Rapid decline of elevated homocysteine level in nitrous oxide use

Abstract

The recreational use of nitrous oxide (N₂O) has increased significantly in recent years, becoming an emerging public health concern owing to its association with severe neurological complications. Vitamin B12 is inactivated by N₂O, and accumulation of homocysteine (Hcy) could be a relevant marker of N₂O toxicity. Kinetics of Hcy after cessation of exposure remain poorly understood. This retrospective study examined the Hcy levels of patients hospitalized consecutively for neurological impairment secondary to N₂O intoxication. Demographic and clinical data were collected, including clinical phenotype and motor deficit severity. Levels, dates, and hours of blood testing for vitamin B12, Hcy, and hospital entrance were collected. The statistical analysis used the Wilcoxon test or the Chi-square test. Among the 86 patients (median age 22 years; 59% woman), 92% exhibited abnormal Hcy levels, with a median Hcy concentration of 69 µmol/L [38; 106] (normal < 15 µmol/L). Vitamin B12 levels were normal in 80% of cases. Hcy levels were significantly lower in patients who had received vitamin B12 supplementation (p = 0.016). When blood testing was performed within the first 8 h after hospital admission, Hcy levels were high (median 106 µmol/L [89; 112]). A rapid decrease was observed within the following hours, reaching normal levels approximately one week after admission. The exponential decline of Hcy underscores its potential as a valuable biomarker for N₂O-induced toxicity under the condition of a very early sampling and patient reference. In current practice, Hcy values that do not increase dramatically could suggest a delay in testing rather than low N₂O usage.

Introduction

The recreational use of nitrous oxide (N₂O) has increased significantly in recent years, becoming an emerging public health concern owing to its association with severe complications, including hematologic, psychiatric, and other systemic effects and particularly neurological dysfunctions in young adults in the Greater Paris area^1–3. However, no biological marker has yet demonstrated clear clinical utility for N₂O use³. Vitamin B12 is inactivated after oxidation of the cobalt ion by N₂O, impairing its role as a cofactor of methionine synthase in the remethylation of homocysteine into methionine. This inhibition leads to the accumulation of homocysteine (Hcy), which could be a relevant marker of use^4–7. However, Hcy levels correlate poorly with the severity of N₂O-induced neurological dysfunction^8,9. The influence of the last intake lag upon Hcy levels at the time of blood testing is unknown, and this lag is difficult to obtain⁸. We investigated how Hcy levels are influenced by delayed blood testing after hospital admission as a proxy for the last N₂O intake. The aim of this study was to provide a descriptive assessment of the temporal dynamics of homocysteine levels in relation to the timing of blood sampling after hospital admission in a real-world clinical setting.

Methods

We examined the Hcy levels of all patients hospitalized consecutively for neurological impairment secondary to N₂O intoxication, between January 1, 2018, and December 31, 2023, in two tertiary hospitals (Henri-Mondor University Hospital, Créteil, and Delafontaine Hospital, Saint-Denis) in the Greater Paris area. At admission to the neurology department demographic characteristics (sex and age), clinical neurological involvement (myelopathy and/or peripheral neuropathy), and motor deficit severity were systematically assessed. Levels, dates, and hours of blood testing for vitamin B12, Hcy, and hospital entrance were collected. Blood samples were processed according to standard laboratory protocols. Plasma homocysteine levels were measured using an immunoturbidimetric assay after refrigerated transport of samples.

All patients were asked about any recent vitamin B12 intake. In practice, most patients reported the last N₂O intake within the hours or days preceding hospitalization, without being able to provide a precise credible time for it. We hypothesized that patients did not consume N₂O after hospital admission; therefore, the time of admission (day 0, hour 0) was considered the first time point at which abstinence from N₂O was ensured. We therefore used the time of hospital admission as a proxy for the last intake. We then leveraged the variability in the time interval between hospital admission and blood sampling. This interval was used as a proxy for the delay between the last probable N₂O exposure and blood sampling. A rapid decline in Hcy levels was expected in the days following admission. Numerical data were expressed as median values with interquartile ranges [IQR 25–75%] because the data were not normally distributed. Continuous variables were compared using the Wilcoxon rank sum test as appropriate, while categorical variables were analyzed using the Chi-square test, or Fisher’s exact test when expected cell counts were too low. A p-value of < 0.05 was considered statistically significant.

Statistical analyses were conducted using R version 4.4.2. The study received ethics committee approval from Hôpital Delafontaine, Saint-Denis (n°0023_NEUROLOGIE_EPI-NOX, November 18, 2021). This study was conducted in compliance with the applicable regulatory framework for university-affiliated hospitals in France conducting research not involving human subjects (RNIPH). This framework requires a declaration of non-opposition rather than written informed consent.

Results

Among the 86 patients, median age was 22 years [20; 30], and 59% were women. Clinical phenotype data were available for 48 patients. Neuropathy was observed in 13 patients (27.1%), myelitis in 18 patients (37.5%), and a combined presentation in 17 patients (35.4%). Overall, 92% exhibited abnormal Hcy levels, with a median Hcy concentration of 69 µmol/L [38; 106] (normal < 15 µmol/L). Despite these significant metabolic abnormalities, vitamin B12 levels were normal in 80% of cases. Vitamin B12 levels were inversely correlated with Hcy abnormality (95% vs. 80%, p = 0.048). Additionally, 23% of patients had already received vitamin B12 supplementation prior to hospitalisation, with 80% of these cases being self-supplemented without any medical prescription. Hcy levels were significantly lower in patients who took supplements on their own (median 82 vs. 48 µmol/L, p = 0.016), with a median level of half that of non-supplemented patients (Table 1).

Table 1.

Comparing homocysteine (Hcy) outcomes in patients with and without vitamin B12 supplementation prior to hospitalisation. Six patients did not have vitamin B12 levels measured.

Characteristics	Overall N = 86a	No supplementation N = 68a	Supplementation by B12 N = 20a	p-valueb
Age (y)	22 [20; 30]	22 [20; 31]	26 [21; 29]	0.3
Women	52/86 (59%)	44/68 (65%)	8/20 (40%)	0.048
homocysteine value (µmol/L)	69 [38; 106]	82 [44; 114]	48 [22; 73]	0.016
Homocysteine abnormality (%)	79/86 (92%)	63/66 (95%)	16/20 (80%)	0.048
Delay between date of hospitalisation and homocysteine sampling (day)	0.8 [0.2; 2.7]	0.7 [0.1; 2.7]	1.8 [0.8; 3.3]	0.2
Vitamin B12 abnormality (%)	16/80 (20%)	9/61 (15%)	7/19 (37%)	0.050
Vitamin B12 value (µmol/L)	222 [176; 312]	214 [173; 260]	288 [182; 945]	0.085

^aMedian [IQR 25–75] or n/N (%).

^bWilcoxon rank sum test, Pearson’s Chi-squared test, or Fisher’s exact test.

Baseline characteristics differed according to hospital and patient profiles (n = 59 and n = 29, respectively). Homocysteine levels and vitamin B12 concentrations were comparable between groups. In contrast, sex distribution differed significantly between groups (68% vs. 41% women, p = 0.018). Prior vitamin B12 supplementation showed a non-significant trend toward higher prevalence in one group (17% vs. 34%, p = 0.065). A trend toward differences in the delay between hospital admission and homocysteine sampling was observed (median 1.3 [0.2; 3.6] vs. 0.5 [0.1; 1.9] days, p = 0.083).

Among the 86 patients initially identified, 20 were excluded from the analysis because of prior vitamin B12 supplementation and 6 were excluded due to missing blood sampling date, leaving 62 patients included in the final analysis. Homocysteine measurements were therefore available only for patients with complete and eligible data. When blood testing was performed within the first 8 h after hospital admission, Hcy levels were high, with a median of 106 µmol/L [89; 112]. A rapid decrease was observed within the following hours (Fig. 1, left panel). This decline continued over the subsequent days, reaching normal levels approximately one week after admission (Fig. 1, right panel).

Fig. 1.

Homocysteine (Hcy) levels over time after admission to hospital. Hcy measurements according to time from hospital admission (n = 62). The left panel shows homocysteine measurements obtained within the first 24 h after hospital admission, whereas the right panel shows measurements obtained on subsequent days after admission. levels drop rapidly from the first day of hospitalization (left panel) and are lower when the test is performed later after admission (right panel). Patients with prior vitamin B12 supplementation (n = 20) or missing sampling date (n = 6) were excluded from analysis.

Discussion

Hcy levels are initially elevated during N₂O use^4,6. We demonstrate that a dramatic decrease occurs within the first hours after hospital admission, and that levels normalize generally within a week. These results highlight the importance of considering the timing of Hcy testing in relation to the cessation of N₂O exposure. While studies on healthy subjects have shown that Hcy levels rise significantly within minutes after experimental N₂O intoxication and normalize within a few hours¹⁰, with peak values reaching around 50 µmol/L, recreational N₂O users present a markedly different profile.

First, in N₂O users with neurological disorders following massive and prolonged N₂O intoxication sometimes lasting several months², we hypothesize that Hcy levels may reach such high values that normalization may take more than a week. They decrease sharply immediately after cessation, as reported by a few teams^8,11,12, and normalize within days. It is therefore crucial to measure Hcy levels as soon as possible, even if N₂O intoxication dates back several days, although the timing of cessation is the determining factor when interpreting the results.

Second, we took hospital admission as Day-0 of exposure cessation, yet the last N₂O intake preceded admission by hours or days in most cases. The rapid decline in Hcy levels within the first hours after cessation suggests that Hcy measurements tested at admission (H0) likely underestimate the maximal Hcy levels at the exact time of cessation (which would be the real H0). In addition, a significant number of patients had taken vitamin B12 as a preventive measure, which seems to have had an impact on Hcy levels.

Our study has several interrelated limitations. The date of cessation of N₂O consumption was estimated to be the first day of hospitalisation. However, this assumption may not be entirely valid, as discussed above. Self-medication with vitamin B12 prior to admission also introduces another layer of variability. Our study did not take into account the exact amount of N₂O consumed by each patient since quantities required to cause neurological damage are always very large², whereas exact individual consumption remains subjective and its relation to Hcy levels is still unclear. Finally, we cannot rule out the possibility that the sampling time may be influenced by the severity of clinical signs, even if this seems unlikely.

Some heterogeneity in patient profiles and management practices across hospitals was observed, particularly regarding the timing of homocysteine sampling. This variability likely reflects differences in clinical pathways and the absence of standardized recommendations for biological assessment in patients with suspected nitrous oxide–related neurological complications. According to our data, existing laboratory chemical markers for N₂O can only be used up to one week after the last intake. Further research is needed to determine the exact dynamics of Hcy depending on the amount of N₂O consumed and its delay. Understanding this relationship could provide valuable insights into the prognosis and management of patients with severe N₂O-related intoxication.

Conclusion

The frequent normality of vitamin B12 levels in N₂O users reduces their utility in assessing intoxication severity. In contrast, the exponential decline of Hcy underscores its potential as a valuable biomarker for N₂O-induced toxicity, provided that sampling and patient referencing are conducted very early. In current practice, Hcy values that are not dramatically elevated could suggest a delay in testing rather than chronic low N₂O use.

Author contributions

YD: Conception, manuscript writing, statistical analysis.MB: Conception, supervision, data collection, manuscript review.TG: Data collection, manuscript review.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

The datasets generated and/or analysed during the current study are not publicly available due to patient confidentiality but are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.