Functional vitamin B₁₂ deficiency is a consistent feature in hospital admissions for neurological disorders due to the use of nitrous oxide

Abstract

Introduction: Misuse of inhaled nitrous oxide is a growing concern in France. It is known to alter concentrations of vitamin B_12, which is required as a cofactor for methionine synthase and methylmalonyl-CoA mutase activity. Hence, measurement of the concentrations of cobalamin metabolism biomarkers, including vitamin B₁₂, homocysteine, and methylmalonic acid, could assist in the management of patients with a complex clinical presentation or in those who deny the consumption of nitrous oxide.

Methods: We retrospectively collected clinical and biological data in patients hospitalized for nitrous oxide use in a university hospital in southern France between January 2020 and October 2022.

Results: Thirty-one patients were identified during 34 months; 79% were men with a median age of 23.7 years. Most (97%) presented with peripheral polyneuropathy and/or myelopathy. The median vitamin B₁₂ concentration was 134.6 pmol/L, with 17 of 31 patients having values less than 145 pmol/L (the lower limit of the normal range). The median plasma folate concentration was 20.1 nmol/L, which is within the normal range. The median plasma homocysteine concentration was 87.7 µmol/L (normal range <15 µmol/L), and the median plasma methylmalonic acid concentration was 3.8 µmol/L (normal range <0.5 µmol/L).

Conclusion: Nitrous oxide use is an emerging public health problem in France, as shown by the number of patients admitted to our hospital. The presence of a functional vitamin B₁₂ deficiency was a consistent feature that could be helpful in diagnosis in complex cases.

Keywords:

• Nitrous oxide
• homocysteine
• methylmalonic acid
• vitamin B₁₂
• cobalamin

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Introduction

Inhalation of nitrous oxide is a known aetiology of vitamin B₁₂ deficiency, observed typically in patients who received high doses of nitrous oxide for analgesic purposes [1]. Nitrous oxide oxidizes the cobalt Co(I) form of vitamin B₁₂ to Co(III) [2], thus blocking the cobalamin intracellular process. Cobalamin acts as a cofactor for two key enzymes: cytoplasmic methionine synthase (responsible for the formation of methionine and S-adenosylmethionine) and mitochondrial methylmalonyl Co-A mutase. Because reduced Co(I) form is needed for cobalamin-dependent enzyme activities, nitrous oxide-induced oxidation of cobalt compromise these pathways (Figure 1), as demonstrated for methionine synthase [3–4]. Physiologically, the reduction of Co(III) and Co(II) to Co(I) is ensured by reductases in the cytoplasm [5] but does not appear to be effective in counteracting nitrous oxide-induced oxidation.

Figure 1. Impact of nitrous oxide on cobalamin metabolism. A potential mechanism of nitrous oxide toxicity is the oxidation (dashed arrows) of cobalamin’s reduced form (cobalamin^I) to more oxidized forms (cobalamin^II and cobalamin^III).

The nitrous oxide-induced decrease of methionine synthase could, therefore, impair S-adenosylmethionine formation. Some authors hypothesize that this could lead to a defect in myelination [2], but S-adenosylmethionine concentrations have never been measured in humans with nitrous oxide intoxication. The mechanism of demyelination is unclear, as an animal study by Dorris [6] showed that the decrease in S-adenosylmethionine was not important during nitrous oxide exposure. Interestingly, methionine concentrations and the ratio of methionine to homocysteine were recently reported to be related to clinical severity [7].

Two main neurological pathologies are observed in cases of nitrous oxide-induced vitamin B₁₂ functional deficiency: subacute combined degeneration of the spinal cord and peripheral neuropathy [8]. Symptoms of subacute combined degeneration of the spinal cord and peripheral neuropathy are overlapping and include general limb numbness and weakness, mainly affecting the lower limbs. Subacute combined degeneration of the spinal cord associated with nitrous oxide abuse mainly involves the cervical posterior spinal cord, sparing the lateral spinal cord. Axonal and/or demyelinating abnormalities can be present in electromyoneurography [9]. Nevertheless, neurological deficits due to nitrous oxide use are probably not only related to vitamin B₁₂ functional deficiency, as nitrous oxide toxicity could be mediated through other pathways such as N-methyl-D-aspartate (NMDA) receptor overstimulation [10]. Indeed, homocysteine and nitrous oxide itself are NMDA receptor ligands. Vodovar et al. [11] have also postulated that recurrent hypoxia induced by the use of pure oxygen-free nitrous oxide is another important mechanism in neurological impairments.

Nitrous oxide use has been reported to the French Addictovigilance Network and also at a European level and worldwide [12–17]. Vitamin B₁₂ functional deficiency occurs after substantial nitrous oxide use, such as the chronic inhalation of hundreds of "whippits" (approximately 8 g of nitrous oxide) or several containers (cylinders or tanks of nitrous oxide 600 g or 2,000 g) of nitrous oxide per day [18]. In some cases, vitamin B₁₂ functional deficiency occurs in the presence of other risk factors or after acute consumption [19–20].

Blood nitrous oxide measurement is feasible [21] but is of very little interest due to its very short half-life. Interestingly, plasma vitamin B₁₂ concentrations are not always decreased in patients with nitrous oxide intoxication, whereas plasma homocysteine and methylmalonic acid concentrations, which reflect vitamin B₁₂ functional deficiency, appear to be more reliably elevated [22,23].

Oral or intramuscular supplementation of vitamin B₁₂ is used to treat these patients, together with cessation of nitrous oxide use and physiotherapy. Although correction of vitamin B₁₂ deficiency and reversibility of symptoms can be achieved, some patients have long-term sequelae such as residual limb numbness [13,24].

To investigate biological markers in the diagnosis of nitrous oxide intoxications, we conducted a single-centre retrospective survey of hospitalized patients presenting with nitrous oxide-induced neurological disorders.

Material and methods

Patients

Data from patients aged 17 or more admitted to our university hospital (emergency unit or neurology department) for nitrous oxide-induced neurological disorders between 1 January 2020 and 31 October 2022 were collected retrospectively. To be included, patients had to have at least one concentration of vitamin B_12, homocysteine, or methylmalonic acid measured. Patients with known inherited metabolic diseases were excluded. Clinical, biological, neuroimaging and sociodemographic data were analyzed. Nitrous oxide inhalation was reported according to the patient’s self-declaration and was considered chronic when used more than twice per week for at least two months. Associated self-reported toxic substance use, including alcohol, cannabis, cocaine, ketamine, and opioids, were recorded. Diagnoses of subacute combined degeneration of the spinal cord and peripheral neuropathy were recorded from the medical records. This study was approved by the local institutional review board and declared under the number 202201305.

Laboratory measurements

Biological data were collected from laboratory assessments performed as routine at the beginning of hospitalization. Concentrations of vitamin B₁₂, homocysteine, methylmalonic acid and folate were measured in plasma collected in a lithium heparin tube after centrifugation 10 min at 2000 times gravity speed. Electrochemiluminescence assays using a Cobas 8000® (Roche Diagnostics, Meylan, France) were employed to measure folate and vitamin B₁₂ concentrations based on competition with intrinsic factors for vitamin B₁₂. Plasma homocysteine and methylmalonic acid concentrations were measured by liquid chromatography coupled with tandem mass spectrometry (Waters, Milford, USA) and gas chromatography coupled mass spectrometry (Thermoscientific, Hennigsdorf, Germany), respectively. The manufacturer’s reference range for folate was 8.8–60.8 nmol/L, and for vitamin B₁₂ was 145–569 pmol/L. The reference range for homocysteine was <15 µmol/L, and for methylmalonic acid was <0.5 µmol/L.

Other parameters, haemoglobin, mean corpuscular volume and estimated glomerular filtration (eGFR) were also collected.

Results

Population and clinical features

Among the 31 included patients, the majority were men (79%) with a median age of 23.7 years (range 17.4–41.2 years). Clinical manifestations included inferior and/or superior limb numbness (51.6%), unsteady gait (35.5%), balance disorders (32.2%) and vesical dysfunction (6.4%). Nineteen of 25 (76%) patients had sensory and/or motor nerve abnormalities on electromyoneurography and were diagnosed with peripheral neuropathy, 14 of 29 (48.3%) patients had cervical and/or thoracic spinal cord lesions on medullar magnetic resonance imaging and were diagnosed with myelopathy, and one patient was diagnosed with a psychotic episode. Guillain-Barre syndrome was suspected in five patients, and two were treated with polyclonal immunoglobulins.

Most (81%) patients reported massive chronic inhalation of nitrous oxide, which ranged from 200 small cartridges (8 g) per month to 10 cylinders (600 g) per day. The duration of nitrous oxide consumption was less than one month in six cases, with vitamin B₁₂ deficiency present in two of them. The first was a 25-year-old woman with a recent decrease in food intake and a weight loss of 14.5 kg over six months who had been diagnosed with peripheral neuropathy and who had a reported consumption of thirty cartridges of nitrous oxide a few days before symptoms onset. Her vitamin B₁₂ concentration was 97.3 pmol/L. The second was a 38-year-old woman with a history of sleeve surgery one year before her hospitalization who presented with subacute combined degeneration of the spinal cord after substantial intake of nitrous oxide during the three weekends preceding hospitalization. Her vitamin B₁₂ concentration was 132.8 pmol/L.

Twenty-four (77.4%) patients were hospitalized for an average of 10 days, and 25.8% of them were admitted for follow-up care to a rehabilitation centre after hospitalization. Seven patients were discharged from the hospital within 24 h. All patients received oral or intramuscular vitamin B₁₂ supplementation during hospitalization and after discharge (1 mg per day for ten days, then 1 mg per week for one to three months). All were advised to discontinue nitrous oxide completely, and eight received physiotherapy re-education.

Clinical outcome

Two patients were readmitted to the emergency unit within a month for clinical worsening of their gait disorders, which had led to falls. Twelve cases were re-evaluated within six months but without available biological data since a part of their follow-up was performed outside the hospital. Two had a normal clinical examination, six had clinically significant improvement, two were stable, and two exhibited clinical worsening. Nineteen cases were without follow-up or did not present to the follow-up visit.

Biological findings

Biological findings on admission are summarized in Figure 2. The median plasma vitamin B₁₂ concentration in 31 patients was 134.6 pmol/L, including 17 cases (54.8%) with a concentration below the lower limit of the reference range (145 pmol/L). In three patients, vitamin B₁₂ concentrations were above the upper limit of the normal range (569 pmol/L); these patients had supplemented themselves with vitamin B₁₂ shortly before being hospitalized. No other cases of self-supplementation were identified. These three vitamin B₁₂ outliers were excluded from descriptive statistics shown in Figure 2. Plasma homocysteine concentrations were assessed in 23 of 31 (74%) patients, and methylmalonic acid concentrations were available in 18 of 31 (58%) patients. The median homocysteine concentration was 87.7 µmol/L, and the median methylmalonicacid concentration was 3.8 µmol/L; these concentrations were, in all cases, above the normal ranges. Minor elevation of the plasma methylmalonic acid concentration (0.51 µmol/L and 0.74 µmol/L) was observed in two patients; both had elevated homocysteine concentrations (86.6 µmol/L and 141.6 µmol/L). The median plasma folate concentration in 87% of patients was 20.1 nmol/L, with values <8.8 nmol/L in only four patients. Kidney function was normal with a median eGFR of 126.6 ml/min/1.73m², and no patient had an eGFR <60 mL/min/1.73m². Macrocytic anaemia occurred in two male patients; the haemoglobin concentration in the first was 119 g/L with a mean corpuscular volume of 109 fL, and in the second, the haemoglobin concentration was 119 g/L with a mean corpuscular volume of 101 fL. Four patients had normocytic anaemia: a woman with a haemoglobin concentration of 113 g/L and mean corpuscular volume of 94 fL; three men with haemoglobin concentrations between 107 g/L and 126 g/L and mean corpuscular volumes between 82 and 88 fL. The median mean corpuscular volume for all patients was close to the upper limit (96 fL), and six patients had a mean corpuscular volume greater than 100 fL with a normal haemoglobin concentration.

Figure 2. Box-plot representations of biological assessments. For all markers, values are represented by a point, mean by a cross, and the following values are indicated by horizontal bars from bottom to top: minimum, first quartile, median, third quartile, maximum. All these values are defined on non-aberrant values only. A value is considered as aberrant if not comprised in the [first quartile-1.5*inter-quartile range; third quartile + 1.5*inter-quartile range) interval. Horizontal dotted bars represent normal ranges of analytes.

Only four patients had biological data available within six months after admission. Their homocysteine concentrations were between 34.7 µmol/L and 156.5 µmol/L and their methylmalonic acid concentrations were from 0.63 µmol/L to 1.55 µmol/L. Their vitamin B₁₂ concentrations ranged from 186 pmol/L to 504 pmol/L.

Discussion

We report a French case series of nitrous oxide-induced neurological disorders. The number of patients, 31 in 34 months, confirms the epidemiological reports of growing nitrous oxide use in France, especially among young patients in the past three years [13,14,25]. The diagnosis of neurological complications induced by nitrous oxide is not easy; patients do not always admit their consumption, and quantification can be inaccurate. Another difficulty is the differential diagnosis with other types of neuropathies, notably with Guillain-Barré syndrome, as occurred in five patients in our cohort, resulting in potentially unnecessary polyclonal immunoglobulin treatments.

Although absolute vitamin B₁₂ deficiency was observed only in 54.8% of patients, 100% had a biological profile corresponding to a functional B₁₂ deficiency characterized by elevation of homocysteine and methylmalonic acid concentrations. A difficulty in interpreting plasma vitamin B₁₂ concentrations is the definition of the cut-off. Manufacturers of analytical equipment use a cut-off for vitamin B₁₂ concentration of 145 pmol/L, whereas we advocate that a grey zone should be considered between 150 pmol/L and 220 pmol/L [26], corresponding to 29% of patients in the present study. Nevertheless, the biological profile of functional B₁₂ deficiency was consistent with that observed in other large case series [7,9,23,24,27]. It is essential that clinicians are aware that normal or elevated B₁₂ concentrations might not exclude exposure to nitrous oxide and may indicate self-supplementation [28]. An explanation for normal plasma vitamin B₁₂ concentrations is that the oxidized vitamin B₁₂ molecule still interacts with the recombinant intrinsic factor used in the electrochemiluminescence assay.

Clinicians should also recognize that in complex cases, homocysteine and methylmalonic acid concentrations have an important role in detecting functional vitamin B₁₂ deficiency. Plasma homocysteine concentrations may increase to greater than 15 µmol/L, reaching 180 µmol/L, due to disturbance of the remethylation pathway. In parallel, an increase of plasma methylmalonic acid concentrations may be observed ranging from 0.51 µmol/L to 28 µmol/L. Of note, the two lowest increases in the concentration of methylmalonic acid (0.51 µmol/L and 0.74 µmol/L) were associated with very high homocysteine concentrations (86.6 µmol/L and 141.6 µmol/L), in agreement with literature data showing that methionine synthase is more sensitive to nitrous oxide exposure than methylmalonyl Co-A mutase. In addition, methylmalonic acid could be normalized with prolonged self-supplementation, while homocysteine concentrations appear not to normalize until discontinuation of nitrous oxide use [29]. Although an increase in homocysteine concentrations is not a specific biomarker of vitamin B₁₂ deficiency, it appears as a sensitive biomarker for nitrous oxide use. However, for a correct interpretation of homocysteine elevation, other factors should be evaluated, such as chronic kidney insufficiency, folate deficiency, iatrogenic interaction, and inherited disorders of one-carbon metabolism [30–31].

To summarize, in complex cases, cobalamin metabolism biomarkers, such as the measurement of vitamin B₁₂, homocysteine, and methylmalonic acid concentrations, are helpful in making the diagnosis. Nevertheless, vitamin B₁₂ supplementation should be initiated prior to the receipt of biological results, as they are not available in all laboratories or 24 hours a day, seven days a week. Vitamin B₁₂ treatment combined with monitoring of improvement in neurological status could be an alternative strategy for centres without access to these markers.

Regarding other biomarkers, we found that a decrease in the plasma folate concentration, a low haemoglobin concentration, or an increased mean corpuscular volume had a poor diagnostic value, as previously shown by a meta-analysis [22]. Macrocytic anaemia was not prevalent in our case series, but macrocytosis was often present. We also observed that neurological disorders were present without macrocytic anaemia.

Inhaled nitrous oxide misuse is becoming commonplace for many reasons: it is legal (only the sale to minors is prohibited in France), easily accessible, and often considered a safe and harmless substance. This is becoming a public health burden by generating a clinically significant number of neurological and rehabilitation hospitalizations involving mainly young patients.

Patients with neurological impairment caused by nitrous oxide can suffer long-term consequences in terms of mobility restrictions, affecting dramatically their quality of life [9,24]. Follow-up of this population is a major issue, as only 12 cases attended a follow-up visit within six months in our cohort. Not all patients recovered clinically and biologically, suggesting that nitrous oxide-induced neurological impairment persists for a clinically significant period, even with vitamin B₁₂ supplementation. An alternative explanation could be that not all patients completely stopped nitrous oxide consumption, even though they claimed to have done so during a follow-up visit. This explanation is in accord with the high homocysteine concentrations observed in the few patients with biological follow-up. Stopping nitrous oxide is critical for recovery, and attendance at an addiction clinic must be integrated into care plans.

Surveys carried out among French students showed a high prevalence of health students abusing nitrous oxide (76.6 and 80% at least one time) [32–33]. In our case series, patients were mainly unemployed (45.2%), some had a professional activity (38.7%), and only two were students. The inhalation of nitrous oxide in France seems not to be limited to students, but data on the general population are lacking, and evaluation seems necessary.

This study has some limitations as electromyoneurography was not always undertaken, and remethylation biomarkers such as methionine or S-adenosylmethionine formation were not always assessed. In addition, nitrous oxide use was based only on self-declared patient data.

Conclusions

Neurological disorders related to nitrous oxide use are increasing in the south of France, as in the whole country. Awareness of nitrous oxide complications must be reinforced, especially in young adults that are of the most concern. In complex cases of nitrous oxide intoxication, the determination of homocysteine and methylmalonic acid concentrations to detect functional vitamin B₁₂ deficiency is helpful in diagnosis.

Author’s contributions

Etienne Mondesert: formal analysis, original draft writing; Céline Eiden: conceptualization, review and editing; Hélène Peyriere: review and editing, Aude Formoso: resources, review and editing; Lucas Corti: resources, review and editing, Jean-Paul Cristol: conceptualization, review and editing, Stéphanie Badiou: supervision, original draft writing.

Acknowledgements

None

Disclosure statement

No potential conflict of interest was reported by the authors.

Correction Statement

This article was originally published with errors, which have now been corrected in the online version. Please see Correction (http://dx.doi.org/10.1080/15563650.2023.2299552)

Additional information

Funding

None to declare.