Serum free thiols in recently diagnosed patients with schizophrenia spectrum disorder: A potentially useful biomarker of oxidative stress

Schizophrenia spectrum disorders (SSD) have been linked to oxidative stress (OS). Recent findings from our group show that serum free thiols (R-SH, sulfhydryl groups) can function as an accurate biomarker of systemic OS, since they are readily oxidized by reactive species (ROS), thereby serving as potent antioxidants. The aim of this study is to investigate if reduced R-SH levels can be demonstrated in recently diagnosed patients with SSD compared to healthy controls (HC). In this study, 102 patients with recently diagnosed SSD ( < three years), and 42 HC were included. Levels of R-SH were quantified and studied for correlations with age, C-reactive protein (CRP) as proxy of inflammation as well as body mass index (BMI) and total cholesterol as indices of metabolic health. R-SH levels were significantly lower in patients when compared to HC. When correcting for age the difference was borderline significant ( p = 0.05 ). Moreover, R-SH correlated significantly with age ( r = -0.29) and CRP ( r = -0.29) in patients with SSD, but not in the HC. R-SH levels are reduced in SSD as compared to HC and correlate negatively with CRP and age in SSD. Future studies are required to further investigate R-SH and its role in SSD.


Introduction
Schizophrenia spectrum disorder (SSD), a complex mental disorder characterized by disruption in thought processes, perceptions, emotional responsiveness, and social interactions, have been linked to oxidative stress (OS) in multiple studies (Ermakov et al., 2021;National Institute of Mental Health, 2018;Tsugawa et al., 2019). OS is suggested to induce structural and functional changes in the brain through several pathways, including oligodendrocyte abnormalities, abnormal metabolism of dopamine and glutamate, hypofunction of N-methyl--D-aspartate (NMDA) glutamate receptors and impairment of fast-spiking gamma-aminobutyric acid (GABA) interneurons (Bitanihirwe & Woo, 2011).
OS is defined as an imbalance between oxidants and antioxidants in favor of the oxidants, leading to a disruption of redox signaling and control and/or molecular damage (Sies, 2015). In general, physiological levels of reactive species fulfill pivotal physiological functions, but overproduction of reactive oxygen species (ROS) may result in OS, which subsequently leads to damage to cellular components such as lipids and proteins (Turrens, 2003). This cellular and/or molecular oxidative damage and tissue destruction is strongly connected to the process of ageing and is associated with many human diseases Damba et al., 2020).
Several markers of OS have been studied for their utility as biomarkers for SSD (Ermakov et al., 2021;Tsugawa et al., 2019), but a gold standard has not yet been established. It has been suggested that some markers of OS are intrinsic to SSD, known as trait markers, while other markers are transient and associated with active episodes, also known as state markers (Flatow, Buckley, & Miller, 2013). Recent findings from our group show that systemic serum free thiols (R-SH, serum sulfhydryl) can function as an accurate indication of OS . R-SH are a direct measure of the in vivo reduction/oxidation (redox) status since they are instantly oxidized by circulating ROS to form disulfide bonds (Banne, Amiri, & Pero, 2003). According to their molecular weight, extracellular R-SH can be divided into low-molecular-weight (LMW) thiols, such as glutathione and cysteine, and cysteine-based protein thiols, which predominate in the serum (Bourgonje et al., 2023;Turell, Radi, & Alvarez, 2013). Taken together, these are referred to as total free thiols. Circulating free thiols are readily oxidized by reactive species, resulting in reduced free thiol levels. After oxidation, thiols in serum are less readily reversed to their reduced state compared to their intracellular counterparts (Koning et al., 2016). As such, relatively lower levels of R-SH are indicative of oxidation events due to the overproduction of reactive species, whereas higher levels of R-SH are indicative of a rather favourable redox status. Chronic OS has been associated with decreased levels of R-SH in a variety of conditions, including inflammatory bowel disease  and cardiovascular disease .
There is evidence of OS in recently diagnosed patients with SSD (Flatow et al., 2013;Fraguas et al., 2019). However, the utility of R-SH as a marker for OS in early SSD has not been investigated. So far, only one study has evaluated R-SH in 77 acute phase schizophrenia patients and 110 healthy controls (Huang, Liou, & Lin, 2010), showing that patients with schizophrenia had significantly lower R-SH levels after adjusting for body mass index (BMI). Here, we hypothesize that recently diagnosed patients (< 3 years after diagnosis of SSD) are already affected by systemic OS as reflected by reduced R-SH levels. The aim of this study is therefore to investigate if reduced R-SH can be demonstrated in this patient population, compared to healthy controls, and to examine potential associations between R-SH and demographic (e.g., age), anthropometric (e.g. body mass index), biochemical measures of low-grade inflammation (e.g. C-reactive protein [CRP]) and metabolic health (e.g., cholesterol).

Study population
This post-hoc study used baseline data and material from the Simvastatin study (ClinicalTrails.gov, NCT01999309, Begemann et al., 2015;Sommer et al., 2021). For this study, 102 patients between 18 and 50 years of age with a DSM-IV diagnosis of schizophrenia (DSM classification code: 295.9), schizoaffective (295.7) or schizophreniform disorder (295.4), or psychotic disorder NOS (not otherwise specified; 298.9), and onset of first psychosis no longer than three years ago were included. Patients were recruited from both inpatient and outpatient settings throughout the Netherlands. Further inclusion criteria required the participants not to have a chronic somatic disorder. In addition, 42 healthy controls (HC) between 18 and 45 years of age with no history of psychiatric disorders were included. HC were recruited via advertisements on notice boards and in newspapers.
The Simvastatin study was approved by the ethics committee of the University Medical Center Utrecht (UMCU), Utrecht. All participants provided written informed consent upon explanation of the study procedures. The study was performed according to the Declaration of Helsinki (64th World Medical Association general assembly; October 2013) and the International Conference on Harmonisation -Good Clinical Practice (ICH-GCP).

Laboratory procedures
R-SH were measured as described previously (Bourgonje et al., 2021a). In short, absorbance was measured with the Clariostar plus microplate reader (BMG Labtech) from the serum samples to determine R-SH concentrations. Finally, R-SH concentrations were adjusted to total serum protein (measured according to standard procedures) by calculating the R-SH/total protein ratio (μmol/g of protein). This adjustment was performed to indirectly account for total thiol status since serum proteins harbor the largest amount of potentially detectable R-SH groups (Cortese-Krott et al., 2017). CRP levels were measured by turbidimetry at the hospital laboratory of the University Medical Center Utrecht (UMCU) or the University Medical Center Groningen (UMCG).

Questionnaires
To assess psychiatric symptom severity the Positive and Negative Syndrome Scale (PANSS, Kay et al., 1987) and the Calgary Depression Scale for Schizophrenia (CDSS, Addington et al., 1993) were used. Neurocognitive functioning was evaluated using the Brief Assessment of Cognition in Schizophrenia (BACS, Keefe et al., 2004). Recreational drug and alcohol use was assessed with the Comprehensive Assessment of Symptoms History part 3 (CASH, Andreasen et al., 1992). Please note that more information regarding the Simvastatin study can be found in the protocol paper (Begemann et al., 2015) and the results paper (Sommer et al., 2021).

Statistical analysis
Data analysis and visualisation were performed in R version 4.1.2 (R Core Team, 2022) and figures were produced using the package ggplot2 (Wickham, 2016).
The distribution of demographic and clinical variables was determined with the use of Shapiro-Wilk test, histograms, and Q-Q plots and are presented in the demographics table as mean ± standard deviation (SD) for normally distributed continuous variables, as median (interquartile range, IQR) for continuous variables with a skewed/non-normal distribution, or as frequencies (n) and corresponding percentages for discrete variables.
We then stratified patients with SSD into three groups based of protein-adjusted R-SH concentrations. Comparison of demographic and clinical characteristics among the tertiles was performed using ANOVA or Kruskal-Wallis rank sum test depending on the distribution of the data. In case of categorical data Pearson's Chi-squared test was used.
To detect if differences exist in the levels of R-SH between patients and controls, a t-test was used. The equality of variances between the groups was tested with Levene's test. Cohen's d was used to estimate the effect size. Moreover, ANCOVA was used to adjust for age when comparing R-SH between HC and patients with SSD. For the comparison of the other biological and demographic variables between HC and patients, an independent sample t-test or the Wilcoxon Rank Sum test was used depending on whether the assumptions for parametric testing were fulfilled.
The correlation between R-SH and age, CRP, cholesterol and BMI for each group were tested with Pearson or Spearman correlation depending on whether the assumptions of parametric testing were fulfilled. Last, to investigate whether age, CRP and cholesterol interact with SSD to predict R-SH levels, we tested the interaction term of each biomarker with diagnosis in a multiple linear regression with R-SH as a dependent variable and age as a covariate. For all statistical tests, alpha was set at 0.05.

Associations between serum free thiols and demographic and clinical parameters
We tested correlations of R-SH with age and known indices of inflammation and metabolic health: CRP, total cholesterol, and BMI.
Age correlated significantly with R-SH levels in patients with SSD (Fig. 2b, r = -0.29, p = 0.003), but not in the HC (r = 0.07, p = 0.7). R-SH levels were significantly correlated with CRP in patients with SSD (Fig. 2a, r = -0.29, p = 0.004), but not in HC (r = -0.06, p = 0.7). When age was included as covariate R-SH levels were still significantly correlated with CRP in patients with SSD (r = -0.27, p = 0.007) and not in HC (r = -0.07, p = 0.64). Total cholesterol and R-SH levels in patients with SSD were negatively correlated, but this correlation was not significant (Fig. 2c, r = -0.17, p = 0.09), but not in HC (r = -0.06, p = 0.7). BMI did not correlate with R-SH in HC (r = -0.07, p = 0.7), nor in SSD (r = -0.05, p = 0.6). Furthermore, PANSS (sub)scores were not correlated with R-SH levels in patients with SSD (Supplementary Table 1) Multivariable regression analyses with R-SH as outcome showed that there was no significant interaction between age and presence of SSD  Table 2), indicating that the associations of age, CRP and total cholesterol with R-SH did not differ significantly between the patients with SSD and HC.

Discussion
In this study we showed that R-SH levels are significantly decreased in recently diagnosed patients with SSD compared to HC. However, when corrected for age R-SH levels were still decreased in the patients with SSD but the p-value was borderline significant with a value of 0.05. In addition, we demonstrated that R-SH levels are negatively correlated with age, CRP, and total cholesterol, but not to BMI in patients with SSD. Collectively, our study shows the potential use of R-SH as a proxy measure of systemic OS in patients with SSD.
To our knowledge, the only other study that investigated R-SH levels in schizophrenia patients was the performed by Huang et al. (2010). They found R-SH to be decreased in schizophrenia patients (n = 77) compared to healthy controls (n = 110) when adjusted for BMI. Our patient group, however, was relatively younger and was diagnosed more recently. In our study, R-SH levels were significantly different, but when corrected for age the difference became borderline significant (p = 0.05). Even though the finding should not be interpreted as conclusive, it should definitely not be dismissed, considering the accessibility of R-SH as a potential biomarker and the long-sought need for biological readouts in these disorders. Taking this finding as the basis, replication studies could expand or dismiss our current understanding." Moreover, we looked into association between R-SH levels and biochemical measures of low-grade inflammation (e.g. C-reactive protein [CRP]) and metabolic health (e.g. cholesterol). Studies with other markers of OS are in line with the presented findings. In meta-analyses, others have shown that the total antioxidant status (TAS) of first episode psychosis patients is decreased (Flatow et al., 2013;Fraguas et al., 2019). On the other hand, both studies also show that GSH levels are not significantly different between first episode psychosis patients and healthy controls.
In this study, we also find a negative correlation between R-SH and age, in the patients with SSD but not in healthy controls. However, the difference in slopes between patients with SSD and HC was not significant. This is perhaps due to a small difference in the mean concentration of R-SH between the two groups. Juchnowicz et al. (2021) did find a negative association between superoxide dismutase-1 (SOD-1), an important antioxidant, and age in subjects with schizophrenia, but not in healthy individuals. Another study also showed a differential effect of aging on plasma antioxidant proteins in patients with schizophrenia, compared to healthy controls (Yao, Reddy, & Van Kammen, 2000). These observations and the fact that many studies have related systemic oxidative stress, as reflected by circulating free thiols, to age related diseases like cardiovascular diseases, chronic kidney diseases and neurodegenerative diseases, might point towards R-SH being an accelerated aging signature in this relatively young patient group (Rebouças et al., 2021;Abdulle et al., 2020;Bourgonje et al., 2022;Bourgonje et al., 2021a;McBean et al., 2015) It is important to mention though, that we cannot exclude the possibility that the lack of a significant correlation between R-SH and age in the healthy group is due to insufficient statistical power, given that the control group in this study was much smaller than the group of patients with SSD (n = 42 compared to n = 102 patients with SSD).
In patients with SSD, we observed a significant negative correlation between R-SH and CRP levels, but not in HC. However, the interaction term between SSD and CRP when tested in a regression model, was not significant. Therefore, we cannot conclude that there is an interaction between the diagnosis of SSD and CRP from this sample. The correlation between R-SH and CRP has not been studied before in SSD. Studies in other OS-related conditions though have shown associations between R-SH levels and CRP Damba et al., 2020). These data confirm that systemic inflammation and OS are two highly intertwined pathophysiological processes. Although multiple studies suggest increased pro-inflammatory mediators and reduced antioxidant capacity in patients with schizophrenia, data on the relation between these two in schizophrenia are scarce. One study found the interaction of malondialdehyde (MDA) with tumour necrosis factor-a (TNF-a) to be a risk factor for schizophrenia (Zhu et al., 2020). Another study found brain-derived neurotrophic factor (BDNF) levels to be positively correlated with copper/zinc-containing SOD levels in drug-naïve first-episode schizophrenia patients (Xiu et al., 2020). In contrast, Dudzińska et al. (2022) did not find any significant correlations between OS variables (SOD, catalase, MDA, lipid hydroperoxide) and ferric reducing ability of plasma. It is however good to keep in mind that the latter study was performed in a small group of 34 schizophrenia patients.
Concerning cardiovascular risk-factors, we reported negative correlation between R-SH and total cholesterol in the SSD subjects, which was however not statistically significant. Furthermore, no correlation was observed between R-SH and BMI in either group. In terms of the relationship between R-SH and cholesterol in SSD, the existing evidence is scarce. In fatty liver disease, associations between hypercholesterolemia and R-SH were found (Damba et al., 2020). Another study found an association of cholesterol with R-SH in a group of 2,980 females (Bourgonje et al., 2021b). Our findings regarding BMI and R-SH are partially in line with other studies. An et al. (2018) found a positive correlation of BMI with plasma MDA, but did not observe differences of the antioxidant enzymes, SOD, catalase and GSH peroxidase between a low and high BMI schizophrenia group. Others found no association between BMI and OS (Juchnowicz et al., 2021). Considering the effect of variability of the data on correlation metrics, it is worth mentioning that both groups in this study are comprised of young individuals that show low BMI variability (Goodwin & Leech, 2006). Especially the patients with SSD, who have a recent diagnosis and have not been treated with antipsychotics for long, have reduced chances of having a higher BMI as a result of (atypical) antipsychotic treatment. This is reflected by the fact that no BMI differences are observed between the groups (Table 1). Last, as shown in the demographics table, the lipid profile (total cholesterol, triglycerides, and LDL) and glucose levels was different in the patients compared to the HC. These biochemical characteristics often co-occur with SSD diagnosis and are not easily disentangled (Malhotra, Grover, Chakrabarti, & Kulhara, 2013).
Regarding psychiatric symptoms, as measured by the PANSS, we found no associations with R-SH. To date, no other studies have investigated the relation between psychiatric symptoms and R-SH. However, there are reports showing associations between distinct oxidative stress markers and their relation to psychiatric symptoms. These results, however, seem to be mixed. One study found malondialdehyde (MDA, a lipid peroxidation marker) to be positively correlated with positive PANSS scores (r = 0.439), but not with any of the other PANSS scores in drug-naïve first episode schizophrenia patients. Moreover, they did not find associations between superoxide dismutase (SOD, antioxidant enzyme), catalase (CAT, antioxidant enzyme), lipid hydroperoxides or total antioxidant and any of the PANSS scores (Dudzińska et al., 2022). Another study did not detect any significant correlation between SOD, GSH, CAT or MDA and PANSS scores in chronic schizophrenia patients (Zhang et al., 2015). These results indicate that the relationship between psychiatric symptoms severity and systemic oxidative stress is not particularly evident, which may be due to insufficient study power, cohort heterogeneity, methodological differences, or any other unknown reason. Based on its pathophysiological position, oxidative stress probably is more related to an underlying biological instability in SSD, thereby fueling the emergence of new psychotic episodes, rather than having a direct relation to symptom severity. However, to validate this theory, studies following patients with SSD for longer period of time are needed. This is, nevertheless, the first paper to study the relationship between R-SH and symptom severity. Future research is in larger, prospective longitudinal cohorts is necessary to further establish its definitive role and potential utility as biomarker of symptom severity. R-SH are considered to reliably reflect the extracellular redox equilibrium and these compounds constitute the main biological targets of reactive species. Whereas most studies focused on low-molecular-weight (LMW) free thiols, such as GSH and cysteine, the R-SH measured in this study include both cysteine-based protein thiols and LMW thiols, which are collectively referred to as total free thiols. Worth mentioning, proteins, especially the redox-activeCys34 sulfhydryl group of albumin, are by far the largest source of free and oxidized thiols. Albumin is the most relevant example not only because it is the most abundant human plasma protein, but also because it confers transporting capacity for LMW-thiols. Aside from ROS, other types of reactive species, including reactive nitrogen species (RNS) and reactive sulphur species (RSS), may also react with R-SH groups. Acting as the central hubs of redox-regulated events, R-SH are able to transduce a number of short-term (e.g. alteration of protein structure and/or activity) and longer-term (e.g., influencing gene regulation or gene expression) biological adaptations and thereby govern multiple protein functions. Hence, the determination of serum R-SH levels may be viewed as an easily reproducible, minimally invasive and robust method to determine the degree of systemic OS (Cortese-Krott et al., 2017).
In future studies it would be relevant to track the R-SH over time in SSD to discover its mechanisms in relation to the disease and see if it perhaps may predict worsening of symptoms. In OS-mediated diseases, R-SH have been proven to be a very sensitive biomarker and often associate strongly with disease severity (Cortese-Krott et al., 2017). Next to that, it would be relevant to study the effects of antipsychotic medication on R-SH and to study if R-SH levels could potentially be a biomarker for treatment efficacy. Especially, since there are a lot of discrepancies still regarding this subject (Yao & Keshavan, 2011).
In line with the previous paragraph, an interesting future perspective would be to investigate R-SH as a possible treatment target for SSD, as it can be therapeutically modulated. For example, N-acetylcysteine (NAC), a thiol-based antioxidant, which has long been used therapeutically for the treatment of acetaminophen (paracetamol) overdose, could be a possible therapeutic modality Lavoie et al., 2008;Bourgonje et al., 2021c;Forman and Zhang, 2021;Rushworth and Megson, 2014). Although NAC is able to directly scavenge reactive species via its own thiol group, its intrinsic reactivity towards oxidants is relatively poor, and instead other mechanisms, such as stimulation of GSH biosynthesis and its H 2 S-generating capacity, may strongly contribute to its therapeutic potential.
The current findings should be viewed through the lens of the study's strengths and limitations. As of the strong point, this is the first study to investigate R-SH in recently diagnosed patients with SSD with the largest sample size to date for this purpose. Moreover, we are the first to study associations between biochemical measures and R-SH in this population. Yet, the association of different antipsychotics with R-SH levels was not investigated in this study, since the respective groups were too small. Additionally, our female group of the patients with SSD was rather small (23.5%). A more balanced sex distribution would have allowed to perform a sex-stratified analysis. As already mentioned previously, this study included a considerably lower number of healthy controls compared to patients. Therefore, the lack of association between R-SH and CRP, age and total cholesterol in the control group, could be attributed to lack of sufficient power and should interpreted with caution until the finding is reproduced in a bigger sample. Last, given the case-control design of this study, the observed differences in R-SH should be replicated in different cohorts in order to confirm that they are explained by the diagnosis of SSD and not due to other unobserved group differences.
In conclusion, we show that R-SH are significantly reduced in recently diagnosed patients with SSD as compared to healthy individuals, indicating OS. Furthermore, we show that R-SH levels negatively correlate with age and CRP in patients with SSD. Future studies are required to further investigate R-SH and its potential as a biomarker and treatment target in SSD.