Aspartic Acid: Properties, Function, Benefits, and Sources

What is aspartic acid?

Aspartic acid (aspartate, Asp) is a biologically non-essential amino acid identified by the hydrolysis of asparagine. Aspartate comprises two isoforms: the predominant form is L-aspartic acid (L-Asp), while D-aspartic acid (D-Asp) is found in somewhat lesser quantities. L-Aspartic acid is critically significant in urea synthesis, the purine-nucleotide cycle, the malate-aspartate shuttle, gluconeogenesis, and neurotransmission. It serves as a substrate for the synthesis of proteins, asparagine, arginine, nucleotides, and various compounds essential for the development of nervous tissue and neurotransmission. D-Aspartic acid holds distinct importance in cerebral development and the modulation of hypothalamic function.

The production of asparagine, arginine, purines, and pyrimidines all begin with aspartate. The majority of human cells rely on mitochondrial aspartate aminotransferase (GOT2) to synthesize aspartate from oxaloacetate since their aspartate/glutamate transporter expression is so low. Under specific metabolic stressors, aspartate synthesis can restrict the development of cancer cells. Because aspartate aminotransferase substrate synthesis involves oxidative steps that are related to NAD+ availability, reduced aspartate synthesis is the consequence of poor cellular NAD+ regeneration. Cytoplasmic aspartate aminotransferase 1 (GOT1) maintains cellular aspartate levels by reducing glutamine carboxylation even when the electron transport chain is inhibited. But GOT1 can't make enough aspartate to sustain cell division. The usage of heterologous production of a NAD+ recycling enzyme, boosting aspartate absorption, supplementation of exogenous electron acceptors such pyruvate or alpha-ketobutyrate, or the blockage of the electron transport chain can all alleviate cell proliferation abnormalities. These results are in line with the notion that cells without mitochondria can't multiply in culture without supraphysiological amounts of pyruvate. It is interesting to note that the response of various cancer cell lines to inhibitors of the electron transport chain (ETC) can be predicted by cellular aspartate levels and the expression of its transporters. Aspartate production occurs in complex II defective kidney tumors when pyruvate carboxylase (PC) converts pyruvate to oxaloacetate. Furthermore, aspartate can be redirected to nucleotide production when cancer cells downregulate mechanisms that use aspartate. To illustrate how alternative metabolic pathways are employed to circumvent an aspartate deficit, consider cancer cells that express low levels of ASS1. These cells build up aspartate to sustain proliferation and nucleotide synthesis. Aspartate aids in the production of oxaloacetate and facilitates NADH recycling for ongoing glycolysis in the context of less severe ETC malfunction. It is possible that cancer cells also employ aspartate carbons to restock the TCA cycle. Indeed, when glutamine levels drop, cancer cells expressing the wild-type p53 gene can continue to proliferate and maintain the TCA cycle by expressing an aspartate transporter, which increases the availability of aspartate.

A number of recent investigations have pointed to aspartate as a limiting metabolite in the progression of certain cancers. The expression of the aspartate plasma membrane transporter SLC1A3 or the enzyme guinea pig asparaginase, which converts intracellular asparagine into aspartate, increases the availability of aspartate and hence the growth of tumors. To our surprise, out of all the metabolites we tested, the amount of aspartate in primary glioblastoma tumors had the strongest correlation with known transcriptional markers of hypoxia, lending credence to the idea that hypoxic tumor settings might hinder aspartate production. The observation that some pancreatic cancer cell lines do not exhibit the growth advantage afforded by increased aspartate levels suggests that aspartate restriction is probably not universal among malignancies. Given that pancreatic malignancies have been found to scavenge extracellular proteins through macropinocytosis, it is important to identify alternative sources of aspartate for these tumors. In malignancies, the synthesis of nucleotides is probably restricted by low amounts of aspartate. Isotope tracking of glutamine or aspartate, which are external substances, shows that purines and pyrimidines incorporate aspartate more readily, especially in environments with low oxygen levels. Remarkably, the discovery that breast cancer cells deficient in mitochondria could not develop tumors because of the respiratory-linked pyrimidine synthesis enzyme dihydroorotate dehydrogenase (DHODH) showed this crucial function in nucleotide synthesis. Since hypoxic malignancies like pancreatic ductal adenocarcinoma and most other cancer types are unable to absorb exogenous aspartate from the environment, they may undergo aspartate synthesis or find alternative sources to fuel their proliferation. Targeting aspartate de novo synthesis in cancers has not been attempted, despite its limiting role in tumor growth. To better understand the efficacy of this anti-cancer approach, additional research into the effects of aspartate metabolism targeting in cancer is required.

Aspartate is a limiting metabolite in the tumor microenvironment. (Garcia-Bermudez J., et al., 2020)

Aspartic acid structure

Like all other amino acids, aspartic acid contains an amino group and a carboxylic acid. It has a side chain with another carboxyl group (-CH₂COOH). Under normal conditions, the majority of L-Asp derived from food is translocated across the apical membrane of enterocytes via SLC1A1 (EAAT3) and employed in the biosynthesis of other amino acids (alanine, glutamate, proline, ornithine, and citrulline), nucleotides, and ATP. Research indicates that fewer than 1% of L-Asp, provided either alone or in conjunction with 18 other amino acids and glucose, was retrieved intact in the intestinal bloodstream. Consequently, L-Asp quantities in the bloodstream are minimal, with the primary source of L-Asp being its production by aspartate aminotransferase (AST) in various tissues. D-Aspartate can be obtained alongside other D-amino acids via dietary intake and gut bacteria or produced from L-Aspartate by a specialized racemase, and subsequently destroyed by a specific oxidase into oxaloacetate, ammonia, and hydrogen peroxide.

D-aspartic acid (Asp) and L-Asp. (D'Aniello A., 2007)

Aspartic acid pKa and charge

Under healthy conditions, the α-amino group of aspartic acid exists in the protonated -NH³⁺ form, whereas its α-carboxylic acid group is in the deprotonated −COO⁻ form. Aspartic acid possesses an acidic side chain (CH2COOH) that interacts with many amino acids, enzymes, and proteins throughout the body. Under physiological conditions (pH 7.4), the side chain of proteins often exists in the negatively charged aspartate form, −COO⁻.

Similar to glutamic acid, aspartic acid is an acidic amino acid. At pH 7.0, the anionic form of aspartic acid, known as aspartate, is formed when the 3-carboxylic acid group of aspartic acid is ionized. The pKa of this group is 3.86. Proteins that dissolve in water often have anionic carboxylate groups on their surface, where they can interact with the liquid. Protein folding is stabilized by these surface contacts. peptides, on the other hand, can have values as high as fourteen, depending on their specific local habitat.

The pKa and pI values of selected free amino acids at 25 ℃. (Bhagavan N V., 2002)

Aspartic acid solubility

L-Aspartic acid possesses a mildly acidic flavor and is soluble in acidic solutions, exhibits little solubility in water, and is challenging to dissolve in ethanol. The solubility of d-aspartic acid and l-aspartic acid in aqueous solutions escalates with temperature. Sodium chloride and potassium chloride enhance the solubility of d-aspartic acid by a salting-in effect. As the molalities of sodium chloride or potassium chloride rise, the solubility of d-aspartic acid in aqueous solution increases. The solubility of l-aspartic acid exhibits a similar effect in sodium chloride solution. Potassium chloride exerts a more pronounced salting-in impact on the solubility of d-aspartic acid compared to sodium chloride. As the molalities of the solutions of the two salts near their saturated molalities, the solubility of the two aspartic acids experiences a modest increase. The solubility of d-aspartic acid in pure water parallels that of l-aspartic acid in pure water.

Parameters for D-Aspartic Acid and L-Aspartic Acid in aqueous solutions. (Wang J., et al., 2010)

Aspartic Acid at Creative Peptides

Aspartic acid synthesis

Aspartate is predominantly produced in the human body by the transamination of oxaloacetate. Aminotransferase enzymes aid the manufacture of aspartate by transferring an amine group from molecules like alanine or glutamine, resulting in the formation of aspartate and an alpha-keto acid.

Aspartate is industrially synthesized through the amination of fumarate, mediated by L-aspartate ammonia-lyase. The enzyme aspartase (EC 4.3.1.1), also known as L-aspartate ammonia lyase, facilitates the addition of ammonia to fumaric acid in the industrial production of L-Aspartic Acid. Increased product concentration and productivity with reduced byproduct formation are two benefits of the enzymatic production method. Therefore, crystallization is a simple method for separating L-aspartic acid from the reaction mixture. Japan was the first country to commercialize a method for continuously producing L-aspartic acid using carrier-fixed aspartase, an enzyme obtained from Escherichia coli. For example, a 4.5 percent solution of aspartase-containing Brevibacterium flavum cells could be recycled in seven repeated batches to produce concentrations up to 166 g/L L-aspartate, making it an economically appealing procedure for L-aspartic acid. A method for mass-producing immobilized cells that used E. coli cells caught in a lattice of polyacrylamide gel was first proposed in 1973. This is the first instance of immobilized microbial cells being used in a fixed-bed reactor for industrial purposes. Biocatalyst half-lives of over two years were the consequence of additional enhancements, namely the immobilization of cells in k-carrageenan, which significantly enhanced operational stability. A theoretical productivity of 140 g/L/h/L-aspartate is possible with a column packed with the k-carrageenan-immobilized system.

Production of amino acids, methods, and production volume. (DRAUZ K., et al., 2007)

Amino acids produced by direct fermentation from carbohydrates. (DRAUZ K., et al., 2007)

What does D-aspartic acid do?

D-Aspartic acid is the most prevalent amino acid in mammalian tissues. In 1977, we identified its existence in the brain and optic lobes of the cephalopod mollusk Octopus vulgaris (common octopus). Subsequently, it was discovered in the peripheral nervous system (stellate ganglia and axoplasmic fluid of the giant axon) of the cephalopods Sepia officinalis (common cuttlefish) and Loligo vulgaris (common squid), as well as in their reproductive system. Subsequently, d-Aspartic acid was identified in the brain and endocrine tissues of various animal phyla.

As the neurotransmitter/neuromodulator

According to a large body of evidence, d-Asp functions as a neurotransmitter or neuromodulator at synapses and enhances the production of proteins that are important in nervous system development. For example, it has been noted that around 230-260 nmol/g tissue of d-Asp occurs in the retina and 13- to 14-day-old chicken embryos, whereas 550-650 nmol/g tissue of d-Asp occurs in the brain. Concentrations of d-Aspartate drop precipitously after this point in the life of the chicken, reaching a steady state of approximately 20–40 nmol/g tissue. The mammalian retina and brain likewise undergo similar processes. The brains of 17–19 day old rat embryos also undergo a high transitory accumulation of d-Asp, with a tissue concentration of about 300 nmol/g. Nonetheless, following this stage, it gradually declines, eventually reaching concentrations as low as 15-20 nmol/g of tissue. In contrast, d-Asp does not appear in mammalian retinas during embryonic development but rather in the early postnatal period, around 5-7 days after birth, when tissue concentrations reach approximately 350 nmol/g. Then, it drops precipitously to extremely low levels (about 30-50 nmol/g tissue) and stays there, just like the other species. The involvement of d-Asp in the development of the central nervous system, especially during fetal life or shortly after birth, is therefore well demonstrated in both rats and chickens. It is worth noting that the peak concentrations of d-Asp in the retina of both rats and chickens happen four to seven days prior to the onset of visual development. Chickens actually have full visual systems from the moment they are born, which is approximately 5-6 days after d-Asp reaches its retinal peak. In contrast, 4-5 days after d-Asp reaches retinal peak concentrations, on days 12–13 of life, the rat starts to open its eyes.

The light response in goldfish retinas can be enhanced by a factor of fifteen when exposed to d-Asp, in contrast to l-glutamate. As a selective NMDA receptor agonist, d-Asp triggers GABA release in cultured cells derived from the retina of goldfish. The l-glutamate transporters that are dependent on Na+ can detect d-aspartate. In addition to carrying l-Glu from glial cells to terminal axons, this carrier protein can also efficiently carry d-Asp from synaptic clefts to neurons.

Hormone regulation

Additional research has shown that D-Asp can enhance the activity of hCG (human chorionic gonadotropin) in the testes, leading to the induction of testosterone synthesis in pure Leydig cells of rats. An increase in steroidogenesis activity may result from D-Asp being taken up into cells. The absorption inhibitor l-cysteine sulfinilic acid reduces intracellular d-aspartate levels and testosterone synthesis. No other amino acid, including l-Asp, d-Glu, l-Glu, etc., was able to stimulate testosterone production in Leydig cells, demonstrating the extreme specificity of D-Asp's activity in hormone release. A regulatory protein known as stimulating steroidogenic acute regulatory protein (mRNA of the StAR gene) controls the biological process by which D-Asp enhances testosterone production. It has also been shown in non-mammalian animals that D-Asp is involved in endocrine action. During the various stages of the reproductive cycle, the concentration of D-Asp, which regulates testosterone production in the gonads of the green frog R. esculenta, fluctuates. Interestingly, d-Asp concentration and testosterone production in this animal's gonads show an inverse association, which goes against what happens in mammals. During the beginning of spring (March), when the ovary's D-Asp concentrations are low (2-4 nmol/g ovary), testosterone levels are high (36.9 ± 4.8 ng/g ovary). On the other hand, testosterone levels are relatively low (1-2 ng/g ovary) in the fall (October), when D-Asp concentrations are quite high (50-60 nmol/g ovary). Therefore, D-Asp prevents amphibians from fertilizing in the winter by reducing testosterone production in October. Testosterone levels are reduced in laboratory trials when D-Asp is injected into the dorsal lymphatic sac of adult females. The results show that D-Asp increases in the ovary and decreases in the testes. In the Podarcis s. sicula lizard, D-Asp has been found to have an additional biological role in the ovaries. In fact, d-Asp can cause a rise in aromatase activity, the enzyme that converts testosterone into 17β-estradiol, in this particular animal.

The role of D-Asp in the hormone release. (D'Aniello A., 2007)

Regulate activity of exocrine glands

There have been reports of rather high amounts of D-Asp in the salivary glands of rats. Rats between 4 and 7 weeks of age showed very high amounts in their parotid glands (PG) and submadibular glands (SMG)—approximately 400 and 250 nmol/g tissue, respectively. A study conducted by Raucci et al. (2005) discovered that the Harderian glands (HG), which are orbital seromucoid glands that exhibit seasonal fluctuations in secretory activity, contain elevated levels of d-Aspartame in the frog R. esculenta. During the spring, the highest concentration of d-Asp was observed. Injecting D-Aspartate into R. esculenta mimics the action of the pituitary gland in rats by increasing its concentration in the HG, which in turn stimulates the production of mucous substances. More RNA is synthesized at the same time as this action, which is mediated by the activation of the transcriptional phosphoprotein kinase ERK1. So, it seems that the secretory effects of D-Asp on exocrine gland activity are unique to this amino acid and not to any other D- or L-amino acid.

Improvement of sperm quality

An endogenous amino acid in animals, D-aspartic acid is produced in the testis and other endocrine glands by an enzyme called a D-aspartate racemase, which converts L-aspartate to D-aspartate. Past research has shown that D-aspartic acid plays a role in the production and secretion of sexual hormones in both humans and animals. It has also been proven that D-aspartate is present in spermatozoa and human seminal plasma, and that the concentration of D-aspartate is directly correlated with the quality of the semen. Based on these findings, we postulated that D-aspartate is involved in steroidogenesis and that supplementing it with it could increase the concentration and motility of spermatozoa, leading to a higher rate of pregnancy for partners of sub-fertile patients (oligo-asthenozoospermic and asthenozoospermic). So, we treated 60 patients for three months with an oral supplement of sodium D-aspartate. These patients had not fertilized their spouses in the preceding two years. In this study, we found that D-aspartate significantly increases spermatozoa count and motility in both oligo-asthenozoospermic and asthenozoospermic patients. Consequently, we also found that women whose partners were treated with D-aspartate had a significantly higher rate of pregnancy. Specifically, six out of thirty partners of oligoasthenozoospermic patients treated with D-aspartate were pregnant (rate = 20%), while ten out of thirty partners of asthenozoospermic subjects treated with Daspartate became pregnant (or 33% of the total). In this respect, two pregnancies happened within one month of their partners using D-aspartate, and four pregnancies happened within two months of their spouses taking the drug. The fact that the patients' health remained unaffected after consuming D-aspartate for over three months is the last intriguing finding of this study. Actually, we have tracked the main serum metabolites that are typically measured in clinics both before and after D-aspartate treatment. We found that all serum parameters were within normal ranges in patients treated with D-aspartate, suggesting that the drug has no negative effects on health. Men who were previously unable to conceive had a far better chance of fertilizing their own spouse after taking oral sodium D-aspartate daily for two to three months. This enhanced sperm quality, including the quantity and mobility of spermatozoa, by a significant margin.

Aspartic acid benefits and applications

(1) For protein synthesis

The single-letter amino acid symbol "D" denotes aspartate in protein sequences, which occurs globally at a frequency of approximately 8%. Due to its polar nature, L-Asp residues predominantly reside on the protein surface, where they affect protein interactions and functionality. The side chains of L-Aspartic acid frequently engage in hydrogen bonding with the main chain NH group, resulting in asx turns or asx motifs (asx denotes L-Aspartic acid or asparagine) that facilitate protein folding. The ASX turn comprises three amino acid residues that establish a hydrogen connection between the side chain carbonyl group and the main chain amine group. The ASX motif has four or five amino acid residues, with either L-Asp or asparagine as the first residue, and is stabilized by two internal hydrogen bonds.

D-Asp, with L-Asp, has been identified in the proteins of various human tissues, including the eye lens, brain, skin, bone, teeth, and aorta. The inclusion of D-Asp residues, likely resulting from racemization during tissue aging, modifies protein structure and function and may contribute to disease progression. The cataract, a translucent region in the eye's lens, is the most extensively studied condition resulting in visual impairment.

Both L-Asp and D-Asp residues can undergo dehydration at the side chain carbonyl, resulting in the formation of L-isoAsp and D-isoAsp, respectively. IsoAsp production has been demonstrated to elicit autoimmune reactions to self-proteins.

(2) For gluconeogenesis

L-Asp transported from the mitochondria to the cytosol is crucial for the synthesis of oxaloacetate, the precursor for glucose synthesis, through two potential mechanisms. The initial option is the direct conversion of L-Aspartate to oxaloacetate via cAST (L-Asp + 2-OG → OA + Glu). The second step involves the entry into the urea cycle, resulting in the formation of fumarate, which is subsequently removed from the cycle and hydrated to produce malate. In settings of elevated NAD+ supply generated during gluconeogenesis by 3-phosphoglyceraldehyde dehydrogenase or lactate dehydrogenase, some malate is redirected from entering the mitochondria.

Oxaloacetate participates in gluconeogenesis through phosphoenolpyruvate carboxykinase (PEPCK), which is activated by elevated glucagon/insulin ratios during fasting or by catecholamines and cortisol during exercise and stress-related illnesses. L-Asp is pivotal in gluconeogenesis as it serves as an intermediary in the production of alanine in muscle tissue.

(3) For neurotransmission

D-Aspartate and L-Aspartate, together with L-Glutamate, are categorized as excitatory neurotransmitters that induce depolarization of the postsynaptic membrane. It is widely believed that pure aspartate-releasing neurons do not exist, and that aspartate and glutamate are co-stored inside the same excitatory nerve terminal, released by a calcium-dependent exocytotic mechanism. L- and D-aspartate bind to NMDA-type L-glutamate receptors but do not activate AMPA-type glutamate receptors. EAATs, chiefly EAAT2, which transport glutamate, L-Asp, and D-Asp with comparable affinity, are accountable for the clearance of aspartate from the synaptic cleft. L-Aspartate serves a modulatory function in certain excitatory circuits, especially inside the hippocampus. Numerous studies have shown that D-Asp can induce the secretion of growth hormone from the pituitary gland, regulate melatonin production in the pineal gland, and activate the hypothalamic–pituitary–gonadal axis by stimulating the release of gonadotropin-releasing hormone from the hypothalamus.

(4) For brain development

D-Aspartate is abundantly available in the embryonic brain and significantly diminishes during the postnatal phase due to the activation of D-Aspartate oxidase. Mouse models exhibiting elevated D-Asp levels have demonstrated that D-Asp augments hippocampal NMDA receptor-dependent synaptic plasticity, dendritic architecture, and memory.

(5) Enhance myocardial function

Myocardial preservation and left ventricular performance are both improved by cardioplegic solutions that are rich in the hydrophilic and basic amino acids aspartate and glutamate. Various animal models of ischemia with and without reperfusion have shown this to be true. Even if there is a lack of extensive clinical evidence, the argument that these amino acids protect the heart is well-founded. Amino acids may reduce ischemia and reperfusion damage through a number of metabolic pathways. In the context of cardiac ischemia, glutamate and aspartate may become preferred fuels. They may also de-inhibit glycolysis by lowering cytoplasmic lactate levels and cardiac ammonia generation. The citric acid cycle may use some amino acids as substrates. Oxidative phosphorylation and energy production rely on reducing equivalents, which are transported from the cytoplasm to the mitochondria via glutamate and aspartate.

References

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