Nutritional summary

Function: The nonessential amino acid L-alanine (Ala) is needed for the synthesis of proteins. It is also used as an energy fuel: its complete oxidation requires thiamin, riboflavin, niacin, vitamin B6, pantothenate, lipoate, ubiquinone, iron, and magnesium. Food sources: Adequate amounts arc consumed when total protein intakes meet recommendations, since dietary proteins from different sources all contain Ala. and the body can produce additional amounts from other amino acids. Requirements: Since adequate amounts can be produced endogenousty, no Ala has to be consumed as long as total protein intake is adequate.

Deficiency: Prolonged lack of Ala due to low total protein intake causes growth failure, loss of muscle mass and organ damage.

Excessive intake: Very high intake of protein and mixed amino acids [more than three times the RDA or 2.4g kg) is thought to increase the risk of renal glomerular sclerosis and accelerate osteoporosis. The consequences of very high intakes of Ala have not been adequately evaluated.

Endogenous sources

Large amounts of Ala are produced in muscles, liver, small intestine, and some other tissues, L-Alanine aminotransferase (EC2.6.1.2) uses L-glutamate to transaminate the glycolysis metabolite pyruvate ami produce Ala. Since the reaction operates near cooh I

hrin -ch

Sulfino-L- ata nine

COOH

Pyruvate

.COOH

HSO,

Aspartate 4-decarboxylase (PLP)

L-Alanine amlno-translerase (PLP)

COOH I

COOH L-Aspartate

L-glutamate k..

-ketogl uta rate

.COOH

3-Hydroxy-ant hrani late

Aspartate 4-decarboxyIase (PLP)

COOH I

4 ch3

L-Ala nine i

Kynureninase (PLP)

R-3-ami no-2-methyl-propionate-pyruvate am i not ran sie rase (PLP)

R-3-ami no-2-methyl-propionate-pyruvate am i not ran sie rase (PLP)

R-3-a mines'melhyl-propanoate pyruvate

R-3-a mines'melhyl-propanoate pyruvate

L-Sel enocy steine selenide lyase (PLP)

COOH 1

H2N -CH

Thymine

COOH

CHj I

L-Seieno-cysteine

H2N -CH

L-Tryptophan

figuro H.ZK Endogenous sources of L-aíanine equilibrium, high availability of glucose (and consequently of pyruvate) can increase Ala production,

Mucli smaller amounts of Ala arise from the metabolism of L-tryptophan (kynureni-nase; EC3.7.13), thymine (R-3-amino-2-methylpropionatc-pyruvatc aminotransferase; EC2.6.1.40), L-aspartate and 3-sullino-l.-alanine (both by aspartate 4-decarboxylase; EC4.1,l.I2). and selenocysteine (L-sclenocysteinc selcnide-lvase; EC4.4.1.16). All of these enzymes use pyridoxal 5-phosphate (PLP) as a prosthetic group.

Dietary sources

Most food proteins contain about 4 6% Ala. Accordingly. Ala intakes depend more on the amount than on the type ofprotein consumed. Small amounts of the D-isomer are present in many plant-derived foods, but typical intakes have twit been characterized.

Cooking promotes the cross-linking of Ala w ith other amino acids in food proteins (generating lysinoalaninc, omithinoalanine. histidinoalaninc, phenylethylaminoala-nine). the formation of dehydroalanine. mclhyldehydroalanine. beta-aminoalanine. and racemization to D-alanine (Friedman, 1999).

Digestion and absorption

1 lydrolysis of Ala-containing proteins begins with mastication of foods in the mouth. However, most of this activity, such as salivary N-acctylmuramyl-L-alanme anudase (EC3.5.1.28) and kallikrein 2, appears to be antibacterial in nature.

Intestinal lumen

Capillary lumen

Brush border membrane

Basolateral membrane

Capillary endothelium

Figure 8.29 lnitscui.il absorption of L-alanifie

A large spectrum of gastric and pancreatic enzymes continues protein breakdown, many of them cleaving peptide bonds between specific amino acids and Ala. Pancreatic endopeptidase E (HO.4,21.70 > specifically cleaves at Ala residues within proteins; the pancreatic carboxypeptidases Al and A2 (EC3.4.2,I) cleave earboxv-terminal amino acid Ala residues.

Alanine aminopeptidase (AAP; EC3.4.11,2) at the small intestinal brush border releases N-termina! Ala from peptides, amides or arylamides preferentially, but not exclusively.

The presence of lysinoalanine and other modilied alanine residues decreases the digestibility of food proteins (Friedman, 1999),

Ala is taken up from the small intestinal lumen mainly via the sodium-amino acid eotransport system B" (Avissar et at,. 20011, The transporter ASC works predominantly by exchanging Ala for another small neutral amino acid, as does the sodium-independent transport system b" •"' .a transporter comprised of a light subunit BAT I (SLC7A9) and a heavy subunit rBAT (SLC3A1). Passive non-mediated uptake has also been suggested. Ala as a component of dt- or tripeptides can also be taken up v ia the hydrogen ion peptide eotransporter (SLC15A1. PepTI |.

Export across the basolatcral membrane uses the sodium amino acid eotransport systems ASCTI (SLC1A4) and ATA2 (Sugawara et al.. 2000), the sodium-independent transporter LAT2/4F2 and possibly a sodium-independent transporter with the properties ofase.

Starvation increases expression of the transport systems A and I (LAT24F2), whereas AS( and non-mediated uptake are not affected (Muniz el at., 1993).

Transport and cellular uptake

Blnod circulation: Plasma concentration of Ala I typically between 270 and 500p.mol I) increases significantly after meals and is lowest during the early morning hours (Tsai and Huang, 1999). Uptake from blood into tissues relics largely on the sodium-dependent transport systems A and ASC. Expression patterns of individual transporters vary greatly between different cell types, ATA2 is present in most cells (Sugawara et at., 2000) while ATA3 is restricted to liver (llatanaka ct at.. 2001).

Blood-brain barrier. LAT I (SLC7A5) and ATA1 (Varoqui etal., 2(HM)> are expressed in brain capillary endothelial cells and certain to contribute to Ala transport, but their locations, relative importance and the role of other transporters is not completely understood. U-alanine is transported in brain by Ase-1, a recently described sodium-independent transporter for neutral amino acids which is associated with the glycoprotein 4F2 (SLC3A2) (Fukasawa et al.. 20(H)).

Materno-fetal transfer: The sodium-amino acid eotransport system A (possibly also ASC and N). and the sodium-independent exchanger LAT1 mediate Ala uptake from maternal blood across Ihe brush border membrane of the syncytiotrophoblast. Transfer across the basolatcral membrane proceeds v ia the sodium-independent transporters LAT 1 and LAT2 (SLC7AH) and (he sodium-amino acid eotransport system A (ATA11. Ibis system A transporter is expressed at high level in placenta (Varoqui et at.. 2000), bul its exact location is not yet known.

CH3 h2n-ch

COOH L-Alanine

[.-Alanine aminotransferase (PLP)

^ "-ketogl uta rate L-gtutamate

COOH Pyruvate rigurc 8,30 L-Alamnr transamination

Metabolism

The amino group of Ala can be used to generate glutamate (alanine aminotransferase; EC2.6.1.2, in cytosol and mitochondria), glutamine (glutamine-pyruvate aminotransferase; EC2.6.1.15). glycine (alantne-glyoxylate aminotransferase: EC2.6.I.44), 1.-phenylalanine(phenylalanine/histidineaminotransferase; EC2.6.1.58), L-serine (serine-pyruvate aminotransferase: EC2.6.1.51) and aminonialonaie (alanine-oxomalonate aminotransferase: EC2.6.I.47). Additional enzymes may transfer the amino group toketo-aeids with low activity. All aminotransferases require PL.P lightly hound to a specific lysine residue at the catalytic center.

As a result of the transamination reaction pyruvate is generated w hich can be utilized via the pyruvate dehydrogenase complex, the citric acid cycle, and oxidative phosphorylation.

D-alaninc probably can be converted to pyruvate by glycine hydroxy me thy It rans-ferase (EC2.1.2.1): the amino group is transferred in this reaction to the enzyme-bound PLP, generating pyridoxamine phosphate in the process (Ogiwa and Fujioka, 1981).

Storage

Most Ala is bound in proteins much of that in musclcs. w hich contain about 0%. As protein is turned o\er Ala becomes available. Ala mobilized from muscle protein can be exported to the liver for urea synthesis and gluconeogcnesis.

Excretion

Filtered free Ala is taken up into proximal renal tubules mainly by the sodium-amino acid colransport system B" (Avissar c(al. 2001). di- and tripeptides via pepTl and pcpT2. Ala is then exported across the basolateral membrane via the sodium-dependent transporters ATA2 and ASCT1. As a result of very efficient rcabgorption ihe loss of Ala into urine is minimal in healthy people. I osses into feces are negligible while gastrointestinal function is normal.

Most nitrogen front metabolized Ala is excreted into urine as urea.

Regulation

Early starvation increases Ala synthesis. When increased amounts of amino acids are released from muscle, especially in response to starvation, severe infection, inflammation. and injury, the amino group is transported preferentially as Ala from muscles to liver {glueose-alanine cycle, see below). Cytokines such asTNF-alpha can acutely alter amino acid transport and thereby deplete muscle Ala concentration (Tayek, 19%),

Function

Protein synthesis: Ala is a constituent of practically all proteins and peptides synthesized in the body. Alanine-tRNA ligase (ECft. 1.1.7) loads Ala onto a specific t-RNA in an ATP'magnesium-dependent reaction.

Energfuel: The pyruvate released by transamination of Ala may be completely oxidized as an energy fuel yielding 3.425 keal g (May and IIill, 1990). The necessary reactions are dependent on thiamin. riboflavin. niacin, vitamin 136, pantothenate, lipoate. ubiquinone, iron, and magnesium

Glucose-alanine cycle: Most of the free Ala from muscles is exported into circulation. In the liver, the amino group is used for urea synthesis and the residual pyruvate is used for gluconeogenesis via conversion to oxaloacetate (pyruvate carboxylase: EC6.4.I.I) and phosphoenolpyruvate (phosphoenolpyruvate carboxylase: EC4.1.1.31). The reactions involved in glucose synthesis from pyruvate depend on niacin, biotin. and magnesium (see Pyruvate in Chapter 7), The cycle is completed when glucose returns to muscles, is metabolized to pyruvate and transaminated to Ala. The glucose alanine cycle facilitates the utilization of muscle amino acids in the liver during fasting, starvation, and traumatic stress. Ala accounts for more than a quarter of total amino acids taken up by liver from circulation.

Heme synthesis: Aminolev ulinatc aminotransferase (EC2.6.1.43, PLP-dcpendcnt) synthesizes the heme precursor delta-aminolevutinate by transferring the amino group from Ala to 4,5-dioxovalerate. It has been suggested that this housekeeping enzyme ensures a minimal level of 5-aminolcvulinic acid in addition to the more variable and regulated amounts produced by aminolevulinate synthase. Other nitrogen compounds: The ammo group of Ala can be used for the synthesis of other amino acids or any of numerous other nitrogen compounds. Clyoxylate metabolism I he transfer of the amino group from Ala to glvoxylate by alanine-glyoxylate aminotransferase (EC2.ft.L44, PLP-dcpendcnt) in liver peroxisomes is critical for the conversion of this metabolite to glycine. The alternative fate of glyoxalate is nonenzymatic conversion into the dead-end metabolite oxalate.

References

Avissar NE, Ryan CK. Ganapathy Y Sax HC. Na' -dependent neutral amino acid trans* porter AT Bo isa rabbit epithelial cell brush border protein. Am J Physiol Cell Physiol 200I:28I:C963 71

Friedman M, Chemistry, biochemistry, nutrition, and microbiology of lysinoalanine, Ian-thionine, and histidinoalanine in food and other proteins. J Agrii Food Client 1999;47:1295-319

FukasawaY. Segawa H, Kim JY. Chatroungdua A, Kim OK, Matsuo H.Cha SH, F.ndou H. Kanai Y. Identification and characterization of a Na(+^independent neutral amino acid transporter that associates with the 4F2 heavy chain and exhibits substrate selecto ity for small neutral I)- and L-amino acids. J Biol Chan 2000:275:9690 8 Hatanaka T. Huang W. Ling R. Prasad PD, Sugawara M. Leibach FH. Ganapathy V Ev idence for the transport of neutral as well ascaiionic amino acids by ATA 3. a novel and liver-spec i lie subtype of amino acid transport system A. Biochim Biophys \ctu 2001:1510:10-17

May ML, Hill JO. Energy content of diets of variable amino acid composition, bu J Clin Nutr 1990;52:770-6

Muni/ R, Burguillo L. del Castillo JR. Effect of starvation on neutral amino acid transport in isolated small-intestinal cells from guinea pigs. Pfl An. li Eur J Physiol 1993;423:59-66 Ogawa II, Fuji oka M. Purification and characterization of cytosolic and mitochondrial serine hydroxy methyl transferases from rat liver. J Biochem 1981:90:381-90 Sugawara M, Nakanishi T, Fei YJ. Huang W. Ganapathy ML. Leibach FH, Ganapathy v: Cloning of an amino acid transporter with functional characteristics and tissue expression pattern identical to thai of system A../ Biol Client 2000:275:16473 7 Tayek JA. Effects of tumor necrosis factor alpha on skeletal muscle amino acid metabolism Studied in vivo. J Am Coll Nutr J 996; 15:164 R Tsai PJ. I luang PC. Circadian variations in plasma and erythrocyte concentrations of glutamate. glutamine. and alanine in men on a diet without and with added monosodium glutamalc. Metab Clin Exp 1999:48:1455 60 Vuroqui 11. Zhu II. Yao D, Ming If, Erickson Jl>. Cloning and functional identification of a neuronal glutamine transporter../ Biol Client 2000:275:4049 54

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