Imidazolelactic Acid Research Articles

Dissecting the effects of paraquat-induced pulmonary injury in rats using UPLC-Q-TOF-MS/MS-based metabonomics.

Paraquat (PQ) is a toxic compound that selectively accumulates in the lungs, inducing severe pulmonary inflammation and fibrosis. However, data on the metabolomic changes induced by the PQ remain scant. This study aimed to determine the metabolic changes in Sprague-Dawley rats subjected to PQ using UPLC-Q-TOF-MS/MS. We established groups of PQ-induced pulmonary injury rats for 14 or 28days. Our data showed that PQ decreased the survival of the rats and induced pulmonary inflammation at day 14 or pulmonary fibrosis at day 28. There was upregulation of IL-1β expression in the inflammation group as well as upregulation of fibronectin, collagen and α-SMA in the pulmonary fibrosis group. OPLS-DA revealed differential expression of 26 metabotites between the normal and the inflammation groups; 31 plasma metabotites were also differently expressed between the normal and the fibrosis groups. There was high expression of lysoPc160-, hydroxybutyrylcarnitine, stearic acid, and imidazolelactic acid in the pulmonary injury group compared to the normal group. Metabolomics analysis confirmed that the PQ-induced lung injury was not only related to the aggravation of inflammation and apoptosis but also to mediated histidine, serine, glycerophospholipid, and lipid metabolism. This study gives insights into the mechanisms of PQ-induced lung injury and highlights the potential therapeutic targets. The effect of PQ on lung injury in rats was detected by metabonomics, and the possible metabolic mechanism was investigated by KEGG analysis. OPLS-DA revealed the differential expression of 26 metabotites and 31 plasma metabotites between the normal and the pulmonary injury groups. Metabolomics analysis confirmed that the PQ-induced lung injury was not only related to the aggravation of inflammation and apoptosis but also to mediated histidine, serine, glycerophospholipid, and lipid metabolism. Oleoylethanolamine, stearic acid, and imidazolelactic acid are potential molecular markers in PQ-induced pulmonary injury.

Urine biomarkers discovery by metabolomics and machine learning for Parkinson's disease diagnoses

Parkinson's disease (PD) is a complex neurological disorder that typically worsens with age. A wide range of pathologies makes PD a very heterogeneous condition, and there are currently no reliable diagnostic tests for this disease. The application of metabolomics to the study of PD has the potential to identify disease biomarkers through the systematic evaluation of metabolites. In this study, urine metabolic profiles of 215 urine samples from 104 PD patients and 111 healthy individuals were assessed based on liquid chromatography-mass spectrometry. The urine metabolic profile was first evaluated with partial least-squares discriminant analysis, and then we integrated the metabolomic data with ensemble machine learning techniques using the voting strategy to achieve better predictive performance. A combination of 8-metabolite predictive panel performed well with an accuracy of over 90.7%. Compared to control subjects, PD patients had higher levels of 3-methoxytyramine, N-acetyl-l-tyrosine, orotic acid, uric acid, vanillic acid, and xanthine, and lower levels of 3,3-dimethylglutaric acid and imidazolelactic acid in their urine. The multi-metabolite prediction model developed in this study can serve as an initial point for future clinical studies.

Metabolomics for improved treatment monitoring of phenylketonuria: urinary biomarkers for non-invasive assessment of dietary adherence and nutritional deficiencies.

Management of phenylketonuria (PKU) requires lifelong restriction of phenylalanine (Phe) intake using specialized medical foods to prevent neurocognitive impairment in affected patients. However, dietary adherence is challenging to maintain while ensuring adequate nutrition, which can lead to sub-optimal clinical outcomes. Metabolomics offers a systematic approach to identify new biomarkers of disease progression in PKU when using urine as a surrogate for blood specimens that is more accurate than self-reported diet records. Herein, the plasma and urine metabolome of a cohort of classic PKU patients (median age = 11 years; n = 22) mainly prescribed (78%) a Phe-restricted diet were characterized using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Overall, there was good mutual agreement between plasma Phe and tyrosine (Tyr) concentrations measured from PKU patients when using an amino acid analyzer based on UPLC-UV as compared to MSI-CE-MS with a mean bias of 12% (n = 82). Longitudinal measurements of recently diagnosed PKU infants (n = 3) revealed good long-term regulation of blood Phe with dietary management, and only occasional episodes exceeding the recommended therapeutic range (>360 μM) unlike older PKU patients. Plasma metabolomic studies demonstrated that non-adherent PKU patients had lower circulating concentrations of Tyr, arginine, 2-aminobutyric acid, and propionylcarnitine (q < 0.05, FDR) that were inversely correlated to Phe (r ≈ -0.600 to -0.830). Nontargeted metabolite profiling also revealed urinary biomarkers associated with poor dietary adherence among PKU patients, including elevated concentrations of catabolites indicative of Phe intoxication (e.g., phenylpyruvic acid, phenylacetylglutamine, hydroxyphenylacetic acid). Additionally, PKU patients with poor blood Phe control had lower excretion of urinary compounds derived from co-metabolism of Tyr due to microbiota activity (e.g., cresol sulfate, phenylsulfate), as well as several metabolites associated with inadequate nutrient intake, including low carnitine and B vitamin status (e.g., folic acid, vitamin B12). Interestingly, an unknown urinary metabolite was strongly correlated with Phe excretion in PKU patients (r = 0.861), which was subsequently identified as imidazole lactic acid when using high resolution MS/MS. Overall, urine profiling offers a non-invasive approach for better treatment monitoring of individual PKU patients, which can also guide the design of novel therapies that improve adherence to Phe-restricted diets without acquired nutritional deficiencies.

Metabolomics analysis of blood identifies potential biomarkers and possible treatment targets for nocturia.

Background:Our aim was to investigate the association between serum metabolites and nocturia.Methods:A total of 66 males aged 65–80 years were enrolled in this study and stratified according to micturition behavior, which was characterized in terms of the 24 h frequency volume chart (FVC) for 3 consecutive days, the International Prostate Symptom Score (IPSS), and quality-of-life score. The nocturia group included participants with any total IPSS and ⩾1.5 micturitions/night as the mean of 3 nights, while the control group included participants with total IPSS < 8 and <1.5 micturitions/night. We conducted a comprehensive capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) study of plasma metabolites. Between-group comparisons of metabolite levels employed the Welch t test. The relationship between nocturia and metabolite profiles was determined using multivariable logistic regression analysis.Results:Of 66 participants, 45 were included in the nocturia group and 21 in the control group. There were no differences in background factors between the two groups. FVC analysis revealed that urine production during night-time, as well as micturition frequency during daytime and night-time were significantly higher in the nocturia group. CE-TOFMS identified eight metabolites whose plasma levels differed between the two groups. Multivariate analysis indicated that increased levels of lauric acid and imidazolelactic acid, as well as decreased levels of thiaproline and glycerol, contribute to the etiology of nocturia in aged men.Conclusions:Our findings suggest that abnormal serum levels of metabolites in several pathways play a role in the pathogenesis of nocturia in aged men.

Metabonomics analysis of kidneys in rats administered with chronic low-dose cadmium by ultra-performance liquid chromatography-mass spectrometry.

This study aimed to investigate the nephrotoxicity in rats administered with chronic low-dose cadmium (Cd) by ultra-performance liquid chromatography-mass spectrometry. A total of 40 male Sprague-Dawley rats were randomly assigned to four groups, namely: control; low-dose (0.13mg/kg·body weight [bw]); middle-dose (0.80mg/kg·bw); and high-dose (4.89mg/kg·bw). The rats received CdCl2 daily via drinking water for 24weeks. Rat kidneys were collected for metabonomics analysis. Principal components analysis and partial least-squares discriminant analysis were used to investigate the metabonomics profile changes in the kidney samples and to screen the potential biomarkers. Ten metabolites were identified in the positive and negative ion modes. Compared with the control group, the intensities of tetranor 12-HETE, uric acid, hypoxanthine, phenylacetylglycine, guanidinosuccinic acid and xanthosine significantly increased (P<0.01), and those of imidazolelactic acid, lactose 6-phosphate, l-urobilinogen and arachidonic acid significantly decreased (P<0.01) in the high-dose group. Results showed that exposure to Cd in rats induced oxidative stress to the kidneys and disrupted amino acid metabolism, fatty acid metabolism and energy metabolism.

Effects of histidine and imidazolelactic acid on various parameters of the oxidative stress in cerebral cortex of young rats

Histidinemia is an inherited metabolic disorder caused by deficiency of histidase activity, which leads to tissue accumulation of histidine and its derivatives. Affected patients usually present with speech delay and mental retardation, although asymptomatic patients have been reported. Considering that the pathophysiology of the neurological dysfunction of histidinemia is not yet understood and since histidine has been considered a pro-oxidant agent, in the present study we investigated the effect of histidine and one of its derivatives, l-β-imidazolelactic acid, at concentrations ranging from 0.1 to 10mM, on various parameters of oxidative stress in cerebral cortex of 30-day-old Wistar rats. Chemiluminescence, total radical-trapping antioxidant potential (TRAP), thiobarbituric acid reactive substances (TBA-RS), and the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were measured in tissue homogenates in the presence of l-histidine or l-β-imidazolelactic acid. We observed that l-histidine provoked an increase of chemiluminescence and a reduction of TRAP at concentrations of 2.5mM and higher, while TBA-RS measurement, GSH-Px, CAT and SOD activities were not affected. Furthermore, l-β-imidazolelactic acid provoked antioxidant effects at high concentrations (5–10mM) as observed by the reduction of chemiluminescence, although this compound enhanced chemiluminescence at low concentrations (0.5–1mM). These results suggest that in vitro oxidative stress is elicited by histidine but only at supraphysiological concentrations.

Studies on the possibility of histidine biosynthesis from histodinol, imidazolepyruvic acid, imidazoleacetica acid, and imidazolelactic acid by mixed ruminal bacteria, protozoa, and their mixture in vitro.

The possibility of histidine (His) synthesis using a main biosynthetic pathway involving histidinol (HDL) and also the recycling capability of imidazolic compounds such as imidazolepyruvic acid (ImPA), imidazoleacetic acid (ImAA), and imidazolelactic acid (ImLA) to produce His were investigated using mixed ruminal bacteria (B), protozoa (P), and a mixture of both (BP) in an in vitro system. Rumen microorganisms were anaerobically incubated at 39 degrees C for 18 h with or without each substrate (2 mM) mentioned. His and other related compounds produced in both the supernatants and hydrolyzates of the incubation were analyzed by high-performance liquid chromatography. B, P, and BP suspensions failed to show His synthesizing ability when incubated with HDL. His was synthesized from ImPA by B, P, and BP. Expressed in units "per gram of microbial nitrogen (MN)", ImPA disappearance was greatest in B (72.7 micromol/g MN per hour), followed by BP (33.13 micromol/g MN per hour) and then P (18.6 micromol/g MN per hour) for the 18-h incubation period. The production of His from ImPA in B (240.0, 275.9, and 261.2 micromol/g MN in 6, 12, and 18 h incubation, respectively) was about 3.5 times higher than that in P (67.3, 83.8, and 72.7 micromol/g MN in 6, 12, and 18 h incubation, respectively). Other metabolites produced from ImPA were ImLA, ImAA, histamine (HTM), and urocanic acid (URA), found in all microbial suspensions. ImLA as a substrate remained without diminution in all microbial suspensions. Although ImAA was found to be degraded to a small extent (3.4-6.3%) only after 18 h incubation, neither His nor other metabolites were detected on the chromatograms. These results have been demonstrated for the first time in rumen microorganisms and suggest that His may be an essential amino acid for rumen microorganisms.

In vitro catabolism of histidine by mixed rumen bacteria and protozoa.

An in vitro study was conducted to examine the metabolism of histidine (His) by mixed rumen bacteria (B), mixed rumen protozoa (P), and a combination of the two (BP). Rumen microorganisms were collected from fistulated goats fed with lucerne cubes (Medicago sativa) and a concentrate mixture twice a day. Microbial suspensions were anaerobically incubated with or without 2 mm each of His, or histamine (HTM), or 1 mm urocanic acid (URA) at 39 degrees C for 12 h. His and other related compounds in both supernatant and microbial hydrolysates were analyzed by HPLC. After 6- and 12-h incubations, the net degradation of His was 26.1% and 51.7% in B, 13.5% and 20.9% in P, and 21.7% and 46.0% in BP, respectively. The rate of the net degradation of His in B (98.0 micromol/g microbial nitrogen/h) was about 2.6 times higher than that of P during a 12-h incubation period. His was found to be degraded into urocanic acid (URA), imidazolelactic acid (ImLA), imidazoleacetic acid (ImAA), and histamine (HTM). Of these degraded His was mainly converted into URA in all microbial suspensions. The production of ImLA and ImAA was higher in B than in P suspensions, whereas the production of HTM was higher in P than in B suspensions. From these results, the existence of diverse catabolic routes of His in rumen microorganisms was indicated.

Determination of Histidine and Related Compounds in Rumen Fluid by Liquid Chromatography

A liquid chromatographic procedure was developed for quantitative determination of histidine (His), histidinol (HDL), histamine (HTM), urocanic acid (URA), imidazolepyruvic acid (ImPA), imidazoleacetic acid (ImAA), and imidazolelactic acid (ImLA) in rumen fluid. The method is based on direct injection analysis by UV absorbance detection at 220 nm. The separation was performed under 2 different chromatographic conditions on a LiChrospher 100 NH2 column. In the first chromatographic system, the mobile phase used for isocratic elution was 67 mM potassium phosphate buffer (monobasic and dibasic) pH 6.45-90% acetonitrile in water (21 + 79); in the second system, an acetonitrile gradient in 63 mM potassium phosphate buffer (monobasic) pH 3.0, obtained by addition of 60 mM phosphoric acid, was used. Analyses of both systems were completed within 32 and 25 min, respectively. The limits of detection of these compounds were (microM): His, 2.8; HDL, 3.7; HTM, 4.0; URA, 0.75; ImPA, 4.7; ImAA, 1.2; and ImLA, 1.3. Recovery of these compounds added to rumen fluid was 97.4-103.0% within a 1-day study and 95.4-99.0% on different day studies. Detectable levels of His were found in the deproteinized rumen fluid of goats, with average concentrations of 16.10, 10.43, 11.14, and 13.62 microM in the rumen fluid collected before the morning feeding and 2, 4, and 6 h after feeding, respectively. HDL, HTM, URA, ImPA, ImAA, and ImLA were not detected in the rumen fluid before and after feeding. Trp, Phe, and Tyr were also identified in the rumen fluid, with average concentrations of 8.25, 29.04, and 12.6 microM, respectively, before the morning feeding.

The coordination of imidazole and its derivatives by aquocobalamin

Formation constants have been determined for the substitution of coordinated water in aquocobalamin (vitamin B 12a) by imidazole and a number of its derivatives at 25 °C, μ = 1.0 M (KCl) in the pH range 5.5 to 10.5. The observed formation constants are all strongly pH dependent. Since the binding constant for 2-methylimidazole (log K = 1.15) is low compared to that for imidazole (log K = 4.59) because of steric repulsion between the annular substituent and the corrin ring, only the five-substituted tautomers of asymmetrically substituted imidazoles are considered to be significantly coordinated by Co(III). Taking this and the state of ionisation of the pendant amino group of histamine and histidine into account, equations were derived to explain the pH dependence of the observed formation constants. From these the following pH independent formation constants (log K) were deduced: imidazole, 4.59; 2-methylimidazole, 1.15; 5-methylimidazole, 4.90; N-methylimidazole, 4.90; 1,5-dimethylimidazole, 4.82; imidazole lactic acid, 4.86; cationic histamine, 4.43; neutral histamine, 4.71; neutral histidine, 4.30; and anionic histidine, 4.60. The magnitude of the formation constant is virtually independent of the basicity of the ligand. HPLC evidence is presented to show that at sufficiently high concentrations, these imidazoles displace 5,6-dimethylbenzimidazole (dbzm) from the fifth coordination position of Co(III) with log K = 1.4 to <−1. At high pH, ionisation of bound imidazole labilises the trans 5,6-dimethylbenzimidazole ligand which is substituted by an imidazole from solution. From fits of the relevant equations to the experimental data, and from direct spectrophotometric titrations, it is shown that coordination decreases the p K a of the imino group of the imidazoles from >14 to c. 10 due to polarisation by the metal ion. Furthermore, the p K a s of the pendant amino groups of histamine and histidine decrease from 9.96 and 9.18 to 8.89 and 7.99, respectively, on coordination. This is ascribed to destabilisation of the conjugate acids by coulombic repulsion with the residual positive charge at the metal centre. The possible biological significance of these results are discussed.

The biological activity of hydrogen peroxide: IV. Enhancement of its clastogenic actions by coadministration of L-histidine

An enhancing effect of L-histidine ( L-His) was detected on the induction by hydrogen peroxide (H 2O 2) of chromosomal aberrations of both the chromosome type and the chromatid type, in human embryonic fibroblasts. The maximum efficiency of induction was about 8-fold higher in the presence of L-His than in the presence of H 2O 2 alone, at a concentration of L-His of 50 μM. D-His and DL-His showed lower enhancing effects than L-His, with approximately 2-fold and 5-fold enhancement of induction of chromosomal aberrations, respectively. L-Histidinol and L-His-methyl ester, among various derivatives of L-His tested, also enhanced this process. However, the effects of these derivatives were smaller than those of L-His in a range of concentrations equivalent to that of the most effective dose of L-His (50 μM), while they produced greater enhancement than L-His at concentrations higher than 200 μM. Other derivatives of L-His, such as L-carnosine, urocanic acid, imidazolepyruvic acid, 1-methyl- L-His, imidazolelactic acid, imidazoleacetic acid and histamine and imidazole itself did not enhance the frequency of chromosomal aberrations induced by H 2O 2. These results indicate that at least both the imidazole ring and the amino group are essential components of the chemical structure of L-His required for the enhancing effect. Moreover, in order to cause such an enhancing effect, L-His had to be applied together with H 2O 2 to cells, because the enhancing effect of L-His was not observed with cells which were washed after pretreatment with L-His. The preliminary study suggested that this enhancing effect depends on the His-peroxide adduct derived from L-His and H 2O 2. None of the amino acids tested other than His produced any enhancing effect on the induction of chromosomal aberrations by H 2O 2.

Reaction of histidine with methyl linoleate: Characterization of the histidine degradation products

AbstractEfforts have been made to characterize the products that result from interactions between L‐histidine (free base) and peroxidizing methyl linoleate (ML) in a model system consisting of reactants dispersed on a filter paper. Imidazole lactic acid and imidazole acetic acid are identified as breakdown products when histidine is incubated with ML, methyl linoleate hydroperoxide (MLHPO), or n‐hexanal over a period of 3 weeks. Two other reaction products are found to give back histidine upon acid hydrolysis. These products are though to be Schiff's base compounds which result from the condensation of the histidyl α‐amino group and carbonyl groups of reactive aldehydes formed during ML peroxidation. Most of the detectable reaction products have the imidazole moiety intact indicating the high relative reactivity of the functional groups, especially the amino group, associated with the α‐carbon. Such high reactivity provides an explanation for the low concentrations of ninhydrin‐positive free amino compounds that are, at best, barely detectable on thin layer chromatography.

Conditions for the formation of complexes between pyridoxal phosphate and some imidazole derivatives

Imidazole derivatives form cyclic compounds with pyridoxal phosphate (PLP). The conditions of the cyclization reaction depend on both the pH of the media and the concentration of the substrate. Imidazole and imidazolelactic acid do not react with PLP. Histamine, histidine and its methyl derivatives each form a Schiff's base which is transformed during incubation into a complex compound with a maximum absorption spectrum at 330 mμ—probably imidazoletetrahydropyridine. The optimal pH for the reaction with histamine is 7.0–9.0; optimal concentration is 5 × 10 −2 M, but the complex is still formed with 10 −5 M of histamine. The optimal pH for reaction of PLP with histidine and its methyl derivatives is 7.2; optimal concentration 10 −1 M; the complex is still formed in 10 −4 M of histidine. Methyl derivatives of histidine ( l-1-methyl and l-3-methylhistidine) form a Schiff's base and cyclic compound. The mechanisms of the cyclization reaction and the importance of the phenomenon in the regulation of the level of the amines in the tissues are discussed.

Automatic column chromatographic analysis of urinary and serum imidazoles in patients with histidinaemia and normals

A 4 1 2 - h automatic column Chromatographic method for the analysis of imidazoles in patients with histidinaemia is reported. Imidazoles are separated on a cation exchanger by a gradient elution procedure. Continuous analysis of the column effluent is performed with a Technicon AutoAnalyzer using diazotized sulphanilic acid as a colour-producing reagent. The scope and the limits of the method are discussed and data for reproducibility and recoveries are given. Values for the urinary excretion of histidine, N-acetylhistidine, imidazolelactic acid, imidazolepvruvic acid and imidazoleacetic acid, together with the corresponding serum histidine concentrations have been determined in even patients with histidinaemia. Also the effect of a load with l-histidine in a patient treated with a diet low m histidine has been investigated. Quantitatively histidine and imidazolelactic acid are the most important excretory products and therefore are the most conclusive parameters for diagnosis when urine is analyzed. Also values for the urinary excretion of histidine, N-acetylhistidine, imidazolelactic acid and imidazoleacetic acid in normals are given. Increased urinary imidazoles in patients with diseases other than histidinaemia are commented upon.

Evidence for Delayed Histidine Transamination in Neonates with Histidinemia

The excretion of metabolites of histidine transamination by histidinemics, specifically imidazolepyruvic acid (IPA), imidazolelactic acid (ILA), and imidazoleacetic acid (IAA) is less in the neonatal and early infancy periods than in later life; whereas, histidine excretion is generally greater in the early months of life. This suggests that early in life the maturation of histidine transaminase may be delayed. In screening for histidinemia it is important to recognize that in neonates with histidinemia, IPA excretion may be very low or absent and thus that the ferric chloride test may be negative. For this reason screening procedures that detect increased histidine in urine are much more effective in the early diagnosis of histidinemia than are those procedures that detect only increased histidine metabolite excretion.

A simple test for histidinuria and histidinemia

A screening test for histidinuria and histidinemia is described based on paper Chromatographic separation and development with diazotized sulfanilic acid reagent. Mean excretion of histidine by 790 subjects was 210 ± 167 μ g/ml. Mean histidine concentration in plasma from 127 normal subjects was 0.96± 0.52 mg/100 ml. Histidine determinations in plasma by paper chromatography and by amino acid analyzer showed a high correlation. Measurement of urinary histidine and detection of histidine metabolites, imidazole lactic acid and imidazole pyruvic acid, permit differentiation of histidinuria associated with histidinemia and histidinuria associated with pathological aminoaciduria.

The effect of the configuration of histidine upon its metabolism

To determine the influence of stereoisomerism upon the course of histidine metabolism, metabolite excretion in 24 hr was assayed in rats previously fed normal and ethionine-supplemented diets for 12 days, then fasted for 12 hr before being given normal or radioactive d- or l-histidine, subcutaneously or by mouth. When histidine-α- 14C was given, contributions to carbon dioxide excretion and protein deposition were also measured. In rats fed normal diets, the greater outputs of histidine, imidazolepyruvic, imidazoleacetic, and imidazolelactic acids induced by d-histidine support its inversion via imidazolepyruvic acid, with imidazoleacetic and imidazolelactic acid produced secondarily by oxidative decarboxylation and reduction. The smaller yields of urocanic, formiminoglutamic, and glutamic acids from d-histidine undoubtedly reflect their production only from the l-histidine formed by inversion. The conclusions drawn are supported by the quantitative differences observed with l-histidine, by differences induced in ethionine-fed animals, and by those found in the carbon dioxide and protein assays. Metabolite separation was effected by thin-layer chromatography. Assays were made spectrophotometrically or by scintillation counting, or both. Specific activities of the metabolites indicated marked dilution only in the glutamic acid. Contributions to the carbon dioxide excreted and to the proteins precipitated were estimated by counting.

Paper chromatography of some weakly basic urine constituents and the estimation of urinary tyrosine

Weak bases in urine were chromatographed after absorption by Dowex 50 at pH 2.0 and elution with aqueous pyridine. Various abnormalities observed in pathological urines included increased excretion of imidazolepropionic acid, particularly in cystic fibrosis, and of imidazolelactic acid in phenylketonuria. The extracts were suitable for quantitative estimation of urinary tyrosine which was exceptionally high in tyrosinosis.

Histidine Metabolism in the Human Adult: Histidine blood tolerance, and the effect of continued free L-histidine ingestion on the concentration of imidazole compounds in blood and urine

Relationships among the intermediary metabolites of histidine were studied in the human adult. Histidine and imidazolelactic acid were the only imidazole compounds identified in the fasting plasma of 3 healthy human adults fed a constant diet. Plasma histidine tolerance studies in which 5 g of L-histidine (free base) were fed, showed the peak increase in histidine to occur one hour after ingestion; imidazolelactic acid changes paralleled that of histidine. Eleven imidazole compounds were quantitatively identified in 24-hour urine samples collected for periods of 3 consecutive days before histidine ingestion, during ingestion of a 10-g daily load of free L-histidine, and after histidine ingestion. Histidine loading caused an increase in the excretion of histidine, urocanic acid, imidazolepropionic acid, imidazoleacetic acid and imidazolelactic acid; none of these returned to the pre-histidine ingestion level by the end of the 3-day post-histidine period. The amounts of 4-amino-5-imidazole carboxamide, carnosine, homocarnosine, anserine, 1-methylhistidine, and 3-methylhistidine either were not affected by histidine loading, or the results were erratic and varied among the subjects.

Synthesis of α-tritiated L-histidine, L-arginine and L-lysine

Tritium was introduced into the alpha position of histidine, arginine, and lysine by racemization using the azlactone rearrangement in an anhydrous tritiated medium. The monoacetyl derivatives of histidine and arginine were resolved with acylase I; lysine was converted into its α,ϵ-dichloroacetyl derivative and resolved with acylase IA. The α-tritiated l-amino acids recovered from these reactions had the expected optical rotations. The specific activities of l-[α 3H]-histidine·HCl·H 2O, l-[α 3H]-arginine·HCl, and l-[α 3H]-lysine·HCl were 3.40 × 10 9, 4.47 × 10 9, and 1.03 × 10 9 dpm/mmole, respectively. l-[α 3H] Arginine·HCl was converted into l-ornithine·HCl and l-citrulline without change in specific activity. l-Arginine and l-citrulline, when subjected to the action of l-amino acid oxidase, gave α-keto-δ-guanidinovaleric and α-keto-δ-carbamidovaleric acids, which were devoid of any activity. ϵ-Cbzo- l-[α 3H]-lysine was prepared and subjected to the action of l-oxidase. The resulting α-keto-ϵ- N-Cbzo-aminocaproic acid had no activity. l-[α 3H]-histidine·HCl·H 2O was oxidized by nonenzymic means to imidazolelactic acid, cyanomethylimidazole, and imidazoleglyoxylic acid; the compounds retained 100, 8, and 2% of the original activity respectively. β-Imidazolyl-pyruvic acid could not be prepared by the action of venom l-amino acid oxidase. Imidazoleacetic acid prepared with this enzyme in the absence of catalase had 6% of the original l-histidine activity. It is concluded that tritium introduced into arginine and lysine by the azlactone rearrangement is exclusively linked to the α-carbon. For histidine, 92% of the label appeared in the α-position, 6% in the β-hydrogen atoms, and 2% in the imidazole ring. The difficulties in the determination of the tritium activity of histidine and its derivatives by liquid scintillation counting can be overcome by the addition of formaldehyde.