**Background:** Trimethylamine N-oxide (TMAO) is a gut microbiome-derived metabolite suspected to enhance platelet responsiveness and promote thrombotic risk. While TMAO has been associated with cardiovascular disease and stroke, its role in subarachnoid hemorrhage (SAH)—a condition where microvascular thrombosis contributes to delayed cerebral ischemia (DCI)—had not been explored. This study aimed to investigate plasma and cerebrospinal fluid (CSF) TMAO levels in SAH patients compared to controls, and to examine their relationship with clinical severity and course over time.
**Methods:** This prospective observational study was conducted at the Department of Neurosurgery, University of Leipzig Medical Center from October 2018 to January 2020. The SAH group included patients with confirmed non-traumatic SAH (aneurysmal or perimesencephalic) treated in the intensive care unit. The control group comprised patients with nerve, nerve root, or plexus disorders without known central nervous system pathology or thrombotic events. Exclusion criteria for both groups were age <18 years; SAH patients with traumatic SAH were also excluded. Blood samples were collected on admission (day 0) from all participants, and daily for up to 15 days from SAH patients in the ICU. CSF was collected from SAH patients with external ventricular drains on days 0, 5, and 10. TMAO and its precursors (betaine, carnitine, choline) were measured by LC-MS/MS. Routine laboratory parameters, including hematocrit, hemoglobin, platelet count, eGFR, prothrombin time, and aPTT, were also assessed. SAH severity was classified using the WFNS classification and Fisher grading. Blood-CSF barrier (BCB) disturbance was defined by an age-dependent upper limit of the CSF/plasma albumin ratio (Q_ALB). Statistical analyses used Mann-Whitney U, Wilcoxon, Chi-square, and Spearman's rank correlation tests as appropriate.
**Key Results:** Thirty-four SAH patients and 108 controls were included. Baseline characteristics were balanced except for hypertension, which was more prevalent in the SAH group (79.4% vs. 52.8%, p=0.009). Plasma TMAO levels at baseline were significantly lower in the SAH group (median 1.7 μmol/L, IQR 0.9–2.8) compared to controls (2.9 μmol/L, IQR 1.9–4.1; p<0.001). Post hoc power analysis yielded a power of 0.90 with effect size d=0.659. TMAO precursors (betaine, carnitine, choline) were also significantly lower in SAH patients. CSF TMAO was detectable in 17 SAH patients (median 0.4 μmol/L, IQR 0.2–0.9) and was significantly lower than plasma levels (p<0.001). Plasma and CSF TMAO levels correlated positively (p<0.001). BCB disturbance was present in 15 SAH patients, but Q_ALB did not correlate significantly with CSF TMAO or the CSF/plasma TMAO ratio. Over the 15-day observation period (390 blood samples total), plasma TMAO levels did not vary significantly, and no characteristic temporal pattern was observed. The eGFR varied significantly but remained above the reference threshold (>90 mL/min/1.73m²). Platelet count increased significantly after approximately day 4. Plasma betaine, carnitine, and choline levels increased significantly from day 5–7 after SAH. Nine patients developed DCI; plasma and CSF TMAO levels did not differ between DCI and non-DCI subgroups. Plasma TMAO levels did not differ significantly across WFNS or Fisher grade subgroups, though a non-significant trend toward lower TMAO in poor-grade (WFNS 4–5) vs. good-grade (WFNS 1–3) SAH was noted. At recovery or end of observation, SAH patients still had significantly lower plasma TMAO (1.9 μmol/L) than controls at baseline (2.9 μmol/L).
**Clinical Implications:** Contrary to the authors' hypothesis, plasma TMAO levels were lower in SAH patients than in controls, and this difference persisted over the observation period. The findings do not support TMAO as a biomarker for SAH risk or its thrombotic complications such as DCI. The decreased TMAO levels may reflect acute gut dysbiosis via the gut-brain axis following SAH, possibly due to sympathetic activation and catecholamine surge altering the gut microbiome. The detection of TMAO in CSF and its correlation with plasma levels, without correlation with BCB disruption, suggests active transport across the blood-CSF barrier rather than passive diffusion. The increase in TMAO precursors from day 5–7 may indicate gut recovery, but TMAO itself did not rise, possibly due to the observation period length. Limitations include the small SAH sample size (n=34), decreasing sample count over time, and inability to determine the exact time from hemorrhage to first measurement. Larger studies incorporating fecal microbiome analysis and including more DCI cases are needed to clarify the role of TMAO in SAH.