What Is Leucine?

Leucine ((2R) -2-amino-3,3-dimethylbutanoic acid) chemical name is L-2-amino-4-methylvaleric acid, white crystal or crystalline powder; odorless, slightly bitter taste. Soluble in formic acid, slightly soluble in water, and slightly soluble in ethanol or ether. The density is 1.038g / cm3, the melting point is greater than 300ºC, and the boiling point is 217.7ºC at 760 mmHg.

Leucine's role includes working with isoleucine and valine to repair muscle, control blood sugar, and provide energy to body tissues.

Leucine, isoleucine, and valine are all branched-chain amino acids that help promote muscle recovery after training. Among them, leucine is the most effective branched chain amino acid, which can effectively prevent muscle loss, because it can be quickly broken down into glucose.

Chinese name: Leucine
Foreign name: Leucine

Molecular formula: C6H13NO2
CAS number: 61-90-5

Introduction to leucine compounds

Chinese name: Leucine, Leucine Chinese alias: -amino--methylvaleric acid, -aminoisocaproic acid

English name: Leucine

Three-letter symbol: Leu

Single letter symbol: L

Leucine

Chemical formula: C ₆ H ₁₃ NO ₂

Molecular formula: (CH ₃ ) ₂ CHCH ₂ CH (NH ₂ ) COOH

Molecular weight: 131.18

Category: amino acid drugs

This product is L-2-amino-4-methylvaleric acid. Calculated on dry basis, containing C ₆ H ₁₃ NO ₂ shall not be less than 98.5%. ^[1]

Physiochemical Properties of Leucine

Appearance and properties: This product is white crystal or crystalline powder; odorless, slightly bitter.

Melting point: 332 ° C (racemic form), 293 to 295 ° C (left-hand form)

Acid and basic: pH is about 5.5 to 6.5

Solubility: This product is easily soluble in formic acid, slightly soluble in water, and slightly soluble in ethanol or ether.

Relative density (water = 1): L-rotor 1.293

Specific rotation: Take this product, accurately weigh, add 6mol / L hydrochloric acid solution to dissolve and release it into a solution containing 40mg per 1ml, and measure it according to law (Appendix VI E). Specific rotation is + 14.5 ° to + 16.0 °

Storage Precautions: This product should be sealed, dry and protected from light. ^[1]

Leucine Pharmacopoeia Standard

The main active ingredient of leucine

L-2-amino-4-methylvaleric acid. Calculated on dry basis, containing C6H13NO2 shall not be less than 98.5%. ^[2]

Leucine traits

White crystal or crystalline powder; odorless, slightly bitter.

Soluble in formic acid, slightly soluble in water, very slightly soluble in ethanol or ether ^[2]

Leucine specific rotation

Take this product, weigh it accurately, add 6mol / L hydrochloric acid solution to dissolve and quantitatively dilute it to make a solution containing about 40mg per 1ml, and measure it according to the law (Appendix VI E of the second edition of the Pharmacopoeia, 2010 edition). + 16.0 ° ^[2]

Leucine identification

(1) Take appropriate amounts of this product and leucine reference substance, dissolve and dilute with water to make a solution containing about 0.4mg per 1ml, as the test solution and reference solution. According to the chromatographic conditions test under other amino acids, the position and color of the main spots displayed by the test solution should be the same as the main spots of the reference solution.

(2) The infrared light absorption spectrum of this product should be the same as that of the control ("Infrared Spectra of Drugs" 987). ^[2]

Leucine check

For acidity, take 0.50g of this product, add 50ml of water, heat to dissolve, let cool to room temperature, measure according to law (Appendix VI H of Pharmacopoeia Part II of 2010 Edition), pH value should be 5.5 6.5. The light transmittance of the solution was 0.50 g of the product, 50 ml of water was added, the solution was heated to dissolve, and allowed to cool to room temperature. The light transmittance was measured at a wavelength of 430 nm according to the ultraviolet-visible spectrophotometry (Appendix IV A of Part Two of the Pharmacopoeia 2010 Edition). , Not less than 98.0%. Take 0.25 g of this product and check it according to law (Appendix A of Part Two of the Pharmacopoeia 2010), compared with the control solution made of 5.0 ml of standard sodium chloride solution, it must not be more concentrated (0.02%). Take 1.0 g of this sulfate and check it according to law (Appendix B of Part Two of the Pharmacopoeia 2010), compared with the control solution made of 2.0 ml of standard potassium sulfate solution, it must not be more concentrated (0.02%). Take 0.10g of ammonium salt and check it according to law (Appendix K of the second edition of the Pharmacopoeia 2010), compared with the control solution made of 2.0ml of standard ammonium chloride solution, it must not be deeper (0.02%). Take the appropriate amount of other amino acids, dissolve and dilute with water to make a solution containing about 20mg per 1ml, as a test solution; take 1ml precisely, place in a 200ml volumetric flask, dilute to the mark with water, shake well, and use as a control solution ; Another appropriate amount of leucine reference substance and valine reference substance were placed in the same volume bottle, dissolved in water and diluted to make a solution containing about 0.4 mg per 1 ml, as a system suitability test solution. According to the thin layer chromatography (2010 Appendix Pharmacopoeia Part II Appendix V B) test, draw 5 l of each of the three solutions, and point them on the same silica gel G thin layer plate with n-butanol-water-glacial acetic acid (3: 1: 1) As a developing agent, after developing, dry it, spray with ninhydrin acetone solution (1 50), heat at 80 ° C until spots appear, and inspect immediately. The control solution should show a clear spot, and the system suitability test solution should show two completely separated spots. If the solution of the test product is a spot of impurities, its color must not be darker than the main spot of the control solution (0.5%). Loss on drying: Take this product and dry it at 105 ° C for 3 hours. The weight loss should not exceed 0.2% (Appendix L of Pharmacopoeia Part II of 2010 Edition). Take 1.0g of this product and check it according to law (Appendix N of Part II of the Pharmacopoeia of 2010 Edition). The remaining residue shall not exceed 0.1%. Take 1.5g of iron salt and check it according to law (Appendix G of Part Two of the Pharmacopoeia 2010), compared with the control solution made of 1.5ml of standard iron solution, it must not be deeper (0.001%). Residues left under the heavy metal burning residues shall be inspected according to law (Appendix H, Second Method of the Pharmacopoeia, 2010 Edition), and the content of heavy metals shall not exceed 10 parts per million. Take 2.0g of arsenic salt, add 5ml of water, add 1ml of sulfuric acid and 10ml of sulfurous acid, heat to a volume of about 2ml on the water bath, add 5ml of water, dropwise add the ammonia test solution to the phenolphthalein indicator solution, and add 5ml of hydrochloric acid. Add water to make 28ml, check according to law (Appendix J of the second edition of the Pharmacopoeia of 2010 Edition J first method), should meet the requirements (0.0001%). Take this product for bacterial endotoxin and check it according to law (Appendix E of Part Two of Pharmacopoeia 2010), the amount of endotoxin in 1g leucine should be less than 25EU (for injection) ^[2]

Determination of leucine

Take about 0.1g of this product, accurately weigh, add 1ml of anhydrous formic acid to dissolve, add 25ml of glacial acetic acid, according to the potentiometric titration method (Appendix A of Part Two of the 2010 Pharmacopoeia), and use perchloric acid ) Titrate and correct the results of the titration with a blank test. Each 1ml of perchloric acid titration solution (0.1mol / L) is equivalent to 13.12mg of C6H13NO2. ^[2]

Leucine analysis method

Method name: Determination of Leucine-Potentiometric Titration

Application range: This method uses the titration method to determine the content of leucine. This method is applicable to leucine.

Principle of the method: The test product is placed in an Erlenmeyer flask, dissolved in anhydrous formic acid and glacial acetic acid, and titrated with perchloric acid titration solution (0.1mol / L) according to the potentiometric titration method. Read out the amount of perchloric acid titrant used and calculate the leucine content.

Reagent:

1. Water (newly boiled to room temperature)

2. Perchloric acid titrant (0.1mol / L)

3. Crystal violet indicator liquid

4. Anhydrous formic acid

5. Glacial acetic acid

6. Reference potassium phthalate

Equipment: Potentiometric Titrator

Sample preparation:

Perchloric acid titration solution (0.1mol / L)

Preparation: Take 750mL of anhydrous glacial acetic acid (calculated with water content, add 5.22mL acetic anhydride per 1g of water), add 8.5mL perchloric acid (70% -72%), shake well, and slowly add acetic anhydride dropwise at room temperature. 23mL, shake while adding, shake evenly after adding, let cool, add an appropriate amount of anhydrous glacial acetic acid to 1000mL, shake well, and leave for 24 hours. If the test sample is easily acetylated, the water content on this page must be determined by moisture measurement, and then the water content of this solution should be adjusted to 0.01% -0.2% with water and acetic anhydride.

Calibration: Take about 0.16g of standard potassium hydrogen phthalate dried to constant weight at 105 , accurately weigh, add 20mL of anhydrous glacial acetic acid to dissolve, add 1 drop of crystal violet indicator solution, and titrate slowly with Blue, and the results of the titration are corrected with a blank test. Each 1mL of perchloric acid titration solution (0.1mol / L) is equivalent to 20.42mg of potassium hydrogen phthalate. Calculate the concentration of this solution based on the consumption of this solution and the amount of potassium hydrogen phthalate taken.

Storage: Place in a brown glass bottle and keep tightly closed.

crystal violet indicator liquid

Take 0.5g of crystal violet, add 100mL of glacial acetic acid to dissolve, and get.

Operation steps: Precisely weigh about 0.1g of the test sample, place it in a conical flask, add 1mL of anhydrous formic acid to dissolve, add 25mL of glacial acetic acid, and titrate with perchloric acid titration solution (0.1mol / L) according to the potentiometric titration method. And correct the titration results with a blank test. Record the volume (mL) of perchloric acid titrant consumed. Each 1mL of perchloric acid titrant (0.1mol / L) is equivalent to 13.12mg of C6H13NO2.

Note 1: "Precision weighing" means that the weighed weight should be accurate to one thousandth of the weight. Precision requirements. ^[2]

Leucine preparation method

The manufacture of amino acids began with the hydrolysis of proteins in 1820. In 1908, the Japanese Ikeda discovered that sodium glutamate was an umami enhancer, and began the history of industrial production of amino acids. In 1957, Japan began to use microorganisms to produce glutamic acid. Since then, it has opened a new chapter in the history of amino acid production by microbial fermentation. Around the 1960s, the biosynthesis of L-leucine and its metabolic regulation mechanism were successively elucidated. This provides a theoretical basis for directional fermentation of L-leucine production by microbial fermentation and L-leucine production by enzymatic methods.

The production methods of L-leucine mainly include extraction, chemical synthesis, enzyme catalysis, and microbial fermentation.

Leucine hydrolysis

Amino acid is a constituent unit of protein. Under acidic conditions, a protein with a higher content of L-leucine is hydrolyzed to obtain a mixture of various amino acids, and the product of L-leucine is obtained through isolation, purification, and refinement.

Most manufacturers in China use the proteolytic method to produce L-leucine and L-cystine. The proteolytic method for producing L-leucine has the advantages of simple production equipment, low technical requirements, and high content of L-leucine in protein. However, the disadvantages of proteolytic production are time-consuming, serious pollution, low yield, product quality cannot be guaranteed, and large-scale production is limited.

Leucine Synthesis

Leucine chemical synthesis methods include A. Strecker, n-halogen acid ammonolysis, phase transfer catalysis and other methods. Although the chemical synthesis method is simple in principle and low in price, it has complicated operations, harsh reaction conditions, many by-products, and low yields, and some methods involve toxic substances. The leucine obtained by chemical synthesis is racemic DL-leucine. In order to obtain L-leucine, the optical isomers must be resolved. Therefore, chemical synthesis is rarely used in the production of L-leucine.

Leucine catalysis

Enzyme-catalyzed production of L-leucine usually uses transaminase to transfer ammonia to a-ketoisocaproic acid to generate L-leucine and histidine. The relevant enzymes and NADH are covalently bonded to the membrane to make the substrate slowly The ground passes through the membrane and undergoes an enzyme-catalyzed reaction to produce L-leucine. For example, in 1981, Wichmann er al. Established a membrane reactor made of ultrafiltration membrane, which covalently combined leucine aminotransferase, formate aminotransferase, and NADH as substrates. After monoketoisocaproic acid passes through the membrane reactor, it can be catalyzed to produce L-leucine, in which NADH is regenerated by simultaneous oxidation of formic acid to CO2.

The advantages of enzymatic amino acid production are its strong transformation ability, which can avoid feedback inhibition and feedback inhibition in metabolic regulation, and its bioreactor is compact, the product components are relatively single, and it is easy to perform post-processing, which can improve product quality and reduce costs . However, the enzymes used in the enzymatic production need to be generated and extracted by microbial fermentation, the process is more complicated, and the cost is higher, so it has not been widely used at present. If low-activity enzymes can be obtained at low cost, enzymatic production of L-leucine is an economically feasible process route.

Microbial fermentation

Leucine fermentation

In 1987, German scholar Groegere adopted the addition of precursors. Monoketoisocaproic acid produces L-leucine when precursors are added to the medium. The concentration of monoketoisohexanoic acid is 20g / L. Fermentation of Corynebacterium glutamicum ATCC 13032 for 57h can produce 16g / LL monoleucine with a mass conversion rate of 91-96%; while the batch fed culture method can be used 32 g / L of a-ketoisocaproic acid was added and fermented for 23 hours to produce 24 g / L of L-leucine. The conversion of monoketoisocaproic acid to L-leucine is generated by transaminase catalysis.

Leucine production process

One. Concentration section

Raw material: steam

The primary mother liquor was passed into the concentration tank, and steam was passed in, the temperature was 120 ° C., the air pressure was -0.09 MPa, and the concentration time was 6 hours, and the crystals were crystallized.

End product: crystallization solution (go to the neutralization stage)

Second, once neutralization section

Excipients: sulfuric acid, pure water

The crystallization solution enters the neutralization tank once, and is passed through sulfuric acid, pure water, the temperature is 80 ° C, and the neutralization time is 4 hours, and filtered.

End products: 1, filtrate (recycling) 2, filter residue (deamination hydrolysis section)

Third, ammonia hydrolysis section

Excipients: ammonia, pure water, steam

The filter residue enters the ammonia hydrolysis tank, and ammonia water, pure water, steam is passed in, the temperature is 80 ° C, the ammonia hydrolysis time is 3 hours, and the filter is filtered.

End product, 1, filtrate (recycling) 2, filter residue (decolorizing section) (cystine)

Fourth, the bleaching section.

Excipients: steam, pure water, activated carbon

The filter residue enters the decolorization tank, and steam (pure) is passed in, pure water, activated carbon, temperature 80 ° C, decolorization time 2h, filtration,

End products: 1, filter residue (recycling) 2, filtrate (to the secondary neutralization stage)

Five and second neutralization

Excipients: ammonia, steam

The filtrate enters the secondary neutralization tank, and ammonia water, steam, temperature 80 ° C, and neutralization time 4h are filtered.

End products, 1, filtrate (recycling) 2, filter residue (ie crude L-leucine, de-refined section)

Six, refined section

Excipients: distilled water, steam (histidine hydrochloride)

Rinse the product from the previous process with distilled water and centrifuge to dry, send it to the dryer, steam it, dry it, pack it, put it in storage, dry it at 100 , and dry it for 3 hours.

End product: finished L-leucine

Use of leucine

Leucine can be used as a nutritional supplement and flavoring flavoring agent. Amino acid infusion and comprehensive amino acid preparation, hypoglycemic agent, plant growth promoter can be formulated.

Leucine's role includes working with isoleucine and valine to repair muscle, control blood sugar, and provide energy to body tissues. It also increases the production of growth hormones and helps to burn visceral fat, which is difficult to have an effective effect on them only through dieting and exercise because it is located inside the body.

Leucine, isoleucine, and valine are all branched-chain amino acids that help promote muscle recovery after training. Among them, leucine is the most effective type of branched chain amino acid, which can effectively prevent muscle loss, because it can be quickly broken down into glucose. Increasing glucose prevents damage to muscle tissue, so it's especially suitable for bodybuilders. Leucine also promotes healing of bones, skin, and damaged muscle tissue, and doctors usually recommend patients to take leucine supplements after surgery.

Because it is easily converted to glucose, leucine helps regulate blood sugar levels. People with leucine deficiency experience symptoms similar to hypoglycemia, such as headache, dizziness, fatigue, depression, insanity, and irritability.

The best food sources of leucine include brown rice, beans, meat, nuts, soy flour, and whole wheat. Since it is an essential amino acid, this means that the body cannot produce it by itself, but only through diet. People engaged in high-intensity physical activity and low-protein diets should consider taking leucine supplements. Although there are independent supplements, it is best to take them with isoleucine and valine. Therefore, it is more convenient to choose a mixed supplement.

But like anything, excessive intake of leucine can cause side effects. Large intakes are known to be related to pellagra, vitamin A deficiency, and other problems, and cause problems such as dermatitis, diarrhea, and mental disorders. Excessive leucine in the diet also increases the amount of ammonia in the body and destroys liver and kidney function. Therefore, unless you have consulted a doctor, patients with impaired liver or kidney function should not take large doses of leucine, which will worsen the condition.

Leucine- related compounds

Leucine drug name:

[Common name] Compound Leucine Granules (3AA)

[English name] Compound Leucine Granules (3AA)

Chinese Phonetic Alphabet Fu Fang Liang An Suan Ke Li (3AA)

Leucine Ingredients:

This product is a compound preparation, and its components are each bag containing 1.375g of leucine (C6H13NO2), 1.125g of isoleucine (C6H13NO2), and 1.050g of valine (C5H11NO2). ^[3]

Leucine traits:

This product is yellow or light yellow particles. ^[3]

Leucine Pharmacology and Toxicology:

Valine, leucine and isoleucine are branched-chain amino acids. After entering the body, it can correct the imbalance of branched-chain amino acids and aromatic amino acids in the plasma and prevent liver coma caused by excessively high concentrations of aromatic amino acids in the brain. Protein synthesis and reduction of protein breakdown are beneficial to promote regeneration and repair of liver cells, and can improve hypoproteinemia. ^[3]

Leucine Pharmacokinetics:

It is directly metabolized in muscle, fat, heart, brain and other tissues to generate energy for the body to use. ^[3]

Leucine indications:

For chronic hepatic encephalopathy, cirrhosis, chronic active hepatitis and chronic persistent hepatitis and other diseases caused by disorders of amino acid metabolism. ^[3]

Leucine Usage and Dosage:

oral.

Hepatic encephalopathy: 2-3 bags once in a coma, three times a day; one bag once awake.

Chronic liver disease: 1 bag at a time. Swallow or flush 3-4 times a day, or as directed by your doctor. ^[3]

Leucine adverse reactions:

No adverse reactions have been reported. ^[3]

Leucine contraindications:

Those who are allergic to this product are prohibited. ^[3]

Leucine precautions:

It is forbidden to use when the properties of the medicine are changed. ^[3]