Transplantation Proceedings
Volume 34, Issue 1, February 2002, Pages 377-378

Oxidation of low-density lipoproteins in renal transplant recipients treated with tacrolimus

F. Cofan, D. Zambon, J. C. Laguna, E. Ros, E. Casals, M. Cofan, J. M. Campistol and F. Oppenheimer

^a Renal Transplant Unit, Lipid Section, Biochemistry Department, and Pharmacology Department, Hospital Clinic, University of Barcelona, Barcelona, Spain

Hyperlipidemia predisposes the patients to chronic rejection and is one of the most important cardiovascular risk factors in renal transplantation.[1] The pathogenesis of hyperlipidemia is multifactorial, but immunosuppressive treatment with cyclosporine and steroids is known to be a contributing factor. [2 and 3]

Several reports have demonstrated that oxidation of the LDL fraction is involved in the initiation and progression of arteriosclerosis.[4 and 5] It has been speculated that the high incidence of cardiovascular disease in renal transplant recipients is due to a greater susceptibility of LDL to oxidation. Various authors have reported that cyclosporine increases and azathioprine reduces the oxidizability of LDL. However, the effect of other immunosuppressants on lipid peroxidation is controversial.

The aim of this study was to analyze low-density lipoprotein (LDL) oxidizability in renal transplant recipients under treatment with tacrolimus.

Patients and methods

Patients

We evaluated the oxidizability of LDL from 18 stable renal transplant recipients (RTR) (mean age 52 ± 8 years, 13 men and five women) treated with tacrolimus-prednisone. The control group included 15 age-and gender-matched healthy subjects. Patients with nephrotic syndrome, creatinine levels over 2.5 mg/dL, or diabetes mellitus were excluded. None of the participants were taking lipid-lowering therapy or antioxidant supplements.

Lipid parameters

The following lipid parameters were determined: total cholesterol, low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), triglycerides (TG), apolipoprotein-B (ApoB), apolipoprotein-AI (ApoAI), lipoprotein (a) [Lp(a)], cholesterol-VLDL, ApoB-VLDL, ApoB-LDL, and TG-VLDL in both groups.

LDL oxidation

Venous blood was taken after an overnight fast and was collected in vacutainers containing EDTA. Plasma was separated by low-speed centrifugation. LDL was separated from plasma by discontinuous density gradient ultracentrifugation and dialyzed overnight against phosphate-buffered saline at 4°C to remove the EDTA. LDL oxidation was carried out according to the method of Esterbauer et al.⁵ The susceptibility of LDL to oxidation was monitored continuously by measuring conjugated diene formation (CD) during copper ion-mediated oxidation. The following parameters were evaluated: the lag phase or interval before start of oxidation (minutes), rate of conjugated diene formation (nmol CD/min/mg LDL protein), and maximum concentration of conjugated dienes (nmol CD/mg LDL protein).

Statistics

Statistical comparison of continuous variables between the two groups was performed using Student's t test for unpaired data. Simple correlations between variables were calculated using the Pearson correlation test. Significance was set at a P value of <.05.

Results

The group of renal transplant recipients had a more atherogenic lipid profile than the control group. Levels of total cholesterol (236 ± 56 mg/dL), LDL cholesterol (133 ± 30 mg/dL), TG (157 ± 79 mg/dL), TG-LDL (32 ± 12 mg/dL), TG-VLDL (104 ± 63 mg/dL), ApoB (127 ± 27 mg/dL), Lp(a) (31 ± 20 g/L), C-VLDL (33 ± 19 mg/dL) and ApoB-LDL (115 ± 25 mg/dL) in RTR were significantly greater when compared to the controls (TC 186 ± 18, P < .01; LDL 110 ± 18, P < .05; TG 84 ± 23 P < .01; TG-LDL 14 ± 2 P < .001; TG-VLDL 38 ± 22, P < .001; ApoB 97 ± 21, P < .01; Lp(a) 12 ± 19, P < .05, C-VLDL 9 ± 4, P < .001, and ApoB-LDL 90 ± 2, P < .05). However, no significant differences were found between the two groups for HDL cholesterol (RTR 60 ± 18 mg/dL vs control 60 ± 13) and ApoAI (RTR 164 ± 28 mg/dL vs control 163 ± 26).

Treatment with tacrolimus produced an LDL oxidation profile similar to that of healthy controls. Comparison between the RTR and control groups showed no significant differences in lag time (39 ± 12 minutes vs 38 ± 6 minutes) or rate of conjugated diene formation (40 ± 9 nmol vs 37 ± 8 nmol CD/min/mg LDL protein). Maximum CD production in the transplanted patients was slightly greater than in the controls (752 ± 108 vs 661 ± 108 nmol CD/mg LDL protein) (P < .1), though results did not reach statistical significance. A positive correlation was observed between rate of CD formation and maximum CD production in both the transplant recipients (r = 0.81, P < .001) and controls (r = 0.79, P < .01).

Discussion

It is well recognized that tacrolimus therapy has a less detrimental effect on the lipid profile than Neoral therapy, by inducing a smaller increase in totoal and LDL cholesterol.[6] Reports have shown that LDL from renal transplant recipients with neoral treatment is abnormally more susceptible to in vitro and in vivo oxidation.

Apanay et al showed a negative correlation between lag time and cyclosporine concentration; that is, patients with higher cyclosporine levels showed significantly higher LDL oxidizability when compared to a control group. Moreover, the authors found no differences in lag phase between renal transplanted patients with low cyclosporine levels and healthy individuals, although it should be mentioned that the number of patients in the series was small.[7] Ghanem et al reported that the lag time of in vitro LDL oxidation was shorter in cyclosporine-treated patients than in controls; they also found that mean LDL diameter was smaller because of a higher frequency of the LDL subclass pattern B. Additionally, concentrations of IgG and IgM autoantibodies against modified malondialdehyde were higher in kidney transplant recipients. [8] Sutherland et al found that lag time was significantly shorter in renal transplant patients when compared to control subjects and hemodialysis patients. No differences were found between azathioprine and cyclosporine treatment. However, paradoxically, the rate of conjugated diene formation and maximum diene production were significantly lower in the transplanted group when compared to healthy subjects. The author also observed substantial individual variation in lag time in renal transplant patients and those with shorter lag time were mainly women. [9]

Van den Dorpel et al analyzed the effect of conversion from cyclosporine to azathioprine treatment on the profile of LDL oxidizability. Treatment with cyclosporine increased the susceptibility of LDL to in vitro and in vivo oxidation. Conversion to azathioprine resulted in a more favorable effect on lipid profile, with a longer lag phase (in vitro oxidation), reduced titers of IgM and IgG autoantibodies against oxidized LDL (in vivo LDL oxidation), increased LDL size, and more frequent LDL subclass pattern B. Definitively, LDL particles were less susceptible to oxidative modification during conventional treatment with azathioprine.[10]

These data suggest that cyclosporine is an important risk factor for accelerating atherosclerosis, not only because of the increase in LDL levels, but also because it enhances LDL peroxidation. There is, however, one study evidencing that cyclosporine is not a direct pro-oxidant. In this interesting work, Devaraj et al reported that several concentrations of cyclosporine had no significant effect on LDL oxidation, as assessed by different systems of evaluation. Moreover, preincubation of LDL with cyclosporine did not affect LDL oxidation.[11]

The effect of tacrolimus on LDL oxidation is not well understood. Apanay et al showed that LDL oxidation in tacrolimus-treated patients was similar that of control subjects, but only five patients under tacrolimus treatment were studied.[7] In contrast, Varghese et al observed that LDL from tacrolimus-treated patients had significantly lower oxidation lag time and serum antioxidant activity when compared to neoral-treated patients. Vitamin C and E supplementation in the tacrolimus group provided protection against oxidation and normalized the lag-time phase. The author speculated that the differences in oxidation lag times between neoral- and tacrolimus-treated patients might be due to the presence of alpha-tocopherol in the formulation of neoral, which provides protection against oxidation. [12] In the present study we found no differences in the in vitro oxidation of LDL between renal transplantation patients receiving tacrolimus and the control group. These results may be explained by the fact that the lipid profile in our patients was more favorable than that of the subjects in other reports.

In conclusion, the in vitro susceptibility to oxidation of LDL from renal transplant recipients treated with tacrolimus was similar to that of the general population.

References

1. B.L. Kasiske, C. Guijarro, Z.A. Massy et al.J Am Soc Nephrol 7 (1996), p. 158.

2. S. Aakhus, K. Dahl and T.E. Wideroe J Intern Med 239 (1996), p. 407.

3. J.A. Kobashigawa and B.L. Kasiske Transplantation 63 (1997), p. 331.

4. V.P. Steinbrecher, H. Zhang and M. Lougheed Free Radic Biol Med 9 (1990), p. 155.

5. R. Ross Nature 362 (1993), p. 801.

6. C.G. Vela, J.P. Cristol, B. Descomps et al.Transplant Proc 32 (2000), p. 398.

7. D.C. Apanay, J.F. Neylan, M.S. Ragab et al.Transplantation 58 (1994), p. 663.

8. H. Ghanem, M.A. van den Dorpel, W. Weimar et al.Kidney Int 49 (1996), p. 488.

9. W.H. Sutherland, R.J. Walker, M.J. Ball et al.Kidney Int 48 (1995), p. 227.

10. M.A. Van den Dorpel, H. Ghanem, J. Rischen-Vos et al.Kidney Int 51 (1997), p. 1608.

11. S. Devaraj, D.J. Li, M. Vazquez et al.Free Radic Biol Med 26 (1999), p. 1064.

12. Z. Varghese, R.L. Fernando, G. Turakhia et al.Transplant Proc 30 (1998), p. 2043.