Elevated serum homocysteine levels and cognitive impairment in methamphetamine use disorder: the impact of attention deficit hyperactivity disorder comorbidity

Introduction

Methamphetamine use disorder (MUD) is defined as a chronic and relapsing clinical condition in which compulsive methamphetamine (METH) use is maintained despite serious physical, psychological, and social consequences (Khan et al. 2025). Methamphetamine, one of the most widely abused psychoactive substances in the world, is considered a growing and deepening public health threat due to its high addiction potential, destructive effects on dopaminergic neurotransmission, and mechanisms leading to persistent neurotoxicity (Wang et al. 2025). A significant body of research demonstrates that individuals with MUD experience broad and clinically significant cognitive deficits affecting domains such as attention, executive function, working memory, verbal and visual memory, language, and social cognition (Potvin et al. 2018). Cognitive impairments in METH users exhibit both acute and chronic components. The persistence of cognitive deficits varies across cognitive domains, with long-term impairments particularly seen in executive function and memory (Weber et al. 2012). Multiple neurotoxic mechanisms contribute to METH-induced brain injury, including oxidative stress, hyperthermia, and excitotoxicity. Chronic METH use causes significant changes in dopaminergic neurotransmission, particularly affecting the striatal dopamine system (Warton et al. 2018). The prefrontal cortex exhibits a marked sensitivity to changes in dopaminergic neurotransmission, and these changes contribute significantly to the observed impairments in executive functions (González et al. 2014).
The comorbidity of attention deficit hyperactivity disorder (ADHD) and METH use disorder represents a complex clinical picture that has profound effects on cognitive functions and treatment processes. Findings indicate that the prevalence of ADHD in METH users ranges from 10% to 30%, which is significantly higher than in the general population. This suggests a bidirectional relationship, with ADHD symptoms increasing susceptibility to METH use, while chronic METH exposure exacerbates existing attention deficits (Mihan et al. 2018; Obermeit et al. 2013). This comorbidity produces combined cognitive deficits that exceed the effects of each disorder individually; therefore, comorbid individuals exhibit more severe impairments in areas such as attention, executive functions, and processing speed than those with only a single diagnosis (Obermeit et al. 2013). The neurobiological basis of this clinical comorbidity is that chronic METH use further disrupts the dysregulated dopaminergic circuits that are the hallmark of ADHD, creating a self-reinforcing cycle that feeds cognitive function loss and continued substance use (Mihan et al. 2018).
Homocysteine is not an amino acid taken directly from the diet but is derived from methionine. Methionine and homocysteine are precursors of each other, and this transformation is at the heart of the methionine cycle, one of the body’s fundamental biochemical processes (Finkelstein and Martin 2000). Hyperhomocysteinemia is considered an independent risk factor for decline in cognitive functions. Therefore, hyperhomocysteinemia is closely linked to both neurodegenerative diseases and low cognitive performance (Öner et al. 2023; Setién-Suero et al. 2016). Research shows an association between high homocysteine levels and cognitive dysfunction in ADHD. A previous study found significantly increased homocysteine levels and decreased vitamin B12 levels in ADHD patients compared to controls (Lukovac et al. 2024). Homocysteine causes neurotoxicity through oxidative stress, inflammation, and impaired neurotransmission, but significant research gaps remain regarding its specific role in substance use disorders. Although research has shown that elevated homocysteine levels are associated with cognitive impairment in ADHD, the specific relationship between homocysteine levels and cognitive dysfunction in both MUD and patients with ADHD comorbid with MUD has not yet been investigated. In this context, the aim of our study was to investigate the relationship between serum homocysteine levels and cognitive functions in individuals diagnosed with MUD, especially in the presence of ADHD comorbidity. Thus, the study aimed to better understand the role of homocysteine in neurobiological processes and to shed light on the pathophysiology of cognitive impairments in MUD.

Material and methods

Study population

A total of 44 male patients who were diagnosed with MUD according to DSM-5 diagnostic criteria and presented to the hospital with complaints of subjective cognitive decline were included in the study. All of these patients were using only METH and had no other substance abuse. Forty-five healthy male individuals with no psychiatric illness or history of substance use were included in the control group. All participants were between 18 and 65 years of age, and demographic data were collected for both groups. The inclusion criteria required participants to be male and aged between 18 and 65; in the patient group, a DSM-5 diagnosis of MUD together with subjective cognitive decline and exclusive METH use was required, whereas in the control group the absence of psychiatric illness and no history of substance use were necessary. Exclusion criteria for both groups included a history of chronic disease, cardiovascular system disease, thromboembolism, mental retardation, neurological disorders that could cause cognitive impairment, smoking, folic acid use, regular medication use, and missing data. The Montreal Cognitive Assessment (MoCA), which was administered to patients during clinical evaluation at the time of presentation, was also examined. The study was conducted in accordance with the principles of the Declaration of Helsinki. The study received approval from the Elazığ Fırat University Non-Interventional Ethics Committee. (No: 2024/12-20).

Hematological analysis for homocysteine, vitamin B12, folate

Blood samples were taken from the antecubital vein (6 ml). Samples were collected and analyzed in vacuum tubes, including 15% K3 ethylene diamine tetraacetic acid-anticoagulation tubes (Sarstedt, Essen, Belgium). The levels of homocysteine were tested using the spectrophotometric method on an Abbott Architect c8000 (Abbott, Abbott Park, USA) Chemistry System. Vitamin B12 and folate levels were measured via chemiluminescence using a Beckman Coulter DxI800 (Beckman Coulter, Inc. CA, USA) device.

Data collection tools

Sociodemographic data form

The form collected data such as age, disease history, educational status, background, and substance use history.

Montreal Cognitive Assessment (MoCA)

This scale is designed to assess mild cognitive impairment by evaluating a broad range of cognitive domains, including visuospatial and executive functions, naming, memory, attention, language, abstraction, and orientation. The highest possible score is 30, with scores below 21 indicating cognitive impairment, while higher scores reflect better cognitive performance. The scale was originally developed by Nasreddine et al. (2005), and its Turkish validity and reliability were subsequently established by Selekler et al. (2010)

Statistical analysis

The data obtained from the study were analyzed using SPSS Statistics for Windows 27.0. Descriptive statistics included numbers, percentages, means, and standard deviations. The Kolmogorov-Smirnov test was applied to evaluate whether the research variables showed normal distribution, and it was determined that the variables were normally distributed. Therefore, parametric tests were used in data analysis. The t-test was used to compare continuous quantitative variables between two independent groups. The distribution of categorical variables between groups was analyzed using the chi-square test. Pearson correlation analysis was performed to evaluate the correlations between continuous variables. To determine which variables were independently associated with homocysteine levels, linear regression analysis was used. Beta coefficients and 95% confidence intervals (CIs) were used for the presentation of results. All p values were two-tailed and values < 0.05 were considered to indicate statistical significance.

Results

All participants in the study were male. There was no significant difference between the groups in terms of marital status or educational status of the participants (p > 0.05). The distribution of comorbid ADHD presence in the MUD group was as follows: those with comorbid ADHD n = 20 (45.5%), those without comorbid ADHD n = 24 (54.5%). There was no significant difference in age between the MUD group (31.636 ±7.959) and the control group (33.533 ±8.308) (p > 0.05). The mean duration of METH use (years) in the MUD group was 3.272 ±2.296. The mean frequency of METH use (per week) in the MUD group was 3.022 ±1.848. The mean MoCA score in the MUD group was 19.863 ±3.921. Some demographic data for the groups are presented in Table 1.
Serum homocysteine levels of the MUD group (x– = 18.477 ±4.374 µmol/l) were found to be significantly higher than those of the control group (x– = 10.977 ±3.816 µmol/l) (p < 0.001) (Table 1, Fig. 1). No significant difference was observed between the serum vitamin B12 levels of the MUD group (x– = 204.636 ±70.044 ng/l) and those of the control group (x– = 221.555 ±89.532 ng/l) (p = 0.324 > 0.05) (Table 1, Fig. 1). No significant difference was observed between the serum folate levels of the MUD group (x– = 6.049 ±1.456 µg/l) and those of the control group (x– = 6.328 ±1.557 µg/l) (p = 0.386 > 0.05) (Table 1, Fig. 1). Some laboratory results and scale scores for the groups are presented in Table 1.
When the distribution of homocysteine levels was examined according to the presence of comorbid ADHD in the MUD group, the homocysteine levels of patients with comorbid ADHD (22.300 ±2.696) were significantly higher than the homocysteine levels of patients without comorbid ADHD (15.291 ±2.544) (p < 0.001) (Table 2, Fig. 2).
When correlation analyses were examined according to homocysteine levels in the MUD group, there was a positive correlation between duration of METH use (years) and homocysteine levels (r = 0.424, p = 0.004). There was no significant correlation between frequency of METH use (per week) and homocysteine levels (p = 0.236 > 0.05). There was a negative correlation between MoCA scores and homocysteine levels (r = –0.464, p = 0.002) (Table 3, Fig. 3).
A linear regression model was used to evaluate the independent effects of the presence and severity of MUD on homocysteine levels. Linear regression analysis showed that the linear regression model was statistically significant and that the variables included in the model explained 67.2% of the variance in homocysteine levels (Nagelkerke R2 = 0.672, p < 0.001). In this model, those with ADHD had homocysteine levels higher by 6.320 µmol/l (Table 4). The results of this regression model showed that the presence and severity of MUD were independent predictors of homocysteine levels (Table 4).

Discussion

The results of our study provide new evidence for significantly higher homocysteine levels in male patients with MUD compared to healthy controls. We found that patients who used METH for a longer period had higher homocysteine levels. Furthermore, our findings indicate an association between elevated homocysteine and cognitive impairment, as evidenced by a significant negative correlation with MoCA scores. Most notably, patients with comorbid ADHD exhibited significantly higher homocysteine levels.
Although some studies have reported low vitamin B12 or folate levels in patients with MUD (Zhai et al. 2018), no significant differences in serum vitamin B12 or folate levels were found between the MUD group and the control group in our study. This finding is important because vitamin B12 and folate are critical cofactors in the methionine-homocysteine pathway that converts homocysteine back to methionine (Yektaş et al. 2019). Although homocysteine levels were high in our MUD group, the lack of significant vitamin B12 or folate deficiencies suggests that other factors (e.g., impaired enzyme activity or increased homocysteine production due to the effects of METH on methylation and oxidative stress) may contribute more to hyperhomocysteinemia in this specific population.
The elevated homocysteine levels observed in MUD patients found in our study can be mechanistically explained by the well-known neurotoxic pathways of METH. METH exposure induces robust oxidative stress, excitotoxicity, and mitochondrial dysfunction, creating a cellular environment that disrupts one-carbon metabolism and methylation chemistry (Northrop and Yamamoto 2015). The effect of METH on dopaminergic terminals produces excessive reactive oxygen species (ROS) that deplete antioxidant systems, including glutathione, a subproduct of homocysteine transsulfuration (Krasnova and Cadet 2009). Psychostimulant exposure specifically alters redox intermediates such as NAD+ and S-adenosyl-L-methionine (SAM), which are critical cofactors in homocysteine metabolism (Sundar et al. 2022). This disruption shifts the flux through homocysteine-based pathways, potentially favoring homocysteine accumulation over its conversion to methionine (remethylation) or cysteine (trans-sulfuration). The positive correlation between duration of METH use and homocysteine levels in our study supports this dose-dependent relationship between chronic METH exposure and metabolic dysregulation.
Recent evidence suggests that MUD is characterized by persistent neuroinflammation with elevation of proinflammatory cytokines, including TNF-α and IL-6 (Permpoonputtana et al. 2025). This neuroinflammatory state can further exacerbate homocysteine elevation through multiple pathways. Inflammatory cytokines can reduce the activity of methionine synthase and cystathionine β-synthase, key enzymes in homocysteine metabolism, leading to substrate accumulation (Škovierová et al. 2016). Additionally, chronic METH exposure compromises blood-brain barrier integrity through mechanisms involving decreased β-catenin expression and altered endothelial morphology (Qiu et al. 2025). This barrier dysfunction may contribute to the systemic elevation of homocysteine with long-term METH use observed in our study, as impaired clearance mechanisms allow the accumulation of toxic metabolites in both the peripheral circulation and brain tissue. A study in chronic METH users showed that longer duration of METH use and higher frequency of use were associated with cognitive impairment (Wang et al. 2017). In our study, no statistically significant correlation was found between the frequency of METH use and homocysteine levels. This result suggests that duration of use may be a more predictive factor than frequency of use in terms of potential effects on homocysteine metabolism.
One of our most striking findings was that we detected a significant negative correlation between homocysteine levels and MoCA scores in the MUD group. This correlation strongly suggests that increased homocysteine levels are associated with decreased cognitive performance. This finding supports the hypothesis that the neurotoxic effects of homocysteine may play a role in the pathogenesis of cognitive dysfunction associated with MUD. The neurotoxicity mechanisms of homocysteine have been explained in various ways. Among these mechanisms, homocysteine interferes with methylation processes, triggers neuroexcitotoxicity, and induces oxidative stress and inflammation (Ansari et al. 2015). High homocysteine concentrations can lead to excitotoxicity by activating N-methyl-D-aspartate (NMDA) receptors. They may also contribute to structural and functional disorders such as DNA damage and increased permeability of the blood-brain barrier (Ho et al. 2002). METH itself can cause severe neurotoxicity through oxidative stress, damage to the blood-brain barrier, and overexpression of pro-inflammatory cytokines (Zhai et al. 2018). Therefore, the increased homocysteine levels we observed in our MUD patients may contribute to cognitive deficits by exacerbating these neurotoxic processes.
Another important finding of our study, which has been examined for the first time in the literature, is that homocysteine levels in patients with comorbid ADHD in the MUD group were significantly higher than those in patients without comorbid ADHD. The relationship between ADHD and homocysteine appears to be complex and bidirectional. Some pediatric studies have reported lower homocysteine levels in children with ADHD who were not taking medication, possibly reflecting nutritional deficiencies in B vitamins and folate (Altun et al. 2018; Karababa et al. 2017). However, other studies have found elevated homocysteine levels in ADHD populations (Lukovac et al. 2024; Namjoo et al. 2020). ADHD itself has been described as an inflammatory condition associated with immunological and oxidative responses. Elevated levels of C-reactive protein (CRP), homocysteine, and interleukin-6 have been associated with inflammation in ADHD (Namjoo et al. 2020). Given the role of homocysteine in inflammation and oxidative stress, higher homocysteine levels in MUD patients with comorbid ADHD may indicate an increased inflammatory and oxidative burden in this subgroup and may reflect additive effects of both conditions on methylation pathways and oxidative stress. Comorbid ADHD may be associated with altered dopaminergic signaling and executive dysfunction, processes that may be exacerbated by METH-induced neurotoxicity (Warton et al. 2018). This may contribute to more pronounced cognitive or psychiatric symptoms. Furthermore, our linear regression analysis showed that the presence of ADHD increased homocysteine levels by 6.320 µmol/l. All these results suggest that the presence of comorbid ADHD uniquely affects homocysteine metabolism in individuals with MUD or that the interaction of these two disorders leads to a distinct neurobiological profile.
In our study, despite significant elevation in homocysteine, no significant difference was found in vitamin B12 and folate levels between the groups, suggesting that different factors beyond simple nutritional deficiency contribute to the observed metabolic dysfunction. However, targeted supplementation with B vitamins (B6, B12, folate) and methylation cofactors may potentially address the underlying enzymatic dysfunction in homocysteine metabolism (Ford et al. 2010). Given the role of neuroinflammation in both METH neurotoxicity and homocysteine elevation, anti-inflammatory interventions offer a promising treatment option. The oxidative stress component of METH neurotoxicity suggests that antioxidant interventions may help normalize homocysteine metabolism.
This study had some limitations. Because the study population was limited to male participants, generalizability to female MUD patients, who may exhibit different metabolic profiles, is limited. Another limitation is that the study was single-centered, and only the MoCA scale was used to evaluate cognitive impairment. The observation of ADHD comorbidity and its relationship with homocysteine levels is a novel finding that warrants replication in larger and more diverse populations. Future studies should include comprehensive neuropsychological assessments, neuroimaging correlations, and prospective longitudinal follow-up to better characterize the temporal relationships between homocysteine elevation, cognitive decline, and response to treatment.

Conclusions

This study provides the first direct evidence linking elevated homocysteine levels to cognitive impairment in male patients with MUD. This increase, which is particularly evident in patients with comorbid ADHD, points to a distinct neurobiological phenotype requiring targeted treatment approaches. Furthermore, we found higher homocysteine levels in MUD patients with longer-term METH use. These findings open new avenues for understanding the metabolic basis of METH-induced cognitive dysfunction and highlight homocysteine as a potential therapeutic target to improve cognitive outcomes in this vulnerable population.

Disclosures

This research received no external funding.
The study received approval from the Elazığ Fırat University Non-Interventional Ethics Committee (No: 2024/12-20).
The authors declare no conflict of interest.

References