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Ann Thorac Surg 2002;74:1553-1557
© 2002 The Society of Thoracic Surgeons

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Original article: cardiovascular

Sustained increases of plasma homocysteine, copper, and serum ceruloplasmin after coronary artery bypass grafting

^a Bristol Heart Institute, Bristol, United Kingdom
^b Department of Clinical Biochemistry, University of Bristol, Bristol Royal Infirmary, Bristol, United Kingdom

Accepted for publication May 19, 2002.

* Address reprint requests to Dr Jeremy, Bristol Heart Institute, Bristol Royal Infirmary, Bristol BS2 8HW, United Kingdom.
e-mail: j.y.jeremy{at}bris.ac.uk

	Abstract

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Background. Homocysteine (Hcy) is an independent risk factor for coronary artery disease, but there are no reports on Hcy levels in patients undergoing coronary artery bypass graft (CABG) surgery. Interactions between Hcy and copper may mediate the vasculopathic impact of Hcy, and this may play a role in vein graft failure. The aim of this study was to assess the perioperative levels of Hcy, copper, ceruloplasmin (CP), folate, and vitamin B₁₂ in patients undergoing myocardial revascularization surgery.

Methods. Blood samples were taken from 55 consecutive patients undergoing elective conventional CABG (43 male; mean age, 63.2 ± 5.2 years) 1 day preoperatively and postoperatively at 1 day, 6 days, and 6 weeks. Hcy, copper, CP, red cell folate, vitamin B₁₂, creatinine, and C-reactive protein (CRP) were then measured using standard clinical chemistry methods. The same protocol was applied to 10 patients (7 male; mean age, 63.3 ± 5.2 years) undergoing off-pump coronary artery bypass (OPCAB) surgery.

Results. In the conventional CABG group, there were significant increases in the plasma concentrations at 6 days and 6 weeks postoperatively of Hcy (from 10.1 to 11.6 and 13.5 µmol/L, respectively), plasma copper (from 13.5 to 20.3 and 18.5 µmol/L), and serum ceruloplasmin (from 0.3 to 0.41 and 0.44 g/L). CRP and vitamin B₁₂ were elevated at 6 days but not 6 weeks after the operation. In contrast, red cell folate and creatinine were not significantly changed. The subgroup analysis for the OPCAB patients showed the same trend as for the conventional group.

Conclusions. Coronary surgery precipitates a significant and sustained increase in the blood concentrations of Hcy and copper, which is not due to a decrease in folate and vitamin B₁₂, altered renal function, or inflammation. Further studies are required to establish whether the concomitant increase in Hcy and copper plays an etiological role in vein graft disease.

	Introduction

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Mild hyperhomocysteinemia has been widely purported to be an independent risk factor for coronary artery disease (CAD), although the underlying pathophysiology is not fully elucidated [1, 2]. High levels of Hcy may be an important risk factor also for vein graft disease because Hcy: 1) impairs endothelial function, including a reduction of nitric oxide (NO) formation; and 2) promotes vascular smooth muscle cell (VSMC) proliferation, both of which are key events in vein graft neointima formation, atherogenesis, and thrombosis [3–5]. Iwama and associates have also reported that elevated plasma levels of Hcy are related to atherosclerotic lesions of saphenous vein grafts after coronary artery bypass graft surgery (CABG), when assessed at 1 to 13 years after surgery [6].

Mechanistically, Hcy may elicit its pathologic effects through the auto-oxidative generation of superoxide and hydrogen peroxide [7]. Several studies have also demonstrated that copper facilitates the auto-oxidation of Hcy and impairs NO formation though its reaction with superoxide [3, 7–10], suggesting that alterations in copper rather than absolute levels of Hcy may determine the vasculopathic impact of Hcy [3, 9]. Indeed, prospective studies have also demonstrated that elevated plasma copper levels correlate positively with CAD [11].

The aim of this prospective study, therefore, was to assess the independent effect of myocardial revascularization techniques on perioperative levels of Hcy, copper, ceruloplasmin, folic acid, vitamin B₁₂, creatinine, and C-reactive protein (CRP).

	Material and methods

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Patient selection
Fifty-five consecutive elective patients undergoing first-time conventional CABG were selected for the study. Eligibility for surgery was based upon the medical history and the most recent angiogram. Exclusion criteria included only emergency or salvage operation. Subsequently, a further group of 10 patients undergoing off-pump coronary artery bypass (OPCAB) surgery were selected according to the same exclusion criteria. These were computer-matched for age, gender, extent of coronary disease, and ejection fraction to 10 patients selected from the whole conventional group, and comparison was carried out. The study was approved by the United Bristol Health Care Trust Ethics Committee, and all patients gave informed consent.

Anesthetic and surgical techniques
Anaesthetic technique was standardized for all patients and consisted of intravenous anesthesia with propofol and remifentanil infusions. Neuromuscular blockade was achieved with pancuronium bromide or vecuronium, and the lungs were ventilated to normocapnia. Heparin was given at a dose of 300 mg/kg to achieve a target activated clotting time (ACT) of 480 seconds or more before commencement of cardiopulmonary bypass (CPB). In the OPCAB subgroup, heparin was given at a dose of 150 mg/kg to achieve a target ACT of 300 seconds or more before the first anastomosis. All cardiac procedures were undertaken through a median sternotomy.

Conventional cABG
CPB was instituted using ascending aortic cannulation and two-stage venous cannulation of the right atrium as previously described [12, 13]. Nonpulsatile flow was used with flow rates throughout bypass of 2.4 L/m² /min. Systemic temperature was kept between 34°C and 36°C. Myocardial protection was achieved by using intermittent anterograde hyperkalemic warm blood cardioplegia [12, 13].

OPCAB surgery
The method of exposure and stabilization to perform the anastomosis consisted of the technique previously described by our group [14]. The target vessel was exposed and snared for few seconds above the anastomotic site using a 4-0 Prolene suture with a soft plastic snugger, to allow insertion of an intracoronary shunt. A pressure stablizer was then used to perform the distal anastomosis.

Fluid management
Extracorporeal circuit was primed with 1,000 mL of Hartmann’s solution, 500 mL of Gelofusine (Braub-Misugen, Misugen, Germany), 0.5 g/kg mannitol, and 6,000 IU heparin. When additional volume was required during CPB, this consisted of Gelofusine (hematocrit > 22%) or red blood cells (RBC) (hematocrit < 22%). Fluid management postoperatively consisted of a 5% dextrose infused at 1 mL/kg/h with additional Gelofusine or blood to maintain normovolemia and a hematocrit more than 24%.

Blood collection and measurements
Blood samples for the measurement of Hcy concentrations were taken from fasted patients and placed on ice into tubes containing EDTA, and the plasma was separated immediately. Blood samples for the measurement of plasma copper concentrations were taken into copper-free, lithium-heparin tubes and samples for serum concentrations of ceruloplasmin, creatinine, and vitamin B₁₂ into plain tubes before centrifugation and separation. EDTA anticoagulated whole blood was used for the measurement of red cell folate. Samples were collected from the central venous line from fasting patients in the morning at the following times: before induction of anesthesia (T0), at 1 and 6 days, and 6 weeks (T1, T2 and T3, respectively) postoperatively. Samples were then analyzed using routine methods. Homocysteine was assayed by reversed-phase high-performance liquid chromatography [15], copper by flame atomic absorption spectroscopy [16], creatinine by the kinetic Jaffé method [17], ceruloplasmin by immunoturbidimetry [18], CRP by latex-enhanced immnoturbidimetry [19], and red cell folate and vitamin B₁₂ by immunoassay [20].

Statistical analysis
Data were analyzed using the computer program Graphpad Instat and Microsoft Excel. Because the blood values followed exponential distributions, the data were logged and geometric means and confidence intervals calculated and the Wilcoxon test for matched pairs then performed. The Spearman rank test for nonparametric data was used for correlation analyses. For the purpose of the subanalysis between on-pump and OPCAB groups, comparisons between preoperative characteristics and postoperative variables were made using Fisher’s exact test or ² test where appropriate. For continuous variables, two-way analysis of variance for repeated measurements was used to assess group differences with interaction of time and treatment effects. Differences with a p value of less than 0.05 were considered significant. All p values are two-tailed.

	Results

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Baseline and intraoperative characteristics are given in Table 1. No deaths occurred. Major postoperative complications included two myocardial infarctions and one transient stroke. In the larger group, there were significant increases in plasma concentrations (geometric means) at 6 days and 6 weeks postoperatively of: homocysteine (from 10.1 to 11.6 and 13.5 µmol/L, respectively) (Fig 1 ), plasma copper (from 13.5 to 20.3 and 18.5 µmol/L, respectively) (Fig 1), and serum ceruloplasmin (from 0.3 to 0.41 and 0.44 g/L, respectively) (Fig 2 ), when compared with baseline levels. In contrast, serum CRP values increased significantly only at 6 days postoperatively but returned to preoperative levels 6 weeks after surgery (Fig 2). Serum vitamin B₁₂ concentrations increased significantly from preoperative levels of 258 to 386 ng/L at 6 days postoperatively, but were not significantly different at 6 weeks (Fig 3). There were no significant changes in red cell folate or serum creatinine concentrations at any time points (Fig 3). One day after surgery, there was a similar reduction in all values, which can be ascribed to a direct impact of hemodilution during CPB (Figs 1–3].

View larger version (31K): [in this window] [in a new window]   Fig 1. Concentrations of (a) plasma homocysteine and (b) plasma copper in 55 patients who have undergone CABG: T0 = preoperative; T1 = 1 day postoperative; T2= 6 days postoperative; T3 = 6 weeks postoperative. Data are expressed as geometric mean values ± 95% confidence intervals. Statistical analyses were carried out on previous postsurgical levels. *p < 0.001; **p < 0.0001.

View larger version (22K): [in this window] [in a new window]   Fig 2. Concentrations of (a) serum ceruloplasmin and (b) serum C-reactive protein (CRP) in 55 patients who have undergone CABG: T0 = preoperative; T1 = 1 day postoperative; T2 = 6 days postoperative; T3= 6 weeks postoperative. Data are expressed as geometric mean values ± 95% confidence intervals. Statistical analyses were carried out on previous postsurgical levels. *p < 0.001; **p < 0.0001.

View larger version (28K): [in this window] [in a new window]   Fig 3. Concentrations of (a) red cell folate, (b) serum vitamin B12, and (c) serum creatinine in 55 patients who have undergone CABG: T0 = preoperative; T1 = 1 day postoperative; T2 = 6 days postoperative; T3= 6 weeks postoperative. Data are expressed as geometric mean values ± 95% confidence intervals. Statistical analyses were carried out on previous postsurgical levels.*p < 0.001.

The subgroup analysis of the OPCAB patients showed the same trend as the conventional CABG population, with the exception of Cu²⁺, which was significantly greater in in the OPCAB group at 6 days (21.9 ± 2.5 vs 19.0 ± 2.6 µmol/L; p = 0.05) (Table 2).

	Comment

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

Several factors are known to promote an increase in plasma Hcy concentrations, most notably, decreased blood folate and vitamin B levels, renal dysfunction, and inflammation [1, 2, 23]. However, in the present study, folate levels were unchanged more than 6 weeks after CABG and were not below normal levels preoperatively. Vitamin B₁₂, on the other hand, actually increased at 6 days and was unchanged 6 weeks after surgery, indicating that folate and B₁₂ did not determine the alterations in Hcy. Because serum creatinine concentrations were unaltered after surgery, the increase in plasma Hcy concentrations could not be ascribed to alterations of renal function. Furthermore, the prolonged elevation of Hcy could not be ascribed to acute-phase inflammation because CRP was elevated at 6 days after surgery but had returned to baseline after 6 weeks. Finally, CPB as a determinant of these increases in Hcy was discounted because Hcy levels were no different between the OPCAB and the conventional groups.

Both plasma copper and serum ceruloplasmin levels were also significantly increased compared with preoperative levels in patients undergoing CABG at 6 days after surgery, an elevation that persisted more than 6 weeks. The strong correlation between copper and ceruloplasmin concentrations reflects the fact that the majority of plasma copper is bound to ceruloplasmin [11]. Blood copper levels have also been correlated with those of Hcy in 65 patients with peripheral arterial disease but not in age-matched controls [24]. Copper levels are also increased in hyperhomocysteinemic patients [25]. In contrast, we found no correlation with Hcy and copper at any time point, indicating that the relationship may be specific for peripheral vascular disease.

Both CABG [21] and surgery, per se [26], are associated with an increase blood levels of copper at 5 to 6 days postoperatively, although this latter study did not investigate copper levels at 6 weeks after surgery. This would seem to indicate that the present changes are not specifically associated with the CABG procedure. Indeed, the only significant difference identified between the two small subgroups of patient undergoing on- and off-pump coronary surgery was in copper levels at 6 days after surgery. It appears, therefore, that CPB, per se, is not a determinant of these longer-term changes changes. Nevertheless, it is possible that the multiplicity of inflammatory humoral responses, involving the complement, coagulation, fibrinolytic, and kallikrein cascades that occur during exposure of the blood to the artificial surfaces of the cardiopulmonary bypass, may interact with Hcy or copper to promote vein graft disease [12, 13, 27].

Notwithstanding the mechanisms responsible for these alterations in circulating values, the increases in Hcy, copper, and ceruloplasmin may be of relevance to the initiation and augmentation of vein graft thickening. There is now considerable evidence that oxygen free radicals (including superoxide and hydrogen peroxide) are central to the vasculopathic impact of Hcy [7], which may be augmented by copper [3, 9]. In turn, superoxide may promote vein graft disease because it promotes VSMC proliferation, endothelial lysis, apopotosis, and lipid oxidation, all key events in the progression of vein graft thickening [5, 28]. Likewise, the increases in Hcy and copper may also exert an impact on arterial conduits, because all studies that have demonstrated an effect of Hcy and copper on oxidant stress and NO were carried out in arterial tissues [3, 9]. Further studies comparing responses to Hcy and copper in arteries versus veins would be valuable.

Hcy has been shown to reduce nitric oxide (NO) formation both in vivo and in vitro [8, 9], possibly through the reaction with superoxide to form peroxynitrite, thereby reducing NO bioavailability [7]. Copper markedly increases the inhibitory potency of Hcy on NO-mediated arterial relaxation through H2O2 and superoxide [9]. Although copper is tightly bound to proteins, ceruloplasmin can catalyze the auto-oxidation of Hcy [29]. Thus, copper need not be dissociated from protein binding sites for vasculopathic redox reactions to take place.

In conclusion, the results of the present study demonstrate that after CABG, there is a significant increase in plasma Hcy, copper, and ceruloplasmin that is manifest within 1 week after the procedure and, more importantly, persists over the ensuing 6 weeks. Given that neointima formation occurs within this time scale, these changes may be of importance to the pathophysiology and treatment of late vein graft disease. The significant increase in Hcy and copper may contribute interactively to vein graft thickening through oxidative stress-mediated mechanisms. It is also suggested that it may not only be the absolute levels of Hcy, but the relative concentrations of copper (and ceruloplasmin) that are of pathologic significance, at least in relation to vein graft failure. If this should be the case, then the administration of antioxidants may prove beneficial in CABG patients. The administration of folate and B vitamins may have little effect, because they are not reduced in this scenario. Longer-term studies are required to determine whether these changes of Hcy and copper actually influence neointima formation in CABG patients. Whether surgery, per se, elicits these changes also warrants investigation.

	Acknowledgments

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We thank the British Heart Foundation and Garfield Weston Trust for their support, Drs Rosemary Greenwood and David Stansbie for their expert advice, and Dr Jeff Scott for his analytical expertise

	References

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Gianni D. Angelini Sudath P. Talpahewa Raimondo Ascione Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jeremy, J. Y. Articles by Ascione, R. Search for Related Content PubMed PubMed Citation Articles by Jeremy, J. Y. Articles by Ascione, R. Related Collections Coronary disease