Clinical epidemiological characteristics of nitrous oxide abusers: A single‐center experience in a hospital in China

Abstract Purpose This study investigated the clinical epidemiological characteristics of nitrous oxide (N2O) abusers in a hospital in China, which have not been systematically reported. Methods The characteristics of patients abusing N2O who were examined and treated at the Affiliated Hospital of Xuzhou Medical University from January 2017 to December 2020 were analyzed. Results A total of 61 patients (average age: 21.7 ± 3.2 years; 42 male and 19 female) were enrolled; 60.7% of the patients had an education level of high school or lower, and most (59.0%) had no stable occupation. The mean exposure time was 8.5 ± 7.7 months (range: 1–36 months). Only 52.5% of the abusers reported the physician of the relevant exposure history at the first time of visiting the doctor. The main clinical type was mixed (49.2%). The most common clinical manifestation was distal limb numbness (80.3%). The most frequent outcome was peripheral neuropathy (59%) and subacute combined degeneration (36%). Serum homocysteine level was elevated in 67.5% (27/40) of the patients, while 44.4% (20/45) showed reduced vitamin B12. Note that 61% (22/36) showed abnormal signals in the posterior or lateral funiculus of the spinal cord, and 97% (31/32) of the patients showed peripheral nerve damage by electromyography. In all cases, symptoms were alleviated after halting N2O intake and receiving nutritional neurotherapy. Conclusions N2O abuse can lead to nervous system damage, especially peripheral nerve and spinal cord damage. A full understanding of its clinical epidemiological characteristics is helpful for clinicians to make a timely and clear diagnosis.


INTRODUCTION
Nitrous oxide (N 2 O) (i.e., laughing gas) is widely used in clinical practice for analgesia, sedation, and anesthesia. In recent years, an increasing number of young people have been abusing N 2 O in their pursuit of stimulation, with a concomitant increase in the incidence of related neurologic diseases and irreversible nerve damage. However, the risks associated with N 2 O abuse-which include subacute combined lesions of spinal cord, toxic encephalopathy, peripheral neuropathy, and mental disorders-are not fully recognized by society and clinicians.
Previous studies have focused on the pathogenesis and clinical characteristics of diseases caused by N 2 O abuse, and there have been few epidemiologic investigations despite the importance of data from such studies for the development and implementation of effective interventions by health authorities (Bao et al., 2020;Garakani et al., 2016;Kaar et al., 2016;Lan et al., 2019;Li et al., 2016;Tuan et al., 2020). In order to collect epidemiologic data on N 2 O abuse, the present study analyzed the general epidemiological and clinical characteristics of 61 patients who were abusing N 2 O and were treated at the Affiliated Hospital of Xuzhou Medical University from January 2017 to December 2020. Our findings can improve the clinical diagnosis and treatment of patients who abuse N 2 O and guide health policies to address this problem.

Patients
Between January 2017 and December 2020, 71 patients who were abusing N 2 O were treated at the Affiliated Hospital of Xuzhou Medical University. After excluding patients with incomplete baseline data and those who met the exclusion criteria, 61 patients were enrolled in the present study (20 outpatients and 41 inpatients).

Inclusion and exclusion criteria
At present, there is no unified diagnostic standard for N 2 O abuse. The inclusion criteria used to select patients for this study were as follows: (1) a clear history of N 2 O inhalation over the course of the disease; (2) clinical manifestations and signs of nervous system damage after N 2 O abuse that could not be attributed to other nervous system diseases; and (3) complete baseline data were available. The exclusion criteria were as follows: (1) a history of neurologic and mental illness before exposure to N 2 O and (2) use of other addictive drugs during the period of N 2 O abuse.

Data collection
Based on studies on methamphetamine-related mental symptoms conducted in China (M. F. Su, Liu, et al., 2018)

Data processing
Quantitative data were presented as means and SD (x ± s). Qualitative data were described as counts or percentages, and intergroup comparisons were performed by the Fisher test. Non-normally distributed data were expressed as medians and interquartile range (IQR). p < .05 was considered statistically significant. Statistical analysis was performed using SPSS 24.0 (SPSS Inc., Chicago, IL, United States).

Epidemiology of patients abusing N 2 O
The number of patients abusing N 2 O treated at our hospital showed a generally increasing trend from January 2017 to June 2020, although it decreased thereafter ( Figure 1).

Diagnosis
The initial diagnosis in 65.6% of the patients was peripheral neuropathy; only 32 (52.5%) informed the physician of their history of exposure at the first visit. The exposure history of nearly half of the patients was revealed only after admission, and four cases were misdiagnosed as Guillain-Barré syndrome and received hormone or gamma globulin treatment (

Main clinical manifestations
Neurologic lesions caused by N 2 O were observed in our cohort. The top symptoms were numbness (80.3%) and limb weakness (65.6%), which was a decrease in distal muscle strength in most instances (  Table 5). Mutation of the methylene tetrahydrofolate reductase (MTHFR) was detected in six cases (TT, n = 3; CC, n = 2; and CT, n = 1).

TA B L E 4 Symptoms and signs of patients in this study
Clinical features (n = 61) N (%) and cerebral atrophy in one case (Figure 3).  showed peripheral nerve damage, and most had motor and sensory involvement (Table 7). Our data showed that the number of patients seeking medical assistance for symptoms related to N 2 O abuse has been increasing yearly at our hospital; however, the number of patients decreased markedly after June 2020. There are a few possible reasons for this decline. In our study population, 89.3% of the patients were registered locally and only a small number came from provinces and cities adjacent to Xuzhou. We also found that there was no significant difference in the number of patients from urban and rural areas. However, given the relatively small sample size in this study, it is possible that there is a difference in the rate of N 2 O abuse according to the type of community (i.e., urban vs. rural).

Treatment and outcome
The data show that the patients with N 2 O abuse have the following demographic characteristics: (1) the overall age is younger, there are more men than women, and the average age of women is lower than that of men; (2) the proportion of unmarried patients is higher; (3) the overall level of education is not high; and (4) most of the patients have no stable occupation. In previous studies, the patients of N 2 O abuse were mainly adolescents (Bao et al., 2020;Garakani et al., 2016;Li et al., 2016;Tuan et al., 2020), and no significant gender differences were mentioned. However, whether there are significant gender differences in patients with N 2 O abuse still needs a large sample study.
Women are more likely to use psychotropic drugs earlier (He et al., 2013), which explains the average age difference between men and women. The fact that there are more unmarried women than men is considered to be related to the fact that the average age of men is higher than that of women. Based on these data, young people who are single, have a low level of education, and lack stable employment are the target demographic for education regarding the risks of N 2 O abuse.
Five of our patients were positive for syphilis by virologic examination.
Previous studies have reported high rates of AIDS and syphilis among drug users as they are more likely to have unprotected sex (S. Su, Mao et al., 2018). This is the first report of the positive rate of syphilis among were admitted to the hospital with myelopathy, and four were misdiagnosed as Guillain-Barré syndrome and underwent related examination and treatment. In many cases, the history of exposure was unknown even after many outpatient visits, possibly because of the refusal of some patients to provide this information and a lack of awareness of symptoms on the part of clinicians.
According to our N 2 O abuse-related disorder classification, peripheral nerve with spinal cord injury is the most common type of injury.
Brain parenchyma lesions have been previously reported, but there were no cases with spinal cord or brain parenchyma involvement in our study (Assaf et al., 2020;Bajaj et al., 2018). Because patients' self-reports of exposure may not be reliable, we were unable to compare the clinical data of different types of patients; therefore, it is unclear whether the peripheral nerve damage caused by N 2 O precedes CNS damage, or whether patients with CNS involvement have worse prognosis. Additionally, the psychiatric manifestations of N 2 O abuse including delusions, hallucinations, and mental confusion should not be ignored (Assaf et al., 2020;Bajaj et al., 2018). Some studies have also reported more severe mental symptoms leading to suicide or violence; professional psychiatric evaluation is recommended in such cases (Chien et al., 2020).
In our study, 91.8% of the patients were ultimately diagnosed with peripheral neuropathy or subacute combined SCD, which is in line with other reports (Garakani et al., 2016;Li et al., 2016). The main neurologic symptoms in our patients were limb weakness, numbness, and sensory abnormalities, while a subset exhibited mental abnormalities and autonomic nervous dysfunction (bladder and sexual dysfunction).
In general, short-term exposure to N 2 O is not associated with neurologic complications unless the patient has vitamin B12 deficiency or an absorption disorder. However, long-term intake of N 2 O >80 g/day can increase the risk of diseases (Alt et al., 2011;Cheng et al., 2013).
It is thought that long-term abuse of N 2 O causes nervous system dysfunction by altering vitamin B12 metabolism (Lassen et al., 1956). Vitamin B12 mainly exists in two active forms: adenosine cobalamin and methylcobalamin. The latter is an important cofactor of methionine synthase (MS), the rate-limiting enzyme in the conversion of HCY to methionine (Met). N 2 O irreversibly oxidizes cobalt ion (Co+) in vitamin B12, thereby inactivating and inducing the excretion of cobalamin from the body, which results in the inhibition of MS activity, accumulation of HCY, and decreased Met synthesis (Flippo & Holder, 1993;Healton et al., 1991;Reynolds, 2006). Under normal circumstances, Met is converted into S-adenosylmethionine (SAM); impairment of SAM and Met synthesis can lead to the failure of myelin methylation and demyelinating changes in the spinal cord (Healton et al., 1991). Additionally, HCY accumulation was shown to exert toxic effects by enhancing oxidative stress and activation of N-methyl-Daspartate receptor in neurons, leading to neuronal death. N 2 O is itself neurotoxic and can affect cortical function. We found that blood HCY was elevated in 67.5% of the patients, and vitamin B12 was decreased in 44.4%, consistent with previous work that elevated blood HCY levels were more diagnostic than vitamin B12 deficiency (Ahn & Brown, 2005 Human MTHFR gene is located on chromosome 1p36.3 and contains 11 exons, which are responsible for encoding important enzymes involved in folic acid/HCY metabolism. Its transcriptional product is a 77 kDa protein that can catalyze the demethylation of 5,10methyltetrahydrofolic acid to 5-methyltetrahydrofolic acid, which can be reduced to 5-methyltetrahydrofolic acid with biological functions.
5-Methyltetrahydrofolic acid as a methyl donor induces HCY remethylation to methionine. The amino acid mutation of MTHFR gene may affect the structure and function of the gene coding products.
So far, many types of MTHFR gene mutations have been reported.
Among them, C667T mutation has a relatively high frequency all over the world. A mutation in codon 667 (CC→CT or TT) decreases MTHFR activity (Goyette et al., 1994), dysregulates folate metabolism, increases plasma HCY level (Liew & Gupta, 2015), and is an independent predictor of coronary heart disease and stroke. It was reported that plasma HCY was higher after N 2 O anesthesia in patients with MTHFR gene mutation than in those with the wild-type gene (Nagele et al., 2008); and neonates with MTHFR deficiency developed severe neurologic symptoms and died after inhaling a small amount of N 2 O (Erbe & Salis, 2003 but are rarely associated with the anterior funiculus, which may reflect how demyelination progresses in the dorsal spinal cord (Jain et al., 2014). In our study, 53% of the patients showed abnormal signals in the cervical spinal cord, mostly associated with only the posterior funiculus, which is in agreement with previous findings about SCD (Cao et al., 2018 There are no guidelines for the treatment of nervous system damage caused by N 2 O abuse; the main recommendations are to halt N 2 O intake and use vitamin B12 supplementation (Keddie et al., 2018). Met can also be used as an adjuvant (Stacy et al., 1992). Methylprednisolone combined with vitamins was shown to alleviate SCD caused by N 2 O abuse (Zhang et al., 2021). Most of the patients have a good prognosis after timely treatment, and only a small percentage of patients continue to experience mild sensory or motor disorder. However, chronic N 2 O exposure can lead to irreversible nerve damage or even sudden death (Layzer et al., 1978). Some studies have demonstrated that vitamin B12 did not prevent demyelination in patients who consistently