Liquid chromatography-tandem mass spectrometric quantification of the dehydration product of tetranor PGE-M, the major urinary metabolite of prostaglandin E(2) in human urine

A LC-MS/MS method has been developed to analyze tetranor PGE-M, the major urinary metabolite of PGE(2), that involves the acid-catalyzed dehydration of tetranor PGE-M and its deuterated (d(6)) analog followed by LC-MS/MS measurement of the dehydrated tetranor PGE-M product (tetranor PGA-M). We also report a method for quantification of creatinine in urine by LC-MS/MS to normalize tetranor PGE-M concentrations with that of urinary creatinine. These methods were used to study the effect of aspirin on urinary tetranor PGE-M levels in healthy male volunteers. Aspirin did not affect urinary creatinine concentrations but decreased urinary tetranor PGE-M concentrations by approximately 44%.     

Accurate quantification of dimethylamine (DMA) in human urine by gas chromatography-mass spectrometry as pentafluorobenzamide derivative: evaluation of the relationship between DMA and its precursor asymmetric dimethylarginine (ADMA) in health and disease

Dimethylamine [DMA, (CH(3))(2)NH)] is abundantly present in human urine. Main sources of urinary DMA have been reported to include trimethylamine N-oxide, a common food component, and asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis. ADMA is excreted in the urine in part unmetabolized and in part after hydrolysis to DMA by dimethylarginine dimethylaminohydrolase (DDAH). Here we describe a GC-MS method for the accurate and rapid quantification of DMA in human urine. The method involves use of (CD(3))(2)NH as internal standard, simultaneous derivatization with pentafluorobenzoyl chloride and extraction in toluene, and selected-ion monitoring of m/z 239 for DMA and m/z 245 for (CD(3))(2)NH in the electron ionization mode. GC-MS analysis of urine samples from 10 healthy volunteers revealed a DMA concentration of 264+/-173 microM equivalent to 10.1+/-1.64 micromol/mmol creatinine. GC-tandem MS analysis of the same urine samples revealed an ADMA concentration of 27.3+/-15.3 microM corresponding to 1.35+/-1.2 micromol/mmol creatinine. In these volunteers, a positive correlation (R=0.83919, P=0.0024) was found between urinary DMA and ADMA, with the DMA/ADMA molar ratio being 10.8+/-6.2. Elevated excretion rates of DMA (52.9+/-18.5 micromol/mmol creatinine) and ADMA (3.85+/-1.65 micromol/mmol creatinine) were found by the method in 49 patients suffering from coronary artery disease, with the DMA/ADMA molar ratio also being elevated (16.8+/-12.8). In 12 patients suffering from end-stage liver disease, excretion rates of DMA (47.8+/-19.7 micromol/mmol creatinine) and ADMA (5.6+/-1.5 micromol/mmol creatinine) were found to be elevated, with the DMA/ADMA molar ratio (9.17+/-4.2) being insignificantly lower (P=0.46). Between urinary DMA and ADMA there was a positive correlation (R=0.6655, P<0.0001) in coronary artery disease, but no correlation (R=0.27339) was found in end-stage liver disease.     

Determination of 3-nitrotyrosine in human urine at the basal state by gas chromatography-tandem mass spectrometry and evaluation of the excretion after oral intake

3-Nitrotyrosine (NO(2)Tyr) is a potential biomarker of reactive-nitrogen species (RNS) including peroxynitrite. 3-Nitrotyrosine occurs in human plasma in its free and protein-associated forms and is excreted in the urine. Measurement of 3-nitrotyrosine in human plasma is invasive and associated with numerous methodological problems. Recently, we have described an accurate method based on gas chromatography (GC)-tandem mass spectrometry (MS) for circulating 3-nitrotyrosine. The present article describes the extension of this method to urinary 3-nitrotyrosine. The method involves separation of urinary 3-nitrotyrosine from nitrite, nitrate and l-tyrosine by HPLC, preparation of the n-propyl-pentafluoropropionyltrimethylsilyl ether derivatives of endogenous 3-nitrotyrosine and the internal standard 3-nitro-l-[(2)H(3)]tyrosine, and GC-tandem MS quantification in the selected-reaction monitoring mode under negative-ion chemical ionization conditions. In urine of ten apparently healthy volunteers (years of age, 36.5+/-7.2) 3-nitrotyrosine levels were determined to be 8.4+/-10.4 nM (range, 1.6-33.2 nM) or 0.46+/-0.49 nmol/mmol creatinine (range, 0.05-1.30 nmol/mmol creatinine). The present GC-tandem MS method provides accurate values of 3-nitrotyrosine in human urine at the basal state. After oral intake of 3-nitro-l-tyrosine by a healthy volunteer (27.6 microg/kg body weight) 3-nitro-l-tyrosine appeared rapidly in the urine and was excreted following a biphasic pharmacokinetic profile. Approximately one third of administered 3-nitro-l-tyrosine was excreted within the first 8 h. The suitability of the non-invasive measurement of urinary 3-nitrotyrosine as a method of assessment of oxidative stress in humans remains to be established.     

Increased asymmetric dimethylarginine (ADMA) dimethylaminohydrolase (DDAH) activity in childhood hypercholesterolemia type II

Asymmetric dimethylarginine (ADMA) systemic concentrations are elevated in hypercholesterolemic adults and contribute to nitric oxide (NO) dependent endothelial dysfunction. Decreased activity of the key ADMA-hydrolyzing enzyme dimethylarginine dimethylaminohydrolase (DDAH) may be involved. Yet, the ADMA/DDAH/NO pathway has not been investigated in childhood hypercholesterolemia. We studied 64 children with hypercholesterolemia type II (HCh-II) and 54 normocholesterolemic (NCh) children (mean ± SD; age, years: 11.1 ± 3.5 vs. 11.9 ± 4.6). Plasma and urine ADMA was measured by GC-MS/MS. Dimethylamine (DMA), the ADMA metabolite, creatinine, nitrite and nitrate in urine were measured by GC-MS. The DMA/ADMA molar ratio in urine was calculated to estimate whole body DDAH activity. ADMA plasma concentration (mean ± SD; nM: 571 ± 85 vs. 542 ± 110, P = 0.17) and ADMA urinary excretion rate (mean ± SD: 7.1 ± 2 versus 7.2 ± 3 μmol/mmol creatinine, P = 0.6) were similar in HCh-II and NCh children. Both DMA excretion rate [median (25th-75th percentile): 56.3 (46.4-109.1) vs. 45.2 (22.2-65.5) μmol/mmol creatinine, P = 0.0004] and DMA/ADMA molar ratio [median (25th-75th percentile): 9.2 (6.0-16.3) vs. 5.4 (3.8-9.4), P = 0.0004] were slightly but statistically significantly increased in HCh-II children compared to NCh children. Plasma and urinary nitrite and nitrate were similar in both groups. In HCh-II whole body DDAH activity is elevated as compared to NCh. HCh-II children treated with drugs for hypercholesterolemia had lower plasma ADMA levels than untreated HCh-II or NCh children, presumably via increased DDAH activity. Differences between treated and untreated HCh-II children were not due to differences in age. In conclusion, HCh-II children do not have elevated ADMA plasma levels, largely due to an apparent increase in DDAH activity. While this would tend to limit development of endothelial dysfunction, it is not clear whether this might be medication-induced or represent a primary change in HCh-II children.     

Regulation of redox forms of plasma thiols by albumin in multiple sclerosis after fasting and methionine loading test

Increases in plasma concentrations of total homocysteine (tHcy) have recently been reported in multiple sclerosis (MS) as the alteration of the methionine cycle for the onset of autoimmune diseases. Homocysteine (Hcy) and cysteine (Cys) are generated by the methionine cycle and transsulfuration reactions. Their plasma levels are subjected to complex redox changes by oxidation and thiol/disulfide (SH/SS) exchange reactions regulated by albumin. The methionine loading test (MLT) is a useful in vivo test to assay the functionality of the methionine cycle and transsulfuration reactions. Time courses of redox species of Cys, cysteinylglycine (CGly), Hcy, and glutathione have been investigated in plasma of MS patients versus healthy subjects after an overnight fasting, and 2, 4, and 6 h after an oral MLT (100 mg/kg body weight), to detect possible dysfunctions of the methionine cycle, transsulfuration reactions and alterations in plasma distribution of redox species. After fasting, the MS group showed a significant increase in cysteine-protein mixed disulfides (bCys) and total Cys (tCys). While plasma bCys and tCys in MS group remained elevated after methionine administration when compared to control, cystine (oxCys) increased significantly with respect to control. Although increased plasma concentrations of bCys and tCys at fasting might reflect an enhance of transsulfuration reactions in MS patients, this was not confirmed by the analysis of redox changes of thiols and total thiols after MLT. This study has also demonstrated that albumin-dependent SH/SS exchange reactions are a potent regulation system of thiol redox species in plasma.     

Manipulation of citrulline availability in humans

To determine whether circulating citrulline can be manipulated in vivo in humans, and, if so, whether citrulline availability affects the levels of related amino acids, nitric oxide, urinary citrulline, and urea nitrogen, 10 healthy volunteers were studied on 3 separate days: 1) under baseline conditions; 2) after a 24-h treatment with phenylbutyrate (0.36 g.kg(-1).day(-1)), a glutamine "trapping" agent; and 3) during oral L-citrulline supplementation (0.18 g.kg(-1).day(-1)), in randomized order. Plasma, erythrocyte (RBC), and urinary citrulline concentrations were determined by gas chromatography-mass spectrometry at 3-h intervals between 1100 and 2000 on each study day. Regardless of treatment, RBC citrulline was lower than plasma citrulline, with an RBC-to-plasma ratio of 0.60 +/- 0.04, and urinary citrulline excretion accounted for <1% of the citrulline load filtered by kidney. Phenylbutyrate induced an approximately 7% drop in plasma glutamine (P = 0.013), and 18 +/- 14% (P < 0.0001) and 19 +/- 17% (P < 0.01) declines in plasma and urine citrulline, respectively, with no alteration in RBC citrulline. Oral L-citrulline administration was associated with 1) a rise in plasma, urine, and RBC citrulline (39 +/- 4 vs. 225 +/- 44 micromol/l, 0.9 +/- 0.3 vs. 6.2 +/- 3.8 micromol/mmol creatinine, and 23 +/- 1 vs. 52 +/- 9 micromol/l, respectively); and 2) a doubling in plasma arginine level, without altering blood urea or urinary urea nitrogen excretion, and thus enhanced nitrogen balance. We conclude that 1) depletion of glutamine, the main precursor of citrulline, depletes plasma citrulline; 2) oral citrulline can be used to enhance systemic citrulline and arginine availability, because citrulline is bioavailable and very little citrulline is lost in urine; and 3) further studies are warranted to determine the mechanisms by which citrulline may enhance nitrogen balance in vivo in humans.     

Quantification of urinary o,o'-dityrosine, a biomarker for oxidative damage to proteins, by high performance liquid chromatography with triple quadrupole tandem mass spectrometry. A comparison with ion-trap tandem mass spectrometry

We recently described an isotope dilution reversed-phase liquid chromatography-atmospheric pressure chemical ionization-ion-trap-tandem mass spectrometry (HPLC-APCI-MS/MS) method for the quantitative determination of oxidized amino acids in human urine, including o,o'-dityrosine, a specific marker of protein oxidation. In the present study, we investigated the possibility to use a triple quadrupole instrument for the analysis of this biomarker in urine. The two instruments were compared in terms of sensitivity, specificity and reproducibility. Results showed that the triple quadrupole instrument reaches 2.5-fold higher sensitivity (LOD=0.01 microM) compared to the previously used ion-trap instrument. Precision of the present assay is as follows: in-day variation is 4.6% and inter-day variation is 17%. The currently developed method was applied to a group of smoker urine samples. The mean urinary o,o'-dityrosine concentration was 0.08+/-0.01 microM. Expressed per urinary creatinine concentration, this corresponds to 10.1+/-0.4 micromol/mol creatinine. This is comparable to the previously reported values of 5.8+/-0.3 micromol/mol creatinine in non-smokers night-time urines, and 12.3+/-5 micromol/mol creatinine in day-time urines measured by the ion-trap instrument.     

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.     

Comparison of plasma and urinary phytoestrogens in Japanese and Finnish women by time-resolved fluoroimmunoassay

Time-resolved fluoroimmunoassays (TR-FIA), with europium labeled phytoestrogens as tracers, were developed for the quantitative measurement of genistein, daidzein and enterolactone in plasma and urine for the purpose of screening large populations and studies on possible correlation between the values in biological fluids and the risk of western diseases. The mean values of the three phytoestrogens in plasma as determined by TR-FIA were similar to those obtained by gas chromatography-mass spectrometry (GC-MS). The urinary excretion levels of total individual phytoestrogens were higher than those obtained by GC-MS, with the exception of the daidzein values. However, comparing the assay results obtained by the present method and those obtained by GC-MS, a strong correlation was evident (r = 0.87 - 0.99, p < 0.001). We measured plasma levels of genistein, daidzein and enterolactone in 111 healthy Japanese women The mean and median levels of genistein were 406.8 and 306.3 nmol/l, respectively, and those of daidzein were 118.4 and 76.8 nmol/l, respectively. These levels are higher than those reported for Americans and Western Europeans. Isoflavone intake as calculated from dietary records (genistein: mean, 86.5 mircomol/day and daidzein: mean, 57.4 micromol/day) was correlated with the plasma concentrations observed (genistein: r = 0.287, p < 0.01 and daidzein: r = 0.313, p < 0.01). Plasma enterolactone levels were low in Japanese women (mean, about 10 nmol/l). The levels of urinary excretions of genistein, daidzein were also measured and it was found that, in the majority, the levels ranged between 5-25 and 5-50 micromol/24 h, respectively. In contrast, healthy Finnish women showed very low values of isoflavones (below 10 nmol/l in plasma (n = 87) and below 0.6 micromol/24 h in urine (n = 126) for both compounds) and high levels of enterolactone in both plasma and urine (plasma: mean, 25 nmol/l and urine: majority range, 1-7 micromol/24 h).     

gamma-Tocopherol biokinetics and transformation in humans

BACKGROUND: The uptake and biotransformation of gamma-tocopherol (gamma-T) in humans is largely unknown. Using a stable isotope method we investigated these aspects of gamma-T biology in healthy volunteers and their response to gamma-T supplementation. METHODS: A single bolus of 100 mg of deuterium labeled gamma-T acetate (d(2)-gamma-TAC, 94% isotopic purity) was administered with a standard meal to 21 healthy subjects. Blood and urine (first morning void) were collected at baseline and a range of time points between 6 and 240 h post-supplemetation. The concentrations of d(2) and d(0)-gamma-T in plasma and its major metabolite 2,7,8-trimethyl-2-(b-carboxyethyl)-6-hydroxychroman (-gamma-CEHC) in plasma and urine were measured by GC-MS. In two subjects, the total urine volume was collected for 72 h post-supplementation. The effects of gamma-T supplementation on alpha-T concentrations in plasma and alpha-T and gamma-T metabolite formation were also assessed by HPLC or GC-MS analysis. RESULTS: At baseline, mean plasma alpha-T concentration was approximately 15 times higher than gamma-T (28.3 vs. 1.9 micromol/l). In contrast, plasma gamma-CEHC concentration (0.191 micromol/l) was 12 fold greater than alpha-CEHC (0.016 micromol/l) while in urine it was 3.5 fold lower (0.82 and 2.87 micromol, respectively) suggesting that the clearance of alpha-CEHC from plasma was more than 40 times that of gamma-CEHC. After d(2)-gamma-TAC administration, the d(2) forms of gamma-T and gamma-CEHC in plasma and urine increased, but with marked inter-individual variability, while the d(0) species were hardly affected. Mean total concentrations of gamma-T and gamma-CEHC in plasma and urine peaked, respectively, between 0-9, 6-12 and 9-24 h post-supplementation with increases over baseline levels of 6-14 fold. All these parameters returned to baseline by 72 h. Following challenge, the total urinary excretion of d(2)-gamma-T equivalents was approximately 7 mg. Baseline levels of gamma-T correlated positively with the post-supplementation rise of (d(0) + d(2)) - gamma - T and gamma-CEHC levels in plasma, but correlated negatively with urinary levels of (d(0) + d(2))-gamma-CEHC. Supplementation with 100 mg gamma-TAC had minimal influence on plasma concentrations of alpha-T and alpha-T-related metabolite formation and excretion. CONCLUSIONS: Ingestion of 100mg of gamma-TAC transiently increases plasma concentrations of gamma-T as it undergoes sustained catabolism to CEHC without markedly influencing the pre-existing plasma pool of gamma-T nor the concentration and metabolism of alpha-T. These pathways appear tightly regulated, most probably to keep high steady-state blood ratios alpha-T to gamma-T and gamma-CEHC to alpha-CEHC.     

Comparison of 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels using mass spectrometer and urine albumin creatinine ratio as a predictor of development of diabetic nephropathy

Abstract Background. Measurement of urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) has recently become more popular as a means of assessing oxidative stress in the human body. The aim of this study is to compare the levels of urine 8-OHdG in patients with type 2 diabetes with and without nephropathy and to evaluate its role as a biochemical marker for distinguishing these patients from healthy and patients without complications. Methods. For this purpose, 52 patients with type 2 diabetes mellitus (32 with nephropathy (DMN), 20 without nephropathy (DM)) and 20 healthy control subjects (C) were included in this study. The urine concentrations of 8-OHdG were measured by modified LC-MS/MS method and compared with the first morning voiding urine albumin/creatinine ratio (UACR) and HbA1c values of the same patients. Results. The concentrations of urine 8-OHdG in DMN and DM patients were higher than those of the control subjects (3.47?±?0.94, 2.92?±?1.73, 2.1?±?0.93 nmol/mol creatinine, respectively). But there was no statistical difference between DMN and DM (p =?0.115). There is significant correlation between urinary 8-OHdG and UACR (r =?0.501, p 相似文献   

An improved method for the measurement of urinary and plasma F2-isoprostanes using gas chromatography-mass spectrometry

We have developed an improved method for the measurement of F2-isoprostanes using stable isotope dilution capillary gas chromatography/electron capture negative ionization mass spectrometry (GC-ECNI-MS). The F2-isoprostane family consists of a series of chemically stable prostaglandin F2 (PGF2)-like compounds generated during peroxidation of arachidonic acid in phospholipids. There is evidence that measurement of F2-isoprostanes represents a reliable and useful index of lipid peroxidation and oxidant stress in vivo. Furthermore, 8-epi-PGF2alpha, which is one of the more abundant F2-isoprostanes, is biologically active, being a potent mitogen and vasoconstrictor of rat and rabbit lung and kidney, as well as a partial agonist of platelet aggregation. Measurement of F2-isoprostanes in biological samples is complex and has involved methods which utilize multiple chromatographic steps, including separation by thin-layer chromatography, leading to poor sample recovery. We now present an improved method for the measurement of plasma and urinary F2-isoprostanes using a combination of silica and reverse-phase extraction cartridges, high-performance liquid chromatography (HPLC), and GC-ECNI-MS. Different approaches to the derivatization of the F2-isoprostanes prior to GC-ECNI-MS are also addressed. The overall recovery of F2-isoprostanes is improved (approx 70% for urine) and the within and between assay reproducibility is 6.7% (n = 23) and 3.7% (n = 3), respectively. The mean urinary excretion of F2-isoprostanes in eight healthy males was 365 +/- 5 pmol/mmol creatinine and in three smokers 981 +/- 138 pmol/mmol creatinine. The mean total (free + esterified) plasma F2-isoprostane concentration was 952 +/- 38 pmol/liter, with a within and between assay reproducibility of 8% (n = 13) and 5.6% (n = 3), respectively. This improved method for the measurement of F2-isoprostanes represents a significant advance in terms of the rapidity and yield in the purification of biological samples. The inclusion of HPLC separation enables improved analysis of F2-isoprostanes by GC-MS. This methodology will assist in defining the role of F2-isoprostanes as in vivo markers of oxidant stress in clinical and experimental settings.     

A LC-MS/MS method for the specific, sensitive, and simultaneous quantification of 5-aminolevulinic acid and porphobilinogen

Accurate determinations of 5-aminolevulinic acid (ALA) and porphobilinogen (PBG) in physiologic fluids are required for the diagnosis and therapeutic monitoring of acute porphyrias. Current colorimetric methods are insensitive and over-estimate ALA and PBG due to poor specificity, while LC-MS/MS methods increase sensitivity, but have limited matrices. An LC-MS/MS method was developed to simultaneously determine ALA and PBG concentrations in fluids or tissues which were solid phase extracted, butanol derivatized, and quantitated by selective reaction monitoring using (13)C(5), (15)N-ALA and 2,4-(13)C(2)-PBG internal standards. ALA was separated from interfering compounds on a reverse phase C8-column. For ALA and PBG, the matrix effects (87.3-105%) and process efficiencies (77.6-97.8% and 37.2-41.6%, respectively) were acceptable in plasma and urine matrices. The assay was highly sensitive for ALA and PBG (LLOQ=0.05 μM with 25 μL urine or 100 μL plasma), and required ～4 h from extraction to results. ALA and PBG accuracy ranged from 88.2 to 110% (n=10); intra- and inter-assay coefficients of variations were <10% for urine and plasma. In clinical applications, patients with mutation-confirmed acute porphyrias had normal to slightly increased urinary ALA and PBG levels when asymptomatic, and high levels during acute attacks, which decreased with hemin therapy. In AIP mice, baseline ALA and PBG levels in urine, plasma, and liver were increased after phenobarbital induction 28-/63-, 42-/266-, and 13-/316-fold, respectively. This LC-MS/MS method is rapid, specific, highly sensitive, accurate, and simultaneously measures ALA and PBG in urine, plasma, and tissues permitting porphyria clinical diagnoses, therapeutic monitoring, and research.     

Urinary deoxynivalenol (DON) and zearalenone (ZEA) as biomarkers of DON and ZEA exposure of pigs

Four diets contaminated with 1.1 to 5.0 mg/kg deoxynivalenol (DON) and 0.4 to 2.4 mg/kg zearalenone (ZEA) were fed to four groups of six growing Large White pigs. Urine samples were collected after 3 to 4 days and again after 6 to 7 days on the diets. On each sampling day, half of the animals were sampled in the morning, after an 8-h fast, and the other half were sampled in the afternoon, after 7 h of ad libitum access to feed. The urinary concentrations of DON, DON-glucuronide, DON-3-sulphate, de-epoxy-DON, as well as of ZEA, ZEA-14-glucuronide, α-zearalenol and α-zearalenol-14-glucuronide, analysed using LC-MS/MS, were used to calculate urinary DON and ZEA equivalent concentrations (DONe and ZEAe). The urinary concentration of DONe (P?<?0.001), but not of ZEAe (P?=?0.31), was lower in the fasted than that in the fed animals. The urinary DONe/creatinine and ZEAe/creatinine ratios were highly correlated with DON and ZEA intake per kg body weight the day preceding sampling (r?=?0.76 and 0.77; P?<?0.001). The correlations between DON intake during the 7 h preceding urine sampling in the afternoon and urinary DONe/creatinine ratio (r?=?0.88) as well as between mean ZEA intake during 3 days preceding urine sampling and urinary ZEAe/creatinine ratio (r?=?0.84) were even higher, reflecting the plasma elimination half-time of several hours for DON and of more than 3 days for ZEA. ZEAe analysed in enzymatically hydrolysed urine using an ELISA kit was highly correlated with the LC-MS/MS data (r?=?0.94). The urinary DONe and ZEAe to creatinine ratios, analysed in pooled urine samples of several pigs fed the same diet, can be used to estimate their exposure to DON and ZEA.     

The methylenetetrahydrofolate reductase 677C-->T polymorphism as a modulator of a B vitamin network with major effects on homocysteine metabolism

Folates are carriers of one-carbon units and are metabolized by 5,10-methylenetetrahydrofolate reductase (MTHFR) and other enzymes that use riboflavin, cobalamin, or vitamin B6 as cofactors. These B vitamins are essential for the remethylation and transsulfuration of homocysteine, which is an important intermediate in one-carbon metabolism. We studied the MTHFR 677C-->T polymorphism and B vitamins as modulators of one-carbon metabolism in 10,601 adults from the Norwegian Colorectal Cancer Prevention (NORCCAP) cohort, using plasma total homocysteine (tHcy) as the main outcome measure. Mean concentrations of plasma tHcy were 10.4 micromol/liter, 10.9 micromol/liter, and 13.3 micromol/liter in subjects with the CC (51%), CT (41%), and TT (8%) genotypes, respectively. The MTHFR 677C-->T polymorphism, folate, riboflavin, cobalamin, and vitamin B6 were independent predictors of tHcy in multivariate models (P<.001), and genotype effects were strongest when B vitamins were low (P相似文献   

Determination of urinary malondialdehyde by isotope dilution LC-MS/MS with automated solid-phase extraction: a cautionary note on derivatization optimization

A highly sensitive quantitative LC-MS/MS method was developed for measuring urinary malondialdehyde (MDA). With the use of an isotope internal standard and online solid-phase extraction, urine samples can be directly analyzed within 10 min after 2,4-dinitrophenylhydrazine (DNPH) derivatization. The detection limit was estimated as 0.08 pmol. This method was further applied to assess the optimal addition of DNPH for derivatization and to measure urinary MDA in 80 coke oven emission (COE)-exposed and 67 nonexposed workers. Derivatization optimization revealed that to achieve complete derivatization reaction, an excess of DNPH is required (DNPH/MDA molar ratio: 893-8929) for urine samples that is about 100 times higher than that of MDA standard solutions (molar ratio: 10-80). Meanwhile, the mean urinary concentrations of MDA in COE-exposed workers were significantly higher than those in nonexposed workers (0.23±0.17 vs 0.14±0.05 μmol/mmol creatinine, P<0.005). Urinary MDA concentrations were also significantly associated with the COE (P<0.005) and smoking exposure (P<0.05). Taken together, this method is capable of routine high-throughput analysis and accurate quantification of MDA and would be useful for assessing the whole-body burden of oxidative stress. Our findings, however, raise the issue that derivatization optimization should be performed before it is put into routine biological analysis.     

Quantification of free and total sialic acid excretion by LC-MS/MS

BACKGROUND: The main purpose for measuring urinary free sialic acid (FSA) is to diagnose sialic acid (SA) storage diseases. Elevated amounts of conjugated sialic acid (CSA) are observed in several diseases indicating the need to quantify CSA as well. A LC-MS/MS method for quantification of FSA and total sialic acid (TSA) in urine is developed and validated. METHODS: FSA is analyzed directly after filtration of urine samples. For determination of TSA an enzymatic (neuraminidase) and a chemical (acid) hydrolysis were compared. 13C3-sialic acid was used as internal standard. LC-MS/MS was performed in negative electrospray ionisation mode with multiple reaction monitoring of transitions m/z 308.2-->87.0 (SA) and m/z 311.2-->90.0 (13C3-SA). CSA was calculated by subtracting FSA from TSA. RESULTS: Limit of detection for FSA and TSA was 0.3 and 1.7 micromol/L, respectively. Limit of quantification for FSA and TSA was 1.0 and 5.0 micromol/L. Intra- and inter-assay variations of FSA were 4.6% and 6.6% (n=10) for FSA and 6.5% and 3.6% (n=10) for TSA. Linearity was tested till 7800 micromol/L (r2=0.9998). Values of SA analyzed after neuraminidase- or acid hydrolysis treatment were comparable. Urine samples from patients with inborn errors of SA (related) metabolism were analyzed and compared with age-related reference values. CONCLUSION: A method has been developed for routine determination of urinary FSA and TSA. The method is rapid, specific, robust and sensitive. Age-related reference values for FSA, TSA and CSA were determined and improved diagnostic efficacy.     

GC–MS determination of creatinine in human biological fluids as pentafluorobenzyl derivative in clinical studies and biomonitoring: Inter-laboratory comparison in urine with Jaffé, HPLC and enzymatic assays

In consideration of its relatively constant urinary excretion rate, creatinine in urine is a useful biochemical parameter to correct the urinary excretion rate of endogenous and exogenous biomolecules. Assays based on the reaction of creatinine and picric acid first reported by Jaffé in 1886 still belong to the most frequently used laboratory approaches for creatinine measurement in urine. Further analytical methods for creatinine include HPLC–UV, GC–MS, and LC–MS and LC–MS/MS approaches. In the present article we report on the development, validation and biomedical application of a new GC–MS method for the reliable quantitative determination of creatinine in human urine, plasma and serum. This method is based on the derivatization of creatinine (d₀-Crea) and the internal standard [methyl-trideutero]creatinine (d₃-Crea) with pentafluorobenzyl (PFB) bromide in the biological sample directly or after dilution with phosphate buffered saline, extraction of the reaction products with toluene and quantification in 1-μl aliquots of the toluene extract by selected-ion monitoring of m/z 112 for d₀-Crea-PFB and m/z 115 for d₃-Crea-PFB in the electron-capture negative-ion chemical ionization mode. The limit of detection of the method is 100 amol of creatinine. In an inter-laboratory study on urine samples from 100 healthy subjects, the GC–MS method was used to test the reliability of currently used Jaffé, enzymatic and HPLC assays in clinical and occupational studies. The results of the inter-laboratory study indicate that all three tested methods allow for satisfactory quantification of creatinine in human urine. The GC–MS method is suitable for use as a reference method for urinary creatinine in humans. In serum, creatine was found to contribute to creatinine up to 20% when measured by the present GC–MS method. The application of the GC–MS method can be extended to other biological samples such as saliva.     

Detection and quantification of α-keto-δ-(N(G),N(G)-dimethylguanidino)valeric acid: a metabolite of asymmetric dimethylarginine

Nitric oxide is an ubiquitary cell signaling substance. Its enzymatic production rate by nitric oxide synthase is regulated by the concentrations of the substrate L-arginine and the competitive inhibitor asymmetric dimethylarginine (ADMA). A newly recognized elimination pathway for ADMA is the transamination to α-keto-δ-(N(G),N(G)-dimethylguanidino)valeric acid (DMGV) by the enzyme alanine-glyoxylate aminotransferase 2 (AGXT2). This pathway has been proven to be relevant for nitric oxide regulation, but up to now no method exists for the determination of DMGV in biological fluids. We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of DMGV. D(6)-DMGV was used as internal standard. Samples were purified online by column switching, and separation was achieved on a porous graphitic carbon column. The calibration was linear over ranges of 10 to 200 nmol/L for plasma and 0.1 to 20 μmol/L for urine. The intra- and interday accuracies and precisions in plasma and urine were better than 10%. In plasma samples, DMGV was present in concentrations between 19.1 and 77.5 nmol/L. In urine samples, concentrations between 0.0114 and 1.03 μmol/mmol creatinine were found. This method can be used as a tool for the scientific investigation of the ADMA conversion to DMGV via the enzyme AGXT2.     

Quantification of acetylcholine, choline, betaine, and dimethylglycine in human plasma and urine using stable-isotope dilution ultra performance liquid chromatography-tandem mass spectrometry

Disorders in choline metabolism are related to disease conditions. We developed a stable-isotope dilution ultra performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of acetylcholine (ACh), betaine, choline, and dimethylglycine (DMG). We used this method to measure concentrations of the analytes in plasma and urine in addition to other biological fluids after a protein precipitation by acetonitrile. The detection limits were between 0.35 nmol/L (for ACh in urine) and 0.34 μmol/L (for betaine in urine). ACh concentrations were not detectable in plasma. Intraassay and interassay coefficient of variation (CVs) were all <10.0% in biological fluids, except for DMG in cerebrospinal fluid (CV=12.44%). Mean recoveries in urine pool samples were between 99.2% and 103.9%. The urinary excretion of betaine, choline, and DMG was low, with approximately 50.0% higher excretion of choline in females compared to males. Median urinary excretion of ACh were 3.44 and 3.92 μmol/mol creatinine in males and females, respectively (p=0.689). Plasma betaine concentrations correlated significantly with urinary excretions of betaine (r=0.495, p=0.027) and choline (r=0.502, p=0.024) in females. Plasma choline concentrations correlated significantly with urinary excretion of ACh in males (r=0.419, p=0.041) and females (r=0.621, p=0.003). The new method for the simultaneous determination of ACh, betaine, choline, and DMG is sensitive, precise, and fast enough to be used in clinical investigations related to the methylation pathway.