Effect of Nitrous Oxide Exposure during Surgery on the Homocysteine Concentrations of Children

• In adults, nitrous oxide inhalation increases plasma total homocysteine, which possibly has cardiovascular consequences, but there are no studies in small children

• Nitrous oxide anesthesia in children increased plasma total homocysteine concentrations the morning after surgery

• The duration of exposure, initial plasma total homocysteine, and serum vitamin B12 concentrations were associated with this increase

HOMOCYSTEINE is an intermediary metabolite in the body produced by demethylation of the essential amino acid methionine. Homocysteine may be remethylated to methionine by the enzyme methionine synthetase, requiring the presence of the reduced form of vitamin B12 and methyltetrahydrofolate as a methyl donor. Homocysteine can be converted to the conditionally essential amino acid cysteine by the enzymes cystathionine ß-synthase and cystathionine γ-lyase with vitamin B6 serving as a cofactor.1Alternatively, homocysteine can be converted to methionine by the enzyme betaine-homocysteine methyltransferase.2 

It has been shown that nitrous oxide increases plasma total homocysteine (tHcy) concentrations in adolescents3and adults4,–,11as a result of the conversion of vitamin B12 to its metabolically inactive form.12,–,14Similarly, nitrous oxide exposure has been shown to be associated with an increased risk of postoperative cardiovascular events in adults.15This has led to speculation that the elevation in plasma tHcy concentration after nitrous oxide exposure may play a specific role in these complications; however, this remains unclear. Indeed, increased postoperative plasma tHcy concentrations after nitrous oxide exposure have been shown to be associated with myocardial infarction and stroke,5myocardial ischemia9and impaired endothelial function6in adults. No evidence exists to suggest an association between nitrous oxide exposure and postoperative cardiovascular events in children. At the population level, both nutritional (folate and vitamins B12 and B6) and genetic factors have been associated with changes in plasma tHcy concentrations.16Synthesis of 5-methyltetrahydrofolate is regulated by the enzyme methylenetetrahydrofolate reductase (MTHFR).17The common single nucleotide polymorphism (C677T) yields an enzyme with reduced activity, often an important genetic determinant of plasma tHcy concentration.17,–,19 

Although there is a large body of literature investigating postoperative plasma tHcy concentrations in adults after exposure to nitrous oxide, limited data exist on children.3Therefore, the purpose of this study was to evaluate if young children exposed to nitrous oxide demonstrate an increase in plasma tHcy concentrations the morning after surgery and whether blood concentrations of folate and vitamins B12 and B6 were associated with the increase in plasma tHcy concentrations.

The study was approved by The Human Research Ethics Committee at The Hospital for Sick Children, Toronto, Ontario, Canada. Informed written consent from parents and assent from the child, where appropriate, were obtained before participation. Children were recruited from preoperative clinics in the Plastics Surgery Division at The Hospital for Sick Children from February to July 2009. Eligible children needed to weigh ≥3 kg, be younger than 11 yr, and be scheduled for surgery with general anesthesia expected to last ≥2 h using nitrous oxide at an inspired concentration ≥50%. Children were not eligible if they were 11 yr of age or older because puberty is known to lead to sex differences in plasma tHcy concentrations.20Children parenterally fed, exposed to nitrous oxide 30 days before surgery, diagnosed with active disease, and/or using medication(s) known to affect plasma tHcy concentrations (e.g. , kidney disease, methotrexate treatment) were excluded.

A blood sample (after more than 8 h of fasting) was obtained 14 ± 7 min after induction of anesthesia (preoperative), and a second specimen was collected 24 ± 3 h thereafter (next morning, after surgery). Children were not fasted before collection of the postoperative blood draw. Given the age and size of the children, a limited volume of blood could be drawn. In the event that there was insufficient plasma or serum to perform all planned biochemical analyses, they were prioritized as follows: (1) plasma tHcy, (2) plasma folate, (3) plasma methylmalonic acid (MMA), (4) serum vitamin B12, (5) MTHFR C677T genotype, and (6) plasma vitamin B6. Plasma tHcy concentrations were determined before and after surgery. Total plasma folate, serum vitamin B12, plasma MMA (for cobalamin status), and plasma pyridoxal-5-phosphate (for vitamin B6 status) were measured in blood collected before surgery. The normative cutoff values for the vitamin biomarkers and citations used to support them can be found in table 1. A whole blood sample was obtained for MTHFR C677T genotyping. Intraoperative data, such as type of surgery and duration of exposure to nitrous oxide, were collected from anesthesia records. Demographic, history of vitamin(s) supplement use, and postoperative dietary intake data were obtained by interviewing parents.

Blood samples were collected in EDTA and serum separator tubes. All samples were immediately cooled and kept on ice until centrifuged (1,250g , 4°C, 15 min). Blood samples were processed within 30 min of the blood draw, and aliquots were stored at −80°C until analyzed. With the exception of plasma folate, all blood samples were analyzed as a single batch for each analyte. Plasma tHcy concentration was determined by liquid chromatography-electrospray tandem mass spectrometry as described by Rafii et al. .21Total plasma folate concentrations were determined by microbiologic assay using the test organism Lactobacillus rhamnosus  (ATCC 7649; American Type Tissue Culture Collection, Manassas, VA), as described by Molloy and Scott.22Serum vitamin B12 was analyzed by solid-phase competitive chemiluminescent enzyme immunoassay and the IMMULITE 2500 Analyzer (Diagnostics Products Corporation, Los Angeles, CA). The concentration of plasma MMA was measured by chemical ionization gas chromatography mass spectrometry according to the method described by Yazdanpanah et al.  23Plasma vitamin B6 (pyridoxal-5-phosphate) concentrations were measured by reversed-phase high-performance liquid chromatography with chlorite postcolumn derivatization, as described by Rybak and Pfeiffer.24Genotyping of MTHFR C677T (CC, CT, TT) was determined by allele-specific, real-time polymerase chain reaction,25using the TaqMan SNP Genotyping Assay for MTHFR (Applied Biosystems, Foster City, CA).

Because there were no available data in children at study initiation, we estimated the sample size from previous studies using adults. A thorough review of the literature pertaining to the use of nitrous oxide of any duration during anesthesia in adults suggested that on average a 0.55 SD increase in plasma tHcy concentration would be expected.4,–,7,9,–,11,26We estimated that a final sample size of 28 children would enable us to observe a 0.55 SD difference in plasma tHcy concentrations before surgery versus  after surgery with 80% power and an α level of 0.05.

Distributions of continuous variables were assessed for normality using the Kolmogorov–Smirnov test, and skewed data were transformed where possible. Parametric data are reported as mean ± SD and nonparametric data as median (first, third quartile). Student t  tests or Wilcoxon signed-rank test were used for two-group comparisons of parametric and nonparametric data, respectively. Linear univariate regression analysis was performed to identify significant associations between various sociodemographic variables and blood biochemical indices and the main outcome variables: preoperative plasma tHcy concentration and increase in plasma tHcy concentration after anesthesia. Multivariate analyses (general linear model) were then performed, including variables from the univariate analyses shown to be associated with the increase in plasma tHcy after anesthesia (P < 0.05). All statistical analyses were performed with SAS software (version 9.1; SAS Institute Inc., Cary, NC) and a two-tailed P < 0.05 was considered statistically significant.

The sample consisted of 18 boys and 14 girls with a median (first, third quartile) age of 11 (5, 40.5) months and weight of 9.2 (7.1, 15.5) kg. Subjects underwent surgery to repair a birth defect but were otherwise healthy: oral cleft, 17; speech repair, 3; brachial plexus repair, 3; facial correction, 4; other, 5. Only three children consumed multivitamin supplements containing folic acid (260 or 400 μg) and vitamins B12 (6 μg) and B6 (2 mg). Results for the vitamin biomarkers can be found in table 1. The median preoperative plasma tHcy concentration of all children was 5.1 (4.1, 6.4) μM. The only measured variables associated with preoperative plasma tHcy were vitamin B12 and MMA concentrations (table 2).

For all children, anesthesia was induced using nitrous oxide. The median (first, third quartile) duration of exposure to nitrous oxide was 180 (142.0, 217.5) min, and the median concentration was 60% (60%, 65%) in the inhaled gas mixture. Six of 28 (21%) children with normal, age-appropriate, preexposure plasma tHcy concentrations had postoperative plasma tHcy concentrations greater than cutoff values (fig. 1). The median increase in plasma tHcy concentration was 1.2 μM (0.1, 2.7 μM; P < 0.0001), an increase of 25% (2%, 42%). This increase remained statistically significant after removing the results for three children whose plasma tHcy concentrations increased more than 2 SD (outliers) above the mean of the other children (6.7 ± 2.8 vs.  5.2 ± 1.5 μM, respectively; P < 0.0003). Two of the children whose tHcy values were outliers had been exposed to nitrous oxide for more than 700 min. The third child presented with a preoperative plasma MMA concentration indicative of vitamin B12 deficiency. All 32 subjects were included in all other statistical analyses. Nine children exposed to more than 3.5 h of nitrous oxide (median 246[225,667] min) experienced an approximate 80% increase in plasma tHcy concentration (median 4.1 [4.0, 6.1]vs.  7.5 [4.7, 19.3]μM, respectively; P = 0.0078).

The duration of nitrous oxide exposure (minutes) was associated with the increase in plasma tHcy concentration before and after surgery (R  ²= 0.696, P = <0.0001; table 2, fig. 2). We observed a greater spread in tHcy concentrations at higher nitrous oxide exposures. To correct for heteroscedasticity of the data and reduce the skewness of both the dependent and independent variables and, consequently, the high leverage of the more extreme values, we reanalyzed our data using a fourth root transformation of both the dependent and independent variables after replacing some small negative values with zero. Our conclusions were unchanged when this model was used.

When expressed as total nitrous oxide exposure (minutes × nitrous oxide concentration), the association between duration of nitrous oxide exposure and the increase in tHcy concentration after surgery remained unchanged (R  ²= 0.687, P = <0.0001). In a similar manner, preoperative plasma tHcy concentrations were associated positively with the increase in plasma tHcy concentrations after nitrous oxide exposure (table 2). Serum vitamin B12 concentrations were associated inversely with the increase in plasma tHcy concentrations after nitrous oxide exposure (R  ²= 0.149; P = 0.046). The impact of nitrous oxide exposure on the increase in Hcy concentration by MTHFR C677T genotype is summarized in table 3.

Multiple linear regression analyses were conducted to determine the influence of more than one associated variable (as determined by univariate analysis) on the change in plasma tHcy concentration from before to after surgery. Potential variables for inclusion were duration of exposure to nitrous oxide, preoperative plasma tHcy concentration, and serum vitamin B12 concentration. Because the serum vitamin B12 concentration was inversely associated with the preoperative plasma tHcy concentration, we ran the multiple linear regression analyses twice, first with the preoperative plasma tHcy concentration and duration of nitrous oxide exposure and then with the serum vitamin B12 concentration and the duration of nitrous oxide exposure. In the first model, the duration of nitrous oxide exposure and preoperative plasma tHcy concentration explained 82% of variance in the increase in postoperative plasma tHcy concentration (R  ²= 0.821; P = < 0.0001). In the second model, the duration of nitrous oxide exposure and serum vitamin B12 concentration accounted for 72% of the increase in postoperative plasma tHcy concentration (R  ²= 0.721; P = < 0.0001).

In this study, we investigated the effects of exposure to nitrous oxide anesthesia on the plasma tHcy concentrations in young children the morning after surgery. In our sample of children exposed to nitrous oxide (≥2 h) at an inspired concentration of ≥50%, postoperative plasma tHcy concentrations increased by 25% (1.2 μM), a small, albeit statistically significant, increase. This magnitude of increase is consistent with reports of an overall average 20–26% increase in plasma tHcy concentrations after exposure to nitrous oxide in adults (∼3 h at ∼70% inspired concentration)5,7but less than the 228% (9.4 μM) increase newly reported for adolescents (0.5–10 h at ∼55% inspired concentration) 6–8 h after the induction of anesthesia.3By 24 h after anesthesia induction in the latter study, plasma tHcy concentrations had returned to baseline.

The duration of nitrous oxide exposure was the main factor associated with the increase in postoperative plasma tHcy concentrations among young children in our study, a relationship first reported in adults by Ermens et al.  4and subsequently confirmed by others.5,–,7,9,10In the current study, children exposed to more than 3.5 h of nitrous oxide (n = 9) presented with postoperative plasma tHcy concentrations approximately 80% greater than initial concentrations. Similarly, Nagele et al.  reported an 80% increase in (median [first, third quartile]) plasma tHcy concentration from baseline (8.1 [7.3, 10.0]μM) to the end of surgery (14 [13.1, 22.8]μM) among 16 adults exposed to nitrous oxide for more than 4 h.7 

Preoperative plasma tHcy concentration was also significantly associated with the increase in plasma tHcy concentration after anesthesia among children in the current study (R  ²= 0.305, P = 0.001), a finding consistent with that reported for adults.27In the current study, both serum vitamin B12 and plasma MMA concentrations were significantly associated with baseline tHcy, and serum vitamin B12 was associated with the increase in tHcy after nitrous oxide exposure. These findings are consistent with the known metabolic role of vitamin B12 as a cofactor in the methyl transfer reactions involved in the conversion of homocysteine to methionine.28At a population level, vitamin B12 has been identified as a determinant of plasma tHcy concentrations in children.29,30Whether supplementation of patients with vitamin B12 before nitrous oxide anesthesia would prevent the postoperative increase in tHcy remains to be determined.8,26 

The clinical significance of an acute increase in plasma tHcy concentrations induced by nitrous oxide is unclear. Five children in our study presented with postoperative plasma tHcy concentrations greater than 13.5 μM. Published studies of adults treated with nitrous oxide found that postoperative plasma tHcy concentrations ≥13.5 μM were associated with myocardial infarction and stroke,5concentrations of 14.1 ± 5.7 μM were associated with endothelial dysfunction,6and concentrations greater than 17 μM were associated with a 2-fold increased risk of myocardial ischemia.9Whether increased plasma tHcy concentrations play a causal role in these adverse cardiovascular outcomes or merely reflect nitrous oxide exposure will need to be clarified in future studies.

We acknowledge a number of limitations in our study. To minimize the need to collect blood samples from small children, we did not include a randomized control group for which an anesthetic without nitrous oxide was used. Rather, we obtained a blood sample before and after surgery from each child. We do not think surgery alone was responsible for the increase in plasma tHcy concentrations in the current investigation because the ENIGMA Trial and other studies in adults have shown that plasma tHcy concentrations do not increase after anesthesia during surgery using other anesthetic agents.5,8All children in this study were recruited from the Plastics Surgery Clinic at The Hospital for Sick Children, where surgery was being arranged for treatment of a birth defect. We can not rule out the possibility that some of the children in our study had underlying genetic defects that could have affected their response to nitrous oxide. Likewise, we are uncertain whether our results in this otherwise healthy cohort of children can be generalized to children exposed to nitrous oxide who are of poor health or have major organ dysfunction. We acknowledge that our sample size was too small to explore the effect of MTHFR C677T genotype. In small children nitrous oxide often is used to induce anesthesia, and this was the case for all subjects in the current study. Therefore, the nitrous oxide used for induction inadvertently may have increased the baseline plasma tHcy concentration, resulting in a lower than expected postoperative increase. We acknowledge several statistical inferences were made in this study without adjustment of the P  value to account for multiple comparisons. Although most comparisons were planned, it does raise the possibility of a type I error. Finally, it is possible that the peak in plasma tHcy concentration after nitrous oxide exposure occurred before collection of the second blood sample in our study. If this was the case, the increase in plasma tHcy concentrations reported in the current study likely is a very conservative estimate of the increase.

In conclusion, children exposed to ≥2 h nitrous oxide anesthesia showed a small but significant increase in plasma tHcy concentrations the morning after surgery (1.2 μM [0.1, 2.7 μM]). The clinical significance of this increase is unknown. The duration of exposure to nitrous oxide and initial plasma tHcy concentrations were associated significantly with the increase in plasma tHcy concentrations after nitrous oxide exposure.

The authors thank Christopher Forrest, M.D., M.Sc., F.R.C.S.(C), F.A.C.S., Division Head, Department of Plastic Surgery, The Hospital for Sick Children, Toronto, Ontario, Canada, and Associate Professor, Department of Surgery, University of Toronto, Toronto, Ontario, Canada.