Addressing the silent epidemic of recreational nitrous oxide use: a position, call to action and recommendations by the European Federation of Clinical Chemistry and Laboratory Medicine Committee on Biological Markers of Nitrous Oxide Abuse

Constitution of the group and methodology

The European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Committee on Biological Markers of Nitrous Oxide Abuse (C-BNOA) was established within the EFLM Science Division in response to the urgent need for guidance in this area. Members representing EFLM were nominated by their National Societies and approved by the Science Division, ensuring geographical diversity and expertise in laboratory medicine. In addition, external experts were invited through collaborations with international societies (ADLM, EUSEM, EUFAS), with nominations based on recognized expertise in toxicology, neurology, emergency medicine, or addiction medicine. In total, the expert panel comprised 15 members from nine countries.

In line with the EFLM publication policy, this manuscript represents an official output of the C-BNOA functional unit. The draft was reviewed and approved by the Committee Chair and subsequently by the EFLM Science Division, including members independent from the Committee. Following EFLM procedures for Type 1 documents, the recommendations were further submitted to the EFLM Executive Board and National Societies for consultation and approval before submission to CCLM, the official journal of the Federation.

To structure its work, the Committee organized activities into five work packages (WP): (1) International Survey, (2) International Network, (3) Detection and Road Safety, (4) Recommendations and Bibliography, and (5) Education and Promotion. The current document is related to WP4 specific work.

The recommendations presented here are based on a narrative review of the existing literature, complemented by the collective expertise and clinical experience of the panel. Drafts were discussed in structured meetings and revised iteratively until consensus was reached (agreement 100 % of members). The final version was externally reviewed and approved by the EFLM Science Division prior to submission.

Although frameworks such as AGREE II and RIGHT provide validated standards for guideline development, their full methodology could not be applied here due to the urgent need for rapid recommendations and the limited amount of robust evidence available. Instead, this document should be considered as a rapid expert consensus, designed to provide immediate clinical and laboratory guidance while serving as a first step toward more comprehensive guidelines that will follow these frameworks in the future.

A silent epidemic and a lack of awareness

Despite the growing prevalence of N₂O toxicity [4], awareness and sense of urgency among healthcare professionals and policymakers remain limited. The absence of robust epidemiological data and standardized surveillance mechanisms complicate risk assessment, prevention, and public health interventions. Additionally, gaps in medical knowledge contribute to misdiagnosis and delayed treatment, underscoring the need for improved education and structured data collection [5].

The absence of specific biomarkers

Unlike many other neurotoxic substances, N₂O intoxication lacks specific and reliable biomarkers for diagnosis and prognosis [5]. Current laboratory investigations rely on indirect markers, such as plasma homocysteine (HCY) and methylmalonic acid (MMA), which reflect vitamin B12 metabolism disruption but are not exclusive to N₂O exposure [6]. Developing novel biomarkers is crucial to improving early detection, risk stratification, and patient monitoring. To achieve this, collaboration between specialists in laboratory medicine, clinicians, and industry partners is essential. The recently established EFLM C-BNOA aims to accelerate research in this area and support the identification of robust biomarkers.

Due to the lack of specific biomarkers for clinical use, detecting and confirming nitrous oxide (N₂O) consumption remains challenging. This also creates an issue for the investigation of prevalence of use and other epidemiological measures. For instance, modern approaches such as wastewater analysis are currently difficult to apply in this context [7]. Considering this we suggest using a technique that has been termed “Drug Information System” utilizing multiple sources of information to gather drug use information [8]. This would include information from authorities, police, customs, postal deliveries, renovation, nightlife industry, social workers, school nurses, teachers, and medical personnel in general, laboratory medicine, primary care, emergency departments and specialized health care (especially neurology). This comprehensive list includes possible rather than compulsory contributors, and is already utilized by the European Drug Agency (EUDA) [9]. However, it is important to emphasize that these are reported or declarative data, which likely result in a substantial underestimation of the actual number of cases.

A structured approach to addressing the issue

To respond effectively to this growing health crisis, our actions have been structured into specific work packages:

1. International survey: Conducting a large-scale international questionnaire to assess the prevalence of N₂O intoxications and evaluate the level of knowledge among healthcare professionals.

2. Knowledge dissemination plan: Developing a structured dissemination strategy to raise awareness and inform the medical community about the risks and clinical implications of N₂O intoxication.

3. Expert team structuring: Establishing expert teams to ensure comprehensive patient care, in collaboration with the international PROTOSIDE network (www.protoside.com/en/).

4. Harmonization of clinical and biological guidelines: Working towards standardized clinical and biological recommendations, which require a deeper understanding of the underlying mechanisms through collaborative scientific efforts.

The role of specialists in laboratory medicine and interdisciplinary collaboration

Specialists in laboratory medicine play a pivotal role in addressing N₂O-related health challenges. Their expertise is essential for improving diagnostic strategies, validating new biomarkers, and standardizing laboratory testing protocols. However, their efforts must be integrated within a broader interdisciplinary framework. To ensure a comprehensive approach to this issue, the EFLM C-BNOA has invited international experts from various disciplines, including emergency medicine, neurology, and addiction medicine from leading scientific societies in the field. This multidisciplinary collaboration is necessary to:

1. Enhance knowledge dissemination among healthcare professionals through education and awareness campaigns.

2. Develop evidence-based clinical guidelines and recommendations for the management of N₂O-related harm.

3. Foster international research initiatives aimed at identifying novel biomarkers and improving diagnostic tools.

Call for urgent action

To tackle the growing threat posed by N₂O intoxications, the scientific and medical communities must take immediate, proactive steps. The establishment of an international collaborative network dedicated to N₂O research and clinical management is imperative. Such a network would facilitate data sharing, promote research on novel biomarkers, and contribute to the development of harmonized recommendations. The EFLM C-BNOA serves as a crucial platform to accelerate these efforts. The integration of laboratory medicine specialists within this initiative will be key to ensuring a comprehensive and multidisciplinary approach.

Research priorities and futures directions

In the long term, our Committee emphasizes the need to invest in dedicated research programs aimed at identifying novel and specific biomarkers that could improve the diagnosis and monitoring of nitrous oxide abuse. It is equally important to support translational studies that explore the metabolic and neurological mechanisms underlying its toxicity. To remain at the forefront of scientific advancement, we propose the establishment of a continuous bibliographic watch system focused on newly emerging biomarkers. This will allow the scientific community to stay informed and responsive as new knowledge becomes available.

We also advocate for the optimization and harmonization of analytical methods used in the measurement of biomarkers such as plasma HCY [10] and MMA. Standardization across laboratories through the adoption of validated protocols and robust quality control procedures will improve the reliability and comparability of results throughout Europe and beyond. These efforts should be supported by both academic initiatives and industry partnerships in collaboration with the EFLM Committee.

At the organizational level, the creation of a European or even global registry for confirmed cases of N₂O intoxication would provide valuable epidemiological insights, inform clinical practice, and help shape public health policy. Parallel to this, the development of harmonized European guidelines is essential. These guidelines should integrate biological, clinical, and therapeutic dimensions, and be drafted in collaboration with stakeholders from various medical specialties and healthcare systems.

Recent developments could include the exploration of neuro-injury biomarkers such as neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP), which may provide objective measures of axonal and glial damage in affected patients.

Finally, the designation and coordination of expert clinical and laboratory centers is crucial to structure and support an efficient and multidisciplinary response. These centers would act as reference hubs for patient care, professional training, methodological innovation, and collaborative research. Such a network as PROTOSIDE (www.protoside.com/en/) would reinforce the collective ability to address the evolving challenges associated with nitrous oxide misuse in a sustainable and evidence-based manner.

Clinical recognition and early identification

From a clinical standpoint, the early identification of patients at risk of N₂O-related complications requires heightened awareness and systematic vigilance among healthcare professionals. Large case series report that >70 % of patients present with sensory disturbances and gait ataxia, while weakness is observed in around half, and psychiatric symptoms are documented in 10–20 % of cases [11], 12]. Young individuals presenting with unexplained neurological symptoms such as paresthesia, gait disturbance, limb weakness, sensory ataxia, sphincter and/or sexual dysfunction should prompt consideration of N₂O as a cause [2], 11], 13], especially when these features are accompanied by Lhermitte’s sign or posterior column dysfunction. Psychiatric manifestations including acute anxiety, hallucinations, mood swings, or cognitive complaints may also be revealing. Clinicians should also be alert to thromboembolic events, particularly in the absence of classical risk factors, and to burns around the mouth and hands that may indicate inappropriate handling of gas cartridges. In all cases, contextual clues such as recreational use or the presence of whipped cream cartridges should reinforce clinical suspicion. Importantly, recent case series indicate that most thrombo-embolic events occur after the onset of neurological symptoms, offering a window of opportunity for prevention through early recognition and treatment [14].

These warning signs are supported by recent clinical series [13], which highlighted that most patients presented after repeated episodes of use, and neurological signs were predominant. In some cases, patients also showed psychiatric manifestations such as acute anxiety or hallucinations [15]. These neuropsychiatric symptoms, often underestimated, may precede or accompany neurological signs and justify early exploration, indeed these cases are often clear, there is a differential diagnosis for some cases and other causes of myelopathy/neuropathy need to be considered [16], 17].

Denial of nitrous oxide use is frequent in clinical practice; therefore, clinicians should maintain a high index of suspicion and proceed with biomarker testing and neurological evaluation even when patients do not report consumption.

Addiction to nitrous oxide is a frequent driver of poor adherence and loss to follow-up; early involvement of addiction specialists and integration of motivational and behavioral approaches are therefore critical to long-term outcomes.

Biological evaluation and diagnostic workflow (Figure 1)

The diagnostic approach must be rapid and systematic. A biomarker panel consisting of HCY, MMA, vitamin B12, and folate (vitamin B9) should be performed as early as possible [5], 6], 12], 18], ideally immediately at the time of patient admission. This is essential given the fast clearance of certain markers, which may quickly return to normal despite ongoing clinical damage. Plasma HCY serves as a sensitive marker of recent exposure, whereas plasma MMA more accurately reflects the severity and chronicity of neurological involvement [6]. Vitamin B12 levels may remain within normal ranges despite significant functional deficiency, highlighting the need to prioritize functional markers [6]. Folate measurement is important due to its potential aggravating role in hyperhomocysteinemia, although supplementation must be discussed individually. Interpretation of these biomarkers should always consider renal function, particularly for MMA, which is sensitive to changes in glomerular filtration [19].

The interpretation of biological markers must rely on a nuanced understanding of their respective kinetic profiles and pathophysiological significance. Plasma total homocysteine (tHcy) concentrations vary with cobalamin and folate status, as well as with age and renal function. Therefore, an elevated tHcy level should always be interpreted in the context of these variables to avoid misclassification. In clinical practice, establishing whether plasma tHcy is increased in a patient suspected of nitrous oxide exposure requires ensuring adequate vitamin status and normal renal function. However, age- and method-specific reference intervals for tHcy are not yet harmonized across laboratories, and there are currently no validated clinical decision limits specific to nitrous oxide exposure. Laboratories should thus interpret elevated tHcy results cautiously and in relation to local reference data, analytical methods, and patient context.

An elevated HCY level and/or an increased MMA concentration in the absence of renal dysfunction are suggestive of folate deficiency or a functional vitamin B12 deficiency, which may result from N₂O-mediated inactivation of cobalamin. HCY is particularly useful for identifying recent exposure, as its levels may rise within hours and are frequently abnormal even in early stages of intoxication. In contrast, MMA correlates more closely with the severity and chronicity of neurological involvement, often reflecting irreversible metabolic injury if elevated persistently [6]. Notably, a normal serum vitamin B12 level does not preclude a functional deficiency, making the simultaneous measurement of HCY and MMA essential for accurate assessment [20]. Folate should also be systematically evaluated, as its deficiency can aggravate hyperhomocysteinemia and may contribute to neuropsychiatric symptoms; however, supplementation should be considered on a case-by-case basis, especially in the absence of documented deficiency. Renal function must always be assessed prior to interpreting MMA, since impaired clearance can lead to false-positive results. Given the rapid normalization of these biomarkers after cessation of use or treatment initiation, blood sampling must be carried out preferably on admission to avoid underestimation of metabolic disturbances. In all symptomatic patients, vitamin B12 supplementation should begin immediately without awaiting laboratory confirmation, in association with the cessation of N₂O consumption. A structured follow-up strategy is also required, including repeat biomarker testing post-supplementation and during withdrawal, to document metabolic recovery and confirm abstinence. This follow-up must continue beyond hospital discharge, with referral to primary care or addiction specialists to support long-term relapse prevention and ensure continuity of care.

Structured care and multidisciplinary coordination

At the local level of an individual hospital, a structured care pathway is essential. Emergency physicians, neurologists, laboratory specialists, psychiatrists, and addiction professionals must work together within local multidisciplinary teams to ensure the continuity and quality of care. These teams should be formally connected to new centers of expertise as we propose above, and as exemplified by existing collaborative frameworks, such as the international PROTOSIDE network [3], to benefit from shared expertise, promote harmonized practices, and contribute to the collective effort against N₂O misuse.

The patient care pathway must integrate all steps from early detection and biological confirmation to neurological and psychiatric follow-up, rehabilitation, and where necessary, addiction management. Existing models, such as the French hospital care coordination system (PROTOSIDE, www.protoside.com), illustrate how centralized expertise and rapid biological confirmation can improve diagnosis and referral. Ultimately, local integration with primary care and addiction support services remains essential to prevent recurrence and support long-term recovery.

Key practical points for suspected nitrous oxide intoxication (Figure 1)

1. Consider N₂O intoxication in young patients presenting with neurological signs (paresthesia, gait disturbances, sensory ataxia), psychiatric symptoms (acute anxiety, hallucinations), or unexplained thromboembolic events.

2. Always ask about potential N₂O exposure, including recreational use (party settings, whipped cream cartridges, gas tanks); it is often important to probe beyond a single question because people may not volunteer information easily.

3. Perform diagnostic testing immediately upon admission to avoid false negatives due to rapid marker normalization.

4. Diagnostic testing:

   1. Plasma homocysteine (HCY) – early marker of recent exposure (Table 1).

      1. Isolated elevation of HCY does not specifically point to N₂O intoxication, given other causes of elevation.

      2. An absence of HCY elevation does not rule out N₂O intoxication, given the marker’s as yet poorly understood kinetics.

      3. To help interpretation, age-specific clinical decision limits have been proposed (21). These thresholds, derived from the 90th percentile of HCY in individuals with normal renal function and adequate vitamin status.

   2. Plasma methylmalonic acid (MMA) – marker of clinical severity.

      1. Isolated elevation of MMA does not specifically point to N₂O intoxication, given other causes of elevation.

      2. An absence of MMA elevation does not rule out N₂O intoxication, given the marker’s as yet poorly understood kinetics.

   3. Serum Cobalamin (Vitamin B12) – may be normal despite functional deficiency.

   4. Serum Folate (Vitamin B9) – assess and supplement if deficient.

5. Interpret HCY and MMA cautiously in case of impaired renal function (check eGFR), vitamin deficiency or biological test performed too late.

6. Stop definitively N₂O use and initiate parenteral vitamin B12 supplementation without delay in symptomatic patients.

7. Consider folate supplementation on a case-by-case basis depending on clinical and biological data.

8. Plan a full biological follow-up (HCY, MMA, B12, B9) to confirm normalization and assess adherence.

9. Refer to addiction specialists and ensure long-term follow-up in primary care.

10. Use collaborative platforms like PROTOSIDE for expert support and case discussion.