Oxidative Stress and Inflammatory Biomarkers in People with Methamphetamine Use Disorder

Abstract

Objective

This study aimed to investigate the blood serum levels of biomarkers specifying oxidative stress status and systemic inflammation between people using methamphetamine (METH) and the control group (CG). Serum thiol/disulfide balance and ischemia-modified albumin levels were studied to determine oxidative stress, and serum interleukin-6 (IL-6) levels and complete blood count (CBC) were to assess inflammation.

Methods

Fifty patients with METH use disorder (MUD) and 36 CG participants were included in the study. Two tubes of venous blood samples were taken to measure oxidative stress, serum thiol/disulfide balance, ischemia-modified albumin, and IL-6 levels between groups. The correlation of parameters measuring oxidative stress and inflammation between groups with sociodemographic data was investigated.

Results

In this study, serum total thiol, free thiol levels, disulfide/native thiol percentage ratios, and serum ischemia-modified albumin levels of the patients were statistically significantly higher than the healthy controls. No difference was observed between the groups in serum disulfide levels and serum IL-6 levels. Considering the regression analysis, only the duration of substance use was a statistically significant factor in explaining serum IL-6 levels. The parameters showing inflammation in the CBC were significantly higher in the patients than in the CG.

Conclusion

CBC can be used to evaluate systemic inflammation in patients with MUD. Parameters measuring thiol/disulfide homeostasis and ischemia-modified albumin can be, also, used to assess oxidative stress.

INTRODUCTION

Methamphetamine (METH) is an amphetamine-derived synthetic substance. It has a stronger effect and longer half-life than amphetamine. This substance can be easily abused due to its strong psychostimulant and addictive properties [1,2].

Due to chronic use of METH, neurotoxicity occurs in the central nervous system (CNS), especially in dopaminergic neurons [3]. As a result, treatment-resistant psychosis, neurodegeneration, and cognitive impairment are detected [4,5].

Oxidative stress, neuroinflammatory processes, and excitotoxicity play a role in the pathophysiology of neurotoxic damage associated with METH use [6]. Oxidative stress and elevated cytosolic dopamine due to chronic METH use may cause microglial cell activation. Microglial cell activation causes neurotoxic damage by releasing proinflammatory cytokines (tumor necrosis factor [TNF]- a, Interleukin [IL]-1b, IL-6, and IL-8) and producing molecules such as reactive oxygen species (ROS) [7]. Besides, activation of microglia leads to the release of high amounts of excitotoxic glutamate, thus neurodegenerations occur as a result of neuroinflammation [8-10]. METH generates hydrogen peroxide and superoxide radicals by stimulating the release of high levels of serotonin and dopamine in the CNS [11,12]. Oxidative stress has a crucial role in METH-mediated neurotoxicity [13]. METH impairs the function of the blood-brain barrier (BBB) by initiating oxidative stress in brain endothelial cells [12,14]. The endothelial barrier disruption increases transendothelial leukocyte migration, contributing to tissue damage and ultimately to neuroinflammation [1].

Thiols form an important part of the antioxidant molecules in the plasma. During oxidative stress, a thiol is oxidized by free oxygen radicals and turns into a disulfide [15]. In this process, thiol prevents tissue damage due to oxidative stress by reducing or inactivating oxidant levels [16,17]. Among the different markers used in the oxidant/ antioxidant balance evaluation, one of the most recent is the thiol/disulfide homeostasis (TDH) developed by Erel and Neselioglu [15]. TDH has critical roles in antioxidant defense, detoxification, apoptosis, regulation of enzyme activities, and mechanisms of transcription and cellular signal transduction [16-18]. In healthy individuals, there is a balance between oxidant and antioxidant mechanisms [15,17]. Oxidative stress is defined as the disruption of this balance. In cases of oxidative stress, it is expected that the level of serum disulfide, which is an oxidation product, will increase, and the levels of serum native thiol and total thiol, which are antioxidants, will decrease [15,17]. Its oxidation shows a high disulfide and disulfide/native thiol ratio.

The formation of oxidative-modified proteins increases as a result of oxidative stress in the brain. Ischemic modified albumin (IMA), one of these proteins, has been suggested to may reflect ischemia and oxidative stress due to tissue hypoxia in many diseases [19,20].

In recent studies, it has been reported that IMA may be a marker of oxidative stress [21]. Normally, albumin passes through the BBB, but the IMA cannot cross the BBB. The BBB disruption due to increased intracranial pressure makes grater the serum IMA level.

We think that there are links between impaired oxidative balance and increased levels of proinflammatory cytokines in individuals with METH use disorder (MUD). In this study, we tested our hypothesis that thiol-disulfide homeostasis and serum IMA level in determining oxidative stress in individuals with MUD and serum IL-6 level in evaluating systemic inflammation may be significantly related compared to the control group (CG). The study aimed to investigate oxidative stress and inflammation biomarkers between individuals with MUD and the CG and to evaluate their relationship with each other. Thus, it will guide us to use oxidative stress and inflammation parameters effectively in clinical practice in the MUD. There is no comprehensive study in the literature on this aspect in individuals with MUD.

METHODS

Study Population

This study was carried out in the psychiatry clinic of a Training and Research Hospital. The study group consisted of male participants over the age of 18, diagnosed with MUD according to the DSM-5 (Diagnostic and Sta-tistical Manual of Mental Disorders, 5th edition) criteria, and using METH for at least one year. Same-sex participants were accepted to the study to control for the confounding effect of sex. It was evaluated that there was evaluated to be no case receiving methylphenidate treatment in cases diagnosed with MUD. The study included 50 people without chronic or mental illness, not taking anti-inflammatory and b-blockers within one month, not having a sexually transmitted infection disease, and using METH in he last one week. The illicit drug use (METH) of the patients was confirmed by measuring the urine samples with the enzyme multiplied immunoassay technique method using a Simens Advia 1800 chemistry analyzer (Siemens Healthineers). Urine samples were taken from the patients before detoxification and studied without waiting. In the CG, 36 people, whose demographic characteristics were similar to the study group, consisted of hospital personnel and their relatives. All of the participants in both groups were smokers. Body mass index was similar between the groups. All participants reviewed the informed consent form and their written consent was obtained. This study was designed in accordance with the 2013 Brazilian version of the Declaration of Helsinki and was approved by the local ethics committee (2011-KAEK- 25 2021/04-23). Good clinical practice principles were followed throughout the study.

Biochemical Parameters

Two tubes of venous blood samples were taken from all participants at the time of enrollment in the study to measure TDH parameters, IMA levels, and serum IL-6 levels. After the blood samples were centrifuged at 1,500 rpm for ten minutes, plasma and serum samples were separated. Serum samples were stored at −80°C. After all the samples of the participants were collected, plasma samples to be tested for thiol/disulfide and IMA levels were sent to the biochemistry laboratory of Ankara City Hospital. Serum samples to be studied for IL-6 levels were sent to the microbiology laboratory of Bursa Yuksek Ihtisas Training and Research Hospital. Besides, all participants’ complete blood count (CBC) and biochemistry examinations were measured simultaneously at the time of enrollment in the study.

Serum thiol/disulfide levels were determined by the spectrophotometric measurement method developed by Erel and Neselioglu [15], using 5.5’-dithiobis-(2-nitrobenzoic) acid as the chromogen. With the help of this method, native thiol and total thiol levels were measured. Disulfide, disulfide/native thiol % ratio, and disulfide/total thiol % ratio were calculated by using native thiol and total thiol levels. Half of the difference between the total amount of thiol and the amount of native thiol gives the amount of disulfide.

The serum IMA level was measured according to the principle of determining the cobalt binding capacity of albumin, defined by Bar-Or et al. [22]. The properties of albumin change under ischemic attacks associated with oxidative stress, production of ROS, and acidosis. Under these conditions, especially for metals such as cobalt, IMA is produced with a low metal binding capacity [23].

While the ratio of IMA to serum albumin is 1−2% in physiological conditions, this ratio increases to 6−8% in pathological conditions [24].

Serum IL-6 (Dia Source) level was analyzed by enzyme-linked immunosorbent assay (ELISA) test. IL-6 is a soluble mediator secreted in response to environmental stressors such as inflammation and tissue injuries [25].

Statistics

In the study, demographic and clinical characteristics of cases with MUD and CG were evaluated with descriptive statistical analyzes such as mean, standard deviation, median, and ratio. Mean data with normal distribution between MUD cases and CG were compared using the Independent Groups ttest, and data non-normal distribution using Mann–Whitney Utest. The relationships between age, age of onset of substance use, duration of substance use, and blood values of MUD cases were analyzed with Pearson correlation analysis for normally distributed data and Spearman Correlation Analysis for data that were non-normally distributed.

Factors that may have an effect on explaining the IL-6 and thiol values of MUD cases (age, duration of substance use [years], neutrophil/lymphocyte ratio [NLR], monocytes/lymphocyte ratio [MLR], platelet/lymphocyte ratio [PLR], IMA) were examined by multivariate simple linear regression analysis. The conformity of the data to the normal distribution was examined by the coefficients of kurtosis and skewness. The significance level for all analyzes was determined as p < 0.05. IBM SPSS 22.0 program (IBM Co.) was used for the analysis.

RESULTS

The mean age of the cases diagnosed with MUD evaluated in the study was 30.62 ± 6.64, the mean age of the participants with CG was 33.28 ± 7.25, and the mean age of the two groups was found statistically similar (t = −1.76, p = 0.082). The age of onset of substance use mean in MUD cases was detected as 25.94 ± 6.63, and the duration of substance use (years) mean was 4.44 ± 2.47. Of the MUD cases evaluated in the study, 33 (66.0%) were stated to use less than 0.5 mg per week in the last year, 13 (26.0%) used 0.5−1 mg, and 4 (8%, 0%) used more than 1 mg. According to the Mann–Whitney Utest, it was found that disulfide (Z = −1.58, p = 0.115), disulfide/total thiol ratio (Z = −1.52, p = 0.130), and IL-6 median values (Z = −0.33, p = 0.741) were not statistically different between cases using less than 0.5 mg (n = 33) and using more than 0.5 mg (n = 17).

Based on the Independent Groups ttest, the mean of the IL-6 values of the cases with MUD were found not to be statistically different from the mean of the cases in the CG (t = 0.95, p = 0.346). According to the Independent Groups ttest, it was found that the mean percentages of native thiol (t = −4.67, p < 0.001), total thiol (t = −4.60, p < 0.001), and disulfide/total thiol ratio (t = −2.30, p = 0.024) of cases with MUD were statistically significantly lower than the cases with CG. Considering the Independent Groups ttest, the disulfide/native thiol percentage ratio (t = 2.31, p = 0.024), white blood cell (WBC) (t = 2.68, p = 0.009), neutrophil (t = 3.22, p = 0.002), and monocyte (t = 4.60, p < 0.001) means of the cases with MUD were determined to be statistically significantly higher than the means of CG.

In accordance with the Mann–Whitney Utest analysis, mean IMA (Z = −3.88, p < 0.001), platelet (PLT) (Z = −2.62, p = 0.009), NLR (Z = −2.00, p = 0.045), and MLR (Z = −3.92, p < 0.001) of MUD cases was found to be statistically significantly higher than mean of CG (Table 1).

Table 1.

Comparison of mean blood values between cases with SUD evaluated in the study and those in the control group

Variable	MUD			Control group		Analysis	pvalue
	Mean (median)	SD (25−75%)		Mean (median)	SD (25−75%)
Native thiol	404.73	55.99		474.50	82.73	t = −4.67	< 0.001
Total thiol	442.13	56.33		510.77	82.15	t = −4.60	< 0.001
Disulfide	18.70	5.62		18.13	5.03	t = 0.48	0.630
Disulfide/native thiol (%)	4.72	1.62		3.96	1.33	t = 2.31	0.024
Disulfide/total thiol (%)	4.28	1.34		3.64	1.14	t = 2.30	0.024
Native thiol/total thiol (%)	91.45	2.68		92.72	2.29	t = −2.30	0.024
IMA	0.84	0.83−0.86		0.83	0.81−0.84	Z = −3.88	< 0.001
IL-6	2.37	0.48		2.28	0.33	t = 0.95	0.346
WBC	9.12	2.64		7.56	2.14	t = 2.68	0.009
Hemoglobin	14.70	14.10−15.40		15.10	14.50−15.70	Z = −1.37	0.172
Hematocrit	43.65	41.40−46.20		44.90	42.10−45.50	Z = −0.90	0.366
Neutrophil	5.50	2.07		4.11	1.33	t = 3.22	0.002
Monocyte	0.70	0.21		0.49	0.17	t = 4.60	< 0.001
PLT	268.00	240.50−316.00		236.00	204.00−290.00	Z = −2.62	0.009
Lymphocyte	2.94	0.83		2.76	0.86	t = 0.88	0.380
Eosinophil	0.21	0.11−0.33		0.14	0.10−0.21	Z = −1.75	0.081
Basophil	0.02	0.01−0.03		0.02	0.02	Z = −0.02	0.987
NLR	1.77	1.40−2.28		1.52	1.27−1.75	Z = −2.00	0.045
MLR	0.23	0.19−0.30		0.16	0.14−0.22	Z = −3.92	< 0.001
PLR	101.30	77.50−121.49		83.83	72.99−105.47	Z = −1.42	0.156

SUD, substance use disorder; MUD, methamphetamine use disorder; IMA, ischemic modified albumin; IL-6, interleukin-6; PLT, platelet; NLR, neutrophil/lymphocyte ratio; MLR, monocytes/lymphocyte ratio; PLR, platelet/lymphocyte ratio; SD, standard deviation; WBC, white blood cell.

According to Pearson correlation analysis, a statistically significant positive correlation was detected between the ages of the cases diagnosed with MUD and disulfide (r = 0.281, p = 0.048), the disulfide-native thiol percent ratio (r = 0.295, p = 0.038), and the disulfide/total thiol ratio (r = 0.286, p = 0.044). Furthermore, a statistically significant negative correlation was found between the percentage of native thiol/total thiol ratio and the age of the cases using MUD (r = −0.286, p = 0.044). There was a statistically significant negative correlation with respect to the Spearman Correlation Analysis between the age values of cases diagnosed with MUD and NLR (rho = 0.343, p = 0.017), MLR (rho = 0.428, p = 0.002), and PLR (r = 0.433, p = 0.002). as well as; a statistically significant negative correlation was found between the age of onset of substance use in cases diagnosed with MUD and NLR (rho = 0.398, p = 0.005), MLR (rho = 0.376, p = 0.008), and PLR (r = 0.519, p < 0.001). With respect to the Pearson correlation analysis, there was a statistically significant positive association between the duration of substance use (years) and percent of disulfide/native thiol ratio (r = 0.288, p = 0.043), and percent of disulfide/total thiol ratio (r = 0.287, p = 0.044) in the MUD cases. A statistically significant negative correlation was, moreover, detected between the percentage of native thiol/total thiol ratio and the age of the cases using MUD (r = −0.287, p = 0.044) (Table 2).

Table 2.

Correlation between age, age of onset and duration of use of patients with SUD in the cases evaluated in the study

Variable	Analysis	Age	Age of onset	Duration of use
Native thiol	r	−0.063	0.032	−0.219
	p	0.665	0.827	0.126
Total thiol	r	−0.006	0.065	−0.175
	p	0.965	0.655	0.225
Disulfide	r	0.281	0.166	0.215
	p	0.048	0.249	0.133
Disulfide/native thiol (%)	r	0.295	0.151	0.288
	p	0.038	0.295	0.043
Disulfide/total thiol (%)	r	0.286	0.143	0.287
	p	0.044	0.323	0.044
Native thiol/total thiol (%)	r	−0.286	−0.143	−0.287
	p	0.044	0.322	0.044
IMA	Rho	0.015	−0.051	0.025
	p	0.920	0.723	0.861
IL-6	R	0.241	0.185	0.240
	p	0.092	0.199	0.093
NLR	Rho	0.343	0.398	−0.220
	p	0.017	0.005	0.133
MLR	Rho	0.428	0.376	0.073
	p	0.002	0.008	0.624
PLR	Rho	0.433	0.519	−0.188
	p	0.002	< 0.001	0.200

SUD, substance use disorder; IMA, ischemic modified albumin; r, Pearson correlation analysis; IL-6, interleukin-6; NLR, neutrophil/lymphocyte ratio; MLR, monocytes/lymphocyte ratio; PLR, platelet/lymphocyte ratio; Rho, Spearman correlation analysis.

According to multivariate simple linear regression analysis, 36% of IL-6 values (F = 2.46, p = 0.031) were evaluated to be explained by age, duration of substance use (years), NLR, MLR, PLR, WBC, and IMA values. Considering the examination of the regression model, only the duration of substance use was found a statistically significant factor in explaining IL-6 values (p = 0.017, 95% confidence interval, 0.013−0.127) (Table 3). Again in the same analysis method, disulfide/native thiol ratio percentage values were not explained statistically significantly by age, duration of substance use (years), NLR, MLR, PLR, WBC, and IMA values (F = 1.08, p = 0.399) found (Table 4). In addition to all these, the disulfide values were found not to be statistically significantly explained by age, duration of substance use (years), NLR, MLR, PLR, WBC, and IMA values (F = 1.12, p = 0.372) (Table 5).

Table 3.

Multivariate linear regression analysis related to explaining interleukin-6 values

Variable	Unstandardized coefficients			Standardized coefficients	t	pvalue	95% CI
	B	SE		β			LL	UL
Constant	5.195	2.240			2.319	0.026	0.667	9.723
Age	0.003	0.010		0.042	0.277	0.783	−0.018	0.024
Year	0.061	0.026		0.334	2.380	0.022	0.009	0.114
NLR	−0.031	0.091		−0.072	−0.347	0.731	−0.215	0.152
MLR	0.579	0.801		0.109	0.722	0.474	−1.041	2.198
PLR	0.005	0.002		0.359	1.904	0.064	0.000	0.009
WBC	0.030	0.028		0.174	1.064	0.294	−0.027	0.088
IMA	−4.802	2.614		−0.239	−1.837	0.074	−10.086	0.482

IMA, ischemic modified albumin; SE, standard error; CI, confidence interval; NLR, neutrophil/lymphocyte ratio; MLR, monocytes/lymphocyte ratio; PLR, platelet/lymphocyte ratio; WBC, white blood cell; LL, lower limit; UL, upper limit.

R² = 0.36, F = 3.19, p = 0.009.

Table 4.

Multivariate linear regression analysis associated with explaining disulfide/native thiol ratio % values

Variable	Unstandardized coefficients			Standardized coefficients	t	pvalue	95% CI
	B	SE		β			LL	UL
Constant	1.375	9.146			0.150	0.881	−17.109	19.859
Age	0.053	0.043		0.213	1.242	0.222	−0.033	0.139
Year	0.186	0.105		0.280	1.765	0.085	−0.027	0.399
NLR	−0.034	0.370		−0.021	−0.091	0.928	−0.782	0.715
MLR	−1.868	3.272		−0.097	−0.571	0.571	−8.480	4.745
PLR	0.009	0.010		0.195	0.918	0.364	−0.011	0.029
WBC	−0.091	0.116		−0.144	−0.780	0.440	−0.325	0.144
IMA	1.562	10.674		0.021	0.146	0.884	−20.010	23.134

IMA, ischemic modified albumin; SE, standard deviation; CI, confidence interval; LL, lower limit; UL, upper limit; NLR, neutrophil/lymphocyte ratio; MLR, monocytes/lymphocyte ratio; PLR, platelet/lymphocyte ratio; WBC, white blood cell.

R² = 0.18, F = 1.27, p = 0.291.

Table 5.

Multivariate linear regression analysis associated with explaining disulfide values

Variable	Unstandardized coefficients			Standardized coefficients	t	pvalue	95% CI
	B	SE		β			LL	UL
Constant	5.627	31.906			0.176	0.861	−58.858	70.111
Age	0.116	0.148		0.138	0.783	0.438	−0.184	0.416
Year	0.452	0.368		0.200	1.231	0.226	−0.290	1.195
NLR	0.467	1.292		0.087	0.362	0.719	−2.143	3.078
MLR	4.074	11.414		0.062	0.357	0.723	−18.995	27.143
PLR	0.021	0.034		0.135	0.620	0.539	−0.048	0.091
WBC	−0.357	0.405		−0.167	−0.882	0.383	−1.175	0.461
IMA	7.878	37.236		0.032	0.212	0.834	−67.378	83.134

IMA, ischemic modified albumin; SE, standard deviation; CI, confidence interval; LL, lower limit; UL, upper limit; NLR, neutrophil/lymphocyte ratio; MLR, monocytes/lymphocyte ratio; PLR, platelet/lymphocyte ratio; WBC, white blood cell.

R² = 0.14, F = 0.93, p = 0.497.

DISCUSSION

In this study, the mean of serum native thiol and total thiol in patients with MUD were found to be statistically significantly lower than the mean of the CG. This finding suggests that antioxidant capacity decreased in the patient group. Besides, the percentage of disulfide/native thiol was significantly higher in the group with MUD, indicating that the oxidant/antioxidant balance was disrupted in favor of oxidants.

There is only one retrospective case-control study in the literature evaluating the oxidative stress status with TDH in patients with MUD [26]. In this study by Hacimusalar et al. [26], the levels of native thiol, total thiol, and disulfide in the patient group with MUD were detected to be significantly higher than in the CG. Besides, only the serum disulfide level continued to increase in the samples taken before and after detoxification [26]. In our study, serum disulfide levels were observed not to change before detoxification. In this study, we think that the high disulfide/native thiol ratio is a significant finding in terms of showing oxidation. However, it is thought that disulfide levels, a second oxidation indicator, did not make a significant difference between the two groups, which contradicts the previous study, but there are not enough studies in the literature on cases with MUD, and new studies will be needed in the future to support our study results. The number of studies showing adequacy in the evaluation of TDH in cases with MUD, which will shed light on our research, is limited.

Moreover, serum disulfide and disulfide/native thiol ratio were not statistically significantly explained by age, years of METH use, and inflammatory parameters, taking into account regression analysis. In order to confirm our study, it was necessary to investigate the changes in TDH parameters, which were disrupted as a result of oxidative stress in the MUD. In addition, demographic characteristics such as age and duration of use provide limited information on addiction severity and dose/amount of meth use. Hence, new studies examining the effects of METH amount used on inflammation in future studies will contribute to the literature.

There are many studies investigating TDH parameters in substance use disorder. In a case-control study conducted by Kotan et al. [27] on heroin addicts, native thiol and total thiol levels were found to be significantly lower in the patient group compared to the CG, while disulfide levels were found to be higher. The duration of substance use was, however, reported not to be significantly associated with these variables [27]. In studies conducted with patients using cannabis and synthetic cannabinoids, an increase in inflammation and oxidative stress has been reported [28,29]. Contrary to the studies in the literature, serum disulfide levels did not differ in both groups. The fact that the mechanism of action of heroin, cannabis, and synthetic cannabinoid substances is different from METH may cause this contradiction.

The brain is sensitive to oxidative stress. Determination of TDH may help to explain the pathophysiology of many diseases, including substance-induced neurotoxicity, schizo-phrenia, bipolar disorder, major depression, and neurodegenerative diseases, and to prevent them with treatment interventions [15]. Studies investigating the effects of antioxidant and anti-inflammatory agents to reduce neurotoxic damage in MUD have increased in recent years. In animal models, melatonin, ketoprofen, crocin, intranasal insulin, and ginkgolide B have been reported to reduce METH-induced neuroinflammation by different mechanisms [30-34]. The use of ibudilast in patients with MUD has also been reported to reduce inflammation in the peripheric nervous system (PNS) and CNS [35]. Antioxidants such as N-acetylcysteine have been found to weaken the neurotoxic effects of METH [36]. Besides, treatments reducing neuroinflammation have been observed to decrease BBB damage in acute METH toxicity [31,37].

All these studies are promising. Patients with MUD, which has been becoming increasingly common and a public health problem, are quite susceptible to the neurotoxic effect of the substance. Oxidation and inflammation have been stated to continue during the detoxification or withdrawal period in patients with MUD [26,38].

We think that these agents should be included more in the treatment algorithms to determine the oxidative and inflammatory status and reduce morbidity during these periods. In the future, further studies on biomarker determination may be useful in planning the follow-up and treatment of patients. Evaluation of the oxidant/antioxidant status due to MUD with TDH parameters is easier and can yield rapid results.

IMA values, known in many studies, may increase in some medical conditions. It has been stated that IMA can be used as an early biomarker in diseases associated with oxidative stress and ischemia, such as ischemic stroke, myocardial infarction, renal failure, and diabetes mellitus [39-42].

In our study, the mean IMA of the patient group was found to be statistically significantly higher than the CG. This result is consistent with increased serum IMA levels in different medical conditions. It was thought that ischemia and disruption of TDH due to METH use may increase IMA levels which can be a crucial marker in determining the oxidative load and ischemia status in the early period of MUD. Thus, it may provide an opportunity for early pharmacological interventions to prevent com-plications. The rapid measurement of IMA with a simple technique also provides an advantage to the clinician. This is the first study to investigate IMA levels in METH users. As long-term ischemia may, however, lead to both oxidative stress and inflammation, it was thought that it could not be a specific marker. More research is needed to support these findings.

Numerous studies in the literature have stated that chronic use of METH increases proinflammatory cytokines [43,44]. There are many studies evaluating the relationship of proinflammatory cytokines with psychopathological symptoms. Serum levels of these cytokines have been indicated to be elevated in depression, schizophrenia, bipolar disorder, and anxiety disorders [45-47].

In addition, elevated serum IL-6 levels have been correlated to cognitive impairment and METH-associated psychosis [48]. However, the relationship between IL-6 levels and psychiatric symptoms was not evaluated in our study.

Reducing inflammation during the METH withdrawal period has been suggested to diminish the craving [49]. It has been indicated that complaints of cognitive impairment in patients with METH during the withdrawal period may be due to the continuation of this inflammation [50]. The level of inflammation can be reduced by using anti- inflammatory agents during the METH withdrawal period. Thus, it can be useful in coping with cognitive impairment and craving, which clinicians frequently encounter in treatment.

Changes in IL-6, IL-10, Cox-2, TNF-a, and IL1-b molecules play an important role in the immune response in METH neurotoxicity [51]. In a case-control study, they emphasized that TNF-a, IL-6, and IL-18 levels of patients using chronic METH were significantly higher than the CG and that pre-inflammatory cytokines would be important in psychopathological symptoms [44].

In our study, the mean IL-6 levels of people with MUD were observed not to differ from the CG; however, when other findings were controlled according to the regression model, the duration of substance use was significantly effective in explaining IL-6 values. As the duration of substance use increases, living on the street, malnutrition, tendency to infections, and sharing of the substance were predicted to may be increased, and these risk factors were thought to may be affected the serum IL6 level. The longer the METH is used, the longer the patients may be exposed to inflammation. The emergence of psychiatric symptoms as a result of METH-induced inflammation was concluded to may be associated with the duration of substance use. Serum IL-6 level is not a quick and simple test. However, it is thought that whether there will be a biomarker showing inflammation in cases with a long duration of METH use should be shown with new studies.

In the current study, IL-6 level was measured in peripheral blood. Whether this cytokine is proportionally increased in the CNS has not been investigated. Cytokines formed in the peripheral blood have been indicated to can cross the BBB by active transport or vagal stimulation [52]. In addition, cytokine transition from BBB dysfunction has been reported [53]. Whether this transition is bidirectional is not clear. In future studies, PNS and CNS cytokine levels can be evaluated simultaneously in the MUD. Thus, the contribution of transmission from peripheral inflammation or release from glial cells to elevated CNS cytokine levels can be investigated.

The level of IMA and thiols in the CNS was not analyzed in this study. These molecule levels were measured by tests related to albumin and proteins. Since there is much less albumin and protein in cerebrospinal fluid (CSF) than in the blood serum level, these tests are not sensitive enough to measure in CSF. Glutathione is the main antioxidant of the CNS, as it is a significant source of thiols [54]. Demonstration of changes in serum TDH levels in CNS-related diseases draws forth that redox activity in serum may be associated with the redox activity of the CNS [55].

Changes in WBC, neutrophil, lymphocyte, NLR, MLR, and PLR parameters in CBC may point out low-grade systemic inflammation [56]. In particular, the high NLR ratio calculated from WBC is used to indicate non-specific systemic inflammation.

In recent years, studies comparing inflammatory parameters between individuals with substance use disorders (SUD) and the CG in CBC have increased [57-60]. In the study of Soder et al. [57], in which patients with cocaine use disorder were included, the NLR values of the patient group were found to be higher than those of healthy controls.

In a retrospective case-control study by Demir et al. [61], NLR and PLR rates were detected to be lower in MUD cases compared to healthy controls. In this study, they concluded that simple blood parameters did not indicate systemic inflammation [61].

In our study, the means of WBC, neutrophil, PLT, monocytes, NLR, and MLR ratios in MUD were found to be statistically significantly higher than the means of the cases with CG. This study supports previous studies investigating the consequences of CBC parameters in indivi-duals with SUD. However, the current study contradicted the study of Demir et al. [61] in MUD cases. Our study is the first prospective study investigating the parameters of CBC in the MUD. Our CBC findings need to be supported by new studies. CBC is a routine examination giving accurate and rapid results. Systemic inflammation can be evaluated with CBC in many psychiatric diseases and MUD.

We consider the limitations of our study as the fact that our study was conducted in the male sex, the biomarkers studied in serum were not examined in the CNS, and the association of the biomarkers measured in the serum with the severity of addiction and frequency of METH use was not evaluated.

Briefly, it has been shown that TDH, IMA, and CBC can be used in the determination of systemic inflammation and oxidant-antioxidant balance in SUD. The fact that these parameters give quick results and can be studied with a simple technique may provide convenience to clinicians. In the future, measuring these biomarkers during the detoxification and withdrawal periods of SUDs may provide an opportunity to evaluate follow-up and treatment.

Funding Statement

Funding None.