Quantification of the major urinary metabolite of PGE2 by a liquid chromatographic/mass spectrometric assay: determination of cyclooxygenase-specific PGE2 synthesis in healthy humans and those with lung cancer

Prostaglandin (PG)E2 is a major cyclooxygenase (COX) product that is important in human physiology and pathophysiology. Quantification of systemic PG production in humans is best assessed by measuring excreted urinary metabolites. Accurate and easy-to-perform assays to quantify the major urinary metabolite of PGE2, 11alpha-hydroxy-9,15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid (PGE-M), do not exist. We now report the development of a robust and facile method to measure urinary PGE-M excretion in humans using stable isotope dilution techniques employing liquid chromatography/tandem mass spectrometry (LC/MS/MS). Concentrations of the metabolite in urine from healthy humans are nearly twofold greater in men than in women (10.4+/-1.5 vs. 6.0+/-0.7 ng/mg creatinine). Levels of PGE-M in healthy humans are suppressed significantly not only by the nonselective COX inhibitor ibuprofen but also by the COX-2 selective inhibitor rofecoxib, suggesting that the majority of PGE2 formed in vivo is derived from COX-2. Increased COX-2 expression and increased PGE2 production are associated with malignancy. Levels of PGE-M were found to be greatly increased in humans with unresectable non-small cell cancer of the lung, and this increase is dramatically reduced by administration of the COX-2 inhibitor celecoxib, implying that COX-2 contributes significantly to the overproduction of PGE2. In summary, quantification of PGE-M using LC/MS/MS provides a facile and accurate method to assess PGE2 formation in human physiological and pathophysiological processes.     

High-throughput and simultaneous measurement of homocysteine and cysteine in human plasma and urine by liquid chromatography-electrospray tandem mass spectrometry

Total homocysteine (tHcy) and cysteine (tCys) concentrations in biological fluids are routinely used in the clinical diagnosis of genetic and metabolic diseases, and this necessitates the development of rapid and sensitive methods for quantification. Liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) was used to measure tHcy and tCys in 23 plasma and 21 urine samples from healthy adults and 14 urine samples from healthy children. The results were compared with a standard high-performance liquid chromatography (HPLC) method. The coefficient of variation (CV) for the LC-MS/MS method ranged from 2.9% to 6.1% for the intraassay and 4.8% to 6.4% for the interassay. Mean recoveries were close to 100% for both plasma and urinary tHcy and tCys. The mean plasma tHcy and tCys concentrations in healthy adults were 8.62 and 261.40 micromol/L, respectively. The mean urinary tHcy and tCys in adults were 0.98 and 22.60 micromol/mmol creatinine, respectively. The mean urinary tHcy and tCys in children were 1.17 and 27.43 micromol/mmol creatinine, respectively. Bland-Altman difference plots of method comparison between LC-MS/MS and HPLC showed good agreement in plasma and urinary tHcy and tCys concentrations. Our method is suitable for rapid measurements, and the reported urinary values in children will help to develop a pediatric reference range for clinical use.     

Evaluation of enzyme-linked immunosorbent assay and liquid chromatography-tandem mass spectrometry methodology for the analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine in saliva and urine

While ELISA is a frequently used means of assessing 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) in biological fluids, differences in baseline urinary 8-oxodG levels, compared to chromatographic techniques, have raised questions regarding the specificity of immunoassays. Recently, ELISA of salivary 8-oxodG has been used to report on periodontal disease. We compared salivary 8-oxodG levels, determined by two commercial ELISA kits, to liquid chromatography-tandem mass spectrometry (LC-MS/MS) with prior purification using solid-phase extraction. While values were obtained with both ELISA kits, salivary 8-oxodG values were below or around the limit of detection of our LC-MS/MS assay. As the limit of detection for the LC-MS/MS procedure is much lower than ELISA, we concluded that the assessment of salivary 8-oxodG by ELISA is not accurate. In contrast to previous studies, ELISA levels of urinary 8-oxodG (1.67 ± 0.53 pmol/μmol creatinine) were within the range reported previously only for chromatographic assays, although still significantly different than LC-MS/MS (0.41 ± 0.39 pmol/μmol creatinine; p = 0.002). Furthermore, no correlation with LC-MS/MS was seen. These results question the ability of ELISA approaches, at present, to specifically determine absolute levels of 8-oxodG in saliva and urine. Ongoing investigation in our laboratories aims to identify the basis of the discrepancy between ELISA and LC-MS/MS.     

Evaluation of enzyme-linked immunosorbent assay and liquid chromatography–tandem mass spectrometry methodology for the analysis of 8-oxo-7,8-dihydro-2′-deoxyguanosine in saliva and urine

While ELISA is a frequently used means of assessing 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) in biological fluids, differences in baseline urinary 8-oxodG levels, compared to chromatographic techniques, have raised questions regarding the specificity of immunoassays. Recently, ELISA of salivary 8-oxodG has been used to report on periodontal disease. We compared salivary 8-oxodG levels, determined by two commercial ELISA kits, to liquid chromatography-tandem mass spectrometry (LC-MS/MS) with prior purification using solid-phase extraction. While values were obtained with both ELISA kits, salivary 8-oxodG values were below or around the limit of detection of our LC-MS/MS assay. As the limit of detection for the LC-MS/MS procedure is much lower than ELISA, we concluded that the assessment of salivary 8-oxodG by ELISA is not accurate. In contrast to previous studies, ELISA levels of urinary 8-oxodG (1.67 ± 0.53 pmol/μmol creatinine) were within the range reported previously only for chromatographic assays, although still significantly different than LC-MS/MS (0.41 ± 0.39 pmol/μmol creatinine; p = 0.002). Furthermore, no correlation with LC-MS/MS was seen. These results question the ability of ELISA approaches, at present, to specifically determine absolute levels of 8-oxodG in saliva and urine. Ongoing investigation in our laboratories aims to identify the basis of the discrepancy between ELISA and LC-MS/MS.     

A simple and robust UPLC-SRM/MS method to quantify urinary eicosanoids

Eicosanoids are key mediators and regulators of inflammation and oxidative stress often used as biomarkers for diseases and pathological conditions such as cardiovascular and pulmonary diseases and cancer. Analytically, comprehensive and robust quantification of different eicosanoid species in a multi-method approach is problematic because most of these compounds are relatively unstable and may differ in their chemical properties. Here we describe a novel ultra-performance liquid chromatography-selected reaction monitoring mass spectroscopy (UPLC-SRM/MS) method for simultaneous quantification of key urinary eicosanoids, including the prostaglandins (PG) tetranor PGE-M, 8-iso-, and 2,3-dinor-8-iso-PGF_2α; the thromboxanes (TXs) 11-dehydro- and 2,3-dinor-TXB₂; leukotriene E₄; and 12-hydroxyeicosatetraenoic acid. In contrast to previous methods, which used time-consuming and complex solid phase extraction, we prepared samples with a simple liquid/liquid extraction procedure. Because collision-induced dissociation produced characteristic product ions for all analytes, no derivatization step for SRM/MS analysis was necessary. Analytes were separated with a short UPLC reversed-phase column (1.7 µm particles), allowing shorter run times than conventional HPLC columns. The method was validated and applied to human urine samples showing excellent precision, accuracy, detection limits, and robustness. In summary, the developed method allows robust and sensitive profiling of urinary eicosanoid species, making it a useful and valuable tool for biomarker profiling in clinical/toxicological studies.     

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.     

Quantification of the major urinary metabolite of the E prostaglandins by mass spectrometry: Evaluation of the method's application to clinical studies

Measurement of 7α-hydroxy-5,11-diketotetranorprostane-1,16-dioic acid, (PGE-M), the major urinary metabolite of prostaglandin E₁ and E₂ in man provides a useful indicator to monitor prostaglandin biosynthesis. For quantitative analysis of this prostaglandin metabolite the stable-isotope dilution technique of selected ion monitoring (SIM) is employed using gas-liquid chromatography-mass spectrometry. The preparation of the (D₃-methyloxime), -methyl ester of PGE-M containing a tritium tracer in position 2 which was used as internal standard for the SIM method is described. The synthesis of this internal standard includes the biosynthetic conversion of 11-hydroxy-9,15-diketoprostanoic acid to PGE-M by the rabbit. The intra-assay coefficient of variation of this SIM method ranged between 4.0 to 6.7 percent. The recovery of authentic, underivatized PGE-M added to urine was 93 ± 3% (mean ± SEM, n=17).The levels of PGE-M excreted in urine were higher (p<0.001) in males than in females (15.2 ± 1.9 μg/24 hours (n=24) and 3.3 ± 0.3 μg/24 hours (n=17), respectively). These levels were in close agreement with values published previously. No significant difference in excretion of PGE-M between the sexes was observed in the pre-pubertal age-group (male: 2.9 ± 0.8 μg/24 hours, n=5; female: 3.1 ± 0.9 μg/24 hours, n=5) or in the age-group of 45–80 years (male: 9.3 ± 1.1 μg/24 hours, n=21; female: 7.3 ± 0.9 μg/24 hours, n=12). The amount of PGE-M excreted decreased significantly after administration of indomethacin or acetyl salicylic acid in therapeutic doses. The concomitant reduction of the urinary excretion of PGE-M (68 to 85% decrease) and prostaglandin E (73 to 100% decrease) after indomethacin treatment in each case (n=8) is evidence that a diminished urinary PGE-M output reflects a decrease in prostaglandin E biosynthesis.     

8-Oxo-2'-deoxyguanosine as a biomarker of tobacco-smoking-induced oxidative stress

7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dGuo) is a useful biomarker of oxidative stress. However, its analysis can be challenging because 8-oxo-dGuo must be quantified in the presence of dGuo, without artifactual conversion to 8-oxo-dGuo. Urine is the ideal biological fluid for population studies, because it can be obtained noninvasively and it is less likely that artifactual oxidation of dGuo can occur because of the relatively low amounts that are present compared with hydrolyzed DNA. Stable isotope dilution liquid chromatography-selected reaction monitoring/mass spectrometry (LC-SRM/MS) with 8-oxo-[(15)N(5)]dGuo as internal standard provided the highest possible specificity for 8-oxo-dGuo analysis. Furthermore, artifact formation was determined by addition of [(13)C(10)(15)N(5)]dGuo and monitoring of its conversion to 8-oxo-[(13)C(10)(15)N(5)]dGuo during the analytical procedure. 8-Oxo-dGuo concentrations were normalized for interindividual differences in urine flow by analysis of creatinine using stable isotope dilution LC-SRM/MS. A significant increase in urinary 8-oxo-dGuo was observed in tobacco smokers compared with nonsmokers either using simple urinary concentrations or after normalization for creatinine excretion. The mean levels of 8-oxo-dGuo were 1.65ng/ml and the levels normalized to creatinine were 1.72μg/g creatinine. Therefore, stable isotope dilution LC-SRM/MS analysis of urinary 8-oxo-dGuo complements urinary isoprostane (isoP) analysis for assessing tobacco-smoking-induced oxidative stress. This method will be particularly useful for studies that employ polyunsaturated fatty acids, in which a reduction in arachidonic acid precursor could confound isoP measurements.     

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.     

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   

Excretion of primary prostanoids and their metabolites during acute volume expansion

Simultaneous determination of urinary excretion rates of primary unmetabolized prostanoids and their enzymatic metabolites were performed by gas chromatography-mass spectrometry (GC/MS) or tandem mass spectrometry (GC/MS/MS). Changes in kidney function were induced by acute (4 h) volume expansion. Despite marked changes in urine flow, GFR, urinary pH, osmolality, sodium and potassium excretion, only a insignificant or transient rise in the enzymatic prostanoid metabolites (2,3-dinor-6-keto-PGF1 alpha, PGE-M, 2,3-dinor-TxB2 and 11-dehydro-TxB2) was observed. The excretion rates of the primary prostanoids were elevated in parallel with the rise in urine flow: PGE2 rose (p less than 0.05) from 14.2 +/- 4.0 to 86.2 +/- 20.7, PGF2 alpha from 60.0 +/- 4.9 to 119.8 +/- 24.0, 6-keto-PGF2 alpha from 7.2 +/- 1.3 to 51.5 +/- 17.0, and TxB2 from 11.2 +/- 3.3 to 13.6 +/- 3.6 ng/h/1.73 m2 (means +/- SEM) at the maximal urine flow. Except for 6-keto-PGF1 alpha and TxB2, this rise in urinary prostanoid levels was only transient despite a sustained fourfold elevated urine flow. We conclude that urine flow rate acutely affect urine prostanoid excretion rates, however, over a prolonged period of time these effects are not maintained. The present data support the concept that urinary levels of primary prostanoids mainly reflect renal concentrations whereas those of enzymatic metabolites reflect systemic prostanoid activity. From the excretion pattern of TxB2 one can assume that this prostanoid represents renal as well as systemic TxA2 activity.     

Detection of DNA adduct 8-hydroxy-2&#x27;-deoxyguanosine (8-OHdG) as a toxicity bioindicator to the effects of nickel on Ni-Cr alloy prosthesis users

Previous studies have suggested that exposure to Ni from Ni-Cr alloys can affect the human body through oxidative stress. The present study discusses the effect of nickel from Ni-Cr alloy prostheses on the formation of DNA Adduct 8-Hydroxy-2′-Deoxyguanosine (8-OHdG), evaluated based on creatinine and 8-OHdG concentrations in urine, determined with LC-MS/MS, for a Ni-Cr alloy user group and a never-user control group. The mean creatinine and 8-OHdG concentrations were not significantly different between the test groups, although highest levels were observed for the in the Ni-Cr user group. It is suggested that samples with relatively high creatinine and/or 8-OHdG levels are further studied in more detail for stability of concentrations and for the effect of contributing factors.     

Analysis of urinary 8-oxo-7,8-dihydro-purine-2'-deoxyribonucleosides by LC-MS/MS and improved ELISA

Non-invasive monitoring of oxidative stress is highly desirable. Urinary 7,8-8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is a biologically relevant and convenient analytical target. However, immunoassays can over-estimate levels of urinary 8-oxodG. Measurement of more than one DNA oxidation product in urine would be advantageous in terms of mechanistic information. Urines samples were analysed for 8-oxodG by solid-phase extraction/LC-MS/MS and ELISA. The solid-phase extraction/LC-MS/MS assay was also applied to the analysis of urinary 7,8-dihydro-8-oxo-2'-deoxyadenosine (8-oxodA). Concurring with previous reports, urinary 8-oxodG measured by ELISA was significantly higher than levels measured by LC-MS/MS. However, apparent improvement in the specificity of the commercially available Japanese Institute for the Control of Ageing (JaICA) ELISA brought mean LC-MS/MS and ELISA measurements of urinary 8-oxodG into agreement. Urinary 8-oxodA was undetectable in all urines, despite efficient recovery by solid phase extraction. Exploitation of the advantages of ELISA may be enhanced by a simple modification to the assay procedure, although chromatographic techniques still remain the 'gold standard' techniques for analysis of urinary 8-oxodG. Urinary 8-oxodA is either not present or below the limit of detection of the instrumentation.     

Determination of aldosterone in serum by liquid chromatography-tandem mass spectrometry

Measurement of serum aldosterone is clinically important in the diagnosis of hypertension. While isotope dilution gas chromatography-mass spectrometry (ID-GC-MS) provides reliable results, it requires derivatization and is lengthy and time-consuming. Detection by liquid chromatography-mass spectrometry (LC-MS) is a potentially superior method. The analysis utilizes 0.5mL of serum. The samples were extracted with dichloromethane-ether. The extract was evaporated to dryness and aldosterone was analyzed by LC-MS/MS operating in the negative mode ESI after separation on a reversed-phase column. Aldosterone was also measured by RIA. The calibration curves for analysis of serum aldosterone exhibited consistent linearity and reproducibility in the range of 60-3000pmol/L. Interassay CVs were 4.3-7.5% at aldosterone concentrations of 97-993pmol/L. The lower limit of quantitation (LOQ) was 30pmol/L (signal to noise ratio=10). The mean recovery of the analyte added to serum ranged from 95 to 102%. The regression equation by LC-MS/MS (x) and RIA (y) method was: y=1.33x+185 (r=0.95; n=124). Sensitivity and specificity of the LC-MS/MS method for serum aldosterone offer advantages over GC-MS by eliminating derivatization. The novel method is rapid, reliable and simple to perform with a routine LC-MS/MS spectrometer. The sensitivity is adequate for patient samples. Aldosterone concentrations reported by nonextraction RIA were consistently higher than those produced by LC-MS/MS.     

Analysis of urinary 8-oxo-7,8-dihydro-purine-2’-deoxyribonucleosides by LC-MS/MS and improved ELISA

Non-invasive monitoring of oxidative stress is highly desirable. Urinary 7,8-8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) is a biologically relevant and convenient analytical target. However, immunoassays can over-estimate levels of urinary 8-oxodG. Measurement of more than one DNA oxidation product in urine would be advantageous in terms of mechanistic information. Urines samples were analysed for 8-oxodG by solid-phase extraction/LC-MS/MS and ELISA. The solid-phase extraction/LC-MS/MS assay was also applied to the analysis of urinary 7,8-dihydro-8-oxo-2’-deoxyadenosine (8-oxodA). Concurring with previous reports, urinary 8-oxodG measured by ELISA was significantly higher than levels measured by LC-MS/MS. However, apparent improvement in the specificity of the commercially available Japanese Institute for the Control of Ageing (JaICA) ELISA brought mean LC-MS/MS and ELISA measurements of urinary 8-oxodG into agreement. Urinary 8-oxodA was undetectable in all urines, despite efficient recovery by solid phase extraction. Exploitation of the advantages of ELISA may be enhanced by a simple modification to the assay procedure, although chromatographic techniques still remain the ‘gold standard’ techniques for analysis of urinary 8-oxodG. Urinary 8-oxodA is either not present or below the limit of detection of the instrumentation.     

Tetranor PGDM, an abundant urinary metabolite reflects biosynthesis of prostaglandin D2 in mice and humans

Prostaglandin D(2) (PGD(2)) is a cyclooxygenase (COX) product of arachidonic acid that activates D prostanoid receptors to modulate vascular, platelet, and leukocyte function in vitro. However, little is known about its enzymatic origin or its formation in vivo in cardiovascular or inflammatory disease. 11,15-dioxo-9alpha-hydroxy-2,3,4,5-tetranorprostan-1,20-dioic acid (tetranor PGDM) was identified by mass spectrometry as a metabolite of infused PGD(2) that is detectable in mouse and human urine. Using liquid chromatography-tandem mass spectrometry, tetranor PGDM was much more abundant than the PGD(2) metabolites, 11beta-PGF(2alpha) and 2,3-dinor-11beta-PGF(2alpha), in human urine and was the only endogenous metabolite detectable in mouse urine. Infusion of PGD(2) dose dependently increased urinary tetranor PGDM > 2,3-dinor-11beta-PGF(2alpha) > 11beta-PGF(2alpha) in mice. Deletion of either lipocalin-type or hemopoietic PGD synthase enzymes decreased urinary tetranor PGDM. Deletion or knockdown of COX-1, but not deletion of COX-2, decreased urinary tetranor PGDM in mice. Correspondingly, both PGDM and 2,3-dinor-11beta-PGF(2alpha) were suppressed by inhibition of COX-1 and COX-2, but not by selective inhibition of COX-2 in humans. PGD(2) has been implicated in both the development and resolution of inflammation. Administration of bacterial lipopolysaccharide coordinately elevated tetranor PGDM and 2,3-dinor-11beta-PGF(2alpha) in volunteers, coincident with a pyrexial and systemic inflammatory response, but both metabolites fell during the resolution phase. Niacin increased tetranor PGDM and 2,3-dinor-11beta-PGF(2alpha) in humans coincident with facial flushing. Tetranor PGDM is an abundant metabolite in urine that reflects modulated biosynthesis of PGD(2) in humans and mice.     

Liquid chromatography-electrospray tandem mass spectrometric method for quantification of monensin in plasma and edible tissues of chicken used in pharmacokinetic studies: applying a total error approach

A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for use in pharmacokinetic studies in order to determine the concentrations of monensin in plasma and edible tissues of chicken. Two sample preparations were performed, one for determining monensin concentrations in plasma using acetonitrile for protein precipitation and another one for determining monensin concentrations in muscle, liver, and fat using methanol-water followed by a clean up on a solid-phase extraction cartridge. Sample extracts were injected into the LC-MS/MS system, and a gradient elution was performed on a C18 column. Narasin was used as internal standard. The LC-MS/MS method was validated using an approach based on accuracy profiles, and applicability of the method was demonstrated for the determination of monensin in chicken plasma, muscle, liver, and fat in a pharmacokinetic study.     

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 <?0.001). According to ROC analysis, the AUC value of HbA1c was higher than the value of the AUC of 8-OHdG (0.882 and 0.771, respectively). Conclusions. This study shows that the urine 8-OHdG levels increase in diabetic patients. However, urinary 8-OHdG is not a useful clinical marker, compared with UACR, to predict the development of diabetic nephropathy in diabetic patients.     

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.     

Production and characterization of transgenic mice harboring mutant human UMOD gene

Familial juvenile hyperuricemic nephropathy is caused by mutations in the UMOD gene encoding uromodulin. A transgenic mouse model was developed by introducing a human mutant UMOD (C148W) cDNA under control of the mouse umod promoter. Uromodulin accumulation was observed in the thick ascending limb cells in the kidney of transgenic mice. However, the urinary excretion of uromodulin in transgenic mice did not decrease and LC-MS/MS analysis indicated it was of mouse origin. Moreover, the creatinine clearance was not different between wildtype and transgenic animals. Consequently, the onset of the disease was not observed in transgenic mice until 24 weeks of age.