Effects of singlet oxygen on the extracellular matrix protein collagen: oxidation of the collagen crosslink histidinohydroxylysinonorleucine and histidine

The reaction of singlet oxygen, a putative agent of skin photodamage, with the dermal collagen crosslink histidinohydroxylysinonorleucine (HHL) and its precursor histidine is reported. Reaction studies were performed with both purified HHL and bovine dermal tissue. We demonstrate that singlet oxygen can selectively oxidize HHL and histidine amino acid residues in dermal tissue and that intermediate oxidation products of histidine lead to new crosslink products. A novel mechanism for crosslink formation was proposed to involve nucleophilic addition to a transient imidazolone intermediate formed from singlet oxygen oxidation of the histidine imidazole moiety. The implication for such adduct formation and histidine oxidation in collagen proteins is the expression of aberrant collagen crosslinks, perturbation of the dermal collagen function, and hence an altered dermal state.     

Collagen crosslinking: isolation of hydroxyaldol-histidine, a naturally-occurring crosslink.

A new trifunctional crosslink, termed hydroxyaldol-histidine, was isolated from cow skin collagen. This compound was not reducible by sodium borohydride; it was characterized by PMR spectroscopy and by low and high resolution mass spectroscopy of volatile derivatives. This crosslink is identical to an unknown amino acid previously detected in pure collagen-derived peptides. We postulate that it arises by condensation of peptidyl allysine, hydroxyallysine and histidine. This is the first example of a non-borohydride reducible crosslink found in collagen.     

Isolation, purification and characterization of histidino-threosidine, a novel Maillard reaction protein crosslink from threose, lysine and histidine

We isolated a novel acid-labile yellow chromophore from the incubation of lysine, histidine and d-threose and identified its chemical structure by one and two-dimensional NMR spectroscopy combined with LC-tandem mass spectrometry. This new cross-link exhibits a UV absorbance maximum at 305 nm and a molecular mass of 451 Da. The proposed structure is 2-amino-5-(3-((4-(2-amino-2-carboxyethyl)-1H-imidazol-1-yl)methyl)-4-(1,2-dihydroxyethyl)-2-formyl-1H-pyrrol-1-yl)pentatonic acid, a cross-link between lysine and histidine with addition of two threose molecules. It was in part deduced and confirmed through synthesis of the analogous compound from n-butylamine, imidazole and d-threose. We assigned the compound the trivial name histidino-threosidine. Systemic incubation revealed that histidino-threosidine can be formed in low amounts from fructose, glyceraldehyde, methylglyoxal, glycolaldehyde, ascorbic acid, and dehydroascorbic acid, but at a much higher yield with degradation products of ascorbic acid, i.e. threose, erythrose, and erythrulose. Bovine lens protein incubated with 10 and 50 mM threose for two weeks yielded 560 and 2840 pmol/mg histidino-threosidine. Histidino-threosidine is to our knowledge the first Maillard reaction product known to involve histidine in a crosslink.     

Reversible reaction via a carbanion intermediate in the elimination of ammonia from l-histidine catalysed by histidine ammonia-lyase

l-[5′-²H₂]Histidine was used as a substrate to investigate the enzymatic reaction mechanism with histidine ammonia-lyase from Pseudomonas fluorescens. The study was performed to determine the exchange rate of deuterium at C-5′ of the imidazole ring with solvent hydrogen relative to the net urocanic acid production. The extent of hydrogen exchange at C-5′ of histidine or urocanic acid was measured by gas chromatography—mass spectrometry—selected ion monitoring, monitoring the molecular ion intensities of the respective gas chromatographic derivatives, at m/z 380 and 379 for histidine and at m/z 267 and 266 for urocanic acid. The observed hydrogen exchange at C-5′ suggested a reversible mechanism via a carbanion intermediate in the reaction with histidine ammonia-lyase.     

A dye-photosensitized reaction that generates stable protein-protein crosslinks

Irradiation of fibrinogen with visible light for 30 s in the presence of 1-1000 microM fluorescein was found to crosslink fibrinogen both inter- and intramolecularly. Optimum crosslinking was achieved at dye concentrations of around 100 microM and the amount of crosslinking was shown to increase with pH. Crosslinking was inhibited in the presence of 50 microM tryptophan or tyrosine and enhanced in the presence of 5 mM histidine. Twice as much crosslinking was found to take place under anaerobic conditions. These observations are consistent with a dye-photosensitized reaction following the hydrogen abstraction pathway. The subunits of eukaryotic ribosomes and those of phosphorylase a were also crosslinked by the method described.     

Alkylation of calf thymus DNA with reductively activated mitomycin C

Mitomycin C (MMC) was catalytically reduced in the presence of a nucleotide or calf thymus DNA. Reaction with 5'-guanylic acid (5'-GMP) gave 1,2-cis-2, 7-diamino-1-(5'-guanylyl) mitosene. Reaction with calf thymus DNA gave modified DNA, which on enzymatic hydrolysis gave two alkylated 5'-deoxyguanylic acid (MG-1 and MG-2) and an alkylated 5'-deoxyadenylic acid (MA). This is the first example of isolation of nucleotides from DNA modified by MMC.     

2,3′-Diamino-4,4′-stilbenedicarboxylic acid sensitizer for dye-sensitized solar cells: quantum chemical investigations

The metal-free organic dye sensitizer 2,3′-diamino-4,4′-stilbenedicarboxylic acid has been investigated for the first time for dye-sensitized solar cell applications. Density functional theory (DFT) and time-dependent DFT (TD-DFT) calculations (performed using the hybrid functional B3LYP) were carried out to analyze the geometry, electronic structure, polarizability, and hyperpolarizability of 2,3′-diamino-4,4′-stilbenedicarboxylic acid used as a dye sensitizer. A TiO₂ cluster was used as a model semiconductor when attempting to determine the conversion efficiency of the selected dye sensitizer. Our TD-DFT calculations demonstrated that the twenty lowest-energy excited states of 2,3′-diamino-4,4′-stilbenedicarboxylic acid are due to photoinduced electron-transfer processes. Moreover, interfacial electron transfer between a TiO₂ semiconductor electrode and the dye sensitizer occurs through electron injection from the excited dye to the semiconductor’s conduction band. Results reveal that metal-free 2,3′-diamino-4,4′-stilbenedicarboxylic acid is a simple and efficient sensitizer for dye-sensitized solar cell applications.     

Reversible stepwise mechanism involving a carbanion intermediate in the elimination of ammonia from L-histidine catalyzed by histidine ammonia-lyase.

L-Histidine labeled with deuterium at the C-5' position of the imidazole ring, L-[5'-2H]histidine (His-5'-D), was used as a probe for investigating a stepwise reversible mechanism via a carbanion intermediate in the elimination of ammonia catalyzed by histidine ammonia-lyase (EC 4.3.1.3). The labeled L-histidine (His-5'-D) (2.45 mM) was incubated with histidine ammonia-lyase (200 units) from Pseudomonas fluorescens at pH 7.0 or 9.0 at 25.0 degrees C for 24 h. The time course of the reaction was examined to determine the rates of enzyme-catalyzed hydrogen exchange at C-5' of L-histidine and urocanic acid. The finding of the enzyme-catalyzed hydrogen exchange at C-5' of both L-histidine and urocanic acid in the presence of L-histidine provided a rational explanation for a stepwise reversible mechanism via a carbanion intermediate in the elimination reaction. The rate of increase in the concentration of urocanic acid exchanged with hydrogen (UA-5'-H) did not depend on the formation rate of urocanic acid and UA-5'-H was continuously formed at a constant rate (25.6 microM/h) even after the completion of urocanic acid formation. These observations suggested the presence of the reversible reaction of urocanic acid and a carbanion intermediate. Since there was only a minor contribution for the formation of UA-5'-H from L-histidine exchanged with solvent hydrogen (His-5'-H), the main pathway in the enzymatic reaction of His-5'-D must be the formation of UA-5'-D via a carbanion intermediate (carbanion-D). Regeneration of the carbanion-D from UA-5'-D by its reverse reaction and subsequent hydrogen incorporation at C-5' would contribute to a large extent for the formation of UA-5'-H. The stability of carbanion was also demonstrated to be approximately three times higher at pH 7.0 than at pH 9.0.     

Proton magnetic resonance studies on Escherichia coli dihydrofolate reductase. Assignment of histidine C-2 protons in binary complexes with folates on the basis of the crystal structure with methotrexate and on chemical modifications.

The effects of pH upon the C-2 resonances of the 5 histidine residues of Escherichia coli MB 1428 dihydrofolate reductase in binary complexes with methotrexate, aminopterin, folate, methopterin, and trimethoprim were studied by 300-MHz 1H nmr spectroscopy. Three of the five histidine residues, labeled 1, 2, and 3, exhibited similar pK' values and chemical shifts for their C-2 protons in the five binary complexes. One histidine, 4, was quite different in the folate complex and the last histidine, 5 was quite different in the trimethoprim complex. For all five binary complexes, each histidine had a pK' which was significantly different from the other 4 histidines of that complex. Titration of the binary methotrexate complex of a 5,5'-dithiobis(2-nitrobenzoate)-modified enzyme showed that 2 histidines were not perturbed by this modification of Cys 152, and that the alkaline form of histidine 2, the acid form of histidine 4, and, to a lesser extent, the acid form of histidine 3 were slightly perturbed. Titration of the binary methotrexate complex of a N-bromosuccinimide-modified enzyme demonstrated that this modification slightly affected all of the histidines and drastically affected histidine 5. Histidines 3 and 5 of the binary methotrexate complex reacted rapidly with the histidine-specific reagent, ethoxyformic anhydride, while histidines 2 and 4 reacted at a moderate rate and histidine 1 reacted slowly if at all. The local electrostatic environments of the 5 histidine residues as deduced from the crystal structure of the binary complex of the enzyme with methotrexate (Matthews, D.A., Alden, R.A., Bolin, J.T., Freer, S.T., Hamlin, R., Xuong, N., Kraut, J., Poe, M., Williams, M.N., and Hoogsteen, K. (1977) Science 197, 594-597) were used as the basis for proposed assignments of the five histidine C-2 nmr resonances. The assignments were: 1, pK' 7.9 to 8.2, His 124; 2, pK' 7.2 to 7.4, His 141; 3, pK' 6.5 to 6.7, His 149; 4, pK' 5.7 to 6.3, His 114; and 5, pK' 5.2 to 5.9, His 45. The effect of the chemical modifications upon the enzyme's histidine residues were consistent with the assignments, but no direct chemical evidence in support of the assignments was obtained. It was proposed that, since the crystallographic data provided consistent assignments of the histidine nmr data for both native and chemically modified enzyme, the local environment of each of the 5 histidine residues was similar in the crystal and in solution.     

Determination of the dissociation constants of pea diamine oxidase.

The activity of diamine oxidase [EC 1.4.3.6] (DAO) isolated from pea cotyledons was measured in Britton-Robinson buffers at pH range 5.0-9.6 by spectrophotometric method with E-1,4-diamino-2-butene as substrate. The enzyme has the highest activity at pH = 7.7 and in pH greater than 8.0 it is irreversible denaturated with time. The dissociation constants of the enzyme and enzyme-substrate complex were calculated by Dixon's method from plots of log Vmax, log KM and log Vmax/KM against pH. The pKEA = 6.5 suggests that histidine is in active site of DAO.     

Factors controlling the expressed activity of histidine ammonia-lyase in the epidermis and the resulting accumulation of urocanic acid.

The synthesis of urocanic acid by histidine ammonia-lyase in guinea-pig epidermis was investigated in various ways. 1. In epidermal homogenates the enzyme obeys Michaelis-Menten kinetics and shows marked dependence of its activity of pH, such that below pH 6 it is inactive. 2. Part-thickness skin samples cultured with radioactive histidine do not accumulate detectable radioactive urocanic acid during 3 days in culture. 3. Very little histidine ammonia-lyase activity can be detected in the living cells of the epidermis. The enzyme is almost completely restricted to the dead cells of the stratum corneum. 4. Radioactive histidine injected into living animals does not result immediately in the accumulation of radioactive urocanic acid. By 3 days after the injection, however, both radioactive urocanic acid and histidine appear, apparently at the expense of radioactive proteins, 5. In isolated stratum corneum, the residual histidine can be converted into urocanic acid by the histidine ammonia-lyase in the tissue only if the natural acidity of the tissue is neutralized. It is concluded from these observations that the biosynthesis of urocanic acid occurs naturally only in the stratum corneum, which contains only dead cells. The amount of urocanic acid accumulated is limited by the availability of free histidine produced by proteolysis of residual protein in these cells and by the penetration into the stratum corneum of the 'acid mantle' of the skin.     

Studies on the production and assessment of experimental histidinemia in the rat.

Intraperitoneal administration to rats of D- or DL-alpha-hydrazinoimidazolylpropionic acid was found to produce a substantial inactivation of hepatic histidine ammonia-lyase (EC 4.3.1.3) in vivo. Proportional to this loss in enzyme activity was an impairment of the ability of treated rats to oxidize L-[ring-2-14C] histidine to 14CO2. Rats in which hepatic histidine ammonia-lyase activity was either depressed by DL-hydrazinoimidazolylproprionic acid injection or elevated by feeding a high protein diet displayed proportionately altered rates of 3H2O release into plasma water following L-[3-3H] histidine administration. Plasma L-histidine clearance following loading with this amino acid was similarly affected by these treatments. Administration of DL-alphal-hydrazinoimisazolylproprionic acid to rats was also found to inactivate non-specifically pyridoxal 5-phosphate enzymes in vivo; pyridoxine injection was found to reverse the DL-alpha-hydrazinoimidazolylproprionic acid-induced inactivation of hepatic aspartate aminotransferase (EC 2.6.1.1) in vivo, but not that of hepatic histidine ammonia-lyase. These findings demonstrate that histidine ammonia-lyase is the rate-limiting factor in L-histidine degradation in the rat. The potential usefulness of DL-hydrazinoimidazolylproprionic acid in the production of an animal model for histidinemia (hereditary histidine ammonia-lyase deficiency) is discussed.     

Mutagenesis and heterologous expression in yeast of a plant Delta6-fatty acid desaturase.

Membrane-bound microsomal fatty acid desaturases are known to have three conserved histidine boxes, comprising a total of up to eight histidine residues. Recently, a number of deviations from this consensus have been reported, with the substitution of a glutamine for the first histidine residue of the third histidine box being present in the so called 'front end' desaturases. These enzymes are also characterized by the presence of a cytochrome b5 domain at the protein N-terminus. Site-directed mutagenesis has been used to probe the functional importance of a number of amino acid residues which comprise the third histidine box of a 'front end' desaturase, the borage Delta6-fatty acid desaturase. This showed that the variant glutamine in the third histidine box is essential for enzyme activity and that histidine is not able to substitute for this residue.     

The mycotoxin patulin induces intra- and intermolecular protein crosslinks in vitro involving cysteine, lysine, and histidine side chains, and alpha-amino groups

As previous studies have indicated a multiple electrophilic reactivity of patulin (PAT) towards simple thiol nucleophiles, we have methodically investigated the ability of PAT to covalently crosslink proteins in vitro. By means of sodium dodecylsulphate polyacrylamide gel electrophoresis, the formation of PAT-induced intermolecular protein-protein crosslinks was clearly demonstrated for bovine serum albumin containing one thiol group per molecule, but also for the thiol-free hen egg lysozyme. Characterization of the crosslink sites was carried out by (1) modulation of the thiol groups with N-ethylimaleimide and 2-iminothiolane; (2) comparison with various known crosslinking agents, i.e. phenylenedimaleimide, glutardialdehyde, and dimethylsuberimidate, and (3) fluorescence incorporation studies using dansyl-labeled amino acids and a fluorescent glutathione derivative. The thiol group of cysteine was preferred for PAT-mediated crosslink reactions, but the side chains of lysine and histidine, and alpha-amino groups also exhibited reactivity. PAT can act both as a homobifunctional as well as a heterobifunctional crosslinking agent. The initial formation of a monoadduct with a thiol group appears to activate PAT for the subsequent reaction with an amino group, but also leads to rapid loss of further electrophilic properties when no second nucleophile for crosslink completion is available. Studies using microtubule proteins as a protein with experimentally controllable quarternary structure and a proposed cellular target for PAT toxicity emphasized the influence of specific sterical conditions on crosslink formation at low protein concentrations. Non-polymerized microtubule proteins, i.e. tubulin alpha,beta-dimers, formed a defined product with PAT consisting of an intramolecularly crosslinked beta-tubulin, whereas guanosine triphosphate- or paclitaxel-induced polymerization to microtubule-like quarternary structures prior to treatment with PAT gave rise to intermolecular crosslink formation between alpha- and beta-tubulin. In contrast, denaturated tubulin yielded none of those two new protein species, but only unspecific intramolecular crosslinks and highly crosslinked aggregates. Thus, in addition to the amino acid composition, the tertiary and quarternary superstructures of proteins appear to markedly influence their reactivity towards PAT. Under appropriate conditions, the generation of protein crosslinks could easily be observed at concentrations of PAT equal to or even below the concentration of the protein. The relevance of these novel reaction pathways of PAT demonstrated in vitro for its in vivo mechanisms of toxicity remains to be investigated.     

The effects of vitamin A deficiency on hepatic folate metabolism in rats

The effects of severe vitamin A deficiency (liver retinol less than 2 micrograms/g) on hepatic folate metabolism in rats were studied. The oxidation of a [ring-2-14C] histidine load or a [14C]formate load to 14CO2 was significantly depressed in vitamin A-deficient rats and those given histidine also excreted more urinary formiminoglutamic acid (FiGlu) than pair-fed controls. The increase in FiGlu excretion was not due to augmented production from histidine, implicating an impairment of FiGlu catabolism. FiGlu formiminotransferase activity was unaltered in vitamin A-deficient rats, but hepatic tetrahydrofolic acid (THF) concentration was decreased by 58% in vitamin A-deficient rats given a histidine load while 5-methyl-THF concentration was increased by 39%. Formyl-THF and total folate levels were similar to controls. A redistribution of folate coenzymes was not found in vitamin A-deficient rats not force fed histidine. A 43% decrease in 10-formyl-THF dehydrogenase activity, which generates both THF and the 14CO2 from the labeled substrates, and an 81% increase in 5,10-methylene-THF reductase activity, which generates 5-methyl-THF, were found in vitamin A-deficient rats. It appears that the production of severe vitamin A deficiency results in selective changes in the activities of hepatic folate-dependent enzymes, so that when a load of a one-carbon donor is given, THF concentration decreases and metabolism of the load is impaired.     

Histamine, histidine, and growth-phase mediated regulation of the histidine decarboxylase gene in lactic acid bacteria isolated from wine

Fermented foods are frequently contaminated by histamine that is generated by microorganisms with histidine decarboxylase activity. The ingestion of large amounts of histamine can cause serious toxicological problems in humans. A study of the effects of histamine, histidine, and growth phase on histamine production by lactic acid bacteria isolated from wine is reported here. With northern blots and specific activity analysis, we observed that histidine induces the expression of the histidine decarboxylase gene (hdc) and that histamine causes a decrease in the expression of this gene. The expression of hdc is also mediated by the bacterial growth phase. Histidine and histamine do not affect histidine decarboxylase activity, whereas pyridoxal 5'-phosphate does. Data on histamine-producing lactic acid bacteria isolated from wine should contribute to the prevention of histamine formation during winemaking and storage.     

Polyamine-linked sepharoses: preparation and application to mammalian spermine synthase.

Seven different polyamine-linked Sepharose derivatives were prepared for the affinity chromatography of spermidine and spermine binding macromolecules: Spermine synthase from rat and hog brain was used as a model protein with a spermidine binding site. Comparative studies of the affinities of the enzymes for the seven matrixes suggested that two negative charges, three to four methylene groups apart, should be present at the decarboxylated S-adenosylmethionine binding site and should improve the binding of the enzyme to the Sepharose derivative. Two negative charges at the spermidine binding site would be expected to do the same. Three affinity matrixes linked with 1,17-diamino-4,9,14-triazaheptadecane, 1,21-diamino-4,9,13,18-tetraazaheneicosane, and 5-spermine carboxylic acid, respectively, had an affinity for spermine synthases higher than that of spermine-Sepharose, which has been used for the purification of spermine synthase. The first of these matrixes was used and proved to be effective for the purification.     

2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate synthases of fungi and archaea

The pathway of riboflavin (vitamin B2) biosynthesis is significantly different in archaea, eubacteria, fungi and plants. Specifically, the first committed intermediate, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate, can either undergo hydrolytic cleavage of the position 2 amino group by a deaminase (in plants and most eubacteria) or reduction of the ribose side chain by a reductase (in fungi and archaea). We compare 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate synthases from the yeast Candida glabrata, the archaeaon Methanocaldococcus jannaschii and the eubacterium Aquifex aeolicus. All three enzymes convert 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate into 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate, as shown by 13C-NMR spectroscopy using [2,1',2',3',4',5'-13C6]2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate as substrate. The beta anomer was found to be the authentic substrate, and the alpha anomer could serve as substrate subsequent to spontaneous anomerisation. The M. jannaschii and C. glabrata enzymes were shown to be A-type reductases catalysing the transfer of deuterium from the 4(R) position of NADPH to the 1' (S) position of the substrate. These results are in agreement with the known three-dimensional structure of the M. jannaschii enzyme.     

Interaction between Glu-219 and His-245 within the a subunit of F1F0-ATPase in Escherichia coli

Oligonucleotide-directed mutagenesis was used to generate mutations in the a subunit gene (uncB) altering the glutamic acid 219 and the histidine 245 codons. Substitutions of aspartic acid, glutamine, histidine, and leucine for glutamic acid at position 219 neither alter the hydrolytic activity of membrane-bound F1 nor the association of F1 with F0. However, the efficiency of F0-mediated proton translocation was reduced to varying degrees. Replacement of glutamic acid 219 with leucine reduced the ATP-driven proton pumping activity of intact F1F0 to undetectable levels. Roughly 5% of normal activity was observed when glutamine occupied position 219. Surprisingly higher activity, approaching 20% of wild type levels, is seen when histidine replaced glutamic acid 219. The aspartic acid substitution resulted in little loss of enzyme function. Substitution of glutamic acid for histidine 245 results in a reduction to about 45% of normal proton translocation. Construction of the double mutant with substitution of histidine at position 219 and glutamic acid at position 245 yields a complex with better proton translocation than with either mutant separately. The possibility that a functionally important interaction between histidine 245 and glutamic acid 219 of the a subunit may be directly involved in the proton translocation mechanism of F1F0-ATP synthase is discussed.     

The role of histidine in the anemia of folate deficiency

The amino acid histidine is metabolized to glutamic acid in mammalian tissue. Formiminoglutamic acid (FIGLU) is an intermediary in this reaction, and tetrahydrofolic acid is the coenzyme that converts it to glutamic acid. A test for folate deficiency concerns the measurement of urinary FIGLU excretion after a histidine load. It was observed that folate-deficient individuals receiving the histidine for the FIGLU test made hematological response that alleviated the anemia associated with this deficiency. This was unusual in that a biochemical test to determine the deficiency results in a beneficial effect for one aspect of the deficiency. The studies reported in this paper give a metabolic explanation for this phenomenon. Urine was collected for 24 hr from 25 folate-deficient subjects, 10 vitamin B(12)-deficient subjects, and 15 normal controls. Urinary excretion of histidine was a mean of 203 mg with a range of 130-360 mg for the folate-deficient subjects; 51.5 mg with a range of 30-76.6 mg for normal subjects; and 60.0 mg with a range of 32.3-93.0 mg for the vitamin B(12)-deficient subjects. All the folate-deficient subjects subsequently made a hematological response to the histidine administered for the FIGLU test. No hematological response was observed in the vitamin B(12)-deficient individuals. When folic acid was given to folate-deficient subjects who received no histidine, urinary histidine levels returned to normal levels rapidly and this was followed by a hematological response. Others have shown that volunteers fed a histidine-free diet developed anemia. In normal subjects, histidine is excreted much more in the urine than other essential amino acids are. Hemoglobin protein contains 10% histidine. Under normal conditions, dietary histidine can supply sufficient histidine to prevent anemia. When the dietary intake is diminished or the urinary excretion is greatly increased, anemia results. It is concluded that folate deficiency causes histidine depletion through increased urinary excretion of this amino acid. Feeding histidine replenishes tissue levels of histidine, resulting in hemoglobin regeneration. Folic acid administration results in return of histidine to normal urinary levels. Thus, a combination of folic acid histidine would be beneficial for folate deficient individuals.