Metabolite of the Week

Alanine is a neutral amino acid and an essential building block for protein synthesis, along with Leucine. Alanine is one of the two most common protein amino acids. Alanine is also of the most common plasma biomarkers for diseases. Alanine is not a dietary essential amino acid, as it can be made within cells.

How is alanine so prevalent as a disease biomarker and in dysregulated in many cell studies? What could be its role as a biomarker and what pathways other than protein synthesis?

• Alanine is a zwitterion, consequently it can act as an osmoprotectant under cellular stress and act as a protein stabilizer.
• Alanine comes in two stereosiomers, D & L, that activate sweet taste receptors, however, L-Alanine with high levels of nucleotides contributes to Umami taste while D-Alanine contributes only sweet.
• Alanine, released from skeletal muscle, is used as a substrate for gluconeogenesis in the liver. A liver enzyme, Alanine aminotransferase(ALT), catalyzes the reaction to form pyruvate from alanine in the liver, pyruvate converting to glucose back in muscle (which is known as the “glucose-alanine cycle”. Hence Alanine plays a critical role in muscle metabolism
• There is more to this story than just Alanine to glucose. ALT catalyzes the transfer of an amino group from alanine to alpha-ketoglutarate in the alanine cycle to form pyruvate but there is a second product, glutamate, a critical amino acid itself and part of the transport of ammonia from muscle to liver to kidney.
• Lymphocytes depend on the import of extracellular alanine as vital for the transition from quiescence to activation of both naïve and memory T cells. Though the role is under study, the mechanism of which is not clear, but gluconeogenesis may be one factor to active cells as well as protein synthesis.
• Alanine racemase is a bacterial enzyme that catalyzes the conversion of L-alanine to D-alanine and is dependent upon Vit B6. This function is critical for the growth of bacteria due to their need for D-alanine as an essential component in the biosynthesis of cell wall peptidoglycan in both gram-positive and gram-negative bacteria thus important for supporting our microbiome.
• Another role for D-alanine is through D-amino acid oxidase (DAO) which catalyzes the oxidation of bacterial D-amino acids, such as D-alanine, and generates hydrogen peroxide, which is linked to antimicrobial activity. D-alanine is made exclusively within the microbiome, where as, DAO is conserved widely in eukaryotes, but not in bacteria hence DAO is considered a potential component of the innate defense in mammals.
• The dipeptide, Ala-Ala, is commonly observed in human blood and believed to be from beef, has a sweet taste and has shown anti-hypertensive activity against ACE-I.
• Alanine can be appropriately referred to as alpha-alanine as compared to beta-alanine, which has a different biochemical profile.

• L-Arginine participates in major pathways such as the Urea Cycle and Nitric Oxide synthesis.
• L-Arginine acts as a vasodilator, opening (dilating) blood vessels. Many people take oral L-arginine to treat heart conditions or for erectile dysfunction. This action presumably is through generation of nitric oxide (NO) from the conversion of Arginine to Citrulline.
• L-Arginine is classified as a basic amino acid. It is classified as a semi-essential or conditionally essential amino acid, depending on the developmental stage and health status of the individual. Hence, while there are dietary requirements, Arginine can also be synthesized within cells. Adults can synthesize Arginine in the urea cycle as part of the disposal of waste ammonia.
• L-Arginine has a bitter taste.
• L-Arginine plays an important role in immunity and wound healing through NO generation and transcription activation.
• L-Arginine is necessary for the synthesis of creatine and phosphocreatine (ATP recycling) and can be used for the synthesis of polyamines through transformation to Ornithine.
• The conversion to asymmetric dimethylarginine (ADMA) inhibits the nitric oxide synthase (NOS) reaction, providing negative feedback control. ADMA is considered a marker for vascular disease as high levels can inhibit NOS.
• L-Arginine stimulates the release of hormones; specifically, growth hormones and prolactin.
• L-Arginine is a known inducer of mTOR and is responsible for inducing protein synthesis through the mTOR pathway.
• disease states such as sepsis, injury, and cancer can cause an increase in L-Arginine utilization, which can exceed normal body production, leading to L-Arginine depletion.
• L-Arginine activates AMPK which then stimulates skeletal muscle fatty acid oxidation and muscle glucose uptake, thereby increasing insulin secretion by pancreatic beta-cells.

• Aside from being an essential building block for protein, L-Cysteine is also a component of the strong antioxidant Glutathione (GSH), which also serves as a reservoir of L-cysteine.
• The liver addresses both the need to have adequate L-Cysteine to support normal metabolism and the need to keep L-Cysteine levels below the threshold of toxicity.
• L-Cysteine has antioxidant properties on its own, while D-Cysteine is known to be toxic to bacteria.
• L-Cysteine is oxidized to a dimer, Cystine, which is readily transported into mammalian cells. In cells, Cystine is reduced back to L-Cysteine, which is an essential substrate for the synthesis of biomolecules such as proteins, Coenzyme A and for mTORC1 signaling.
• L-Cysteine is classified as a non-essential amino acid. Mammalian cells utilize the trans-sulphuration pathway for the synthesis of L-Cysteine from L-Methionine, most of which occurs in the liver.
• The L-Cysteine moiety of GSH can be liberated via γ-glutamyl cycle in which exported GSH is cleaved sequentially by γ-Glutamyl Transpeptidase (GGT) and DiPeptidases (DP) to release L-Cysteine which is then imported into the cells.
• The first and rate-limiting step of GSH synthesis is catalyzed by Glutamate-Cysteine Ligase (GCL), which is regulated by L-Cysteine availability at the transcriptional and translational level.
• The thiol functionality in L-Cysteine can undergo oxidation/reduction (redox) reactions. It can also undergo nucleophile addition and substitution reactions with oxygen, methyl transfer and acylation producing metabolites such as L-Cysteic acid, Lactoyl-L-Cysteine, S-Methyl L-Cysteine and many others. These metabolites may act as reservoirs for L-Cysteine or detoxification end products or oxidative products, and as such can be biomarkers for changes in oxidation state, transulfation and methylation pathways
• N-Acetyl-L-cysteine (NAC) is a powerful antioxidant. As a drug, NAC is used by healthcare providers to treat acetaminophen (Tylenol) poisoning.

• L-Aspartic acid is the Swiss army knife of amino acids, being versatile and participating in a variety of critical roles.
• -Aspartic acid is one of only 2 amino acids (the other one Serine) that exists as a common D-amino acid.
• The D form, D-Aspartic acid, is linked to neurotransmission, steroid hormones and appears to be regulated.
• L-Aspartate participates in the Malate Aspartate Shuttle to provide additional NADH from the cytosol to the electron transport chain in mitochondrial matrix.
• L-Aspartate reacts with L-Citrulline leading to L-Arginine synthesis and the release of Fumarate to TCA cycle.
• L-Aspartate reacts with bicarbonate leading to synthesis of UMP.
• L-Aspartate combines with IMP to lead to synthesis of AMP.
• L-Aspartate can act as a neurotransmitter and immunostimulant.
• Both L- and D- Aspartate activate the sweet receptor, however, when present with nucleic acids, L Aspartate can contribute to a UMAMI taste response.
• L-Aspartate is a non-essential amino acid in that it is synthesized mainly from OAA in the cytosol, but also can be generated from glutamic acid with Vit B6 as co-factor.
• Bacteria can use L-Aspartic acid to generate beta-Alanine leading to CoA synthesis and carnosine.
• Bacteria can use L-Aspartic acid to lead to synthesis of several amino acids, homoserine and ectoine.

• Histidine is classified as an aliphatic, positively charged, and basic amino acid.
• Histidine forms complexes with many divalent metal ions as a chelator within small peptides and proteins.
• Histidine is an essential amino acid in humans and other mammals.
• Histidine is a precursor for histamine and carnosine biosynthesis.
• Histamine regulates functions in the gut and acts as a neurotransmitter for the brain and spinal cord. Histamine is produced by basophils and by mast cells and increases the permeability of capillaries to white blood cells.
• Carnosine is a dipeptide of beta-alanine and histidine that is highly concentrated in muscle and brain. Carnosine is naturally produced in the liver. Carnosine scavenges reactive oxygen species (ROS) as well as alpha-beta unsaturated aldehydes formed from peroxidation of fatty acids during oxidative stress. It also acts as a neurotransmitter in the brain.
• Histidine and other imidazole compounds have antioxidant properties. The efficacy of L-histidine in protecting inflamed tissue is attributed to the capacity of the imidazole ring to scavenge reactive oxygen species (ROS) generated by cells during acute inflammatory response.
• can inhibit cytokines and growth factors involved in cell and tissue damage. Histidine appears to suppress pro-inflammatory cytokine expression, possibly via the NF-kappa-B pathway in adipocytes.
• The main pathway of catabolism begins with production of trans-urocanate and ammonia; in the skin considered a significant source of blood ammonia in the systemic circulation. The urocanate produced in the liver is hydrolyzed to formiminoglutamate (FIGLU), and FIGLU is converted to glutamic acid.
• The major histidine derivatives are 3-methylhistidine, whose plasma concentration and urine excretion are markers of myofibrillar protein degradation, 1-methylhistidine, produced from anserine in muscle, and ergothioneine, produced by cyanobacteria, mycobacteria, and fungi.

• It is classified as an aliphatic, non-polar amino acid.
• In humans, glycine is a nonessential amino acid
• It is the only achiral proteinogenic amino acid.
• The name comes from the Greek word glucus or “sweet tasting”.
• Glycine is biosynthesized in the body from the amino acid serine, which is in turn derived from 3-phosphoglycerate.
• In addition to being synthesized from serine, glycine can also be derived from threonine, choline or hydroxyproline via inter-organ metabolism of the liver and kidneys.
• The predominant degradation pathway catalyzes the oxidative conversion of glycine into carbon dioxide and ammonia, with the remaining one-carbon unit transferred to folate as methylenetetrahydrofolate. It is the main catabolic pathway for glycine and it also contributes to one-carbon metabolism.
• Most proteins incorporate only small quantities of glycine, a notable exception being collagen, which contains about 35% glycine.
• In higher eukaryotes, delta-aminolevulinic acid, the key precursor to porphyrins (needed for hemoglobin and cytochromes), is biosynthesized from glycine and succinyl-CoA by the enzyme ALA synthase.
• Glycine provides the central C2N subunit of all purines, which are key constituents of DNA and RNA.
• Glycine is an inhibitory neurotransmitter in the central nervous system, especially in the spinal cord, brainstem, and retina.

• Methionine is classified as an aliphatic, non-polar amino acid.
• Methionine is a sulfur-containing, essential amino acid, and a precursor of succinyl-CoA, homocysteine, cysteine, creatine, taurine and carnitine.
• In addition to being a substrate for protein synthesis and an intermediate in transmethylation reactions, it serves as the major methyl group donor in vivo, including the methyl groups for DNA and RNA intermediates.
• It is a methyl acceptor for 5-methyltetrahydrofolate-homocysteine methyltransferase (Methionine synthase); the only reaction that allows for the recycling of this form of folate, and is also a methyl acceptor for the catabolism of betaine.
• Methionine is the metabolic precursor for Cysteine (CYS). Only the sulfur atom from Methionine is transferred to Cysteine; the carbon skeleton of Cysteine is donated by serine!
• Acute doses of Methionine can lead to increases in plasma Homocysteine (hCys), which can be used as an index of the susceptibility to cardiovascular disease.
• Can regulate metabolic processes, the innate immune system, and digestive functioning in mammals.
• Intervenes in lipid metabolism, activates endogenous antioxidant enzymes such as methionine sulfoxide reductase A, and the biosynthesis of glutathione to counteract oxidative stress.
• Methionine restriction prevents altered methionine/transmethylation metabolism, thereby decreasing DNA damage and carcinogenic processes and possibly preventing arterial, neuropsychiatric, and neurodegenerative diseases.

• A non-essential amino acid with a polar side group, naturally produced largely in the liver from phenylalanine.
• One of the few amino acids that readily passes through the blood-brain barrier.
• Starting precursor to a number of well-known neurotransmitters including: Dopamine, L-DOPA, adrenaline, noradrenaline, additionally, thyroid hormones (T3,T4) and the pigment melanin:
• Three structural isomers of L-tyrosine are known. In addition to the common amino acid L-tyrosine, which is the para isomer, there are two additional regioisomers, namely meta-tyrosine (also known as 3-hydroxyphenylalanine, L-m-tyrosine, and m-tyr) and ortho-tyrosine (o-tyr or 2-hydroxyphenylalanine), that occur in nature. The m-tyr and o-tyr isomers, which are rare, arise through non-enzymatic free-radical hydroxylation of phenylalanine under conditions of oxidative stress.
• Plasma tyrosine as a precursor of noradrenaline in the brain might be a valuable biomarker to predict brain glycogen dynamics.
• Tyrosine is mainly degraded in the liver where alterations in degradation pathways can be indicative of liver disease.
• The degraded product, homogenistic acid, can be further broken down to Acetyl-CoA and fumaric acid to feed into the TCA cycle.
• Tyrosine is not found in large concentrations throughout the body, probably because it is rapidly metabolized.
• Tyrosine is needed to synthesize the benzoquinone structure which forms part of coenzyme Q10.
• Altered Tyrosine in serum has been associated with insulin resistance as an inflammatory marker and indicative of impaired glucose metabolism prior to diagnosis of metabolic syndrome.
• Blood tyrosine levels can be a biomarker of non-alcoholic fatty liver disease.
• High tyrosine concentrations have also been detected in septic patients.

• Citrulline was first isolated from watermelon in 1914 by Japanese researchers Yotaro Koga and Ryo Odake and further codified by Mitsunori Wada of Tokyo Imperial University in 1930.
• Citrulline is a neutral, non-essential amino acid and a major precursor of Arginine in the nitric oxide (NO) cycle.
• Citrulline can be biosynthesized from carbamoyl phosphate and Ornithine.
• Associated with several diseases such as ulcerative colitis, Parkinson’s, dementia, ED, high blood pressure, diabetes and rheumatoid arthritis.
• Proteins that normally contain citrulline residues include myelin basic protein (MBP), filaggrin, and several histone proteins.
• Citrulline is also produced as a byproduct of the enzymatic production of nitric oxide from the amino acid arginine, catalyzed by a nitric oxide synthase (iNOS, eNOS, or nNOS).
• Mainly synthesized in the liver and intestinal membranes and can be a biomarker for gut health.
• CItrulline prevents neuronal cell death and protects cerebrovascular injury, therefore,  Citrulline may have a neuroprotective effect to improve cerebrovascular dysfunction
• Citrulline supplements have been claimed to promote energy levels, stimulate the immune system and help detoxify ammonia.

• Ornithine is one of the key reactants in the urea cycle, responsible for 80% of the nitrogen excretion in the body.
• Ornithine enhances liver function and helps detoxify harmful substances.
• L-Ornithine is one of the products of the action of the enzyme Arginase on L-Arginine, creating urea. In mammalian non-hepatic tissues, the main use of the urea cycle is in Arginine and Citrulline biosynthesis.
• Ornithine is converted into citrulline by action of ornithine transcarbamylase. Ornithine binds with carbamoyl phosphate which requires ammonia to be produced and then is converted into Citrulline giving off urea as a byproduct. Due to this, the conversion is one that reduces ammonia concentrations in the blood and concomitantly increases urea.
• In the liver, human ORC1 catalyzes the citrulline/ornithine exchange across the mitochondrial inner membrane, which is required for the urea cycle. Human ORC1, ORC2, and SLC25A29 are likely to be involved in the biosynthesis and transport of Arginine, which can be used as a precursor for the synthesis of NO, Agmatine, polyamines, Creatine, Glutamine, Glutamate, and Proline, as well as in the degradation of basic amino acids.
• Associated with Hepatic encephalopathy, Ornithine is dysregulated in people with chronic liver disease, such as cirrhosis or hepatitis.
• ORN may have immunomodulatory and wound-healing activities in disease (by virtue of its metabolism to L-arginine).
• Ornithine decarboxylase (ODC) initiates the polyamine biosynthetic pathway. The amount of ODC is altered in response to many growth factors, oncogenes, and tumor promoters and to changes in polyamine levels. Ornithine decarboxylase (ODC) catalyzes the first step in the polyamine biosynthetic pathway forming putrescine, which is then converted into the polyamines spermidine and spermine. Polyamine content plays important roles in both normal and neoplastic growth and alterations of polyamine synthesis via changes in ODC content occur in response to tumor promoters and carcinogens.
• OAT is a nuclear-encoded pyridoxal phosphate (vitamin B6) requiring enzyme that catalyzes the conversion of excess ornithine – generated from arginine in the urea cycle – into pyrroline-5-carboxylic acid (P5C). As ornithine is a precursor of proline, a deficiency in OAT leads to decreased formation of Proline.

• Threonine is an essential amino acid.
• Threonine is an amino acid that is both glucogenic (converted to pyruvate) and ketogenic (converted to Acetyl-CoA).
• A common pathway of Threonine degradation involves the formation of Acetyl-CoA and Glycine in the mitochondria in a two-step sequence. L-threonine dehydrogenase (Tdh) oxidizes Thr to 2-amino 3-ketobutyrate which in turn combines with CoA to form Glycine and Acetyl-CoA.
• Glycine is subsequently converted into Serine by serine hydroxymethyl transferase (ShT) , and then Serine in transformed into Pyruvate by serine dehydratase (SDh).
• However, the main flux of Thr catabolism occurs in the cytosol with generation of Propionyl-CoA from α-ketobutyrate.
• α-ketobutyrate can be converted to succinyl-CoA for oxidation in the citric acid cycle or serve as a precursor to Ophthalmic acid.  Ophthalmate can be biologically synthesized from α-ketobutyrate through consecutive reactions with gamma-glutamylcysteine synthetase and glutathione synthetase. Ophthalmic acid can be used as a biomarker for oxidative stress when the depletion of glutathione occurs.
• Acetyl-CoA is required for carbon recyclization, whereas Glycine and its subsequent degradation are required for nitrogen recyclization.
• The deaminase/dehydratase or Propionyl-CoA pathway is an anaerobic pathway that produces Propionyl-phosphate, which contributes to ATP synthesis.
• Threonine is a limiting amino acid (the others are Lysine, Methionine, and Tryptophan). Limiting amino acids are found in the shortest supply from digested proteins.
• An alternative pathway from 2-amino-3-oxobutanoate is the Glyoxalase pathway which increases with high Glutathione concentrations, while the preference to α-ketobutyrate to Ophthalmic acid increases with low glutathione concentration.
• Most nitrogen from metabolized Thr is excreted into urine as urea. Aminoacetone and d-lactate are minor metabolic products whose urinary excretion increases disproportionately with high Thr intake and when availability of free CoA is limited.
• Threonine is an immunostimulant which promotes the growth of thymus gland. It also can probably promote cell immune defense function.

• Lysine is an essential basic amino acid.
• Lysine is often used as a supplement for muscle growth.
• Methylated forms of lysine lead to carnitine synthesis (minor pathway, as most of carnitine is from diet).
• Common modifications are acetylation, methylation, hydroxylation (Collagen) and O-glycosylation. Other modified forms may reflect protein aging (advanced glycation end products or AGES).
• Modification of histones includes Lysine modifications as a major regulatory factor.
• The major degradation pathway is the Saccharopine pathway within liver mitochondria leading to Glutaryl-CoA and finally to Acetyl-CoA.  Lysine and Leucine are the only two solely ketogenic amino acids.
• A secondary degradation pathway, the Pipecolate pathway, converges with the Saccharopine pathway. This pathway primarily occurs within the peroxisome, the site of β-oxidation of fatty acids, biosynthesis of phospholipids and metabolism of reactive oxygen species.
• Metabolites of Lysine, alpha amino adipic acid (AAA) from the Saccharopine pathway and pipecolic acid (PA) from the Pipecolate pathway are potential biomarkers. AAA is a potential predictor of the development of diabetes and modulator of glucose homeostasis. PA has been described as a biomarker for peroxisome dysfunction and certain cancers.

• Proline is a nonessential, sweet tasting amino acid. It contains a secondary α-imino group and is sometimes called an α-imino acid.
• Proline, and its secondary metabolite hydroxyproline, constitute a third of the total amino acids found in collagen. Lysine, proline, hydroxyproline, and vitamin C are all important in the synthesis of collagen for skin, bones, tendons, and cartilage. Proline is also commonly found in antimicrobial peptides, salivary proteins and cornified epidermis.
• In addition to dietary sources, proline can be synthesized from glutamate/glutamine, arginine, and ornithine. It can also be synthesized within enterocytes from degradation of small peptides.
• Proline has many physiologic functions, including:
  • regulation of gene expression via mTOR activation (integrating nutrient and growth factor signaling in cells)
  • cellular redox reactions
  • amino acid (hydroxyproline and arginine) generation, and protein synthesis
  • ROS scavenging (antioxidant activity)
• Small proline peptides can be bioactive: Pro-Pro induces NGF, Gly-Pro-Glu, Gly-Pro are anti inflammatory, while Pro-Gly-Pro has been found to be neuroprotective.
• Proline can be an energy source for bacteria and cancer cells by conversion to 2-OG and entering the TCA cycle.
• In addition to its role as ROS scavenging, Proline can act as an osmolyte and protein chaperone, further abating cellular stress
• Proline can participate in cell signaling by affecting levels of Glu and Arg (mTOR pathway) and promote Histone methylation and levels of 5-methylcytosine and 5-Hydroxymethylcytosine.
• Proline can stimulate growth of stem and tumor cells.