Amino Acid Disorders

Amino Acids -- Cofactors and Relationships

Introduction

All tissues have some capability for synthesis of the non-essential amino acids, amino acid remodeling, and conversion of non-amino acid carbon skeletons into amino acids and other derivatives that contain nitrogen. However, the liver is the major site of nitrogen metabolism in the body. In times of dietary surplus, the potentially toxic nitrogen of amino acids is eliminated via transaminations, deamination, and urea formation; the carbon skeletons are generally conserved as carbohydrate, via gluconeogenesis, or as fatty acid via fatty acid synthesis pathways. In this respect amino acids fall into three categories: glucogenic, ketogenic, or glucogenic and ketogenic. Glucogenic amino acids are those that give rise to a net production of pyruvate or TCA cycle intermediates, such as a-ketoglutarate or oxaloacetate, all of which are precursors to glucose via gluconeogenesis. All amino acids except lysine and leucine are at least partly glucogenic. Lysine and leucine are the only amino acids that are solely ketogenic, giving rise only to acetylCoA or acetoacetylCoA, neither of which can bring about net glucose production.

A small group of amino acids comprised of isoleucine, phenylalanine, threonine, tryptophan, and tyrosine give rise to both glucose and fatty acid precursors and are thus characterized as being glucogenic and ketogenic. Finally, it should be recognized that amino acids have a third possible fate. During times of starvation the reduced carbon skeleton is used for energy production, with the result that it is oxidized to CO₂ and H₂O. back to the top

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Essential vs. Nonessential Amino Acids

Nonessential	Essential
Alanine	Arginine*
Asparagine	Histidine
Aspartate	Isoleucine
Cysteine	Leucine
Glutamate	Lysine
Glutamine	Methionine*
Glycine	Phenylalanine*
Proline	Threonine
Serine	Tyrptophan
Tyrosine	Valine

*The amino acids arginine, methionine and phenylalanine are considered essential for reasons not directly related to lack of synthesis. Arginine is synthesized by mammalian cells but at a rate that is insufficient to meet the growth needs of the body and the majority that is synthesized is cleaved to form urea. Methionine is required in large amounts to produce cysteine if the latter amino acid is not adequately supplied in the diet. Similarly, phenyalanine is needed in large amounts to form tyrosine if the latter is not adequately supplied in the diet.

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Amino Acid Biosynthesis

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Glutamate and Aspartate

Glutamate and aspartate are synthesized from their widely distributed a-keto acid precursors by simple 1-step transamination reactions. The former catalyzed by glutamate dehydrogenase and the latter by aspartate aminotransferase, AST.

Reactions of glutamate dehydrogenase

Aspartate is also derived from asparagine through the action of asparaginase. The importance of glutamate as a common intracellular amino donor for transamination reactions and of aspartate as a precursor of ornithine for the urea cycle is described in the Nitrogen Metabolism page. back to the top

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Alanine and the Glucose-Alanine Cycle

Aside from its role in protein synthesis, alanine is second only to glutamine in prominence as a circulating amino acid. In this capacity it serves a unique role in the transfer of nitrogen from peripheral tissue to the liver. Alanine is transferred to the circulation by many tissues, but mainly by muscle, in which alanine is formed from pyruvate at a rate proportional to intracellular pyruvate levels. Liver accumulates plasma alanine, reverses the transamination that occurs in muscle, and proportionately increases urea production. The pyruvate is either oxidized or converted to glucose via gluconeogenesis. When alanine transfer from muscle to liver is coupled with glucose transport from liver back to muscle, the process is known as the glucose-alanine cycle. The key feature of the cycle is that in 1 molecule, alanine, peripheral tissue exports pyruvate and ammonia (which are potentially rate-limiting for metabolism) to the liver, where the carbon skeleton is recycled and most nitrogen eliminated.

There are 2 main pathways to production of muscle alanine: directly from protein degradation, and via the transamination of pyruvate by alanine transaminase, ALT (also referred to as serum glutamate-pyruvate transaminase, SGPT).

glutamate + pyruvate <-------> a-KG + alanine

The glucose-alanine cycle is used primarily as a mechanism for skeletal muscle to eliminate nitrogen while replenishing its energy supply. Glucose oxidation produces pyruvate which can undergo transamination to alanine. This reaction is catalyzed by alanine transaminase, ALT (ALT used to be called serum glutamate-pyruvate transaminase, SGPT). Additionally, during periods of fasting, skeletal muscle protein is degraded for the energy value of the amino acid carbons and alanine is a major amino acid in protein. The alanine then enters the blood stream and is transported to the liver. Within the liver alanine is converted back to pyruvate which is then a source of carbon atoms for gluconeogenesis. The newly formed glucose can then enter the blood for delivery back to the muscle. The amino group transported from the muscle to the liver in the form of alanine is converted to urea in the urea cycle and excreted.

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Cysteine Biosynthesis

The sulfur for cysteine synthesis comes from the essential amino acid methionine. A condensation of ATP and methionine catalyzed by methionine adenosyltransferase yields S-adenosylmethionine (SAM or AdoMet).

Biosynthesis of S-adenosylmethionine, SAM

SAM serves as a precurosor for numerous methyl transfer reactions (e.g. the conversion of norepinephrine to epinenephrine, see Specialized Products of Amino Acids). The result of methyl transfer is the conversion of SAM to S-adenosylhomocysteine. S-adenosylhomocysteine is then cleaved by adenosylhomocyteinase to yield homocysteine and adenosine. Homocysteine can be converted back to methionine by methionine synthase, a reaction that occurs under methionine-sparing conditions and requires N⁵-methyl-tetrahydrofolate as methyl donor. This reaction was discussed in the context of vitamin B₁₂-requiring enzymes in the Vitamins page.

Transmethylation reactions employing SAM are extremely important, but in this case the role of S-adenosylmethionine in transmethylation is secondary to the production of homocysteine (essentially a by-product of transmethylase activity). In the production of SAM all phosphates of an ATP are lost: one as P_i and two as PP_i. It is adenosine which is transferred to methionine and not AMP.

In cysteine synthesis, homocysteine condenses with serine to produce cystathionine, which is subsequently cleaved by cystathionase to produce cysteine and a-ketobutyrate. The sum of the latter two reactions is known as trans-sulfuration.

Cysteine is used for protein synthesis and other body needs, while the a-ketobutyrate is decarboxylated and converted to propionyl-CoA. While cysteine readily oxidizes in air to form the disulfide cystine, cells contain little if any free cystine because the ubiquitous reducing agent, glutathione effectively reverses the formation of cystine by a non-enzymatic reduction reaction.

Utilization of methionine in the synthesis of cysteine

The 2 key enzymes of this pathway, cystathionine synthase and cystathionase (cystathionine lyase), both use pyridoxal phosphate as a cofactor, and both are under regulatory control. Cystathionase is under negative allosteric control by cysteine, as well, cysteine inhibits the expression of the cystathionine synthase gene.

Genetic defects are known for both the synthase and the lyase. Missing or impaired cystathionine synthase leads to homocystinuria and is often associated with mental retardation, although the complete syndrome is multifaceted and many individuals with this disease are mentally normal. Some instances of genetic homocystinuria respond favorably to pyridoxine therapy, suggesting that in these cases the defect in cystathionine synthase is a decreased affinity for the cofactor. Missing or impaired cystathionase leads to excretion of cystathionine in the urine but does not have any other untoward effects. Rare cases are known in which cystathionase is defective and operates at a low level. This genetic disease leads to methioninuria with no other consequences. back to the top

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Tyrosine Biosynthesis

Tyrosine is produced in cells by hydroxylating the essential amino acid phenylalanine. This relationship is much like that between cysteine and methionine. Half of the phenylalanine required goes into the production of tyrosine; if the diet is rich in tyrosine itself, the requirements for phenylalanine are reduced by about 50%.

Phenylalanine hydroxylase is a mixed-function oxygenase: one atom of oxygen is incorporated into water and the other into the hydroxyl of tyrosine. The reductant is the tetrahydrofolate-related cofactor tetrahydrobiopterin, which is maintained in the reduced state by the NADH-dependent enzyme dihydropteridine reductase (DHPR).

Biosynthesis of tyrosine from phenylalanine

Missing or deficient phenylalanine hydroxylase results in hyperphenylalaninemia. Hyperphenylalaninemia is defined as a plasma phenylalanine concentration greater than 2mg/dL (120mM). The most widely recognized hyperphenylalaninemia (and most severe) is the genetic disease known as phenlyketonuria (PKU). Patients suffering from PKU have plasma phenylalanine levels >1000mM, whereas the non-PKU hyperphenylalaninemias exhibit levels of plasma phenylalanine <1000mM. Untreated PKU leads to severe mental retardation. The mental retardation is caused by the accumulation of phenylalanine, which becomes a major donor of amino groups in aminotransferase activity and depletes neural tissue of a-ketoglutarate. This absence of a-ketoglutarate in the brain shuts down the TCA cycle and the associated production of aerobic energy, which is essential to normal brain development.

The product of phenylalanine transamination, phenylpyruvic acid, is reduced to phenylacetate and phenyllactate, and all 3 compounds appear in the urine. The presence of phenylacetate in the urine imparts a "mousy" odor. If the problem is diagnosed early, the addition of tyrosine and restriction of phenylalanine from the diet can minimize the extent of mental retardation.

Because of the requirement for tetrahydrobiopterin in the function of phenylalanine hydroxylase, deficiencies in DHPR can manifest with hyperphenylalaninemia. However, since tetrahydrobiopterin is a cofactor in several other enzyme catalyzed reactions (e.g. see the synthesis of the tyrosine- and tryptophan-derived neurotransmitters as well as nitric oxide in Specialized Products of Amino Acids), the effects of missing or defective DHPR cause even more severe neurological difficulties than those usually associated with PKU caused by deficient phenylalanine hydroxylase activity. back to the top

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Ornithine and Proline Biosynthesis

Glutamate is the precursor of both proline and ornithine, with glutamate semialdehyde being a branch point intermediate leading to one or the other of these 2 products. While ornithine is not one of the 20 amino acids used in protein synthesis, it plays a significant role as the acceptor of carbamoyl phosphate in the urea cycle. Ornithine serves an additional important role as the precursor for the synthesis of the polyamines. The production of ornithine from glutamate is important when dietary arginine, the other principal source of ornithine, is limited.

The fate of glutamate semialdehyde depends on prevailing cellular conditions. Ornithine production occurs from the semialdehyde via a simple glutamate-dependent transamination, producing ornithine. When arginine concentrations become elevated, the ornithine contributed from the urea cycle plus that from glutamate semialdehyde inhibit the aminotransferase reaction, with accumulation of the semialdehyde as a result. The semialdehyde cyclizes spontaneously to D¹pyrroline-5-carboxylate which is then reduced to proline by an NADPH-dependent reductase. back to the top

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Serine Biosynthesis

The main pathway to serine starts with the glycolytic intermediate 3-phosphoglycerate. An NADH-linked dehydrogenase converts 3-phosphoglycerate into a keto acid, 3-phosphopyruvate, suitable for subsequent transamination. Aminotransferase activity with glutamate as a donor produces 3-phosphoserine, which is converted to serine by phosphoserine phosphatase. back to the top

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Glycine Biosynthesis

The main pathway to glycine is a 1-step reaction catalyzed by serine hydroxymethyltransferase. This reaction involves the transfer of the hydroxymethyl group from serine to the cofactor tetrahydrofolate (THF), producing glycine and N⁵,N¹⁰-methylene-THF. Glycine produced from serine or from the diet can also be oxidized by glycine cleavage complex, GCC, to yield a second equivalent of N⁵,N¹⁰-methylene-tetrahydrofolate as well as ammonia and CO₂.

Glycine is involved in many anabolic reactions other than protein synthesis including the synthesis of purine nucleotides, heme, glutathione, creatine and serine. back to the top

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Aspartate/Asparagine and Glutamate/Glutamine Biosynthesis

Glutamate is synthesized by the reductive amination of a-ketoglutarate catalyzed by glutamate dehydrogenase; it is thus a nitrogen-fixing reaction. In addition, glutamate arises by aminotransferase reactions, with the amino nitrogen being donated by a number of different amino acids. Thus, glutamate is a general collector of amino nitrogen.

Aspartate is formed in a transamintion reaction catalyzed by aspartate transaminase, AST. This reaction uses the aspartate a-keto acid analog, oxaloacetate, and glutamate as the amino donor. Aspartate can also be formed by deamination of asparagine catalyzed by asparaginase.

Asparagine synthetase and glutamine synthetase, catalyze the production of asparagine and glutamine from their respective a-amino acids. Glutamine is produced from glutamate by the direct incorporation of ammonia; and this can be considered another nitrogen fixing reaction. Asparagine, however, is formed by an amidotransferase reaction.

Aminotransferase reactions are readily reversible. The direction of any individual transamination depends principally on the concentration ratio of reactants and products. By contrast, transamidation reactions, which are dependent on ATP, are considered irreversible. As a consequence, the degradation of asparagine and glutamine take place by a hydrolytic pathway rather than by a reversal of the pathway by which they were formed. As indicated above, asparagine can be degraded to aspartate. back to the top

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Amino Acid Catabolism

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Glutamine/Glutamate and Asparagine/Aspartate Catabolism

Glutaminase is an important kidney tubule enzyme involved in converting glutamine (from liver and from other tissue) to glutamate and NH₃⁺, with the NH₃⁺ being excreted in the urine. Glutaminase activity is present in many other tissues as well, although its activity is not nearly as prominent as in the kidney. The glutamate produced from glutamine is converted to a-ketoglutarate, making glutamine a glucogenic amino acid.

Asparaginase is also widely distributed within the body, where it converts asparagine into ammonia and aspartate. Aspartate transaminates to oxaloacetate, which follows the gluconeogenic pathway to glucose.

Glutamate and aspartate are important in collecting and eliminating amino nitrogen via glutamine synthetase and the urea cycle, respectively. The catabolic path of the carbon skeletons involves simple 1-step aminotransferase reactions that directly produce net quantities of a TCA cycle intermediate. The glutamate dehydrogenase reaction operating in the direction of a-ketoglutarate production provides a second avenue leading from glutamate to gluconeogenesis. back to the top

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Alanine Catabolism

Alanine is also important in intertissue nitrogen transport as part of the glucose-alanine cycle. Alanine's catabolic pathway involves a simple aminotransferase reaction that directly produces pyruvate. Generally pyruvate produced by this pathway will result in the formation of oxaloacetate, although when the energy charge of a cell is low the pyruvate will be oxidized to CO₂ and H₂O via the PDH complex and the TCA cycle. This makes alanine a glucogenic amino acid. back to the top

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Arginine, Ornithine and Proline Catabolism

The catabolism of arginine begins within the context of the urea cycle. It is hydrolyzed to urea and ornithine by arginase.

Ornithine, in excess of urea cycle needs, is transaminated to form glutamate semialdehyde. Glutamate semialdehyde can serve as the precursor for proline biosynthesis as described above or it can be converted to glutamate.

Proline catabolism is a reversal of its synthesis process.

The glutamate semialdehyde generated from ornithine and proline catabolism is oxidized to glutamate by an ATP-independent glutamate semialdehyde dehydrogenase. The glutamate can then be converted to a-ketoglutarate in a transamination reaction. Thus arginine, ornithine and proline, are glucogenic. back to the top

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Serine Catabolism

The conversion of serine to glycine and then glycine oxidation to CO₂ and NH₃, with the production of two equivalents of N⁵,N¹⁰-methyleneTHF, was described above. Serine can be catabolized back to the glycolytic intermediate, 3-phosphoglycerate, by a pathway that is essentially a reversal of serine biosynthesis. However, the enzymes are different. Serine can also be converted to pyruvate through a deamination reaction catalyzed by serine/threonine dehydratase. back to the top

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Threonine Catabolism

There are at least 3 pathways for threonine catabolism. One involves a pathway initiated by threonine dehydrogenase yielding a-amino-b-ketobutyrate. The a-amino-b-ketobutyrate is either converted to acetyl-CoA and glycine or spontaneously degrades to aminoacetone which is converted to pyruvate. The second pathway involves serine/threonine dehydratase yielding a-ketobutyrate which is further catabolized to propionyl-CoA and finally the TCA cycle intermediate, succinyl-CoA. The third pathway utilizes threonine aldolase. The products of this reaction are both ketogenic (acetyl-CoA) and glucogenic (pyruvate). back to the top

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Glycine Catabolism

Glycine is classified as a glucogenic amino acid, since it can be converted to serine by serine hydroxymethyltransferase, and serine can be converted back to the glycolytic intermediate, 3-phosphoglycerate or to pyruvate by serine/threonine dehydratase. Nevertheless, the main glycine catabolic pathway leads to the production of CO₂, ammonia, and one equivalent of N⁵,N¹⁰-methyleneTHF by the mitochondrial glycine cleavage complex. back to the top

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Cysteine Catabolism

There are several pathways for cysteine catabolism. The simplest, but least important pathway is catalyzed by a liver desulfurase and produces hydrogen sulfide, (H₂S) and pyruvate. The major catabolic pathway in animals is via cysteine dioxygenase that oxidizes the cysteine sulfhydryl to sulfinate, producing the intermediate cysteinesulfinate. Cysteinesulfinate can serve as a biosynthetic intermediate undergoing decarboxylation and oxidation to produce taurine. Catabolism of cysteinesulfinate proceeds through transamination to b-sulfinylpyruvate which then undergoes desulfuration yielding bisulfite, (HSO₃^-) and the glucogenic product, pyruvate. The enzyme sulfite oxidase uses O₂ and H₂O to convert HSO₃^- to sulfate, (SO₄^-) and H₂O₂. The resultant sulfate is used as a precursor for the formation of 3'-phosphoadenosine-5'-phosphosulfate, PAPS.

PAPS is used for the transfer of sulfate to biological molecules such as the sugars of the glycosphingolipids.

Other than protein, the most important product of cysteine metabolism is the bile salt precursor taurine, which is used to form the bile acid conjugates taurocholate and taurochenodeoxycholate.

The enzyme cystathionase can also transfer the sulfur from one cysteine to another generating thiocysteine and pyruvate. Transamination of cysteine yields b-mercaptopyruvate which then reacts with sulfite, (SO₃^2-), to produce thiosulfate, (S₂O₃^2-) and pyruvate. Both thiocysteine and thiosulfate can be used by the enzyme rhodanese to incorporate sulfur into cyanide, (CN^-), thereby detoxifying the cyanide to thiocyanate. back to the top

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Methionine Catabolism

The principal fates of the essential amino acid methionine are incorporation into polypeptide chains, and use in the production of a-ketobutyrate and cysteine via SAM as described above. The transulfuration reactions that produce cysteine from homocysteine and serine also produce a-ketobutyrate, the latter being converted to succinyl-CoA.

Regulation of the methionine metabolic pathway is based on the availability of methionine and cysteine. If both amino acids are present in adequate quantities, SAM accumulates and is a positive effector on cystathionine synthase, encouraging the production of cysteine and a-ketobutyrate (both of which are glucogenic). However, if methionine is scarce, SAM will form only in small quantities, thus limiting cystathionine synthase activity. Under these conditions accumulated homocysteine is remethylated to methionine, using N⁵-methylTHF and other compounds as methyl donors. back to the top

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Valine, Leucine and Isoleucine Catabolism

This group of essential amino acids are identified as the branched-chain amino acids, BCAAs. Because this arrangement of carbon atoms cannot be made by humans, these amino acids are an essential element in the diet. The catabolism of all three compounds initiates in muscle and yields NADH and FADH₂ which can be utilized for ATP generation. The catabolism of all three of these amino acids uses the same enzymes in the first two steps. The first step in each case is a transamination using a single BCAA aminotransferase, with a-ketoglutarate as amine acceptor. As a result, three different a-keto acids are produced and are oxidized using a common branched-chain a-keto acid dehydrogenase, yielding the three different CoA derivatives. Subsequently the metabolic pathways diverge, producing many intermediates.

The principal product from valine is propionylCoA, the glucogenic precursor of succinyl-CoA. Isoleucine catabolism terminates with production of acetylCoA and propionylCoA; thus isoleucine is both glucogenic and ketogenic. Leucine gives rise to acetylCoA and acetoacetylCoA, and is thus classified as strictly ketogenic.

There are a number of genetic diseases associated with faulty catabolism of the BCAAs. The most common defect is in the branched-chain a-keto acid dehydrogenase. Since there is only one dehydrogenase enzyme for all three amino acids, all three a-keto acids accumulate and are excreted in the urine. The disease is known as Maple syrup urine disease because of the characteristic odor of the urine in afflicted individuals. Mental retardation in these cases is extensive. Unfortunately, since these are essential amino acids, they cannot be heavily restricted in the diet; ultimately, the life of afflicted individuals is short and development is abnormal The main neurological problems are due to poor formation of myelin in the CNS. back to the top

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Phenylalanine and Tyrosine Catabolism

Phenylalanine normally has only two fates: incorporation into polypeptide chains, and production of tyrosine via the tetrahydrobiopterin-requiring phenylalanine hydroxylase. Thus, phenylalanine catabolism always follows the pathway of tyrosine catabolism. The main pathway for tyrosine degradation involves conversion to fumarate and acetoacetate, allowing phenylalanine and tyrosine to be classified as both glucogenic and ketogenic.

Tyrosine is equally important for protein biosynthesis as well as an intermediate in the biosynthesis of several physiologically important metabolites e.g. dopamine, norepinephrine and epinephrine (see Specialized Products of Amino Acids).

As in phenylketonuria (deficiency of phenylalanine hydroxylase), deficiency of tyrosine aminotransferase (TAT) leads to hypertyrosinemia and the urinary excretion of tyrosine and the catabolic intermediates between phenylalanine and tyrosine. The adverse neurological symptoms are similar for phenylalanine hydroxylase and TAT deficiencies. In addition, hypertyrosinemia leads to painful corneal eruptions and photophobia.

The first genetic disease ever recognized, alcaptonuria, was demonstrated to be the result of a defect in phenylalanine and tyrosine catabolism. Alkaptonuria is caused by defective homogentisic acid oxidase. Homogentisic acid accumulation is relatively innocuous, causing urine to darken on exposure to air, but no life-threatening effects accompany the disease. back to the top

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Lysine Catabolism

Lysine catabolism is unusual in the way that the e-amino group is transferred to a-ketoglutarate and into the general nitrogen pool. The reaction is a transamination in which the e-amino group is transferred to the a-keto carbon of a-ketoglutarate forming the metabolite, saccharopine. Unlike the majority of transamination reactions, this one does not employ pyridoxal phosphate as a cofactor. Saccharopine is immediately hydrolyzed by the enzyme a-aminoadipic semialdehyde synthase in such a way that the amino nitrogen remains with the a-carbon of a-ketoglutarate, producing glutamate and a-aminoadipic semialdehyde. Because this transamination reaction is not reversible, lysine is an essential amino acid. The ultimate end-product of lysine catabolism is acetoacetyl-CoA

Genetic deficiencies in the enzyme a-aminoadipic semialdehyde synthase have been observed in individuals who excrete large quantities of urinary lysine and some saccharopine. The lysinemia and associated lysinuria are benign. Other serious disorders associated with lysine metabolism are due to failure of the transport system for lysine and the other dibasic amino acids across the intestinal wall. Lysine is essential for protein synthesis; a deficiencies of its transport into the body can cause seriously diminished levels of protein synthesis. Probably more significant however, is the fact that arginine is transported on the same dibasic amino acid carrier, and resulting arginine deficiencies limit the quantity of ornithine available for the urea cycle. The result is severe hyperammonemia after a meal rich in protein. The addition of citrulline to the diet prevents the hyperammonemia.

Lysine is also important as a precursor for the synthesis of carnitine, required for the transport of fatty acids into the mitochondria for oxidation. Free lysine does not serve as the precursor for this reaction, rather the modified lysine found in certain proteins. Some proteins modify lysine to trimethyllysine using SAM as the methyl donor to transfer methyl groups to the e-amino of the lysine side chain. Hydrolysis of proteins containing trimethyllysine provide the substrate for the subsequent conversion to carnitine back to the top

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Histidine Catabolism

Histidine catabolism begins with release of the a-amino group catalyzed by histidase, introducing a double bond into the molecule. As a result, the deaminated product, urocanate, is not the usual a-keto acid associated with loss of a-amino nitrogens. The end product of histidine catabolism is glutamate, making histidine one of the glucogenic amino acids.

Another key feature of histidine catabolism is that it serves as a source of ring nitrogen to combine with tetrahydrofolate (THF), producing the 1-carbon THF intermediate known as N⁵-formiminoTHF. The latter reaction is one of two routes to N⁵-formiminoTHF.

The principal genetic deficiency associated with histidine metabolism is absence or deficiency of the first enzyme of the pathway, histidase. The resultant histidinemia is relatively benign. The disease, which is of relatively high incidence (1 in 10,000), is most easily detected by the absence of urocanate from skin and sweat, where it is normally found in relative abundance.

Decarboxylation of histidine in the intestine by bacteria gives rise to histamine. Similarly, histamine arises in many tissues by the decarboxylation of histidine, which in excess causes constriction or dilation of various blood vessels. The general symptoms are those of asthma and various allergic reactions. back to the top

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Tryptophan Catabolism

A number of important side reactions occur during the catabolism of tryptophan on the pathway to acetoacetate. The first enzyme of the catabolic pathway is an iron porphyrin oxygenase that opens the indole ring. The latter enzyme is highly inducible, its concentration rising almost 10-fold on a diet high in tryptophan.

Kynurenine is the first key branch point intermediate in the pathway. Kynurenine undergoes deamniation in a standard transamination reaction yielding kynurenic acid. Kynurenic acid and metabolites have been shown to act as antiexcitotoxics and anticonvulsives.

A second side branch reaction produces anthranilic acid plus alanine. Another equivalent of alanine is produced further along the main catabolic pathway, and it is the production of these alanine residues that allows tryptophan to be classified among the glucogenic and ketogenic amino acids.

The second important branch point converts kynurenine into 2-amino-3-carboxymuconic semialdehyde, which has two fates. The main flow of carbon elements from this intermediate is to glutarate. An important side reaction in liver is a transamination and several rearrangements to produce limited amounts of nicotinic acid, which leads to production of a small amount of NAD⁺ and NADP⁺

Aside from its role as an amino acid in protein biosynthesis, tryptophan also serves as a precursor for the synthesis of serotonin and melatonin. These products are discussed in Specialized Products of Amino Acids back to the top

Amino Acids -- Cofactors and Relationships

Amino acid profiling clinical guidelines for determination of preferred specimen choice.

From: Townsend Letter for Doctors and Patients | Date: 12/1/2003 | Author: Feinerman, Judy

Introduction

Profiling of amino acids in plasma and urine has been used to elucidate a rapidly growing number of aminoacidopathies since the introduction of partition chromatography methods in 1945. (1) The question of whether plasma or urine may be the preferred specimen choice for amino acid testing is a frequent clinical concern in the evaluation of a patient's amino acid status. An informed decision must involve what principal clinical answers are sought and which amino acids are being tested. To state categorically that profiling of amino acids is best performed on plasma or urine is to oversimplify. The question of preferred specimen can be answered only when it is addressed to specific amino acids or to the specific type of information desired.

One commonly practiced method to judge the relative value of results from two specimen types is to ask which specimen has been most used for scientific studies. The majority of published studies have used plasma as the specimen for analysis (approximately a 3:1 plasma/urine ratio). (2) This is primarily because most investigations have been concerned with essential amino acid status. Urine is typically reserved for studies of dietary protein intake, digestive adequacy, bone loss and muscle protein catabolic states. The aminoacidemias and aminoacidurias associated with metabolic disorders are approximately equally divided in the published research. Inherited metabolic disorders generally result in extreme elevations, and the abnormality is easily detected in either specimen type. The branched chain amino acids (BCAAs), for example, are elevated in both plasma and urine in maple syrup urine disease. The newer application of amino acid profiling of older children and adults to determine amino acid status in chronic degenerative diseases is more pertinent for this article.

Amino Acid Dynamics

Plasma

A fasting plasma specimen reflects the state of the dynamic flux of amino acids leaving sites like skeletal muscle and flowing into sites of utilization in liver, brain, and other tissues (Figure 1). Amino acid levels in plasma reach their homeostatic balance point making a fasting specimen ideal for repeated measures to monitor progress. The principal factors effecting changes over time are dietary intake, digestive efficiency, hepatic uptake, and the ability of skeletal muscle to maintain sufficient rates of transamination. The amount of an essential amino acid in plasma determines the rate of any dependent process in the tissues. For example, low plasma tryptophan results in reduced formation of serotonin in the brain. (3)

[FIGURE 1 OMITTED]

Urine

Twenty-four hour urinary amino acids have been measured in the evaluation of specific clinical conditions. In many cases the research represents disruption of normal amino acid metabolism as a result of the disease process and the shortterm changes in plasma amino concentration. A 24 hour urine amino acid analysis reveals amino acid metabolism throughout the period of metabolic stress of digestion and daily activity. This aspect is of particular value for evaluating those amino acids that primarily reveal tissue degradation, such as hydroxylysine and hydroxyproline, which are released from collagen of connective tissue and bone.

Clinical Categories Assessed via Amino Acid Profiling

Gastrointestinal Function

Amino acids and their derivatives provide some useful markers that can reflect gastrointestinal function, specifically protein digestion capacity. The normal digestion of dietary protein results in free-form amino acids and short-chain peptides. Recent (i.e. 3 days) dietary protein intake has little influence on plasma amino acid profiling. A fasting plasma specimen highlights the dynamics of homeostatic maintenance of the free form amino acid pool, which is remarkably stable, independent of diet. In contrast, 24-hour urine analysis of amino acids more clearly elucidates recent protein intake based on the activities of the previous 24-48 hours. In feeding young men a protein mixture (patterned after egg protein) specifically devoid of methionine and cystine for eight days, fasting plasma methionine and cystine levels showed little change during the eight-day period. Urinary levels of methionine decreased markedly within a few days after feeding of the experimental diet, suggesting urinary amino acids are more useful to monitor short-term changes in protein intake. However, plasma levels are the preferred way to assess long-term adequacy and dynamics of amino acid utilization. (4), (5)

Abnormal amino acid patterns can correspond to what may be wrong in protein nourishment or digestion. The patterns seen may reflect dietary protein deficiency, and/or maldigestion. Hyperaminoacidemias and hyperaminoacidurias typically indicate genetically inherited metabolic enzyme impairments or transport problems, not digestive enzyme impairments or insufficient stomach acid secretion. Low levels measured among the essential and some of the semi-essential amino acids reflect dietary and uptake problems. For example, the essential amino acid histidine is required to make histamine, an important digestive function, which occurs early in the stomach. Low plasma or urinary histidine may then suggest impaired ability for optimal protein digestion. Low levels of the aromatic amino acids--tryptophan, phenylalanine, and tyrosine--may indicate inadequate stomach acid (HCl) secretion as this is critical to activate pepsin-mediated protein digestion. Clinicians must remember to consider renal function in evaluation of urinary amino acids, however, as patients with renal failure may show decreased creatinine measurements, resulting in skewed levels upon measurement and subsequent interpretation.

In select circumstances, elevations in urine amino acids can serve as disease markers. For example, hydroxyproline appears to be a hallmark for celiac disease and other malabsorption states, with the greatest hydroxyproline excretion occurring in those patients with the most pronounced steatorrhea. (6) This is believed to reflect an increased turnover of collagen and may be related to the osteomalacia sometimes accompanying malabsorption.

Cellular Energy Production

Fatigue may be one of the most commonly reported medical complaints heard by clinicians today. Amino acid deficits may be related to the cause of fatigue. Amino acids undergo transamination reactions which supply intermediates to the citric acid cycle in order to facilitate mitochondrial oxidative phosphorylation; or more meaningful to the patient, cellular energy production. (7) Citric acid cycle intermediates are produced from aspartate, tyrosine, phenylalanine, isoleucine, valine, methionine, glutamine, histidine, arginine, proline, glutamate, and beta-alanine. Despite a significant lack of clinical research on urinary amino acids for assessment of fatigue syndromes, one study of interest has emerged in which strong associations of beta-alanine in urine with chronic fatigue symptom expression has suggested a possible molecular basis in the development of an objective test for chronic fatigue syndrome. (8)

There has been increasing interest in the mechanisms behind central (brain-related) fatigue, particularly in relation to changes in brain monoamine metabolism and the influence of specific amino acids on fatigue. (9) Central fatigue has been implicated in both chronic fatigue syndrome (10) and postoperative fatigue. (11) Evidence continues to emerge demonstrating increased ratios of plasma tryptophan to branched-chain amino acids may be responsible for the central fatigue seen in long, sustained exercise and post-surgery. (12-14) The literature abounds with clinical studies on fatigue, with an overwhelming preponderance of these studies utilizing measurements of plasma amino acids.

Detoxification

Determination of detoxification capacity is an important clinical issue for many patients with chronic illness, especially if suspected to be environmentally induced. While the role of amino acids in phase II hepatic conjugation reactions is well established, assessment of amino acid availability for optimal conjugation warrants further clarification. Of particular interest are the amino acids, glycine, cysteine, glutamic acid, taurine, methionine, glutamine, and aspartate. As urinary levels are best reserved for evaluation of short-term dietary changes or protein digestion capability, profiling of plasma pool availability is relevant to detoxification capacity. Highly targeted urinary amino acid derivatives however, such as hydroxyproline, may serve as useful biomarkers of exposure to pollution. (15), (16)

Detoxification of ammonia is an important responsibility of the liver. The urea cycle involves a series of biochemical steps in which ammonia, a waste product of protein metabolism, is removed from the blood, converted to urea, and excreted in urine. In urea cycle dysfunction, ammonia (a highly toxic substance) accumulates, and is not removed from the body efficiently. Ammonia accumulation in the general circulation may go on to reach the brain, where it may cause neurologic damage and in severe cases can lead to irreversible brain damage and/or death. Mild hyperammonemia conditions are often seen as low plasma glutamic acid levels and high glutamine levels. (17) Symptoms include headache, irritability, fatigue, mental confusion, poor concentration, and food intolerance reactions, particularly to high protein foods. At the other end of the spectrum of urea cycle dysfunction are inherited urea cycle disorders. A urea cycle disorder is a distinct genetic disease caused by a deficiency of one of the enzymes in the urea cycle, which is responsible for removing ammonia from the bloodstream.

Removal of ammonia via the urea cycle can be an important clinical issue. A case of infantile autism has been associated with inefficient ammonia detoxification as evidenced by elevated plasma ammonia and elevated plasma and urine levels of gamma-aminobutyric acid (GABA). It was postulated that elevated ammonia levels may result in higher GABA concentrations and that a link between plasma ammonia and plasma GABA exist where the concentration of GABA in the plasma is directly related to plasma ammonia concentration. (18) Meanwhile, in elderly subjects, patients with Alzheimer's disease (vs. healthy controls) exhibited altered plasma ornithine and arginine concentrations, (19) perhaps highlighting the long term effect of altered urea cycle function on neurodegeneration.

Neurotransmitter Metabolism

The aromatic amino acids--phenylalanine, tyrosine, and tryptophan--are converted to catecholamines and serotonin by enzymes in adrenal, intestinal, and neronal tissue. GABA and glutamic acid exert CNS-active neurotransmission effects without any modification of their chemical structures. Plasma levels of these amino acids are known to influence CNS concentrations of the respective neurotransmitters. Schizophrenia treatments (and etiologic mechanisms) have been linked to the glutamatergic and dopaminergic excitatory amino acid systems. (20) Alterations in plasma levels of aspartate, glutamate, glycine, and taurine have been suggested as neurochemical markers of epilepsy. (21)

Plasma tyrosine has been proposed as a useful assessment of thyroid function. Low plasma levels of tyrosine have been associated with hypothyroidism. (22), (23) Tyrosine has been used as a treatment for depression and blood pressure modulation. (24) Possible additional symptoms of low plasma tyrosine would be chronic fatigue, learning, memory or behavioral disorders, and autonomic dysfunction. (1) High levels of stress lead to depletion of phenylalanine. (25) The inherited metabolic disorder of phenylketonuria results in greatly elevated phenylalanine in plasma and urine. Excessive protein intake or a metabolic block in the conversion of phenylalanine to tyrosine can also elevate phenylalanine in plasma or urine.

Numerous studies have demonstrated that plasma tryptophan is an indirect marker of changes in brain serotonin synthesis. (26) Tryptophan has been shown to help induce sleep in insomniacs due to increased serotonin production in the brain stem. Plasma tryptophan levels are increased with sleep deprivation because of decreased utilization. (27-29) Low plasma levels of tryptophan have been reported in depressed patients (30) and are correlated with the degree of depression. (31) Used alone or with amitryptyline, the amino acid is effective against depression in general practice. (32)

Serine is also a critical component in the biosynthesis of acetylcholine, an important CNS neurotransmitter used in memory function and mediator of parasympathetic activity. Patients suffering from episodic acute psychosis display a disturbance of serine-glycine metabolism, (33) and a higher serine/ glycine ratio is observed in depressed individuals. (34)

Muscle Catabolism

Specific amino acids measured in urine provide insight into protein catabolism. Urinary 1-methylhistidine (1-MeHis) is a marker of beef, chicken and poultry consumption. (35-37) High urinary excretion of 3-methylhistidine (3-MeHis), a component of muscle, indicates active catabolism of muscle and is an abnormal marker for excessive muscle breakdown. It has been used as such a marker in studies of clinical conditions associated with nitrogen loss, including trauma, surgery, (38) infection (39) and in uncontrolled diabetes. (40) A study in Sweden looked at 3-MeHis levels to evaluate effect of alphaketoglutarate-enriched enteral feeding on protein metabolism after major surgery. (41) Other numerous studies utilized urinary 3-MeHis in cases where limiting catabolism is the outcome being studied. Urine 3-MeHis was used to evaluate the anabolic effectiveness of supplementation with exercise. Muscle breakdown in resistance exercisers trying various post-exercise beverages was assessed via urinary 3-MeHis. (42)

Collagen

Proline is required for protein synthesis and is metabolized into hydroxyproline, an important component in connective tissue. Therefore, high urinary levels may reflect inadequate connective tissue synthesis. Low levels of proline can indicate a poor quality protein diet and consequently prevent optimal connective tissue maintenance. Hydroxyproline is a component of collagen. High levels in 24-hour urine or plasma correlate with the increased osteocalcin secretion that is characteristic of high bone turnover. (43) Also involved with collagen synthesis in connective tissue is the amino acid hydroxylysine (HLys), a derivative of lysine. HLys and Hydroxyproline are indicators of liver disease, however elevated HLys seems to be a stronger index of hepatic collagen metabolism in chronic liver disease. (44)

Nutritional Markers

Abnormal levels of amino acids in plasma and urine can also indicate insufficiencies of nutrients. Specific vitamins and minerals are required for amino acid metabolism. Abnormal results from amino acid profiling may be due to deficiencies of the nutrients required as cofactors for transformation into other compounds. Low levels of essential amino acids may indicate inadequate pancreatic enzyme activity. Because zinc is required as a cofactor in several digestive enzymes, a deficiency of this element can affect overall plasma amino acid levels. (45), (46) Individual amino acid abnormalities are indicators of specific nutrient insufficiencies.

Because the catabolism of amino acids is a heavily utilized pathway in the liver, breakdown of branched chain amino acids (BCAAs) affords an opportunity for detecting interruptions in the pathway caused by inadequacy of vitamin B6, thiamin and/ or other B vitamins. Leucine, isoleucine and valine are initially metabolized utilizing a pyridoxal-5-phosphate dependent enzyme. Continued deamination into keto-acids requires vitamins B1, B2, B3, B5 and lipoic acid. Plasma homocysteine elevations indicate a demand for vitamins B6, B12 and folate, necessary cofactors for the metabolism of this amino acid. A limitation of homocysteine as a marker for any one component in this vitamin triad is the fact that homocysteine will rise in the absence of B6, B12 and/or folate.

One study performed on cobalamin deficient rats, serine (Ser) and threonine (Thr) levels in plasma and urine were significantly elevated. After two weeks of B12 supplementation, in addition to decreased urinary methylmalonic acid, was normalization of plasma Ser and Thr. It appears that cobalamin deficiency results in impaired metabolism of Thr and Ser due to minimization of the enzymes responsible for the conversion of Ser and Thr to pyruvate. (47)

Vitamin C is the main cofactor involved in collagen synthesis-namely the conversion of proline to HPro. Acute or chronic deficiency of vitamin C produces a significant increase in the proline /HPro ratio in urine. (48) Supplementation with vitamin C has been used to successfully treat certain types of collagen disorders and to stimulate collagen synthesis. (49)

Vascular Function

Vascular tension involves the cell regulator nitric oxide (NO) and its precursor arginine. A sequence of events in the endothelial cells results in NO release. NO penetrates into the underlying layer of muscle cells where it elicits release of the final modulator of muscle relaxation, cyclic guanosine monophosphate. Many of the reported effects of arginine in human health are due to NO-related cell responses. Impairment of endothelium-dependent coronary microvascular function due to aging in particular, can be restored by Larginine supplementation. (50) NO plays a role in vascular homeostasis influencing vascular tone and structure. (51) NO-mediated pathways are also investigated in understanding erectile dysfunction. (52) In evaluating vascular function plasma arginine and/or urinary nitrates are measured. (53-55) Plasma asymmetric-dimethylarginine, a NO inhibitor is another index used in similar studies. (56-58) However, measurement of urine amino acids in assessment of vascular health is minimal. Homocystinuria, a genetic disorder caused by a cystathione beta-synthase deficiency, is associated with vascular events as a result of markedly elevated circulating homocysteine. (59) Human studies have shown that high levels of homocysteine are associated with impaired endothelial-dependent vasodilation in healthy subjects indicating that the bioavailability of NO is decreased in those with hyperhomocysteinemia. (60) Plasma homocysteine levels are preferred in studies investigating related disorders. (61-64)

Other Conditions

Urinary amino acids have been measured in the evaluation of specific clinical conditions. In many cases the research represents disruption of normal amino acid metabolism as a result of the disease process and the short-term changes in plasma amino concentration.

Patients with Cushing's disease exhibit changes in urinary and serum concentrations, and renal clearance of amino acids with relationship to glucose tolerance. Normalization of cortisol levels restores amino acid status. (65) Investigation of aminoaciduria of subjects with different types and severity or traumatic injuries shows that many amino acids are involved and that the aminoaciduria is correlated with a reduced total serum calcium. (66) Changes in plasma and urinary amino acids were seen during diabetic keto-acidosis (DKA). A strong correlation was found between the urinary excretion of several amino acids and that of the beta-2-microglobin characterizing tubular dysfunction, thus reflecting altered metabolic state and renal function due to DKA. (67) Urinary phosphoethanolamine (PEA) is typically elevated in the first few weeks of life and declines throughout childhood and adolescence. Higher than normal levels of urinary PEA were seen in infants and children with impaired central nervous systems, systemic skeletal affections and hepatopathies. (68) Urinary beta-aminoisobutyric acid has been used in several studies as a marker of urinary tract tumors and at helping to predict recurrences, (69,70) while other studies have correlated this amino acid derivative in urine with leukemias and lymphomas. (71,72)

Clinical Application

For evaluation of overall amino acid body status, plasma testing emerges as the method of choice. Urine amino acid assays appear to be most commonly used to diagnose genetic metabolic disorders. Muscle protein and collagen catabolism and integrity are evaluated by certain amino acids elevated in urine. Urine amino acids are typically not measured to indicate nutrient demands. For example, folate deficiency leads to increased catabolism of histidine (73,74) and consequent increased urinary histidine excretion and/or its metabolites. Although an elevated histidine may indicate need for folate, the urinary organic acid formiminoglutamate is a more specific marker for folate status within the tissues. (75,76)

Organic Acids in Urine

There are various methods of acquiring data about vitamin status. Concentrations of vitamins can be measured in serum or blood cells. The excretory products formed from vitamins may be measured in urine. Thirdly, functional adequacy of a particular vitamin can be revealed by the urinary levels of specific metabolic intermediates controlled by the action of the vitamin. For routine clinical purposes, the most useful assay gives a clear answer to the question of whether body pools are adequate to meet current tissue demands.

To demonstrate, increased plasma or urine isoleucine or appearance of significant levels of the branched chain keto acids (not BCAAs) in urine, are markers of thiamin deficiency. (77) Ultimately, the combination of markers most useful in assessing an individual need for a specific nutrient such as thiamin is plasma or urine isoleucine, urine pyruvate, alpha-ketoisovalerate, alpha-ketoisocaproate, and alpha-keto-betamethylvalerate. In addition, urinary levels of organic acids formed from amino acid catabolism can be extremely useful as markers of functional adequacy of amino acids. This should be considered when answering the question of specimen selection for direct testing of amino acids. The combination of amino acids in plasma with organic acids in urine provides a more complete picture of amino acid abnormalities and becomes an exciting prospect to further assess an individual's specific nutritional needs.

Conclusion

The overall conclusion to be drawn from this discussion is that a great majority of reports documenting clinically useful information from evaluation of essential amino acids have evaluated plasma levels. We can also say that for most, but not all clinical situations, the greatest array of useful information is derived from the measurement of plasma amino acids. Plasma is especially favored when the prime consideration is the supply of the essential amino acids for optimum balance to maintain or restore health. Amino acid testing is extremely valuable in establishing nutritional therapies and understanding cellular and metabolic needs of a patient. The choice of specimen for testing should be based upon what clinical information is being investigated.

Glutamate "Blockers?"

Article that asks what things might be taken to block excess glutamates

Mike: Here's a practical question that's actually been burning in my head for about eight years: Is there anything that a person can take to block the absorption of MSG or glutamate as a defensive supplement?

Dr. Blaylock: Well, not necessarily to block it. You have other amino acids that can't compete for glutamic acid absorption. So that may be one way to help reduce the rate at which it would be absorbed.

Mike: Which aminos would those be?

Dr. Blaylock: Those would include leucine, isoleucine and lysine. They would compete for the same carrier system, so that would slow down absorption. There are a lot of things that act as glutamate blockers. You know, like silimarin, curcumin and ginkgo biloba. These things are known to directly block glutamate receptors and reduce excitotoxicity. Curcumin is very potent. Most of your flavonoids.

Magnesium is particularly important, because magnesium can block the MNDA glutamate type receptor. That's its natural function, so it significantly reduces toxicity. Vitamin E succinate is powerful at inhibiting excitotoxicity, as are all of your antioxidants. They found combinations of B vitamins also block excitotoxicity.