REVIEW Origin, Microbiology, Nutrition, and Pharmacology ...szolcsanyi/education/files/Organicka...

REVIEW

Origin, Microbiology, Nutrition, and Pharmacology of d-Amino Acids1)

by Mendel Friedman

Western Regional Research Center, Agricultural Research Service, USDA, Albany CA 94710, USA(phone: þ1-510-5595615; fax: þ1-510-5595777; e-mail: [email protected])

Exposure of food proteins to certain processing conditions induces two major chemical changes:racemization of all l-amino acids (LAAs) to d-amino acids (DAAs) and concurrent formation of cross-linked amino acids such as lysinoalanine (LAL). The diet contains both processing-induced andnaturally-formed DAA. The latter include those found in microorganisms, plants, and marineinvertebrates. Racemization impairs digestibility and nutritional quality. Racemization of LAA residuesto their d-isomers in food and other proteins is pH-, time-, and temperature-dependent. Althoughracemization rates of LAA residues in a protein vary, relative rates in different proteins are similar. Thenutritional utilization of different DAAs varies widely in animals and humans. Some DAAs may exertboth adverse and beneficial biological effects. Thus, although d-Phe is utilized as a nutritional source ofl-Phe, high concentrations of d-Tyr in such diets inhibit the growth of mice. Both d-Ser and LAL inducehistological changes in the rat kidney. The wide variation in the utilization of DAAs is illustrated by thefact that, whereas d-Meth is largely utilized as a nutritional source of the l-isomer, d-Lys is not. Similarly,although l-CysSH has a sparing effect on l-Meth when fed to mice, d-CysSH does not. Since DAAs areconsumed as part of their normal diet, a need exists to develop a better understanding of their roles infoods, microbiology, nutrition, and medicine. To contribute to this effort, this overview surveys ourpresent knowledge of the chemistry, nutrition, safety, microbiology, and pharmacology of DAAs. Alsocovered are the origin and distribution of DAAs in food and possible roles of DAAs in human physiology,aging, and the etiology and therapy of human diseases.

1. Introduction. – Microorganism-synthesized d-amino acids (DAAs) are estimatedto constitute approximately one third of the human DAA burden [1]. During foodprocessing, naturally occurring l-amino acids (LAAs) may be transformed to theirmirror-image configuration d-isomers. A better understanding of the dietary signifi-cance of DAAs requires knowledge about the factors that induce racemization of LAAto DAA in food proteins during food processing. Processing-induced amino acidracemization includes those formed during exposure of food proteins to high pH, heat,and acids. The nutritional effectiveness of protein-bound essential DAAs depends onthe amino acid composition, digestibility, and physiological utilization of releasedamino acids. Since an amino acid must be liberated by digestion before nutritionalutilization can occur, the decreased susceptibility to digestion by proteolytic enzymes ofd-d, d-l, and l-d peptide bonds in DAA-containing proteins is a major factor adverselyaffecting the bioavailability of protein-bound DAAs. In addition, some DAAs may betoxic. Some DAAs have been shown to impart beneficial effects in vivo. It is not known

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010) 1491

� 2010 Verlag Helvetica Chimica Acta AG, Z�rich

1) Proceedings of First International Conference of d-Amino Acid Research, Awaji Island, Japan, July1–4, 2009.

whether the biological effects of DAAs vary, depending on whether they are consumedin the free state or as part of protein that contains processing-induced DAAs. To cross-fertilize information among several disciplines and to stimulate further needed studies,this review attempts to integrate and correlate the widely scattered literature on theorigin, formation, chemistry, microbiology, nutrition, pharmacology, and toxicology,and medical aspects of DAA. This review is intended to stimulate interest in furtherresearch to optimize beneficial effects of DAAs.

Origin of Chirality. Although the formation of l-amino acid (LAA) and d-sugars inthe primordial environment during evolution of life is generally considered to be aresult of chance [2], definitive proof of the origin of homochirality is still elusive, asillustrated by the following selected observations listed chronologically.� A mathematical model was used to describe the parity-violating energy

differences between enantiomeric molecules [3]. The model predicts that anenantiomeric excess (ee) is expected in chiral products resulting from reactions ofachiral or racemic substrates. A non-equilibrium racemic reaction system such as thepolymerization of d,l-amino acids to a protein spontaneously switches to a homochiralreaction channel, giving only the l-product. Calculations show that polypeptides of thel-series are preferentially stabilized by electroweak interactions. Such polypeptideshave lower energies due to parity violation than the corresponding d-isomers. Since theformation of optically active biomolecules may be �energy-driven�, the creation ofhomochiral biochemistry may not be accidental.� Based on crystallization studies, Cintas [4] suggests that homochirality may be the

result of differences in energy and growth rates of crystal surfaces to which d- or l-amino acids bind. The autocatalytic cycles during crystallization and oligomerizationmay account for homochirality of living organisms.� A review of the literature on cycles of solvation, crystallization, and adsorption

processes in the primordial environments (geochromatography) suggests that verysmall differences in the physical properties of some enantiomers may account for theenhanced formation of pure l-enantiomers, as well as for the preferential incorporationof LAAs and d-carbohydrates into early life forms [5].� Another plausible idea is that the asymmetric earth rotation caused (induced) at

dawn and dusk circularly polarized UV light of opposite polarity and reversedtemperature profiles in the oceans [6]. Destruction of the d-isomer by the polarizedUV light in a sea surface hotter than at dawn created a daily l-isomer excess protectedfrom radiation by nightfall. The LAAs are then preserved by mechanical diffusion intocold, darker regions of the sea leading to an LAA excess in early marine life. Theauthors suggest that these postulated events can be subjected to computer simulation.� Although the selection of d-ribose in RNA and d-deoxyribose leads to the

biosynthesis of proteins exclusively from l-amino acids [7], Bolik et al. [8] provideexperimental evidence for the preferential stabilization of the natural d- over thenonnatural l-configuration in nucleic acids. The energy differences in electronic levelsof d- and l-RNA may have led to the preferential stabilization of the naturallyoccurring d-RNA over the l-configuration.� Nonenzymatic aminoacylation of an RNA minihelix occurred preferentially with

l-amino acids [9]. This finding suggests that tRNA aminoacylation, a process by whichamino acids first encounter RNA, may have been a critical step in determining LAA

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)1492

homochirality. However, it should be noted that cells contain defenses against d-aminoacyl-tRNAs [10].

It is likely that more than one physical-chemical event in the primordialenvironment occurred simultaneously or consecutively that resulted in the creationof homochirality favoring LAAs. The creation of l-homochirality is not the end ofcreation, however, because special needs seem to have induced nature to also deviseenzyme-catalyzed alternate biosynthetic pathways to DAAs [11] [12]. Enzymes that areknown to be involved in DAA synthesis and metabolism include DAA oxidases [13],deacylases [14], dehydrogenases [15], epimerases, [16] [17], proteases [18], andracemases [15].

Because DAAs from natural sources are part of the human diet, we will first brieflyreview their formation and function in plants, microbes, and other natural sources,followed by a discussion of factors that favor racemization by conditions encounteredduring home and commercial processing of food.

2. Analysis. – Because all of the LAA residues (Fig. 1) in a protein and additionalformed during food processing (Fig. 2) can undergo racemization simultaneously, butat differing rates, assessment of the extent of racemization in a protein is a difficult taskthat requires quantitative measurement of ca. 40 l- and d-optical isomers [19].Reported methods include the use of chiral-phase gas chromatography [20], HPLC[21], HPLC with enzyme reactors [22], LC/MS [23] [24], capillary electrophoresis[25] [26], electrokinetic chromatography with detection by laser-induced fluorescence[27], and biosensors [28] [29]. Detailed discussion of these and other methods foranalysis of DAA in food and biological samples is beyond the scope of this review [30–40].

3. Natural Occurrence of DAAs. – Plants. Plants appear to be able to synthesizeDAA derivatives and peptides de novo [41 – 43], as illustrated by the followingexamples. Germinating pea seedlings (Pisum sativum) contain high concentrations ofN-malonyl-d-Ala and g-l-glutamyl-d-Ala [44]. The concentration of the latterincreased to 2.5 mm/seedling after 8 d of germination. d-Ala-d-Ala was found to bepresent in tobacco leaves [45]. The relative concentrations of three d-Ala peptides (d-Ala-Gly, d-Ala-d-Ala, and d-Ala-l-Ala) produced in rice plants (genus Oryzae)appears to be related to the nature of the rice strain [46]. Approximately 7% of thetotal alanine content in grass leaves consisted of d-Ala [47]. Exogenously supplied d-Ala, possibly of microbial origin, is required for the plant to synthesize the dipeptides.Ono et al. [48] demonstrated the presence of alanine racemase and of DAAs in alfalfaseedlings (Medicago sativa L.). Studies by Forsum et al. [49] indicate that transgenicplants can be engineered to utilize DAAs from the soil as a nitrogen source. The plantracemase resembled racemases of bacterial origin. Hydrolyzed proteins used as plantfertilizers also contain significant amounts of DAAs [25].

3.1. Microbes. Microbes provide a potential source of dietary DAAs. They produce,metabolize, and utilize DAAs [50] [51]. Since microbial DAAs enter the food chain, Iwill briefly examine some aspects of the microbiology of DAAs.

Br�ckner et al. [52] determined the DAA content produced by several classes ofbacteria (Acetobacter, Bifidobacterium, Brevibacterium, Lactobacillus, Micrococcus,


Propionibacterium, and Streptococcus) used in starter cultures for the production offermented foods and beverages. d-Ala and d-Asp were found at the highestconcentrations in all bacteria. d-Glu was present in several classes of the micro-organisms. Lower but significant amounts of d-Leu, d-Lys, d-Met, d-Orn, d-Phe, d-Pro, d-Ser, d-Thr, and d-Tyr were also detected in some of the bacteria. DAAs infermented foods may largely originate from bacteria. Bacteria from oral and intestinalfloras and rumen microorganisms are also potential sources of dietary DAAs [53] [54].

3.2. Peptidoglycans in Bacterial Cell Walls. Bacterial cell walls may constitute amajor source of dietary DAAs. The thick cell wall of a Gram-positive bacteriumconsists of peptidoglycan (a sugar– amino acid polymer) and teichoic acid. Bothpolymers contain DAAs. The much thinner cell wall of Gram-negative bacteria consistsentirely of peptidoglycan and associated proteins. The composition of the peptidogly-cans and teichoic acids may vary among different classes of bacteria. The DAAs in thebacterial cell walls contribute to their resistance to digestion by proteolytic enzymes.Related studies showed that:� d-Ala is required for the synthesis of the mucopeptide component of nearly all

bacterial cell walls [55];


Fig. 1. Structures of genetically coded amino acids subjected to racemization

� the peptidoglycan structure of Lactobacillus casei contained asparagine cross-links between d-Ala and l-Lys [56];� glutamate racemase in E. coli provides d-glutamate, an indispensable component

of peptidoglycans in bacteria [57];� mutations leading to increased resistance of Enterococcus faecalis to antibiotics

are governed by the amount of d-Ala-d-Ala and d-Ala-d-lactate incorporated intopeptidoglycan precursors [58];� d-Ala-d-Ala contributes to the resistance of antibiotics [59];� the gene encoding d-Thr aldolase (an enzyme that catalyzes the inversion of d-

Thr to d-allo-Thr in E. coli) is responsible for the incorporation of d-Ala into cell wallteichoic acid in Bacillus subtilis [60] [61];� d-Ser dehydratase from E. coli catalyzes the conversion of d-Ser, d-Thr, and allo-

d-Thr to the corresponding a-keto acids and NH3 [62];� d-Ser inhibits the action of bacterial transaminase, whereas d-Ala partially

protects against this inhibition [63];� d-Asp and d-Glu can serve as indicators for proteins of bacterial origin [64];� anaerobic bacteria are capable of using DAAs and l-sugars for growth [65];


Fig. 2. Structures of post-translational amino acids present in foods and tissues. Amino acids with oneasymmetric C-atom can exists as two, those with two as four, and those with three as eight isomers,

respectively.

� d-Asp and d-Glu levels correlated with growth of probiotic bacteria duringfermentation in milk and whey powders [66].

3.3. Peptide Antibiotics. DAA-Containing synthetic and natural peptides possessstrong antimicrobial properties. Synthetic peptides that inactivated Clostridiumbotulinum include glycyl-d-Ala, myristoyl-d-Asp, sorbyl-d-Ala, sorbyl-d-Trp [67].DAAs in natural antibiotics include d-Asp and d-Glu (bacitracin, mycobacillin); d-Cys(malformin); d-Leu (circulin); d-Orn (bacitracin); d-Phe (funigsporin, polymixin,tyrocidine); d-Ala, d-Leu, d-Val (gramicidin); and d-Ala, d-Leu, d-Val (actinomycin,valinomycin) [68 – 70]. Substitution of DAAs in the peptides designed to reducesusceptibility to digestion enhances the activity of pleurocydin [71], bombinins H [72],lacticin [73], and other antibiotic peptides [74 – 76]. The mechanism of action of thesepeptide antibiotics involves the induction of ionic channels that lead to the disruption ofbacterial cell membranes [77].

The cited studies suggest that an understanding of the biochemistry of genes andenzymes involved in the synthesis and metabolism of DAA-containing functional andstructural microbial molecules that are targets of antibiotics may help efforts designedto discover improved bactericidal agents.

4. Food Processing-Induced Formation of DAAs. – Historical Perspective. Since theearly part of the 20th century, alkali treatments have been known to racemize aminoacids [78 –80]. The early authors used changes in optical rotation to determine thealkali-induced racemization, and Kjeldahl N analysis of NH2 groups of liberated aminoacids and peptides to detect concurrent hydrolysis of peptide bonds. They reportedthat:� l-Arg was racemized to the d-isomer, and that the protein clupein was

concurrently partly hydrolyzed and racemized in Ba(OH)2 and NaOH solution at408 [79];� optically active hydantoins prepared from a-amino acids underwent spontaneous

racemization at room temperature, presumably as a result of keto – enol tautomerisminvolving the asymmetric C-atom [78] [81];� racemization of amino acids in gelatin occurred on treatment with 0.1 and 1.0n

NaOH, but not on treatment with 3.0n alkali, presumably because in the latter case therate of peptide-bond hydrolysis is higher than that of racemization [80] [82 –84];� heating casein with 1.0n NaOH at 1258 resulted in complete racemization of all

amino acids, which occurred at a faster rate than analogous treatment of gelatin [83] oredestin [84].

The field of amino acid racemization in proteins lay largely dormant forapproximately half century because of lack of suitable analytical methods to determinethe formation of specific protein-bound DAAs. The development of GC and HPLCtechniques along with chiral columns that can separate d- and l-isomers stimulatedwidespread interest in this subject. The observation by Masters and Friedman [85– 89]that widely consumed processed foods contain DAAs stimulated worldwide interest inthe distribution of DAAs in the human diet. They reported the following levels of d-Asp in widely consumed foods (d/l ratio; % as d-isomer): coffee-mate (0.208; 17);Fritos potato chips (0.164; 14); simulated bacon breakfast strips (0.134; 13); Isomilbaby formula (0.108; 10); and textured soy protein meat analogue (0.095; 9). These


authors were also apparently the first to relate racemization rate constants of protein-bound DAAs to inductive parameters associated with free LAAs (linear free-energyrelationships), and to describe the relationship between DAA content of racemizedcasein and in vitro digestibility by proteolytic enzymes [86]. First-time studies byFriedman and Gumbmann [90] on the biological utilization of DAA as source of the l-isomer using all amino acid diets and the predicted formation of cross-linked aminoacids such as histidinoalanine that may accompany racemization [91] that were laterrealized are also of historical interest. Some of these aspects are further examinedbelow.

Racemization Mechanisms. Fig. 3 offers a mathematical derivation of the first-orderkinetic equations that govern reversible in vitro amino acid racemization. Fig. 4 depictsthree different mechanisms that have been postulated to govern racemizations ofamino acids during food processing. Fig. 5 depicts a postulated mechanism for the invivo inversion of an LAA to its d-isomer catalyzed by pyridoxal phosphate. Figs. 6– 9illustrate the kinetics of racemization, and Fig. 10 (see below) shows susceptibilities ofsome amino acids at high pH that may accompany racemization. Tables 1 – 3 list theDAA contents of racemized proteins. The cited observations indicate that heat andhigh pH widely used in processing of food induce the racemization of all amino acidresidues by the indicated pathways.

Alkali-induced peptide-bond cleavage of soy proteins determined by a ninhydrinassay [35] influenced the extent of racemization [92]. Racemization rates of the sameamino acid in polyamino acids were much lower than in soy proteins. Related studies[93] showed that:� free amino acids racemized approximately ten times slower than did protein-

bound ones;� alkali treatment of zein induced racemization of amino acids residues and caused

nutritional damage without the presence of lysinoalanine (LAL) [94];� serine residues in lactalbumin, leaf protein concentrate, and soy protein were

highly susceptible to heat- and alkali-induced racemization [95];� racemization of protein-bound amino acids, especially of lysine, induced by heat

extrusion was greater in soy than in corn proteins [96];� melanoidins formed during heating of glucose or fructose with LAA or DAA had

similarly shaped but different absorption spectra [97].

5. Distribution of DAAs in Food. – As already mentioned, the observation byMasters and Friedman [88] that processed commercial foods contained DAAs wasfollowed by numerous worldwide studies on the content of DAAs in a variety of foods.The aspect is briefly examined below for several food categories listed in alphabeticalorder.

5.1. Alfalfa Seeds. Alanine racemase catalyzes formation of d-Ala in alfalfa seeds(Medicago sativa L.) [48].

5.2. Animal Skins. Peptides and proteins isolated from animal skin are reported tocontain DAAs [98] [99].

5.3. Bread. Use of lactic acid bacteria and yeast in the fermentation of sourdoughbefore baking results in the introduction of d-Ala and d-Glu into the dough [100].Baking of the dough into bread induces a 44% decrease in the total free DAA content.


5.4. Coffee. Roasted coffee contained 10 –40% of d-Asp, d-Glu, and d-Phe [101].Higher amounts of DAAs were formed during roasting of Robusta than of Arabicacoffees [102]. Liquid coffee contained ca. 20 mg of total DAA/100 ml [103].

5.5. Dairy Products. DAAs in cow�s milk originate from the digestion (autolysis) ofpeptides and proteins containing DAAs originating from microbial cell walls


Fig. 3. Derivations of first-order kinetic equations for reversible amino acid racemization. Adapted from[87] [88].

(peptidoglycan) proteins in the rumen of the cows [104]. Raw milk from ruminants(cows, goats, and sheep), but not human milk, contains the following DAAs: d-Ala, d-Asp, d-Glu, d-Lys, and d-Ser [66] [105] [106]. Relatively high concentrations of theseDAAs were also present in widely consumed ripened cheeses [21]. Emmental cheesecontains as much as 70 mg of total DAAs per 100 g [103]. The DAA content of varied


Table 1. DAA Content [(d/dþd)�100)¼% d] of Eight Alkali-Treated Proteins. Adapted from[20] [85] [92] [199] [255].

Aminoacid

Casein Lactalbumin Wheatgluten

Cornprotein

Fishprotein

Soybeanprotein

Bovinealbumin

Hemoglobin

Ala 15.2 14.4 18.6 22.2 19.3 15.8 22.1 17.1Val 2.6 2.7 4.0 4.9 3.1 2.5 3.5 4.0Leu 7.4 5.0 7.2 7.8 6.8 6.3 8.2 6.6Ile 3.3 3.1 4.0 5.5 3.6 3.9 5.7 5.0Cys – 32.1 32.0 43.7 22.8 21.0 23.0 30.0Met 24.7 32.3 33.1 29.8 29.2 24.3 30.0 26.2Phe 24.4 24.3 24.4 32.4 28.0 25.5 28.1 30.0Lys 8.1 7.2 9.4 8.0 11.5 11.3 13.3 9.9Asp 29.2 22.6 25.6 41.6 25.0 30.8 27.0 18.9Glu 19.7 19.5 32.3 35.0 18.9 21.1 18.4 19.8Ser 41.0 47.1 42.2 44.0 42.1 44.2 43.0 44.5Thr 29.3 29.1 30.0 36.3 32.8 27.8 28.3 31.2Tyr 15.0 18.9 19.5 35.5 16.3 13.7 15.3 22.6LALa) 4.4 5.4 0.9 0.3 2.8 3.2 8.5 4.4

a) Mixture of (ldþll) LAL isomers in g/16 g N.

Table 2. Variation in LAL Isomer Content of Alkali-Treated Proteins. Adapted from [20] [199].

Protein LAL [g/16 g N] [ll-LAL]/([ll-LAL]þ [ld-LAL]), Isomer ratio

Zein 0.32 Not detectedWheat gluten 0.95 0.50Fish protein concentrate 2.75 0.40Bovine hemoglobin 3.36 0.50Casein 4.40 0.51Lactalbumin 5.38 0.51Bovine serum albumin 8.52 0.50

Table 3. Aspartic Acid Racemization and LAL Content of Alkali-Treated Casein and Acetylated Casein.Adapted from [88].

Protein d/l Asp LAL [mol-%]

Casein, untreated 0.023 0.00Caseinþalkali 0.387 2.35Acetylated caseinþalkalia) 0.336 0.00

a) Acetylation of e-NH2 groups of lysine side chains prevents LAL but not d-AA formation.


Fig. 4. Base-catalyzed racemization: Proton abstraction–addition mechanism. a) An OH� ion abstractsan Hþ from the R-CH a-C-atom of an amino acid to form a negatively charged carbanion which has lostits original asymmetry [256] [257]. The carbanion can then recombine with a proton from the solvent toregenerate the original amino acid which is now racemic (dl). Elimination–addition mechanism: Thecarbanion can also undergo an elimination reaction to form dehydroalanine side chain. Thedehydroprotein can then react with H2O to form racemized amino acid side chains (e.g., dl-Ser)[85] [258]. The concurrent formation of lysinoalanine is also shown. b) Acid-catalyzed racemization:Protonation of the carboxy group of an l-amino acid facilitates removal of a proton to form thedehydroalanine. Proton addition at two sides (re and si faces) of the double bond generates both d- andl-isomers. Adapted from [259]. c) Racemization of a compound (fructose-l-phenylalanine) intermedi-

ate of d-Phe. Adapted from [23].

among cheeses and changed during cheese production [107]. Peptide-bond hydrolysisof casein during storage of cheeses may also influence their DAA content [104].


Fig. 4 (cont.)

Fig. 5. Isomeric inversion in vivo: catalysis of inversion of an l-amino acid by the pyridoxal phosphate(PLP) coenzyme of a microbial racemase involving stereoselective shifts of H-atoms. Adapted from [260].

The cited authors also suggest that:� because the d-Ala content of raw milk increased during storage at 48, d-Ala

could serve as an indicator of contamination by d-Ala-producing psychotropicbacteria;� because mastitis is an infection of the udder caused by bacteria, milk from

infected cows should have a higher DAA content originating from the microorganisms;� pasteurization of milk does not increase its d-amino content.5.6. d-Glutamic Acid in Processed Foods. Monosodium glutamate (MSG), the

sodium salt of the naturally occurring nonessential amino acid, is often added at levelsof 0.2 – 0.9% to foods to improve flavor and palatability. A survey showed that a varietyof processed foods contained significant amounts of d-Glu (which does not possessflavor-enhancing properties) [108]. The d-% ranged from 0.25 for pure MSG to 0.8 forsoups (highest value), 2.9 for crackers, 7.9 for sauces, 18 for vinegars, 36 for sauerkrautjuices, 1.6 for tomato products, and 6.2 for milk products.

The d-Glu could originate from microbial sources as well as from food-processing-induced racemization of free and protein-bound l-Glu. Table 2 shows that protein-bound l-Glu is one of the fastest racemizing amino acids. The possible impact of the d-isomer of MSG on the �Chinese Restaurant Syndrome� has apparently not beenevaluated.

5.7. Eggs. Alkali pickling of duck eggs in a 4.2% NaOH/5% NaCl solution for 20 d atroom temperature used to prepare the traditional Chinese pidan resulted in extensiveracemization of amino acid residues and concurrent formation of lysinoalanine (LAL)[109]. The relative racemization rates appear to be similar to those observed with eggalbumin and other pure proteins.

5.8. Fish. Heating of laboratory-made herring meals at 1258 induced time-dependent formation of d-Asp [110]. The d-Asp content of twelve fishmeals from


Fig. 6. Effect of temperature on content d-Ser, d-Tyr, and LAL of soybean protein. Adapted from[92] [199] [261].

various sources ranged from 0.7 to 3.7%. The d-Asp content may indicate the severityof the thermal treatment during cooking or drying of fish meals [111].

5.9. Food (Dietary) Supplements. Widely used dietary supplements are alsoreported to contain DAAs [112] [113].

5.10. Fruits and Vegetables. Fruits (apples, grapes, oranges) and vegetables(cabbage, carrots, garlic, tomatoes) as well as the corresponding juices contain variablebut measurable amounts of DAAs including d-Ala, d-Arg, d-Asp, and d-Glu[21] [105]. These amino acids could originate from plant sources, from soil, and othermicroorganisms, and/or from heat treatments used to kill pathogens.

5.11. Fruit and Vegetable Juices. Br�ckner and Westhauser [114] reported thefollowing total DAA amounts in fruit and vegetable juices (in mm): carrot juice, 8.5;orange and other fruit juices, 28 –57; tomato juice, 14.5. Other investigators [22] [27]confirmed that DAAs occur naturally in fruit juices, beers, and wines. Specific DAAscould permit differentiating juices from biologically dissimilar fruits, and could serve asan indicator for detecting bacterial activity and shelf-life of fruit juices.

5.12. Honey. The d/l ratios of Leu, Phe, and Pro could serve as indicators of age,processing, and storage histories of honeys [115].

5.13. Maize (corn grain). Significant differences were observed in the DAA contentof some transgenic maize (corn) cultivars compared to standard varieties [36]. Theauthors suggest that this finding opens up new approaches in comparing thecomposition of transgenic plant foods to their conventional counterparts.

5.14. Meat Products. Low levels of DAAs (1.25 – 13.79 mg/g) were detected in bothirradiated and non-irradiated cooked hams [116]. There was no correlation betweenradiation dose and DAA content.

5.15. Spices. Liquid spices contain very high amounts of DAAs, up to 600 mg/100 ml[103].

5.16. Vinegars. Several investigators reported that fermentation conditions used toprepare different vinegars (balsamic, cider, sherry, white wine) affect their DAAprofiles [117 – 120].

5.17. Wines. Autolysis of yeast (Saccharomyces cervisiae) produces compounds thatbeneficially influence the quality of sparkling wines. Giuffrida et al. [121] found that thepattern of release of DAAs from conventional yeast strains differ from transgenic ones.The content of several DAAs of 26 wines did not correlate with storage times [122].Wine marinades can be used to inactivate bacteria [123] that may produce DAAs.Other potential dietary sources of naturally occurring DAAs include fungi, insects, fish,and other marine organisms [13].

6. Nutrition, Pharmacology, and Toxicology of DAA. – Twenty naturally occurringamino acids and several hundred amino acid derivatives formed in vivo post-translationally in plants and animals, and in vitro during food processing play afundamental role in nutrition and other aspects of animal and human health. Thegeneral requirements are adequate amounts of the essential amino acids with areasonable balance among all [124]. Bioavailability and biological utilization of bothLAAs and DAAs vary widely and depend on source, chemical and metabolicinteractions, and on the diet and health of the consumer. To develop a betterunderstanding of the nutritional significance of DAAs and alkali-treated food proteins,



Fig. 7. Relationship between racemization rates (k) relative to Ala, and the inductive constant (s*) of theamino acid side chain R in RCH(NH2)COOH. Adapted from [199] and [93].

we compared the weight gain in mice fed free amino acid diets in which the test LAAwas replaced with the d-isomer. The results obtained reflect the ability of mice toutilize DAA in the complete absence of the l-form. In the case of essential amino acids,the mice must meet the entire metabolic demand from the d-isomeric forms.

Biological utilization by weanling male mice of DAAs was tested in a 14-day growthassay by using an all-free amino acid diet in which each l-amino acid was replaced bythe d-isomer. The growth assay was standardized with six mice per group, with potencyestimation being based of response of two to seven groups. Statistical evaluation ofpotencies of the DAA was calculated as the slopes of growth curves (weight gain after14 d per unit of dietary concentration) where linearity was approximated. Mean bodyweight gains were compared by Duncan�s multiple range test using individual values.The results illustrated in Figs. 10 and 11, and Table 4 show wide variations in theutilization of DAAs as a nutritional source of the corresponding l-isomers.

Digestion and Nutrition. A number of studies described the adverse and beneficialeffects of alkali treatment on protein nutrition [86] [125 – 132]. Friedman et al. [86]attempted to quantitatively define the susceptibility of alkali-treated casein to in vitrodigestion by trypsin and chymotrypsin (Fig. 11,a). Monitoring on a pH-stat revealed anapproximately inverse relationship between DAA and LAL content on one hand andthe extent of proteolysis measured on the other. The decreased susceptibility of alkali-treated casein to digestion could arise from loss of susceptible sites to enzyme cleavage.Sarwar and Paquet [133] reported that, although tripeptides composed of LAAs werecompletely hydrolyzed by intestinal mucosal peptidases, the following DAA-containingtripeptides largely resisted digestion: Ala-d-Met-Ala, Val-d-Met-Phe, Ala-d-Glu-Ala,Val-d-Asp-Ala, and Ala-d-Phe-Leu. Rat feeding studies also showed that d-Met in thetripeptides was less available for growth than l-Met. Alkali treatment by Jenkins et al.


Fig. 7 (cont.)

induced formation of DAA and nutritional damage in corn proteins (zein) withoutformation of LAL [94].

To obtain additional information on this aspect, we evaluated nutritional andhistopathological consequences of feeding-toasted and alkali-treated soy flours tobaboons. Fig. 11, b, shows the growth curves for seven pre-adolescent male baboonseach fed soy control and alkali-treated soy diets. There was no difference in growthrates over a 150-day period, since the slopes of the growth curves are not significantlydifferent from each other. However, in absolute terms, weight gain of the baboons fedthe treated soy diet (containing 370 mg of LAL/100 g air-dry diet) was ca. 20% lowerthan the gain observed with the control soy diet (containing 80 mg of LAL/100 of air-


Fig. 8. Arrhenius Plots for the racemization of protein amino acid residues in proteins: a) casein; b) soyprotein. Adapted from [85] [199].

dry diet). The decreased weight gains may be due to the decreased digestibility andutilization of the alkali-treated soy protein compared to the control.

Using a 15N-tracer technique, Heine et al. [134] investigated the utilization of DAAsby infants fed parenterally. Retention of 15N in the protein pool ranged from 23.2% ford-[15N]Val to 48.6% for d-[15N]Ala. DAAs were utilized by the infants for proteinsynthesis only to some and variable extent.

Possible causes for the reduction in digestibility – nutritional quality includedestruction of proteolytic enzyme substrates such as Arg and Lys, isomerization ofLAAs to less digestible d-forms, formation of inter- and intramolecular cross-links thathinder access of proteolytic enzymes, inhibition of proteolytic enzymes [135– 139], andalkali-induced formation of trypsin- and chymotrypsin-inhibiting peptides [132].

7. Pharmacology, Toxicology, and Potential Medical Uses of Individual DAA. – Tofacilitate understanding the complex nature of biological activities of DAAs, we brieflysummarize results of published studies on the following nutrition- and health-relatedaspects of DAA.

7.1. d-Ala. Because it is part of the structure of bacterial cell walls, d-Ala derivedfrom microbial sources is present in many foods as well as in hydrolyzed proteinfertilizers [25]. d-Ala is structural component of bacterial cell membranes [140] [141]and is a possible indicator of bacterial contamination, heat treatment, and shelf life offruit juices and other foods [22] [105]. It can also be used in studies of molecularimaging [142], to induce cytotoxic oxidative stress in brain tumor cells [143], and totreat schizophrenia [144]. Changes in d-Ala content of the rat pancreas are related totheir diurnal and nocturnal (circadian) habits [145]. The submandibular gland and oralepithelial cells, not ingested food or oral bacteria, appear to be the source of high levelsof d-Ala and d-Asp in human saliva [146].


Fig. 9. Effect of temperature on DAA content in coffee. Adapted from [102].

7.2. d-Arg. Both l- and d-Arg protected against oxygen-radical-induced injury ofrat heart tissue [147] and against endotoxin shock in rabbits [148]. d-Arg and otherDAAs inhibited cell proliferation and tumor growth in rats [149], acted as a centralnervous system stimulant, and exhibited anticonvulsant activity in humans [150].

7.3. d-Asp. d-Asp aggravated the nephritis in rats induced by Staphylococcus aureusbacteria [151] and prevented K and Mg depletion in rats induced by diuretics [152].

7.4. d-Cysteine (d-CysSH). Although l-CysSH had a sparing effect of l-Metconsumed by mice, d-CysSH did not [153]. In fact, d-CysSH imposed a metabolicburden as indicated by depressed growth when fed to mice with less than optimal levelsof d-Met (Fig. 12, a). The 24% decrease in weight gain of the d-CysSH plus l-Metamino-acid diet compared to l-Met alone implies that d-Cys is nutritionallyantagonistic or toxic.

d-CysSH but not N-acetyl-d-cysteine lowered rat blood cyanide levels derived fromacrylonitrile [154]. d-CysSH is also reported to be involved in the detoxification and/orprevention of toxicities caused by cyanides [155], the drug paracetamol [156], and


Fig. 10. Effect of pH on the extent of degradation of susceptible amino acids. Adapted from [89].

other drugs [157 –159]. l-Cysteine-glutathione disulfide, but not the d-cysteine analog,protected mice against acetaminophen-induced liver damage [160]. d-Cysteinedesulfhydrase catalyzes the elimination of H2S form d-cysteine, presumably to formdehydroalanine [161].

7.5. d-Cystine (d-Cys). Although l-Cys is not an essential amino acid for rodents,less l-Met is needed for growth if the diet contains l-Cys [153]. Our results also showthat l-Cys is somewhat more efficient in sparing d-Met than in sparing l-Met in diets


Fig. 11. Nutritional utilization of alkali-treated proteins by mice and baboons. Adapted from [262].

containing low levels (0.29%) of the two isomers (Fig. 12, b). Supplementation of d-Met with an equal sulfur equivalent of l-Cys doubled growth. Thus, the overallresponse was equal to that produced by l-Met in the presence of l-Cys. In contrast,supplementation of suboptimal levels of l-Met with increasing concentrations of d-Cysreduced the growth rate of mice. Excess d-Cys in the diet is toxic.

7.6. Dehydroalanine. Dehydroalanine is an intermediate in the racemization ofprotein-bound amino acids (Fig. 4). We found that its content (in g/16 g N) in alkali-treated casein was 0.33 and in alkali treated acetylated casein 1.39 [162]. Dehydroa-lanine can, in principle, act as a biological alkylating agent similar to that suggested forprocessing-induced acrylamide [163] [164].

7.7. d-His. d-His enhanced Zn accumulation and reduced the fraction of Zn that wasretained and absorbed by fish [165]. Both d- and l-His enhance DNA degradation byH2O2 and Fe2þ ions [166]. d-His-induced cell injury is mediated by an Fe-dependentformation of reactive oxygen species [167] [168].

7.8. d-Homocysteine. Both d- and l-homocysteine inhibit transmethylations andprevent neural tube defects in rat embryos [169] [170].

7.9. Lanthionine (LAN) Isomers. LAN Isomers are formed during the biosynthesisof microbially derived peptide antibiotics [171] and during exposure of proteins toalkali and heat. Reaction of the SH group of CysSH and the double bond of


Table 4. Growth Responses to DAA and Peptides in Mice Fed Amino-Acid Diets. Adapted from[90] [153] [172] [219–221] [255].

DAA and peptides Relative nutritional value to l-form [%]

d-Amino acidsd-Methionine 79.5d-Phenylalanine 51.6d-Tryptophan 24.7d-Leucine 12.4d-Histidine 8.5d-Valine 5.1d-Threonine 3.1d-Isoleucine 1.2d-Lysine �8.2l,lþd,l-Lysinoalanine 3.8

Peptides and sulfoxidesN-Acetyl-l-methionine 89.6N-Acetyl-d-methionine 22.9l-Methionyl-l-methionine 99.2l-Methionyl-d-methionine 102.9d-Methionyl-l-methionine 80.4d-Methionyl-d-methionine 41.0l-Methionine sulfoxide 87.5d-Methionine sulfoxide 26.8N-Acetyl-l-methionine sulfoxide 58.7N-Acetyl-d-methionine sulfoxide 1.7l-Methionine hydroxyl analogue 55.4d-Methionine hydroxyl analogue 85.7

dehydroalanine gives rise to one pair of optically active d- and l-isomers, and onediastereoisomeric (meso) form of LAN [33]. The mixture of dlþmeso-LAN has asparing effect on l-Met, as evidenced by a 27% greater weight gain when the twoamino acids were fed together, than when fed suboptimal l-Met [153] [172]. Base-


Fig. 12. Effects of d-amino acids added to mice diets. a) Sparing effects of l- and d-Cys, and other sulfuramino acids in methionine-deficient diets. b) Effect of d-cystine in low methionine diets. c) Sparingeffect of d-Tyr on growth of mice on l-Phe-deficient diets. d) Effect of d-Tyr on growth of mice on high-

and low-protein diets. Adapted from [90] [153].

catalyzed formation of LAN and LAL does not affect bioactivity in bovinesomatotropin [173].

7.10. d-Lys. d-Lys is not utilized as a nutritional source of l-Lys by chicks, dogs,mice, rats, or humans, presumably because d-amino acid oxidase does not metabolized-Lys [174]. The extent of nutritional damage of alkali treatment associated with loss oflysine may depend on the original lysine content of a protein. Decrease in lysine due toracemization and LAL formation in a high-lysine protein such as casein, high-lysinecorn protein, or soy protein isolate may have a less adverse effect than in a low-lysineprotein such as wheat gluten or corn protein, where lysine is a nutritionally limitingamino acid [175] [176]. Because of its low toxicity, d-Lys may be a better candidate toreduce radioactivity uptake by the kidneys during cancer therapy with radionuclidesthan is l-Lys [177 – 179].

7.11. Lysinoalanine (LAL) Isomers. The cross-linked amino acid LAL, formedconcurrently with DAA in alkali-treated proteins, has two asymmetric C-atoms, makingpossible four diastereoisomeric forms: dd, dl, ld, and ll. The nutritional value of theindividual isomers as a source of l-Lys is not known. Table 2 shows that a mixture of ll-and ld-isomers has a value for the mouse equivalent on a molar basis to 3.8% of l-Lys.

We have examined the affinity of LAL towards a series of metal ions, of which CuII

was chelated the most strongly. The four LAL isomers differ in their ability to chelatemetal ions such as Cu [180] [181]. On this basis, we suggested a possible mechanism forkidney damage in the rat involving LAL�s interaction with Cu within the epithelial cellsof the proximal tubules. The direct relationship between the observed affinities of thetwo LAL isomers for CuII ions in vitro and their relative toxic manifestation in the ratkidney suggests that LAL exerts its biological effect through chelation of Cu in bodyfluids and tissues. The observed binding of ll- and ld-LAL to CoII, ZnII, and othermetal ions imply that LAL could also influence Co utilization.

LAL, ornithinoalanine (OAL), and LAN are formed during alkaline treatment ofwool during skin unhairing of sheep and other processes designed to enhance thestrength of wool, and possibly also human hair, via cross-linking reactions [91] [182].Serine racemase contains a catalytically active LAL residue [183].

7.12. d-Met. The nutritional value of d-Met in mice approaches that of l-Met(Table 2). By contrast, d-Met appears to be poorly utilized by humans when consumedeither orally or during total parenteral nutrition. One factor giving rise to incon-sistencies in the utilization of d-Met is the dose dependency of the apparent potency ofd-Met relative to its l-isomer, i.e., the dietary level of the d-form for any given growthresponse relative to that of the l-form that would produce the same growth response.This dose-dependency is a result of the non-linear nature of the dose – response curves(Fig. 13). This complicates attempts to compare results from mice with those of otheranimal species. The latter studies often provided data based on a single substitution ofthe d- for the l-isomer. The figures show that high levels of l-Met (but not of d-Met)are toxic since they inhibited growth of mice [184].

d-Met-Containing solutions inhibited tumor cell growth in vitro [185], andprotected against cisplatin ototoxicity in humans [186– 189] and against oral radiation[190]. A cascade of enzymes transformed d-Met to the l-form [191].

7.13. Orn Isomers. Mart�nez-Giron et al. [113] [192] developed analytical methodsfor the separation of unnatural ornithine isomers that may be present in processed foods.


7.14. d-Phe. The relative growth rate of mice fed d-Phe replacing the l-isomer in afree-amino-acid diet is concentration-dependent, ranging from 28.3 to 81.3% whencompared to control diets containing the same amounts of l-Phe (Figs. 12 and 13, andTable 4) [90]. The data suggest the absence of any antinutritional effects or toxicityfrom feeding either Phe isomer at twice the optimum dietary level. d-Phe andhydroxyproline were reported to define the native structure and presumably virulenceof conotoxin, a toxic peptide obtained from the venom produced by cone snails [193].

7.15. d-Pro. Oral feeding of an aqueous solution of d-Pro for one month to ratsinduced fibrosis and necrosis of kidney liver cells and elevation of serum enzymes[194]. By contrast, orally fed d-Pro and d-Asp induced did not induce acute toxicity inrats [195].

7.16. Selenomethionine Isomers. Selenomethionine is a major source of Se in thediets of both animals and humans. Comparison of the effects of oral consumption ofseleno-l-Met, seleno-dl-Met, and selenized yeast on the reproduction of mallardducklings revealed that, although both seleno-Met preparations were of similar toxicity,their potency was greater than that of Se present in yeast [196]. Heinz et al. [196] foundthat the survival of day-old ducklings consuming l-seleno-Met after two weeks wassignificantly lower (36%) than that of ducklings consuming the dl-isomer (100%). d-seleno-Met protected against adverse effects induced by space radiation [197] [198].

7.17. d-Ser. Protein-bound l-Ser racemizes faster to the d-isomer than the otheramino acids (Table 1, and Figs. 6 and 7) [199]. d-Ser has been reported to enlarge ratkidney cells (cytomegaly) similar to that observed with LAL. Several approaches wereused in an attempt to elucidate possible mechanisms of d-Ser renal toxicity [200– 202].Sodium benzoate [200] [201], protein-deficient diets [203], and a-aminoisobutyric acid[204] attenuated d-Ser nephrotoxicity in rats.

d-Ser-induced kidney damage may be due to a lowering of the concentration ofrenal glutathione (GSH) that protects the kidneys against kidney-damaging reactiveoxygen species [204]. The decrease in glutathione concentration takes place during themetabolism of d-Ser by DAA oxidase. Because nephrotoxicity induced by d-Ser issimilar to that caused by LAL, the question arises as to whether effects of these twoamino acids on the rat kidney are competitive, additive, or synergistic?

Because d-Ser also acts as an agonist of N-methyl-d-aspartate (NMDA) receptor-mediated neurotransmission in the brain, it may be useful as �innovative pharmacologicstrategy in schizophrenia� and pain [205]. A strain of protozoa (Drosophilamelanogaster) has developed a transport mechanisms with adaptive specialization inthe absorption and brain functions of d-Ser and other DAA that may be involved inneuronal functions [206]. d-Ser can be enzymatically transformed to l-Trp [207]. Thed-configuration of Ser maintains the toxic conformation of the mushroom poisonviroisin [208].

7.18. d-Thr. l-Thr is the second-limiting amino acid in maize (corn) proteins. Theutilization of d-Thr by the chick, rat, mouse, or human as a nutritional source of the l-isomer is insignificant [174].

7.19. d-Trp. The relative nutritional potency of d-Trp compared to l-Trp in mice isstrongly dose-dependent, being inversely related to the dietary concentration andranging from 29 to 64% (Fig. 11) [209]. The maximum growth obtainable for l-Trpoccurred at 0.174% in the diet. By increasing the dietary concentration of d-Trp up to



Fig. 13. Top four plots: weight gain in mice fed increasing dietary levels of l-Met, d-Met, and isomericmethionine derivatives and analogs for 14 d. Asterisk indicates significant difference from l-Met at thesame dietary concentration (top four plots). Adapted from [153] [172]. Lower two plots: relationships ofweight gains to percent of l- and d-Phe, and l- and d-Trp isomers in amino acid diets fed to mice. Adapted

from [90] [209] [216].

0.52%, growth passed through a maximum at 82% of that achieved with the l-isomer.This occurred at 0.44% of d-Trp.

Considerable species variation exists for the nutritive value of d-Trp [174]. Inchicks� diets, relative potency of the d- to the l-isomer has been reported to be 20%. d-Trp was well-utilized by growing pigs [210]. The value for humans is ca. 10%. Ratsutilize d-Trp as efficiently as l-Trp. d-Trp can serve as a niacin precursor in rats to thesame extent as does the l-form [211] [212]. These studies also showed that d-Trp hasone sixth of the activity of niacin. Both d-Phe and d-Trp taste sweet [209] [213– 215].

7.20. d-Tyr. Nutritionally, l-Tyr is classified as a semi-essential amino acid [7].Combinations of l-Tyr and l-Phe are complementary in supporting growth of mice[90]. Thus, under conditions where l-Phe may be limiting, l-Tyr may supply half therequirement of l-Phe alone for chicks, mice, rats, and humans. Our mice-feedingstudies showed that with d-Tyr in an amino acid diet, growth inhibition was severe at ad-Tyr/l-Tyr ratio of 2 :1, but was less so when the ratio was 1 : 1 (Fig. 12,c). Similarresults were obtained with a casein diet supplemented with d-Tyr (Fig. 12,d). Theantimetabolic manifestation of d-Tyr may be ascribed to interference with thebiosynthesis of vital neurotransmitters and proteins in vivo [90] [216].

d-Tyr has no sparing effect for l-Phe in mice. Growth inhibition may becomeevident when d-Tyr is present in the diet at a level equal or greater than l-Phe. Thepotential for chronic toxicity following exposure to lower levels of d-Tyr remainsunknown. Formation of d-tyrosyl-tRNATyr may be responsible for the toxicity of d-Tyrtoward E. coli [217].

7.21. d-Val. Administration of total parenteral nutrition (TPN) containing d-Leu,d-Met, d-Phe, and d-Val to hepatoma-bearing rats showed that d-Val inhibited tumorgrowth without negative effects on the host. d-Leu and d-Met also improved thenutritional status of the sick rats [218]. These observations suggest that some DAAdiets may benefit cancer patients.


Fig. 13 (cont.)

In summary, mice provide a good animal model to study the biological utilizationand biological effects of DAAs, both free and protein-bound. A major advantage ofmouse bioassays is that they require approximately one-fifth of the test substanceneeded for rats and can be completed in 14 days [90] [153] [172] [219– 221].

8. Role of DAA in Humans. – Indicators of Aging. There appears to be a directrelationship between the age of the collagen proteins in rat teeth and their d/l-Asp ord/l-hydroxyproline ratios, presumably because of little or no metabolic activity in themolar teeth after their formation [222] [223]. The extent of Asp racemization can alsobe used to estimate the age at death [224 – 226]. The use of spontaneous racemization ofl- to d-Asp and of other amino acids to assess the age of tissues such as dentin, bone,and eye lens [227– 231] has been questioned by Clarke and co-workers [232] [233].Their objection is based on their findings that red-cell protein aspartyl/asparagineresidues racemize much more slowly in erythrocyte and other proteins than in proteinsof the lens, tooth enamel, and dentin. The in vivo rates of d-Asp formation inerythrocyte proteins were similar to the in vitro rates. It is not known whetherbiochemical processes exist to degrade or repair deamidated and isomerized aspartyland asparagine residues in tissue proteins. However, Chinese scientists [14] reportedthat human d-Tyr-tRNA (Tyr) deacylase protected cells and neurons against adverseeffects of excess DAA by deacylating d-aminoacyl-tRNAs into free DAA and tRNA.

The in vivo formation of d-Asp may not be the result of a racemization process forwhich the d/l ratio cannot exceed 1.0, but rather of an inversion of configuration of l-to d-Asp, where the ratio can and does exceed 1.0 [234]. Age-related accumulationof both DAAs and advanced glycation end products, and the decreased func-tional capacities of structural and bioactive proteins contribute to the developmentof diabetic complications and eye diseases [235]. Moreover, because the accumula-tion of d-Asp during aging has been implicated in the pathogenesis of Alzheimer�sdisease, arteriosclerosis, and cataracts, the question arises as to whether the enzyme d-aspartyl endopetidase reported to degrade d-Asp-containing peptides and proteins inmammalian mitochondria and nuclei [236] can slow down or reverse the aging oftissues.

Role of DAA in Disease. Changes in the concentrations of DAAs may be related tothe pathogenesis and therapy of diseases [237]. The total content of d-Phe and d-Tyrwas significantly greater in patients suffering from chronic renal failure than in normalhumans [238]. The increase in DAAs in renal failure may arise from preferentialretention of DAAs in the kidneys (low glomerular filtration compared to l-isomers),depletion, and inhibition of DAA oxidase activity, and the consumption of DAA-containing foods and antibiotics. High levels of d-Ser and d-Pro are also reported toaccumulate during renal failure [239]. Down-regulation of energy metabolism after d-Ser treatment may be related to the mechanism of its nephrotoxicity [240].

The free d-Ser in normal human brain tissue (d/l ratio 0.086) was comparable tothat in Alzheimer�s-affected human brain (d/l ratio 0.099). Because d-Ser (like the l-isomer) passes through the blood – brain barrier, d-Ser in the brain probably originatesfrom the diet. d-Ser is metabolized by d-amino oxidase in the brain. In contrast, otherstudies suggested that synthesis of d-Ser in the brain occurs by in vivo racemization ofl-Ser [241]. These conflicting suggestions need to be resolved in view of the fact that,


although at high pH protein-bound l-Ser racemizes rapidly (Table 2), the amount of d-Ser in food proteins that have not been exposed to alkaline conditions are too low toaccount for the relatively high amounts detectable in the brain.

An understanding of d-Ser signaling in the human brain may facilitate developmentof therapies for brain disorders [242]. In contrast to the apparent beneficial role inneurotransmission [16] [17], d-Ser concentrations above physiological levels inducedlipid and protein oxidative damage and decreased glutathione levels in brain cortex ofrats [243]. d-Ser availability in the nervous system may be altered in schizophreniabecause of increased DAA degradation by DAA oxidase [244].

The involvement of d-Ser in neurotransmission is supported by a double-blind,placebo-controlled trial with 31 schizophrenic patients. This study revealed that d-Ser(30 mg/kg/day) added to an antipsychotic regimen had significant beneficial effects oncognitive function and performance [245]. The d-Ser was well tolerated by the patientswith no apparent side-effects. Will protein-bound d-Ser induce similar improvements?

The following additional observations suggest that DAAs may benefit diseasetherapy:� d-Ser can contribute to the therapy of post-traumatic stress disorder [246];� DAA oxidase oxidizes d-DOPA in the brain, which may then be converted to

dopamine into an alternate biosynthetic pathway for the treatment of Parkinson�sdisease [247];� DAA inhibit tumor growth in rats, and may be useful in cancer gene therapy

[143] [248];� Parenteral administration of d-Val hepatoma-bearing rats induced significant

improvement in nutritional status and tumor-inhibition compared to control diets[249];� d-Phe induces analgesia in mice and humans [250];� The observed clinical relief in chronic-pain patients given oral doses of 750 to

1000 mg of d-Phe is ascribed to inhibition of carboxypeptidase and the accompanyingincrease in the levels of brain enkephalin. Whether dietary levels of d-Phe present inprocessed food proteins can induce analogous pain relief merits study.

Although healthy young adults can invert approximately one third of d-Phe to its l-isomer [251], and human plasma contained high concentrations of d-Ser, d-Ala, and d-Pro (d/l¼0.24, 0.21, and 0.1, resp.), no DAAs were present in plasma proteins[155] [252]. The total DAA content of the plasma correlated with the serum creatininelevels. The possible significance of high levels of DAAs in human saliva for oral healthis not known [146]. The possible value of d-Tyr in treating hypertension [253] and thepossible significance for human reproduction of the observed high content of d-Asp inrat testis fluids and tissues, and spermatozoa [254] are also not known. It appears thatDAAs possess both adverse and health-promoting attributes. The challenge is tooptimize the beneficial ones.

9. Conclusions and Outlook. – There is a need to standardize methods designed toascertain the role of DAAs in nutrition. Because the organisms are forced to use theDAAs as the sole source of the l-forms, the use of all-amino-acid diets in which the l-isomer is completely replaced with different levels of the corresponding DAA may bepreferable to supplementation of proteins with DAA. DAAs along a peptide chain may


be less utilized than the l-forms. The utilization of any DAA may be affected by thepresence of other DAA in the diet. Largely unresolved are the following questions:� Can analytical methods be developed for all four possible isomeric forms (ll, ld,

dd, and dl) of cross-linked amino acids with two asymmetric C-atoms (Fig. 2).� How do biological effects of DAAs vary, depending on whether they are

consumed in the free state or as part of a food protein?� Can poorly digestible racemized food proteins serve as dietary fiber in the

digestive tracts of humans?� Do DAAs and d-peptides alter the normal microflora of the intestine?� Investigate differences in the interactions of DAAs as compared to LAAs in the

binding to the active sites of proteases, such as trypsin, chymotrypsin, and pepsin in thedigestive tract.� Do metabolic interactions, antagonisms, or synergisms among free and protein-

bound DAAs occur in vivo?� Do proteins and peptides acquire antibiotic competence upon racemization?� Does racemization alter protein conformations and charge distributions (iso-

electric points) resulting in beneficial protective effects against protein-induced allergy,celiac disease, and bacterial, plant, and venom toxic proteins?� Does food processing-induced formation of DAAs adversely or beneficially

affect the safety of concurrently formed potentially toxic acrylamide?� Does pasteurization and other heat treatments widely used to kill pathogenic and

spoilage bacteria and fungi in liquid (milk, fruit juices) and solid (meat) foods result inreduced levels of DAAs produced by the foodborne microorganisms?� Will DAAs prevent biofilm formation by bacteria on or in contaminated food

[263]?

I thank Patricia M. Masters (Scripps Institution of Oceanography, La Jolla, CA), Remy Liardon(Nestle Research Center, CH-Lausanne), Kevin Pearce (New Zealand Dairy Research Center, PalmerstonNorth), and Michael R. Gumbmann (this laboratory) for excellent scientific collaboration, and Carol E.Levin for assistance with the preparation of the manuscript. I am grateful to Professor Hans Br�ckner forhis invitation to submit a contribution to this Topical Issue on d-amino acids.

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[147] A. Suessenbacher, A. Lass, B. Mayer, F. Brunner, �Antioxidative and myocardial protective effectsof l-arginine in oxygen radical-induced injury of isolated perfused rat hearts�, Naunyn. Schmiede-berg�s Arch. Pharmakol. 2002, 365, 269–276.

[148] E. Wiel, Q. Pu, D. Corseaux, E. Robin, R. Bordet, N. Lund, B. Jude, B. Vallet, �Effect of l-arginineon endothelial injury and hemostasis in rabbit endotoxin shock�, J. Appl. Physiol. 2000, 89, 1811–1818.

[149] B. Szende, �The effect of amino acids and amino acid derivatives on cell proliferation�, Acta Biomed.Ateneo Parmense 1993, 64, 139–145.

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[151] H. Koyuncuoglu, M. G�ngçr, . Ang, D. InanÅ, M. Ang-K�Å�ker, H. Sagduyu, V. Uysal,�Aggravation by morphine and d-aspartic acid of pyelonephritis induced by i.v. inoculation ofStaphylococcus aureus in rats�, Infection 1988, 16, 42–45.

[152] I. N. Iezhitsa, A. A. Spasov, N. V. Zhuravleva, M. K. Sinolitskii, S. P. Voronin, �Comparative study ofthe efficacy of potassium magnesium l-, d- and dl-aspartate stereoisomers in overcoming digoxin-and furosemide-induced potassium and magnesium depletions�, Magnesium Res. 2004, 17, 276–292.

[153] M. Friedman, M. R. Gumbmann, �The utilization and safety of isomeric sulfur-containing aminoacids in mice�, J. Nutr. 1984, 114, 2301–2310.

[154] F. W. Benz, D. E. Nerland, W. M. Pierce, C. Babiuk, �Acute acrylonitrile toxicity: studies on themechanism of the antidotal effect of d- and l-cysteine and their N-acetyl derivatives in the rat�,Toxicol. Appl. Pharmacol. 1990, 102, 142–150.

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[158] M. Friedman, �The Chemistry and Biochemistry of the Sulfhydryl Group in Amino Acids, Peptides,and Proteins�, Permagon Press, Oxford, UK, 1973, 485 pages.

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[160] L. I. Berkeley, J. F. Cohen, D. L. Crankshaw, F. N. Shirota, H. T. Nagasawa, � Hepatoprotection by l-cysteine-glutathione mixed disulfide, a sulfhydryl-modified prodrug of glutathione�, J. Biochem.Mol. Toxicol. 2003, 17, 95 –97.

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[168] R. A. Yokel, �Blood-brain barrier flux of aluminum, manganese, iron and other metals suspected tocontribute to metal-induced neurodegeneration�, J. Alzheimers Dis. 2006, 10, 223–253.

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[171] H.-G. Sahl, G. Bierbaum, �Lantibiotics: biosynthesis and biological activities of uniquely modifiedpeptides from gram-positive bacteria�, Annu. Rev. Microbiol. 1998, 52, 41 –79.

[172] M. Friedman, M. R. Gumbmann, �Nutritional value and safety of methionine derivatives, isomericdipeptides and hydroxy analogs in mice�, J. Nutr. 1988, 118, 388–397.

[173] J. S. Tou, B. N. Violand, Z. Y. Chen, J. A. Carroll, M. R. Schlittler, K. Egodage, S. Poruthoor, C.Lipartito, D. A. Basler, J. W. Cagney, S. B. Storrs, �Two novel bovine somatotropin species generatedfrom a common dehydroalanine intermediate�, Protein J. 2009, 28, 87 –95.

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