Trimethylglycine

Trimethylglycine (TMG) is a betaine molecule, and the first betaine to be discovered as a component of beetroot sugars but later found to be a component of the body under standard conditions.^[2] It is simply the glycine molecule with three additional methyl groups added to it, which gives the name of trimethyl- (three methyl groups) glycine.

Due to its origin, the terms trimethylglycine and betaine are commonly used interchangeably despite betaine technically being a category of molecules of which TMG belongs to. For the purpose of this article the terms will be used interchangeably, and if betaine (as a category of molecules) or any specific betaine molecule that is not trimethylglycine is referred to it will be made note of.

Trimethylglycine (TMG) is molecule in the 'betaine' class of molecules. It was the first discovered betaine and the entire category of betaine was named after its initial source (beets). Due to this the molecule is commonly referred to as betaine. (the more technically correct, but less common name for TMG is 'glycine betaine')

The term 'betaine' refers to any molecule with a cationic (positively charged) group not bearing any free hydrogens (in the case of TMG, this is the nitrogenous ammonium group) connected to an anionic (negatively charged) group. TMG has the amino acid glycine as the negatively charged functional group, and on the nitrogen of glycine (which forms the core of the ammonium group) are three methyl groups; hence the name trimethylglycine.

The term 'glycine betaine' is commonly used to refer to dietary TMG, and other betaines include 'proline betaine' (trimethylproline) found in fruits;^[3] and technically trigenolline (found in coffee^[4] and alfalfa^[5]) as well as dimethylsulfoniopropionate (DMSP; mostly in microalgae^[6]) are dietary betaines.

Whereas DMSP is structurally similar to TMG except for a sulfur replacing the nitrogen, trigenolline and proline betaine are both cyclical betaine structures. Other names for trimethylglycine beyond glycine betaine include lycine^[2] (not to be confused with the amino acid L-lysine) and oxyneurine.^[2]

Betaine can be found in the following foods (dry weight unless otherwise specified for solid foods):

Grains

• Wheat bran (13,390µg/g^[2][7])

• Wheat germ (12,410µg/g^[2][7])

• White bread (360-520µg/g^[3])

• Wholemeal bread (670-790µg/g^[3])

• Wholegrain (560-620µg/g^[3])

• Bran cereal (2,300-7,200µg/g) with trace levels in corn flakes^[3]

• Muesli (270-440µg/g^[3])

• Biscuits of wheat (1,900-2,500µg/g), chocolate (160µg/g), or plain (290-430µg/g)^[3]

• Fettucini pasta (1,300-1,400µg/g^[3])

• Instant noodles (990-1,400µg/g^[3])

• Plain crackers (1,000-1,300µg/g^[3]) with trace levels in rice crackers

• Cakes (170-270µg/g^[3])

• Muffin (120-160µg/g^[3])

• Scone (440-500µg/g^[3])

Vegetables

• Spinach (6,000-6,450µg/g^[2][7])

• Beets (1,140-2,970µg/g^[2][7])

• Asparagus (33-45µg/g^[3])

• Potatoes (26-39µg/g^[3])

• Bell peppers including green (24-31µg/g), orange (10-11µg/g), and yellow (10-26µg/g) with limited in red (12µg/g or trace)^[3]

• Trace levels in corn, baked beans, onions, and tomatoes^[3]

Fruits

• Avocadoes (3-35µg/g^[3]), with the best sources of betaines in fruits being proline betaine in mandarins (920µg/g^[3]) and orange juice (700-780µg/g^[3]) with all other fruits essentially devoid of betaines

• Wine (processed from grapes) at an average 10-11mg per liter of wine (range of 21-211µM; no difference between white and red apparent)^[8]

Meat and Alternatives

• Shrimp (2,190µg/g^[2][7])

• Salmon (20-23µg/g^[3])

• Tuna (33-45µg/g^[3])

• Fish (battered product) at 71µg/g^[3]

• Corned beef (76-140µg/g^[3])

• Mutton (62-180µg/g^[3])

• Bacon (49-97µg/g^[3])

• Ham (81-95µg/g^[3])

• Sausage (320µg/g^[3])

• Meat pie (240µg/g^[3])

• Peanut butter (9-10µg/g^[3])

Dairy

• Milk (Homo) at 7-28µg/g^[3]

• Milk (Soy) at 11µg/g or less^[3]

• Brie (Cheese) at 54-67µg/g^[3]

• Blue (Cheese) at less than 1µg/g^[3]

• Edam (Cheese) at 33-35µg/g^[3]

• Feta (Cheese) at less than 1µg/g^[3]

The New Zealand diet appears to have a daily intake of 298+/-4mg of TMG daily, mostly via grain products.^[3]

Betaine is frequently referred to as a zwitterionic molecule (or an inner salt) due to it possessing both a positive and negative charge on different parts of the same molcule; this zwitterionic property does not apply to its parent molecule (choline) which possesses only a positive charge.

Boiling food products results in a significant decrease in betaine concentrations.^[9]

The human body can convert dietary choline into betaine in the liver, initially due to choline being metabolized into betaine aldehyde (via mitochondrial choline oxidase) and then again in the mitochondria by betaine aldehyde dehydrogenase to form betaine.^[10][11] This conversion is largely seen as irreversible, and is the principle metabolic role of choline in the body^[2][12] with the remainder of choline being used towards synthesis of acetylcholine and phospholipids (such as phosphatidylcholine).^[2]

Betaine is the major active metabolite of choline (although choline itself can influence more than just what betaine influences)

The synthesis of Phosphatidylcholine in the human body requires the conversion of three molecules of S-Adenosyl Methionine (SAMe) into S-adenosylhomocysteine^[11] via the PEMT enzyme (phosphatidylethanolamine N-methyltransferase). This appears to be the most important quantitative usage of SAMe in the body and the major contributor of de novo synthesis of homocysteine.^[13]

Betaine is thought to be relevant to this process since, via the betaine-homocysteine methyltransferase (BHMT) enzyme, it reduces homocysteine into methionine which is one of the two pathways homocysteine can be reduced (the other being a folate dependent mechanism);^[11] deficiencies of dietary choline or betaine cause a greater need for dietary folate to compensate.^[14][15] This enzyme is also suppressed in instances of high salt intake^[16] thought to be due to preventing excessive cellular swelling.

Alongside folate, dietary choline (via the metabolite betaine) or betaine itself are able to reduce homocysteine back into methionine and preserve whole-body methyl donation processes.

Similar to some other molecules (such as taurine, creatine, glycerol, and trehalose^[17]) betaine is an osmolyte and positively influences the hydration status of a cell.^[17] Although its synthesis (from choline) is not altered by the tonicity of a cell,^[18] it seems that the uptake of choline into the mitochondria (required to produce betaine) is,^[19] suggesting an intracellular role in osmoregulation.

Similar to other osmolytes, intracellular betaine can influence hydration status by affecting tonicity. It can be moved into or out of a cell to maintain hydration status, and increasing cellular concentrations with supplementation) can increase cell volume.

Betaine is absorbed in the intestines via a sodium and chloride dependent mechanism^[2] mostly in the duodenum (chick studies^[20][21]).

Absorption of betaine appears to be rapid,^[22]wtih an absorption half-life of around 17 minutes following oral ingestion in humans.^[23] Plasma levels peak after 40-60 minutes,^[23][22][24] which is based on circulating concentrations following a single dose peaking in the range of 933-1014μM (from a basal concentration of less than 100μM).

Betaine is taken up in the intestines and released into the blood stream quite fast. Absorption of betaine is near complete.

Under normal dietary fasting conditions betaine concentrations tend to be in the range of 20-70μM (without extra supplementation).^{[25][23][26][2][23]} No significant differences betaine levels are noted in insulin resistant (type II diabetic) patients despite having higher urinary excretion rates.^[23] Betain levels have been found to be in the lower range of normal in persons with elevated fasting homocysteine concentrations.^[23]

Fasting betaine concentrations in serum (independent of supplementation) do not appear to be perturbed in states of insulin resistance of type II diabetes, but appear to be lower when homocysteine is higher.

The absorption of betaine appears to be rapid as, following an acute dose of 50mg/kg, it appears in the blood with an absorption half-life of 17 minutes.^[23] This remained unchanged following repeated dosing.^[23] This dose reached a C_max of 940+/-190μM at a T_max of 54 minutes, and while the T_max remained unchanged following repeated doses the C_max progressively rose, thought to be due to accumulation of betaine.^[23]

Elsewhere, dosing in the range of 1-6g betaine has been noted to have a similar T_max of 40-60 minutes but a dose-dependent C_max increasing serum betaine from a baseline of 47+/-10μM to 284+/-131µM, 599+/-190μM, and 1015+/-231μM.^[22] This has been noted elsewhere in athletes given betaine, where the increase in serum betaine (reaching 933μM) was not different between consumption of betaine with water or with a 6% carbohydrate/electrolyte solution.^[24]

Supplementation of 1,000mg betaine in otherwise healthy persons appears to increase steady state serum from a baseline concentration of 31.4+/-13.6µM to 52.5+/-26.5µM (67%).^[27] In this same population 3,000mg betaine increased serum levels from 31.4+/-13.6µM to 109+/-41µM (247%). 6,000mg betaine increased serum levels from 31.4+/-13.6µM to 255+/-136µM (712%).^[27]

In otherwise healthy humans, serum betaine dose-dependently increases in response to oral supplementation up to 6,000mg. Increased serum betaine levels are gave been noted on an acute both (when measuring the C_max value) and steady state basis.

It seems that tissue levels of betaine are higher than plasma levels in all measured tissues except for skeletal muscle and brain, where they tend to be comparable to and lower than plasma respectively.^[28] Intestines, lungs, the heart, and spleen showed high dependence on and correlation with plasma TMG^[28] whereas other tissues that were less dependent (liver, testes and kidneys) tended to be sites of synthesis and have highest concentrations of betaine.^[28]

Elevations in tissue betaine (in rats) are known to be dietary rather than due to endogenous synthesis^[29] and when rats (already consuming betaine at 0.9% of the diet) are measured betaine has been noted to accumulate mostly in the liver (1.6-9.5mM per liter of tissue water; 3.21-3.22µM/g tissue wet weight), kidneys (2.0-5.4mM; 2.78-3.24µM/g) and testes (2.4-3.4µM; 2.63µM/g) while the brain contained lowest levels of betaine (190µM or less; 0.02-0.04µM/g).^[28] Betaine does not appear to be present in the female reproductive tract when consumed at normal dietary levels in rats.^[28]

Orally ingested betaine causes accumulation in tissues. Betaine appears to be present at higher levels in tissue relative to in plasma for all measured tissues except muscle and brain. Tissues with low synthetic capacities or lower betaine concentrations can more readily accumulate it from plasma.

Betaine is taken up into a cell mostly by the betaine/GABA transporter designated BGT-1^[30] (similar to rat BGT-1, but 97% homologous^[31] to mouse mGAT2.^[32]) It also has two other less specific transporters that it can be taken up by, although these are not expressed in humans, suggesting that BGT-1 is the only betain tranporter relevant for humans.^[33][18]). BGT-1 (SLC6A12^[34]) is in the same receptor superfamily as the creatine transpoters (major one is SLC6A8^[35] and the testicle specific one is SLC6A10^[36]), and similar to the taurine transporter (SLC6A6) which also has low affinity for GABA as well.^[37] BGT-1 doesn't appear to have too much affinity for GABA^[38] and may be predominately a betaine transporter.

BGT-1 appears to be regulated by hypertonicity, designed to take up betaine from outside of a cell when said cell requires the osmolytic properties of betaine.^[30] It should be noted that the other GABA transporters (GAT1-3) are not known to mediate betaine uptake.

Betaine can readily be taken up by a cell from the plasma via the BGT-1 transporter, which is more active in periods of cellular hypertonicity in order to preserve cellular hydration status.

On the intracellular level, it appears that choline synthesis into betaine is controlled by uptake into the mitochondria (where betaine is produced) since the influx into the mitochondria responds to cellular tonicity (specifically, in a hypertonic state^[19]) although synthesis per se is unaffected.^[18]

The transport of choline into mitochondria for betaine synthesis is induced by a hypertonic state in order to preserve the cellular hydration status.

The first stage of choline metabolism (the one stage not highly relevant to betaine supplementation) is the irreversible conversion of choline into betaine via mitochondrial oxidation.^[2][12] Beyond that, metabolism of betaine involves its methyl donation process, since in the process of converting homocysteine into methionine betaine itself is degraded into N,N-dimethylglycine (DMG) via the enzyme betaine homocysteine methyltransferase (BHMT).^[2]

DMG itself can be subject to the dimethylglycine dehydrogenase enzyme, a folate binding protein (protein that can bind folate, which serves as a coenzyme in this process),^[39][40] which converts DMG to N-methylglycine.^[41] N-methylglycine is more commonly referred to as sarcosine, and metabolism of sarcosine via sarcosine dehydrogenase will produce free glycine since the last methyl group is removed.^[42][43] Both of these stages, which lose a methyl group, donate it to the active folate molecule known as tetrahydrofolate which then becomes 5,10-methylenetetrahydrofolate and is itself a methyl donor.^[41]

The general metabolic pathway goes from choline which produces (via an irreversible two step oxidation) betaine, which produces (via methyl donation to homocysteine) DMG followed by producing (removing yet another methyl group) sarcosine and finally (removing the final remaining methyl group) the amino acid glycine.

Both betaine and the newly synthesized 5,10-methylenetetrahydrofolate can convert homocysteine into L-methionine, which is how they both influence homocysteine levels.^[44][41] Specifically, 5,10-methylenetetrahydrofolate is substrate to the MTHFR enzyme to create 5-methyltetrahydrofolate (Levomefolic acid or Metafolin).

5,10-methylenetetrahydrofolate can also donate a methyl group to glycine if need be, to synthesize serine (the racemic amino acid mixture of D-serine) via serine transhydroxymethylase^[45] or it can be oxidized to form 10-formyltetrahydrofolate and support purine synthesis via thymidylate synthase.^[46] Administration of pure 5-methyltetrahydrofolate (Levomefolic acid) circumvents these two options.

The methyl groups 'lost' during the metabolism of DMG into glycine are actually given to the bioactive form of folate known as tetrahydrofolate (THF). THF then goes off to donate these methyl groups to other processes.

The elimination half-life for betaine (50mg/kg oral intake) has been noted to be 14.38+/-7.17 hours after a single dose, and this was significantly increased to 41.17+/-13.50 after repeated dosing.^[23]

There appears to be a very long elimination half-life with betaine, in accordance with its high bodily retention.

Urinary elimination of trimethylglycine (TMG) supplementation tends to be in the form of dimethylglycine (DMG), which is the methylation byproduct of trimethylglycine. Overall urinary excretion appears to be low as it has been measured at around 4% of oral intake (50mg/kg).^[23] In the dosage range of 1,000-6,000mg in otherwise healthy persons when measured over the next 24 hours the urine accounts for 3.2-7.4% of orally ingested betaine (and excreted in the form of betaine, which increased with dose as DMG excretion was fairly stable).^[22]

Both betaine and DMG are found in the urine, and DMG excretion rates do not seem to be responsive to changes in dietary supplementation (perhaps due to a limit on methylation), whereas betaine elimination goes up linearly with supplementation. It seems urinary excretion consists of a small amount of overall excretion when measured acutely, perhaps due to high bodily retention or fecal elimination.

Out of all organs which tend to hyperaccumulate trimethylglycine (TMG) to levels higher than observed in serum (and skeletal muscle which is approximately equal), the brain tends to have nearly undetectable levels of TMG^[28] which may be related to how, at least in mice, the transporter that mediates TMG uptake (BGT-1 in humans and rats, mGAT2 in mice) is present at the blood brain barrier^[31] and in the brain at quite low concentrations (at least relative to other GABA transporters).^[34] While high levels of inhibitors for other GABA transporters (0.5mM) can reduce GABA uptake across the blood brain barrier by around 81.4-89.1%,^[31] high levels of TMG only suppress uptake by 22.2% in vitro.^[31]

mGAT-2 colocalizes with P-glycoprotein (a known efflux protein) at the blood brain barrier^[31] and does have high affinity for GABA,^[47] but due to GABA not readily be taken up from the periphery into the brain (rather, most GABA is synthesized locally^[48][49]) and the low accumulation in the brain relative to serum TMG,^[28] it suggests that this transporter is mostly an efflux transporter (removing GABA from the brain into the periphery) rather than an influx transporter.

Finally, the enzyme of synthesis for TMG from choline known as betaine-homocysteine methyltransferase (BHMT) is not appreciably localized in the brain^[50] and there only appears to be a minor expression of a secondary gene for its transcription;^[51] this suggests minimal local production of TMG from choline, which is in accordance with the measured brain tissue measurements (rats) being 0.02-0.04µM per gram wet weight.^[28]

The transporter to mediate TMG uptake into the brain does exist, but TMG does not accumulate in the brain to a large degree when peripheral (blood not in the brain) levels increase; this may be because the transporter that mediate betaine is thought to be more involved in its efflux rather than influx

Despite the above, the transporter still has a role (astrocytes) in regulating osmotic balance to a degree^[52] although other osmolytes that are known to accumulate in the brain (taurine and creatine, for example) may play a more relevant role than TMG.

The transporter, within the brain, still can have a role in osmoregulation (maintaining hydration status of a cell in response to its environment) although practical significance of this related to oral supplementation of betaine is not known yet unlikely (under normal conditions)

Homocysteine is known to be an agonist of NMDA receptors at the 10-100μM range^[53] which is a concentration seen in some instances of blood brain barrier disturbances (ie. stroke^[54]). It is thought reducing serum homocysteine can alleviate glutaminergic neurotoxicity in these states, but the hypothesis has not been tested.

The BGT-1 transporter appears to be involved in seizures in the medial entorhinal cortex (mEC) as its inhibition reduces the seizure threshold,^[55] but it is unsure what this means for dietary supplementation of trimethylglycine (TMG) due to the brain being nonresponsive to changes in serum TMG.

The betaine transporter has a role in epilepsy and convulsions (in which its inhibition is desired to reduce convulsions), but practical significance to TMG supplementation is not known

The heart tissue of rats appears to accumulate trimethylglycine (TMG) to the concentration of 224-372µM per liter of tissue water (78% water content in the heart) or to the level of 0.19-0.32µM per gram wet weight of the heart at the serum level of 101-186µM; it appears to be highly responsive to changes in serum TMG.^[28]

250mg/kg of TMG daily for a period of 30 days in rats then subject to cardiotoxicity (isoprenaline) was able to greatly attenuate the damage as assessed by preventing changes in LDH (prevented 83.2% of the increase) and CPK (90.6%) while fully preventing the rise in homocysteine;^[56] TMG without cardiotoxicity showed nonsignificant protective effects as assessed by enzyme leakage (indicative of cellular membrane damage)^[56] and has been replicated elsewhere where lysozymal enzymes and lipid peroxidation were normalized to the same degree in heart tissue.^[57]

Preliminary evidence shows a cardioprotective effect, and since it appears to be similar to creatine in a way it is possible this is secondary to the osmolyte properties of TMG; more research is needed to evaluate the mechanisms and see if this is relevant to human ingestion

A meeting abstract (preliminary evidence) has made note that trimethylglycine (TMG) supplementation, at 6g daily in a pilot study of 12 subjects, has increased nitric oxide levels in the blood from 28.8+/-3.4µM to 82.3+/-13.2µM (185% increase) after one week of supplementation.^[58] A later study using a lower dose (2.5g TMG in two divided doses) in otherwise healthy persons failed to replicate any influence on nitric oxide metabolism in otherwise healthy subjects.^[59]

Preliminary evidence suggesting a large increase in nitric oxide has failed to be replicated in otherwise healthy subjects who were exercising; more research is needed to see under what conditions an increase in nitric oxide may occur

Betaine does not actually appear to be correlated with homocysteine in a fasted state (while folate is)^[27] and despite low betaine plus choline intake (ie. low methylation) is thought to promote atherogenesis via reduced methylation of DNA^[60][61] and betaine insufficienct being a risk factor for acute cardiovascular complications in those with metabolic syndrome^[62] typical dietary betaine plus choline intake does not appear to be associated with reduced risk of coronary heart disease.^[63]

A relative deficiency of betaine appears to be a risk factor for acute cardiovascular complications in those with metabolic impairments, but there does not appear to be a dose-dependent protective association seen with betaine when looking at persons not in betaine insufficiency

High doses of betaine (6g or more) have been frequently used for the treatment of homocysteine^[11][64][65] since it appears that patients with high homocysteine levels (hyperhomocysteinemia) due to a genetic defect in homocysteine metabolism only respond to such a high level of intake.^[66][67][68] Homocysteine may work in reducing homocysteine after a single dose^[69] and time-dependently increases in potency up until day five^[27] where it then persists in magnitude for as long as supplementation is continued.

Both folic acid and betaine are known to decrease fasting homocysteine concentrations, with more potency coming from the former^[70][71] although in response to a methionine load betaine is effective whereas folic acid is not.^[72]

High dose betaine supplementation, similar to folic acid (folate), is usually used to lower homocysteine concentrations in pathological conditions characterized by high homocysteine concentrations

In otherwise healthy adults (fasting plasma homocysteine of 8.4-22.2μM), supplemental betaine (1,500-3,000mg daily) is able to reduce plasma homocysteine after six weeks by 12-15% (slightly less than 6,000mg daily which reached 20%) which was of similar magnitude to when homocysteine was measured after two weeks.^[69] Elsewhere, 3,000mg was confirmed to be effective (10% reduction) while 1,000mg failed in otherwise healthy persons (baseline homocysteine of 10.4-13.2μM),^[27] 6,000mg was noted to reduce homocysteine by 8% (pilot study)^[73] and 9% in obese humans,^[74] and one study noted an 11% decrease relative to placebo after six weeks.^[72]

In response to a methionine load in otherwise healthy humans, a single dose of betaine in the range of 1,500-6,000mg is able to reduce the increase in homocysteine by 16-35% which persisted in magnitude when tested again after two and six weeks.^[69] This has been noted to the degree of 40% (AUC) or 49% (increment after six hours) following 6g over six weeks of supplementation, where 800μg of folic acid was ineffective.^[72]

Finally, low doses of betaine (500-800mg) appear to also be able to reduce homocysteine concentrations in otherwise healthy men but only after an L-methionine load;^[75] the dose appears to be too low to influence fasting homocysteine concentrations.^[75]

Betaine supplementation appears to acutely reduce plasma homocysteine in otherwise healthy persons in a dose-dependent manner, and appears to persist in magnitude when measured over the course of six weeks. It is nonsignificantly less potent than the active dose of folic acid at doing this, but appears to be significantly more effective when measuring increases in homocysteine following an L-methionine load

The donation of a methyl group from betaine towards homocysteine results in L-methionine being produced (as L-methionine is methylated homocysteine, and homocysteine is demethylated L-methionine), and an increase in L-methionine is seen with supplementation of 6,000mg of betaine (not 1,500-3,000mg) over six weeks in otherwise healthy humans by 60% (fasting) and 12% (post methionine load).^[69] Betaine also appears to increase L-methionine oxidation as well^[76] due to increasing methionine-mediated methylation.^[76]

As evidence of the direct methyl donation in plasma, betaine supplementation increases plasma L-methionine in accordance with the decrease in plasma homocysteine

Genetically speaking, persons who are homozygotes for the T-allele of methylenetetrahydrofolate reductase (MTHFR) respond more to betaine supplementation, and even in healthy persons without any problems in homocysteine levels in serum the whole-group decrease (10.4-14.2%) is increased when only looking at these persons (15.4-21.9%).^[27]

Trimethylglycine (TMG) supplementation has been noted to, in animal studies (rodent and swine), increase cholesterol relative to control.^[77][78]

Supplementation of betaine at 6g daily for 12 weeks has once been associated with stasis (no change) in total cholesterol and LDL cholesterol levels in obese subjects where the placebo experienced a decline, causing a relative increase.^[74] In this scenario, HDL-C was unaffected in both groups.^[74]

One study conducting three trials on TMG supplementation at 6g daily^[79] noted that there was a significant increase in total cholesterol in otherwise healthy humans (0.36mM: 8%) which was almost solely accounted for by an increase in LDL cholesterol (0.14mM; 11%) detectable within two weeks of supplementation;^[79] this increase in cholesterol was not seen in the trials on folic acid, appears to increase in magnitude from the 1,500-6,000mg dosage range, and did not influence HDL-C production.^[79]

Trimethylglycine (TMG) supplementation is proposed^[79][77] to increase triglyceride levels in serum due to an increased synthesis of vLDL cholesterol and efflux of triglycerides from the liver into peripheral circulation (tissue outside of the liver and not inclusive of the brain), since TMG can contribute to local synthesis of phosphatidylcholine (PC).

PC synthesis in the liver will stimulate vLDL synthesis and efflux per se^[80][81] as PC is a component of vLDL cholesterol, and the process of converting phosphatidylethanolamine into PC is initiated by the BHMT enzyme^[82] and finalized by S-adenosyl methionine which TMG also supports^[83].

Methylation from TMG appears to be able to stimulate phosphatidylcholine synthesis, and it is thought that production of phosphatidylcholine in the liver stimulates vLDL production which then causes efflux of triglycerides from the liver into the blood (good in reducing liver triglycerides, potentially negative for serum triglycerides)

Supplementation of 6g trimethylglycine (TMG) to obese individuals over the course of 12 weeks has failed to significantly influence triglyceride concentrations in serum despite an increase in LDL and total cholesterol (as the decrease seen in placebo was not present with TMG supplementation);^[74] this study did note a nonsignificant trend to increase triglycerides which reached 12% in magnitude relative to placebo.^[74]

One study conducting three trials^[79] each with TMG at 6g daily for six weeks of which measured confirmed an increase of 0.14mM (95% confidence interval of 0.04–0.23mM) or 13% relative to baseline in otherwise healthy subjects, evident within two weeks of supplementation.^[79] This was also seen with dietary supplementation of 2.6g of supplemental phosphatidylcholine (PC) over the course of two weeks, where an 8% increase was noted.^[79]

At this moment in time, it appears that supplementation of the therapeutic dosage of TMG (6g) to reduce homocysteine by around 10-20% is also met with an increase in triglycerides of around 10% in otherwise healthy human subjects

Supplementation of 6g trimethylglycine (TMG) daily for 12 weeks in obese persons has failed to significantly influence the metabolic rate.^[74]

At this moment in time, there does not appear to be an increase in the metabolic rate seen with high doses of TMG supplementation in obese humans

Supplementation of 6g betaine daily for 12 weeks in obese individuals has failed to significantly influence weight loss nor fat mass as assessed by calipers.^[74]

Although preliminary evidence suggested a possible role for trimethylglycine (TMG) supplementation in increasing nitric oxide concentrations in serum^[58] supplementation of 2.5g TMG (via 500mL gatorade) in two divided doses daily for two weeks prior to fasted exercise training in otherwise healthy subjects has failed to modify circulating NOx levels (biomarker for nitric oxide) and failed to influence the perception of a pump,^[59] there was a higher muscle tisssue oxygen saturation (StO₂) before training and lower levels after indicative of more oxygen consumption during training.^[59]

This was hypothesized^[59] to be due to increased cellular swelling with TMG, which is known to protect from urea-induced inactivation of muscle myosin adenosine triphosphatase (ATPase)^[84] and generally protect the cell from stressors (well established with creatine^[85]); this was thought to then enhance the overall work capacity seen (6.5% in the fasted state as assessed by bench press^[59]) to promote more muscle cell metabolism.

One of the hypothesized mechanisms for TMG's ergogenic effects are secondary to cellular swelling and causing a more robust cellular survival in the presence of damaging stimuli such as exercise; this appears to work well with the observations that there is increased muscle oxygen consumption alongside the increased workload, and less biomarkers of damage (lactate) but not alongside an increase in nitric oxide which could be another explanation for increased oxygen consumption

Another possible hypothesis^[86] is that provision of TMG as a methyl donor favors phosphatidylcholine (PC) synthesis (known to require a lot of methylation from TMG^[13]). Similar to PC, methyl donation can promote the synthesis of creatine since S-adenosyl methionine (SAMe) is required to synthesize creatine and SAMe is replenished when TMG methylates homocysteine into L-methionine (although the one trial to investigate phosphocreatine stores in muscle with 2g betaine failed to find any effecti^[87]).

The ability of TMG to act as a methyl donor is thought to possibly play a role vicariously through phosphatidylcholine synthesis, and this exact same role is thought to play another role in promoting the synthesis of creatine

Trimethylglycine (TMG) is detectable in rat skeletal muscle at the concentration of 124-217µM per liter of tissue water (0.10-0.18µM per gram wet weight, due to a 76% water content of skeletal muscle) at the serum level of 101-186µM; it is one of the few organs that does not hyperaccumulate betaine above serum concentrations.^[28]

TMG is found in skeletal muscle, and under normal conditions (standard dietary conditions) it appears to be at levels comparable to that seen in serum

Supplementation of TMG at 1,250mg twice daily (dissolved in gatorade) for two weeks prior to an acute exercise session (in the morning in a fasted state) failed to alter resting Akt and p70S6K phosphorylation, but after exercise Akt phosphorylation dropped in placebo and was maintained in the betaine group (resulting in a significant relative increase).^[88]

Phosphorylation of AMPK, which is reduced during exercise, is not affected by TMG supplementation at the aforementioned dosage of 1,250mg twice daily.^[88]

The Akt/mTOR signalling pathway, involved in protein synthesis, appears to be increased to a greater degree following exercise when the subject consumes TMG supplementation. This was found in a fasted state, and it is not known if it applies to a fed state

In studies using the standard supplemental protocol (1.25g of trimethylglycine (TMG), usually in 250mL of carbohydrate solution like gatorade, to be taken twice daily for two weeks) there has been an increase in total work conducted over the course of a bench press workout (6.5%) despite no significant increase in the volume conducted in any individual set; this study was conducted in a fasted state in resistance trained men while leg exercises saw not benefit.^[59] Similar to that, one study reported a lesser chance in fatigue state during exercise with TMG relative to placebo yet failed to find any whole group differences in fatigue^[86] suggesting minimal practical benefits.

For exercises where near maximal weight was used, one study noted that two weeks supplementation increased the amount of training volume conducted by the legs at 90% average power until fatigue;^[89] exercises at 90% peak power to fatigue saw benefit at week one but became nonsignifcant after two weeks, and the chest was not benefitted^[89] and elsewhere in minimally trained men given TMG there was no benefit to exercises (bench and squat) conducted at 85% of their one rep maximum until fatigue relative to placebo.^[90]

There appears to be an antifatigue effect when looking at the workout as a whole, but since many parameters of fatigue measurement also come back negative and (when fatigue reduction does occur) the magnitude of fatigue reduction is minor and may not be practically relevant

In response to the standard supplemental protocol (1.25g of trimethylglycine (TMG), usually in 250mL of carbohydrate solution like gatorade, to be taken twice daily for two weeks) betaine has failed to increase power output in the bench or leg press exercises in resistance trained men exercising in the fasted state,^[59] no changes in eccentric or concentretic power in weight training (recreationally active men in a nonfasted state),^[86] and no changes to peak power on leg or chest exercises in college aged men at one or two weeks (nor changes in power on a wingate test).^[89] One study conducted in sedentary men given 2g TMG for 10 days either in isolation or alongside 20g of creatine failed to note any power enhancement either inherently with betaine or an enhancement of the creatine-induced increase in power output.^[87]

The two positive tests using the standard protocol (2.5g TMG in two divided doses over two weeks) in minimally active men where an increase in power output occurred as assessed by an isometric (but not jump) squat test and both isometric and bench throw tests^[90] and a study where power output was assessed by sprinting tests on a cycle erogmeter in recreationally active persons (four 12 second sprints against resistance) noted improvements in the average and maximum peak power output (3.4% and 3.8%) and average and maximum mean power output (3.3% and 3.5%) in the TMG group relative to placebo.^[91]

One study has noted an increase in power output in a gym setting and one on a cycle ergometer, while the latter test has failed to be replicated and two other studies in a gym setting have failed to find any significant improvement relative to placebo. At this moment in time possible power enhancement from TMG supplementation seems unreliable

One study has been conducted in trained runners who were dehydrated to 2.7% reduced bodyweight (exercise for 90 minutes in a heated environment) and then were allowed a rehydrating beverage of either a carbohydrate/electrolyte solution (or water) with or without additional trimethylglycine (TMG) at 5g/L until they reached less than 1.4% of a deviation from normal bodyweight, and then they were subject to a performance test.^[24] This study noted a serum level of TMG at the peak levels (933μM) which somewhat similar to supplementation of 50mg/kg (940+/-190μM)^[22] or 6g (1015+/-231μM)^[23] TMG elsewhere.

TMG supplementation alone (relative to water alone) or TMG with carbohydrates (relative to carbohydrate) failed to significantly improve sprinting to volitional fatigue although a nonsignificant trend was evident^[24] and there appeared to be increased whole body oxygen uptake in the carbohydrate plus TMG (relative to carbohydrate) of 5% when measured during the final sprint only.^[24]

There was no significant influence on the rate of percieved exertion of any biomarker of hydration between betaine groups and their respective controls.^[24]

Supplementation of trimethylglycine (TMG) at 1,250mg twice daily (dissolved in gatorade) for two weeks prior to an acute exercise session (in the morning in a fasted state) was noted to be associated with nonsignificantly higher growth hormone (6.1%; P=0.089) while IGF-1 was significantly increased (7.8%) concentrations after exercise.^[88]

May be able to increase IGF-1 in a fasted state, with the increase in growth hormone being very minor (significantly less than arginine and creatine) and only measured acutely rather than over 24 hours; weak evidence to support practically relevant increases in growth hormones

Supplementation of trimethylglycine (TMG) at 1,250mg twice daily (dissolved in gatorade) for two weeks prior to an acute exercise session (in the morning in a fasted state) was noted to be associated with reduced cortisol levels (6.1%) after exercise.^[88]

Minor reductions in cortisol with supplemental TMG when measured around fasted physical training

Compounds that enhance methylation (via SAMe) are thought to be able to augment the anti-viral efficacy of interferon A (IFNα) therapy. This is in part due to how protein phosphatase 2A (PP2Ac; overexpressed in hepatitis C infected cells^[92][93]) is known to negatively regulate the signalling of IFNα^[93] by reducing levels of a protein known as protein arginine methyltransferase 1 (PRMT1) which methylates (positively influences) PIAS1^[94] and STAT1^[95] using SAMe as substrate, and it is though that superloading methyl donating molecules can circumvent this reduction in methylation and preserves IFNα signalling.

At least when looking at in vitro evidence, SAMe can inhibit hepatitis C (HCV) replication^[96] and potentiate and/or preserve IFNα signalling in isolated Huh-7 cells infected with HCV.^[97]

It is thought that hepatitis C viral replications is 'preserved' in part by a mechanism involving reduced methyl donation to a few proteins, resulting in a weakining of interferon alpha (IFNα) signalling and less antiviral effects from IFNα. Supporting methylation appears to preserve the potency of IFNα signalling

The combination of trimethylglycine (TMG; 3g twice daily) paired with S-adenosylmethionine (SAMe; 400mg thrice daily) in addition to standard antiviral therapy (pegylated IFNα and ribavirin) appeared to enhance the early virological response but failed to significantly improve sustained virological response.^[98]

Preliminary evidence supports limited efficacy for TMG supplementation, but is confounded with the inclusion of SAMe supplementation as well

Betaine is thought to be beneficial for dry mouth symptoms due to its inclusion in toothpaste (by itself) appears to reduce dry mouth symptoms at 4% of the toothpaste by weight in subjects with chronic dry mouth (usually Sjögren's syndrome) when used twice daily over two weeks (pilot study) with more potency in those with worse symptoms of dry mouth.^[99] It also seems effective when paired with sodium lauryl sulfate (SLS)^[100][101] but less so than betaine by itself.^[101]

Brushing of the teeth with a betaine containing toothpaste does not appear to significantly alter the microbiotica (bacterial colonies) or the mouth nor the appearance of the mucosa.^[99]

Sodium lauryl sulfate (SLS) is a common detergent in mouthwashes and toothpastes that may irritate the mucosa of the dry mouth, and betaine has been noted to reduce SLS induced mucosal irritation (1.0%)^[102] with a second study failing to note an inhibition of SLS (1.2%) induced irritation^[100] and one of the previous studies noting benefit against 1% also noting a failure against 0.5% and 2.0% SLS concentration.^[102] Another irritant used in some oral cosmetics, cocoamidopropylbetaine or CAPB, at a concentration of 2.0% was not protected against with betaine.^[102]

Betaine by itself in a toothpaste at around 4% concentration appears to alleviate dry mouth symptoms, and while it has the potential to also reduce irritation by SLS (a detergent that is also a mucosal irritant) it is unreliable in its protection and its efficacy against dry mouth may be reduced in the presence of SLS

A mouthwash containing betaine (1.33% betaine solution alongside olive oil and xylitol, the latter at 3.30%) over the course of four weeks in persons with dry mouth induced by medications (xerostomia) when 12mL is used for 30s thrice daily after meals appears to be effective in reducing symptoms as assessed by OHIP‐14 scores where 63% of persons reported improvement from baseline to a small (40%) or moderate (23%) degree;^[103] this has been noted previously within a week of supplementation in an unblinded pilot study,^[104] and other minor constituents of this mouthwash (Vitamin E and B5) may also be active.

Betaine containing mouthwash (1.33%) also appears to reduce symptoms of dry mouth, but at the same time all the studies conducted on this product include other potential confounds which may add to the clinical efficacy

TMG is detectable in the rat kidney at a concentration of 1.6-9.5mM (per liter of tissue water) and 3.21-3.22µM/g (per gram of organ wet weight), and due to its high concentration relative to plasma (101-186µM) and other organs under resting conditions is not highly responsive to changes in serum.^[28]

Betaine is readily synthesized in the liver and is present in high but variable levels, it does not appear to readily respond to plasma alterations of betaine in otherwise normal conditions

Oral supplementation of betaine glucuronate at 150mg (alongisde diethanolamine glucuronate and nicotinamide ascorbate at 30mg and 20mg respectively) twice daily over eight weeks in persons with fatty liver (non-alcoholic steatohepatitis) was able to reduce liver fat by 25% whereas placebo saw a 3% reduction whereas liver enzymes were reduced by 11% (ALT), 14% (AST) and 15% (γ-GT).^[1] There was also a reduction in liver size seen with supplementation, and while there was significantly less reported pain in the upper left quadrant of the torso (45-57%) dyspepsia was not different between groups and supplementation was well tolerated.^[1]

A pilot study using betaine in isolation (3,000mg twice daily) over the course of a year in persons with fatty liver noted significant reductions in ALT and AST where three (out of seven) patients had normalized values and another three had over a 50% reduction; the three dropouts had a 38-39% reduction in those two enzymes and there was one nonresponders.^[105] This study also noted significant reductions in liver necrosis area (assessed by necroinflammatory grade), fibrosis, and fat buildup.^[105] These benefits were later seen in a study on a larger sample (n=23) in a cohort design where 10g twice daily reduced liver fat and inflammation in over 90% of subjects (and four subjects reporting a reduction in liver fibrosis of over two stages) alongside reductions in homocysteine.^[106]

Despite the aforementioned preliminary evidence, the large trial has been conducted on this topic^[107] failed to note a reduction in S-adenosylhomocysteine (the methylation target of betaine) or in fibrosis, and while the betaine group actually underperformed relative to placebo in improving steatosis by over one grade (29% vs 61%) there was less overall changes with betaine (71% vs 22%) suggesting a possible role in stasis of disease progression.^[107] There was an increase in plasma L-methionine and SAMe noted however, with all other serum inflammatory biomarkers and adiponectin unaffected, and subject self-reported health and well being was not different between groups.^[107] It was noted elsewhere^[108] that this study had some limitation such as a high dropout rate (associated with intestinal distress from high dose betaine) and high levels of persons with advanced fibrosis.

Preliminary evidence for betaine in the treatment of nonalcoholic steatohepatitis (NASH) noted remarkable benefits with high dose supplementation, but the most recent and well controlled trial has failed to note many of the benefits although a reduction in liver fat still appeared to exist. There does appear to be a role for betaine in this disease state, but its exact role requires more research to pinpoint

TMG is detectable in the rat kidney at a concentration of 2.0-5.4mM (per liter of tissue water) and 2.78-3.24µM/g (per gram of organ wet weight), and due to its high concentration relative to plasma (101-186µM) and other organs under resting conditions is not highly responsive to changes in serum.^[28]

TMG is one of the major osmolytes of the kidneys alongside sugars (inositol and sorbitol^[109]), taurine,^[110] and glycerophosphocholine (GPC)^[111] with TMG and GPC (the methylamines) being known to counteract the protein destabilizing effects of urea in renal cells without perturbing cellular function.^[112]

It appears that urinary TMG excretion is increased in the type II diabetic state despite plasma levels remaining stable^[113] which has been known to correlated with plasma retinol binding protein (RBP) and glucose^[114] (although acute spikes in glucose in nondiabetics do not increase urinary TMG^[115]). This elevation is also noted in patients with kidney diseases not associated with diabetes^[113] and is thought to be a biomarker for proximal tubular dysfunction.^[114]

TMG is known to accumulate in the intestines to the level of 524-920µM per liter of tissue water (75% water content of tissue) or 0.43-0.75µM/g tissue wet weight under normal rat dietary conditions (serum TMG at 101-186µM), and appears to be responsive to changes in serum TMG.^[28]

TMG is known to accumulate in the lungs to the level of 290-500µM per liter of tissue water (79% water content of tissue) or 0.25-0.43µM/g tissue wet weight under normal rat dietary conditions (serum TMG at 101-186µM), and appears to be responsive to changes in serum TMG.^[28]

TMG is known to accumulate in the skin to the level of 305-412µM per liter of tissue water (65% water content of tissue) or 0.22-0.30µM/g tissue wet weight under normal rat dietary conditions (serum TMG at 101-186µM), and appears to be responsive to changes in serum TMG.^[28]

TMG is detectable in the rat testes at a concentration of 2.4-3.4mM (per liter of tissue water) and 2.63µM/g (per gram of organ wet weight), and due to its high concentration relative to plasma (101-186µM) and other organs under resting conditions is moderately responsive to changes in serum (less than other organs, more than the liver and kidneys).^[28]

Betaine is thought to be beneficial for the treatment of Alzheimer's disease due to correlations with elevated homocysteine and risk of Alzheimer's Disease.^[116][117] A causative or biomarker role is not known for homocysteine, as since it does potentially have excitotoxic roles via the NMDA receptors^[53] there are also reduced S-adenosyl methionine levels in the brain of those with Alzheimer's^[118] and their cerebrospinal fluid^[119] suggesting impairments in overall methylation in general.

Betaine is not known to accumulate in the brain following oral ingestion^[28] nor does its enzyme of synthesis (BHMT) seem to be located in the brain at high levels^[51][50] but it is hypothesized^[120] that reductions in peripheral homocysteine may play a protective role assuming homocysteine is playing a causative role.

On the assumption that homocysteine plays a causative role in Alzheimer's pathology, it is hypothesized that reductions in peripheral homocysteine (perhaps resulting in less homocysteine influx into the brain) could reduce symptoms of Alzheimer's disease

In a small 24-week pilot study, supplementation of betaine (3g twice daily for a total of 6g) to seven persons with Alzheimer's Disease concurrently using an acetylcholinesterase inhibitor, but results were too variable and the sample too small to come to any conclusions on treatment efficacy relative to baseline; Homocysteine levels were not measured in this study.^[120] 

The evidence at this point in time in humans cannot be used to evaluate the efficacy of betaine supplementation

Supplementation of betaine with other factors thought to improve methylation in the body (creatine, B12, and levomefolic acid) to subjects with angelman syndrome has failed to exert therapeutic effects.^[121]

2,400mg choline bitartrate (1g choline) daily for twelve weeks in postmenopausal women (may have higher requirements for dietary choline^[122]) has been noted to not only increase plasma choline at weeks six and twelve (from a medium value of 7.33µM to 11.1-11.7µM) but also plasma TMG (from a median value of 30.7µM to 54.6-65µM, or 77-111%) and dimethylglycine (from 3.53µM to 3.92-4.63µM) under steady state conditions.^[123]

Interestingly, 1g of betaine itself has increased plasma steady state concentrations from 31.4+/-13.6µM to 52.5+/-26.5µM (67%)^[27] suggesting that choline and betaine are actually equipotent in increasing plasma betaine in a resting state.

Oral supplementation of choline appears to be sufficient in increasing plasma betaine concentrations, and at least at the 1,000mg dosage it appears to be just as potent as betaine at increasing plasma betaine concentrations

In rats supplemented with an additional 0.5% betaine in their feed, it appears that SAMe production in the liver is increased two-fold relative to control; this is increased to five-fold if the rats were pretreated with ethanol (which normally impairs this pathway)^[124] and injections of betaine have demonstrated dose-dependent increases in red blood cell concentrations of SAMe in rats.^[125]

Creatine is perhaps the most well renowned ergogenic aid (performance enhancer) known as a dietary supplement, and it appears that the synthesis of creatine requires methyl donation; supplementation of betaine to a diet without supplemental creatine can accelerate creatine synthesis in the rat^[126] probably just via increasing SAMe concentrations (which are directly required for creatine synthesis).^[127]

Supplementation of betaine at 2g to otherwise healthy adults over the course of ten days has failed to increase muscle phosphocreatine content (the active form of creatine)^[87] and the addition of 2g betaine to supplementation of 20g creatine failed to augment the increase noted with creatine.^[87] Alongside the failure to increase intramuscular phosphocreatine levels was a failure of betaine to augment the power enhancement seen with creatine supplementation.^[87]

Folate and betaine are interrelated as both molecules are known to improve methylation status of the body, and betaine can preserve methylation in periods of low folate status while a reduction in bioavailable betaine (via reduced dietary choline intake) increases requirements for folate.^[14][128]

Supplementation of betaine (1,000mg) appears to be able to acutely suppress folic acid concentrations in serum by 23-28% after the first week of supplementaton, but this is readily normalized back to baseline levels within a week afterwards.^[27] Similar to that, when folic acid supplementation is added to betaine there is an increase in serum folic acid due to its supplementation (41-59%) which is then normalized to control within a week^[27] and other studies measuring serum folic acid levels after prolonged supplementation fail to note any significant changes.^[72][129]

When 1mg folic acid is added to 6,000mg betaine, the reduction in homocysteine seen with betaine (14.2%) is added upon by the folic acid (an additional 5.1%).^[27]

Folic acid appears to be additive with betaine in reducing plasma homocysteine

Cobalamin (Vitamin B12) does not appear to have its serum concentrations altered with ingestion of betaine at doses of 1,000mg up to 6,000mg, even if the highest dose has an additional 1mg of folic acid added to it of periods of up to six weeks of supplementation.^[27][129]

One study noted an increase in plasma B12 relative to placebo, but there was a decrease noted in placebo which may have caused the relatively significant increase.^[72] Similar to this, a relative increase has been noted with TMG relative to folic acid in one study^[129] where B12 nonsignificantly decreased with folic acid supplementation (800µg).

It seems that chronic alcohol ingestion (rats) impairs the function of the methionine synthetase enzyme, but liver concentrations of SAMe are not affected acutely since the liver pool of betaine is used up when the betaine:homocysteine methyltransferase (BHMT) enzyme is upregulated;^[124] supplementation of betaine at 0.5% of the rat diet, normally enough to two-fold increase SAMe, increases it five-fold in rats subject to ethanol.^[124]

Trimethylamine (TMA) is a metabolite that is known to have a fishy odour, which is normally at low levels and undetectable unless the person has high levels in their urine and body secretions (Trimethylaminuria) which is a condition known as "Fish Odour Syndrome";^[130] it is caused by either a mutation in the liver enzyme flavin containing mono-oxygenase type 3 (FMO3^[131]) which metabolizes TMA known as 'Primary trimethylaminuria' or from excess production of TMA in the intestinal tract from bacteria known as 'Secondary trimethylaminuria'.^[132][133] It is medically benign (ie. not a medical concern), but adherence to supplementation goes down since many subjects would rather not smell like fish and it mild trimethylaminuria could affect anywhere between 1 in 20 or 1 in 100 of persons.^[134]

The main cause of trimethylaminuria is high dietary choline intake, but it appears that betaine administration may also be capable of inducing Fish Odour Syndrome or at least the scent of fish in bodily secretions when used in therapeutic doses of 20g.^[135] Although one study has noted that dimethylglycine (DMG) was associated with a fishy smell^[136] it is thought that this isn't the factor since it was later found to not be correlated, and an increase in TMA was noted in its place.^[135]

It is possible that, similar to choline, that betaine can cause a fishy smell in the breath and bodily secretions when taken at high therapeutic doses in some persons who are genetically susceptible to it

It appears that oral ingestion of riboflavin (Vitamin B2) at the dose of 100mg taken twice daily has, at least in one case study where fishy odor was a side-effect of 16-20g betaine ingestion, been able to reduce nearly abolish the fishy odour in breath and body secretion within a few days and to reduce TMA in the urine when measured after 30 days;^[135] this was thought to be due to increased FMO3 activity, of which riboflavin is a cofactor for.

At least one case study has supported the usage of riboflavin (Vitamin B2) at 100mg twice daily in eliminating the fish odour associated with betaine supplementation