Simple method for the quantitative analysis of endogenous folate catabolites p-aminobenzoylglutamate (pABG) and its acetamido (apABG) derivative in human serum and urine by liquid chromatography-tandem mass spectrometry

OBJECTIVE: To develop a routine method for quantitative measurement of the folate catabolites p-aminobenzoylglutamate (pABG) and acetamidobenzoylglutamate (apABG) in serum and urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS). DESIGN AND METHODS: Urine, serum and aqueous standards were thawed. Two microliters of d3-glutamic acid (d3-Glu; 1 mmol/L) was added to 200 uL of specimen as internal standard. The samples were acidified with 4 uL 6N HCL, and aliquots were precipitated with 2 volumes (412 uL) of acetonitrile. For urine specimens 30 volumes (6.18 mL) of acetonitrile was used. Samples were centrifuged at 1900 x g for 10 min and the supernatant (10 microL) injected into a Biorad CAT/MET analytical column fitted to the LC-MS/MS. Detection of the catabolites was by selective multiple ion monitoring (multiple SRM) of the respective transitions. Urine and serum samples were analysed in a group of healthy volunteers and in anonymous samples from patients being tested for PTH and urinary catecholamines. RESULTS: pABG and apABG eluted at 5.2 and 4.74 min, respectively while the d3-glutamic acid eluted at around 7 min. Limit of quantitation (LOQ) for both catabolites was 10 nmol/L (which is equivalent to 33.3 fmol for a 10 microL injection). Limit of detection (LOD) was 1 nmol/L based on a signal to noise ratio of 5:1. A linear calibration curve was obtained from 10 to 100 nmol/L for serum specimens and from 10 to 200 micromol/L for urines. Imprecision for spiked serum samples (n=10) was between 2.5 and 20% for apABG and 4.5 and 21% for pABG (at 10 and 100 nmol/L, respectively). Imprecision for spiked urine samples (n=10) was between 2.9 and 4.0% for apABG and 6.0-12.7% for pABG. Recoveries were between 80 and 122% for serum samples and between 92 and 102% for urine specimens. Total folate catabolites in random urine samples from volunteers (n=5) are 2.9+/-2.3 umol/L (mean+/-S.D.). This group also had total serum catabolites of 11.9+/-7.6 nmol/L and serum folate of 35.3+/-5.8 nmol/L. Serum from patients being tested for PTH (n=11) had serum folate levels of 27.0+/-10.4 nmol/L with total serum catabolites of 20.4+/-23.8 nmol/L. Levels of serum folate and total catabolites in pregnant women (n=18) were 33.9+/-22.7 and 11.4+/-8.7 nmol/L, respectively. Mean urinary folate catabolites in patients being tested for urinary catecholamines (n=19) was 581.8+/-368.4 nmol/L. CONCLUSION: A simple, reliable and highly specific method by LC-MS/MS for detecting and quantifying the folate catabolites pABG and apABG was developed. This enables, for the first time, the routine clinical analysis of folate utilization in patients.     

Presence of endogenous prednisolone in human urine

The possibility of an endogenous presence of the glucocorticoid prednisolone has already been demonstrated in bovine and horse urine, with the aim of clarifying its origin in this matrix, which is used by official agencies for the control of illicit treatments. From this point of view, the endogenous nature of prednisolone could be a major topic in doping control of both amateur and professional human athletes. A study was therefore made on 34 human volunteers (13 males and 21 females; aged 22–62) to detect the presence of prednisolone in their urine by HPLC–MS³. One of the volunteers underwent vernal allergy treatment with betamethasone for two subsequent years. An investigation was carried out with the aim of verifying if the suppression, and the circadian rhythm, of cortisol urinary levels could also apply to prednisolone. The results of the study show that prednisolone was present in the urine of all 34 volunteers, with a concentration very close to 100-times lower that of cortisol, with no dependence on gender. The same ratio (1/100) was observed in the prednisolone and cortisol levels detected during the 24 h together with the suppression of prednisolone by betamethasone treatment.These data demonstrate the endogenous nature of low concentrations of prednisolone in human urine, and motivate further studies about the biosynthetic pathways of this corticosteroid and its relationship with stress in humans, as already described in cows.     

The identification of p-acetamidobenzoate as a folate degradation product in rat urine.

Within 48 h of administration of radiolabelled 10-formylfolate, folic acid and the polyglutamate derivative 10-formylfolate tetraglutamate to the rat, fragmentation products are found in the urine. The major catabolite was identified as p-acetamidobenzoate by chromatography and reverse isotope-dilution analysis.     

Evidence for the presence of endogenous 19-norandrosterone in human urine

In 1997, in the scope of antidoping control in sport, a not inconsiderable number of urine analysed by official laboratories revealed the presence of 19-nortestosterone (19-NT: 17β-hydroxyestr-4-en-3-one) metabolites: 19-norandrosterone (19-NA: 3α-hydroxy-5α-estran-17-one) and 19-noretiocholanolone (19-NE: 3α-hydroxy-5β-estran-17-one). These repeated results on a short period of time generated some investigations and especially the verification of the possible production of these metabolites by an unknown endogenous route in adult entire male. Some experiences were led on different persons known to be non-treated with steroids and more precisely with nandrolone. Extractive methods were developed focusing on their selectivity, i.e. searching to eliminate at best matrix interferences from the target analytes. Gas chromatography coupled to mass spectrometry (quadrupole and magnetic instruments) was used to detect, identify and quantify the suspected signals. Two types of derivatization (TMS and TBDMS), a semi-preparative HPLC as well as co-chromatography proved unambiguously the presence, in more than 50% of the analysed urine (n=40), of 19-NA at concentrations between 0.05 and 0.60 ng/ml. 19-NE was not detected with the developed methods (LOD<0.02 ng/ml). Experiments led on athletes showed that after a prolonged intense effort, the 19-NA concentration can be increased by a factor varying between 2 and 4. Even if some complementary researches have to be done in order to determine the maximal physiological level of 19-NA and 19-NE, these results should considerably change the strategy of antidoping laboratories.     

Liquid chromatographic/mass spectrometric procedure for measurement of NAD catabolites in human and rat plasma and urine

Monitoring level of the metabolites of the coenzyme NAD such as nicotinamide and its oxidized and methylated derivatives is important due to therapeutic applications of these compounds and monitoring of oxidative stress. We evaluated feasibility of using HPLC with electrospray ion-trap mass detection for single run separation and quantitation of all the NAD metabolites. We achieved good separation and retention of all the metabolites of interest using reversed-phase with ion-pairing. Single ion monitoring or tandem MS were used for detection and quantitation of the specific compounds with good linearity. The method was able to detect all the physiological metabolites in plasma samples of rats and humans or in urine. However, full validation is necessary before this method could be routinely applied.     

Excretion of endogenous boldione in human urine: Influence of phytosterol consumption

Boldenone (17-hydroxy-androsta-1,4-diene-3-one, Bol) and boldione (androst-1,4-diene-3,17-dione, ADD), are currently listed as exogenous anabolic steroids by the World Anti-Doping Agency. However, it has been reported that these analytes can be produced endogenously. Interestingly, only for Bol a comment is included in the list on its potential endogenous origin. In this study, the endogenous origin of ADD in human urine was investigated, and the potential influence of phytosterol consumption was evaluated.We carried out a 5-week in vivo trial with both men (n = 6) and women (n = 6) and measured α-boldenone, β-boldenone, boldione, androstenedione, β-testosterone and α-testosterone in their urine using gas chromatography coupled to multiple mass spectrometry (GC–MS–MS). The results demonstrate that endogenous ADD is sporadically produced at concentrations ranging from 0.751 ng mL⁻¹ to 1.73 ng mL⁻¹, whereas endogenous Bol could not be proven. We also tested the effect of the daily consumption of a commercially available phytosterol-enriched yogurt drink on the presence of these analytes in human urine. Results from this study could not indicate a relation of ADD-excretion with the consumption of phytosterols at the recommended dose. The correlations between ADD and other steroids were consistently stronger for volunteers consuming phytosterols (test) than for those refraining from phytosterol consumption (control). Excretion of AED, bT and aT did not appear to be dependent on the consumption of phytosterols.This preliminary in vivo trial indicates the endogenous origin of boldione or ADD in human urine, independent on the presence of any structural related analytes such as phytosterols.     

Multipoint analysis of human quantitative genetic variation.

A unique method of partitioning human quantitative genetic variation into effects due to specific chromosomal regions is presented. This method is based on estimating the proportion of genetic material, R, shared identical by descent (IBD) by sibling pairs in a specified chromosomal region, on the basis of their marker genotypes at a set of marker loci spanning the region. The mean and variance of the distribution of R conditional on IBD status and recombination pattern between two marker loci are derived as a function of the distance between the two loci. The distribution of the estimates of R is exemplified using data on 22 loci on chromosome 7. A method of using the estimated R values and observed values of a quantitative trait in a set of sibships to estimate the proportion of total genetic variance explained by loci in the region of interest is presented. Monte Carlo simulation techniques are used to show that this method is more powerful than existing methods of quantitative linkage analysis based on sib pairs. It is also shown through simulation studies that the proposed method is sensitive to genetic variation arising from both a single locus of large effect as well as from several loosely linked loci of moderate phenotypic effect.     

Characterization and quantification of endogenous fatty acid nitroalkene metabolites in human urine

The oxidation and nitration of unsaturated fatty acids transforms cell membrane and lipoprotein constituents into mediators that regulate signal transduction. The formation of 9-NO₂-octadeca-9,11-dienoic acid and 12-NO₂-octadeca-9,11-dienoic acid stems from peroxynitrite- and myeloperoxidase-derived nitrogen dioxide reactions as well as secondary to nitrite disproportionation under the acidic conditions of digestion. Broad anti-inflammatory and tissue-protective responses are mediated by nitro-fatty acids. It is now shown that electrophilic fatty acid nitroalkenes are present in the urine of healthy human volunteers (9.9 ± 4.0 pmol/mg creatinine); along with electrophilic 16- and 14-carbon nitroalkenyl β-oxidation metabolites. High resolution mass determinations and coelution with isotopically-labeled metabolites support renal excretion of cysteine-nitroalkene conjugates. These products of Michael addition are in equilibrium with the free nitroalkene pool in urine and are displaced by thiol reaction with mercury chloride. This reaction increases the level of free nitroalkene fraction >10-fold and displays a K_D of 7.5 × 10⁻⁶ M. In aggregate, the data indicates that formation of Michael adducts by electrophilic fatty acids is favored under biological conditions and that reversal of these addition reactions is critical for detecting both parent nitroalkenes and their metabolites. The measurement of this class of mediators can constitute a sensitive noninvasive index of metabolic and inflammatory status.     

A high-affinity folate binding protein in human urine. Radioligand binding characteristics,immunological properties and molecular size

High-affinity binding of [³H]folate in human urine displayed characteristics, e.g. apparent positive cooperativity, which are typical of specific folate binding. By means of a two-site enzyme-linked immunosorbent assay (ELISA) with rabbit antibodies against the low molecular weight folate binding protein from human milk, we measured folate binding protein concentrations in the range of 0.51 to 4.13 nM in urine samples from 16 apparently healthy individuals. Ultrogel AcA 44 chromatography of the urine showed that immunoreactive and radioligand bound folate binding protein coeluted in one large peak (M_r25,000).     

Detection and analysis of endogenous polar volatile organic compounds (PVOCs) in urine for human exposome research

Background: The human exposome, defined as ‘…everything that is not the genome’, comprises all chemicals in the body interacting with life processes. The exposome drives genes x environment (GxE) interactions that can cause long-term latency and chronic diseases. The exposome constantly changes in response to external exposures and internal metabolism. Different types of compounds are found in different biological media.

Objective: Measure polar volatile organic compounds (PVOCs) excreted in urine to document endogenous metabolites and exogenous compounds from environmental exposures.

Methods: Use headspace collection and sorbent tube thermal desorption coupled with bench-top gas chromatography-mass spectrometry (GC-MS) for targeted and non-targeted approaches. Identify and categorize PVOCs that may distinguish among healthy and affected individuals.

Results: Method is successfully demonstrated to tabulate a series of 28 PVOCs detected in human urine across 120 samples from 28 human subjects. Median concentrations range from below detect to 165?ng/mL. Certain PVOCs have potential health implications.

Conclusions: Headspace collection with sorbent tubes is an effective method for documenting PVOCs in urine that are otherwise difficult to measure. This methodology can provide probative information regarding biochemical processes and adverse outcome pathways (AOPs) for toxicity testing.     

An endogenous carrier-mediated uptake system for folate in oocytes of Xenopus laevis.

We investigated the existence of an endogenous uptake system for folate in Xenopus laevis oocytes. This was done by performing uptake measurements using [3H]folic acid. Uptake of folic acid was linear with time for 4 h of incubation, and was similar in collagenase-treated and non-treated oocytes. The uptake process was carrier-mediated, as suggested by the saturation of folic acid uptake with concentration, and by the ability of unlabelled folic acid and its related compounds to significantly inhibit the uptake of [3H]folic acid. The apparent Km and Vmax of the uptake process were 42 +/- 7 nM and 10.56 +/- 0.46 fmol per oocyte per 2 h, respectively. The uptake of folic acid was independent of the presence of Na+ in the incubation medium, but was highly pH dependent with severe inhibition occurring at pH lower than 6.5. Folic acid uptake was energy- and temperature-dependent, and was significantly inhibited by the anion transport inhibitors DIDS and SITS. These results demonstrate the existence of an endogenous carrier-mediated system for folic acid uptake in Xenopus oocytes. Further characterization of the molecular mechanism of folic acid uptake and its regulation in this non mammalian in vitro unicellular system may prove useful in furthering our understanding of folate movement across biological membranes.     

Reutilization of DNA catabolites in granulocytopoiesis.

Different DNA labelling procedures for the proliferating granulocyte precursor compartments of rat bone marrow were evaluated by studying the appearance of labelled granulocytes in the blood stream by means of autoradiography. 3-H-thymidine was administered by single injection and continuous infusion. 125-I-Iododeoxyuridine, a DNA precursor showing a neglegible extent of reutilization, was studied in comparison. Labelling patterns of blood neurtrophils were identical after single injection and continuous infusion of 3-H-thymidine, while a different pattern was observed after use of a single injection of 125-I-Iododeoxyuridine. The results provide evidence that reutilization of label is an important event to be considered when kinetics of granulocytopoiesis are studied after application of 3-H-thymidine and indicate that reutilization occurs at the level of thymidinemonophosphate in this cell system.     

Glycoasparaginase in human urine.

Glycoasparaginase was purified 15,000-fold from human urine. The enzyme is a tetrameric protein of 86 kDa, composed of two heavy chains (25 kDa) and two light chains (18 kDa). Its structure and properties are very similar to those of human leukocyte glycoasparaginase. Glycoasparaginase activity is totally absent from urine of aspartylglycosaminuria patients.     

Endogenous nandrolone metabolites in human urine: preliminary results to discriminate between endogenous and exogenous origin.

When administered to human subjects, nandrolone is metabolized into two main products, 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE). Recent studies demonstrated the endogenous production of these compounds in man at concentrations very close to the threshold of the International Olympic Committee (IOC), i.e. 2 ng/ml. Because the possibility of reaching or exceeding this fateful limit is difficult to exclude, a complementary biochemical parameter is necessary for the differentiation of endogenous 19-NA and 19-NE production from residues resulting from nandrolone consumption. We measured the endogenous concentrations of 19-NA and 19-NE in 385 urine samples from professional football players, and we studied the phase II metabolite composition in individuals excreting the highest concentrations. The results showed that around 30% of endogenous 19-norandrosterone was sulfo-conjugated, whereas 100% of 19-norandrosterone was excreted conjugated to a glucuronic acid when nandrolone was administered. This significant qualitative difference appears to be a promising complementary criterion to more definitively conclude about an athlete's culpability, especially when nandrolone metabolites are found in the low ng/ml range.     

The indirect analysis of 17-oxosteroid glucosiduronates and sulphates in human urine

1. An analytical procedure has been developed for the determination of urinary 17-oxosteroid sulphates and 11-deoxy-17-oxosteroid glucosiduronates. 2. By submitting urine to the following operations: (a) extraction of the 17-oxosteroid conjugates into chloroform (in the presence of pyridinium sulphate), (b) solvolysis (in the absence of acid) of the steroid sulphates, (c) separation of the steroid glucosiduronates, (d) hydrolysis of the glucosiduronates, (e) oxidation with tert.-butyl chromate of the liberated steroids and (f) analysis of the final extracts by gas-liquid chromatography, the 17-oxosteroid conjugates are converted into and measured as androstanediones and androstane-(ene)triones. 3. Values for the daily excretion of 17-oxosteroid glucosiduronates and sulphates in normal men and women are given.     

Immunoaffinity purification of 11-dehydro-thromboxane B2 from human urine and plasma for quantitative analysis by radioimmunoassay

11-Dehydro-thromboxane B2 is now considered to be a reliable parameter of thromboxane A2 formation in vivo. An immunoaffinity purification method was developed for radioimmunoassay of this compound contained in human urine and plasma. Monoclonal anti-11-dehydro-thromboxane B2 antibody was prepared and coupled to BrCN-activated Sepharose 4B. Human urine or plasma was applied to a disposable column of the immobilized antibody. After the column was washed with water, 11-dehydro-thromboxane B2 was eluted with methanol/water (95/5) with a recovery of more than 90%. The purified extract was subjected to a radioimmunoassay utilizing 11-[3H]dehydro-thromboxane B2 methyl ester and the monoclonal anti-11-dehydro-thromboxane B2 antibody. The detection range of the assay was 10-600 fmol (IC50 = 90 fmol). The cross-reactivities of the antibody with thromboxane B2, 2,3-dinor-thromboxane B2, and other arachidonate metabolites were less than 0.05%. These compounds were efficiently separated from 11-dehydro-thromboxane B2 by the immunoaffinity purification. This procedure also allowed the separation of 11-dehydro-thromboxane B2 from unidentified urinary and plasma substances which interfered with the radioimmunoassay. Validity of the results obtained by the radioimmunoassay was confirmed by GC/MS employing selected ion monitoring for quantification.