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Abstract

Medicines containing citicoline (cytidine-diphosphocholine) as an active principle have been marketed since the s as nootropic and psychostimulant drugs available on prescription. Recently, the inner salt variant of this substance was pronounced a food ingredient in the major world markets. However, in the EU no nutrition or health claim has been authorized for use in commercial communications concerning its properties. Citicoline is considered a dietetic source of choline and cytidine. Cytidine does not have any health claim authorized either, but there are claims authorized for choline, concerning its contribution to normal lipid metabolism, maintenance of normal liver function, and normal homocysteine metabolism. The applicability of these claims to citicoline is discussed, leading to the conclusion that the issue is not a trivial one. Intriguing data, showing that on a molar mass basis citicoline is significantly less toxic than choline, are also analyzed. It is hypothesized that, compared to choline moiety in other dietary sources such as phosphatidylcholine, choline in citicoline is less prone to conversion to trimethylamine (TMA) and its putative atherogenic N-oxide (TMAO). Epidemiological studies have suggested that choline supplementation may improve cognitive performance, and for this application citicoline may be safer and more efficacious.

Keywords:

citicoline, choline, health claims, toxicity, trimethylamine oxide, procognitive effects

1. Introduction

Citicoline is the international nonproprietary name (INN) for cytidine-diphosphocholine (CDP-Cho). The substance is commercially available in two forms, sodium salt and inner salt. Citicoline sodium salt, classified as a nootropic and psychostimulant [1], is an active principle of a variety of prescription drugs, either injectables or oral formulations. In in the USA, citicoline (inner salt) was self-affirmed by the Japanese company Kyowa-Hakko as GRAS (generally regarded as safe) [2], and in it was announced as a novel food ingredient by the appropriate Implementing Decision of the Commission of the European Union [3].

The aforementioned EU Implementing Decision states that citicoline may be placed on the EU market, where it is intended to be used in food supplements aimed at a target population of middle-aged to elderly adults at a maximum level of 500 mg/day, and in dietary foods for special medical purposes with a maximum dose of 250 mg per serving and with a maximum daily consumption level of mg from these types of foods.

2. Citicoline in Food Supplements: The Issue of Health Claims

Classifying citicoline as a food ingredient suitable for food supplements should make it widely available, but in the highly regulated market of the European Union its marketing is problematic. According to the EU Regulation EC No / [4], all nutrition and health claims made in commercial communications concerning food supplements must be formally authorized following scientific assessment performed by the European Food Safety Agency (EFSA). Citicoline does not have any nutrition or health claim authorized up to date. Moreover, application for authorization of a health claim (related to citicoline and maintenance of normal vision) was turned down by the EFSA because it was concluded that a cause and effect relationship has not been established between the consumption of citicoline and the maintenance of normal vision [5]. Does this mean that, although it is legal to introduce citicoline to the EU market in a food supplement, information provided about this supplement should not contain any information about its specific nutritional and/or functional value?

Looking through the positive EFSA Scientific Opinion on citicoline issued prior to the aforementioned implementing decision [6], we find the reference to the observation that, both in humans and in rats, upon ingestion citicoline undergoes quick hydrolysis, breaking down to choline and cytidine [7], which then undergo further metabolism and incorporation into normal pathways of metabolism [8]. Cytidine, a pyrimidine nucleoside which in humans interconverts with uridine [9], undergoes intracellular phosphorylations to cytidine triphosphate (CTP), which participates in phospholipids synthesis via the Kennedy pathway, and may also be incorporated into nucleic acids. Choline is either phosphorylated to phosphocholine and participates in phosphatidylcholine synthesis, or oxidized to betaine, which serves as a methyl donor in the betaine-homocysteine methyltransferase reaction. Also, in cholinergic neurons, choline is acetylated to form the neurotransmitter acetylcholine.

We may, therefore, consider citicoline as a source of choline and cytidine. Whereas there is no nutrition or health claim authorized for cytidine either, there are three such claims authorized for choline. These are so-called functional claims relating to the beneficial effects of a nutrient on certain normal bodily functions. The first two state that choline contributes to normal lipid metabolism and to the maintenance of normal liver function. These claims were accepted because they were substantiated by observations that choline deficiency is associated with signs of liver damage (elevated serum alanine aminotransferase activity) and the development of fatty liver (hepatosteatosis) in humans fed choline-free total parenteral nutrition solutions, whose effects can be reversed by the administration of dietary choline [10,11]. The third claim, stating that choline contributes to normal homocysteine metabolism, was substantiated by the observations that choline-depleted diets tend to increase plasma concentrations of homocysteine [12], whereas human observational [13,14] as well as intervention [15] studies supported the inverse association between dietary choline and blood concentrations of homocysteine. Of note is that in the aforementioned intervention study, choline was supplied orally in the form of phosphatidylcholine (lecithin).

At the same time, health claims stating that choline contributes to the maintenance of normal neurological function and normal cognitive function were rejected by the EFSA because cause and effect relationships have not been established between the consumption of choline and the claimed effects [16]. One of the reasons was that some references that presented support for the claimed effects described studies that did not evaluate choline, but, for example, citicoline. A possible explanation of this paradox is that at the date of issuing scientific opinion on the health claims concerning choline (i.e., year ), citicoline was not yet appreciated by EFSA experts as the dietary source of choline. Indeed, natural foods do not contain any significant amount of this substance.

There is no direct proof that citicoline intake can reverse either elevated serum alanine aminotransferase activity or the development of fatty liver in people who are choline-deficient. There is also no direct proof that citicoline intake may lower homocysteine in blood. On the contrary, single oral administration of a high dose of citicoline (1 g/kg b.w.) to rats resulted in a transient increase of plasma homocysteine, but when a lower dose was supplemented in the diet for two months, plasma homocysteine remained unchanged [17]. At the same time there is no reasonable doubt that oral intake of citicoline is a safe and efficient method of delivery of choline to the human body.

It might perhaps be concluded that the issue of the applicability to citicoline of health claims pertaining to choline (and apparently also to some of its derivatives, such as phosphatidylcholine) is merely a legal problem that shall be settled accordingly by the appropriate authorities. On the other hand, a health claim authorized almost a decade ago may not be supported in its entirety by the contemporary scientific data. Current guidelines for the management of fatty liver do not mention supplementation with choline or its derivatives [18]. Likewise, folic acid, vitamin B6, vitamin B12, and betaine, but not cholines, are listed among nutrients that may counteract hyperhomocysteinemia [19].

3. Citicoline as a Source of Choline: The Issue of Acute Toxicity

It is well established that following ingestion citicoline is fully absorbed and catabolized to cytidine and choline, which enter their respective metabolic pools in the body [20,21,22]. However, the particulars of its absorption, hydrolysis, and dephosphorylation(s) are a bit unclear. Citicoline contains equimolar amounts of choline and cytidine. Following citicoline ingestion in rats, the increase in both plasma cytidine and choline occurred quickly, but the molar increase in plasma choline was markedly smaller [23]. In a human study [24], oral citicoline resulted in increases in plasma choline and uridine that were similar in timing and magnitude, but in the other human study, the increase in plasma choline following citicoline ingestion was biphasic and delayed [25]. It has been suggested that citicoline is absorbed intact and its hydrolysis occurs in the liver and is coupled with a selective withdrawal of choline from blood [26]. Following oral citicoline intake in humans, the quantitative transformation of cytidine to uridine occurring in the intestine or liver was also postulated [24].

Absorption of intact citicoline molecules from the intestine to blood could also be helpful for explaining differences of acute toxicity of citicoline versus choline upon different routes of administration ( ).

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The classical measure of acute toxicity is LD50, the median lethal dose of the tested compound expressed in milligrams per kilogram body weight. The lower the LD50 value, the more toxic the substance. For any route of administration (oral, intraperitoneal, intravenous), the LD50 of citicoline is higher than the corresponding LD50 of choline, indicating that citicoline is much less toxic than choline. This difference is certainly not unexpected when we consider that the molecular weight of choline moiety (MW = 104) contributes less than 30% to the molecular weight of citicoline (MW = 489), whereas the acute toxicity of cytidine is probably lower than that of choline. However, when we express the aforementioned LD50 values on a molar basis, citicoline is still substantially less toxic than choline. The difference in molar toxicity between citicoline and choline is more than 20-fold when the substances are applied intravenously. Apparently intact citicoline molecules do not evoke acute cholinergic toxicity, probably because they are not substrates for acetylcholine synthesis.

When the compounds are given per os, the difference in toxicity is several times lower, but it still is quite significant. Two possible explanations can be proposed for the aforementioned differences. One could be that when cytidine appears in blood concomitantly with choline, it somehow attenuates acute choline toxicity. The other, which seems more plausible, could be that upon oral application choline is not liberated from citicoline in the intestinal lumen, preventing its conversion to TMA. Compared with phosphatidylcholine and other choline derivatives encountered in food (e.g., carnitine, glycerophosphocholine), citicoline may be less prone to enzymatic hydrolysis inside the intestinal lumen because it is the only compound containing pyrophosphate group (it should, however, be noted that according to one study [32], the distribution of radioactivity in tissues, urine, and expired air following oral and intravenous administration of methyl-14C-labeled citicoline in rats showed metabolic differences which suggested that the compound is, at least partially, metabolized to TMA prior to its gastrointestinal absorption).

4. Does Resistance to Hydrolysis in the Intestine Make Citicoline a Safer Choline Supplement?

The issue of hypothetical citicoline resistance to intraintestinal hydrolysis is of importance when we consider that the intestinal microbiome metabolizes a significant fraction of choline and its derivatives to trimethylamine (TMA), a gaseous metabolite readily taken up and oxidized in the liver to its N-oxide, TMAO.

TMAO has been implicated in the etiology of various diseases, such as kidney failure, diabetes, and cancer [33]. There is a large and growing amount of literature on the atherogenicity of TMAO resulting in increased incidence of myocardial infarction, stroke, or death [34]. A meta-analysis published recently led to the conclusion that higher plasma TMAO correlates with a 23% increase in risk for cardiovascular events and a 55% increase in all-cause mortality [35]. Two recent reports showed that higher TMAO levels were associated with increased risk of first ischemic stroke and worse neurological deficit [36], and that patients suffering from atrial fibrillation who developed cardiogenic stroke displayed approx. 4 times higher TMAO levels in plasma than patients with atrial fibrillation who did not develop stroke [37]. Another recent report suggested a link between TMAO and Alzheimer&#;s disease [38]. It has even been suggested that supplementation with choline esters prone to be metabolized to TMA and TMAO, such as phosphatidylcholine, may be dangerous to human health [39].

On the other hand, several observations cast doubt on the pivotal role of TMAO in atherosclerosis. First of all, nutritional intakes of TMAO and its precursors do not always correlate with cardiovascular disease risk. For example, high fish intake increases TMA/TMAO while being cardioprotective. Some hypotheses have been proposed recently to resolve this paradox, employing inter alia a phenomenon of reverse causality, a possible role of insulin resistance and diabetes mellitus in activating N-oxidation of TMA, etc. [40].

Nonetheless, many authors still take it as having been proven that TMAO is a causative factor in the development of atherosclerosis and cardiovascular diseases. For example, in a recent review on TMAO and stroke [41], several reports are quoted that show the importance of TMAO as a risk factor and prognostic marker for this disease, and indicate the pathomechanisms involved. These include increased TMAO generation promoting atherosclerosis, platelet activation, and inflammation. The author concludes that TMAO may be a central molecule in the re­lationship of diet, genetics, the gut microbiota, and cardiovascular disease.

It may be concluded that until the place of TMAO in the chain of events leading to cardiovascular diseases and mortality is ultimately clarified, citicoline could be a more reasonable choice than other choline compounds, when choline supplementation is indicated.

5. Citicoline: A &#;Procognitive&#; Form of Choline

In two population studies, significant associations were found between choline intake or free choline level in blood and the cognitive performance of adult and elderly people. In a community-based population of non-demented individuals ( subjects, mean age 60.9 years), higher concurrent choline intake was related to better cognitive performance [42] ( ). In another cross-sectional study ( subjects aged 70&#;74 years), low plasma free choline concentrations were associated with poor cognitive performance [43]. A possible explanation for the effect of choline intake on cognition in adults has been sought in its function as a precursor of phosphatidylcholine (PC), a major constituent of all biological membranes, and acetylcholine, a neurotransmitter involved in cognition [44].

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Therefore, it might be expected that supplementation with choline will improve cognitive performance. However, trials in which the effects of oral supplementation of humans with choline or phosphatidylcholine on cognition were investigated yielded mixed, mostly negative results (see [45] and references cited therein). On the other hand, in a recent small placebo-controlled study, adolescent males treated with citicoline showed improved attention and psychomotor speed and reduced impulsivity [46]. In other recent controlled studies, citicoline seemed to be efficacious in adult patients suffering from cognitive impairments, especially of vascular origin [47]. These newer studies corroborated results obtained previously when citicoline as a prescription drug had been tested in several placebo-controlled trials for cognitive impairment due to chronic cerebral disorders in the elderly. The review of those early trials led to the conclusion that there was some evidence of a positive effect of citicoline on memory and behavior in at least the short to medium term [48]. Moreover, it was recently shown that in patients suffering from dementia concomitant oral intake of citicoline improved the efficacy of cholinesterase inhibitors [49,50].

6. Conclusions

Altogether, whereas the jury may still be out on the issue whether, or to what extent, citicoline taken orally is metabolized to TMA and TMAO, there are reasons to believe that procognitive effects of citicoline supplementation are superior over those of choline or phosphatidylcholine.

Author Contributions

P.G. and K.S. wrote the paper.

Funding

This research received no external funding.

Conflicts of Interest

The authors declare no conflict of interest.

Front Endocrinol (Lausanne).

; 14: .

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Choline supplements: An update

1 , &#; , 2 , &#; , 1 , 3 , 1 , 4 , * and 1 , 2 , 5

and

Urna Kansakar

1 Department of Medicine, Division of Cardiology, Einstein Institute for Aging Research, Montefiore Health System, New York, NY, United States

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Valentina Trimarco

2 University of Naples &#;Federico II&#;, Naples, Italy

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Pasquale Mone

1 Department of Medicine, Division of Cardiology, Einstein Institute for Aging Research, Montefiore Health System, New York, NY, United States

3 ASL Avellino, Montefiore Health System, New York, NY, United States

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Fahimeh Varzideh

1 Department of Medicine, Division of Cardiology, Einstein Institute for Aging Research, Montefiore Health System, New York, NY, United States

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Angela Lombardi

4 Department of Microbiology and Immunology, Montefiore Health System, New York, NY, United States

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Gaetano Santulli

1 Department of Medicine, Division of Cardiology, Einstein Institute for Aging Research, Montefiore Health System, New York, NY, United States

2 University of Naples &#;Federico II&#;, Naples, Italy

5 Department of Molecular Pharmacology, Einstein-Sinai Diabetes Research Center (ES-DRC), Montefiore Health System, New York, NY, United States

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Author information Article notes Copyright and License information PMC Disclaimer

1 Department of Medicine, Division of Cardiology, Einstein Institute for Aging Research, Montefiore Health System, New York, NY, United States

2 University of Naples &#;Federico II&#;, Naples, Italy

3 ASL Avellino, Montefiore Health System, New York, NY, United States

4 Department of Microbiology and Immunology, Montefiore Health System, New York, NY, United States

5 Department of Molecular Pharmacology, Einstein-Sinai Diabetes Research Center (ES-DRC), Montefiore Health System, New York, NY, United States

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Corresponding author.

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Edited by: Sergio Davinelli, University of Molise, Italy

Reviewed by: Paola Di Pietro, University of Salerno, Italy; Mario Cioce, Campus Bio-Medico University, Italy; Alfonso Baldi, University of Campania Luigi Vanvitelli, Italy

*Correspondence: Angela Lombardi,

&#;These authors have contributed equally to this work

This article was submitted to Cardiovascular Endocrinology, a section of the journal Frontiers in Endocrinology

Copyright © Kansakar, Trimarco, Mone, Varzideh, Lombardi and Santulli

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Abstract

In this comprehensive review, we examine the main preclinical and clinical investigations assessing the effects of different forms of choline supplementation currently available, including choline alfoscerate (C8H20NO6P), also known as alpha-glycerophosphocholine (α-GPC, or GPC), choline bitartrate, lecithin, and citicoline, which are cholinergic compounds and precursors of acetylcholine. Extensively used as food supplements, they have been shown to represent an effective strategy for boosting memory and enhancing cognitive function.

Keywords:

choline, choline alfoscerate, choline bitartrate, choline supplementation, cognitive dysfunction, GPC, lecithin, supplements

Introduction

Choline is an important nutrient essential for proper functioning of liver, muscle, and brain (1&#;5). It is a main constituent of cell and organelle membranes and plays a vital role in numerous physiological processes including signal transduction, DNA and histone methylation, and nerve myelination (6, 7). Choline is a precursor of different metabolites including the neurotransmitter acetylcholine (ACh), the membrane phospholipids phosphatidylcholine (PC) and sphingomyelin, and the methyl donor betaine.

Choline can be obtained from the diet and via de novo biosynthesis from the methylation of phosphatidylethanolamine (PE) to PC (6, 8). The demand for choline increases particularly during pregnancy inasmuch as it is important for placental function, fetal growth, and brain development (7). Choline deficiency can cause serious medical conditions such as premature birth, cystic fibrosis, and hepato-steatosis. Therefore, a sufficient choline intake is necessary for growth and homeostasis.

The US Food and Drug Administration (FDA) identified choline as an essential nutrient in . The National Academy of Medicine (NAM) of the USA and the European Food Safety Authority (EFSA) both specified adequate intake (AI) values for choline. Of course, age, sex, life conditions (pregnancy, breastfeeding), and genetic polymorphisms represent central factors in determining AI (9). In , the European Food Safety Authority (EFSA) set an AI of 400 mg/day for all healthy adults. Similarly, the AIs for pregnant and lactating women are 480 mg/day for and 520 mg/day respectively. The US Institute of Medicine (IOM) has a slightly different choline AIs set for nonpregnant, pregnant, and lactating women: 425 mg/day, 450 mg/day, and 550 mg/day, respectively. Low AIs were set for infants of various ages: AI recommendations for infants 0&#;6 months are 125 mg choline/day whereas for infants 7&#;12 months are 150 mg choline/day. These AI values are set according to choline concentrations in human milk (160 mg/L) and estimated average volume of human milk intake (0.78 L/day) for a whole group of infants (aged 0&#;6 months) with a default body weight of 7 kg (approximately 18 mg/kg), and extrapolation for default body weight (aged 7-12 months) (10, 11). Plasma choline concentrations are three times higher in newborn infants than in their mothers as human milk is rich in choline (12&#;16).

Choline alfoscerate

In addition to choline intake from food, there are several forms of choline supplementation currently available (2). Choline alfoscerate (C8H20NO6P), also known as alpha-glycerophosphocholine (α-GPC, or GPC), is a cholinergic compound and ACh precursor extensively used as a food supplement. Its molecular weight is 257.22 g/mol. GPC is considered one of the most used sources of choline due to its high choline content (41% of choline by weight) and its ability to cross the blood-brain barrier. The content of choline and GPC in common foods is reported in .

Table 1

NDB No.DescriptionFree CholineGPC Fish, steelhead trout, dried, flesh (Shoshone Bannock)15..0 Fish, salmon, red (sockeye), smoked (Alaska Native)46..0 Fish salmon, king (chinook), raw (Alaska Native)20.050.0 Fish, salmon, sockeye (red), raw (Alaska Native)20.053.0 Fish, sheefish, dried (Alaska Native)12.074.0 Seal, bearded (oogruk), meat, air-dried (Alaska Native)17.052.0 Fish, salmon, chum, raw (Alaska Native)23.041.0 Fish, salmon, Atlantic, farmed, cooked, dry heat7.841.0 Fish, salmon, Atlantic, farmed, raw9.943.0 Candies, milk chocolate9.122.0 Milk, reduced fat fluid, 2% milk fat, with added vitamin A2.810.0 Yogurt, plain, low fat, 12 g protein per 8 oz2.39.1 Cereals, Quaker, Oat Bran, Quaker/Mother&#;s Oat Bran, dry4.433.0 Candies, milk chocolate pieces, sugar coated9.622 Frozen yogurts, vanilla, fat free3.713.0 Leavening agents, yeast, baker&#;s, active dry6.116.0 Crackers, cheese, sandwich-type with cheese filling6.715.0 Cake, snack cakes, cupcakes, chocolate, with frosting, low- fat5.010.0 Cheese food, pasteurized process, American, without sodium phosphate7.914.0 Candies, milk chocolate coated wafer bars7.916.0Open in a separate window

After oral administration, GPC can be readily metabolized to PC, the active form of choline that is able to increase the release of the neurotransmitter ACh (17, 18) and brain-derived neurotrophic factor (BDNF) (19, 20). GPC enhances memory and cognitive function and is well-known to be effective in the treatment of several neurodegenerative and vascular diseases such as Alzheimer&#;s disease and dementia (21&#;23). GPC has been shown to be more effective when combined with cholinesterase inhibitors (24, 25). Numerous studies have identified the favorable effects of GPC in the treatment of the sequelae of cerebrovascular accidents (26&#;28).

Nevertheless, GPC can be a friend or a foe depending on the doses and length of its administration. Uncovering a safe therapeutic window is essential to prevent adverse reactions.

Preclinical studies

GPC has been shown to exhibit a favorable action in experimental models of the aging brain as well as in a rat model of pilocarpine-induced seizure (29, 30), and to promote neuronal differentiation in a rat model of noise-restraint stress (29). In vitro assays performed in the SH-SY5Y human cell line have revealed that this cholinergic compound antagonizes neurotoxicity triggered by the fragment Aβ ( 25&#;35) of the Alzheimer&#;s amyloid β-peptide and attenuates the Aβ-induced phosphorylation of the Tau protein (31), by sustaining the expression level of synaptic vesicle proteins, such as synaptophysin (32&#;34). GPC was also shown to increase hippocampal neurogenesis, providing protection against seizure-induced neuronal death and cognitive impairment (26) and to antagonize scopolamine-induced amnesia enhancing hippocampal cholinergic transmission, suggesting that the behavioral effects of GPC could be related to its property to increase hippocampal synthesis and release of ACh (35&#;38).

Although GPC does not seem to be directly involved in the modulation of inflammatory responses (39), it has been shown to improve mitochondrial function and to reduce oxidative and nitrosative stress (40).

Chronic treatment of aged rats with GPC restored the number of muscarinic M1 receptors to levels found in the striatum and hippocampus from young animals (41). In young but not old rats, GPC significantly potentiated K+-stimulated intra-synaptosomal Ca2+ oscillations in purified synaptosomes derived from the hippocampus (17). Repeated injections of GPC significantly increased basal formation of [3H]inositol monophosphate in hippocampal, cortical, and striatal slices of male rats (42). Consistently, GPC potentiated receptor-stimulated phosphatidylinositol hydrolysis in cortical synapto-neurosomes (17).

In a model of acute cerebral ischemia in rats, GPC increased the tolerance of neurons to ischemic damage and slowed the execution of the cell death program (43). Consistent with these findings, in vitro assays in astroglial cell cultures have shown that GPC increases proliferation (44).

Clinical investigations

Cholinergic precursors have represented one of the first approaches attempting to relief cognitive impairment in dementia-related disorders. However, controlled clinical trials failed to show significant improvements with choline or PC, choline-containing phospholipids, alone or in association with cholinesterase inhibitors (tacrine plus choline, or physostigmine plus choline) (44, 45). Luckily, the lack of clinical benefits obtained with choline or lecithin are not shared by other phospholipids involved in choline biosynthetic pathways, including GPC and citicoline (cytidine 5&#;-diphosphocholine, also known as CDP-choline), which are able to increase ACh content and release (44, 46).

A study in male young adults demonstrated that the ingestion of mg GPC significantly increases plasma free choline levels (47). Numerous clinical reports suggest that GPC can improve memory and attention in patients with Alzheimer&#;s disease and dementia (26, 36, 48&#;54)

GPC advances physical and psychomotor performance in the context of muscle strength and conditioning (55&#;58). For instance, in a group of 13 college-aged male subjects, the administration of 600 mg GPC resulted in an increase of 98.8 N during an isometric mid-thigh pull assessment (55). Similarly, maximum velocity and maximum mechanical power were improved by the administration of 250 mg GPC (56) and nutritional supplements containing 300 mg or 150 mg GPC were shown to improve reaction time and vertical jump power (59), indicating the ergogenic properties of GPC.

The effects of GPC on cerebrovascular events remain controversial. Indeed, some investigators have conducted a multicenter clinical trial (daily intramuscular dose of mg for 28 days and oral dose of 800 mg during the following 5 months) that revealed the excellent tolerability and the therapeutic role of GPC on cognitive recovery of patients with acute stroke or transient ischemic attack (TIA) (18); on the other hand, a recent retrospective study has shown that GPC is associated with a higher 10-year incident stroke risk in a dose-response manner after adjusting for traditional cerebrovascular risk factors (60). A potential explanation for these different findings could be the diverse effects of GPC supplementation on the gut microbial community structure: in this sense, a recent preclinical report demonstrated that GPC can cause a shift in the murine microbiota, characterized by increased abundance of Bacteroides, Parabacteroides, and Ruminococcus, and decreased abundance of Lactobacillus, Akkermansia, and Roseburia (61).

Most recently, in a prospective study, GPC was suggested to enrich listening comprehension in older adults using hearing aids (62). Due to its action on the parasympathetic nervous system, GPC has also shown beneficial effects in patients with dry eye (keratoconjunctivitis sicca) and its combination with D-Panthenol accelerated and modulated the repair of the corneal innervation after cataract surgery (63&#;66).

Other forms of choline supplementation

In addition to GPC, other supplements are available to ensure an adequate intake of choline ( ). One of the most used is choline bitartrate, which has shown favorable effects both in preclinical and clinical studies, especially in terms of improved cognitive function (67&#;73).

Table 2

GPCCholine bitartrateLecithinCiticoline IUPAC Name [(2R)-2,3-dihydroxypropyl] 2-(trimethylazaniumyl)-ethyl phosphate(2-hydroxyethyl)-trimethylazanium (2R,3R)-3-carboxy-2,3-dihydroxypropanoate[(2R)-3-hexadecanoyloxy-2-[(9E,12E)-octadeca-9,12-dienoyl]oxypropyl] 2-(trimethylazaniumyl)-ethyl-phosphate[[(2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] 2-(trimethylazaniumyl)-ethyl phosphate Molecular Formula C8H20NO6PC9H19NO7 C42H80NO8PC14H26N4O11P2 Molecular Weight (g/mol) 257....32 Color/Form SolidWhite crystalline powderYellow-brownish powderWhite crystalline powder Odor OdorlessOdorless or faint trimethylamine-like odorOdorless or has nut-like smellHigh doses can cause fishy odor Taste No tasteAcidic tasteNutty tasteNeutral Melting Point 142.5°C149-153°C236.1°C240-242°C Solubility Very soluble in waterFreely soluble in water; slightly soluble in alcohol; insoluble in ether, chloroform, and benzeneLow solubility in water, but serves as an excellent emulsifierVery soluble in waterOpen in a separate window

Importantly, a prospective randomized cross-over study was designed to compare four different choline supplements in terms of their impact on plasma concentration and kinetics of choline; participants received a single dose of 550 mg/d choline equivalent in the form of choline chloride, GPC, egg-PC, and choline bitartrate, in randomized sequence at least 1 week apart; the analysis of these revealed no difference in the area-under-curve of choline plasma concentrations after intake of the different supplements (74).

The main clinical trials assessing the effects of choline supplementation, in different formulations, are reported in .

Table 3

TrialSupplementationSubjectsResultsRef.Randomized cross-over study (DRKS)choline chloride, choline bitartrate, GPC, egg-PC6 healthy adult men-All supplements promptly raised choline and betaine levels to a similar extent, with egg-PC showing the latest peak. Considering TMAO may have unfavorable effects, egg-PC might be the best choline supplementation in adults.(74)Randomized double-blind placebo-controlled parallel clinical trial
(IRCTN25)500 mg/d choline and 500 mg/d magnesium co-supplementation96 patients with type 2 diabetes mellitus-Combination of choline and magnesium intake have better outcomes in improving endothelial dysfunction and inflammation as compared to single supplementation alone(75)Randomized partially blinded single-center trial
(

{"type":"clinical-trial","attrs":{"text":"NCT","term_id":"NCT"}}

NCT)enteral choline (30 mg/kg/day), DHA (60 mg/kg/day), or both24 inborn preterm infants&#;<&#;32-week postmenstrual age-Co-supplementation may enhance DHA utilization. However, choline supplementation did not increase trimethylamine-N-oxide (TMAO) levels(76)Clinical open multicenter trial mg i.m. for 28 days and orally at the dose of 400 mg t.i.d. during the following 5 months after the first phase patients suffering from recent stroke or transient ischemic attacks-Excellent tolerability and therapeutic role of GPC on cognitive recovery of patients with acute stroke or transient ischemic attack(18)Randomized, double-blind, controlled feeding study (NCT-)480 or 930 mg choline/d29 women (&#;21y) entering their 3rd trimester of pregnancy, 24 eligible infants-Infants with higher maternal choline intake demonstrated high information processing speed which lasted for at least the first year of postnatal life(77)Single-center, randomized, double-blind, parallel-group study (

{"type":"clinical-trial","attrs":{"text":"NCT","term_id":"NCT"}}

NCT)550 mg choline/dHealthy pregnant person in their second trimester (21-40y)-Maternal plasma choline metabolome (especially betaine) is very receptive to prenatal choline supplementation(78)Randomized, double-blind, placebo-controlled trial (PACTR)2 g of choline/d52 infants born to heavy-drinking women who consumed choline supplementation during pregnancy-Gestational choline supplementation alleviates alcohol exposure effects on neonatal brain volumes, choline may be neuroprotective against brain structural deficits related to prenatal alcohol exposure(79)Single-center, randomized, double-blind, parallel-group study (

{"type":"clinical-trial","attrs":{"text":"NCT","term_id":"NCT"}}

NCT)500 mg/d choline and 200 mg docosahexaenoic acid30 pregnant women-Prenatal choline supplementation (administered across the second and third trimesters of pregnancy) improved hepatic export of docosahexaenoic acid(80)Randomized, double&#;blind, parallel&#;group controlled trial (

{"type":"clinical-trial","attrs":{"text":"NCT","term_id":"NCT"}}

NCT)480 or 930 mg choline/dChildren born to women during their 3rd trimester of pregnancy-Prenatal choline supplementation enhances child sustained attention (7-year follow up)(81)Randomized, double&#;blind, parallel&#;group controlled trial (

{"type":"clinical-trial","attrs":{"text":"NCT","term_id":"NCT"}}

NCT)480 or 930 mg choline/d26 healthy third-trimester pregnant women-Maternal choline supplementation modulates biomarkers of vitamin B12 status in pregnancy(82)Randomized, Double-Blind, Placebo-Controlled Clinical Trial (

{"type":"clinical-trial","attrs":{"text":"NCT","term_id":"NCT"}}

NCT)500 mg/d citicoline100 healthy men and women aged between 50 and 85y with age-associated memory impairment-Regular consumption of citicoline improved attention and may be beneficial against memory loss due to aging(83)Randomized double-blind, placebo-controlled trial (

{"type":"clinical-trial","attrs":{"text":"NCT","term_id":"NCT"}}

NCT)20 g of lecithin60 women having open gynecological surgery-No analgesic benefit with oral choline supplementation between groups at rest or with movement.(84)Randomized controlled trial500 mg and 250 mg GPC48 healthy college-aged males-Increased maximum velocity and maximum mechanical power(56)Double-blind, placebo-controlled crossover600 mg GPC13 healthy college-aged males-Enhanced strength and performance especially the lower body force production(55)Randomized double-blind Placebo-controlled clinical trial (

{"type":"clinical-trial","attrs":{"text":"NCT","term_id":"NCT"}}

NCT)5.25 ml of liquid GPC (~ mg GPC), equivalent to 625 mg of choline5-10y children with FASD-General neurocognitive processes such as memory and attention, executive functioning, and hyperactivity pre- and post-intervention were not enhanced questioning the therapeutic window of choline for its efficacy(85)Randomized, double-blind, placebo-controlled trial (

{"type":"clinical-trial","attrs":{"text":"NCT","term_id":"NCT"}}

NCT)500 mg/d choline bitartrate18 children aged 2.5-5y with FASD (after 7-year follow-up)-Improved processing speed of lower-order executive tasks and better corpus callosum white matter microstructure and neurocognitive outcomes.(86)Randomized, controlled cross-over clinical trial (

{"type":"clinical-trial","attrs":{"text":"NCT","term_id":"NCT"}}

NCT)Three eggs/d, 400 mg/d choline as choline bitartrate23 men and women aged 35-70y with metabolic syndrome-Plasma lutein and zeaxanthin were increased but plasma TMAO did not elevate eggs intake or choline bitartrate supplementation for 4 weeks
-no significant effects on gut microbiota(87)Randomized, double-blind, placebo-controlled intervention trial (ISRCTN)1 g choline per day as choline bitartrate42 healthy postmenopausal women aged 49-71y-Choline supplementation in postmenopausal women increases circulating free choline as well as methyl donor betaine(88)Placebo-controlled double-blind study2&#;g of choline bitartrate30 healthy individuals-Enhanced visuomotor performance(70)Open in a separate window

Choline bitartrate

Choline bitartrate (C9H19NO7) is a white crystalline powder with no odor. Its molecular weight is 253.25 g/mol with 41.1% choline (104 g/mol choline in 253.25 g/mol choline bitartrate); 2&#;g of choline bitartrate administration provides 800&#;mg of choline action (70). It is freely soluble in water, slightly soluble in alcohol and insoluble in ether, chloroform, and benzene.

Choline bitartrate is widely used in dietary supplements. One of the main advantage of bitartrate is its lower hygroscopicity (89), a feature that in the last years has triggered an increase of its use. The methyl donor betaine, a choline derivative, has been shown to facilitate the cytosolic re-methylation of homocysteine to methionine in a reaction catalyzed by the enzyme betaine-homocysteine S-methyltransferase (BHMT). The same reaction is also catalyzed by the methionine synthase, which uses methyl-cobalamin as a co-factor and is a vitamin B12 dependent enzyme (82, 90). Preclinical studies have reported a choline-sparing effect of vitamin B12 supplementation (91&#;93) and patients deficient in vitamin B12 have lower blood concentrations of choline (94). These aspects provide a strong rationale for the preparation of formulations in which choline, especially choline bitartrate, is associated with vitamin B12.

Lecithin

Lecithin is a mixture of fats and can be obtained from food such as egg yolks (actually, the term lecithin derives from the Greek word λέκιθος, lekythos, which means &#;egg yolk&#;), soybeans, and nuts (95, 96). PC represents one of the main components of lecithin, albeit the two terms are sometimes used interchangeably. Lecithin is essential to cells in the human body. Since lecithin is converted into ACh, its consumption increases ACh concentrations in the brain (97). Several studies have been carried out showing the effects of consumption of lecithin on hypercholesterolemia and cardiovascular disorders (98, 99).

Citicoline

Citicoline is a brain chemical that occurs naturally in the cells, especially organs, of human and animals. It is a natural precursor of phospholipid synthesis, chiefly PC, and serves as a source of choline in the metabolic pathways for biosynthesis of ACh in the body (100). Citicoline enhances cerebral metabolism and has neuroprotective properties in animals and humans (101&#;103). Citicoline is effective in facilitating cognitive improvement in various conditions, including vascular and degenerative dementias, cerebrovascular diseases, amyotrophic lateral sclerosis, Alzheimer&#;s disease, and also Parkinson&#;s disease (104, 105); indeed, citicoline increases brain dopamine levels and may inhibit dopamine reuptake (104).

Choline supplementation and endothelial dysfunction

Endothelial cells play a crucial role in the exchange of choline and other nutrients between plasma and brain tissue (75, 106&#;108). Thus, choline must be incorporated into endothelial cells to be transported to the blood-brain barrier (109). Choline supplementation was shown to be effective against hypoxia-induced endothelial dysfunction by Zhang and co-workers, who demonstrated that choline enhanced rat aortic endothelial cell proliferation during hypoxia by secreting vascular endothelial growth factor (VEGF) (6). Moreover, choline supplementation activated the α7 non-neuronal nicotinic ACh receptors (nAChRs) and served as a key function in regulating blood vessels. Thus, choline can be protective against hypoxia-induced endothelial dysfunction (6, 110). Although the benefits of choline have been reported, the exact mechanisms in protecting endothelial function are yet to be fully defined.

Some investigators have reported that endothelial dysfunction is linked with various cardiovascular diseases (111, 112). Several studies have demonstrated the role of high choline intake and its metabolite trimethylamine N&#;oxide (TMAO) in endothelial dysfunction and atherosclerosis (111, 113&#;117). Instead, phloretin, a flavonoid extracted from apple leaves, plays a protective role, and improves vascular endothelial dysfunction and liver injury (111).

Models of endothelial dysfunction like hypoxia or oxygen and glucose deprivation (OGD) were used to evaluate the effects of citicoline on human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (bEnd.3s) (105, 118&#;120). Citicoline attenuated the hypoxia/OGD-induced increase in endothelial permeability via upregulating the expression of tight junction proteins including zonula occludens-1, occludin, and claudin-5. Thus, citicoline could be an efficient therapeutic drug for targeting diseases characterized by endothelial barrier breakdown (105).

Choline supplementation and cardio-metabolic disorders

Choline plays a protective role in the heart and may be a promising candidate to improve doxorubicin-induced cardiotoxicity via vagal activity and Nrf2/HO-1 pathway (121). Moreover, choline exhibits protective effects against cardiovascular disorders, including arrhythmias, cardiac hypertrophy, and ischemia/reperfusion (I/R)-induced vascular injury by inhibiting the ROS-mediated Ca2+/calmodulin-dependent protein kinase II pathway (122&#;124). Citicoline acts as a myocardial protector from I/R injury via inhibiting mitochondrial permeability transition (125). Choline was also shown to ameliorate cardiovascular damage by slowing the progression of hypertension and enhancing cardiac function in spontaneously hypertensive rats (126).

Low amounts of choline can reduce cardiovascular risks and inflammatory markers as they have lowering effect on plasma homocysteine (127). In contrast, a choline&#; or carnitine&#;rich diet was reported to promote atherosclerosis in mice as it increased the formation of TMAO produced by gut microbiota-related metabolite of choline (128). Similarly, other papers have reported the association of TMAO with an increased risk of cardiovascular disease and mortality (60, 113, 129&#;131). Dietary lecithin has shown favorable results with potential application in the treatment of dyslipidemia associated with metabolic disorders (132). Obesity is linked with several cardio-metabolic chronic diseases, such as non-alcoholic fatty acid liver disease (NAFLD), type-2 diabetes, and cardiovascular disease. Numerous studies have also investigated the beneficial effects of lecithin on obesity-related dyslipidemia (132&#;135). Lecithin-rich diets have hypocholesterolemic effects and display anti-atherogenic properties (136).

Intake of choline and betaine co-supplementation was not associated to cancer or cardiovascular disease; however, an adverse cardiovascular risk factor profile was linked with high choline and low betaine levels in plasma. Therefore, choline and betaine demonstrated opposite relationships with major components of metabolic syndrome (92). Choline and betaine supplementation has not been extensively studied in clinical trials for treating obesity and maintaining normal systemic metabolism. Notwithstanding, Sivanesan and co-workers revealed that choline and betaine administration is favorable for obese and insulin resistant Pcyt2+/- mice; they suggested that choline and betaine supplementations could be beneficial for the treatment of obesity and diabetes due to their participation in mitochondrial oxidative phosphorylation (137).

Choline supplementation and cognitive dysfunction

Environmental factors may contribute to the pathological progression of neurodegenerative diseases and epilepsy. Remarkably, dietary nutrients play an important part in facilitating mechanisms related to brain function (138). As mentioned above, ACh receptors orchestrate the immune response in the central nervous system, and their dysregulation plays a part in the pathogenesis of Alzheimer&#;s disease (139&#;144). In fact, Velasquez and collaborators demonstrated that a lifelong choline supplementation may have beneficial cognitive effects such as decreasing amyloid-β plaque load and improving spatial memory in the APP/PS1 mouse model of Alzheimer&#;s disease. Moreover, consumption of healthy diet throughout life may reduce Alzheimer&#;s disease pathology (139). In another paper, the same group reported that maternal choline supplementation has profound benefits in Alzheimer&#;s disease pathology by reducing brain homocysteine levels across multiple generations (145). Several studies have been carried out to investigate the impact of choline supplementation on cognitive functioning in the Ts65Dn mouse model of Down syndrome; for instance, perinatal choline supplementation was reported to enhance emotion regulation in Down syndrome (146). Other studies revealed that maternal choline supplementation improves spatial learning, increases adult hippocampal neurogenesis and basal forebrain cholinergic neurons (147, 148). Bottom and colleagues demonstrated that co-supplementation of choline protects against effects of prenatal ethanol exposure in fetal alcohol spectrum disorder (FASD) offspring (149). Increasing the intake of choline may also reduce spatial memory deficits due to the exposure of chemotherapeutic agents such as cyclophosphamide and doxorubicin in cancer patients (150).

Several researchers have tested the effects of high uptake of dietary choline in elderly patients suffering from impaired memory. A cross-sectional study conducted on ~ elderly patients demonstrated that choline intake, defined as the combination of dietary and supplement intake, correlates with cognitive performance (151). Choline supplements in the form of lecithin and choline chloride did not significantly improve memory performance in humans although some papers have reported positive outcomes in cognitive function of animal models (152&#;156). However, other choline supplements such as citicoline, choline bitartrate, and GPC appear to be very promising in the treatment of elderly patients suffering from dementia (49, 52, 54, 157, 158).

Conclusions

In summary, preclinical and clinical investigations have shown that GPC and other forms of choline supplementation have beneficial effects especially in terms of improved endothelial function and cognitive performance. Notwithstanding, further dedicated studies are warranted to compare the different effects of the currently available forms of choline supplementation.

Author contributions

All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

Acknowledgments

The authors thank Dr. Wang for the valuable support.

Funding Statement

The Santulli&#;s Lab is supported in part by the National Institutes of Health (NIH): National Heart, Lung, and Blood Institute (NHLBI: R01-HL, R01-HL, R01-HL, T32-HL), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK: R01-DK, R01-DK), National Center for Advancing Translational Sciences (NCATS: UL1TR-06) to G.S., by the Diabetes Action Research and Education Foundation (to G.S.), and by the Monique Weill-Caulier and Irma T. Hirschl Trusts (to G.S.). U.K. is supported in part by a postdoctoral fellowship of the American Heart Association (AHA-23POST). F.V. is supported in part by a postdoctoral fellowship of the American Heart Association (AHA-22POST).

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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