Male C57BL/6 mice (25 ± 0.5 g) were randomly assigned to four groups (n = 6–8 per group): pair-fed (PF), alcohol-fed (AF), PF with betaine (BT/PF), and AF with betaine (BT/AF). Betaine was administered at 0.5% (w/v) in the liquid diet. Animals were fed for 5 weeks using a modified Lieber-DeCarli liquid diet with stepwise ethanol increase (30% to 36% of total calories). [2]
- At the end of the study, mice were euthanized after 4 h of food deprivation. Plasma, liver, and epididymal fat pads were collected for analysis. [2]

Absorption, Distribution and Excretion
Betaine is rapidly absorbed and distributed. In healthy volunteers (n=12), after administration of 50 mg/kg betaine, Cmax, tmax, and AUC0,∞ were 0.939 mmol/L, 0.90 h, and 5.52 mmol⋅h/L, respectively. No significant changes in absorption kinetics were observed after repeated administration of betaine (100 mg/kg/day for 5 consecutive days). The absolute bioavailability of anhydrous betaine has not been determined. Betaine is primarily excreted via metabolism. Assuming a slow elimination rate and 100% bioavailability, its renal clearance is negligible (accounting for only 5% of systemic clearance). A study in healthy volunteers (n=12) showed a volume of distribution of 1.3 L/kg after administration of 50 mg/kg betaine. Another study in healthy volunteers (n=12) showed that after administration of 50 mg/kg betaine, the total plasma clearance was 0.084 L/h⋅kg. Betaine is absorbed into intestinal cells via the small intestine, then released into the portal circulation and transported to the liver. In the liver, betaine undergoes significant first-pass extraction and metabolism. The main metabolic reaction is the transfer of methyl groups from betaine to homocysteine via betaine-homocysteine methyltransferase. The products of this reaction are L-methionine and dimethylglycine. Betaine hydrochloride is converted to betaine in the alkaline environment of the small intestine. It is currently unclear whether betaine is secreted into breast milk. However, its metabolic precursor choline is present in high concentrations in human milk. The authors measured serum/plasma levels of homocysteine, betaine, folic acid, vitamin B6, and related compounds in 500 healthy men and women aged 34 to 69 years, both before (fasting) and 6 hours after a standard methionine loading test. In a multiple regression model adjusted for age and sex, choline, dimethylglycine, and folic acid were the determinants of plasma betaine levels. Elevated homocysteine levels after the loading test were significantly negatively correlated with plasma betaine levels, and weakly negatively correlated with folic acid and vitamin B6. Fasting homocysteine levels were significantly negatively correlated with folic acid, weakly correlated with plasma betaine, and not correlated with vitamin B6. Notably, the dose-response curves of betaine in post-methionine loading homocysteine elevation or fasting homocysteine, adjusted for age and sex, showed that this negative correlation was most significant at low serum folate levels. This observation was also confirmed by interaction analysis… In summary, these results indicate that plasma betaine is a significant determinant of post-methionine loading homocysteine elevation, especially in subjects with low folate levels. In 500 healthy subjects, the negative correlation between betaine and folate was stronger for post-methionine loading total homocysteine (tHcy) elevation than for folate and vitamin B6, while the effect of betaine on fasting total homocysteine (tHcy) was weaker than that of folate. For both methods of total homocysteine (tHcy) measurement, the correlation between betaine and subjects with low folate levels was most significant. Thirty-four healthy men and women received 1 gram, 3 grams, and 6 grams of betaine, respectively, followed by four consecutive weeks of 6 grams of betaine plus 1 milligram of folate. Supplementation with 1 gram, 3 grams, and 6 grams of betaine resulted in a decrease in mean plasma total homocysteine (tHcy) concentrations of 1.1% (not statistically significant), 10.0%, and 14.0%, respectively (P<0.001). Combining 1 mg of folic acid with 6 grams of betaine further reduced plasma tHcy by 5% (P<0.01). Plasma betaine concentrations increased from 31 (standard deviation 13) μmol/L to 255 (standard deviation 136) μmol/L in a dose-dependent manner (R² = 0.97)... The authors concluded that daily administration of 3 or 6 grams of betaine in healthy men and women rapidly and significantly reduced plasma total homocysteine (tHcy) levels. ... The authors investigated changes in plasma choline and betaine levels during normal pregnancy and their relationship with plasma total homocysteine (tHcy)... Blood samples were collected monthly; the initial blood sample was collected at week 9 of gestation, and the last blood sample was collected approximately 3 months postpartum. The study population included 50 West African women. Most participants did not take folic acid regularly… Plasma choline (geometric mean; 95% reference range) steadily increased during pregnancy, from 6.6 (4.5, 9.7) μmol/L at week 9 to 10.8 (7.4, 15.6) μmol/L at week 36. Plasma betaine decreased in the first half of pregnancy, from 16.3 (8.6, 30.8) μmol/L at week 9 to 10.3 (6.6, 16.2) μmol/L at week 20, and then remained stable… The authors confirmed a decrease in plasma total homocysteine (tHcy) levels, with the lowest concentration observed in mid-pregnancy. A negative correlation between plasma tHcy and betaine was observed starting at week 16 of pregnancy. Multivariate regression analysis showed that from week 20 of gestation, plasma betaine was a strong predictor of plasma total homocysteine (tHcy)... The sustained increase in choline during pregnancy may ensure that choline is available for placental transport for fetal growth and development. Betaine is a strong predictor of tHcy during pregnancy.

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Metabolism/Metabolites
Betaine is mainly metabolized in the mitochondria of hepatic and renal cells. Betaine is transmethylated by betaine homocysteine methyltransferase (BHMT) to produce dimethylglycine.
Betaine is absorbed by intestinal cells in the small intestine. Intestinal cells release it into the portal circulation, and the portal vein transports it to the liver, where significant first-pass extraction and first-pass metabolism occur.
The main metabolic reaction is the transfer of methyl groups from betaine to homocysteine via betaine-homocysteine methyltransferase. The products of this reaction are L-methionine and dimethylglycine. Betaine hydrochloride is converted to betaine in the alkaline environment of the small intestine.
Betaine is a metabolite of choline…

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Biological Half-Life
In healthy volunteers (n=12), the elimination half-life was 14.38 hours after administration of 50 mg/kg betaine. In volunteers who received 100 mg/kg betaine daily for 5 consecutive days, the distribution half-life was significantly prolonged, indicating that the transport and redistribution of betaine had reached saturation.

Hepatotoxicity
In small, open-label trials of betaine for homocystinuria and small controlled trials for other diseases (Alzheimer's disease, non-alcoholic steatohepatitis), no elevated serum enzymes or clinically significant liver injury were reported. In fact, in some studies, betaine has been associated with a significant decrease in previously elevated serum enzymes in some patients with non-alcoholic fatty liver disease. Probability Score: E (Unlikely to be a cause of clinically significant liver injury). Interactions This study aimed to evaluate the pharmacokinetics of oral betaine and its acute effects on plasma total homocysteine (tHcy) concentrations. Ten healthy volunteers (3 men, 7 women) with normal weight (mean weight ± standard deviation: 69.5 ± 17.0 kg) and mean age 40.8 ± 12.4 years were included. Betaine doses were 1 g, 3 g, and 6 g. All doses were mixed with 150 mL of orange juice and administered to each volunteer after a 12-hour overnight fast, following a randomized, double-blind, crossover design. Blood and urine samples were collected over 24 hours. Oral betaine had an immediate and dose-dependent effect on serum betaine concentrations. Single doses of 3 g and 6 g of betaine reduced plasma total homocysteine (tHcy) concentrations (P = 0.019 and P < 0.001, respectively), while a 1 g dose had no such effect. Following the highest dose, plasma tHcy concentrations remained low during the 24-hour monitoring period. Changes in plasma total homocysteine (tHcy) concentrations were linearly correlated with betaine dose (P = 0.006) and serum betaine concentration (R² = 0.17, P = 0.025). Both absorption and elimination of betaine were dose-dependent. Urinary betaine excretion appears to increase with increasing betaine dose, although only a very small amount of ingested betaine is excreted in urine. In summary, a single oral dose of betaine has an acute and dose-dependent effect on serum betaine concentrations and reduces plasma total homocysteine (tHcy) concentrations in healthy subjects within 2 hours. This study investigated the preventive effect of taurine or betaine pretreatment on lipopolysaccharide-induced hepatic oxidative damage in rats… Rats pretreated with taurine (1.5%, w/v) or betaine (1.5%, w/v) in their drinking water were intraperitoneally injected with lipopolysaccharide (10 mg/kg) for 4 weeks. Plasma transaminase activity, as well as liver levels of malondialdehyde, diene conjugates (DC), glutathione, α-tocopherol, and ascorbic acid, and superoxide dismutase (SOD) and glutathione peroxidase activities were measured… Results showed that plasma transaminase activity and liver malondialdehyde and DC levels were significantly increased, while liver… Six hours after lipopolysaccharide treatment, glutathione and α-tocopherol levels, as well as SOD and glutathione peroxidase activities, were measured. Compared with the control group, this treatment did not change the level of ascorbic acid in the liver. In rats injected with lipopolysaccharide (LPS), pretreatment with taurine or betaine significantly reduced plasma transaminase activity and hepatic malondialdehyde (MDA) and dendritic cell (DC) levels, and significantly increased glutathione and α-tocopherol (but not betaine) levels, while hepatic ascorbic acid levels and superoxide dismutase (SOD) and glutathione peroxidase activities remained unchanged…/According to the authors/ Taurine or betaine pretreatment effectively prevented LPS-induced hepatotoxicity and pro-oxidative state. The combined use of betaine and folic acid may have an additive effect on reducing serum homocysteine levels. …Homocysteine remethylation to methionine can occur via the folic acid-dependent methionine synthase pathway or the betaine-dependent betaine-homocysteine methyltransferase pathway. The role of betaine as a determinant of fasting total homocysteine (tHcy) remains unclear, as is the relationship between the two remethylation pathways… This study aimed to investigate the relationship between plasma betaine concentration and fasting plasma tHcy concentration, and to assess the effect of folic acid supplementation on betaine concentration in healthy subjects… A double-blind randomized trial was conducted in 308 Dutch men and postmenopausal women (aged 50–75 years), who received either six escalating doses of folic acid (50–800 μg/day) or a placebo daily. Fasting serum concentrations of total homocysteine (tHcy), betaine, choline, dimethylglycine, and folic acid were measured at baseline and after 12 weeks of supplementation… tHcy concentration was negatively correlated with betaine concentration (r = -0.17, P < 0.01), and this correlation was independent of age, sex, and serum folic acid, creatinine, and cobalamin concentrations. Folic acid supplementation dose-dependently increased betaine concentration (trend P = 0.018); the increase in betaine concentration was greatest (15%) at daily doses of 400 to 800 micrograms… The authors concluded that plasma betaine concentration is an important determinant of fasting tHcy concentration in healthy individuals. The increase in betaine concentration due to folic acid supplementation suggests an interrelationship between the two remethylation pathways.

[1]. Betaine Effects on Morphology, Proliferation, and p53-induced Apoptosis of HeLa Cervical Carcinoma Cells in Vitro. Asian Pac J Cancer Prev. 2015;16(8):3195-201.

[2]. Rectification of impaired adipose tissue methylation status and lipolytic response contributes to hepatoprotective effect of betaine in a mouse model of alcoholic liver disease. Br J Pharmacol. 2014 Sep;171(17):4073-86.

[3]. Reconsidering betaine as a natural anti-heat stress agent in poultry industry: a review. Trop Anim Health Prod. 2017 Oct;49(7):1329-1338.

Therapeutic Uses
The authors measured blood lipids in four placebo-controlled, randomized intervention studies examining the effects of betaine (three studies, n = 151), folic acid (two studies, n = 75), and phosphatidylcholine (one study, n = 26) on plasma homocysteine concentrations. They summarized the blood lipid data from each study and calculated the weighted average change in blood lipid concentrations relative to placebo. Compared to placebo, after 6 weeks of betaine supplementation (6 g/day), blood LDL cholesterol concentrations increased by 0.36 mmol/L (95% confidence interval: 0.25 to 0.46), and triglyceride concentrations increased by 0.14 mmol/L (0.04 to 0.23). The total cholesterol to high-density lipoprotein cholesterol ratio increased by 0.23 (0.14 to 0.32). High-density lipoprotein cholesterol (HDL-C) concentrations were unaffected. Daily intake of less than 6 grams of betaine also increased low-density lipoprotein cholesterol (LDL-C), but these changes were not statistically significant. Furthermore, the effect of betaine on LDL-C was evident after 2 weeks of intervention. After 2 weeks of phosphatidylcholine supplementation (approximately 2.6 grams of choline daily), triglyceride concentration increased by 0.14 mmol/L (from 0.06 mmol/L to 0.21 mmol/L), but had no effect on cholesterol concentration. Folic acid supplementation (0.8 mg/day) had no effect on blood lipid concentration. Anhydrous betaine has been used to treat homocystinuria, and betaine may also be helpful for other diseases characterized by elevated plasma homocysteine levels. Betaine hydrochloride is used as a digestive aid in some diseases. Animal studies have shown that betaine may have hepatoprotective effects in certain situations. Cystadane (anhydrous betaine oral solution) is indicated for the treatment of homocystinuria to reduce elevated blood homocysteine levels. Homocystinuria includes a deficiency or defect in cystathionine β-synthetase (CBS), 5,10-methylenetetrahydrofolate reductase (MTHFR), and cobalamin cofactor metabolism (CBL). Veterinary Drug: Betaine glucuronic acid, together with 2-aminoethanol glucuronic acid, is used as an active ingredient in a product for the treatment of symptoms of acute or chronic liver disease, such as endogenous metabolic disorders, exogenous poisoning, or parasitic infections. This product is administered by injection to animals such as cattle, horses, sheep, goats, and pigs. /Glucuronide Betaine/ For more complete data on the therapeutic uses of betaine (14 in total), please visit the HSDB record page.

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Drug Warning
It is currently unknown whether betaine is excreted into human milk (although its metabolic precursor choline is present in high concentrations in human milk). Because many drugs are excreted into human milk, caution should be exercised when breastfeeding women take Cystadane.
Cystadane treatment should be directed by a physician familiar with the management of patients with homocystinuria.
FDA Pregnancy Risk Category: C / Risk cannot be ruled out. There is a lack of adequate, well-controlled human studies, and animal studies have not shown any risk to the fetus or lack relevant data. There is a possibility of fetal harm if this medication is taken during pregnancy; however, the potential benefit may outweigh the potential risk. /
Patients with homocystinuria due to cystathionine β-synthetase (CBS) deficiency may also experience elevated plasma methionine concentrations. Treatment with cystadane may further increase methionine concentrations because homocysteine is remethylated to methionine. Cerebral edema has been reported in patients with hypermethioninemia, including a small number of patients treated with cystadane. Plasma methionine concentrations should be monitored in patients with CBS deficiency. Plasma methionine concentrations should be maintained below 1,000 μmol/L through dietary adjustments and, if necessary, reduction of the cystathionine dose.
For more complete data on drug warnings for betaine (11 in total), please visit the HSDB record page.

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Pharmacodynamics
Betaine can reduce plasma homocysteine concentrations in patients with homocystinuria caused by deficiency or defect of cystathionine β-synthetase (CBS), 5,10-methylenetetrahydrofolate reductase (MTHFR), and cobalamin cofactor (cbl). It is estimated that homocysteine levels can be reduced to 20-30% of pre-treatment levels. Betaine supplementation in patients with homocystinuria can also improve metabolic abnormalities in cerebrospinal fluid. Reports indicate that, depending on the type of homocystinuria, the therapeutic effect of betaine alone may be limited and insufficient to reduce total homocysteine levels and prevent clinical symptoms. For patients with homocystinuria caused by cystathionine β-synthetase (CBS) deficiency, betaine should be used if serum total homocysteine levels remain high after dietary therapy. No tolerance has been observed in patients taking betaine for several years. Furthermore, betaine concentration is not correlated with homocysteine concentration.
In patients with MTHFR deficiency and CBL deficiency, betaine may increase plasma methionine and S-adenosylmethionine (SAM) levels. CBS-deficient patients without methionine intake restriction may accumulate excess methionine. Clinical data show that elevated plasma methionine levels are associated with cerebral edema in CBS-deficient patients.

C5H11NO2

117.15

117.078

107-43-7

590-46-5 (hydrochloride)

247

White to off-white solid powder

1.00 g/mL at 20 °C

301-305 °C (dec.)

-3.25

0

2

1

8

87.6

0

[O-]C(C([H])([H])[N+](C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])=O

KWIUHFFTVRNATP-UHFFFAOYSA-N

InChI=1S/C5H11NO2/c1-6(2,3)4-5(7)8/h4H2,1-3H3

2-(trimethylazaniumyl)acetate

Abromine LycineBetaine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)