Schizophrenia is a chronic and severe mental disorder affecting approximately 1% of the population (Barnett 2018). Currently, the first-line therapy for schizophrenia is atypical antipsychotic medication, such as olanzapine, risperidone and ziprasidone (Lieberman 1996). However, the therapeutic response to antipsychotics is heterogeneous between individuals. Current symptom-driven treatment leads to the poor outcome in patients with schizophrenia, especially in the early stage of treatment (Liu-Seifert et al., 2005).

Metabolites are the final products of the biochemical pathways in the human body, and their abnormalities can better reflect the disruption of functional status of the patients (Lindon et al., 2003). Yet, most of small-molecule metabolites are currently difficult to separate and detect (Rojo et al., 2012). Therefore, their roles in the pathophysiology of schizophrenia remain unclear. Recently, liquid chromatography tandem mass spectrometry (LC-MS) based metabolomics provided an opportunity to understand the pathological role of metabolites and develop new predictive biomarkers that can monitor the response to antipsychotics (Kaddurah-Daouk et al., 2008; Paredes et al., 2014; Pickard 2015). Our previous study by metabolomics method showed that olanzapine treatment for 1 month significantly increased the plasma levels of several types of lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) (Liu et al., 2021).

LPC and LPE are the prominent parts of lysophospolipid and play key roles in physiological and pathological processes of nervous system (Steinbrecher et al., 1984). Increasing evidence shows that LPC and LPE are involved in the function of cell membrane, apoptosis, oxidative stress and inflammatory responses. In the body, the peripheral circulating LPE and LPC are produced from hepatic secretion following the hydrolysis of cellular membrane phosphatidylcholine (PC) and phosphatidylethanolamine (PE) catalyzed by both acyltransferases and phospholipase (PLA) (Schmitz and Ruebsaamen 2010). In contrast, the majority of plasma LPC and LPE species may originate from liver secreted lecithincholesterol acyltransferases (LCAT) reaction (Sekas et al., 1985). In mechanism, LPC and LPE serve as a reservoir for arachidonic acid and as a central precursor for lipid-associated signaling molecular. In addition, LPC and LPE are bioactive lipids involved in monocyte recruitment and activation (Park et al., 2015). For example, studies found that LPC and LPE have protective role on ischemic neurons during the oxidative stress (Blondeau et al., 2002; Chen et al., 2019).

Schizophrenia shows complex and unique abnormal characteristics of glycerophospholipids in serum, plasma or brain tissues (Horrobin et al., 1994; Schmitt et al., 2004; He et al., 2012; Ghosh et al., 2017). Non-targeted LC-MS based metabolic profiling studies in schizophrenia spectrum disorders suggest altered glycerophospholipid species and levels in the brain of patients (Fukuzako et al., 1999; Matsumoto et al., 2011). Metabolomics study of prefrontal cortex from schizophrenia patients reported significantly lower levels of PCs in white and grey matter than healthy controls (Schwarz et al., 2008). Further, some studies revealed that after antipsychotics treatment, schizophrenia patients showed increased levels of 22:5, 20:5, and 20:3 within the PC and PE glycerophospholipid classes (Kaddurah-Daouk et al., 2012; Castillo et al., 2016). Particularly, a study comparing the lipid profiles of antipsychotics-free and drug-naïve (ANFE) patients before and after several types of antipsychotics medication for 7 mo showed that 11 glycerophospholipids (nine PCs and two LPCs) were significantly up-regulated after treatment, suggesting that glycerophospholipids may be used for predictive markers to monitor the treatment in the early stage of this disorder (Leppik et al., 2020).

Based on the aforementioned literatures regarding the phospholipids in schizophrenia, we hypothesized that the LPC and LPE levels were associated with the improvement of clinical symptoms after 1-month treatment. Therefore, this study aimed to investigate whether the plasma LPC and LPE levels identified by LC-MS based metabolomics were the predictive markers for the treatment response to olanzapine in the early treatment of ANFE patients with schizophrenia.

This study found that schizophrenia patients showed increased concentrations of most of LPC and LPE after olanzapine monotherapy for 1 mo. Increased LPC(18:3) and LPC(20:2) levels were correlated with the improvement of positive symptoms. Baseline levels of LPE(20:5), LPE(18:3) and LPE(22:5) also were associated with the reduction rate of positive symptom and may serve as a predictive biomarker for therapeutic response.

Using LC-MS based untargeted metabolomics approach, abnormalities in levels of LPE and LPC have been consistently reported in schizophrenia, indicating that abnormal metabolism of LPE and LPC is a pathophysiological characteristic of this disorder (Law et al., 2019). Lysoglycerophospholipid plays a fundamental role in the neuronal mechanisms underlying the pathophysiological feature of several psychiatric disorders. Moreover, atypical antipsychotic medication has been shown to alter the levels of LPE and LPC in mental disorders (Ask et al., 2016; Cao et al., 2019; Leppik et al., 2020; Canfrán-Duque et al., 2021). In line with our findings, Cao et al. found that after 8-wk treatment with antipsychotics, serum amino acids and LPC levels were increased in first-episode (FEP) patients with schizophrenia (n = 122) (Cao et al., 2019). Leppik et al. profiled 14 LPCs and 76 PCs in serum from 53 FEP patients and found two LPCs and nine PCs levels were significantly increased after antipsychotic treatment for 7 mo (Leppik et al., 2020). Kaddurah-Daouk et al. found that PE levels were reduced in schizophrenia patients, and this change was reversed by olanzapine treatment (Kaddurah-Daouk et al., 2007). Particularly, Qiao et al. reported that decreased LPC (20:3) and LPC (14:0) levels were significantly increased after treatment with olanzapine for 1 mo in female patients with ANFE schizophrenia. However, only 13 metabolites were analyzed in this study (Qiao et al., 2016). The underlying mechanism of increase in the LPC and LPE levels after treatment with olanzapine in patients with schizophrenia is unclear. One possible explanation may be linked with the aberrant activity of phospholipase A₂ (PLA₂) after treatment. It is well known that PLA₂ is a class of enzyme that catalyzes cleavage of fatty acids from the sn-2 position of phospholipids and hydrolyze phospholipids into LPE and LPC (Hishikawa et al., 2014). Several antipsychotics have been reported to influence the activities or levels of PLA₂ in animal models and patients with schizophrenia (Gattaz et al., 1995; Smesny et al., 2005; Smesny et al., 2011; Kim et al., 2012; Shen et al., 2018). Our findings provide further evidence for the regulation of LPE and LPC concentrations by olanzapine treatment in patients with schizophrenia.

Our second finding is that the improvements of clinical symptoms after treatment were associated with the changes of LPC and LPE. The potential mechanism may be related to the physiologic functions of LPC and LPE, such as the formation of cell membranes, vesicle trafficking and bioactive signaling molecules in various cells including neuron (van Meer et al., 2008). As a component of lysophospholipid, LPC and LPE are water-soluble and amphiphilic molecules. They are essential precursors of signaling pathway molecules that activate second messenger to enhance the biological functions. Previous studies have shown that lysophospholipid can stabilize and enhance G protein-coupled receptor G2A signaling pathway for calcium flux by preventing its reuptake or altering its surface expression and localization on the cell surface (Wang et al., 2005; Frasch et al., 2007). There is robust evidence that inflammation and redox imbalance play a crucial role in the pathophysiology of schizophrenia (Zhang et al., 2017; Zhang et al., 2018; Xiu et al., 2019; Xiu et al., 2020). LPC and LPE have also been found to be associated with the regulation of redox system and inflammatory response systems. For example, Cui et al. found that plasma PC (36:4) was correlated with concentration of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) in septic rat models (Cui et al., 2020). Carneiro et al. found that LPC triggered Toll-like receptors 2 (TLR2)- and TLR4-mediated signaling pathways but counteracted LPS-induced NO synthesis by inhibiting nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) translocation and mitogen activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) phosphorylation (Carneiro et al., 2013). In particular, a recent randomized, double-blind and placebo-controlled trial showed that a decomposition product of pig liver, which is a rich source of LPC and LPE, has been identified as a nutrient to improve cognitive function in the healthy individuals over the age of 40 (Matsuda et al., 2020). Moreover, administration with LPC and LPE on the lipopolysaccharides (LPS)- stimulated SIM-A9 microglia cells significantly reduced the expression of IL-6 and the production of reactive oxygen species (ROS) (Tsukahara et al., 2021). However, it remains unclear whether the increased levels of LPC and LPE are the causes or results of the improvement of clinical symptoms. The exact mechanism needs further study.

Specifically, we found that the different classes of lysophospholipids were involved in the improvement of clinical symptoms in this study. Baseline LPE levels including (20:5), (18:3) and (22:5) and the increase in LPC levels including (18:3) and (20:2) were associated with the clinical symptom improvements. In line with our findings, previous studies also supported that lysophospholipid with different group has different functional role. In the studies of prehypertension, increased LPC(16:0) was found to cause oxidative stress, thereby increases inflammation and arterial stiffness (Kim et al., 2014). LPE (18:0) can increase Ca²⁺ concentration in nerve cells through the G2A receptor pathway, resulting in calcium overload (Lee et al., 2015). While, in myocardial infarction induced by a high isoprenaline dose in rat, the enhancement of oxidative stress was observed to be related to the decrease of LPCs [(18:0) and (20:3)] levels (Liu et al., 2013). Increased PC(38:4) levels were found in good responders treated with olanzapine and risperidone. All these above-mentioned studies support our findings that different classes of LPC and LPE were associated with the outcome of treatment in schizophrenia.

This study has a few limitations. First, the sample size is relatively small, which reduces the statistical power of the present study. Second, further study recruiting healthy controls is warranted to verify the clinical value of these predictive biomarkers for the therapeutic efficacy of olanzapine. Third, considering that some patients have been treated with antipsychotics for a short time (less than 14 days) in our study, we cannot completely rule out the possible impact of previous antipsychotics on the results. Fourth, the metabolites were analyzed in plasma rather than in CNS. Future study is warranted to compare the LPC and LPE levels between peripheral (plasma) and CNS (cerebrospinal fluid). Fifth, in a previous study, we published the same cohort of patients and same metabolomic methods (Liu et al., 2021). Although part of methods is similar, the two studies have focused on different clinical problems. Liu et al. focused on the severe side effect of weight gain after treatment with olanzapine. This study focused on the therapeutic response to olanzapine.

Overall, this study provides new evidence that the changes in LPC(18:3) and LPC(20:2) levels were associated with the improvement of positive symptoms, suggesting that LPC may be a potential therapeutic target for olanzapine. Moreover, the baseline levels of LPE(20:5), LPE(18:3) and LPE(22:5) might be highly useful as novel plasma biomarkers for the prediction of therapeutic response in the early stage of schizophrenia. However, this study is limited by the lack of the healthy controls and a small ample size. Replication is warranted in further longitudinal studies with a large sample size to introduce LPC and LPE biomarkers into clinical practice.