Introduction

Type 2 diabetic patients experience a 2–4 fold increased risk for coronary heart disease (CHD) compared with non-diabetics (Haffner & Lehto, 1998). The lipid profile in type 2 diabetes is characterized by increased plasma triglycerides, a reduced high-density lipoprotein (HDL) cholesterol, and an increased concentration of small, dense low-density (LDL) lipoproteins (Steiner, 1995). Supplementation with fish oil has been proposed as a non-pharmacological way to correct these atherogenic lipid abnormalities. However, there is some concern about the possibility of enhanced in vivo lipid peroxidation with fish oil supplementation. Diabetic patients may be particularly vulnerable in this regard, given the increased oxidative stress associated with this disease (Giugliano et al, 1996). One hypothesis for the accelerated atherosclerosis in diabetes is related to the increased oxidative vulnerability of glycosylated LDL (Lyons & Jenkins, 1997) and small, dense LDL particles (Tribble et al, 1992). If fish oil supplementation further impairs the oxidation resistance of LDL, this might lead to accelerated atherogenesis in diabetic patients. So far the effect of fish oil on LDL particles susceptibility to oxidation in type 2 diabetes has not been investigated. In the present study we quantified the impact of vitamin E-enriched fish oil on LDL oxidation resistance in type 2 diabetic patients. The glycemic control of the patients was also monitored.

Subjects and methods

Subjects

Forty-nine type 2 diabetic patients, between 38 and 85 y of age, were recruited from Steno Diabetic Center out-patient clinic. We only included patients that had: (1) diagnosed type 2 diabetes for more than one year; (2) fasting plasma triacylglycerol >1.5 mmol/l at screening; (3) diabetes onset at age greater than 30. Exclusion criteria were: (1) use of lipid-lowering drugs; (2) use of antioxidant, fish oil or garlic supplements; (3) high alcohol intake (>4 drinks/day); (4) use of hormone replacement therapy (females); (5) serum creatinin >150 mol/l. Forty-four patients completed the intervention. Two left the study during the run-in period, one for personal reasons, the other was hospitalized, and one withdrew during the intervention period due to pneumonia. Blood samples from a further two subjects were lost due to electric power supply failure. The Ethical Committee of Frederiksberg and Copenhagen approved the protocol (file no. 01-089/99) and written consent was obtained from each participant prior to beginning of the study. Nine subjects were smokers.

Study design

The study was double-blinded and placebo-controlled and had two parallel arms. During a 4 week run-in period all participants consumed four corn oil (CO) capsules (1 g oil and 13.4 mg -tocopherol in each capsules) daily. During the following 8 week intervention period, participants took four daily capsules of either fish oil (FO; Futura, Dansk Droge, Ishøj, Denmark) or CO. Subjects were randomly assigned to FO or CO after blocking for smoking habits (yes/no) and pre-study plasma triglycerides (3, or <3 mmol/l). Equal group sizes were secured. Both oil supplements were supplied by Dansk Droge (Ishøj, Denmark). Their fatty acid composition is presented in Table 1. The daily intake of four FO capsules contributes 2.6 g of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which correspond to a daily intake of approximately 50–60 g fatty fish. Both oil supplements supplied approximately 53.6 mg vitamin E in 4 g oil consumed daily. Both oil supplements contained a maximum of 5 milliequivalents peroxides/kg.

Table 1 - Fatty acid composition of corn oil(CO) and fish oil (FO) supplements given as mol%.

Full table (18K)

Participants were asked to adhere to their usual lifestyle and diet for the whole study period. We checked fatty acid fish consumption with a simple food frequency questionnaire (five categories) at the beginning of the run-in and after the intervention. Compliance was assessed from LDL fatty acid profiles. In addition, leftover capsules in returned containers were counted after completion of the intervention period. In the present paper oxidation and basal lipid data is presented. Data on lipoprotein and lipoprotein subclasses will be published separately.

Blood collection

Blood samples were obtained after run-in and after the 8-week intervention period. The day before blood sampling, intake of alcohol and participation in sports were not allowed. Venous blood samples were collected into 10 ml tubes containing 0.1% EDTA after an overnight fast (>10 h). Samples were kept in the dark and on ice until centrifugation. The samples were centrifuged for 15 min at 3000 rpm and at 4°C. Plasma samples were immediately aliquotted and overlaid with nitrogen. The samples collected after run-in were quick frozen at -50°C, whereas samples collected after intervention were frozen in liquid nitrogen (-173°C). Both were stored at -80°C. In a separate study we found that the two different freezing methods had no impact on the oxidation parameters (unpublished data). Plasma samples were thawed at 37°C at the time of assay.

Preparation and oxidation of LDLs

Isolation of LDL

LDL (1.019–1.063 g/ml) was isolated by density ultracentrifugation. Briefly, 3 ml plasma stored from each subject was thawed rapidly at 37°C. Thereafter plasma was adjusted with solid KBr to a density of 1.5 g/ml and layered on the bottom of a centrifuge tube (Sw40, ultra clear, 1489, Ramcon). This layer was then successively overlaid with 1.6 ml of 1.21g/ml; 3.0 ml of 1.10 g/ml, and 2.0 ml of 1.09 g/ml KBr solutions. On top was added 1.5 ml of distilled water. All solutions contained 10mol/l EDTA. Tubes were centrifuged for 18 h, at 40 000 rpm, at 4°C in a Beckman, L8-70M Ultracentrifuge (Beckmann, Palo Alto, CA, USA) with a Beckman SW 40-ti rotor (Beckmann, USA). After centrifugation, the LDL fraction was removed from the LDL band and transferred to a dialysis membrane (Spectra/Por 4, MWCO 12–14; 10 mm width). Plasma was dialyzed over night in 6 l phosphate buffered saline (PBS, pH 7.4). The PBS had been chelated with Chelex Resins (Bio-Rad) to remove contaminating transition metal ions before dialysis.

Susceptibility of LDL to oxidation in vitro

In vitro LDL oxidation was performed as described by Esterbauer et al (1989) with modifications. The LDL cholesterol concentration was determined enzymatically using the Chol MPR 1 kit supplied by Boehringer-Mannheim. LDL oxidation was initiated by adding freshly prepared CuSO₄ (5 mol/l) to the LDL fraction at a LDL cholesterol concentration of 75 g cholesterol/ml. The kinetics of LDL oxidation was followed by continuous monitoring of conjugated diene (CD) concentration in the sample as assessed from light absorbance at 234 nm at 37°C with a thermostat-controlled spectrophotometer in an automatic sample changer (UV-2101 PC Spectrofotometer, Shimadzu Scientific Instruments Inc., USA) every 2 min. LDL oxidation lag time, propagation rate, and maximal CD concentrations were determined from the resulting graphs. Lag time (min) was defined as the time from copper addition to the sample until the time corresponding to the point of intersection of the tangent of the lag phase and the tangent to the steepest part of the propagation phase. Propagation rate is defined as the slope of the propagation phase's tangent and is expressed as OD/min. The maximal diene concentration (max optical density (OD)) was defined as the difference between peak OD and baseline OD.

It should be noted that the physiological relevance of the in vitro LDL oxidation assay is still a matter of debate, eg due to the unphysiologically high Cu⁺-concentrations that are applied. Readers are referred to method papers for further discussions (Giesig & Esterbauer, 1994; Yoshida et al, 1994; Abuja & Albertini, 2001).

Determination of malondialdehyde in LDL (LDL-MDA)

MDA concentration (pmol MDA/mg protein) in LDL was determined by a very specific MDA-HPLC method described previously (Lauridsen & Mortensen, 1999). Briefly, the antioxidant BHT and NaOH were added to the LDL sample and heated for 30 min at 60°C to release protein-bound MDA by alkaline hydrolysis (Carbonneau et al, 1991). Reaction with TBARS was performed as previously described (Wallin et al, 1993) and after a centrifugation for 5 min at 10 000g, the supernatant was transferred to the HPLC analysis. Samples were chromatographed with a linear gradient of H₂O/ acetonitrile with 0.1% trifluoroacetic acid (0–60%, 17 min). The HPLC analysis was performed on a Hewlett Packard 1100 system (Waldbronn, Germany) equipped with a diode array detector, using a Purospher RP-18 column (4250 mm, 5 m, Hewlett-Packard), and with detection at 532 nm. Four MDA standards were included in each TBARS procedure and HPLC-run, and the concentration of MDA in the samples was calculated from a standard curve.

The MDA-HPLC method has a greater specificity and sensitivity compared to the TBARS assay. However, MDA can arise from other reactions than oxidative processes and MDA data should therefore also be interpreted with some caution.

LDL fatty acid composition

LDL lipids were extracted from the aqueous LDL fraction by dissolving a 100 l LDL sample in chloroform:methanol (2:1), to which 100 l standard fatty acid (C21:0) was added. The solvent was evaporated in a vacuum centrifuge, and the sample was reconstituted in 0.5 mol NaOH/l in methanol. The fatty acids were methylated with 20% boron trifluoride in methanol in 80°C water bath (Morrison & Smith, 1964). To avoid fatty acid oxidation 0.5 ml 0.1% hydroquinon was added to the samples. Then the heptane and methanol:water (90:10) were added. The top phase was transferred to 10 ml glasses and was evaporated in a vacuum centrifuge. Finally, the samples were reconstituted in 200 l heptane.

The fatty acid composition was determined with a gas chromatograph (HP5890; Hewlett-Packard; Ingelsheim, Germany) with a flame ionization detector on a 30 m 0.32 m internal diameter, fused silica column (model SP2380; Supelco, Bellefonte, PA, USA). Helium was used as carrier gas. Aliquots of 5 l were injected with an autoinjector (HP7673; Hewlett-Packard) with a flow of 12 ml/min and a split ratio of 1.2:10. The column oven was temperature programmed to 120°C initially, raised by 4°C/min to 160°C, by 8°C/min to 200°C, and finally by 30°C/min to 225°C. The fatty acids were identified by comparison with two commercial standards (Standard 85 and 411; Nu-Check-Prep, Elysian, MN, USA) and quantified by the peak area. The quantity of the fatty acids was calculated as mol%.

The fatty acid composition of the oil supplements

A drop of oil was dissolved in 10 ml heptane. The sample was methylated with 60 l KOH (2 M in MeOH; Christopherson & Glass, 1969). The sample was evaporated in a vacuum centrifuge and reconstituted in 1 ml heptane. The fatty acid composition was determined with a gas chromatograph as described above.

Plasma lipids

The concentration of cholesterol and triglycerides were determined in plasma by enzymatic colorimetric methods, using commercially available kits (Boehringer Mannheim, Mannheim, Germany), on a COBAS MIRA auto-analyzer (Roche, Basel). LDL and HDL cholesterol were determined after lipoprotein separation by ultra centrifugation (Petersen et al, 2002) in the same way as in plasma.

Measurement of blood glucose and glycosylated hemoglobin (HbA_1c)

The blood glucose concentration in a drop of blood taken from the ear was measured on a One-Touch Instrument (Life Scan Inc., Milpalaz, CA, USA). Glycosylated hemoglobin was measured with ion exchange HPLC (Bio-Rad Variant).

Other measurements

Blood pressure was measured digitally using instrument from A&D (Japan). Body weight was measured in the morning with the subjects wearing light clothing on a calibrated digitally scale. The same person measured waist and hip circumference according to guidelines throughout the study.

Statistics

t-Tests were used for comparison of all parameters: paired-sampled t-test within the groups and independent-sample t-test between the groups. Differences were considered to be significant at P<0.05. Results are expressed as meanss.e.m. Simple correlation between selected study variables were calculated using Pearson's correlation analysis.

Results

Characteristics of the subjects

The initial characteristics of the participants are presented in Table 2. The two groups were similar with respect to all measured characteristics at the beginning of the study. The average habitual fish intake was 1–2 times a week in the fish oil group, and 2–3 times a month in the corn oil group (group difference: N.S.).

Table 2 - Baseline characteristics of the subjects.

Full table (34K)

Compliance assessment

The fatty acid composition of LDL was determined before and after intervention (Table 3). The saturated and monounsaturated fatty acids were unaffected in both groups. After 8 weeks of fish oil supplementation, EPA (20:5n-3) and DHA (22:6n-3) increased significantly (both P<0.001), while linoleic acid (18:2n-6) and dihomogammalinolenic acid (20:3n-6) decreased significantly (P=0.007 and P=0.001, respectively). No changes were detected in the corn oil group. The unsaturation index increased significantly in the fish oil group (P<0.001), but did not change in the corn oil group. For all these variables the effect of fish oil group corn oil were significantly different.

Table 3 - Effect of corn oil or fish oil supplementation on fatty acid composition of low density lipoprotein particles.

Full table (71K)

Effect of fish oil on LDL oxidation

After fish oil supplementation, lag time and propagation rate were significantly reduced (-16%, P<0.001 and -10%, P<0.001, compared with baseline; Table 4), whereas CO had no effect on neither lag time (+1%) nor propagation rate (+1%). The changes in the FO group were significantly different form those of the corn oil group (lag time, P<0.001; propagation rate, P<0.001). Maximum diene concentration was not affected in either group. Neither lag time nor propagation rate correlated with blood glucose, HbA_1c, age, diabetes duration, or habitual fish intake.

Table 4 - Effect of corn oil or fish oil supplementation on LDL oxidation parameters, blood glucose, HbA_1c and weight.

Full table (51K)

Due to inadequate amount of available plasma, MDA concentration in LDL was only measured for a subgroup (FO, n=18; CO, n=17). LDL-MDA was significantly increased in the FO group after intervention (12%, P=0.004), whereas no changes were detected in the corn oil group (1%, P=0.875; Table 5). LDL-MDA changes differed between groups at borderline significance (P=0.057).

Table 5 - Effect of corn oil or fish oil supplementation on MDA concentration in LDL particles.

Full table (14K)

Effect of fish oil on lipids, blood glucose, and HbA_1c

Serum triglyceride decreased by 23% in the fish oil group after intervention (before, 2.35 mmol/l; after 1.81 mmol/l, P<0.001). No changes were seen for total and LDL cholesterol after fish oil supplementation, while HDL cholesterol increased significantly (before, 1.22 mmol/l; after, 1.28 mmol/l, P=0.019). No significant changes were detected in the corn oil group for any of the variables (Table 4).

Blood glucose and HbA_1c did not differ between the supplement groups after intervention (Table 4). There was a slight increase in blood glucose in the FO group after intervention (8%, P<0.05), but it was not significantly different from the change in the CO group (P=0.775). HbA_1c was not affected in any of the two groups.

Discussion

The present study was designed to investigate the effect of a daily vitamin E-enriched fish oil supplement containing 2.6 g EPA and DHA on selected CHD risk markers in type 2 diabetes. In the present paper we describe the impact on LDL susceptibility to oxidation and on glycemic control.

We found that fish oil supplementation resulted in a significant reduction of LDL lag time and propagation rate, 16 and 18%, respectively, while no changes were observed in the corn oil group. As a marker of in vivo LDL peroxidation we measured MDA concentrations in LDL. LDL-MDA (Table 5) clearly tended to increase after FO supplementation compared to CO supplementation (P=0.057). In the FO group LDL-MDA increased significantly (12%, P=0.004), while no differences were observed in the CO group. This marked decrease in both lag time and propagation rate and the increase in LDL-MDA suggests a pronounced effect of a moderate fish oil dose on LDL susceptibility to oxidation in type 2 diabetes. Earlier studies of nondiabetic subjects also found fish oil consumption to be associated with shortened LDL lag time and lowered propagation rate (Suzukawa et al, 1995; Tsai & Lu, 1997; Sørensen et al, 1998; Wander et al, 1996; Foulon et al, 1999). As the only exception, Brude et al (1997), observed neither lag time nor propagation rate changes, possibly due to insufficient statistical power (n=10). Some studies using alternative methods for assessing fish oil effects on LDL oxidation resistance reported no effect (Nenseter et al, 1992; Frankel et al, 1994; Brude et al, 1997). However, these studies may have been underpowered due to the relatively large analytical variance of the assays that they employed. Other studies also using alternative methods found similar to us a decreased resistance of LDL to oxidation (Lussier-Cacan et al, 1993; Suzukawa et al, 1995; Higdon et al, 2001).

Studies in which plasma concentrations of lipid peroxidation products were determined as a measure for in vivo lipid peroxidation found either no change or increased peroxidation after fish oil consumption. Eritsland et al. (1995) found no effect of fish oil on plasma TBARS, while others showed increased plasma TBARS concentrations after fish oil supplementation (Lussier-Cacan et al, 1993; Suzukawa et al, 1995; Tsai & Lu, 1997). Higdon et al (2000) found no effect of fish oil supplementation on plasma MDA in nondiabetic subjects. Finally, plasma F₂-isoprostanes were unaffected by fish oil supplementation in diabetic patients (Mori et al, 1999) and nondiabetics (Higdon et al, 2000). Of note is the fact that plasma TBARS, MDA, and F₂-isoprostanes all are influenced by the availability of specific PUFAs and thus may reflect changes in concentration of specific PUFAs rather than changes in in vivo lipid peroxidation.

Overall, we find it justified to conclude from well-powered studies using the CD assay that LDL oxidation lag time and propagation rate are both significantly reduced after FO supplementation even after vitamin E enrichment of FO. This conclusion apparently also holds for type 2 diabetics as shown in our study. Our finding of increased LDL-MDA plasma concentrations after fish oil supplementation further supports that fish oil consumption leads to augmented in vivo LDL oxidation, which certainly deserves attention.

We found a shorter lag time and a slower propagation rate after fish oil supplement, which have also been reported by others (Suzukawa et al, 1995; Wander et al, 1996; Sørensen et al, 1998; Foulon et al, 1999). The finding of a shorter lag time and concomitant reduced propagation rate after fish oil supplement may seem paradoxical: a shortened lag time is believed to reflect enhanced LDL oxidizability, while a lower propagation rate is commonly considered an indicator of decreased oxidation. As an explanation for this Brude et al (1997) suggest that EPA+DHA enrichment of the LDL particle leads to a more tight packing of the lipids, which might make the double bonds more resistant to free radical chain reactions attack, thus reducing the propagation rate. Wander et al (1998) speculate that the concentration of linoleic acid in LDL determines the rate of oxidation. Studies show that EPA and DHA are oxidized slower than linoleic acid in aqueous solutions despite their higher number of double bonds (Bruna et al, 1989). Thus, as EPA and DHA mainly replace linoleic acid in LDL the propagation rate would be reduced. As a third alternative we propose that the reduced propagation rate might be an artifact caused by accelerated CD degradation in FO-enriched LDL samples. This would reduce the accumulation of CD in the sample and thus lead to a less steep propagation rate.

Some studies have observed an increase in blood glucose in type 2-diabetes after fish oil supplementation (Friday et al, 1989; Vessby et al, 1992). However, a newly published meta-analysis based in 12 studies conducted on type 2 diabetic patients concluded that fish oil did not raise either blood glucose level or HbA_1c (Montori et al, 2000). Our findings do not support that FO supplementation deteriorates the glycemic control. Both groups experienced small and similar increases in blood glucose, which may be ascribed to the small body weight increases. The body weight increase may be explained by the oil supplement and/or a seasonal change in diet or physical habits.

In conclusion, our study demonstrates that LDL particles from type 2 diabetic patients become significantly less resistant to oxidation after FO supplementation. Our finding of borderline significant LDL-MDA changes further supports that in vivo LDL peroxidation is increased after FO supplementation. Those effects must be considered proatherogenic according to our current understanding of atherosclerosis and as supported by some epidemiological findings (Regnström et al, 1992; van de Vijver et al, 1999). However, fish oil may have beneficial effects on a variety of other mechanisms involved in atherosclerosis such as inflammation, thrombosis, and lipid metabolism (De Deckere et al, 1998) and therefore it is hard to conclude on the net effect of fish oil supplementation on atherosclerosis and primary prevention of CHD. Clinical studies on this topic are highly warranted. When it comes to secondary prevention of CHD the evidence is already there: fish and fish oil intake both lower heart mortality in patients with known CHD, but it seems to be mediated by mechanisms unrelated to atherosclerosis (Burr et al, 1989; GISSI-Prevenzione Investigators, 1999).

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