Metabolome Profiling of Yokukansan in Preventing Postoperative Delirium in Elderly Cancer Patients: A Reverse Translational Study

Abstract

Aims: Postoperative delirium (PD) is a common and severe complication in older adult patients undergoing invasive cancer resections. This study explored the plasma metabolome associated with PD and evaluated the efficacy of Yokukansan (YKS), a traditional Japanese Kampo medicine, in preventing PD.

Methods: An ancillary study was conducted alongside a double-blind, placebo-controlled randomized clinical trial involving patients 65 years and older, focusing on patients older than 75 years as a primary analysis population. Plasma samples were analyzed using targeted and nontargeted metabolomics. An in vivo study using aged mice assessed the effects of YKS on plasma and brain metabolites.

Results: A total of 83 patients, including 21 patients older than 75 years, were enrolled. Patients with PD had lower levels of several lipid mediators, free fatty acids, and phospholipids. YKS administration led to increased nine phospholipids and four hydrophilic metabolites in patients older than 75 years, including phosphatidylcholine (40:7) and phosphatidylcholine (42:8), which were also altered in delirium patients. In the patients older than 65 years, only two metabolites increased in the YKS administration group. In aged mice, YKS elevated plasma phospholipids, similar to findings in patients older than 75 years, and influenced brain citrulline and creatine, which related to oxidative stress and cognitive function. Correlation analyses revealed associations between plasma and brain metabolite changes.

Conclusion: Our findings suggest that the plasma metabolome provides insight into the pathophysiology of PD and the potential mechanism underlying the preventive effect of YKS against PD.

Keywords: Yokukansan; cancer surgery; metabolome profile; postoperative delirium; reverse translational study.

Fig. 1

Flowchart of participants and metabolome analysis. (a) Flowchart of the participants. The study was an ancillary study of a double‐blind, placebo‐controlled, randomized controlled trial (RCT) to examine the efficacy and safety of Yokukansan (YKS) for the prevention of postoperative delirium (PD). (b) Flowchart of metabolome analysis. Targeted metabolite analysis using plasma samples collected from patients was performed to identify plasma metabolites associated with PD and the efficacy of YKS (Figs. 2, 3 and 6). Additionally, nontargeted metabolite analysis was performed to identify YKS‐derived metabolites in plasma and to investigate whether YKS‐derived metabolites influence the efficacy of YKS (Figs. 3b and 6). Based on patient results, targeted metabolite analysis was conducted using plasma and brain samples from mice to validate the reproducibility of YKS effects in plasma and to identify metabolites associated with YKS in the central nervous system (Fig. 4). Target analysis measured lipid mediators (liquid chromatography–tandem mass spectrometry [LC–MS/MS]) and phospholipids (LC–MS/MS), and hydrophilic metabolites (gas chromatography–tandem mass spectrometry [GC–MS/MS] and LC–MS/MS). For overlapping hydrophilic metabolites in both GC–MS/MS and LC–MS/MS, the one with the lower coefficient of variation value was adopted based on the results of the QC samples. The instruments adopted in hydrophilic metabolites are listed in Table S1 (patients) and Tables S4–S7 (mice). Nontarget analysis measured YKS‐derived components/metabolites (LC–high‐resolution MS). Further details are described in Supplementary Material. Abbreviation: CV, coefficient of variation.

Fig. 2

Plasma metabolome profiling of postoperative delirium (PD) and non‐PD patients in the placebo group. Targeted metabolite analysis using plasma samples collected from patients was performed to identify plasma metabolites associated with PD. (a) Volcano plot of the plasma metabolome analysis. Plots were mapped using the log₂ fold‐change value of PD/non‐PD patients vs the log₁₀ P‐value obtained from the Wilcoxon signed rank test. The colored points met the criterion of P < 0.05 from Wilcoxon signed rank test. Red, yellow, pink, and blue indicate phospholipids, lipid mediators, free fatty acids, and hydrophilic metabolites, respectively. (b) Clustering analysis of plasma metabolites that met the criterion of P < 0.05. Data were scaled, and distances were calculated using Spearman correlation coefficient.

Fig. 3

Plasma metabolome profiling in the Yokukansan (YKS) group. (a) Nontargeted metabolite analysis was performed using plasma samples collected from patients and identified the YKS‐derived components and metabolites. Box plots depict the normalized intensities of the YKS‐derived components and metabolites in plasma, with each sample peak height normalized to that of the quality‐control sample. Geissoschizine methyl ether (GM) and hirsuteine are represented as a mixture because the peaks could not be separated. (b) Targeted metabolite analysis was performed using plasma samples collected from patients, and plasma metabolites associated with YKS were identified. Volcano plot comparing the metabolomic profiles between the YKS and placebo groups in patients older than 75 years. The plot corresponds to Fig. 2a.

Fig. 4

Metabolome analysis of the plasma and brain of aged mice. Targeted metabolite analysis was performed using plasma and brain tissue collected from aged mice and identified metabolites associated with Yokukansan (YKS). (a, b) Volcano plot of plasma (a) and the whole brain (b) depicts the metabolomic differences between the YKS and the control. The plots correspond to that presented in Fig. 2a. The colored plots met the criterion of P < 0.1. (c) The correlation network was constructed for the metabolites (P < 0.1) and the combination of plasma and brain metabolites in which the absolute correlation coefficient was >0.5 in both the YKS group and all patients. Red and blue nodes represent plasma and brain metabolites, respectively. Red and blue edges represent positive and negative correlations, respectively. The associations between brain and plasma metabolites found in this analysis are represented in the scatter plots. Black and gray circles represent the YKS and control (Ctrl) groups, respectively. Data were collected from n = 6 in the YKS group and n = 7 in the Ctrl group. Abbreviation: PC, phosphatidylcholine.

Fig. 5

Summary of plasma metabolites in the three comparison groups. The results of the comparisons (postoperative delirium [PD] vs non‐PD in the placebo group, Yokukansan [YKS] vs placebo in patients older than 75 years, and YKS vs control [Ctrl] in aged mice) are summarized as heat maps. The blue and red color gradients indicate the logarithm of the fold‐change in each comparison. Gray indicates undetected metabolites. Candidate metabolites selected to characterize PD and YKS in each comparative analysis are shown in black in the left column.

Fig. 6

Comparison of plasma metabolites between the occurrence of postoperative delirium (PD) in the Yokukansan (YKS) group. To investigate the response to YKS, we compared targeted and nontargeted metabolite analyses in PD patients and non‐PD patients and examined metabolites associated with the onset of PD in the YKS group. (a) Volcano plot of the plasma metabolites comparing positive and negative PD (patients older than 75 years). The plots correspond to Fig. 2a. (b) Plots of representative metabolites. The crossbar indicates the median. Open and closed circles represent the placebo and YKS groups. Abbreviations: 5‐iPF2a‐VI, (8β)‐5,9α,11α‐trihydroxy‐prosta‐6E,14Z‐dien‐1‐oic acid; SM, sphingomyelin.