Nontargeted modification-specific metabolomics study based on liquid chromatography-high-resolution mass spectrometry.

Modifications of genes and proteins have been extensively studied in systems biology using comprehensive analytical strategies. Although metabolites are frequently modified, these modifications have not been studied using -omics approaches. Here a general strategy for the nontargeted profiling of modified metabolites, which we call "nontargeted modification-specific metabolomics", is reported. A key aspect of this strategy was the combination of in-source collision-induced dissociation liquid chromatography-mass spectrometry (LC-MS) and global nontargeted LC-MS-based metabolomics. Characteristic neutral loss fragments that are specific for acetylation, sulfation, glucuronidation, glucosidation, or ribose conjugation were reproducibly detected using human urine as a model specimen for method development. The practical application of this method was demonstrated by profiling urine samples from liver cirrhosis patients. Approximately 900 features were identified as modified endogenous metabolites and xenobiotics. Moreover, this strategy supports the identification of compounds not included in traditional metabolomics databases (HMDB, Metlin, and KEGG), which are currently referred to as "unknowns" in metabolomics projects. Nontargeted modification-specific metabolomics opens a new perspective in systems biology.

Urinary metabolomic analysis for the identification of renal injury in patients with acute heart failure.

OBJECTIVES: Worsening renal function in patients admitted with heart failure is associated with increased morbidity. These changes are not usually apparent initially and often take up to 48 hours to be detected. Using the novel technique of metabolomic analysis, this study aims to determine if markers of renal injury are identifiable at presentation that are associated with the development of worsening renal function in high-risk patients with heart failure. METHODS: A prospective exploratory study enrolled a convenience sample of patients with suspected heart failure. Eligible patients had to be older than 18 years, have a B-type natriuretic peptide (BNP) level over 100 pg/mL, have a history of diabetes or hypertension, meet Boston criteria for heart failure (>8), and require hospital admission as judged by the treating physician. Patients receiving no more than one dose of diuretic prior to enrollment were excluded. Urine was collected during the emergency department (ED) stay. Initial creatinine and the peak value between 24 to 48 hours were used to determine worsening renal function as defined by a change of >0.3 mg/dL or absolute 25% increase. Urine samples underwent gas chromatography/mass spectrometry (GC/MS) profiling. Peak metabolite values were measured and data were log-transformed. Partial least squares-discriminant analysis (PLS-DA) was used to identify metabolites associated with worsening renal function. Specific urinary metabolites were ranked based on their regression coefficients. RESULTS: The 24 enrolled subjects had a median age of 58 years (interquartile range [IQR] = 49.5 to 67.5 years) with 58% being male. Worsening renal function occurred in 10 subjects (41.7%). A total of 156 metabolites were identified. The optimal number of metabolites for class discrimination as determined by PLS-DA was three, with a classification accuracy of 78%. These metabolites were taurine, sulfuric acid, and talose. CONCLUSIONS: Urinary metabolites found at the time of presentation may be markers of early renal injury. It is therefore possible that the process of renal injury is initiated prior to ED arrival in patients with suspected heart failure, and these may be used to identify a high-risk patient population.

Extraction and high-performance liquid chromatographic separation of selected pyrene and benzo[a]pyrene sulfates and glucuronides: preliminary application to the analysis of smokers' urine.

In the study of the complex mixture of urinary metabolites derived from polycyclic aromatic hydrocarbon compounds, it is desirable to simplify the analysis through separation of classes of compounds. We have developed a liquid chromatography (LC) method for the separation of selected sulfate and glucuronide conjugate isomers derived from hydroxybenzo[a]pyrenes (OH-BaP) and hydroxypyrenes. This LC method was utilized in the preliminary analysis of the urine of smokers by combining it with an extraction technique employing tetra-n-butyl-ammonium ion as a coupling agent to generate a 1:1 complex, extractable in chloroform at low pH prior to LC analysis.

Usefulness of the hydrogen--deuterium exchange method in the study of drug metabolism using liquid chromatography-tandem mass spectrometry.

The usefulness of the hydrogen-deuterium (H-D) exchange method in the study of drug metabolism was investigated. Metabolite samples of denopamine and promethazine prepared in vitro were introduced to a triple stage quadrupole tandem mass spectrometer via a high performance liquid chromatography (HPLC) system using a deuterated mobile phase. Mass spectra by various ionization modes and collisionally induced dissociation (CID) mass spectra were obtained. A metabolite of denopamine was observed to have a shift of 7 mass units by the H-D exchange method, and this shift proved to be a glucuronidated metabolite. Discrimination between N- or S-oxide formation and hydroxylation observed in the metabolism of promethazine was also easily accomplished by this method. In this manner, the structures of the metabolites were elucidated unequivocally by determining the number of labile hydrogen atoms by the use of the H-D exchange method, since various reactions in drug metabolism are accompanied by an increase or a decrease in the number of labile hydrogen atoms. Additional information on the structures was obtained by analysis of the CID spectra of the molecular ion species. Thus, the combination of the H-D exchange method and tandem mass spectrometry was found to be very useful for the study of drug metabolism.

A screening method for mucopolysaccharidoses with increased urinary excretion of sulfated N-acetylhexosamines.

Patients with mucopolysaccharidosis have been reported to excrete elevated amounts of sulfated N-acetylhexosamines in their urine. Based on this finding, a new and simple colorimetric screening test for these disorders is presented. Chromatography of whole urine on Dowex AG 1-X8, from each of 23 normal controls, 5 patients with mucopolysaccharidosis and one patient with multiple sulfatase deficiency, was used to separate the sulfated hexosamines. The fractions eluted with 2M NaCl were analyzed according to the method of Reissig. Patients with Sanfilippo syndrome, type A, Sanfilippo syndrome, type D, Maroteaux-Lamy syndrome, Morquio syndrome, type A, and multiple sulfatase deficiency were clearly distinguished from normal controls. The procedure appeared most sensitive for Sanfilippo syndrome, type D, and multiple sulfatase deficiency, each of which involves deficient activity of N-acetylglucosamine 6-sulfate sulfatase.

Characterization of serum and urinary bile acids in patients with primary biliary cirrhosis by gas-liquid chromatography-mass spectrometry: effect of ursodeoxycholic acid treatment.

We have studied the effect of ursodeoxycholic acid on the serum and urinary bile acids in seven patients with moderate to severe primary biliary cirrhosis. Bile acids were characterized by gas-liquid chromatography-mass spectrometry and quantified by capillary gas-liquid chromatography. Serum bile acids were elevated 26-fold over control values, with 2.2 times more cholic acid than chenodeoxycholic acid. Urinary bile acid output was elevated 22-fold over control values with a cholic acid:chenodeoxycholic acid ratio of 1.6. In addition, lithocholic acid, deoxycholic acid, ursodeoxycholic acid, 1 beta-hydroxycholic acid, 1 beta-hydroxydeoxycholic acid, and hyocholic acid were identified in both serum and urine; the proportions of the 1- and 6-hydroxylated bile acids were much higher in urine than in serum of the patients (32.1% versus 4.2%). Three months of placebo administration did not change the serum and urinary bile acid composition. In contrast, ursodeoxycholic acid feeding (12-15 mg/kg body weight per day) for 6 months resulted in a 25% decline in the total serum bile acid concentration from the pretreatment values. The proportion of ursodeoxycholic acid increased from 2.1 to 41.2% of total bile acids, so that total fasting serum endogenous bile acid levels decreased 62.4%. Ursodeoxycholic acid feeding substantially increased urinary bile acid output, with ursodeoxycholic acid comprising 58.1%. The proportion of 1- and 6- hydroxylated endogenous bile acids was reduced by 45.5% from pretreatment levels and approximately 4.5% of the urinary bile acids were omega-muricholic acid, 1 beta-hydroxyursodeoxycholic acid, and 21-hydroxyursodeoxycholic acid. These results demonstrate significant changes in the serum and urinary bile acid pattern in primary biliary cirrhosis during ursodeoxycholic acid treatment. The beneficial effect of ursodeoxycholic acid may be due to reduction of the hydroxylated derivatives of endogenous bile acids together with the appearance of hydroxylated derivatives of ursodeoxycholic acid or it may be due to displacement of the more hydrophobic endogenous bile acids by the hydrophilic ursodeoxycholic acid.

Thermospray mass spectrometry and tandem mass spectrometry of polar, urinary metabolites and metabolic conjugates.

This paper describes the characterization of glucuronide and sulfuric acid conjugates and alkyl phosphates by thermospray tandem mass spectrometry (MS/MS). Primary thermospray mass spectra were generated using ammonium acetate buffer and filament-on ionization with both positive and negative ion detection. In positive ion mode molecular weight information was obtained for glucuronic, phosphoric and other weak acids. Under these conditions, however, spectra were not obtained for the sulfate adducts or phosphate salts. Negative ion thermospray mass spectrometry was more versatile, providing spectra of all metabolic structures examined. Positive and negative ion mass spectra provided complementary information for glucuronic acids. Collisionally activated dissociation (CAD) mass spectra of the glucuronide [M + NH4]+ or [M - H]- ions exhibited characteristic glucuronide 'fingerprints' as well as prominent aglycone ions. The aryl sulfates were hydrolyzed to the corresponding phenols under buffer/thermospray conditions and for these analytes CAD mass spectra were obtained from [phenate]- or [phenol + acetate]- parent ions. The M- of dimethyl thiophosphate underwent sequential loss of alkyl and alkoxy radicals and formaldehyde with collisional activation. MS/MS greatly enhances the power of the thermospray interface by providing fragmentation information useful both in the identification of unknowns and for improved mass spectrometry specificity.

Quantitative aspects of the urinary excretion of meptazinol and its metabolites in human volunteers.

1. The urinary excretion of meptazinol and its metabolites has been studied in five healthy human volunteers following oral administration of 200 mg of the 3H-labelled drug. 2. Meptazinol was well absorbed, with 90% of the radioactivity excreted in the urine; elimination of drug-related material took place rapidly. 3. Metabolism was extensive, no unchanged material being detected in the urine. Following selective enzymic hydrolysis the major metabolites were identified as glucuronide and sulphate conjugates of the parent drug present in an approximate ratio of 4:1. 4. A number of minor metabolites, also present in conjugated form, were tentatively identified.

Gas chromatographic determination of sulfuric acid and application to urinary sulfate.

A new gas chromatographic method for the determination of sulfate was developed. In this method, sulfate was quantitatively converted to a volatile derivative, dimethyl sulfate, by a two-step procedure. First, sulfate was converted to silver sulfate by reaction with silver oxide, and then to dimethyl sulfate by reaction with methyl iodide. The derivative was analyzed by gas chromatography. Methyl methanesulfonate was used as an internal standard. The method was applied to the determination of total urinary sulfate. Phosphate and chloride ions, which interfered with the present method, were eliminated with the use of basic magnesium carbonate and an excess of silver oxide, respectively. Recovery was over 96% when 5 to 40 mumol/ml of sulfate was added to human urine samples.

A mass spectrometric approach to the identification of conjugated drug metabolites.

For the identification of intact underivatized drug conjugates, the mass spectrometric technique of choice is fast atom bombardment (FAB); the combined use of both positive and negative ion FAB usually provides information on the molecular mass and nature of conjugates under study, while the number of exchangeable hydrogen atoms can be determined using trideuterated glycerol as the FAB-matrix. Electron impact and desorption chemical ionization spectra can be used to study the aglycone part of the conjugated metabolites. With this approach metabolites conjugated with glucuronic acid, sulphuric acid and amino acids have been identified. The identification was supported by analysis of reference compounds, prepared by chemical synthesis. The examples given are selected from current metabolism studies on drug candidates in development within Organon's research.

Significance of urinary phenyl sulfate and phenyl glucuronide as indices of exposure to phenol.

Urine samples from subjects, who had been using phenol for treatment of chemical fiber in factories, were analysed for urinary phenyl sulfate (PhS) and phenyl glucuronide (PhG) by high-performance liquid chromatography (HPLC). The urinary levels of phenol metabolites (PhG and PhS) had a significant correlation with the environmental concentration of phenol. The urinary concentration of phenol metabolites, as a total of PhS and PhG, corresponding to 5 ppm of environmental phenol was 251 mg phenol/g creatinine.

Major urinary metabolites in hamsters and rats treated with N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine.

Rats and hamsters were administered a single dose of N-[1-14C]nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP), and their urinary metabolites were examined at various time intervals. In both species, urinary excretion of radiolabeled metabolites reached a plateau at 6 h following injection. At this time, 35 and 28% of the total dose was found in the urine of rats and hamsters, respectively. Separation by liquid chromatography and subsequent characterization by nuclear magnetic resonance, gas chromatography-mass spectroscopy, and infrared showed that the major metabolites in rat urine were HPOP, N-nitrosobis(2-hydroxypropyl)amine (BHP), and their glucuronic acid conjugates. The conjugates accounted for 30 and 9%, while free HPOP and BHP accounted for 42 and 16% of the total metabolites, respectively. Hamster urine, on the other hand, contained free HPOP, BHP, their glucuronic acid conjugates, and a sulfate ester of HPOP not found in rat urine. Six h following administration of HPOP, hamster urine contained BHP, BHP glucuronide, HPOP, HPOP glucuronide, and HPOP sulfate ester at levels of 35, 9, 16, 9, and 14%, respectively. These data suggest that hamsters reduce HPOP to BHP more efficiently than rats, while rats are more effective in forming their glucuronic acid conjugates. Hamsters differ significantly from rats in their capacity to form and excrete the sulfate ester of HPOP.

Excretion of (sulfated) steroids in the urine and excretion of cholesterol sulfate in the feces of boys with recessive X-linked ichthyosis.

The excretion of sulfated steroids was investigated in the urine and feces of six boys aged 9 months to 7 years and 10 months who had recessive X-linked ichthyosis. Profiles of urinary total steroids as well as sulfated steroids were normal. Cholesterol sulfate excretion in the urine was not elevated. In the feces 2-20% of total cholesterol was cholesterol sulfate, whereas in the feces of 28 healthy children no cholesterol sulfate was demonstrable. In the 6 patients total cholesterol excretion (500-2,500 mumol/kg feces) was also elevated in comparison with the 28 healthy controls (150-700 mumol/kg feces, mean 365 mumol/kg feces).

2,4-Difluoroaniline and 4-fluoroaniline exposure: monitoring by methaemoglobin and urine analyses.

Two possible methods for monitoring exposure to 2,4-difluoroaniline and 4-fluoroaniline have been investigated: measurement of methaemoglobin content in blood and measurement of urinary metabolites. Experiments using rats dosed by the oral route as a model system show that measurement of methaemoglobin content provides a very rapid and simple monitoring method, but is not very sensitive. Measurement of the ortho-hydroxy metabolites of the two compounds, as their benzoxazole derivatives, provides a much more sensitive, but complicated technique. Details of both methods are presented.

Analysis of intact steroid conjugates by secondary ion mass spectrometry (including FABMS) and by gas chromatography.

The analysis of intact steroid conjugates by two different methods is described. One method employed secondary ion mass spectrometry (SIMS) using a Cs+ beam for ionisation, although comparable data were obtained by fast atom bombardment (FAB) using a Xe0 beam. In both of these mass spectrometric techniques the samples were analysed in a liquid matrix (glycerol). Positive and negative ion spectra have been obtained, the latter being most useful for steroid sulphate and glucuronide analysis. The negative ion spectrum of each steroid is dominated by a pseudomolecular ion at m/z [M - H]- (M of free acid) and a lack of marked fragmentation. Mixtures of steroids can be resolved in a single spectrum, providing the individual steroids differ in mass. The second method was gas chromatography. The carboxylic acid moieties of the steroid glucuronides were derivatised with diazomethane and the remaining functional groups in the steroids were thermally protected by methyloxime formation (for carbonyls) and trimethylsilylation (for all steroidal and glucuronic acid hydroxyls). Satisfactory analysis of steroid glucuronides was achieved through the use of glass or fused silica columns stable at high temperature (330 degrees C). Conveniently, trimethylsilylation resulted in exchange of the sulphate in 3 beta-hydroxy-5-ene steroid sulphates for a trimethylsilyl group so these could effectively be analysed as "free" steroids.

The excretion of sulfur compounds in the urine of newborn infants.

The excretion of sulfur-containing compounds was determined on the third and sixth day of life in male infants and the results were compared with those previously obtained on fed and fasting men. The output of total sulfur and inorganic sulfate was very low on the third day of life but had increased by the sixth day to levels found in the fasting men, whereas the excretion of mercaptolactate in the newborns decreased from the third to the sixth day of life. These results may be explained by the initial fasting state of neonates followed by an anabolic phase. Neonates excreted acid-labile ester sulfate and mercaptoacetate at levels similar to those found in adults, but the neonatal urine also contained sulfate esters (probably steroid sulfates) that required more drastic acid conditions for hydrolysis. Raised concentrations of sulfur-containing amino acids (methionine, cystathionine, cyst(e)ine and taurine) were found in neonatal urine in confirmation of earlier reports. The excretion of thiosulfate could only be determined in newborns on the sixth day and was low in comparison with that of adults. High urinary thiocyanate concentrations were found in newborns fed by tobacco-smoking mothers, whereas the excretion of thiocyanate was low in other newborns. The possible medical hazards from the exposure of neonates to thiocyanate are emphasized.

Urinary excretion of free and conjugated 3',5'-diiodothyronine and 3,3'-diiodothyronine.

RIAs for the estimation of 3',5'-diiodothyronine (3',5'-T2) and 3,3'-diiodothyronine (3,3'-T2) in human urine have been established. The urinary excretion of both glucuronide and sulfate conjugates of T2 and of T4, T3, and rT3 were estimated by means of enzymatic deconjugation. In healthy controls, the mean excretion (picomoles per 24 h) of free T4 was 1820, that of free T3 was 813, that of free rT3 was 77, that of free 3',5'-T2 was 13, and that of free 3,3'-T2 was 674. The total excretion of free and conjugated T4 was 2941, that of T3 was 1283, that of rT3 was 791, that of 3',5'-T2 was 709, and that of 3,3'-T2 was 2688. Significant amounts of sulfated T4 and T3 could not be demonstrated, amounts of sulfated T4 and T3 could not be demonstrated, whereas the excretion of sulfated rT3 was higher than that of glucuronidated rT3 (P less than 0.001). In contrast, glucuronidated and sulfated 3',5'-T2 as well as glucuronidated and sulfated 3,3'-T2 were found in the urine in equal amounts. In hyperthyroidism, the excretions of free and glucuronidated iodothyronines were increased, whereas the increase of the excretions of sulfated iodothyronines were less pronounced, only reaching statistical significance for 3,3'-T2 (P less than 0.02). In hypothyroidism, the excretions of both free, glucuronidated and sulfated iodothyronines were reduced. Significant amounts of sulfated T4 and T3 could not be demonstrated in urine from hyperthyroid or hypothyroid patients. Our data demonstrate that the amounts of free iodothyronines excreted in the urine vary considerably, suggesting active renal handling. The amounts of urinary glucuronidated and sulfated conjugates of the different iodothyronines studied vary considerably and are affected by thyroid function.