Metabolic profiling by reversed-phase/ion-exchange mass spectrometry.

Metabolic profiling is commonly achieved by mass spectrometry (MS) following reversed-phase (RP) and hydrophilic interaction chromatography (HILIC) either performed independently, leading to overlapping datasets, or in a coupled configuration, requiring multiple liquid chromatography (LC) systems. To overcome these limitations, we developed a single, 20-minute chromatographic method using an in-line RP-ion-exchange (IEX) column arrangement and a single LC system. This configuration separates clinically significant polar and non-polar compounds without derivatization or ion-pairing reagents, allowing ionization in both polarities. An in-house library was created with 397 authentic standards, including acylcarnitines, amino acids, bile acids, nucleosides, organic acids, steroid hormones, and vitamins. Analysis of pooled plasma and urine samples revealed 5445 and 4111 ion features, leading to 88 and 82 confirmed metabolite identifications, respectively. Metabolites were detected at clinically relevant concentrations with good precision, and good chromatographic separation was demonstrated for clinically significant isomers including methylmalonic acid and succinic acid, as well as alloisoleucine and isoleucine/leucine. Evaluation of the samples by unsupervised principal component analysis showed excellent analytical quality.

The effect of casein, hydrolyzed casein, and whey proteins on urinary and postprandial plasma metabolites in overweight and moderately obese human subjects.

BACKGROUND: Casein and whey proteins differ in amino acid composition and absorption rate; however, the absorption rate of casein can be increased to mimic that of whey proteins by exogenous hydrolysis. In view of these compositional differences, we studied the metabolic responses to intake of casein, hydrolyzed casein, and whey proteins in overweight and moderately obese men and women by investigating select urinary and blood plasma metabolites. RESULTS: A total of 21 urinary and 23 plasma metabolites were identified by nuclear magnetic resonance spectroscopy. The postprandial plasma metabolites revealed a significant diet-time interaction for isoleucine (P = 0.001) and tyrosine (P = 0.001). The level of isoleucine and tyrosine peaked 90 min postprandially with a 1.4-fold difference following intake of whey proteins compared with either casein or hydrolyzed casein. A 1.2-fold higher urinary level of lactate was observed after intake of whey proteins compared with intake of intact casein (P < 0.01). CONCLUSION: The plasma metabolites revealed different amino acid profiles reflecting the amino acid composition of casein and whey proteins. Furthermore, the results support that casein hydrolysates neither affect the postprandial amino acid absorption rate nor the amino acid level compared with that of intact casein. The urinary lactate increases following whey protein intake might indicate a higher metabolism of glucogenic amino acids. © 2018 Society of Chemical Industry.

CE-MS for the Analysis of Amino Acids.

Amino acids play an important role in clinical analysis. Capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) has proven to possess several characteristics that make it a powerful and useful tool for the analysis of amino acids in clinical studies. Here we present a method for the separation and quantitative analysis of 27 amino acids in urine based on CE-ESI-MS. The method presents an improved resolution between the isomers Leu, Ile, and aIle, in comparison to other CE-ESI-MS methods in the literature. This method is fast, selective, and simple and has improved sensitivity by applying a pH-mediated stacking strategy, showing that it can be successfully used for amino acid analysis and probably for other small cationic metabolites.

[Metabonomic analysis of the urine from rat model with abnormal sapra syndrome].

OBJECTIVE: To investigate the correlation between the change in metabolic components of urine and the abnormal sapra syndrome by using a rat model of abnormal sapra syndrome.
 Methods: Multiple factors, such as dry environment, dry feed, and chronic electrical stimulation, were used to establish the abnormal sapra syndrome in Wistar rats by Uyghur medicine. The differences in metabolites were detected through the metabonomics method.
 Results: The urine of rats in abnormal sapra syndrome group showed significant high abundance metabolites as follows: Leucine, isoleucine, and glycoprotein. And that significant low abundance metabolites as follows: Glutamine, creatine, citric acid, and phenylalanine.
 Conclusion: The urine of rats with the abnormal sapra syndrome displays abnormal energy metabolism. It is likely that the dysfunctional metabolisms of three major nutrients might be the molecular basis for the abnormal sapra syndrome.

Potential urinary biomarkers of nephrotoxicity in cyclophosphamide-treated rats investigated by NMR-based metabolic profiling.

The anticancer-drug cyclophosphamide (CP) is known to have nephrotoxicity. The aim of this study was to identify urinary biomarkers indicating CP-induced nephrotoxicity. We investigated the urine metabolic profiles using nuclear magnetic resonance spectrometry of rats administered with single high-doses of CP (0, 30, and 100 mg/kg body weight) and daily low-doses over a 4-week period (0, 1, 3, and 10 mg/kg body weight). Among 18 identified urinary metabolites, 2-oxoglutarate, citrate, hippurate, formate, valine, and alanine for short-term and 2-oxoglutarate, citrate, hippurate, isoleucine, leucine, allantoin, valine, and lysine for long-term were selected as potential biomarkers. Pathway-enrichment analysis suggested that the urinary metabolism of CP is related to valine, leucine, and isoleucine biosynthesis; taurine and hypotaurine metabolism; glyoxylate and dicarboxylate metabolism; citrate cycle; and alanine, aspartate, and glutamate metabolism, with high pathway impact. The potential biomarkers obtained in this study could be used to monitor CP-induced nephrotoxicity relative to dose and treatment time.

Valproic acid utilizes the isoleucine breakdown pathway for its complete ß-oxidation.

UNLABELLED: Valproic acid (VPA) is a simple branched medium-chain fatty acid with expanding therapeutic applications beyond its prime anticonvulsant properties. AIMS: (1) To resolve the underlying basis for the interference of valproate with the isoleucine degradative pathway and (2) to shed new light on the enzymology of the ß-oxidation pathway of valproate. METHODS: Urine organic acids were analyzed by gas chromatography/mass spectrometry. In vitro studies were performed with heterologously expressed human 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) and fibroblasts from controls and a patient with MHBD deficiency using 2-methyl-3-hydroxybutyryl-CoA and 3-hydroxyvalproyl-CoA as substrates. The respective enzymatic activities were measured using optimized HPLC procedures. Short-chain enoyl-CoA hydratase (ECHS1) immunoprecipitation in a human liver homogenate was performed and hydratase activity was measured in the supernatants by HPLC, using crotonyl-CoA and Δ(2(E))-valproyl-CoA as substrates. RESULTS: Patients on valproate therapy had a moderately increased urinary excretion of the isoleucine metabolite 2-methyl-3-hydroxybutyric acid. MHBD was found to convert 3-hydroxyvalproyl-CoA into 3-ketovalproyl-CoA. MHBD activity in control fibroblasts was comparable using both 2-methyl-3-hydroxybutyryl-CoA and 3-hydroxyvalproyl-CoA as substrates. In fibroblasts of a patient with MHBD deficiency, there was no detectable MHBD activity when 3-hydroxyvalproyl-CoA was used as substrate. Samples with immunoprecipitated crotonase had no detectable hydratase activity using both crotonyl-CoA and Δ(2(E))-valproyl-CoA as substrates. DISCUSSION: This work demonstrates for the first time, that MHBD is the unique enzyme responsible for the dehydrogenation of 3-hydroxyvalproyl-CoA. Furthermore, we show that crotonase is the major, if not the single hydratase involved in VPA ß-oxidation, next to its role in isoleucine catabolism.

Clinical variability in 3-hydroxy-2-methylbutyryl-CoA dehydrogenase deficiency.

We report the identification of two new 7-year-old patients with 3-hydroxy-2-methylbutyryl-CoA dehydrogenase deficiency, a recently described inborn error of isoleucine metabolism. The defect is localized one step above 3-ketothiolase, resulting in a urinary metabolite pattern similar to that seen for deficiency of the latter. One patient has progressive neurodegenerative symptoms, whereas the clinical phenotype of the other patient is characterized by psychomotor retardation without loss of developmental milestones. A short-term biochemical response to an isoleucine-restricted diet was observed in both children.

Evaluation of circulating levels and renal clearance of natural amino acids in patients with Cushing's disease.

Although the hypercortisolism-induced impairment of protein homeostasis is object of several studies, a detailed evaluation of the complete amino acid profile of patients with Cushing's syndrome (CS) has never been performed. The aim of the current open transversal controlled study was to evaluate serum and urinary concentrations as well as renal clearance of the complete series of natural amino acids and their relationship with glucose tolerance in patients with Cushing's disease (CD). Twenty patients with CD (10 active and 10 cured) and 20 sex- and age-matched healthy controls entered the study. Measurement of serum and urinary levels of the complete series of natural amino acids was performed in all patients analyzed by cationic exchange high performance liquid cromatography (HPLC) after 2 weeks of a standardized protein intake regimen. The renal clearance (renal excretion rate) of each amino acid was calculated on the basis of the serum and urinary concentrations of creatinine and the specific amino acid. Fasting glucose and insulin levels, glucose and insulin response to standard glucose load, insulinogenic and homeostasis model insulin resistance (Homa-R) indexes were also evaluated and correlated to the circulating levels and renal clearances of each amino acid. Significantly higher serum (p<0.01) and urinary (p<0.05) levels of alanine and cystine, lower serum and higher urinary levels of leucine, isoleucine and valine (p<0.05) and higher renal excretion rates of leucine, isoleucine and valine (p<0.01) were found in patients with active CD than in patients cured from the disease and in controls. No difference was found between cured patients and controls. Creatinine clearance was similar in active and cured patients and in controls. In patients with active CD, urinary cortisol levels were significantly correlated to urinary cystine levels (r=0.85; p<0.01) and renal excretion rate of leucine (r=-0.76; p<0.05), isoleucine (r=-0.76; p<0.05) and valine (r=-0.66; p<0.05). Fasting blood glucose levels were significantly correlated to serum alanine levels (r=0.70; p<0.05). Although Homa-R was significantly correlated to BMI in active patients (r=0.74 p<0.05), it was not correlated to amino acid levels. In conclusion, the results of the current study demonstrate that patients with CD have significant changes in serum and urinary concentration of several amino acids and changes in renal clearance of some specific amino acids. Normalization of cortisol levels restored the amino acid profile.

Product equivalence study comparing the tolerability, pharmacokinetics, and pharmacodynamics of various human immunoglobulin-G formulations.

In this randomized, double-blind, parallel-group study, a commercially available human immunoglobulin-G product, IVIG, was compared with two test formulations: (1) IVIG-N, which is a nanofiltered formulation of IVIG, and (2) IVIG-L, which is a nanofiltered, liquid, ready-for-use IgG formulation containing nicotinamide, L-proline, and L-isoleucine as stabilizers. Three groups of 10 healthy subjects each received a single 0.6 g/kg dose of one of the formulations infused over 3.5 to 6.8 hours, depending on the total volume to be infused. Blood samples were obtained over a 6-week period to assess pharmacokinetics, immunogenicity, and the pharmacodynamic effects on leukocytes and TNF-alpha. A blood sample was taken at 6 months for a viral safety check. Administrations were generally well tolerated with only one reference IVIG infusion stopped prematurely due to headache. The IgG Cmax and AUC over the 6-week blood sampling period from both test formulations satisfied equivalence criteria compared with the reference formulation. In subjects receiving IVIG-L, peak concentrations of the stabilizer nicotinamide ranged from 0.34 to 0.47 mmol/L and of nicotinamide-N-oxide from 0.03 to 0.04 mmol/L, which are below those reported to cause adverse events. During the infusion of IVIG, leukocyte counts initially declined from a baseline of 5.7 +/- 1.1 x 10(9)/L to 3.7 +/- 0.8 x 10(9)/L at 2 to 4 hours and returned to baseline by 24 hours. TNF-alpha levels, reflecting activation of the monocyte-macrophage system by the infused IVIG, rose from a baseline of 13 +/- 4 pg/mL to a peak of 272 +/- 324 pg/mL at 2 to 4 hours and returned to baseline by 24 hours. These patterns were generally similar for the test formulations, with the exception that the increase in TNF-alpha levels was dampened for IVIG-N, although this was not statistically significant. There was no evidence of immunogenicity or viral transmission from any of the formulations. Hence, these three formulations were generally well tolerated, yielded similar systemic exposure to IgG over a 6-week period after administration, and did not give rise to immunogenicity or viral safety concerns.

Renal clearance of branched-chain L-amino and 2-oxo acids in maple syrup urine disease.

In maple syrup urine disease (MSUD), branched-chain L-amino (BCAA) and 2-oxo acids (BCOA) accumulate in body fluids owing to an inherited deficiency of branched-chain 2-oxo acid dehydrogenase complex activity. In MSUD, little information is available on the significance of urinary disposal of branched-chain compounds. We examined the renal clearance of leucine, valine, isoleucine and alloisoleucine, and their corresponding 2-oxo acids 4-methyl-2-oxopentanoate (KIC), 3-methyl-2-oxobutanoate (KIV), (S)-(S-KMV), and (R)-3-methyl-2-oxopentanoate (R-KMV), using pairs of plasma and urine samples (n = 63) from 10 patients with classical MSUD. The fractional renal excretion of free BCAA was in the normal range (< 0.5%) and independent of the plasma concentrations. The excretion of bound (N-acylated) BCAA was normal and not significantly dependent on the BCAA plasma concentrations. The fractional renal excretion of BCOA was in the order KIC << KIV < R-KMV < or = S-KMV (range (%): KIC 0.1-25; KIV 0.14-21.3; S-KMV 0.26-24.6; R-KMV 0.1-35.9), significantly correlated with the KIC plasma concentrations, and generally higher than that of the related BCAA. The results show that the renal excretion of free BCAA as well as of the acylated derivatives is negligible. The renal excretion of BCOA, however, to some extent counteracts increases in BCAA concentrations and thus contributes to the lowering of total BCAA pools in MSUD.

Excretion of amino acids by humans during space flight.

UNLABELLED: We measured the urine amino acid distribution patterns before, during and after space flight on the Space Shuttle. The urine samples were collected on two separate flights of the space shuttle. The first flight lasted 9.5 days and the second flight 15 days. Urine was collected continuously on 8 subjects for the period beginning 10 d before launch to 6 d after landing. RESULTS: In contrast to the earlier Skylab missions where a pronounced amino aciduria was found, on shuttle the urinary amino acids showed little change with spaceflight except for a marked decrease in all of the amino acids on FD (flight day) 1 (p<0.05) and a reduction in isoleucine and valine on FD3 and FD4 (p<0.05). CONCLUSIONS: (i) Amino aciduria is not an inevitable consequence of space flight. (ii) The occurrence of amino aciduria, like muscle protein breakdown is a mission specific effect rather than part of the general human response to microgravity.

15N conservation in the metabolic conversion of isoleucine to alloisoleucine in the rat.

15N,13C6-L-Isoleucine was given by stomach tube to a pair of rats and the urine excreted over the following 6 h period was collected. The urinary amino acid fraction showed that the majority of the L-alloisoleucine produced from the labeled isoleucine was formed with the 15N label intact. This fails to support the commonly held supposition that L-isoleucine and L-alloisoleucine interconversion occurs through the reversible enolization of the 2-keto-3-methylvaleric acids formed by their transamination. In contrast to 15N label conservation in L-alloisoleucine, the majority of the 15N in the administered L-isoleucine underwent exchange with 14N.

Mild form of beta-ketothiolase deficiency (mitochondrial acetoacetyl-CoA thiolase deficiency) in two Japanese siblings: identification of detectable residual activity and cross-reactive material in EB-transformed lymphocytes.

Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inherited metabolic disorder of isoleucine and ketone body catabolism. We report the cases of two siblings who showed clinically mild forms of this disorder. They did not excrete tiglylglycine in urine. Their EB-transformed lymphocytes contained residual T2 activity, which was confirmed by immunotitration analysis. In immunoblot analysis, the bands corresponding to T2 in the samples of the cell lines from two patients were the most intensely detected among those from 19 T2-deficient cell lines tested.

Partial substitution of amino acids of a parenteral solution with tripeptides: effects on parameters of protein nutrition in baboons.

We previously showed that triglycine and trileucine are efficiently utilized when infused intravenously (IV) in baboons who are fed a complete diet orally. In the present experiments we investigated the utilization of these tripeptides in the context of total parenteral nutrition. A group of subhuman primates (baboon) was subjected sequentially to two forms of total parenteral nutrition, each for a period of six days. The only difference between the two periods was that in one, all amino acids were given in free form, and in the other, the glycine and leucine components of the amino acid mixture were replaced with triglycine and trileucine, respectively. During both experimental periods the infusion solution provided daily 100 calories/kg body weight and 2.5 g amino acids/kg body weight. There were no significant differences between nitrogen balance, plasma amino acid concentrations, or urinary excretion of amino acids for the two forms of parenteral nutrition. The only exceptions were a greater plasma concentration of isoleucine and a greater urinary excretion of leucine during infusion of the partial peptide solution. Assimilation of triglycine and trileucine under the conditions of total parenteral nutrition was assessed by determining concentrations of these peptides in plasma and urine. Trileucine was not found in plasma, and only 1.8% of the amount of trileucine infused appeared as trileucine and dileucine in urine. Triglycine was detected in plasma and 17.1% of the amount of triglycine infused was excreted in urine as triglycine and diglycine. These data suggest that assimilation of trileucine and triglycine under the conditions of our experiment was sufficiently efficient to meet the daily need for an essential amino acid (leucine) and for nitrogen (glycine).

Higher excretion of urinary bound amino acids in infants: probably related to protein-anabolic effect of growth hormone.

Concentrations of bound amino acids in urine were found to be markedly higher in infants than in children over 4 years of age. And it was also found that bound amino acid levels in plasma were increased in infants and young children than in adults. Urinary excretion of bound amino acids was increased in hypopituitary dwarf after one day of intramuscular injection of human growth hormone. A possibility was advanced that an increased excretion of bound amino acids of the overflow type observed in infants was due to active protein biosynthesis stimulated by growth hormone of which plasma levels have been reported to be high in infancy.

[Laboratory tools in the diagnosis of prostatic cancer (author's transl)]. / Labordiagnostische Möglichkeiten beim Prostatakarzinom.

The most reliable laboratory test for prostate cancer remains prostatic phosphatase determination. With the spectrophotometric method, however, falsely negative results are to be expected in 40% of stage D lesions. In only one third of patients with localized disease results are correctly positive. This poor specificity and sensitivity can be improved by radioimmunoassay (RIA). Using this technique the prostatogenic isoenzyme is elevated in 50% of stage A and in 80% of stage B carcinoma, suggesting RIA for screening. Erythrocyte sedimentation rate or serum iron and copper are not necessarily of prognostic value. Phosphatase determination of bone marrow aspirates also requires the RIA method if differentiation of stage C and D is to be expected. Serum hormone assays are not yet introduced into routine staging programs. Serum and urinary markers such as CEA, polyamines of LDH isoenzymes are unspecific and of uncertain value in prostatic carcinoma. Measurement of urinary hydroxyproline seems a reliable method for the search of osseous spread; other bone diseases have to be excluded. In patients with prostate cancer laboratory tests still represent adjunctive measures in connection with the clinical diagnostic armamentarium of urologists.

[Electro-clinical aspects of leucinosis]. / Aspects électro-cliniques des leucinoses.

Maple syrup urine disease, or leucinosis usually presents between the 4th and 10th day of life with neurological and gastro-intestinal symptoms. A distinctive odour of maple syrup on the child's skin or of his urine enables diagnosis to be made clinically, confirmation coming from amino-acide chromatography which reveals increased levels of the branched amino acids, leucine, valine and isoleucin in the blood and urine. The disease, untreated, is fatal, death occurring rapidly. With early diagnosis and treatment with a diet deficient in branched amino acids and purification methods the prognosis is quite good. E.E.G. appearances are clearly, therefore, of considerable interest: --burst suppression or "pseudo burst suppression" in the acute phase, suggesting severe cerebral insult and warranting amino acid chromatography. --rolandic "picket fence" waves seen between the 2nd and 4th weeks of life, which are strongly suggestive of the disease. --good correlation between clinical, biochemical and E.E.G. parameters, improvement of the last two indicating a good prognosis. --E.E.G. observation at regular intervals is justified after the acute phase to aid forecasts concerning the eventual outcome.