course of the test they sat, walked for less than one hour or laid in bed but did not carry out any strenuous physical exercise. They received a standard fat-rich breakfast and after 3.5 hours a standard lunch. The meal composition is shown in Table 1.


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  		Table 1. Composition of the Standard Meals

 
During the duration of the postprandial test, subcutaneous interstitial fluid glucose was also monitored continuously via an infrared port. This allowed immediate identification of the post-breakfast and post-lunch glucose peaks and dales. Blood samples were extracted at fasting (F), just after (with an interval of maximum 10 minutes) the post-breakfast peak (BP), 3.5 hours after breakfast and just before lunch (B3.5), just after the post-lunch peak (LP), just after the post-lunch dale (LD) and 5 hours after lunch (L5). One patient did not complete the test because of difficulties in the blood extraction. The data presented refers to the 23 patients with complete blood analyses.

The experimental protocol was in accord with the Helsinki declaration and was approved by the ethical commission of the University Hospital.

Analytical Methods
Subcutaneous interstitial fluid glucose was monitored every 3 minutes via the GlucoDay®. The data was transformed into plasma glucose equivalents by using the linear regression comparing the subcutaneous and the intravenous data obtained at 7–9 points in time during the course of the test. All blood glucose data presented in the present article correspond to those derived from the computation of the values obtained by the GlucoDay® device. This allowed calculation of parameters such as area under the curve for a given period, time to peaks and dales, maximum and minimum glucose, spikes and deltas for each patient, as described elsewhere [26].

Routine blood tests including serum total cholesterol, HDL-cholesterol and triglycerides were analysed in the laboratory of the Antwerp University Hospital. Total analytical variability, expressed as coefficient of variation CV was 2%, 1.9% and 0.9% respectively. LDL-cholesterol was calculated according to the Friedewald equation [27]. Glycated hemoglobin (HbA1c) was measured by using a HPLC cation exchange column (Modular Diabetic Monitoring System; Bio-Rad, Richmond, CA); the CV was 1.5%.

Oxidative stress status was evaluated by measuring blood concentrations of individual antioxidants, global plasma antioxidant capacity and products of lipid peroxidation. Vitamin E and A in serum were measured by HPLC (Shimadzu, Kyoto, Japan) with a reversed phase C18 column LiChrospher RP C18 (Alltech, Deerfield, IL) with 100% methanol mobile phase and detection at 292 and 325 nm respectively [28] with CVs of 4.8 and 4.1% respectively. Vitamin C in plasma was measured by HPLC isocratic delivery using a reverse phase column LiChrospher RP C18 (Alltech, Deerfield, IL) with 2 mM KCl mobile phase and electrochemical detection at 1000 mV; the CV was 6% [29]. Glutathione (GSH) in whole blood and protein thiols in plasma were measured by a colorimetric method using Ellman’s reagent and expressed relative to haemoglobin concentration for GSH (measured using the Drabkin reagent and calibrated against commercial standards) and to plasma protein concentration for thiols (measured using the Biuret reagent). CV were 7% and 6% respectively [30,31]. Global plasma antioxidant capacity was evaluated by measuring the inhibition of chemiluminescence after addition of plasma (anticoagulated with Li-heparin and diluted two-fold with Na PO4 pH 8.6) to a reaction mixture containing 75 µM Luminol and peroxyl radicals liberated by the thermal decomposition of 20 mM ABAP (2,2-azo-bis(2-amidinopropaan) hydrochloride) [32]. Two antioxidant effects were distinguished in the plasma samples: 1) the scavenging of the peroxyl radicals (by antioxidants such as GSH, uric acid and ascorbic acid) which cause a lagtime before the appearance of the chemiluminescent signal. The duration of the lagtime was compared to a calibration obtained in parallel with different concentrations of the water-soluble analogue of vitamin E, Trolox, and expressed as Trolox equivalents (TACTE). 2) the inhibition of the maximum chemiluminescence peak, due to antioxidant components which quench the chemiluminescence (for example albumin and transferrin). This antioxidant component was expressed as percent inhibition (TAC-PI); CV was < 9%. Whole blood d-ROM (determinable reactive oxygen metabolites) was measured using a commercial kit (Pharmalab d-ROMs, Parma, Italy). The blood sample was incubated in the presence of Fe2+ ions to produce radicals via a Fenton-like reaction. These radicals were chemically trapped by a phenolic reagent (n,n-diethyl-p-phenyldiamine) to produce a red coloured molecule (absorption at 512 nm). The intensity of the colour produced is proportional to the concentration of peroxides and is calibrated against known concentrations of tert-butyl hydroperoxide; CV was 7%. Plasma total malondialdehyde was analysed by pre-treating plasma samples with thiobarbituric acid in ortho-phosphoric acid containing butyl-hydroxytoluene as antioxidant. The pink-coloured product was separated by HPLC using reverse phase LiChrospher RP C18 (Alltech, Deerfield, IL), methanol/KH2PO4 10 mM (40/60 v/v) as mobile phase and detection at 532 nm [33]; CV was 9%.

Quantitative analysis of energy, nutrient and antioxidant content in the standard meals was done using the BECEL programme [34] based on values obtained from the Belgian and Netherlands food composition tables [35,36].

Statistical Analysis
Data were analysed using SPSS software (version 11.0, Chicago, IL). Results are expressed as means ± SD and two-tailed p values < 0.05 were considered significant. Between group comparisons at baseline was done by T-test or Mann-Whitney test for the non-gaussian variables. The changes in the various parameters were analysed by Repeated measures ANOVA and the Friedman test. Data was analysed for the change during the course of the day (within group comparison) and for differences between groups (male versus female; smoker etc) as regards these time-related changes. Analysis of contrasts was applied to identify the significance of the change when compared to the fasting value and to the value in the preceding time-point.


   RESULTS
TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
ACKNOWLEDGMENTS
 REFERENCES
 
The patient characteristics are shown in Table 2. Two male patients had moderate hypertension (> 135/80 mm Hg). With respect to fasting serum lipids, thirteen patients had total cholesterol > 4.8, three had HDL < 1.2 and two had triglycerides > 1.7 mmol/L. The complication profile included no antecedents of cardiovascular disease, nine patients had mild background retinopathy and four had neuropathy. Albuminuria ranged from 0 to 100 µg/min (median 6.2 µg/min). Five men and nine women used insulin analogues (Novorapid® or Humalog®) and six took ACE inhibitors.


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  		Table 2. General Characteristics of the T1DM Patients

 
The time course of postprandial glucose changes is illustrated in Fig. 1A. There were big inter-individual differences both in the magnitude as well as in the time course of the postprandial glucose changes. The postprandial hyperglycaemic peaks did not differ after breakfast BP (17.4 ± 5.8 mmol/L) and after lunch LP (16.9 ± 6.6 mmol/L) but both were significantly higher than the blood glucose at fasting F (10.5 ± 5.2 mmol/L, p < 0.005). Since the time to reach these peaks varied from 0 to 180 (median 78) minutes post-breakfast and from 3 to 297 (median 57) minutes for the post-lunch peak, the mean values shown in Fig. 1A for each time point are composites of individual values taken at different stages of change and thus under-represent the full extent of the individual changes. Three to four hours after lunch there was also a clear postprandial dale in most patients with a decrease to 4.7 ± 3.2 mmol/L (p < 0.0005 when compared to F).



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  		Fig. 1A. Daytime continuous monitoring profile of the postprandial and postabsorptive changes in blood glucose obtained using the GlucoDay® device that measures glucose concentration in the dyalisate of the interstitial fluid from the subcutaneous tissue. These data was transformed into plasma glucose equivalents by calibration with intravenous glucose samples obtained at 7–9 time points during the test period. Shown are the mean ± 1 SD of the postprandial glucose values of 23 patients obtained at intervals of 3 minutes. Because of differences in the individual time courses, the extent of glucose changes depicted on this graph are less outspoken than the means of the maximum and minimum glucose values reached by the individual patients. B and C. Time course of changes in serum cholesterol (1B) and triglycerides (1C). Values were monitored at fasting F, just after the post-breakfast glucose peak BP, just before lunch B3.5, just after the post-lunch glucose peak LP, just after the post-lunch glucose dale LD and 5 hours after lunch L5. Shown are means ± SEM of 23 patients. The horizontal error bars depict the SEM of the time taken to reach the blood glucose peaks at BP and LP and dale LD. P-value denotes the significance of the overall change over time, calculated by repeated measures analysis of variance. * denotes p < 0.05 when contrasted to the value at fasting. § denotes p < 0.05 when contrasted to the preceding blood sample.

 
The changes in serum lipids followed differing patterns (Fig. 1B and C). Cholesterol (both LDL and HDL) decreased by 10–12% in BP (p < 0.0005 versus the fasting value), was maintained low throughout the post-lunch peak and dale and only returned to the fasting values in the postabsorptive phase. Triglycerides increased gradually in the postprandial periods and reached a peak in LP (1.60 ± 0.87 versus 1.03 ± 0.40 mmol/L at F, p = 0.001). Levels gradually decreased towards fasting values in LD and L5 (1.18 ± 0.56 mmol/L, ns from F).

Fasting oxidative stress status did not correlate with parameters of metabolic control such as HbA1c and duration of diabetes. There was only a positive correlation between BMI and TACPI (r = 0.56, p = 0.007), TACTE (r = 0.45, p = 0.036) and uric acid (r = 0.48, p = 0.022). Fasting serum triglycerides correlated positively with insulin dose (r = 0.57, p = 0.007) and serum vitamin A and E (r = 0.54, p = 0.008 and r = 0.42, p = 0.048 respectively). Serum vitamin E also correlated with serum cholesterol (r = 0.57, p = 0.004).

The time course of changes of the various components of oxidative stress status during the 8–9 hours of the test was compared. Antioxidant defence was monitored by measuring both the total antioxidant capacity in plasma as well as the concentration of the individual antioxidants. One aspect of the total antioxidant capacity in plasma is TACPI which measures the percent inhibition of peroxyl-induced chemiluminescence and is attributed to the presence of antioxidants such as proteins. It decreased significantly but only in the postabsorptive phase L5 (p = 0.038) (Fig. 2A). In contrast, the plasma protein-thiols tended to increase during the course of the day (Fig. 2B) and reached significantly higher values in L5 (3.59 ± 1.40 µmol/g protein versus 3.00 ± 1.33 in F, p = 0.048). Thiol concentrations in whole blood were monitored by measuring reduced glutathione (GSH) in hemolysates, virtually all of which originates from the red blood cells. Concentrations of GSH changed significantly in the course of the day (p = 0.005) (Fig. 2C) by increasing from 6.52 ± 1.20 at fasting F to 7.08 ± 1.45 µmol/g Hb during the post-breakfast hyperglycaemic peak (BP) and remaining high during the postlunch period (LP). In the postabsorptive period L5 (5 hours after lunch), GSH decreased to 5.93 ± 1.52 µmol/g Hb (p = 0.01 when compared to the fasting value).



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  		Fig. 2. Time course of changes in plasma total antioxidant capacity TACPI (2A), plasma protein-thiols (2B) and whole blood reduced glutathione (2C) taken at fasting F, just after the post-breakfast glucose peak BP, just before lunch B3.5, just after the post-lunch glucose peak LP, just after the post-lunch glucose dale LD and 5 hours after lunch L5. Shown are means ± SEM of 23 patients. The horizontal error bars depict the SEM of the time taken to reach the blood glucose peaks at BP and LP and dale LD. p-value denotes the significance of the overall change over time, obtained by repeated measure analysis of variance. * denotes p < 0.05 when contrasted to the value at fasting. § denotes p < 0.05 when contrasted to the preceding value.

 
A second aspect of plasma antioxidant capacity consists of the TACTE which is the global result of water soluble antioxidants such as uric acid and ascorbate. In the same way as Trolox, these molecules scavenge free radicals and thus can prolong the lagtime before the appearance of chemiluminescence. TACTE did not change significantly in the course of the test (Fig. 3A). In contrast, uric acid fluctuated by first decreasing from 215 ± 64 µmol/L at F and 213 ± 77 µmol/L at BP to 204 ± 68 µmol/L at B3.5 (p = 0.01) and then increasing in the LP (231 ± 70 µmol/L) and LD to values higher than F (p = 0.01). There was a return to baseline values in the postabsorptive phase L5 (221 ± 66 µmol/L, ns from F); p < 0.001 for the overall change in time (Fig. 3B). Plasma ascorbate, in contrast, decreased gradually from F (44 ± 17 µmol/L) to a minimum of 39 ± 17 at LD and returned to 42 ± 15 µmol/L at L5 (p = 0.015 for the overall change in time, Fig. 3C).



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  		Fig. 3. Time course of changes in plasma total antioxidant capacity TACTE (3A), serum uric acid (3B) and plasma ascorbate (3C) taken at fasting F, just after the post-breakfast glucose peak BP, just before lunch B3.5, just after the post-lunch glucose peak LP, just after the post-lunch glucose dale LD and 5 hours after lunch L5. Shown are means ± SEM of 23 patients. The horizontal error bars depict the SEM of the time taken to reach the blood glucose peaks at BP and LP and dale LD. P-value denotes the significance of the overall change over time, obtained by repeated measure analysis of variance. * denotes p < 0.05 when contrasted to the value at fasting. § denotes p < 0.05 when contrasted to the preceding value.

 
For plasma retinol, the decrease occurred immediately after breakfast (from 2.47 ± 0.60 at F to 2.31 ± 0.58 µmol/L at BP, p = 0.007) and the lower concentrations were maintained till the post-lunch dale, after which there was a significant return to fasting values in L5 (2.55 ± 0.59 µmol/L) (Fig. 4A).



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  		Fig. 4. Time course of changes in plasma retinol (4A), plasma {alpha}-tocopherol (4B) and plasma malondialdehyde (4C) taken at fasting F, just after the post-breakfast glucose peak BP, just before lunch B3.5, just after the post-lunch glucose peak LP, just after the post-lunch glucose dale LD and 5 hours after lunch L5. Shown are means ± SEM of 23 patients. The horizontal error bars depict the SEM of the time taken to reach the blood glucose peaks at BP and LP and dale LD. P-value denotes the significance of the overall change over time, obtained by repeated measure analysis of variance. * denotes p < 0.05 when contrasted to the value at fasting. § denotes p < 0.05 when contrasted to the preceding value.

 
Similarly, plasma {alpha}-tocopherol decreased from 27.1 ± 6.98 at F to 25.3 ± 5.23 µmol/L at BP (p = 0.010), decreased further 3.5 hours after breakfast B3.5 (24.0 ± 5.71 µg/mL) and then remained low till LD and returned to values similar to fasting in the postabsorptive phase L5 (25.0 ± 5.6 µmol/L) (Fig. 4B). Even when expressed relative to lipids, {alpha}-tocopherol decreased from 4.43 ± 0.76 µmol/mmol lipid at F to 4.12 ± 0.82 at B3.5 (p = 0.006) and then returned to 4.43 ± 0.82 µmol/mmol lipid at L5 (ns from F).

Levels of peroxidation products accumulated in vivo were monitored by measuring reactive oxygen metabolites (d-ROM) which give an indication of blood peroxide levels and plasma malondialdehyde which is an end-product of lipid peroxidation. There was no significant change in d-ROM levels during the course of the day (data not shown). In contrast, plasma malondialdehyde increased gradually from 1.02 ± 0.36 µmol/L at F to a maximum of 1.14 ± 0.40 at LP (p = 0.028) and then decreased to 0.92 ± 0.29 at L5 (p = 0.044 when compared to F; p = 0.12 for the overall change in time). When expressed relative to total lipids, there was a significant overall change in time (p = 0.029) with increases at B3.5 and LP and a significant decrease from LD to L5 (p = 0.023) (Fig. 4C).

As a measure of total postprandial load, the 8.5 hour-areas under the curve (AUC) of glucose, lipids and the OS parameters were compared. AUC-glucose tended to correlate with AUC-GSH (r = 0.39, p = 0.08) but there were no significant correlations with the AUC of antioxidants, total antioxidant capacity or products of peroxidation. AUC-triglycerides correlated positively with AUC-retinol (r = 0.54, p = 0.027) and AUC-total cholesterol with AUC-{alpha}-tocopherol (r = 0.53, p = 0.029). Surprisingly, no inter-correlations were found between the AUC of the various antioxidants.

Men had significantly higher fasting MDA (1.17 ± 0.38 vs 0.86 ± 0.26 µmol/L in women, p = 0.034), uric acid (255 ± 52 vs 172 ± 48 µmol/L in women, p = 0.001), TACTE (159 ± 24 vs 124 ± 28 µmol/L Trolox equivalents, p = 0.004). Smokers had lower GSH (5.46 ± 1.07 vs 6.83 ± 1.11 µmol/g Hb in non-smokers, p = 0.024). Patients treated with insulin analogues had significantly lower MDA (0.87 ± 0.25 vs 1.22 ± 0.43 µmol/L in those treated with regular insulin, p = 0.03) but also lower plasma ascorbate (39 ± 15 vs 54 ± 13 µmol/L, p = 0.048). Use of ACE-inhibitors was associated with higher uric acid concentrations and TACTE (165 ± 19 vs 135 ± 31 µmol/L Trolox equivalents, p = 0.039). These sub-group differences in levels were maintained throughout the day so that the AUC of the above-mentioned parameters were also significantly different. However, the pattern and extent of change of OS parameters was the same for all sub-groups.


   DISCUSSION
 
In this study we aimed to describe the detailed time course of the changes in oxidative stress status occurring in the 8–9 hours encompassing the fasting, postprandial and postabsorptive periods in T1DM. The possible relationship with changes in blood glucose and lipids was also investigated.

As already described for type 1 diabetic patients [37,38], glucose changes varied greatly from patient to patient both as regards timing as well as intensity of the glucose fluctuations [26]. Nevertheless, there were consistent postprandial peaks (BP and LP) reaching pathological values of > 16.7 mmol/L and a decrease to 4.7 mmol/L glucose in the late post-lunch period (L5, postabsorptive phase). There is convincing evidence that high glucose causes an increase in free radical production by several mechanisms. Accelerated metabolism due to the thermic effect of food and to the increase in intracellular glucose leads to increased mitochondrial respiration and release of superoxide [3,39]. Increased glycolysis inside the cells leads to de novo synthesis of diacylglycerol [40] and activation of protein kinase C [41]. One of the many consequences of this activation is exaggeration of the respiratory burst by monocytes, which has been shown to increase in the postprandial phase [42]. Glucose auto-oxidation [43,44] and protein glycation [45] are important additional sources of free radicals during hyperglycemia.

With respect to antioxidant defenses, there is evidence that high glucose, by channelling glucose metabolism along the sorbitol pathway would impair the NAD(P)H/NAD(P) balance [46,47], thus slowing down the recycling of oxidised glutathione and ascorbate [48–50]. However, most of the evidence on antioxidant recycling has been collected from experimental exposure to high glucose lasting days. Much less is known about the biochemical mechanisms mediating acute (time span of hours) effects of high glucose on the oxidant-antioxidant balance.

In order to answer this question we monitored several parameters of oxidative stress during the post-breakfast and post-lunch periods which were associated with considerable blood glucose fluctuations in our group of type 1 patients. Surprisingly, the patterns of change during these postprandial and postabsorptive periods differed in the various oxidative stress parameters. For example, both the thiol (SH)-containing molecules, GSH in erythrocytes and plasma protein-thiol content increased in parallel with the post-breakfast blood glucose peaks. Thereafter GSH remained high throughout the postprandial period only to drop to sub-fasting levels in the postabsorptive phase. In contrast the plasma protein thiols continued to increase and remained high in the postabsorptive phase. These observations contrast with the consistent decreases in plasma protein-thiols found in T2DM patients but not in healthy controls, 2 to 3 hours after a standardised liquid breakfast [20] and after an oral glucose tolerance test (OGTT) [51]. Other authors did not detect any changes in GSH [22]. In elderly T2DM patients, glutathione in neutrophils and serum did not change after OGTT, but the oxidised glutathione (GSSG) increased so that the GSH/GSSG ratio decreased, indicating increased intracellular oxidative stress in these cells [52]. The positive relationship we found between blood glucose and GSH levels suggests a beneficial rather than detrimental effect of the postprandial phase on this antioxidant. It should be noted that our GSH measurements are conducted in hemolysates of whole blood and thus primarily reflect the levels of reduced glutathione in red blood cells, which are determined both by its synthesis and by its recycling. It is thus plausible that in our study conditions, the increased supply of precursor nutrients such as glutamic acid and cysteine in the meals [53] and a beneficial increase in the ATP/energy/redox status during hyperglycemia both contributed to shift the balance in favour of GSH synthesis. As regards recycling, we have previously shown that a 90 minute incubation in high glucose did not affect the capacity of erythrocytes from diabetic patients to recycle oxidised to reduced glutathione (a reaction catalysed by glutathione reductase) after oxidant attack [54]. These observations on the effects of acute exposure to high glucose cannot explain the well-documented lower levels of fasting GSH seen in diabetic patients by us and others [55–57]. The effects of chronic exposure to high glucose are mediated by mechanisms operating over weeks/months with accumulatory effects. For example, non-enzymatic glycation inhibits the enzymes responsible for GSH synthesis ({gamma}-glutamyl cysteine synthetase [57] and recyling (glutathione reductase) [58].

In addition to the antioxidant defense provided by the GSH in the erythrocytes and by the thiols in the plasma proteins, there is also a substantial contribution from water-soluble antioxidants in plasma such as ascorbic, uric acid and bilirubin. Their combined effect can be measured globally as total antioxidant capacity (TACTE). The changes in TACTE during the course of the test did not reach statistical significance. In T2DM patients, but not in healthy controls, total antioxidant capacity (TRAP) decreased 2 to 3 hours after a standardised liquid breakfast [20] and after an oral glucose tolerance test (OGTT) [51]. Other authors did not detect any changes in total antioxidant capacity [22]. Again, the individual antioxidants changed following different patterns. In concordance with the OGTT studies, plasma ascorbate and tocopherol also decreased in the postprandial period in our patients. In T2DM patients this decrease could be prevented by increasing the ascorbate content of the meal to 100 mg [20]. The ascorbate content in our meals was lower (<50 mg) and was probably not sufficient to counteract the increased postprandial oxidative load. Some pathophysiological consequences of this deficiency are illustrated by the postprandial impairment of endothelial function which can be normalised by giving vitamin C and E supplements [59].

The difference in the evolution of GSH when compared to ascorbate and tocopherol is in conflict with the well documented role of GSH in recycling oxidised ascorbate and eventually {alpha}-tocopherol [60]: one would expect that the changes in these three antioxidants would evolve in parallel. Our observation thus suggests that other factors might interfere in the interrelationship between the three antioxidants. One such factor might be the exchange of antioxidants between the plasmatic and the intracellular compartments. For example, an acute rise in insulin (e.g. after an injection) is known to accelerate the entry of {alpha}-tocopherol from the plasma into the cells [61]. On the other hand, glucose and ascorbate compete for entry into the cells, so that postprandial increases in blood glucose would inhibit passage of ascorbate from the plasma into the cells [62,63].

In contrast to ascorbate and tocopherol, uric acid decreased immediately after breakfast but then increased after lunch. The post-breakfast decrease is possibly due to its consumption because uric acid has a strong free radical scavenging activity. There is no clear explanation for the post-lunch increase. Purine catabolism is considered to be the main source of uric acid with little contribution from dietary sources [64]. The relative hypoxemia occurring postprandially might increase ATP catabolism, adenosine formation and activity of xanthine oxidase and thus result in the release of both free radicals and uric acid by the microvascular endothelium [65,66].

When comparing the evolution of the three main plasma antioxidants, one can summarise that a post-breakfast increase in free radical production led to consumption of uric acid followed by a consumption of {alpha}-tocopherol (exaggerated by the insulin-induced increased entry into cells) and a gradual decreased in ascorbate (dampened by its decreased transport into cells). Further studies are needed to identify the specific radicals involved because these determine the type of antioxidant consumed [67]. For example, iron-induced peroxidation is best inhibited by transferrin and ceruloplasmin; lipid peroxidation caused by cigarette smoke is best inhibited by ascorbate; uric acid protects against nitrogen dioxide but not against hypochlorous acid [68]. In vitro exposure of plasma to peroxyl radicals leads to oxidation of ascorbate and albumin-thiol first, followed by bilirubin, uric acid and tocopherol. Exposure to activated neutrophils, in contrast, leads to a rapid depletion of ascorbate followed by partial depletion of urate but no change in bilirubin or tocopherol [69,70].

In comparison, our in vivo observations are the end-result of both the release of so far unidentified radicals and the interference by factors such as meal content and the insulin injections. Meal content is known to be pro-oxidant because of the presence of oxidised lipids [71], advanced glycation end products [18] and lipids. It has recently been postulated that postprandial accumulation of triglycerides and subsequently of intracellular long chain fatty acids leads to release of electrons in the mitochondrial transfer chain. This can be an additional important source of free radicals in the post-meal period [72,73]. Insulin increases hydrogen peroxide production by adipocytes [74,75] and promotes tocopherol clearance from the plasma [61].

It should also be mentioned that the degree of glucose fluctuations and the sources of oxidative stress might differ in type 1 and 2 patients. For example, mononuclear cell respiratory burst is higher in type 1 than type 2 which in its turn is higher than in nondiabetic controls [10].

In our group of patients there was a postprandial increase in a product of oxidative damage, namely MDA, which followed a pattern of evolution closely mirroring the changes in blood glucose and opposite to those of ascorbate and tocopherol. Increases of the various products of lipid peroxidation such as F2 isoprostanes [22], MDA [20,20] and dienes [19] were also observed in the above-mentioned studies conducted on T2 patients. Increases in products of protein oxidative damage such as nitrotyrosine [21] and {alpha}-dicarbonyls [23] have also been confirmed. More recently, oxidative-stress-triggered cell-signalling events such as NFkB and MAPK activation have been demonstrated during acute hyperglycemia [76].

The prolonged 8–9 hours monitoring of our patients permitted us to observe that, with the exception of the thiols, OS parameters reversed to values similar to those at fasting in the post-absorptive phase, which, in these patients was characterised by a fall in blood glucose and triglycerides. The patterns of change of all the OS markers were similar regardless of the difference in levels found in men, smokers, users of insulin analogues or ACE-inhibitors. Both these observations point out to the role of high glucose and triglycerides as the main sources of oxidative stress.

The unexpected differences in the pattern of changes in the various components of oxidative stress illustrate the complexity of assessing in vivo oxidative stress in a near real-life situation. In order to dissect out the separate contribution of diabetes per se, hyperglycemia, hyperlipidemia, food intake and exogenous insulin, further investigations will require strict case/controlled study designs.


   ACKNOWLEDGMENTS
TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
REFERENCES
 
The authors would like to thank the patients and the technical and nursing staff of the university hospital and laboratory of endocrinology. This work was supported by Menarini Belgium and by the Belgian Diabetes Study Group.


   FOOTNOTES
TOP
 FOOTNOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Grants: Belgian Diabetes Study Group and Menarini Belgium.

Received October 14, 2004. Accepted July 19, 2005.


   REFERENCES
TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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