Choline is a vitamin-like essential quaternary ammonium salt present in the diet as free choline and phospholipid-bound forms. Dietary choline is partially absorbed by intestinal cells or metabolized by gut bacteria (5, 6) ( Figure 1 ). While choline does not compete with other nutrients for enterocyte transport, gut microbes can limit its bioavailability (7). In the large intestine, gut microbiota, including Firmicutes and Proteobacteria, convert choline into trimethylamine (TMA) (8), which is subsequently absorbed and oxidized in the liver by flavin monooxygenase 3 (FMO3) into trimethylamine-N-oxidase (TMAO) (9). In Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii, choline uptake is mainly mediated by the BetT transporter, whose expression and transport activity are upregulated by hyperosmotic stress (10). Bacteria use choline primarily as a precursor to glycine betaine, a potent osmoprotectant (11), and as a carbon and nitrogen source (12). In contrast, in eukaryotic cells, choline transport is more complex and relies on various protein transport systems, including choline transporter-like proteins (CTLs), high-affinity choline-specific transporters (CHTs), and organic cation transporters (OCTs) (13) ( Figure 2 ). The preference for a specific transport system is cell-dependent and aligns with the anticipated metabolic fate of choline. Thus, CTLs facilitate choline transport across the plasma and mitochondrial membranes, aiding in the production of phosphatidylcholine (PC) and betaine, respectively (14). CHTs mainly mediate sodium-dependent choline uptake in nervous tissues and aid the synthesis of the neurotransmitter acetylcholine (ACh) (15), while OCT2 plays a role in ACh recycling in cholinergic neurons in the presynaptic terminals (16). Recent evidence has identified mitochondrial choline import via the orphan solute carrier SLC25A48, leading to the production of betaine (17–19), a choline-derived methyl donor, and the synthesis of purine nucleotides (18–20). Loss or a single nucleotide polymorphism on the SLC25A48 gene inhibits mitochondrial choline import, increases reactive oxygen species, disrupts lipid balance, and impairs cell proliferation (18–20).

Once intracellular, choline is directed into various metabolic pathways, including 1) its oxidation to form mitochondrial betaine, 2) its acetylation to produce ACh, and 3) its initial phosphorylation followed by mobilization in the Kennedy pathway to generate cholie-containing phospholipids ( Figure 2 ). Choline undergoes irreversible oxidation to betaine through a two-step process mediated by choline dehydrogenase or choline oxidase. Betaine serves as an osmolyte and methyl group donor, playing a role in the re-methylation of homocysteine into methionine, a precursor of S-adenosylmethionine (SAM), essential for numerous methylation reactions, including DNA epigenetics (21–24). In the human body, choline is primarily utilized for synthesizing essential lipids such as sphingomyelin and phosphatidylcholine (PC), which are major components of cellular membranes (25). In particular, PC accounts for approximately 95% of the total cellular choline pool in most cells. It is synthesized from choline via the Kennedy Pathway, involving cytidine diphosphate-choline and a lipid anchor such as diacylglycerol (26). De novo PC synthesis begins with the phosphorylation of choline by the enzyme choline kinase (ChoK) into phosphocholine (27) ( Figure 2 ). Phosphocholine is then converted into cytidine diphosphate-choline by phosphocholine cytidylyltransferase (CTP), which, using either diacylglycerol or alkyl-acylglycerol as a lipid anchor, is ultimately converted into PC (26). Additionally, choline contributes to the production of lipid mediators such as lysophosphatidylcholine (lysoPC), sphingomyelin, and platelet-activating factor (28).

Beyond lipid metabolism, choline plays a critical role in non-metabolic functions. Choline is also the precursor of the neurotransmitter ACh, whose synthesis is catalyzed by choline acetyltransferase (ChAT), transferring an acetyl group from acetyl-coenzyme A to choline, resulting in ACh and coenzyme A production ( Figure 2 ). After being released and bound to its receptors, ACh is rapidly hydrolyzed into acetate and choline by acetylcholinesterase. Then, free choline is transported back by CHTs for further ACh synthesis (29). Cholinergic signaling in immune cells appears to regulate cytokine synthesis, influencing both the initiation and termination of inflammation (e.g., IL-2 in T cells, TNFα in macrophages, and IL-8 in dendritic cells). Modulation of immune cell cholinergic activity, by regulating ChAT activity, ACh breakdown or choline reuptake, regulates physiological responses such as blood pressure control or anti-viral immune reaction (17, 30–34).

Choline is an essential nutrient throughout life, with particularly high demands during periods of rapid cell proliferation, such as pregnancy. Choline is actively transported across the placenta, leading to fetal plasma choline levels six to seven times higher than maternal blood levels, and amniotic fluid concentrations nearly ten times higher (35). This underscores the critical role of choline in fetal development, particularly in neural tube formation and hippocampal development (36, 37). To prevent deficiency-related complications, health organizations, including the National Institutes of Health (NIH) and the European Food Safety Authority (EFSA), recommend a daily choline intake of 550 mg/day for men and 425 mg/day for women, increasing to 450 mg/day during pregnancy and 550 mg/day during lactation (22). Deficiencies during pregnancy can impair long-term potentiation and memory, while prenatal and early-life choline supplementation has been proposed as an intervention to enhance cognitive outcomes, such as improving cognitive function and mitigating age-related memory decline, enhancing long-term memory, and sustained attention (38–46). Choline supplementation (5 g choline chloride/kg) in rats during pregnancy also protects against gestational inflammation mediated by LPS challenge, reducing the frequency of loss of fetuses, normalizing placenta weights, and attenuating LPS-induced NF-κB activation and TNF-α, IL-1β, IL-6, and IL-17A levels in the placenta (47). Maternal dietary choline can be delivered to the offspring through lactation. Pups from phosphatidylcholine (PC)-fed (egg lecithin) dams have increased concentrations of PC in the plasma and spleen and a lower frequency of antigen-presenting cells (48). However, splenocytes from pups from PC-fed dams produced more IL-2, IL-6, and IFN-γ after stimulation with concanavalin A and LPS (48). Additionally, postnatal choline supplementation (100 mg/kg/day) for 20 consecutive days has been shown to mitigate the long-term effects of prenatal ethanol exposure on hippocampal inflammation and peripheral immune responses in rats (49, 50).

In females, as estrogen levels decline, the choline metabolism and utilization undergo significant changes. A study examining choline intake in 664 subjects enrolled in the cross-sectional study Nonalcoholic Steatohepatitis (NASH) Clinical Research Network (NCT00063622 and NCT00063635) showed that postmenopausal women with self-reported choline intake less than 50% of the adequate intake, had faster progression of NASH as shown by increased liver fibrosis, while no associations were found in children, men, and premenopausal women (51). Dietary choline deprivation led to fatty liver and muscle damage in 77% of men and 80% of postmenopausal women, whereas only 44% of the premenopausal women exhibited signs of organ dysfunction (52). These findings highlight that choline deficiency may become more severe in certain populations, such as postmenopausal women. This is partly due to the loss of ability to maintain the expression of genes with estrogen-response elements involved in choline metabolism. One such gene is phosphatidylethanolamine N-methyltransferase (PEMT) (53), which encodes the enzyme that catalyzes the conversion of phosphatidylethanolamine to PC, through a three-step methylation process using S-adenosylmethionine (SAM) as methyl donor.

In the same line with prenatal choline intake and its effect on cognition and memory, in adult individuals, sustained choline intake between 187.6-399.50 mg/day, is linked to reduced risk of cognitive decline, improved learning ability, verbal fluency, working memory, mental processing speed, and attention span, compared to individuals consuming less than 187.6 mg/kg of total choline (54). Other epidemiological studies have revealed that plasma choline levels are inversely correlated with anxiety (55) and risk of depressive symptoms (56), supporting a potential positive role of choline in mental health as well. While insufficient choline intake is linked to liver dysfunction, neurodegeneration, and muscle damage, excessive intake primarily results in mild cholinergic side effects such as sweating, diarrhea, hypotension, and fishy body odor, with a tolerable upper intake level of 3.5 g/day in adult individuals (57–59). Despite recommendations, a significant portion of the population consumes insufficient choline, and both patients with cirrhosis and those critically ill on parenteral nutrition often experience severe choline deficiencies negatively affect their outcomes (60–62). However, even while consumption levels are optimal, choline depletion may occur locally in specific disease contexts, such as within the tumor microenvironment or at sites of infection by choline-consuming pathogens (63) or systemically, such as in critically ill patients on parenteral nutrition (62). It remains uncertain whether dysregulated choline distribution affects disease progression, and significant gaps exist in understanding how cells adapt to impaired choline availability or metabolism locally.

Myeloid cells are frontline immune defenders that restore tissue homeostasis after infection or tissue damage. Dysfunction of these cells contributes to chronic inflammatory and autoimmune diseases such as gout, rheumatoid arthritis (RA), osteoarthritis (OA), cryopyrin-associated periodic syndromes (CAPS), inflammatory bowel disease, and neurodegeneration. Their functional plasticity, shifting between inflammatory, repair, or anti-inflammatory states, depends on metabolic adaptation and lipid remodeling, including choline uptake and phospholipid reorganization (66, 76). Therefore, disrupting choline metabolism can significantly alter myeloid cell function and disease outcomes.

The phospholipid remodeling and cholinergic anti-inflammatory system play a well-established role in immune homeostasis and the regulation of inflammatory and autoimmune diseases. Most immune cells, including CD4⁺ T cells, B cells, and NK cells, upregulate genes related to the cholinergic system synthesis in response to inflammatory cues, which is pivotal for the maintenance of immunological homeostasis (31–33, 86, 102). Below, we summarize studies that remark the importance of the cholinergic immune signaling and circulating choline-containing lipids on lymphocyte differentiation, activation, and function.

Cardiometabolic diseases include conditions such as diabetes, fatty liver disease, and cardiovascular diseases. Epidemiological studies on circulating choline metabolites and the incidence of diabetes in 3,133 individuals aged 33–45 years found that TMAO levels positively and betaine inversely correlated with the 15-year risk of incident diabetes (147). In a randomized, doubled blind placebo placebo-controlled parallel trial in 96 diabetic patients subjected to dietary supplement intervention that included choline, magnesium or both for 2 months (IRCT20110123005670N25), circulating levels of the inflammation and endothelial factors IL-6 and VCAM-1 decreased significantly in the choline and magnesium group compared to the other groups even after adjusting for potential cofounders (148).

The impact of choline and trimethylamine-N-oxidase (TMAO) on cardiovascular diseases has been investigated in both experimental and human studies. TMAO enhances cholesterol accumulation in macrophages, has been associated with increased cardiovascular disease risk (6, 149), and has a negative impact on disease outcome (150–153). Patients diagnosed with pulmonary hypertension (n=272) with high circulating choline levels (based on the 50th quartile of circulating choline levels, defined as 12.6 µM), exhibit worse key indicators of severe disease progression, including WHO functional class, higher N-terminal pro-B-type natriuretic peptide levels, and reduced cardiac output index, and predicts their prognosis (140). Plasma choline as a diagnostic biomarker for hypertension and artery stenosis was evaluated in 193 individuals, revealing that plasma choline levels were high in patients with hypertension without artery stenosis, and even higher in patients with hypertension with artery stenosis compared to healthy controls (154). The study of the impact of the Dietary Approaches to Stop Hypertension (DASH) diet on plasma choline, choline metabolites, and ceramides in obese older adults revealed a connection between choline metabolites and cardiometabolic outcomes (NCT04127240) (155). The participants in this study consumed either 3 or 6 oz of beef, with rich choline content, within a standardized DASH diet for 12 weeks (155). In response to the DASH diet, with beef intake groups combined, significant changes were observed in plasma biomarkers, including the decreased of plasma choline (by 9.6%); dimethylglycine (10%); phosphatidylcholine (PC) (51%); and triglycerides (18%); and the increase of total lysoPC (by 281%); TMAO (26.5%); total ceramide (22.1%). Around 20 LysoPC species were significantly increased, with lysoPC 16:0 being the most pronounced response. In addition, sphingomyelin 16:0, 18:0, and 18:1 increased by 10.4%, 22.5%, and 24%, respectively, and ceramide 24:1 by 36.8%; whereas sphingomyelin 24:0 significantly decreased by 10%, and ceramides 22:0 and 24:0 declined by 27.6% and 10.9%, respectively (155). These changes in choline and choline metabolites correlated with cardiometabolic outcomes, underscoring the importance of choline in older humans and the role of diet in modulating circulating lysoPC, sphingomyelin, and ceramide species (155). Recently, the study of the effects of L-alpha glycerophosphocholine, a nutritional supplement that has been demonstrated to improve neurological functions, on cardiovascular events in mice has revealed that glycerophosphocholine increases the risk of stroke by shifting the gut microbiota towards abundant Parabacteroides, Ruminococcus, and Bacteroides, while reducing the abundance of Akkermansia, Lactobacillus, and Roseburia (156). These changes reflected an increased relative abundance of choline TMA lyase (cutC). Moreover, glycerophosphocholine supplement also increased the proinflammatory effectors CXC13 and TIMP-1, and activated NF-κB and MAPK signaling pathways in human coronary artery endothelial cells (156). In mice, oral glycerophosphocholine supplementation promotes increased plasma TMAO, phenylalanine, betaine, leucine, and valine (156).

The incidence of acute myocardial infarction was examined in 1981 male patients with stable angina pectoris follow-up during 7.5 years, and subjected to a 169-item food frequency questionnaire to monitor the dietary choline intake, showed that increased intakes of energy-adjusted choline, PC, and sphingomyelin were associated with a higher risk of incident acute myocardial infarction (135). Similarly, a study of 4007 participants (average age of 63) undergoing elective diagnostic cardiac catheterization (NCT04255368) revealed higher baseline TMAO levels in individuals experiencing major adverse cardiovascular events (5.0 μM) compared to those individuals without events (3.5 μM) (157). Participants in the highest TMAO quartile (>6.18 μM) had a 2.54-fold increased hazard ratio compared to controls (157). The prognostic value of elevated plasma TMAO for cardiovascular risk remained significant in various subgroups associated with a reduced overall risk of major cardiovascular events. Notably, a three-year follow-up confirmed elevated plasma TMAO as a significant predictor of major adverse cardiovascular events (157). Another study on cardiometabolic risk factors in a diabetes case-control study (NCT02588898) and a vitamin-supplementation trial (NCT02586181) found high plasma concentrations of metabolites, including TMAO and choline, correlated with lower cholesterol and plasma phospholipid levels, suggesting TMAO may aid in cholesterol solubilization and macrophage cholesterol (158).

Several studies have reported an inverse association between dietary choline supplementation and both the incidence and severity of non-alcoholic fatty liver disease (NAFLD) or steatohepatitis (NASH) (159, 160). Steatosis, or fat accumulation in the liver, which is a direct effect of choline deficiency, can lead to inflammation and cause more severe conditions like fibrosis, cirrhosis and liver cancer (161). Findings from the Framingham Heart Study, a large community-based cohort including offspring and third-generation participants, revealed that choline intake, calculated as the sum of dietary choline-containing compounds including phosphocholine, sphingomyelin, free choline, glycerophosphocholine, and PC, was inversely associated with NAFLD risk (159). A randomized controlled clinical trial (NCT01934777) involving children with non-alcoholic steatohepatitis (NASH) evaluated the effects of combined supplementation with docasahexaenoic acid, choline, and vitamin E (DHA-CHO-VE) (160). Participants underwent a lifestyle counseling along with either a daily supplement containing DHA-CHO-VE (250 mg of DHA, 39 UI of VE, 201 mg of choline) or placebo for six months. The DHA-CHO-VE group showed a marked reduction in severe hepatic steatosis (ranging from 5-50%), alongside improved serum ALT and fasting glucose levels (159). While DHA-CHO-VE supplementation did not affect bile acid concentrations, it did increase intestinal fibroblast growth factor 19 (FGF19), a key regulator of bile acid synthesis and metabolism (160). The role of dietary choline supplementation in adult patients with metabolic syndrome was examined in a randomized crossover intervention in 23 individuals supplemented with either 3 eggs/day or 400 mg choline per day for 3 weeks, followed by a 4-week washout, and then the alternate interventions (NCT03877003) (162). During the egg-derived choline feeding phase, there was a stronger effect on metabolic and inflammatory plasma biomarkers, including an increase in monounsaturated fatty acids, vitamin D, selenium, and a decrease in CRP, IL-6, and insulin (162), while during choline bitartrate feeding, only a decrease in IL-6 was observed (162). This distinct effect may be caused by differences in the lower intestinal absorption of choline bitartrate compared to the choline from eggs (including egg-PC), or by the effect of additional nutrients in the eggs. Collectively, these findings support the idea that different sources of choline have distinct effects, suggesting that a more personalized selection of choline forms, based on individual’s underlying conditions and characteristics, may lead to greater health benefits.

Recent human and animal research has emphasized the importance of choline intake in preventing neurodegenerative diseases such as Alzheimer’s disease (163). High choline intake in early life has been shown to improve outcomes in a mouse model of Alzheimer’s disease, regulating hyperexcitability, preserving hilar neurons, and enhancing spatial memory (164). Further evidence comes from studies using the 3xTg-Alzheimer’s disease mouse model, which replicates key features of human Alzheimer’s disease progression. In this model, choline-deficient diet from 3 to 12 months of age disrupted liver and heart normal function, and altered neural networks associated with microtubule stability and postsynaptic membrane regulation in the hippocampus, elevated soluble and insoluble Amyloid-β levels, increased Thioflavin S structures, and tau hyperphosphorylation at various pathological epitopes in the hippocampus and cortex (163). This system-wide dysfunction in mice fed with choline-deficient choline was observed in the circulation, as it modulates plasma proteins associated with inflammation, immune response, and metabolic processes, including insulin metabolism, mitochondrial function, inflammation, and fructose metabolic processing (163). Lifelong choline supplementation in another Alzheimer’s disease mouse model (APP/PS1 transgenic mice), significantly diminished amyloid-β plaque load and decreased activated microglia, thereby mitigating the detrimental effects of brain inflammation associated with Alzheimer’s disease (44). The use of the cholinergic neurotransmission-enhancing agent choline alphoscerate has also shown protection from antibody-mediated neurotoxicity, directly activating the a7nAChR receptor in microglia, leading to phenotype switching towards a less inflammatory state (165). Human studies further support the neuroprotective role of choline. A study with 3,224 participants found that low choline intake was associated with an increased risk of incident dementia and Alzheimer’s disease (136). Similarly, a study with 125,594 participants showed that moderate dietary choline intake, ranging from 332.89 mg/d to 353.93 mg/d, is associated with lower odds of dementia and better cognitive performance (166).

In the context of fetal alcohol spectrum disorders (FASDs), 625 mg of choline was administered to school-aged children (5–10 years old, n=29 choline and n=26 placebo) for six months (NCT01911299), showing no effect on cognitive performance (167). However, in another study in younger children with weight-adjusted dosing, the dietary choline intake increased the memory scores by 12-14% (NCT01149538 and NCT02735473) (168, 169). Indeed, an inverse relation between choline dose (mg/kg) and memory improvement suggested weight-adjusted doses as preferable to fixed doses (168). Follow-up studies for 4 and 7 years after the intervention with 500 mg/day for 9 months showed that sustained cognitive benefit was associated with potential improvements in associated white matter microstructure in the choline group (170, 171), which suggests that choline supplementation has the potential to alter brain architecture. The participant in the group supplemented with choline showed an 12-25% increase of the elicited imitation memory paradigm, 8% higher verbal IQ, 29% higher visual-spatial reasoning, 36% higher crossmodal learning, 27% higher non-verbal working memory 4 years after the treatment at a mean of 8.6 year of age. Moreover, an improvement in lower-order executive function skills (eg, information processing speed) was found in a subset of participants who returned 7 years after completing a choline trial, at a mean age of 11 years (170, 171). While these findings are promising, other studies in pediatric patients (aged 7–12 years) diagnosed with ADHD did not show statistically significant differences between the use of citicoline (cytidine diphosphate-choline supplement) and placebo. If the absence of effect is due to the choline form used, or the age of the individual is not known (172).

In the adult population, a double-blind randomized clinical trial examined choline dietary supplementation on cognitive and immune function in 97 healthy older adults over six months (NCT01672359) (173). The participants were divided into three groups: group 1 (n=33) received 120 mg/day of Ginkgo Synergy plus 700 mg/day of choline; group 2 (n=31) received 100 mg/day OPC Synergy plus Catalyn; group 3 (n=33) received a placebo (173). While no significant changes were observed in cytokine levels (IL-2, IL-6, IL-8, IL-10, IL-1α, IFN-γ, TNF-α, VEGF, and MCP1), the Ginkgo Synergy plus choline group exhibited a significant reduction (57%) in epidermal growth factor (EGF), a protein that is often overexpressed in individuals with mild cognitive impairment or Alzheimer’s disease (173). However, the absence of follow-up studies limited the ability to conclude the effect on the disease progression.

The effect on cognitive function has also been examined in 36 individuals with Type 2 diabetes mellitus and mild cognitive impairment assessed by the Mini-Mental State Examination (MMSE) score. The group provided 1200 mg/day of choline alfoscerate for 12 months, significantly showed better physical health and an increase mean difference in the MMSE score (+1.7 between the two groups), which would support its use as an adjunct therapy for managing early cognitive decline (174). The effect on physical performance has also been observed in a recent randomized, double blind, placebo-controlled crossover approach in 20 resistance-trained young males (31.3 ± 11 years) who consumed either a placebo, 630 mg alpha-glycerophosphocholine, or 315 mg of alpha-glycerophosphocholine (NCT06690619). Both groups taking alpha-glycerophosphocholine increased the cognitive performance (Stroop total score and time of completion) (175).

In inflammatory disorders, the increased metabolic demands for immune activation and cellular proliferation necessitate an elevated supply of phospholipids, such as phosphatidylcholine (PC). High plasma and tissue choline concentrations are common in diseases characterized by chronic low-grade inflammation, supporting the role of choline in modulating immune cell activation and tissue damage (176–182).

The role of choline metabolism in cancer progression and growth has been extensively studied and revised elsewhere (201, 202). Enhanced choline uptake and metabolism, and increased serum choline are hallmarks of many cancers and correlate with a higher risk of overall cancer with a poor prognosis (203, 204). Notably, elevated choline kinase alpha (ChoKα) expression and activity are often associated with malignant transformation, invasion, and metastasis in some human cancers, making ChoKα a promising therapeutic target in oncology and choline radiotracers a reasonable tool for monitoring cancer growth and therapy response (205–213). Moreover, ChoKα has been recognized as a prognostic marker in various cancers (208, 214–216). High ChoKα expression correlates with early-stage non-small-cell lung cancer (NSCLC) patients at risk of recurrence, whereas lower expression identifies patients with favorable outcomes, potentially allowing for less aggressive treatment approaches (215). Over recent decades, numerous ChoKα inhibitors have been developed and tested for cancer therapy (217–219). However, the efficacy and predictive value of choline metabolism-related signatures for patients’ prognosis, immune microenvironment, and chemotherapy response remain incompletely understood. Recent evidence using several public datasets from The Cancer Genome Atlas (TCGA), Kyoyo Encyclopedia of Genes and Genomes (KEGG), AmiGO (2) and Reactome Pathways databases has identified two choline metabolism-related genes (choline kinase β, CHKB, and phosphatidylethanolamine N-methyltransferase, PEMT) as key genes involved in the pathogenesis of human colorectal cancer (220, 221). Patients were stratified into high- and low-risk groups based on the optimal cutoff value of the choline metabolism-related risk score to assess the prognostic accuracy of the choline metabolism-related signature (222). The overall survival of patients in the high-risk group was significantly worse than that of patients in the low-risk group. The examination of sc-RNAseq revealed that CHKB expression was mainly in endothelial cells, while PEMT was highly expressed in CD4⁺ and CD8⁺ T cells (220). Indeed, there were notable differences in immune microenvironment composition, immune checkpoint gene expression, and chemotherapy response between the two risk groups (220).

Oncogenic MYC drives aberrant choline metabolism by transcriptionally upregulating CTP:phosphocholine cytidylyltransferase-α (PCYT1A) (223), a key enzyme in phosphatidylcholine (PC) de novo biosynthesis. In patients with diffuse large B-cell lymphoma (DLBCL), elevated PCYT1A expression, accompanied by increased MYC levels and decreased serum PC, correlates with a higher international prognostic index risk classification, suggesting that co-expression of MYC and PCYT1A may serve as a biomarker for disease progression (223). The use of histone deacetylase inhibitors (HDACI) modulates lipid metabolism and survival pathways in DLBCL, particularly by gaining dependency on the choline pathway and PI3K signaling activation (224), which results in a decline in the antineoplastic effects of the HDACI (224). In part, the aberrant choline metabolism in cancer is driven by molecular alterations in enzymes such as phospholipases C and D, ethanolamine kinase-α, glycerophosphocholine phosphodiesterases, and choline transporters (211). Tumors exhibit elevated phospholipid levels, characterized by an increase in phosphocholine and total choline-containing metabolites, along with an altered glycerophosphocholine/phosphocholine ratio (208, 210, 225, 226). As the major phospholipid in eukaryotic membranes, PC is essential for cancer cell proliferation, tumor progression, and invasion (209). Increased total choline signal detected by 1H nuclear magnetic resonance spectroscopy (1H-NMR) is currently being considered as a diagnostic marker in multiple cancers (137, 210, 227, 228). Choline plays a critical role in cancer diagnosis and monitoring. The use of (11) C-choline positron emission tomography/computed tomography (PET/TC) has provided insights into choline metabolism in tumors, including lung, liver, ovarian, and prostate cancers (216, 229–234). Both (1)H-NMR and choline PET imaging are being explored to evaluate treatment responses. The increased expression and activity of choline transporters and enzymes, such as CTL1 and ChoKα, respectively, have led to the development of radiolabeled choline analogs as PET imaging tracers (211).

In the INSPIRE cohort of 106 patients with advanced solid tumors treated with pembrolizumab (anti-PD-1 antibody) (NCT03702309) (235), higher serum choline levels were associated with augmented CD8^- T cells and B cells in the tumor stroma, as well as an elevated CD3⁺CD8⁺ cytotoxic T cells to CD3⁺CD8^-FOXP3⁺ T regulatory cells ratio in the tumors (235). A validation study on 51 patients receiving anti-PD-1 therapy alone or in combination (NCT02644369) (235), found no association between choline levels and changes in absolute neutrophil count (ΔANC), lymphocytes, and neutrophil-to-lymphocyte ratio (ΔNLR) (235). Notably, higher serum choline levels correlated with improved progression-free survival and a trend toward better overall survival (235). A case-control study (NCT00794885), suggested that high serum choline levels were linked to overall cancer risk, particularly among older male smokers (204). A case-control study investigating the differences in circulating concentrations of choline metabolites between elderly men with prostate cancer or benign prostatic hyperplasia (236). While no significant differences were found in choline, betaine, dimethylglycine, folate, total homocysteine, cystathionine, methylmalonic acid, S-adenosylhomocysteine (SAH), S-adenosylmethionine (SAM), the presence of 11 sphingomyelin species was significantly reduced in patients with prostate cancer as compared to benign hyperplasia (236).

In triple-negative breast cancer, a very aggressive disease with a poor prognosis, the commensal microbiota Clostridiales and its metabolite trimethylamine-N-oxide (TMAO) colonize the mammary gland, being more abundant in tumors exhibiting an activated immune microenvironment (237). TMAO was identified as a driver of antitumor immunity, providing a foundation for potential TMAO-based therapeutic strategies. Patients with higher plasma TMAO levels achieved better responses to immunotherapy due to TMAO-induced pyroptosis in tumor cells and enhanced CD8⁺ T cell-mediated antitumor immunity (237). An unbiased metabolic screening using liquid chromatography-tandem mass spectrometry identified TMAO, as a key gut microbiome-derived metabolite that enhances antitumor immunity in pancreatic ductal adenocarcinoma (PDAC) (238). Administration of TMAO, either intraperitoneally or via dietary choline supplement, in PDAC-bearing mice significantly reduced tumor growth. This antitumor effect was associated with a shift toward an immunostimulatory tumor-associated macrophage (TAM) phenotype and an enhanced effector T cell response within the tumor microenvironment, mediated by activation of the type I Interferon pathway (238). Consistently, delivering intravenously TMAO-primed macrophages yields similar tumor-suppressive effects (238). Indeed, combining TMAO with immune checkpoint blockade therapy, such as anti-PD1 and anti-TIM3 antibodies, further reduced tumor burden and improved survival compared to monotherapies alone. Additionally, higher levels of gut bacteria expressing CutC, the enzyme responsible for generating trimethylamine, correlated with longer survival in PDAC patients and enhanced responses to anti-PD1 immunotherapy in melanoma patients (238). Collectively, these findings highlight TMAO as a promoter of antitumor immunity across multiple cancer types. By enhancing immune cell function, reshaping the tumor microenvironment, and boosting responses to immunotherapy, TMAO holds promise as a potential adjuvant to improve the efficacy of cancer immunotherapy.

Sepsis is a life-threatening clinical condition arising from an excessive immune response to infection, leading to high morbidity and mortality rates (239). A major challenge in intensive care medicine is managing severe infections with multiple organ dysfunction, commonly referred to as sepsis shock (240). The Systemic Inflammatory Response Syndrome (SIRS) is triggered by endotoxin, which stimulates the release of inflammatory cytokines, such as TNF-α; IL-1α/β, and IL-17 into the circulation. These cytokines, in turn, prompt hepatic cells to release acute-phase proteins such as CRP for immunological regulation (241). Matrix metalloproteinases (MMPs) are up-regulated by pro-inflammatory cytokines, as well as by acute phase proteins like serum amyloid A (SAA), with counter-regulatory inhibition by IL-4 and IL-13, and released into the circulation from damaged vascular endothelium. An increased MMPs/tissue inhibitor MMPs (TIMPs) ratio is more strongly associated with tissue response to LPS-linked injury than with the acute-phase reaction (242, 243). Choline-deficient diet has been linked to increased hepatic injury and mortality in endotoxemic shock (239). In a canine model of sepsis induced by intravenous injection of 0.2 mg/kg LPS from E. coli, choline intravenous administration suppressed the elevation of circulating MMPs and TIMPs, preserved serum IgG and IgM levels, and was associated with reduced acute-phase reaction and multi-organ failure (244).

In a study using the gold-standard experimental model of polymicrobial sepsis, the cecal ligation and puncture (245), metabolic analysis of plasma and urine identified 14 plasma and 11 urine metabolites related to central carbon and choline metabolism (including choline, betaine, methylamine, and creatinine). These metabolites significantly differed between septic mice with and without signs of acute kidney injury (AKI), as defined by Kidney Disease Improving Global Outcomes (KDIGO), as creatinine levels tripled the average of sham mice. These were further validated in 7 pediatric patients with severe sepsis and AKI, and 13 with sepsis with stage 1 or no AKI. Plasma analysis revealed 10 differentially expressed metabolites more abundant in sepsis-associated AKI patients, including succinate, pyruvate, lactate, betaine, dimethylglycine, and choline, resembling the findings in mouse plasma (245). These metabolic changes were supported by alterations in the gene expression of choline metabolism-related enzymes, such as choline dehydrogenase (Chdh), betaine-homocysteine methyltransferase (Bhmt), and DMG dehydrogenase (Dmgdh), and glycerophosphocholine phosphodiesterase 1 (Gpcpd1). Notably, flavin-containing monooxygenase 3 (Fmo3), the enzyme responsible for TMAO production in the liver, was reduced in sepsis-associated AKI. Mice that received choline intraperitoneally before and during sepsis exhibit lower levels of plasma blood urea nitrogen (BUN) and creatinine, as well as urine neutrophil gelatinase-associated lipocalin (NGAL) levels compared to septic mice treated with vehicle. While these findings support that choline may protect the kidneys during sepsis, the survival rate was not affected by choline supplementation (245). A metabolic study using NMR spectroscopy found distinct metabolic profiles between sepsis survivors and non-survivors, those who succumbed at day 0 of hospitalization. Non-survivors exhibited significantly higher levels of creatine, phosphocreatine, choline, betaine, tyrosine, histidine, and phenylalanine compared to survivors (246). However, another study reported that individuals with sepsis had lower concentrations of phosphatidylcholine (PC), phosphatidylserine, lysophosphatidylethanolamine, and lysoPC but higher creatinine and C17-sphinganine, compared to healthy controls (247).

Animal models have demonstrated that choline supplementation improves survival and reduces TNF-α production (248). In endotoxin-infused mice, free serum choline levels dropped by 49% at low endotoxin administration but increased by 98% within 48 hours after high endotoxin exposure (1 mg/kg). Despite these fluctuations, phospholipid-bound choline levels increased regardless of the endotoxin dose, accompanied by an increase in biochemical markers of tissue injury and organ dysfunction (249). However, another study using the CLP mouse model found that administering choline before and after sepsis initiation reduced serum inflammatory mediators such as TNF-α and HMGB1, and improved survival via the α7nAChR-dependent mechanism (250, 251). The anti-inflammatory effect of choline was confirmed ex vivo, where endotoxin-activated human whole blood and macrophages exposed to supraphysiological choline concentrations (between 1–50 mM) exhibited reduced TNF-α production (250). Additionally, elevated plasma choline acetyltransferase (ChAT) concentrations were observed in patients with sepsis, with the highest levels detected in those who succumbed to infection (252). The increase in circulating ChAT coincides with the decline in TNF-α (252), showing its potential anti-inflammatory. However, ChAT activity did not correlate with circulating choline or acetylcholine (ACh) concentration, which suggests additional regulatory mechanisms.

Sepsis is characterized by profound metabolic alterations, with choline metabolism playing a key role in inflammatory regulation and organ function. While experimental and clinical data suggest that choline supplementation may protect against sepsis-associated AKI and modulate inflammation, its effect on overall sepsis survival remains inconclusive. Further research is needed to determine the therapeutic potential of choline in sepsis management, particularly in modulating immune responses and metabolic homeostasis.