Human Serum Phenylpyruvate Quantification Using Responsive 2D Photonic Crystal Hydrogels via Chemoselective Oxime Ligation: Progress toward Developing Phenylalanine-Sensing Elements.

There is a need to develop at-home phenylalanine (Phe) test kits, analogous to home glucose meters, for phenylketonuria patients who must measure their blood Phe levels frequently to adjust their diet. Unfortunately, such test kits are not available yet because of the lack of simple and inexpensive Phe-sensing elements. With the goal of developing a Phe-sensing element, we fabricated two-dimensional photonic crystal (2DPC) hydrogels that quantify human serum phenylpyruvate (PhPY), which is the product of the reaction between Phe and the enzyme phenylalanine dehydrogenase. The PhPY-sensing hydrogels have oxyamine recognition groups that link PhPY to the hydrogel polymer network via chemoselective oxime ligation. This structural modification induces the hydrogel to swell, which then increases interparticle spacings within the embedded 2DPC. The PhPY-induced particle spacing changes are measured from light diffraction and used to quantify the PhPY concentrations. The estimated limit of detection of PhPY in human serum for a detection time of 30 min is 19 µM, which is comparable to the minimum blood Phe concentrations of healthy people. Besides the potential application for developing Phe-sensing elements, this new hydrogel sensing approach via chemoselective oxime ligation is generalizable to the development of other chemical sensors working in complex biological environments.

Interference of hydroxyphenylpyruvic acid, hydroxyphenyllactic acid and tyrosine on routine serum and urine clinical chemistry assays; implications for biochemical monitoring of patients with alkaptonuria treated with nitisinone.

OBJECTIVES: We have assessed the effect of elevated concentrations of hydroxyphenylpyruvic acid (HPPA), hydroxyphenyllactic acid (HPLA) and tyrosine, on a range of chemistry tests in serum and urine to explore the potential for chemical interference on routine laboratory analyses in patients with alkaptonuria (AKU) treated with nitisinone and similarly implications for patients with hereditary tyrosinemia type 1 (HT-1). MATERIALS AND METHODS: HPPA, HPLA and tyrosine were added separately to pooled serum from subjects without AKU in a range of assays with Roche Modular chemistries. Effects on urine were assessed by changes in urine strip chemistries after mixing a positive control urine with various amounts of the test compounds and reading on a Siemens urine strip meter. RESULTS: No significant effect (p > 0.1) was observed up to 225 µmol/L of HPPA and HPLA, and up to 5000 µmol/L tyrosine, on any of the serum-based assays including those with peroxidase-coupled reaction systems of enzymatic creatinine, urate, total cholesterol, HDL cholesterol and triglyceride. Both the monohydroxy HPPA, and the dihydroxy homogentisic acid (HGA), at increased urine concentrations typical of nitisinone-treated AKU and non-treated AKU respectively, did however show marked negative interference in strip assays for glucose and leucocytes; i.e. those with peroxide-linked endpoints. The effect of increased HPLA was less marked. CONCLUSIONS: In patients with AKU or on nitisinone treatment and HT-1 patients on nitisinone, urine strip chemistry testing should be used sparingly, if at all, to avoid false negative reporting. It is recommended that urine assays should be organised with a suitable specialist laboratory.

Reliability of fasting plasma alkylresorcinol metabolites concentrations measured 4 months apart.

Alkylresorcinols (AR) have been suggested as specific dietary biomarkers of whole-grain wheat and rye intake. AR are metabolised to 3,5-dihydroxybenzoic acid (DHBA) and 3-(3,5-dihydroxyphenyl)-1-propanoic acid (DHPPA), which have longer apparent half-lives and were recently proposed to better reflect long-term whole-grain consumption than the intact AR. The objective of this study was to analyse the reliability--expressed by the intraclass correlation coefficient (ICC)--of AR metabolite concentrations among 100 participants from the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam study who provided two fasting plasma samples 4 months apart. DHBA and DHPPA concentrations were not significantly different between the first and second measurement over the 4-month period (P>0.05). The ICC was 0.32 (95% confidence interval (CI)=0.13-0.49) for DHBA and 0.37 (95%CI=0.19-0.53) for DHPPA. These results suggest that AR metabolites cannot be considered to be better biomarkers of whole-grain wheat and rye intake than the intact AR in fasting plasma (ICC=0.42).

Novel potential markers of nasopharyngeal carcinoma for diagnosis and therapy.

OBJECTIVE: To search for markers of nasopharyngeal carcinoma (NPC) for diagnosis. DESIGN AND METHODS: Using gas chromatography and mass spectrometry, we evaluated 51 serum metabolites in 49 NPC, 37 throat cancer patients and 40 healthy controls. High metabolites were selected and confirmed in NPC tissues. Sensitivity and specificity were appraised for 53 NPC diagnoses. RESULTS: Metabolic profiling revealed that kynurenine, N-acetylglucosaminylamine, N-acetylglucosamine and hydroxyphenylpyruvate increased in NPC patient sera. Their sensitivity and specificity were respectively 79% and 71%, 78% and 69%, 83% and 68%, 84% and 73% for NPC diagnosis. These increases were confirmed in NPC cells. Four metabolites gradually increased from stage I to stage III. After radiotherapy, four metabolites decreased gradually, and tended to a normal level, and were associated with rate of tumor reduction. CONCLUSION: The results reveal that kynurenine, N-acetylglucosaminylamine, N-acetylglucosamine and hydroxyphenylpyruvate are potentially markers of NPC for diagnosis and therapy.

Plasma pharmacokinetics of alkylresorcinol metabolites: new candidate biomarkers for whole-grain rye and wheat intake.

BACKGROUND: Alkylresorcinols are phenolic compounds that are present almost exclusively in rye and wheat fiber. Alkylresorcinols are absorbed and thereafter metabolized to 3,5-dihydroxybenzoic acid (DHBA) and 3-(3,5-dihydroxyphenyl)-1-propanoic acid (DHPPA), which have been detected in human urine and plasma. OBJECTIVE: The objective was to determine the plasma pharmacokinetics of DHBA and DHPPA in human subjects to estimate whether they show potential as biomarkers for whole-grain rye and/or wheat intake. DESIGN: Fifteen human volunteers followed a low-alkylresorcinol diet for 2 d before ingesting a single dose of high-fiber rye bread containing 100 mg alkylresorcinols [corrected]. Plasma samples were collected for 25 h, and the alkylresorcinol metabolites were quantified by HPLC with coulometric electrode array detection. RESULTS: Maximum concentrations were reached at approximately 6 h for both metabolites, although interindividual variation was found. The half-life was significantly (P < 0.0002) longer for DHPPA (16.3 h) than for DHBA (10.1 h). No significant differences were discovered between women and men in the half-life of each metabolite, which, from a pharmacokinetic point of view, is the most important parameter. The area under the curve differed significantly between DHBA and DHPPA (P < 0.0001) and between women and men (P = 0.03 for DHBA and P = 0.01 for DHPPA). However, when corrected for body weight, the difference between sexes was no longer significant. CONCLUSIONS: Our results suggest that DHBA and DHPPA are both good candidate biomarkers for whole-grain rye and/or wheat intake; however, DHPPA is the better indicator because of its longer half-life. This could provide a practical tool when investigating the association between diet and diseases.

[Fluorescence quenching method for the determination of p-hydroxyphenylpyruvic acid].

A fluorescence quenching method for the determination of p-hydroxyphenylpyruvic acid with trytophan in the medium pH 11.01 NH3-NH4Cl is studied. The calibration curve is linear for p-hydroxyphenylpyruvic acid from 0 to 15 micrograms.mL-1 with lambda ex/lambda em = 285/356 nm. The detection limit is 0.37 microgram.mL-1. Twenty replicate determinations of solutions containing 10 micrograms.mL-1 p-hydroxyphenylpyruvic acid show a relative standard deviation of 1.2%. This method can be applied to the determination of p-hydroxyphenylpyruvic acid in serum with satisfactory results.

Determination of p-hydroxyphenylpyruvate, p-hydroxyphenyllactate and tyrosine in normal human plasma by gas chromatography-mass spectrometry isotope-dilution assay.

The synthesis and purification of [13C2]p-hydroxyphenyllactic acid from [13C2]p-hydroxyphenylpyruvic acid, the characterization of tert.-butyldimethylsilyl-derivatized tyrosine, p-hydroxyphenylpyruvic acid and p-hydroxyphenyllactic acid, and an isotope-dilution assay for these substances in normal human plasma using gas chromatography-mass spectrometry (GC-MS) are described. Using this method plasma p-hydroxyphenylpyruvate, p-hydroxyphenyllactate and tyrosine levels of 68 +/- 42 ng/ml, 118 +/- 45 ng/ml and 16.6 +/- 6.3 micrograms/ml, respectively, were found in 9 normal adults. Isotope-dilution assays are sensitive enough to determine tyrosine, p-hydroxyphenylpyruvate and p-hydroxyphenyllactate content in normal subjects, and may be useful for studying disorders of tyrosine metabolism, including inborn errors of metabolism, liver disease and ascorbic acid deficiencies.

[The safety problem and a physiological strategy for using glucocorticoid hormones]. / Problema bezopasnosti i fiziologicheskaia strategiia primeneniia gliukokortikoidnykh gormonov.

Principal problems of glucocorticoid hormone application in non-endocrine diseases are discussed proceeding from the mechanism of their action and the role in organism's life. It appeared that the hazard of glucocorticoid therapy is mainly due to the absence of a test for sufficiency and real needs in these hormones. It was shown that the test has to be introduced reflecting on the whole body level the glucocorticoid activity in the cell, that is similar to blood glucose in relation to insulin. The introduction of free blood tyrosine as such test appears to allow the development of safe individual strategy for the usage of glucocorticoids.

Determination of alpha-keto acids including phenylpyruvic acid in human plasma by high-performance liquid chromatography with chemiluminescence detection.

A highly sensitive method for the determination of alpha-keto acids including phenylpyruvic acid in human plasma is investigated. The method employs high-performance liquid chromatography with chemiluminescence detection. The acids and alpha-ketocaproic acid (internal standard) in human plasma are isolated by anion-exchange chromatography on a Toyopak DEAE cartridge, and then converted into the corresponding chemiluminescent derivatives with 4,5-diaminophthalhydrazide dihydrochloride, a chemiluminescence derivatization reagent for alpha-keto acids. The derivatives are separated within 50 min on a reversed-phase column, TSKgel ODS-120T, with isocratic elution, followed by chemiluminescence detection; the chemiluminescence is produced by the reaction of the derivatives with hydrogen peroxide in the presence of potassium hexacyanoferrate(III). The detection limits for the acids are in the range 9-92 pmol/ml in plasma (signal-to-noise ratio = 3). This sensitivity permits precise determination of several alpha-keto acids including phenylpyruvic acid, which cannot be determined by other HPLC methods, in 10 microliters of normal human plasma. The chemiluminescent product from phenylpyruvic acid was characterized as 3-benzyl-7,8-dihydropyridazino[4,5-g]quinoxaline-2,6,9(1H)-trione.

Quantitative metabolic profiling of alpha-keto acids in Friedreich's ataxia.

The plasma distribution of alpha-keto acids was measured in 26 subjects including 8 patients with Friedreich's ataxia, 8 with the recessive spastic ataxia of Charlevoix-Sageunay and 10 healthy volunteers. The groups were matched with regards to age, sex, weight and the study was conducted under standardized dietary intake. The result indicate significant differences in the alpha-keto acids distribution between the groups.

Determination of phenylpyruvic acid in urine and serum by high-performance liquid chromatography with fluorescence detection.

A highly sensitive and simple method for the determination of phenylpyruvic acid in urine and serum is described which employs high-performance liquid chromatography with fluorescence detection. Phenylpyruvic acid, after extraction with ethyl acetate, is reacted with 4'-hydrazino-2-stilbazole in aqueous methanol to give the corresponding fluorescent hydrazone which is separated by reversed-phase chromatography on muBondapak Phenyl. The lower limits of detection are 25 and 32 pmol for phenylpyruvic acid in 0.2 ml of urine and serum, respectively. This sensitivity permits the determination of the acid in urine or normal adults and newborn infants.

Elevation of blood keto acids in cerebrocortical necrosis.

Values of various alpha-keto acids in whole blood were determined from cases of cerebrocortical necrosis (CCN), a thiamine deficiency disease, and were compared with those from normal animals. Thiamine pyrophosphate is an essential cofactor in the decarboxylation of many alpha-keto acids and this was reflected in elevated values not only of pyruvic acid but of glyoxylic, alpha-keto glutaric, phenyl pyruvic and hydroxyphenyl pyruvic acids in the CCN cases.

Renal transport of aromatic acids in patients with phenylketonuria.

Renal clearance of phenylpyruvic acid is maximal at a plasma concentration of 40-60 mumol/l. This concentration is obtained with plasma phenylalanine concentrations of 1.0-1.2 mmol/l, the threshold for separating classical phenylketonuria from phenylketonuria variants.

Characterization of experimental phenylketonuria. Augmentation of hyperphenylalaninemia with alpha-methylphenylalanine and p-chlorophenylalanine.

Phenylalanine in conjunction with p-chlorophenylalanine or alpha-methylphenylalanine was administered to suckling rats to induce hyperphenylalaninemia reminiscent of untreated phenylketonuria, and developmental parameters were monitored. The experimental model utilizing p-chlorophenylalanine was found to be unsatisfactory, in that the drug had general deleterious effects on growth, numerous side effects including increased mortality, and affected brain levels of biogenic monoamine neurotransmitters. The model utilizing alpha-methylphenylalanine was relatively free from nonspecific effects and thus, changes observed in the animals were attributable to experimental phenylketonuria. The latter animals had slightly decreased body and brain weights, and exhibited grossly elevated serum phenylalanine and urinary excretion of phenylketone metabolites. Hyperphenylalaninemia produced greatly disrupted brain amino acids at 10 days of age (prior to the formalization of the blood-brain barrier and specific transport systems) which was limited by 30 days of age to changes in glycine, gamma-aminobutyric acid and the aliphatic and aromatic amino acids which compete for uptake in the brain by a common carrier. These animals also exhibited a myelin deficit and changes in proteins from isolated nerve cell preparations. Mature animals which had daily treatment up to 60 days of age exhibited a long-term learning impairment. These observations are consistent with many aspects of the clinical picture of untreated phenylketonuric patients, and suggest that this animal model will be beneficial in studying the disease.

Tyrosine metabolism in cirrhosis.

The aim of this study was to define the enzyme defect responsible for tyrosinemia in cirrhotic patients. The principal hepatic degradation pathway for tyrosine, tyrosine leads to p-hydroxyphenylpyruvic acid equilibrium homogentisic acid leads to CO2 was studied in 18 cirrhotic patients and eight controls. The classic method employed in elucidation of hereditary tyrosinosis was sued. Metabolic intermediates on the pathway were measured in the basal state, and following oral loading doses (50 mg/kg BW) of tyrosine, PHPA, and homogentisic acid. Cirrhotic patients showed a significant increase (p = 0.005) in fasting plasma tyrosine and in basal PHPA excretion and impaired tolerance to all three metabolites when compared to normals. Fifteen of the 18 cirrhotic patients showed tyrosine intolerance which was not accompanied by change in distal metabolites compared to their basal levels. Nevertheless 13 of the 18 did exhibit intolerance of either PHPA or homogentisic acid. We conclude that in contrast to the single complete defect in hereditary disorders of tyrosine metabolism, cirrhotic patients have partial defects at tyrosine transaminase, PHPA oxidase, and homogentisic acid oxidase, the initial step being rate-limiting.

[Animal experiments on the pharmacokinetics of 4-amidinophenylpyruvic acid (APPA) (author's transl)]. / Tierexperimentelle Untersuchungen zur Pharmakokinetik von 4-Amidinophenylbrenztraubensäure (APPA).

The pharmacokinetics of the synthetic protease inhibitor APPA was studied in rabbits. After intravenous or oral application of 1 and 10 mg of (carboxyl-14C)-4-amidinophenylpyruvic acid hydrochloride/kg to rabbits, the plasma concentration was determined over several days and the excretion in urine during 24 hours. The course of the plasma concentration curves showed no dose-dependence of the pharmacokinetic behaviour. A three-phase course of the plasma concentration curves was observed after intravenous application, the slowest phase having a half-life of about 20 h. This very slow elimination is considered to be caused by tubular re-absorption of APPA. There is neither accumulation in organs, tissues or erythrocytes nor binding with plasma proteins. The thin-layer chromatographic analysis of the plasma revealed no labelled metabolites, but only unchanged APPA. APPA, when applied orally (in the form of an aqueous solution), is absorbed by more than 80%, the maximum plasma level being reached within 2--3 h. Intravenous and oral applications at 24-h intervals result in a cumulative increase in APPA concentration in the plasma. If APPA is applied to pregnant rabbits, it passes into the fetal circulation.

Phenylalanine metabolism in uremic and normal man.

The metabolism of phenylalanine and tyrosine was evaluated in six normal men, five chronically uremic men, and three men undergoing maintenance hemodialysis. Phenylalanine, tyrosine, and 13 acidic metabolites of those amino acids were measured in plasma postabsorptively and in plasma and urine after a phenylalanine load of 100 mg/kg. In addition, five normal subjects and five dialysis patients ingested L-[14C]-phenylalanine (uniformly labeled) with the load. In uremic and dialysis patients, plasma phenylalanine rose higher and fell more gradually after the load, and tyrosine rose more slowly. The 24-hr urinary concentrations of phenylalanine and tyrosine were similar in the three groups. At 24 hr, cumulative expiration of 14CO2 was 20.2% in the dialysis patients and 28.4% in the normal subjects. Plasma phenylalanine levels and 14CO2 expiration varied with protein intake in normal subjects. In uremic and dialysis patients, plasma phenyllactic acid, p-hydroxyphenylacetic acid, and p-hydroxybenzoic acid were elevated, the last one markedly so. Moreover, plasma phenylpyruvic acid (PPA) and mandelic acid were detected only in dialysis patients. After the phenylalanine load, plasma conjugated phenylacetic acid rose in uremic patients, and PPA increased transiently in some dialysis patients. In urine of dialysis patients, concentrations of benzoic acid and conjugated o-hydroxyphenylacetic acid were decreased, and PPA was sometimes increased. The data suggest a mild impairment in the hydroxylation of phenylalanine which does not result in marked changes in plasma or in urinary metabolites after a phenylalanine load.