The Association between Homocysteine Level and Balance Disorder among US Adults Varied In Different Sex Groups: A Cross-Sectional Study

Homocysteine (Hcy) is a sulfated amino acid derived from ingested methionine and is a component of the homocysteine-methionine cycle. High levels of Hcy can cause toxic effects and lead to neuron and neuronal cell death [1]. A growing body of evidence suggests that elevated levels of Hcy may be associated with an increased risk of cardiovascular, neurological, and psychiatric disorders in adults, including coronary artery disease, stroke, dementia, and depression [2,3]. However, there are few findings on balance disorder with Hcy levels in middle-aged and older adults. However, there is limited research on the association between Hcy levels and balance disorder in middle-aged and older adults. 

Maintaining balance involves a complex series of sensorimotor control system [4], and a person's ability to maintain balance is crucial for almost all activities of daily living. The maintenance of human balance mainly relies on sensory input from the vestibular and visual systems, neural processing centers throughout the central nervous system, and motor output from the proprioceptive system. A decline in the functioning of any of these systems can lead to a balance deficit [5]. Imbalance leading to falls is the most common cause of serious injury in older adults and increases the risk of hospitalization [6]. While the prevalence of balance dysfunction in the U.S. is unknown, data from the National Health and Nutrition Examination Survey (NHANES) suggests that 35% of U.S. adults (69 million people) aged 40 years or older have objective evidence of vestibular dysfunction [7]. 

Currently, the exact mechanism between Hcy and balance disorder remains unclear and requires further exploration. This study aimed to assess the relationship between Hcy and balance disorder in individuals over 40 years old in the United States using data from the 1999-2004 NHANES. The findings of this study may provide more accurate and effective methods for the prevention and treatment of this condition.

• Study population and design
  

NHANES is a population-based cross-sectional survey that collected nationally representative data on the health and nutrition of the U.S. household population using a complex, stratified, multistage probability design. The survey comprised of an interview portion that included demographic, socioeconomic, dietary, and health-related questions and a physical examination part that included physiological measurements and laboratory examinations. The NHANES protocol was approved by the National Center for Health Statistics (NCHS) Research Ethics Review Board, and participants provided written informed consent. 

For this study, we obtained data from the 1999-2004 NHANES database, including a total of 31,126 individuals over the age of 40. We excluded 25,599 individuals with missing balance disorder information, 162 individuals who did not have homocysteine measurements, and 734 individuals who did not have complete covariate information. Ultimately, our analysis included 4,631 participants (Figure1).

• Variables
• Balance examination

The Romberg Test of Standing Balance on Firm and Compliant Support Surfaces (CDC 2001b) was used to measure participants' ability to maintain balance under four test conditions that were ordered in increasing levels of difficulty [8]. Balance testing was scored on a pass/fail basis, with test failure defined as (a) the subject needing to open their eyes, (b) the subject moving their arms or feet to achieve stability, or (c) the subject beginning to fall or requiring operator intervention to maintain balance within a 30-second interval. As each successive test condition from 1 to 4 was progressively more difficult than the condition preceding it, the balance testing component was ended whenever a subject failed to pass a test condition (either during the initial test or in the re-test if the participant opted for one). For this study, we focused on test condition 4, which was designed to distinguish participants who could not stay standing when relying primarily on vestibular input. We categorized participants as having balance disorder if they did not pass test condition 4. The severity of vestibular balance disorder was defined by the time that condition 4 was unsuccessful (20 to < 30, 10 to < 20, and 0 to < 10 seconds). The time to failure, which was the duration until the loss of balance, was also recorded for test condition 4, with those who passed the test assigned the maximum value of 30 seconds.

• Serum homocysteine and HHcy 

To measure plasma homocysteine levels, a fasting blood sample was taken from each participant. The sample was collected in the morning after a fast of at least 8 hours but not more than 24 hours. The specimen collection and processing instructions followed the NHANES Laboratory/Medical Technologists Procedures Manual. The blood samples were then frozen and stored at a temperature of −20°C. The samples were transported to the Division of Environmental Health Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention for analysis. Hyperhomocysteinemia(HHcy) was defined as a plasma homocysteine level greater than 15 µmol/L [9].

• Covariates 

Demographic variables included age, race, educational level, marital status, Body Mass Index (BMI), smoking status, drinking status, and disease (hypertension, diabetes, stroke, and arthritis). Participants were categorized as “nonsmokers” (lifetime use of < 100 cigarettes), “former smokers” (previous history of smoking but no longer a smoker at the time of interview), or “current smokers” (lifetime use of ≥100 cigarettes and who currently smoke cigarettes) [10]. Body mass index (BMI) was calculated as a person's weight (mass) in kilograms divided by the square of height in meters. According to World Health Organization classification, participants were classified as underweight (<18.5), normal weight (18.5–24.99 kg/m2), overweight (25.00–29.99 kg/m2) or obese (≥30 kg/m2) [11].

• Statistical analysis

Categorical variables were presented as numbers and percentages calculated using sample weights, while continuous variables were presented as mean±SE. To examine group differences on all continuous measures, independent-samples t-tests were used. The distribution of the categorical variables between the balance disorder group and the normal vestibular function group was compared using Pearson chi-squared test. The suggested weighting method was used in the data analysis, considering significant differences. Since the distribution of the Hcy level is skewed, Hcy was included in the logistic regression model after logarithmic transformation [12]. Logistic regression analysis was performed to evaluate the association between homocysteine and balance dysfunction. 

All statistical analyses were performed using Stata, and P < 0.05 was considered statistically significant.

The characteristics of the subjects are presented in (Table 1). Of the 4631 participants, 2323 were male and 2308 were female. The mean homocysteine level was 8.95 ± 0.06 umol/L for those with normal balance function and 10.59 ± 0.15 umol/L for those with balance disorder. Participants with balance dysfunction were more likely to be older than 60 years old, female, have less than a high school education, be unmarried, smoke, have previously consumed alcohol, have a BMI indicating overweight (25.00-29.99kg/m²), and have associated diseases such as diabetes, hypertension, stroke, and arthritis.

Table 1: Weighted characteristics of participants by Balance Disorder from NHANES 1999–2004 (n = 4631).

N (Weighted %): Categorical variables are presented as number and percentage (calculated using sample weights). Continuous variables are presented as mean±SE. Independent-samples t tests were used to examine group differences on all continuous measures. Pearson chi-squared test was performed to compare the distribution of the categorical variables between the vestibular dysfunction group and the normal vestibular function group. p < 0.05 indicates a statistically significant difference.

Table 2: Association of homocysteine with Balance Disorder from 1999 to 2004 National. 

Health and Nutrition Examination Survey (N = 4,631).

Logistics regression analyses were conducted using sample weights, with significance set at p < 0.05.  p < 0.05*, p < 0.001**.

^a log transformed

Model 1: unadjusted for any potential confounders;

Model 2 adjusted for age, gender, and race;

Model 3 adjusted for age, gender, race, educational level, marital status, body mass index (BMI), smoking status, drinking status, vitamin B₁₂ (pmol/L), and folate (nmol/L).

Model 4 adjusted for age, gender, race, educational level, marital status, body mass index (BMI), smoking status, drinking status, vitamin B₁₂ (pmol/L), folate (nmol/L) and disease (hypertension, diabetes, stroke, and arthritis). 

The results of the multiple regression analysis are presented in (Table 2). In the unadjusted model, there was a positive association between log Hcy and balance disorder (OR=2.69, 95% CI: 1.95-3.72, p < 0.001). This association remained significant after adjusting for confounders in model 2 (OR=2.01, 95% CI: 1.41-2.84, p < 0.001) and model 3 (OR=1.68, 95% CI: 1.19-2.37, p < 0.001). To further analyze the relationship, we transformed the continuous variable (log Hcy) into categorical variables. The results showed that the risk of balance disorder in patients with HHcy was 68% higher than the risk in patients with normal Hcy levels (OR=1.68, 95% CI: 1.18-2.38, p < 0.05). 

In further examining the relationship between log Hcy and balance disorder, we investigated potential interaction effects while adjusting for confounding factors. Our findings revealed a significant difference in the relationship between log Hcy and balance disorder based on sex stratification (P for interaction <0.05). As presented in (Table 3), after adjusting for confounders, we found that log Hcy (OR=1.73, 95%CI: 1.15-2.60, p < 0.05) and HHcy (OR=1.74, 95%CI: 1.10-2.73, p < 0.05) were positively associated with balance disorder in women, resulting in a 74% increased risk. However, among men, the risk increase was 63%, and the positive correlation was not statistically significant (p > 0.05). Nevertheless, the interaction test indicated a significant difference between the risk increases in women and men, indicating that gender significantly impacted the relationship between Hcy and vestibular function. 

Table 3: Association of homocysteine with Balance Disorder by gender difference from 1999 to 2004 National Health and Nutrition Examination Survey (N = 4,631). 

Logistics regression analyses were conducted using sample weights, with significance set at p < 0.05. p < 0.05*, p < 0.001**.

^a log transformed

Model 1: unadjusted for any potential confounders;

Model 2: adjusted for age, gender, and race;

Model 3: adjusted for age, gender, race, educational level, marital status, body mass index (BMI), smoking status, drinking status, vitamin B₁₂ (pmol/L), and folate (nmol/L).

Model 4: adjusted for age, gender, race, educational level, marital status, body mass index (BMI), smoking status, drinking status, vitamin B₁₂ (pmol/L), folate (nmol/L) and disease (hypertension, diabetes, stroke, and arthritis).

In this cross-sectional study, we investigated the relationship between Hcy levels and balance disorders in individuals over the age of 40. Our results indicate a positive correlation between Hcy levels and the risk of balance disorders, with a stronger correlation observed in women based on subgroup analysis. 

While few studies have examined the association between Hcy and balance disorder, previous research has produced mixed results. One prospective study of 41 patients with various vestibular disorders found no relationship between Hcy levels and Peripheral balance Disorder (PVD) [13]. Similarly, a Chinese case-control study of 240 individuals with Benign Paroxysmal Positional Vertigo (BPPV) found no significant difference in Hcy levels between BPPV patients and control groups [14]. However, a retrospective analysis of 90 patients with vestibular neuritis found that elevated Hcy may play a role in preventing recovery after a recent vestibular neuritis [15]. Additionally, a case report describes a 68-year-old woman with severe symptoms of Ménière's syndrome and increased fasting serum homocysteine [16]. The inconsistent results among these studies could be due to differences in the populations investigated and the presence of confounding factors. Further research is needed to better understand the relationship between Hcy and balance disorder and to determine if Hcy levels could be a potential biomarker or therapeutic target for balance disorders. 

Our cross-sectional study conducted from 1999 to 2004 revealed a significant positive correlation between Hcy levels and balance disorder, with a stronger association in women. After adjusting for confounding factors, including disease, our multiple logistic regression model found no significant interaction between Hcy and gender. However, the impact of disease on balance function was greater in men. We further analyzed the reason for the gender difference and found that the decrease in estradiol during menopause may lead to an increase in Hcy and cysteine, resulting in endothelial dysfunction in postmenopausal women [17]. Previous studies have reported that the concentration of Hcy increases in postmenopausal women, and estrogen levels are negatively correlated with Hcy levels in women [18,19]. Hence, sex hormones may play a role in the relationship between balance disorders and Hcy. Additionally, Hcy levels can increase due to inadequate intake of folic acid (vitamin B9), vitamin B12, and vitamin B6, which are essential cofactors for enzymes involved in Hcy metabolism. To account for this, we adjusted for the effects of folic acid and vitamin B12 on Hcy levels. 

Hcy is a sulfur-containing amino acid found in the human body, and it serves as an important intermediate in the metabolism of methionine and cysteine. One key function of homocysteine is its involvement in the methylation cycle, during which methyl is transferred to various substrates [20]. Homocysteine can be utilized in two ways: 1) it can be methylated to methionine through the catalytic activity of N5 force n10-methylenetetrahydrofolate reductase, and 2) it can be converted to cysteine through the catalysis of Cystine β Synthase (CBS) [21]. HHcy, or an abnormally high level of homocysteine in the blood, can result from an imbalance of processes and factors involved in homocysteine metabolism. 

HHcy is a condition characterized by elevated levels of homocysteine in the blood, typically defined as homocysteine levels exceeding 15umol/l. Homocysteine is known to damage blood vessels through several mechanisms. For instance, high levels of homocysteine can induce hypermethylation of DNA promoter regions, which increases the expression of p66shc in endothelial cells and results in oxidative stress [22].This, in turn, can reduce the availability of nitric oxide, a potent vasodilator [23], leading to endothelial cell damage and increased lipid peroxidation and oxidation [24]. Consequently, HHcy is linked to a prothrombotic state characterized by increased platelet aggregation, production of Tissue Factor (TF) [25], and activation of coagulation factors such as VMagneX and XII, as well as decreased activation of protein C and cell surface thrombomodulin [26-28]. Taken together, these findings suggest that HHcy may contribute to vascular inflammation, hypercoagulability, and the development of atherosclerosis and associated thrombotic events [29]. 

The common causes of peripheral vertigo are known to be BPPV, vestibular neuritis, and Meniere's disease. However, the pathophysiological mechanism underlying peripheral vertigo remains unclear. The link between homocysteine and balance disorder can be explained through various mechanisms. Firstly, the blood-labyrinth barrier protects the inner ear from abnormalities in the circulating blood, but metabolic disorders can still affect vestibular function [30]. Additionally, labyrinthine branches receive less collateral flow from the internal carotid artery, making them vulnerable to the effects of atherosclerosis [31]. Thus, vascular endothelial injury caused by homocysteine may lead to balance disorder. Secondly, homocysteine can lead to oxidative stress and the accumulation of free radicals and other oxidizing substances. These oxygen free radicals can cause vestibular-cochlear poisoning and result in permanent damage to sensory hair cells and neurons [32]. Homocysteine may also affect the function of the vestibular system by regulating the level of neurotransmitters. For example, Acetylcholine (ACh) has long been identified as the primary neurotransmitter of the efferent vestibular system in most animals [33]. Studies have shown that an increase in homocysteine level is related to a decrease in acetylcholine sensitivity [34]. However, the specific molecular mechanisms of this regulation are currently unknown. 

In summary, the association between Hcy and balance disorder appears to be multifactorial, involving vascular endothelial injury, oxidative stress, and regulation of neurotransmitters. Further investigation into these mechanisms can enhance our understanding of the link between homocysteine and balance disorder, leading to the development of more effective strategies for prevention and treatment. 

Our study has several strengths, including the use of a nationally representative sample and a large sample size that allowed for subgroup analysis. We believe that our findings are highly relevant to the population as a whole, especially as this is the first study to investigate the relationship between homocysteine and balance disorder in individuals over 40. However, we must also acknowledge the limitations of our study. First, our study is cross-sectional, and thus we cannot establish a causal relationship between homocysteine and balance disorder. Further research is needed to determine the exact nature of this relationship. Second, we cannot rule out the possibility of biases from unmeasured confounding factors.

In summary, our study suggests that there is a significant association between elevated Hcy levels and an increased risk of balance disorder in Americans over the age of 40, particularly in women. This highlights the importance of promoting a healthy lifestyle that includes a well-balanced diet rich in vitamins and antioxidants to reduce Hcy levels and decrease the risk of developing balance disorders. Future studies should aim to explore the underlying mechanisms and establish a causal relationship between Hcy and balance disorder, which can further inform effective prevention and treatment strategies.

This study was approved by the ethics review board of the National Center for Health Statistics and written informed consents were obtained from each participant.

Y Hu, G Li and H Luo conceived and designed the study. Y Hu, M Song, D Wu and Y Zhang conducted the formal analysis and developed the methodology. Y Hu and D Lin wrote the initial drafts. D Wu and Y Zhang helped draft the manuscript. G Li and H Luo are the corresponding authors of this work and supervised work on the entire manuscript. All the authors read and approved the final manuscript.

We would like to thank our colleagues from the Department of Neurology, The Second Affiliated Hospital of Chongqing Medical University, who provided valuable advice regarding the manuscript

The authors have no conflicts of interest to declare.

No funding was received for this study.

The survey data are publicly available on the internet for data users and researchers throughout the world (www.cdc.gov/nchs/nhanes/).

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