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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.

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., (© 2012 by the Society for Academic Emergency Medicine.)

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.

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.

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.

This report describes the mechanism of origin and the quantity of estrogen produced in a prepubertal boy who developed severe feminization at 8 yr of age as the result of a heretofore undescribed metabolic abnormality. The clinical findings were gynecomastia and accelerated linear growth and bone maturation. At the time feminization developed, there were no signs of growth or development of the otherwise normal prepubertal male external genitalia or any increase of muscle mass that normally accompanies male puberty. The hyperestrogenism was found to be the consequence of massive extraglandular conversion of plasma androstenedione to estrone. During a 6-mo period of study, the plasma production rate of androstenedione ranged from 1.2 to 1.6 mg/day. More than 55% of plasma androstenedione was metabolized by aromatization to estrone which, in turn, was extensively sulfurylated in the tissue sites of aromatization before its entry into the blood. Thus, estrone sulfate was the final product in the aromatizing sites, and the plasma production rate of estrone sulfate derived from plasma androstenedione was 782 mug/24 h. The extent of extraglandular conversion of plasma androstenedione to estrone measured in this boy was 50 times that observed in two normal prepubertal boys. Moreover, 94% of the extraglandular aromatization occurred in extrahepatic sites. The metabolic clearance rate of plasma androstenedione, 2,380 liters/day per m(2), was markedly increased in this boy. Approximately 1,500 liters of plasma androstenedione clearance was accounted for by extrahepatic, extraglandular aromatization. The fractional conversion of testosterone to estradiol, 0.16, was 50 times greater in this boy than that observed in normal young adult men. The total extent of aromatization of plasma prehormones was even greater in this boy inasmuch as evidence was obtained that aromatization of 16-hydroxysteroids, e.g. 16alpha-hydroxy androstenedione and 16alpha-hydroxy dehydroisoandrosterone (sulfate), resulted in estriol formation independent of estrone formation. Thus, extensive extrahepatic, extraglandular aromatization resulted in advanced feminization in this prepubertal boy by a previously undescribed metabolic abnormality.

The use of the Henderson-Hasselbalch equation and the relationships between bicarbonate levels and the pCO2 or carbonic acid concentration in evaluating acid-base abnormalities are explained. The etiology, pathophysiology, diagnosis and treatment of respiratory alkalosis and acidosis and metabolic alkalosis and acidosis are discussed. The results of laboratory tests should be examined in relation to the patient's condition and consistency with other laboratory tests. Therapy is directed at correcting the underlying problems and, secondarily, at correcting the numbers. Patients respond primarily to rate of change and not absolute numbers. Therefore, problems should be corrected at approximately the rate they develop. Treatment should be guided by continued patient observation and serial laboratory studies.

Taurine metabolism in man was defined by an isotope dilution technique during normal taurine intake (five subjects) or increased taurine intake (two subjects). A tracer dose of [35S]taurine was administered intravenously, and the amount and chemical form of radioactivity were determined in blood, urine, bile, and feces. Analysis of plasma specific activity decay curves indicated that taurine metabolism can be described by two exchangeable pools: a small (2 mmoles), rapidly exchanging pool (t1/2 approximately equal to 0.1 hour); and a large (98 mmoles), very slowly exchanging pool (t1/2 approximately equal to 70 hours). A small amount of [35S]isethionic acid was detected in urine, possibly the result of deamination of taurine by tissues; but otherwise no evidence of tissue biotransformation was obtained. Taurine was excreted predominantly (95%) in urine, about 70% as taurine and 25% as sulfate. The sulfate was considered to be formed in the intestine by bacterial degradation of taurine and then absorbed. Supplemental taurine (given orally) was well absorbed, caused a transient increase in plasma taurine levels, was excreted in urine without equilibration with the slowly exchangeable pool, and caused only a modest increase in total body taurine. Thus, taurine resembles other amino acids in having large tissue pools but differs strikingly in being metabolically inert with an extremely slow turnover rate.

The urinary excretion of inorganic sulfate, ester sulfate, taurine and total sulfur has been estimated in 18 patients with malignancy, and compared with those of an age-matched reference group. The absolute amount of inorganic sulfate was significantly lower in the cancer group, whereas an increased ester sulfate excretion was observed. Reduced excretion of total sulfate (inorganic plus ester sulfate) and increased excretion of "neutral sulfur" (total sulfur minus total sulfate) in the cancer group was calculated from these data. If these values were recalculated with respect to creatinine, no significant differences were found for inorganic and total sulfate between the two patient groups, whereas ester sulfate and the "neutral sulfur" fractions were still increased. The enlarged "neutral sulfur" could to a large extent be explained by an increased excretion of taurine. The mechanism behind these findings is discussed.

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.

The phytoestrogen, diadzein, was injected intramuscularly as [4-14-C]daidzein into two laying hens. The radioactive materials in the urine for the succeeding 23 (hen 1) or 14 (hen 2) days were fractionated on a DEAE-Sephadex column by a gradient of NaCl and the fractions thus separated were further analyzed by solvent partition, susceptability to enzymic cleavage and thin-layer chromatography. The sic following components were identified and quantitated: [14-C]diadzein, [14-C]equol, [14-C]diadzein monosulfate, [14-C]equol monosulfate, [14-C]diazein disulfate, and [14-C]equol disulfate. The urine from hen 2 yielded also the sulfate of an unidentified conversion product of [14-C]daidzein. Repeared tests for glucuronides of [14-C]daidzein or its conversion products gave negative results, excluding the possibility that any appreciable proportion of the radioactivity in the urine was in the form of beta-glucuronide. It is concluded that the diadzein and the equol excretion in the urine of the laying hen are present for the most part as monosulfates and disulfates.

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.

We have assessed individual variations in urinary acidic metabolite excretion and the effects of extreme alterations in dietary composition on these metabolites in selected normal persons who we considered representative of the general ambulant normal population. Extreme dietary alterations produced relatively small changes in the patterns or amounts of metabolite excretion, but large individual within-subject variations were observed. Our results indicate that variation in the ranges of excretion for the normal population mainly depend on individual metabolic variations rather than on dietary factors, and provide a basis for the assessment of the normal ranges determined from population surveys. Our results are discussed in relation to previous studies on the variability of urinary acidic metabolite excretion in man.

The urinary production rate of pregnenolone has been determined for a male subject using 7,7-d2-pregnenolone as an isotopic tracer.

Serum and urine from chronically uremic patients and normal individuals were subjected to gel filtration of Sephadex-G10. The effects of the eluted fractions on the uptake of urate and para-aminohippurate by isolated cortical tubules of rabbit kidney were investigated. According to the origin of the samples, one to three major groups of fractions inhibiting both urate and para-aminohippurate transport were disclosed. The first eluted group occurred for all the samples under study. The second one was demonstrated in both sera and urines from uremic patients but only in urines from normal individuals. The third one was exclusively detected in uremic sera and urines. Among all the compounds identified, only hippuric acid, eluted in the fractions of the second group, was capable of inhibiting the uptake of urate and para-aminohippurate in vitro. The concentration for which this inhbiitory effect of hippuric acid occurred was in the range of that existing in uremic sera. Indoxyl sulfate, which accumulates to very high concentrations in uremic serum, could not be disclosed in the above-mentioned fractions. This is explained by the strong adsorption of this indole derivative to Sephadex gel. Potassium indoxyl sulfate, when tested in vitro at the concentration existing in uremic serum, substantially inhibited the uptake of both urate and para-aminohippurate. In normal subjects, ingestion of hippuric acid or potassium indoxyl sulfate significantly increased fractional urinary excretion of uric acid. On the basis of these results, it is suggested that progressive retention of hippuric acid, indoxyl sulfate, and other yet unidentified inhibitors may explain the gradual increase in urinary fractional excretion of urate observed in uremia. The present results may be viewed as an example of a mechanism in which retention of normally excreted end products is responsible for adaptation of tubular transport in uremic subjects.

We describe simple and precise methods for the determination of inorganic sulfate, ester sulfate, and total sulfur in urine. The methods are based on turbidimetry of sulfate as barium sulfate in the presence of a small amount of preformed barium sulfate and with polyethylene glycol as a stabilizing agent. Inorganic sulfate is directly determined, whereas ester sulfate is measured after removal of inorganic sulfate, followed by acid hydrolysis. Total sulfur is determined after wet oxidation of the sample with nitric acid and perchloric acid in the presence of vanadate as a catalyst. We also report excretion values for healthy persons on a self-selected diet. Men excrete significantly higher amounts of inorganic sulfate and total sulfur than women, but this sex-related difference becomes insignificant if excretion values are expressed relative to creatinine.

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.

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.

Phenol sulfate esters have been analyzed by paired-ion reversed-phase high-performance liquid chromatography. The method provided direct, rapid chromatography of phenol sulfates in crude extracts of the red alga (Polysiphonia lanosa (2,3-dibromo-4,5-dihydroxybenzyl alcohol 1',4-disulfate), of the brown alga Ascophyllum nodosum (1,2,3,5-tetrahydroxybenzene 2,5-disulfate), and in rat urine (resorcinol mono- and disulfates). Detector response (254 nm) was linear within the approximate range from 30--125 ng to 5--10 microgram. Semipreparative scale chromatography provided sufficient amounts of purified phenol sulfates for further analysis by paper electrophoresis.

A procedure was developed for the separation and determination of dopamine-3-O-sulfate (DM-3-S) and dopamine-4-O-sulfate (DM-4-S) in the urine of subjects administered L-DOPA. The method consists of sample preparation using cation- and anion-exchange resins followed by determination of the sulfates by high-performance liquid chromatography. The addition recoveries were 96 +/- 2.9% (S.D.) for DM-3-S and 93 +/- 3.0% (S.D.) for DM-4-S. Twenty samples could be measured per day. When every 2-h urine specimen from normal subjects was analyzed after L-DOPA administration (0.5 g), the maximum excretion of each sulfate was observed in the second 2-h specimen. For the first 6 h 7.5 +/- 1.5% (S.D.) of the administered L-DOPA was excreted as DM-O-sulfates. During this time, the ratio of DM-4-S to the DM-O-sulfates was 11.7 +/- 0.58% (S.D.).

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.

By starting with 4 1 of rat urine, it was possible to obtain a sulfate ester of hexosamine in crystalline form. A series of identification procedures including chemical analyses, enzymatic digestion, proton magnetic resonance spectroscopy and infrared spectroscopy showed that this substance is 2-acetamido-2-deoxy-D-galactose 4,6-bissulfate. The trivial name for this compound is N-acetylgalactosamine 4,6-bissulfate; Quantitation by isotopic techniques indicated the urine possessed an average concentration of 8 muM N-acetylgalactosamine 4,6-bissulfate. Further extension of these studies necessitated the chemical synthesis of N-acetylgalactosamine 4,6-bissulfate and related compounds to be used for references or as biological substrates. Direct sulfation of N-acetylgalactosamine was attempted first, and strong preference for attack on the primary hydroxyl group (position 6) was found for chlorosulfonic acid. Thus, the reaction with 2.2 molar equivalents of the sulfating agent gave N-acetylgalactosamine 6-sulfate and its derivatives bearing a second sulfate at either position 1 (minor) or position 3 (major). The lack of sulfation at position 4 could be attributed to steric effects of the sulfate group preferentially attached to position 6. Another experiment in which UDP-N-acetylgalactosamine 4-sulfate was used in place of the free sugar led to the formation of a bissulfated sugar-nucleotide which, on subsequent hydrolysis with mild acid, afforded N-acetylgalactosamine 4,6-bissulfate, the same compound as that obtained from rat urine.

The excretion of corticosteroid sulfates and free cortisol in urine, and the total plasma cortisol, have been determined in 41 women receiving an estrogen-containing oral contraceptive and 53 age-matched female controls. The contraceptive steroids caused increases in the urinary corticosteroid sulfates and plasma cortisol similar to those observed in pregnancy; urinary free cortiscol was unchanged.

Amberlite XAD-2 was used to extract bile acids from urine or diluted serum of patients with hepatobiliary diseases. Columns containing Sephadex LH-20 were then used to separate the sulfated and nonsulfated bile acids. Thin-layer chromatography of the sulfated bile acid fraction obtained from urine revealed several spots with R(F) values different from those of the taurine or glycine conjugates. According to thin-layer chromatographic mobilities, gas-liquid chromatographic analyses, infrared spectra, and elementary analysis of the sulfated material, one of these sulfated bile acids was identified as glycochenodeoxycholic acid monosulfate, and the others were presumed to be taurochenodeoxycholic acid sulfate and glycocholic acid sulfate. A large amount of bile acid sulfate was found in urine of patients with hepatobiliary diseases. They accounted for 35.5-93.3% of total urinary bile acids and consisted of both di- and trihydroxycholanoic acids, with chenodeoxycholic acid as the major acid. Sulfated bile acids were also found in serum, and accounted for 1.8-21.2% of the total bile acids. Only dihydroxycholanoic acids (mainly chenodeoxycholic) were identified.

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.