Early and Delayed Increase in Plasma Homocysteine Levels in Heart Transplanted Patients P.E. Lazzerini, P.L. Capecchi, M. Maccherini, F. Diciolla, M.R. Massai, F. Guideri, G.F. Lisi, A. Cuomo, M. Acampa, A. Giordano, M. Toscano, and F. Laghi Pasini
IN THE LAST FEW YEARS homocysteine (tHcy) has become recognized as an independent risk factor for atherosclerosis and thrombosis. Because the atherosclerotic process leading to coronary artery disease in some way resembles the features of cardiac allograft vasculopathy (CAV) in patients having undergone heart transplantation (HTX), some authors have suggested the possibility of a link between tHcy and CAV in HTX. Ambrosi et al1 first showed that HTX patients usually display higher plasma levels of tHcy than normal subjects. Other studies subsequently confirmed this finding.2– 6 Indeed, higher rates of hyperhomocysteinemia have been observed among HTX patients presenting evidence of graft vasculopathy, strongly suggesting a role of tHcy in the development of CAV.6,7 No predisposing factor has been correlated with hyperhomocysteinemia3–5 except positive correlation with serum creatinine, stressing the essential role of renal function in determining hyperhomocysteinemia.3–5 In contrast, no definite evidence suggests an association between folate, vitamin B6, or vitamin B12 deficit, and hyperhomocysteinemia in HTX patients.3– 6 However a possible limitation of available studies is the lack of sequential observations of plasma tHcy levels. To characterize the long-term time course of plasma concentrations of tHcy and to assess total load of tHcy in HTX patients, tHcy levels were sequentially measured in the plasma of HTX recipients. Moreover, factors known to be associated with hyperhomocysteinemia were also evaluated, including levels of folate3,4; vitamin B6 and B12; cholesterol and triglycerides8; as well as creatinine.3–5 Finally, the pharmacological parameters of cyclosporine blood levels4 as well as cyclosporine5 and prednisone dosage were examined for their influence on these observations. MATERIALS AND METHODS Forty-five patients (34 males, 11 females; mean age 54.1 ⫾ 10.3 years), who underwent HTX at the Cardiosurgery Institute of University of Siena, Italy, between 1994 and 2000, were studied at routine follow-up clinical visits from 0.25 to 66 months after transplantation. Transplantation was performed for coronary artery disease in
62% and for dilated cardiomyopathy of other origins in the remaining 38% (Table 1). The following biological parameters were measured: Total homocysteine after reduction or liberation from plasma proteins using tri-n-butylphosphine; and derivatization with a thiol-specific fluorogenic reagent, ammonium 7-fluoro-benzo-2 oxa-1,3-diazole-4-sulphonate. The derivatives were separated by reversed-phase high-performance liquid chromatography: The range in normal subjects is 5 to 15 mol/L. Serum folate and vitamin B12 levels using RIA method with a double antibody (Ab) in conjunction with polyethylene glycol I125-Co57 (Nuclear Laser Medicine, Settala; Milan, Italy). The ranges in normal subjects are 3 to 17 ng/mL and 200 to 950 pg/mL, respectively. Vitamin B6 concentrations were estimated by HPLC using an isocratic method (25°C) on a “reversed phase” column with a mobile phase of sodium azide: The range in normal subjects is 4.3 to 17.9 ng/ml. The serum creatinine, cholesterol and triglyceride contents were measured by standard methods (Syncron Chemical System LX 20, Beckman). The ranges in normal subjects were 0.6 to 1.2 mg/dL, 130 to 220 mg/dL and 40 to 190 mg/dL, respectively. Trough whole blood cyclosporine concentrations were assessed using an RIA method with a monoclonal Ab and I125 tracer (Cyclo-Trac, Incstar Corp.). In addition aspects of the immunosuppressive therapy included cumulative dose (CUD) of cyclosporine and prednisone ie the total amount of drug administered up to the time of each measurement. The reference group for the study of tHcy plasma levels was matched for age and gender among a large group of ambulatory outpatients (103 males, 32 females; mean age 55.8 ⫾ 19.0 years).
Statistical analysis Comparisons of values for HTX patients versus control subjects were performed using Student’s “t” test for unpaired data. Among From the Department of Internal Medicine, Section of Clinical Immunology (P.E.L., P.L.C., F.G., A.C., M.A., A.G., F.L.P.), Institute of Thoracic and Cardiovascular Surgery (M.M., F.D., M.R.M., G.L., M.T.), and the Department of Biomedical Technology, (M.R.M.), University of Siena, Siena, Italy. Address reprint requests to Franco Laghi Pasini, Istituto di Semeiotica Medica, U.O. Immunologia Clinica, Universita` di Siena, Policlinico “Le Scotte,” 53100 Siena, Italy. E-mail: [email protected]
HTX patients, the influence of “time” on tHcy levels was evaluated by a one-way analysis of variance (ANOVA). The difference in tHcy plasma levels at different times was assessed by the StudentNeuman-Keuls’ (SNK) test for multiple parameters. Comparison between data of patients within and over 24 months after heart transplant was performed by the Student’s t test for unpaired data. Values of probability less than 5% were considered as significant. Correlations between tHcy and examined parameters were evaluated by Spearman’s coefficient.
During the whole observation period a total of 111 blood samples were obtained including a mean number of 2.47 per patient (Table 1). The mean plasma levels of tHcy in the entire cohort of HTX patients were significantly higher than those in control subjects (20.6 ⫾ 7.9 mol/L and 13.2 ⫾ 6.4 mol/L, respectively; P ⬍ .001)(not shown), with 88.9% of patients presenting with hyperhomocysteinemia (⬎15 mol /L) (Table 2). The 90th percentile below which the control values lie was 17.5 mol/L 53.2%. At this value of HTX recipients and 10% of controls presented with hyperhomocysteinemia. Among heart transplant recipients, mean plasma levels of folate, vitamin B12, and vitamin B6 were 7.64 ⫾ 3.86 ng/mL, 410.9 ⫾ 181.8 pg/mL, and 7.28 ⫾ 3.07 ng/mL, respectively. Plasma vitamin concentrations that were so low as to be consistent with a true deficit occurred in 6.7%, 22.2%, and 20.0%, respectively (Table 2). The mean serum creatinine concentration was 1.37 ⫾ 0.52 mg/dL, with 55.6% displaying values ⬎1.2 mg/dL which we define as “renal failure”(Table 2). Mean serum levels of total cholesterol and triglycerides were 223.8 ⫾ 36.1 mg/dL and 158.9 ⫾ 57.2 mg/dL, respectively with incidences of 57.8%, as hyTable 2. Mean Plasma Concentration of the Parameters Under Study, and Percent Patients out of the Normal Range
percholesterolemic (⬎220 mg/dL) and 40.0%, hypertriglyceridemic (⬎190 mg/dL) (Table 2). Mean cumulative dose of cyclosporine and prednisone were 50.5 ⫾ 46.8 and 2.98 ⫾ 0.76 g, respectively; mean blood cyclosporine concentration was 374.2 ⫾ 94.4 ng/mL. Table 3 shows the correlation coefficients between tHcy and each of the other parameters. The probability values were only significant for a correlation with creatinine and time after transplantation. Scatter plot analyses allowed construction of a regression line only for tHcy/creatinine (Fig 1). Over time post-transplant tHcy plasma levels showed a statistically significant increase (P ⬍ .001, ANOVA) (Fig 2). However, tHcy plasma concentrations did not show a linear increase throughout the observation period. In fact, an initial, statistically significant (P ⬍ .05, SNK) increased level was observed as early as 3 to 6 months after transplantation, followed by a long-lasting steady state period. A second, delayed increase in tHcy occurred after 24 months posttransplant (Fig 2) that achieved a higher level of significance (P ⬍ .001, SNK). The occurrence of the second peak in tHcy plasma concentration suggested the opportunity to assess whether a change in the biological parameters under study had occurred after 24 months. Table 4shows a statistically significant difference between data obtained within and after 24 months posttransplantation for values of tHcy (p ⬍ .01, Student’s “t” test), and creatinine (P ⬍ .01, Student’s “t” test) but not for folate, vitamin B12, vitamin B6, cholesterol, or triglycerides. DISCUSSION
Hyperhomocysteinemia is an independent risk factor for coronary artery disease, stroke, and peripheral occlusive arterial disease.9 –17 More recently, a high prevalence of hyperhomocysteinemia has been observed in HTX patients,1 particularly in association with CAV.7 However, partially conflicting results have been obtained in a series of studies seeking to identify risk factors putatively involved in the pathogenesis of hyperhomocysteinemia. Previous observations suggest an association between tHcy and creatinine, folate, and age in one report,3 creatinine and cumulative dose of cyclosporine in another report,5 and finally creati-
INCREASE IN PLASMA HOMOCYSTEINE LEVELS
Fig 1. Regression between total homocysteine (tHcy) plasma levels and creatinine serum levels.
nine, cyclosporine blood concentration, folate, and time after transplantation in a third report.4 The present study observed a strong association between renal function and plasma levels of tHcy but ruled out the possible role of other additive factors including vitamins and lipid profile. In our opinion, possible limitations of previous single measurement studies may relate to the lack of sequential observations in single patients, leading to a “static” view of the phenomenon. In these reports, the value of tHcy represented as a snapshot of the whole period of observation, irrespective of changes in plasma levels of tHcy during different times after transplantation. The usefulness of a “dynamic” view of the phenomenon is also
supported by the importance of the time factor in the development of CAV, which becomes the leading cause of death after the first posttransplant year.6,18 –22 We report herein that tHcy plasma levels in HTX recipients show a progressive and biphasic increase, with an initial, early peak within 6 months and a second, delayed peak after 24 months posttransplantation. However, our attempt to identify factors possibly accounting for the further tHcy increase again only documented the role of creatinine. In conclusion, the novel finding of an initial early, and a second, delayed peak of tHcy separated by a long-lasting period of hyperhomocysteinemia provides evidence that HTX recipients undergo a progressively increasing load of
Fig 2. Time course of total homocysteine (tHcy) plasma levels after transplantation. Oneway anlysis of variance (ANOVA) F ⫽ 5.041, P ⬍ .001; n ⫽ 111. Student-Neuman-Keul’s test (SNK) ⴱ ⫽ P ⬍ .05; ⴱⴱ ⫽ P ⬍ .001.
LAZZERINI, CAPECCHI, MACCHERINI ET AL
Table 4. Mean Plasma Concentration of the Parameters Under Study Within and Over 24 Months After Transplant ⬍24 months
Student’s “t” test for unpaired data. NS, not significant.
tHcy, thus suggesting a possible association between tHcy burden and long-term complications of HTX such as CAV. Moreover, the finding that hyperhomocysteinemia develops early after HTX suggests that tHcy may play a role in the pathogenetic mechanisms of vascular derangement from the initial time after transplantation. These findings suggest the early and long-lasting therapeutic approach to hyperhomocysteinemia by folate supplementation which has been shown to reduce and maybe abolish, the increase in homocysteinemia even in subjects not presenting evidence of vitamin deficiency.23 REFERENCES 1. Ambrosi P, Barlatier A, Habib G, et al: Eur Heart J 15:1191, 1994 2. Berger PB, Jones JD, Olson IJ, et al: Mayo Clin Proc 70:125, 1995 3. Gupta A, Moustapha A, Jacobsen DW, et al: Transplantation 65:544, 1998
4. Cole DEC, Ross HJ, Evrovski J, et al: Clin Chem 44:2307, 1998 5. Cook RC, Tupper JK, Parker S, et al: J Heart Lung Transplant 18:420, 1999 6. Cooke GE, Eaton GM, Whitby G, et al: J Am Coll Cardiol 36:509, 2000 7. Ambrosi P, Garcon D, Riberi A, et al: Atherosclerosis 138:347, 1998 8. Tonstad S: Eur J Clin Invest 27:1025, 1997 9. Wilcken DEL, Wilken B: J Clin Invest 57:1079, 1976 10. Stampfer MJ, Malinow MR, Willett WC, et al: JAMA 268:877, 1992 11. Robinson K, Mayer EL, Miller DP, et al: Circulation 92: 2825, 1995 12. Mayer EL, Jacobsen DW, Robinson K: J Am Coll Cardiol 27:517, 1996 13. Coull BM, Malinow MR, Beamer N, et al: Stroke 21:572, 1990 14. Brattstrom L, Lindgren A, Israelsson B, et al: Eur J Clin Invest 22:214, 1992 15. Perry IJ, Refsum H, Ebrahim SB, et al: Lancet 346:1395, 1995 16. Eikelboom JW, Hankey GJ, Anand SS, et al: Stroke 31:1069, 2000 17. Malinow MR: J Nutr 126(suppl):1238S, 1996 18. Bieber CP, Hunt SA, Schwinn DA, et al: Transplant Proc 13:207, 1981 19. Gao SZ, Schroeder JS, Alderman EL, et al: Circulation 80:(suppl III):III-100 –III-105, 1989 20. O’Neill BJ, Pflugfelder PW, Singh NR, et al: Am J Cardiol 63:1221, 1989 21. Hosenpud JD, Shipley GD, Wagner CR: J Heart Lung Transplant 11:9, 1992 22. Miller LW: J Heart Lung Transplant 11:S1, 1992 23. Naurath HJ, Joosten E, Riezler R, et al: Lancet 346:85, 1995