Histidine Amino Acids: Properties, Function, Benefits, and Sources

What is histidine?

Histidine (His) is an important amino acid that has a specific role in the histaminergic system, erythropoiesis, proton buffering, metal ion chelation, and reactive oxygen and nitrogen species scavenging. Various proteins that contain HIS (such as haemoproteins, HIS-rich glycoproteins, histatins, HIS-rich calcium-binding protein, and filaggrin), dipeptides that contain HIS (especially carnosine), and derivatives of HIS that contain methyl and sulfur (such as 3-methylhistidine, 1-methylhistidine, and ergothioneine) serve specific purposes. Supplementing with HIS is theoretically justified in a variety of contexts because to its unique chemical characteristics and physiological actions. From some mammalian elastins to the histidine-rich protein of Plasmodium lophurae, the histidine content in proteins can range from almost none to 73% of total amino acids. One of the amino acids found in the least quantity in human body protein is histidine. Tessari determined the whole protein concentration of different amino acids in human bodies. Histidine was present in low concentrations at 245 g, second only to tryptophan at 88 g, in contrast to the copious proline (1328 g) and glycine (1247 g), which play crucial roles in structural proteins.

Histidine structure

Aside from an imidazole side chain, histidine also has an α-amino group and a carboxylic acid group. Amino acids are protonated and carboxylic acids are deprotonated under physiological circumstances. The antioxidant, metal ion chelating, and proton buffering characteristics are all attributed to the imidazole ring.

Histidine structure. (Holeček M., 2020)

Histidine solubility

Histidine is an amino acid that is moderately soluble in water. The gravimetric method, carried out at atmospheric pressure, was used to measure the solubility of two α-amino acids over a temperature range of 293.15-233.15 K. We examined the α-amino acids L-arginine and L-histidine. A salting-out effect was found to influence the solubility of the amino compounds under study. It became quite apparent that the solubility in water-based chloride solutions improved with rising temperature. The findings were deduced by considering the salt hydration shells and the solute's capacity to form hydrogen bonds with water. The solubility data was connected to a precise semiempirical equation. The Gibbs free energy of transfer from pure water to aqueous solutions of chloride salts for certain α-amino compounds (∆trGo) has been calculated considering the solubility data. The decrease in solubility is linked to the enthalpic origin, which is primarily indicated by the positive (∆trGo) value. When it comes to water solubility, L-histidine is superior to DL-histidine.

Mole fraction solubility of L-arginine and L-histidine in water at different temperature. (Abualreish M J., et al., 2020)

Solubility curves in mg/g of L- (red) and DL-histidine (blue) in water. (Harfouche L C., et al., 2021)

Histidine sources

In contrast to other EAAs, an HIS-deficient diet does not cause a rapid loss of protein balance. For this reason, HIS was formerly considered an optional, non-essential amino acid. Then, research has demonstrated that the body makes up for a long-term HIS food deficit by increasing the catabolism of hemoglobin and CAR, which means that blood haemoglobin levels drop and muscle CAR content rises. Adult people given an HIS-deficient diet for at least one month and rats studied by Nasset and Gatewood both shown that HIS is needed for positive nitrogen balance maintenance. Furthermore, HIS-deficient individuals often exhibit atopic dermatitis and lower HIS levels in both plasma and urine. For individuals aged 19 and up, the estimated average requirement for HIS is 11 mg/kg/day, but the recommended dietary allowance is 14 mg/kg/day. Dietary protein is the primary source of HIS. Plant proteins (soybean, kidney bean, pea, oat, and wheat) contain 20-30 mg/g, but animal proteins (meat, poultry, fish, etc.) contain 25-30 mg/g. Because of their higher protein content, animal sources are preferable.

A popular element in Japanese cuisine, dried bonito broth (dashi) contains a high concentration of HIS (109 mg/g). Bonito, also known as skipjack tuna or Katsuwonus pelamis, is a popular fish that is often used as a soup stock instead of beef or chicken bouillon. HIS-CD, which includes proteins including CAR and anserine, is also found in the muscle of mammals. Chicken breast extract (CBEX^TM), which is mostly utilized in Japan, is a rich source of CAR and anserine. The chicken breast is heated in water to extract CBEX^TM, which has a CAR content of around 0.6 g/100 mL and anserine content of approximately 1.4 g/100 mL. A number of research looking at the effects of HIS and HIS-CD supplementation have utilized both dashi and CBEX^TM.

pKa of histidine

One of the most basic amino acids, histidine, has an imidazole side chain. The free amino acid's side chain has a pK of 6.0, which means that at physiological pH, there are both neutral and protonated forms. In the catalytic triad of serine proteases, histidine's imidazolium side chain provides capabilities not seen in other amino acids, such as general base catalysis. In physiological conditions, the oxygenation, rather than the oxidation, of hemoglobin is dependent on the proximal and distal histidines of the β-globin chains.

The imidazole ring of histidine is unique among amino acid side chains in that it may buffer pH. The two nitrogens on this ring can bind or release a proton, allowing it to take on either an acidic or basic form. Binding to proteins results in pKa values of 6.2 and 6.5 for the imidazole group of free histidine, while CAR and anserine give pKa values of 7.0 and 7.1, respectively. Consequently, during anaerobic activity, histidine-CD like CAR and anserine serve as potent buffers, reducing fluctuations in intracellular pH in the muscles. Since histidine is an effective H⁺ buffer, it is used in solutions that protect myocardial tissue during heart surgery and organs before transplantation.

The pKa of histidine. (Li S., et al., 2011)

Histidine pH and charge

Histidine is classified as a basic amino acid owing to its positive charge at physiological pH (i.e., pH 6.7 - 7.4). The imidazole group possesses a pKa of approximately 6.0, which is near the physiological pH of about 7.4. Histidine can exist in both protonated and deprotonated states at physiological pH, enabling its involvement in acid-base catalysis and metal ion coordination inside proteins. At pH levels below 6, the imidazole group becomes protonated, acquiring a positive charge. At pH levels exceeding 6, the imidazole group becomes deprotonated and assumes a neutral charge. The imidazole side chain's capacity to acquire or relinquish a proton at physiological pH renders histidine an efficient buffer in biological reactions. Histidine is frequently located in enzyme active sites due to its role in stabilizing pH levels.

Phosphorylation of histidine

In prokaryotes, histidine phosphorylation plays a significant role, while in eukaryotic organisms, it accounts for 6% of total phosphorylation. However, proteins rarely contain phosphohistidine residues because the phosphoryl group hydrolyzes quickly in acidic environments. The phosphoenolpyruvate:sugar phosphotransferase system (PTS), bacterial two-component systems, and enzyme-catalyzed reactions like succinyl-CoA synthetase and nucleoside diphosphate kinase all make use of phosphohistidines. No X-ray structures of PTS proteins containing phosphohistidine have been solved, while the NMR structure of the phosphohistidine moiety of phosphohistidine-containing proteins has been solved. Nucleoside diphosphate kinase, succinyl-CoA synthetase, a cofactor-dependent phosphoglycerate mutase, and the protein PAE2307 from the hyperthermophilic archaeon Pyrobaculum aerophilum are among the phosphohistidine-containing proteins whose crystal structures have been identified. The presence of ion-pair hydrogen bonds (salt bridges) connecting the acidic amino acid side chain to the non-phosphorylated nitrogen atom of the histidine imidazole ring is a common feature among these stable phosphohistidines.

Phosphorylation states of histidine. (Puttick J., et al., 2008)

Enzymatic phosphorylation and dephosphorylation of histidine residues in proteins. (Hunter T., 2022)

Timeline of key discoveries in the field of protein-histidine phosphorylation. (Hunter T., 2022)

Histidine biosynthesis

In plants, his biosynthesis follows the identical metabolic pathway as in microorganisms, commencing with the condensation of 5'-phosphoribosyl 1-pyrophosphate (PRPP) and ATP. His biosynthesis is closely associated with nucleotide metabolism, as PRPP, which is produced from ribose-5-phosphate provided by the pentose phosphate pathway, is essential for the de novo synthesis and salvage of purines, pyrimidines, and the pyridine nucleotide cofactors NAD and NADP. Furthermore, the intermediate 5'-phosphoribosyl-4-carboximide-5-aminoimidazole (AICAR) generated at the branch point of the histidine biosynthetic pathway, proceeds into the de novo purine biosynthesis pathway. ATP-phosphoribosyl transferase (ATP-PRT, EC 2.4.2.17) catalyzes the initial reaction in the histidine biosynthetic pathway, namely the condensation of ATP and PRPP to produce N'-5'-phosphoribosyl-ATP (PRATP). The second and third steps in the pathway are catalyzed by PRATP pyrophosphohydrolase (PRA-PH, EC 3.6.1.31) and phosphoribosyl-AMP cyclohydrolase (PRA-CH, EC 3.5.4.19), respectively. Imidazole glycerol-phosphate synthase (IGPS, EC 2.4.2.-) catalyzes the pivotal step in the histidine biosynthetic pathway, leading to the production of IGP and 5′-phosphoribosyl-4-carboximide-5-aminoimidazole (AICAR), which then integrates into the de novo purine biosynthesis pathway. This procedure consists of two steps: the transfer of an amide group from a glutamine donor, followed by a cyclization reaction to form the imidazole ring. In bacteria, the glutamine amidotransferase and cyclase functions reside on distinct polypeptides, hisH and hisF, which assemble into a heterodimer, but the HIS7 gene in S. cerevisiae generates a singular bifunctional protein. The dehydration of IGP to imidazole acetol-phosphate (IAP) is facilitated by imidazole glycerol-phosphate dehydratase (IGPD, EC 4.2.1.19). IAP is subsequently transaminated, utilizing Glu as a nitrogen donor, to form histidinol-phosphate by histidinol-phosphate aminotransferase (HPA, EC 2.6.1.9). Histidinol-phosphate phosphatase (HPP, EC 3.1.3.15) was the final histidine biosynthetic enzyme found in plants, despite the initial discovery of HPP activity in plant extracts occurring about 40 years prior. The concluding processes of the His biosynthetic route involve the two-step oxidation of histidinol to histidine through the aldehyde intermediate histidinal, catalyzed by histidinol dehydrogenase (HDH, EC 1.1.1.23). HDH activity was initially identified in extracts from various plant species, and an HDH protein from wheat germ was partially isolated by Wong and Mazelis.

The histidine biosynthetic pathway. (Ingle R A., 2011)

Histidine amino acids at Creative Peptides

What does histidine do?

Since it cannot be produced in the body, histidine must be consumed in order to meet the amino acid requirements of mammals, fish, and fowl. Histidine shortage causes these creatures to lose weight. In addition, a diet deficient in histidine makes it impossible to keep nitrogen balances. Kriengsinyos et al. found that if total protein consumption is more than the current recommendation (0.75 g/kg), histidine insufficiency may not impact nitrogen balance. Nevertheless, AA oxidation and protein turnover are both reduced in histidine deprivation. Histidine is viewed as an essential amino acid in adults when these additional factors of protein metabolism are taken into consideration. According to some research, histidine is absolutely necessary for ruminants. Red meat and fish are good sources of histidine, however the amount of histidine in different fish species varies. For example, white muscle fish have less histidine than dark muscular fish. A variety of histidine-containing dipeptides, including carnosine and anserine, as well as free L-histidine, N (alpha)-acetylhistidine (NAH), and histidine-containing proteins are found in the bodies of various species (HRC).

(1) For metal ion chelation

Numerous studies have documented the capacity of HIS and HIS-CD, especially CAR, along with HIS-rich proteins, to form complexes with metal ions, including Fe²⁺, Cu²⁺, Co²⁺, Ni²⁺, Cd²⁺, and Zn²⁺. HIS is specifically responsible for the binding of iron in hemoglobin and myoglobin molecules and is often found in the active sites of metalloenzymes, including carbonic anhydrase, cytochromes, heme peroxidases, nitric oxide synthase, and catalases, where it regulates their activity. Histidine-rich glycoprotein found in vertebrate plasma interacts with several ligands, including zinc, and plays a significant role in immunity. Various metal ions facilitate the generation of free radicals via the Fenton reaction and impose harmful effects on organisms, which can be mitigated by HIS or HIS-CD. Research has demonstrated that CAR provides protection against neurotoxicity generated by copper and zinc.

(2) As an antioxidant

The antioxidant efficacy of HIS is facilitated through metal ion chelation, the scavenging of reactive oxygen species (ROS) and reactive nitrogen species (RNS), as well as the sequestration of advanced glycation end products (AGE; e.g., glyoxal and methylglyoxal) and advanced lipoxidation end products (ALE; e.g., malondialdehyde and acrolein). Elevated levels of AGE/ALE are acknowledged as detrimental agents associated with numerous problems, particularly microangiopathy and diabetic retinopathy. HIS-CD, especially CAR, is a more efficient scavenger of ROS/RNS and AGE/ALE compared to free HIS. The fundamental processes behind the antioxidant properties of imidazole-containing drugs are still unclear.

(3) Regulate memory disorders

Geliebter's team investigated the impact of daily histidine dosages, ranging from 24 to 64 g, incorporated into orange juice, on healthy individuals over a four-week period. Subjects reported subsequent headaches, fatigue, lethargy, and nausea. Two patients administered 64 g per day had ocular discomfort and difficulty in focusing. One individual exhibited cognitive disorientation after consuming 64 g daily, along with impaired memory and depressive symptoms characterized by spells of weeping. Sasahara and colleagues found that a daily histidine consumption of 1.65 g lowered fatigue, enhanced efficiency in memory activities, and facilitated clear thinking and focus in individuals exhibiting high fatigue and sleep disruption scores. The significant disparity in histidine dosages likely accounts for the conflicting outcomes observed in the trials conducted by Geliebter and Sasahara.

(4) Improve skin dysfunction

The status of histidine appears to be significant in skin dysfunction and many dermatological disorders. A shortage in histidine, similar to other important amino acids (Ile, Leu, Lys, Met, Cys, Phe, Tyr, Thr, Trp, Val, Arg, or Gln), markedly reduced hyaluronan levels in human dermal fibroblasts. Hyaluronan is crucial for tissue repair, cellular proliferation, and migration in the skin. Furthermore, Voorhees et al. identified an elevated concentration of labeled histidine within keratohyalin, a "histidine-rich" protein (HRP) that has been extracted and described in human necropsy epidermis. No alterations were noted in HRP synthesis within psoriatic lesions. Tan and colleagues discovered that a daily oral intake of histidine (4 g per day) enhanced filaggrin synthesis in relation to dermatitis illness. Filaggrin is a protein found in the granular layer of keratinocytes that aids in barrier function by promoting skin hydration and maintaining the pH of the stratum corneum. The elevation of filaggrin resulted in a reduction of atopic dermatitis severity.

(5) Regulate metabolic syndrome

Dietary histidine may correlate with factors that enhance metabolic syndrome linked with obesity. A cross-sectional study conducted online in a northern Chinese population by Yan-Chuan Li and colleagues found a correlation between elevated dietary histidine intake (1400 mg/d), exceeding dietary requirements (8 to 12 mg/kg of body weight per day in adults as per FAO guidelines, equating to 560 to 840 mg/day for a healthy adult weighing 70 kg), and a reduced prevalence of overweight and obesity, as well as lower BMI, waist circumference, and blood pressure. Histidine supplementation appears to enhance insulin resistance, a significant characteristic of metabolic syndrome, in overweight and obese individuals.

Inflammation biomarkers and pro-inflammatory cytokines are both elevated in obese people. A number of pro-inflammatory cytokines, including TNF-α, IL-1, IL-6, and the inflammation biomarker CRP, were found to have an inverse association with dietary histidine in the research conducted by Li's team. These findings corroborate those of Lee's group. People who are overweight or obese may find that taking a supplement containing histidine reduces inflammation. Another adipocytokine that may be implicated in histidine's impact on obesity is adiponectin, which seems to be involved in the onset of insulin resistance. Supplemental histidine was also associated with elevated blood adiponectin levels in people who were overweight or obese. By reducing inflammation and oxidative stress in fat cells, histidine may enhance adiponectin release.

(6) Regulate food intake

Histidine influences feeding behavior, according to numerous research. According to the findings of these rat experiments, consuming more dietary histidine causes a decrease in food consumption. After eight days of taking 20% casein with either 2.5% or 5% histidine (25 g/kg or 50 g/kg of diet), Kasaoka and colleagues found that subjects consumed less food than those who took 20% casein alone. An activation of histamine neurons can explain the decrease in food intake caused by dietary histidine. Injecting L-histidine into the peritoneum or cerebroventricular space also reduces hunger. Further evidence that histidine inhibits hunger via converting it into histamine in the hypothalamus is provided by the fact that this action is reversed by suppression of histidine decarboxylase (HDC). Through histamine H1 receptors, the secreted histamine influences food intake. A lower caloric intake was associated with reduced food consumption in all of these studies that measured histidine intake. Just like in people, it appears that histamine is responsible for this reduction in food consumption. A study conducted by Holeček showed that rats given histidine-enriched water actually ate more food than rats in the other experiments that showed a decline in food intake. In previous research, the histidine-enriched diet was thought to have caused an AA concentration imbalance, which in turn caused competition for brain cellular transporters, according to their hypothesis. Several AA levels dropped in the brain, which led to a drop in food consumption. The rats in the study ate more food to counteract the higher histidine levels in the water. Perhaps a change in the water or the meal that was supplemented with histidine would have produced different outcomes.

(7) Exhibit neuroprotection

One of the leading causes of mortality in adults is cerebral ischemia. Astrocytes are activated six hours following brain ischemia to promote neuronal survival. A glial scar forms when astrocytes clump together to prevent the lesion from spreading and to stimulate the local immune response. Even when brain ischemia has healed, this barrier can prevent new neurons from being born. Histidine, when converted to histamine, appears to produce neuroprotection at an early stage following cerebral ischemia, according to an early study by Liao and colleagues. Histidine inhibits the recruitment of inflammatory cells and protects neurons, particularly astrocytes, from oxygen-glucose deprivation-induced damage. This same group of researchers has just shown that the late stage following cerebral ischemia is not unaffected. Astrocytes migrated to the infarct location in response to a dose-and stage-dependent therapy with histidine, starting at a high dose of 1000 mg/kg in the early stages and decreasing to 500 mg/kg in the later stages. The fact that histidine allows for neurogenesis to occur in the lesion location following cerebral ischemia suggests that it offers long-term neuroprotection.

Histidine may provide protection against epileptic seizures, according to certain research. In a dose-dependent way, histidine was able to reverse seizures in rats generated by the central nervous system (CNS) stimulant pentylenetetrazole, according to a study conducted by Chen and colleagues. A significant rise in histamine entering the cortex, hippocampus, and amygdala was observed in rats given 500 mg/kg of histidine, according to the study. It appeared that the observed effects were caused by the fact that histidine, a precursor of histamine, increased its synthesis. It appears that epileptic seizures are correlated with brain histamine concentration, since a reduction in histamine lengthened the duration of the seizures. Presynaptic H3-receptors and postsynaptic H1-receptors may mediate histamine's effects on convulsions. In addition, anserine may be an active peptide, according to one study. It is soluble in water. Memory capabilities in Alzheimer disease model mice were enhanced by anserine supplementation, which protected the neurovascular units (endothelial cells, pericytes, and supporting glial cells) in the brain. Thus, it is possible that histidine, through the formation of anserine, also has a neuroprotective impact.

(8) Regulate mineral metabolism

Ions of metal can be chelated by histidine, as mentioned above. Researchers have looked into how histidine affects the absorption and retention of iron, copper, and zinc in a few research. Taking histidine did not change either the iron levels in tissues or the amount of iron lost in feces. The absorption of ascorbic acid was enhanced by adding histidine to a ⁵⁹Fe solution. This fits with the idea that an AA-iron chelate is created and then absorbed, and it implies that there is some direct reaction between the iron and histidine. Regarding copper, rats did not show any effect of histidine consumption. Histidine was discovered to aid copper uptake in liver, placental, and brain cells in vitro. There has been some research on the effects of histidine supplementation on zinc levels, and the results have been mixed. An acute course of histidine supplementation (250 mg/hr intravenously) and a chronic course (500 mg/day gavagely for 43 days) were both studied by Freeman and Taylor. They found that after both short-term and long-term histidine treatment, zinc excretion increased three- to sixfold. On the other hand, prolonged supplementation has not been associated with a drop in plasma zinc levels. Zinc chloride supplemented with L-histidine (40 mg/kg of diet) was found to be more efficient than zinc salt alone in correcting cognitive impairment caused by zinc deficiency in young adult rats. The addition of histidine to perfusion may have stimulated ⁶⁵Zn transport over the brain endothelium, which would account for this impact. According to Snedeker and Greger, protein levels in the diet had a greater effect on zinc absorption and utilization than histidine levels. Still, some research suggests that histidine might lead to zinc shortage. Plasma zinc levels were significantly lower in rats given histidine at levels more than 4 g/kg BW/day for seven to forty-six weeks. Zinc turnover rate (65Zn from two to four weeks after a single tracer injection) was unaffected by 50 g/kg histidine supplementation in rats, but a severe zinc deficiency (a 50% reduction in plasma zinc content) was induced by an 8-glycine diet supplement. The correlation between histidine supplementation and zinc level may be influenced by the amount of zinc consumed through food. The zinc status (zinc concentrations, ⁶⁵Zn tissue distribution, and tissue-specific activity) remained unchanged when rats were given a zinc-adequate diet and histidine supplementation. But histidine supplementation reduced ⁶⁵Zn retention, which in turn increased fecal excretion and decreased the biological half-life, when zinc intake was inadequate.

(9) Inhibit cancer development

New evidence suggests that histidine consumption and catabolism may influence methotrexate sensitivity in cancer cells. Although methotrexate is effective against some solid tumors and blood cancers, it has the potential to damage healthy cells as well. After reducing methotrexate sensitivity by depleting amidohydrolase domain containing 1, histidine ammonia lyase (HAL), and formimidoyltransferase cyclodeaminase (FTCD), CRISPR/Cas9-mediated gene targeting was used. In addition, the combination of methotrexate and histidine significantly reduced tumor growth in the mice, in comparison to the other treatment groups. Taken together, these datasets show how methotrexate's chemotherapeutic efficacy is influenced by the histidine degradation pathway.

Fates of histidine in the human body. (Brosnan M E., et al., 2020)

FAQ

Is histidine in DNA?

Direct incorporation of histidine into DNA does not occur. DNA is composed of sugar, phosphate groups, and nitrogenous bases (adenine, thymine, cytosine, or guanine) that are called nucleotides. Histidine and other amino acids do not occur in DNA but rather in proteins. Nevertheless, histidine plays a crucial role in proteins that are encoded by DNA genes.

Is Histidine polar or nonpolar?

Histidine is categorized as a polar amino acid because its imidazole side chain contains a nitrogen atom that can form hydrogen bonds and engage with water. The imidazole group in histidine has two nitrogen atoms, one of which can undergo protonation or deprotonation based on the pH level. This enables the side chain to engage in hydrogen bonding and interact with water molecules, rendering it polar. The imidazole ring exhibits notable hydrophilicity, particularly when the nitrogen is protonated at lower pH levels. Histidine is frequently located in enzyme active sites, where its polarity facilitates interactions with substrates or other molecules. Histidine's capacity for protonation or deprotonation, attributable to the pKa of its imidazole ring, renders it advantageous in enzyme catalysis and protein-protein interactions.

References

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2. Abualreish M J., et al., Experimental measurement and correlation of two α-amino acids solubility in aqueous salts solutions from 298.15 to 323.15 K, Korean Chemical Engineering Research, 2020, 58(1): 98-105.
3. Harfouche L C., et al., Nucleation behaviour of racemic and enantiopure histidine, CrystEngComm, 2021, 23(47): 8379-8385.
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8. Brosnan M E., et al., Histidine metabolism and function, The Journal of Nutrition, 2020, 150: 2570S-2575S.