Notorious Oxide

Nitrous oxide has an unusual pharmacological side-effect that is unrelated to its anesthetic action: it inactivates vitamin B₁₂. The inactivation of vitamin B₁₂ is irreversible and causes a subsequent accumulation of homocysteine in the cell, because methionine synthase, an important enzyme that converts homocysteine to methionine, depends on the active form of vitamin B₁₂ and is therefore also inactivated by nitrous oxide. The accumulation of homocysteine can be measured clinically by an increase in plasma total homocysteine (bound and unbound homocysteine), the magnitude of which is tightly correlated with the duration and dose of nitrous oxide exposure. This effect of nitrous oxide has long been known and repeatedly shown in animals and adult humans, but not in young children – until now. Together with a recent report in adolescents,¹ the study by Pichardo et al. in this issue of Anesthesiology,² provides the first evidence regarding the effects of nitrous oxide on homocysteine in young children, for which the authors should be congratulated. Not surprisingly, the authors found a 25% increase in plasma total homocysteine after nitrous oxide anesthesia in their cohort of 32 children aged 3 – 126 months. The absolute increase was small (+1.3 μmol/L) and substantially lower than found in a previous report (+9.4 μmol/L),¹ but this discrepancy may be explained by the substantially longer nitrous oxide exposure times in the latter study. Furthermore, Pichardo et al. obtained only a single homocysteine measurement 24 hours after nitrous oxide exposure, which may have missed the plasma homocysteine peak which typically occurs immediately after the cessation of nitrous oxide administration.

The study by Pichardo et al. thus confirms – not unexpectedly – that nitrous oxide causes a dose-dependent increase in plasma homocysteine in children. But what is the clinical relevance of this finding? What is the clinical relevance of an acute increase in plasma homocysteine? What is homocysteine?

Homocysteine is a non-essential amino-acid whose main role is in the re-methylation pathway of methionine. Homocysteine rose to prominence as a potential causative risk factor for early-onset atherosclerosis and cardiovascular disease after McCully published his seminal report in 1969³ on two children who died of premature atherosclerosis due to homocystinuria with massively elevated plasma homocysteine concentrations. Normal plasma total homocysteine concentrations in children range between 4–8 μmol/L (adults 8–12 μmol/L); children with homocysteinuria, if untreated, have 10-fold higher concentrations. The question then became are these homocysteine elevations confined to rare diseases such as homocystinuria, or was there a general association, perhaps even a causal one, between plasma homocysteine elevation, atherosclerosis and cardiovascular risk. Was homocysteine a cardiovascular risk factor?

To answer this scientific question, several well-conducted observational studies with thousands of patients were performed in the 1990s which unanimously showed that a chronic elevation of plasma homocysteine is a strong predictor of cardiovascular mortality in adult patients.^4,5 The studies showed a strong, graded risk increase with plasma homocysteine concentrations < 9 μmol/L conferring the lowest and concentrations > 20 μmol/L the highest cardiovascular risk. By then it was long known that elevated homocysteine can be fairly easily lowered by diet supplementation with B-vitamins (vitamin B₂, B₆, B₁₂, and folic acid [= vitamin B₉]), so several research groups instituted large homocysteine-lowering clinical trials with the goal to ameliorate the cardiovascular risk associated with elevated plasma homocysteine concentrations. The expectation was clear: lowering plasma homocysteine to “normal” levels would reduce cardiovascular morbidity and mortality. Unfortunately, everyone was wrong. Eight randomized controlled trials with more than 37,485 patients and a follow-up period of more than 5 years showed absolutely no benefit of B-vitamin supplementation on a reduction in cardiovascular morbidity and mortality (relative risk 1.01; 95% CI 0.97 – 1.05), despite a strong homocysteine-lowering effect.⁶ The homocysteine hypothesis has become a homocysteine controversy; in fact, most researchers now believe that homocysteine is a (bio-)marker rather than a mediator of atherosclerosis and cardiovascular disease.⁷

Now back to the question of nitrous oxide and the acute increase in plasma homocysteine. It is an irrefutable fact that nitrous oxide anesthesia will cause a dose- and duration-dependent increase in plasma homocysteine in all patients. However, is there evidence that acute hyperhomocysteinemia is of clinical consequence for patients? The answer to this question is actually more difficult and complicated than it may seem. At present, a few studies in human volunteers found that experimentally induced acute hyperhomocysteinemia by the way of methionine loading causes endothelial dysfunction.^8,9 This finding was replicated by Myles et al. who investigated nitrous oxide-induced hyperhomocysteinemia¹⁰, but the clinical relevance of the observed endothelial dysfunction is unknown.

Before we all rush to the conclusion that nitrous oxide-induced hyperhomocysteinemia is potentially harmful, a word of caution. Thus far, not a single study has conclusively proven that it is actually homocysteine that causes endothelial dysfunction. It may well be that homocysteine is nothing but an innocent bystander, a biomarker, for the reduced activity of the folate and methionine pathway and for the temporary vitamin B₁₂-inactivation caused by nitrous oxide. It is telling that the first publication linking nitrous oxide and homocysteine was in 1998¹¹ right at the peak of the “homocysteine hype”; all studies in the three decades before that investigated nitrous oxide’s effects on vitamin B₁₂ – and there were many! – focused exclusively on the reduction in methionine.^12,13 At present, even the evidence regarding nitrous oxide and potential adverse cardiovascular outcomes is weak and ambiguous. In adults, two clinical trials are currently investigating the risk of perioperative myocardial infarction related to nitrous oxide (ENIGMA-2 and Vitamins in Nitrous Oxide [VINO]-trial), and it appears prudent to wait until definitive evidence from these trials is available before evaluating the risk associated with nitrous oxide in adults. In children, perioperative myocardial infarction is of no concern and the only potential adverse cardiovascular outcome related to acute hyperhomocysteinemia is venous thrombosis. However, there is currently zero evidence to support this notion.

So, how should we therefore interpret the findings of Pichardo et al. in this issue of Anesthesiology when serious cardiovascular outcomes related to nitrous oxide are unlikely to be expected in children? The small increase in plasma homocysteine observed in the study is probably of little clinical relevance and we should not attempt to change our clinical practice based on that. What we should be doing, however, is to look at outcomes beyond the operating room. Does the temporary vitamin B₁₂-inactivation caused by nitrous oxide and indicated by an acute increase in plasma homocysteine result in adverse outcomes typically seen in vitamin B₁₂-deficiency such as neuropathy or megaloblastic anemia in children? This question has never been investigated and is unanswered.

Isn’t it quite remarkable how little definitive evidence we have regarding nitrous oxide, the oldest anesthetic drug in our armamentarium? Many questions are still unanswered. Is the use of nitrous oxide safe in vulnerable patient populations such as patients with coronary artery disease or children? Or may nitrous oxide actually confer significant benefits to patients as a very interesting recent study suggest, indicating a significantly lower risk of chronic pain after surgery?¹⁴ Is an acute increase in plasma homocysteine of any clinical relevance, recognizing that just because we can easily measure a metabolite in the blood such as homocysteine does not necessarily mean it is important and/or related to observed clinical effects. Answers to these questions and definitive evidence are crucial before we can fully assess the benefit/harm ratio of nitrous oxide in children and adults.