Although there is substantial evidence that hyperhomocysteinaemia (HHcy) is a risk factor for cardiovascular disease (CVD; Refsum et al., 1998), the mechanisms underlying this association have not been elucidated. It has been demonstrated, however, that copper augments the inhibitory effect of homocysteine (Hcy) on nitric oxide (NO)-mediated relaxation of the rat aorta through increased superoxide (O₂^▪-) formation in vitro (Emsley et al., 1999). O₂^▪- reacts with NO thereby reducing the bioavailability of NO. It was suggested, therefore, that copper status may determine the vasculopathic impact of Hcy, in vivo (Emsley et al., 1999). In order to test this hypothesis, the effect of penicillamine (a copper chelator) administration on NO-dependent relaxation and O₂^▪- formation in the aortae of HHCy rabbits induced with a methionine (Me)-enriched diet was studied.

New Zealand White rabbits (3 kg) were fed a Me-rich (20 g / kg chow) diet for one month. In another group of Me-fed rabbits, penicillamine was administered orally (1 mg / kg / day). After one month, plasma samples were taken, rabbits killed with a barbiturate overdose and aortae excised and rings prepared. Rings were mounted in an organ bath and contraction elicited with phenylephrine and relaxation with acetylcholine (Ach). Superoxide dismutase (SOD)-inhibitable O₂^▪- release was measured spectrophotometrically. Data are expressed as mean ± SEM; n = 6, for all experiments. One-way ANOVA was used for statistical analysis. All studies were carried out in accordance with Home Office approval.

After one month, the Me-enriched diet elicited a marked increase in plasma Hcy levels compared to controls (214 ±35 vs 16 ±2 mmoles/l, p<0.001). Penicillamine administration had no effect on Hcy levels but significantly reduced plasma total copper in Me-fed rabbits (6.3 ±0.6 µmoles/l) compared to controls (10.2 ±1.2 mmoles/l). Ach-induced relaxation was significantly impaired in the Me-fed group (38.4 ±4%) which was significantly reversed by penicillamine administration (58 ±7%) compared to controls (68 ±6%). O₂^▪- release was significantly enhanced by Me-rich diet (0.63 ± 0.2 nmoles / mg tissue/hr) compared to controls (0.08 ±0.002 nmoles / mg tissue / hr), an effect again reversed by the administration of penicillamine (0.04 ±0.002 nmoles / mg tissue / hr).

This study consolidates that HHcy augments the formation of arterial O₂^▪- which in turn reduces NO bioavailability. Although a direct effect of methionine on these systems cannot be excluded, it has yet to be established whether methionine is a risk factor for CVD in its own right. Notwithstanding this possibility, the present study demonstrates that the in vivo chelation of copper prevents the induction of arterial oxidative stress by HHcy, consolidating the hypothesis that copper plays a role in mediating Hcy-induced vasculopathy. Further studies are required to determine whether this effect is mediated by copper-Hcy or copper-methionine interactions, or indeed both.

Refsum , H., et al., (1998) Annu Rev Med, 49: 31-62.
Emsley , A., et al., (1999) Br J Pharmacol, 126 : 1034-1040.