Analysis of clinical characteristics and prognostic factors in 110 patients with nitrous oxide abuse

Abstract Purpose To review the clinical symptoms, auxiliary examination findings, and outcomes of patients with nitrous oxide (N2O) abuse, and analyze the factors that affect outcomes. Methods Patients with N2O abuse treated in the Department of Neurology between January 2018 and December 2020 were included. The clinical data of these patients were collected, and follow‐up was conducted to determine the outcomes. Results The average age of the 110 patients with N2O abuse was 21.4 ± 4.2 years (range: 14–33 years). Clinical presentation primarily included neurological symptoms, such as limb numbness and/or weakness (97%), psychiatric symptoms, changes in appetite, and skin hyperpigmentation. Laboratory test results were characterized by vitamin B12 deficiency (60%, 34 out of 57 cases) and high homocysteine level (69%, 31 out of 45 cases). Electromyography indicated mixed axonal and demyelination injury (92%, 80 out of 87 cases). Motor and sensory nerves were simultaneously involved, and injury primarily involved the lower limbs. One hundred and seven (97%) patients were clinically diagnosed with peripheral neuropathy, of whom 26 (24%) exhibited spinal abnormalities on magnetic resonance imaging, supporting a diagnosis of subacute combined degeneration. Treatment included N2O withdrawal and vitamin B12 supplementation. Reexamination of six patients indicated that treatment was effective. Follow‐up was completed for 51 patients. Thirty‐four patients (67%) recovered completely, 17 patients (33%) had residual limb numbness, and only one patient experienced relapse. Sex was an independent prognostic factor; the outcomes of female patients were better than that of male patients. Conclusion The recreational use of N2O has largely expanded among youth in recent decades, which has become a growing public health concern in China. It highlights the importance of the recognition of various clinical symptoms, particularly limb numbness and/or weakness related to the cases of N2O abuse. The therapeutic administration of vitamin B12 supplementation and N2O withdrawal can make the overall prognosis good, especially for female patients.


INTRODUCTION
Nitrous oxide (N 2 O) is a colorless gas that is often mixed with 30% oxygen for use as an anesthetic for dental and surgical operations, and its anesthetic effect is characterized by rapid onset and high safety.
In recent years, recreational N 2 O use has increased significantly both in China and abroad (Randhawa & Bodenham, 2016; The GDS Core Research, Team; Zheng et al., 2020). Currently, surveys on the prevalence and demographics of N 2 O abuse in China have not been comprehensive. The United States National Survey on Drug Use and Health estimates that 21% of teenagers who start using inhaled drugs start with N 2 O [OAS, 2009]. Short-term use of high doses or long-term use of N 2 O can lead to vitamin B 12 deficiency, resulting in a series of neurological diseases, which primarily include peripheral neuropathy, myelopathy, and encephalopathy. Some patients may also experience psychiatric, emotional, and psychological changes, or even death (Blanco & Peters, 1983;Garakani et al., 2016;Keddie et al., 2018;Mancke et al., 2016;Patel et al., 2018;Pema et al., 1998;Sahenk et al., 1978;Van Amsterdam et al., 2015).
The use of N 2 O is not restricted in China. Although Internet search results for N 2 O are blocked in China, other options exist for finding suppliers online. N 2 O is becoming an increasingly important substance of abuse, although the public still lacks a more formal understanding of its adverse effects, which is also a major reason for its popularity.
This study aimed to review the clinical characteristics and outcomes of patients with N 2 O abuse in order to improve the understanding of this condition by physicians and increase public awareness of the harm caused by N 2 O abuse.

Research subjects and characteristics
Patients with N 2 O abuse treated in the Shengjing Hospital of China Medical University Department of Neurology (Clinic and Ward) between January 2018 and December 2020 were included in the study.
This study complies with the Declaration of Helsinki and was approved by the Institutional Review Board of Shengjing Hospital (2020PS047K).
The clinical data of patients were collected and included history of N 2 O exposure, clinical presentation, and results of laboratory, imaging, and electrophysiological examinations. Patients were followed up by telephone to determine outcomes. The content of the followup included treatment plan, treatment duration, degree of recovery, whether relapse occurred, and changes in the results of laboratory, imaging, and electromyographic examinations. Patients were divided into a complete recovery group and an incomplete recovery group based on their status of recovery, and further analysis of factors that may affect outcomes was performed (Table 1).

General demographic data and clinical presentation
Demographic data and clinical presentation of the patients are shown in Table 2. A total of 110 patients (57 males and 53 females) were included. Of the 110 patients, 96 patients came from the neurology clinic. Fourteen patients came from the neurology ward and they were admitted to the ward after going through the neurology clinic. The average patient age was 21.4 ± 4.2 y (range: 14-33 y). None of the patients had a personal or family history of neurological or psychiatric diseases. All patients had a history of N 2 O exposure; the average duration of exposure was 0.5-72 (12.5 ± 4.2) mo. The most common clinical presentations were limb numbness and/or weakness (97%), of which the symptoms involved all four limbs in 64% of patients. Other neurological symptoms included difficulty walking (12%), headache or dizziness (9%), involuntary movements (7%), constipation (5%), urinary retention (3%), urinary incontinence (2%), memory loss (3%), epilepsy (2%), and foot drop (2%). Some patients experienced psychiatric symptoms, such as mood disorders (2%), presenting as anxiety, irritability, or depressed mood; sleep disorders presenting as insomnia (3%) or hypersomnia (1%); and a few patients even developed hallucinations (3%) and behavioral abnormalities. The most common neurological signs were decreased muscle strength (83%), followed by superficial sensory disturbances (80%), decreased tendon reflex (71%), deep sensory disturbances (64%), and positive Romberg's sign (62%), with increased tendon reflex (9%) and positive Babinski sign (7%) in a small number of patients.

Laboratory, imaging, and electromyographic examinations
The results of laboratory, imaging, and electromyographic examinations are shown in Table 3. Vitamin B 12 level was tested in 66 patients (333.6 ± 396.7 pg/ml). Among them, nine patients had selfadministered vitamins before seeking medical help, and their vitamin B 12 levels were very high (1152 ± 527.8 pg/ml). In reality, 60% (34 out of 57 cases) of patients were deficient in vitamin B 12 . Of the 45 patients tested for high homocysteine level, 69% (31 out of 45 cases) exhibited high homocysteine levels (44.6 ± 42.1 µmol/L). Of the 71 patients tested for hemoglobin (133.3 ± 20.7 g/L), 35% (25 out of 71 cases) exhibited anemia. In these patients, folic acid levels were within the normal range, and 20% (five out of 25 cases) of these patients presented with hypochromic macrocytic anemia, with a mean corpuscular volume of 107.1 ± 5.2 fl (normal value 83-101 fl). Tests for thyroid function, rheumatoid arthritis-associated antibodies, antineutrophil cytoplasmic antibodies, tumor biomarker, and human immunodeficiency virus (HIV) were performed in some patients, and all results were normal or nonspecific. Spinal magnetic resonance imaging (MRI) revealed a long T2 signal on the dorsal side of the spinal cord in 52% (26 out of 50 cases) of patients. The signal was generally located in the neck, in a "V," inverted "V" triangle, or elliptical shape ( Figure 1). Head MRI revealed abnormalities in 55% (six out of 11 cases) of patients, of whom three exhibited brain atrophy, and the other three exhibited demyelination, primarily distributed in the frontal lobe. A total of 87 patients underwent electromyography (EMG) examination of four sensory nerves and four motor nerves. Prolonged distal latency and decreased amplitude indicate axonal injury, and decreased nerve conduction velocity indicates demyelination. The results of the examination showed that peripheral neuropathy associated with N 2 O abuse TA B L E 3 Laboratory, imaging, and electromyography results

Diagnosis and treatment
Based on clinical presentation and signs, 107 patients (97%)

Follow-up
During the treatment period, six patients underwent re-examination: at 1 mo after treatment, four patients underwent blood tests only, one underwent blood tests and cervical spine MRI, and one underwent   Figure 2). Cervical spine MRI reexamination indicated that the abnormal signal of the spinal cord was reduced from before ( Figure 3).
One patient who underwent EMG reexamination underwent the initial EMG examination after limb weakness at 10 d; the result peripheral nerve damage, sensory motor fiber involvement, myelin sheath and axon involvement, and the lower limbs as the principal site affected.
Symptoms were not significantly improved after 1 month of treatment, and EMG reexamination indicated that the amplitude and nerve conduction velocity of the motor nerves were still significantly decreased from before. Symptoms improved significantly after 4 months of treatment, and EMG reexamination indicated that nerve conduction velocity and amplitude of the motor nerves had improved to varying degrees ( Figure 4). All patients underwent follow-up by telephone after data collection. The most common reason for loss to follow-up was the inability to reach the patient by telephone. Eventually, telephone follow-up was completed in only 51 patients (46%

DISCUSSION
N 2 O has euphoric effects in addition to anesthetic effects, and has been used as a substitute for benzodiazepines to relieve withdrawal syndromes caused by cocaine, alcohol, and other substances. It has also been recommended for the treatment of refractory depression [Daynes & Gillman, 1994;Gillman & Lichtigfeld, 1985;Gillman et al., 2006;Gillman et al., 2007;Nagele et al., 2015]. The mechanism by which N 2 O produces euphoria remains poorly understood and may be due to the inhibition of N-methyl-D-aspartate ( (Chiang et al., 2013;Gilliam & Cox, 1973). The clinical manifestations of patients with N 2 O abuse vary and may involve neurological, psychiatric, autonomic nervous system, and even dermatological symptoms. For clinicians, the ability to identify the possible clinical manifestations of N 2 O abuse will prevent delays in diagnosis and treatment.
In addition to affecting vitamin B 12 , N 2 O may also exert its neurotoxic effects through an antagonism of NMDA receptors (Maze M, Fujinaga, 2000;Garakani et al., 2016;Keddie et al., 2018). Moreover, homocysteine accumulation, oxidative stress damage, and changes in cytokine and growth factor levels are also believed to be associated  Singh et al., 2015). The causes of the psychiatric symptoms remain poorly understood. Hutto et al. believed that increased synthesis of tetrahydrobiopterin (BH 4 ) affected the rate of monoamine (dopamine, norepinephrine, and serotonin) synthesis, thereby inducing psychiatric symptoms, whereas others believed psychiatric symptoms to be associated with cerebral hypoxia, methemoglobinemia, low arterial partial pressure of oxygen, and acidosis (Brodsky & Zuniga, 1975;Hutto, 1997;Scalabrino, 2005).
As mentioned above, N 2 O irreversibly induces the oxidation of vitamin B12, which not only blocks methylation and DNA synthesis, but also leads to homocysteine accumulation. The findings of the present study also suggest that elevated homocysteine level is very common in patients with N 2 O abuse, as 69% of the patients exhibited hyperhomocysteinemia. Decreased vitamin B 12 was also a common abnormal test result, but it was not as high as the rate of abnormal level of homocysteine, consistent with the findings of previous studies (Hannibal et al., 2016;Li et al., 2016;Oussalah et al., 2019]. This may be because many patients have learned about the possible adverse consequences of N 2 O inhalation and its prevention through their social circles and take vitamin B 12 supplementation on their own before initial consultation. Some patients with normal serum vitamin B 12 levels have functional vitamin B 12 deficiency and clinical symptoms. Serum vitamin B 12 levels do not represent intracellular levels of vitamin B 12 nor do it represent the existence of functional vitamin B 12 deficiency. Cyclocobalamine is the gold standard test to evaluate vitamin B 12 deficiency, but it is impossible, because it is a retrospective study and was limited by conventional test methods. Increased homocysteine or methylmalonic acid levels indicates a lack of functional vitamin B 12 at the cellular level (Chiang et al., 2013). Therefore, homocysteine is more suitable as a test indicator than vitamin B 12 in patients who are  Based on p < 0 .1, the three independent variables of age, sex, and duration of medical history were selected for univariate analysis (Table 1 and Table 4). Further logistic regression analysis (Table 5) showed that sex was the N 2 O-related independent factor among the three variables influencing neurological disease outcomes; female patients were more likely to achieve complete recovery than male patients (OR = 5.803, 95% CI: 1.289-26.125; p = 0.022).
density of myelinated fibers in the fasciculus gracilis is highest in the cervical spinal cord (Ohnishi et al., 1976). Abnormal brain MRI was uncommon; three patients presented with brain atrophy, but only one patient presented with mild memory loss, and the other three patients exhibited demyelination, primarily in the frontal lobe. This may be explained by impaired methylation of myelin phospholipids caused by N 2 O abuse leading to demyelination of nerves in the central nervous system [Pema et al., 1998;Maze & Fujinaga, 2000]. In this study, only 11 patients underwent MRI examination of brain, but the abnormal rate was relatively high (55%), which may be related to the symptoms of brain injury in these patients before the examination. However, we should recognize that it is necessary to perform brain/spinal MRI to help determine whether there is brain/spinal cord injury, and the severity and prognosis of diseases.
EMG is of great significance for the diagnosis of peripheral neuropathy. In the present study, EMG of 87 patients with N 2 O abuse revealed abnormalities of varying degrees, characterized by mixed axonal and demyelination injury. Motor and sensory nerves were often simultaneously involved, and there was significant injury in the lower limbs.
These results are consistent with those of previous reports of N 2 Orelated peripheral neuropathy and the characteristics of most electromyographic changes in peripheral neuropathy (Vasconcelos et al., 2006;Li et al., 2016;Zheng et al., 2020 numbness, but their daily living activities were largely unaffected. Sex was an independent prognostic factor, but the specific reasons remain to be studied further. There was only one case of relapse among the patients who underwent follow-up, which may indicate that N 2 O withdrawal is relatively easy. The follow-up rate of the present study was only 46%, which may be insufficient to reflect the true outcomes and relapse rate of patients with N 2 O abuse.
As this study was retrospective, it had the following limitations: the clinical data is incomplete; the quantity of N 2 O intake is uncertain; lack of cyclocobalamin testing; low rate of MRI examination; low reexamination rate; low telephone follow-up rate. These are the limitations of this study, which need to be further improved in future prospective research.

CONCLUSION
N 2 O abuse is a noteworthy emerging public health problem that primarily affects young people. Clinicians should be fully aware of the various clinical symptoms that may develop in cases of N 2 O abuse, particularly in young patients with limb numbness and/or weakness, and history of N 2 O exposure should be ascertained in such patients. N 2 O withdrawal and vitamin B 12 supplementation result in good outcomes, especially for female patients.

CONFLICT OF INTEREST
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

ACKNOWLEDGMENTS
We would like to thank Editage for English language editing and Peng Chen for proofreading the article.

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1002/brb3.2533

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.