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1.  25-hydroxyvitamin D and parathyroid hormone are not associated with carotid intima-media thickness or plaque in the Multi-Ethnic Study of Atherosclerosis 
Objective
Observational evidence supports independent associations between 25-hydroxyvitamin D (25-OHD), parathyroid hormone (PTH) and cardiovascular risk. A plausible hypothesis for these associations is accelerated development of atherosclerosis.
Approach and Results
We evaluated cross-sectional and longitudinal associations of 25-OHD and PTH with carotid intima-media thickness (IMT) and carotid plaques among 3251 participants free of cardiovascular disease in the Multi-Ethnic Study of Atherosclerosis. 25-OHD and PTH were measured at baseline by mass spectrometry and immunoassay, respectively. All subjects underwent a carotid ultrasound exam at baseline and 9.4 years later (median, range 8–11.1y). Multivariable linear and logistic regressions were used to test associations of 25-OHD and PTH with the extent and the progression of IMT and the prevalence and incidence of carotid plaque. Mean (SD) 25-OHD and PTH were 25.8ng/ml (10.6) and 44.2pg/ml (20.2). No independent associations were found between 25-OHD or PTH and IMT at baseline [increment of 1.9µm (95%CI −5.1 to 8.9) per 10ng/ml lower 25-OHD; increment of 0.8µm (95%CI −3.2 to 4.8) per 10pg/ml higher PTH] or progression of IMT [increment of 2.6µm (95%CI −2.5 to 7.8) per 10ng/ml lower 25-OHD, increment of 1.6µm (95%CI −1.9 to 5.2) per 10pg/ml higher PTH]. No associations were found with the baseline prevalence of carotid plaque or the incidence of new plaques over the study period. We did not observe any interaction by race or ethnicity (White, Chinese, Black and Hispanic).
Conclusions
The consistent lack of association of vitamin D and PTH with carotid IMT and plaque suggests that these hormones may influence cardiovascular risk through pathways not reflected by carotid atherosclerosis.
doi:10.1161/ATVBAHA.113.301781
PMCID: PMC3956469  PMID: 23814117
vitamin D; PTH; mineral metabolism; intima-media thickness; plaque; atherosclerosis; carotid
2.  Characterizing and harnessing antibody cross-reactivity for the immunoaffinity purification of analytes prior to multiplexed liquid chromatography-tandem mass spectrometry 
Clinical chemistry  2012;58(12):1711-1716.
Background
Immunoassays for 1α,25-dihydroxyvitamin D [1α,25(OH)2D] lack specificity. We aimed to characterize the cross-reactivity of an anti-1α,25(OH)2D antibody using purified vitamin D metabolites and to use these data to map the chemical features of 1α,25(OH)2D that are important for antibody binding. Additionally, we hypothesized that when combined with isotope dilution-liquid chromatography-tandem mass spectrometry (LC-MS/MS), antibody cross-reactivity could be used to semi-selectively enrich for structurally similar metabolites of vitamin D in a multiplexed assay.
Methods
Sample preparation consisted of immunoaffinity enrichment with a solid-phase anti-1α,25(OH)2D antibody and derivatization. Analytes were quantified using LC-MS/MS. Spike-recovery studies were performed using eleven vitamin D metabolites. A novel method for quantifying 25(OH)D2, 25(OH)D3, 24,25(OH)2D3, 1α,25(OH)2D2 and 1α,25(OH)2D3 simultaneously was developed and evaluated, which included deuterated internal standards for each analyte.
Results
The important chemical features of vitamin D metabolites for binding to the antibody were (1) native orientation of the hydroxyl group on carbon C3 in the A-ring, (2) the lack of substitution at carbon C4 in the A-ring, and (3) the overall polarity of the vitamin D metabolite. The new multiplexed method had lower limits of quantification (20% CV) of 0.2 ng/mL, 1.0 ng/mL, 0.06 ng/mL, 3.4 pg/mL and 2.8 pg/mL for 25(OH)D2, 25(OH)D3, 24,25(OH)2D3, 1α,25(OH)2D2 and 1α,25(OH)2D3, respectively. Method comparisons to three other LC-MS/MS methods were acceptable (r2>0.9, intercept
Conclusions
LC-MS/MS can be used to characterize antibody cross-reactivity. We developed and evaluated a multiplexed assay for five vitamin D metabolites using immunoenrichment in a targeted metabolomic assay.
doi:10.1373/clinchem.2012.185827
PMCID: PMC3731945  PMID: 22968104
Liquid chromatography-tandem mass spectrometry; immunoaffinity enrichment; multiplexed assay; specificity; hapten mapping; vitamin D metabolism
Clinical endocrinology  2013;79(3):429-436.
Objective
High circulating concentrations of parathyroid hormone (PTH) have been associated with increased risks of hypertension, left ventricular hypertrophy, congestive heart failure, and cardiovascular mortality. Impaired arterial function is a potential mechanism for these associations. We tested whether serum PTH concentration is associated with measures of arterial function.
Design
Cross-sectional study.
Participants
6,545 persons without clinical cardiovascular disease participating in the community-based Multi-Ethnic Study of Atherosclerosis.
Measurements
Brachial artery flow-mediated dilation as well as aortic pulse pressure and arterial pulse parameters derived from Windkessel modeling of the radial pressure waveform.
Results
Higher serum PTH concentration was associated with lower brachial artery flow-mediated dilation (mean difference −0.09% per 10 pg/mL PTH), higher aortic pulse pressure (0.53 mmHg per 10 pg/mL), and reduced Windkessel capacitive index C1 (large artery elasticity, −0.12 ml/mmHg X 10 per 10 pg/mL), adjusting for potential confounding variables (all p-values ≤ 0.001). These relationships were independent of serum calcium concentration, serum 25-hydroxyvitamin D concentration, and estimated glomerular filtration rate and were consistent across relevant participant subgroups. Associations of PTH with aortic pulse pressure and capacitive index C1 were attenuated after adjustment for blood pressure. Serum PTH concentration was not associated with the oscillatory index C2 (small artery elasticity).
Conclusions
Higher serum PTH concentration was associated with impaired endothelial function, increased aortic pulse pressure, and decreased capacitive index C1 in a large, diverse, community-based population. These relationships may help explain previously observed associations of elevated PTH with cardiovascular disease.
doi:10.1111/cen.12163
PMCID: PMC3664253  PMID: 23402353
parathyroid hormone; calcium; vitamin D; arterial function; epidemiology
IMPORTANCE
Low circulating concentrations of 25-hydroxyvitamin D (25[OH]D) have been consistently associated with an increased risk of coronary heart disease (CHD) in white populations. This association has not been rigorously evaluated in other races or ethnicities, in which the distributions of 25(OH)D concentration and possibly other aspects of 25(OH)D metabolism differ.
OBJECTIVE
To examine the association of serum 25(OH)D concentration with risk of CHD in a multiethnic population.
DESIGN, SETTING, AND PARTICIPANTS
We studied 6436 participants in the Multi-Ethnic Study of Atherosclerosis (MESA), recruited from July 2000 through September 2002, who were free of known cardiovascular disease at baseline. We measured baseline serum 25(OH)D concentrations using a mass spectrometry assay calibrated to established standards. We tested associations of 25(OH)D with adjudicated CHD events assessed through May 2012.
MAIN OUTCOME AND MEASURES
Primary outcome measure was time to first adjudicated CHD event, defined as myocardial infarction, angina, cardiac arrest, or CHD death.
RESULTS
During a median follow-up of 8.5 years, 361 participants had an incident CHD event (7.38 events per 1000 person-years). Associations of 25(OH)D with CHD differed by race/ethnicity (P for interaction < .05). After adjustment, lower 25(OH)D concentration was associated with a greater risk of incident CHD among participants who were white (n = 167 events; hazard ratio [HR], 1.26 [95%CI, 1.06–1.49] for each 10-ng/mL decrement in 25(OH)D) or Chinese (HR, 1.67 [95%CI, 1.07–2.61]; n = 27). In contrast, 25(OH)D was not associated with risk of CHD in participants who were black (HR, 0.93 [95%CI, 0.73–1.20]; n = 94) or Hispanic (HR, 1.01 [95%CI, 0.77–1.33]; n = 73).
CONCLUSIONS AND RELEVANCE
Lower serum 25(OH)D concentration was associated with an increased risk of incident CHD events among participants who were white or Chinese but not black or Hispanic. Results evaluating 25(OH)D in ethnically homogeneous populations may not be broadly generalizable to other racial or ethnic groups.
doi:10.1001/jama.2013.7228
PMCID: PMC4150653  PMID: 23839752
Diabetes Care  2013;36(8):2423-2429.
OBJECTIVE
People with type 1 diabetes are at high risk of premature atherosclerosis. Existing evidence suggests that impaired vitamin D metabolism may contribute to the development of atherosclerosis. We tested associations of circulating vitamin D metabolite concentrations with subclinical atherosclerosis among 1,193 participants with type 1 diabetes in the DCCT/EDIC study.
RESEARCH DESIGN AND METHODS
We measured plasma concentrations of 25-hydroxyvitamin D [25(OH)D], 1,25-dihydroxyvitamin D, and 24,25-dihydroxyvitamin D by mass spectrometry at the end of the DCCT. In a staggered cross-sectional design, we tested associations with coronary artery calcium (CAC), measured by computed tomography a median of 10 years later, and with common and internal carotid intima-media thickness (IMT), measured by B-mode ultrasonography on two occasions a median of 4 years later and a median of 10 years later. We hypothesized that lower concentrations of each vitamin D metabolite would be associated with increased risk of CAC and greater carotid IMT.
RESULTS
At the time metabolites were measured, mean age was 32.4 years and mean duration of diabetes was 7.5 years. The prevalence and severity of CAC tended to be lower—not higher—with lower concentrations of each vitamin D metabolite. For instance, in a fully adjusted multinomial logistic model, a 25 nmol/L lower 25-hydroxyvitamin D was associated with a 0.8-fold decrease in the odds of having higher CAC (95% CI 0.68–0.96, P = 0.01). No vitamin D metabolite was associated with either common or internal mean IMT.
CONCLUSIONS
We did not find evidence linking impaired vitamin D metabolism with increased subclinical atherosclerosis in type 1 diabetes.
doi:10.2337/dc12-2020
PMCID: PMC3714470  PMID: 23530012
Clinical chemistry  2013;59(6):982-990.
Background
Measurement of serum thyroglobulin (Tg) is used to monitor patients after treatment for differentiated thyroid carcinoma (TC). Difficulty in using Tg as a biomarker of the recurrence of TC in many patients stems from the presence of endogenous anti-Tg autoantibodies (Tg-AAb), which can interfere with immunoassays (IA) and cause false-negative results.
Methods
Tg was enriched from serum samples using rabbit polyclonal anti-Tg antiserum and protein precipitation. Unrelated proteins were partially depleted in the process. Enriched proteins were then denatured, reduced, and digested with trypsin after the addition of a winged internal standard peptide. A Tg-specific tryptic peptide was purified by immunoaffinity extraction and analyzed by 2D-LC-MS/MS. Instrument cycle time was 6.5 min per sample.
Results
The lower limit of quantification was 0.5 ng/mL (0.76 fmol/mL of dimer). Total imprecision of triplicate measurements in serum samples over five days was less than 10%. Comparison with a commercial IA using serum samples free of Tg-AAb (n=73) showed Deming regression, IA= 1.00*LC-MS/MS-2.35, r=0.982, Sy,x=9.52. In a set of Tg-AAb positive samples tested negative for Tg using IA (n=71), concentrations determined by LC-MS/MS method were at or above 0.5 in 23% of samples (median 1.2, range 0.7–11 ng/mL).
Conclusions
The method has acceptable performance characteristics for use in clinical diagnostic applications. The most substantial disagreement between the methods was observed in Tg-AAb positive samples with concentration below 2 ng/mL (determined with LC-MS/MS). The affinity assisted enrichment strategy used for Tg in this method is applicable to other biomarkers that have endogenous autoantibodies.
doi:10.1373/clinchem.2012.195594
PMCID: PMC4016991  PMID: 23396140
thyroglobulin; thyroid cancer; mass spectrometry; thyroglobulin auto-antibody
Statistical process control (SPC) is a robust set of tools that aids in the visualization, detection, and identification of assignable causes of variation in any process that creates products, services, or information. A tool has been developed termed Statistical Process Control in Proteomics (SProCoP) which implements aspects of SPC (e.g., control charts and Pareto analysis) into the Skyline proteomics software. It monitors five quality control metrics in a shotgun or targeted proteomic workflow. None of these metrics require peptide identification. The source code, written in the R statistical language, runs directly from the Skyline interface which supports the use of raw data files from several of the mass spectrometry vendors. It provides real time evaluation of the chromatographic performance (e.g., retention time reproducibility, peak asymmetry, and resolution); and mass spectrometric performance (targeted peptide ion intensity and mass measurement accuracy for high resolving power instruments) via control charts. Thresholds are experiment- and instrument-specific and are determined empirically from user-defined quality control standards that enable the separation of random noise and systematic error. Finally, Pareto analysis provides a summary of performance metrics and guides the user to metrics with high variance. The utility of these charts to evaluate proteomic experiments is illustrated in two case studies.
doi:10.1007/s13361-013-0824-5
PMCID: PMC4020592  PMID: 24496601
Quality Control; Statistical Process Control; Proteomics; Mass Spectrometry; Shewhart Control Charts
Background
The implementation of mass spectrometry to measure serum 25-hydroxyvitamin D [25(OH)D] concentrations has led to concerns regarding the measurement and reporting of the C3-epimer of 25-hydroxyvitamin D3 [3-epi-25(OH)D3], for which there is a near-total lack of data regarding its clinical significance.
Methods
We developed a chromatographic method to resolve (>90%) 3-epi-25(OH)D3 from 25(OH)D3 using a pentafluorophenyl propyl chromatographic column. Using LC-MS/MS, we determined the serum concentrations of 25(OH)D3 and 3-epi-25(OH)D3 in 626 patients aged 3 days to 94 y undergoing routine vitamin D testing.
Results
Comparison between DiaSorin RIA and the new LC-MS/MS method for total 25(OH)D had acceptable agreement. Our data indicate an increase in 25(OH)D3 rather than a reduction in epimer concentration. An average of 3.3 ng/ml of 3-epi-25(OH)D3 was detected in adolescents and adults. Inclusion of 3-epi-25(OH)D3 in the total 25(OH)D3 concentration resulted in 9% (< 1 y) and 3% (1 to 94 y) potential misclassification of patients as vitamin D sufficient.
Conclusions
The new LC-MS/MS method is capable of chromatographically separating 25(OH)D3 and 3-epi-25(OH)D3. It was used to confirm that the contribution of 3-epi-25OHD3 to total 25OHD3 concentrations decreases with age in infants and is detectable in adults.
doi:10.1016/j.cca.2011.09.028
PMCID: PMC3236660  PMID: 21983164
Vitamin D; epimer; Liquid chromatography-tandem mass spectrometry; 25-hydroxy vitamin D
Clinical Chemistry  2011;57(9):1279-1285.
Background
1α,25-dihydroxy vitamin D [1,25(OH)2D] is the active metabolite of vitamin D. Antibody-based detection methods lack specificity, but when combined with isotope dilution-UPLC-tandem mass spectrometry, immunoextraction provides an attractive method for 1,25(OH)2D. We developed a method for simultaneous quantification of 1,25(OH)2D2 and 1,25(OH)2D3 with a 4.6 min instrument cycle time. Results are available 36 h after sample preparation begins.
Methods
Sample preparation consisted of protein precipitation, immunoextraction with solid-phase anti-1,25(OH)2D antibody, and derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione. Analytes were resolved using reversed-phase UPLC and quantified using positive ion electrospray ionization-tandem mass spectrometry. Hexadeuterated 1,25(OH)2D3 and 1,25(OH)2D2 were used as internal standards. Method comparisons were performed against the DiaSorin RIA and an LC-MS/MS method available at a reference laboratory.
Results
1,25(OH)2D3 intra-assay and inter-assay imprecision was 5.6% and 8.0% (120 pmol/L) and 8.7% and 13% (48 pmol/L). Limits of detection and quantification were 1.5 pmol/L and 3.0 pmol/L, respectively. 1,25(OH)2D2 intra-assay and inter-assay imprecision was 8.7% and 11% (186 pmol/L) and 11% and 13% (58 pmol/L). Limits of detection and quantification were 1.5 pmol/L. Comparison with RIA had a proportional bias of 0.75, constant bias of −4.1 and Pearson correlation (r2) of 0.31. Comparison with a reference LC-MS/MS assay had a porportional bias of 0.89, constant bias of 3.7 and Pearson correlation (r2) of 0.88.
Conclusion
Protein precipitation with antibody-based extraction is effective for sample preparation prior to LC-MS/MS analysis of derivatized 1,25(OH)2D. This method appears to have improved specificity over a clinically-used RIA with low imprecision and limits of detection.
doi:10.1373/clinchem.2010.161174
PMCID: PMC3261234  PMID: 21768219
Liquid chromatography-tandem mass spectrometry; vitamin D; dihydroxy vitamin D; immunoaffinity; PTAD; protein precipitation
Adoption of targeted mass spectrometry (MS) approaches such as multiple reaction monitoring (MRM) to study biological and biomedical questions is well underway in the proteomics community. Successful application depends on the ability to generate reliable assays that uniquely and confidently identify target peptides in a sample. Unfortunately, there is a wide range of criteria being applied to say that an assay has been successfully developed. There is no consensus on what criteria are acceptable and little understanding of the impact of variable criteria on the quality of the results generated. Publications describing targeted MS assays for peptides frequently do not contain sufficient information for readers to establish confidence that the tests work as intended or to be able to apply the tests described in their own labs. Guidance must be developed so that targeted MS assays with established performance can be made widely distributed and applied by many labs worldwide. To begin to address the problems and their solutions, a workshop was held at the National Institutes of Health with representatives from the multiple communities developing and employing targeted MS assays. Participants discussed the analytical goals of their experiments and the experimental evidence needed to establish that the assays they develop work as intended and are achieving the required levels of performance. Using this “fit-for-purpose” approach, the group defined three tiers of assays distinguished by their performance and extent of analytical characterization. Computational and statistical tools useful for the analysis of targeted MS results were described. Participants also detailed the information that authors need to provide in their manuscripts to enable reviewers and readers to clearly understand what procedures were performed and to evaluate the reliability of the peptide or protein quantification measurements reported. This paper presents a summary of the meeting and recommendations.
doi:10.1074/mcp.M113.036095
PMCID: PMC3945918  PMID: 24443746
Objective
Insulin resistance and obesity are hallmarks of type 2 diabetes and the metabolic syndrome, which confer an increased risk of cardiovascular disease. Recent studies suggest that the protein cargo of high density lipoprotein (HDL) makes important contributions to the lipoprotein’s cardioprotective effects. We used targeted proteomics to determine if obesity and insulin resistance associate with changes in HDL’s protein content in two different groups of men.
Methods and Results
In a discovery study, we used isotope dilution mass spectrometry to quantify the relative concentrations of five proteins previously implicated in HDL’s cardioprotective effects in three groups of healthy subjects: lean insulin-sensitive, lean insulin-resistant, and obese insulin-resistant. We validated our findings in a different group of subjects. Clusterin concentration in HDL strongly and negatively associated with insulin resistance and body mass index in both populations. HDL clusterin levels were lower in subjects with low HDL and high triglycerides, key components of the metabolic syndrome. There was an inverse correlation between clusterin levels in HDL and VLDL/LDL.
Conclusions
Clusterin levels in HDL are lower in men with reduced insulin sensitivity, higher body mass index, and an unfavorable lipid profile. Our observations raise the possibility that clusterin depletion contributes to the loss of HDL’s cardioprotective properties.
doi:10.1161/ATVBAHA.110.212894
PMCID: PMC2988100  PMID: 20847305
High density lipoprotein; atherosclerosis; apolipoprotein J; inflammation; intra-abdominal fat
Background
The unmitigated rise in demand for the assessment of vitamin D status has taxed the ability of clinical mass spectrometry laboratories to preserve turn-around times. We aimed to improve the throughput of liquid-liquid extraction of plasma/serum for the assay of 25-hydroxy vitamin D.
Methods
We designed and fabricated a flexible rubber gasket that seals two 96-well plates together to quantitatively transfer the contents of one plate to another. Using the transfer gasket and a dry-ice acetone bath to freeze the aqueous infranatant, we developed a novel liquid-liquid extraction workflow in a 96-well plate format. We applied the technology to the mass spectrometric quantification of 25-hydroxy vitamin D.
Results
Cross-contamination between wells was ≤0.13%. The interassay imprecision over 132 days of clinical implementation was less than 10%. The method compared favorably to a standard liquid-liquid extraction in glass tubes (Deming slope=1.018, Sx|y=0.022). The accuracy of the assay was 102-105% as assessed with the recently released control materials from NIST.
Conclusions
The development of a plate-sealing gasket permits the liquid-liquid extraction of clinical specimens in a moderate-throughput workflow and the reliable assay of vitamin D status. In the future, the gasket may also prove useful in other sample preparation techniques for HPLC or mass spectrometry.
doi:10.1016/j.jchromb.2010.04.010
PMCID: PMC2877171  PMID: 20444657
Vitamin D; Liquid chromatography-tandem mass spectrometry; assay; 96-well plate; liquid-liquid extraction
Journal of immunological methods  2009;347(1-2):3-11.
Immunoassays have made it possible to measure dozens of individual proteins and other analytes in human samples for help in establishing the diagnosis and prognosis of disease. In too many cases the results of those measurements are misleading and can lead to unnecessary treatment or missed opportunities for therapeutic interventions. These cases stem from problems inherent to immunoassays performed with human samples, which include a lack of concordance across platforms, autoantibodies, anti-reagent antibodies, and the high-dose hook effect. Tandem mass spectrometry may represent a detection method capable of alleviating many of the flaws inherent to immunoassays. We review our understanding of the problems associated with immunoassays on human specimens and describe methodologies using tandem mass spectrometry that could solve some of those problems. We also provide a critical discussion of the potential pitfalls of novel mass spectrometric approaches in the clinical laboratory.
doi:10.1016/j.jim.2009.06.003
PMCID: PMC2720067  PMID: 19538965
Immunoassays; tandem mass spectrometry; autoantibody interference; hook effect; heterophile anti-reagent antibodies; standardization
Kidney international  2012;83(2):323-330.
Patients with chronic kidney disease are often insulin resistant and glucose intolerant; abnormalities that promote cardiovascular disease. Administration of 1,25-dihydroxyvitamin D (calcitriol) has improved glucose metabolism in patients with end stage renal disease. We conducted a randomized, placebo-controlled clinical trial to test whether paricalcitol, a 1,25-dihydroxyvitamin D analogue, changes glucose tolerance in earlier stages of chronic kidney disease. In a cross-over design, 22 non-diabetic patients with estimated glomerular filtration rates of stage 3-4 chronic kidney disease and fasting plasma glucose 100-125 mg/dL were given daily oral paricalcitol for 8 weeks and matching placebo for 8 weeks, separated by an 8-week washout period. The order of interventions was random and blinded to both participants and investigators. Paricalcitol significantly reduced serum concentrations of parathyroid hormone, 1,25-dihydroxyvitamin D, and 25-hydroxyvitamin D while significantly increasing serum concentrations of fibroblast growth factor-23 and 24,25-dihydroxyvitamin D. Paricalcitol, however, had no significant effect on glucose tolerance (the primary outcome measure), insulin sensitivity, beta-cell insulin response, plasma free fatty acid suppression, or urinary F2-isoprostane excretion. Thus, despite substantial effects on vitamin D metabolism, paricalcitol did not improve glucose metabolism in non-diabetic patients with stage 3-4 chronic kidney disease.
doi:10.1038/ki.2012.311
PMCID: PMC3509239  PMID: 22913981
Skeletal Muscle  2013;3:20.
Background
Presently, there is no effective treatment for the lethal muscle wasting disease Duchenne muscular dystrophy (DMD). Here we show that increased sphingosine-1-phoshate (S1P) through direct injection or via the administration of the small molecule 2-acetyl-4(5)-tetrahydroxybutyl imidazole (THI), an S1P lyase inhibitor, has beneficial effects in acutely injured dystrophic muscles of mdx mice.
Methods
We treated mdx mice with and without acute injury and characterized the histopathological and functional effects of increasing S1P levels. We also tested exogenous and direct administration of S1P on mdx muscles to examine the molecular pathways under which S1P promotes regeneration in dystrophic muscles.
Results
Short-term treatment with THI significantly increased muscle fiber size and extensor digitorum longus (EDL) muscle specific force in acutely injured mdx limb muscles. In addition, the accumulation of fibrosis and fat deposition, hallmarks of DMD pathology and impaired muscle regeneration, were lower in the injured muscles of THI-treated mdx mice. Furthermore, increased muscle force was observed in uninjured EDL muscles with a longer-term treatment of THI. Such regenerative effects were linked to the response of myogenic cells, since intramuscular injection of S1P increased the number of Myf5nlacz/+ positive myogenic cells and newly regenerated myofibers in injured mdx muscles. Intramuscular injection of biotinylated-S1P localized to muscle fibers, including newly regenerated fibers, which also stained positive for S1P receptor 1 (S1PR1). Importantly, plasma membrane and perinuclear localization of phosphorylated S1PR1 was observed in regenerating muscle fibers of mdx muscles. Intramuscular increases of S1P levels, S1PR1 and phosphorylated ribosomal protein S6 (P-rpS6), and elevated EDL muscle specific force, suggest S1P promoted the upregulation of anabolic pathways that mediate skeletal muscle mass and function.
Conclusions
These data show that S1P is beneficial for muscle regeneration and functional gain in dystrophic mice, and that THI, or other pharmacological agents that raise S1P levels systemically, may be developed into an effective treatment for improving muscle function and reducing the pathology of DMD.
doi:10.1186/2044-5040-3-20
PMCID: PMC3750760  PMID: 23915702
Journal of Toxicology  2013;2013:329407.
An important role of the clinical toxicology laboratory is to provide continuous diagnostic testing for patients with altered mental status and for other medical indications. To meet these needs, we have developed a new Gas Chromatography-Mass Spectrometry (GC-MS) platform that facilitates routine screening and automated reporting of 212 drugs by laboratory technologists around the clock without the need to sign out by an on-site mass spectrometry-trained toxicologist. The platform uses a programmable temperature vaporizer (PTV) injector for large sample volume injection and the free software Automated Mass Spectral Deconvolution and Identification System (AMDIS) for data reduction and spectral matching that facilitates rapid library searching and analyte identification. Method comparison with 118 patient samples demonstrated that this platform and data searching algorithm independently provided improvements in sensitivity compared to an established GC-MS platform. Further examination of the role of the data processing software and the in-house databases used in the established versus the new platform demonstrated that the improved analytical sensitivity of the new platform was attributed to both the technical superiority of the new GC-MS instrumentation and the use of AMDIS in conjunction with the newly generated in-house library for data processing.
doi:10.1155/2013/329407
PMCID: PMC3723246  PMID: 23935615
Bioanalysis  2012;4(3):281-290.
For decades, immunoassays have provided the framework for protein biomarker studies in clinical medicine and in therapeutic monitoring for drug development. At the same time, investigators have uncovered many issues that make immunoassays unreliable in many human serum and plasma samples. LC-MS/MS after tryptic digestion of proteins is potentially an attractive solution, but the sensitivity of the method is not sufficient to measure many important low-abundance proteins directly. The use of antipeptide antibodies to immunoenrich peptides of interest can improve the sensitivity of the approach, greatly simplify the matrix enabling shortened chromatographic runs, and facilitate the multiplexed quantification of analytes, which could reduce the costs of quantitative protein measurements in complex specimens. We provide an overview of the method and the steps needed to develop an assay. In addition, we review the efforts to make this method generally more applicable.
doi:10.4155/bio.11.319
PMCID: PMC3699856  PMID: 22303832
Clinical chemistry  2012;58(4):777-781.
Background
Mass spectrometric assays have the potential to replace protein immunoassays in basic science, clinical research, and clinical care. Previous studies have demonstrated the utility of assays using multiple-reaction monitoring mass spectrometry (MRM-MS) for the quantification of proteins in biological samples and many examples of the accuracy of these approaches to quantify spiked analytes have been reported. However, a direct comparison of multiplexed assays using liquid chromatography-tandem mass spectrometry with established immunoassays to measure endogenous proteins has not been reported.
Methods
We purified the HDL from the plasma of 30 human subjects enrolled in a clinical nutrition research study and used label-free shotgun proteomics approaches to analyze each sample. We then developed two different 6-plex assays that used isotope dilution MRM-MS: one assay used stable isotope labeled peptides and the other used stable isotope labeled apolipoprotein A-I (apoA-I), the most abundant protein in HDL, as internal standards to control for matrix effects and mass spectrometer performance. The shotgun and MRM-MS assays were then compared with commercially available immunoassays for each of the six analytes.
Results
Quantification by shotgun proteomics approaches correlated poorly with the six protein immunoassays. However, the MRM-MS approaches that used internal standard peptide or a single internal standard protein correlated well. In addition, MRM-MS approaches had good repeatability (<10% CV) and linearity.
Conclusions
Multiplexed MRM-MS assays correlate well with immunochemical measurements and have acceptable operating characteristics in complex samples. Our results support the proposal that MRM-MS could be used to replace immunoassays in a variety of settings.
doi:10.1373/clinchem.2011.173856
PMCID: PMC3665768  PMID: 22307200
Mass spectrometry; multiple reaction monitoring; endogenous; proteins; high density lipoprotein; targeted proteomics
Steroids  2012;77(5):454-460.
Exogenous androgens can lower HDL-cholesterol (HDL-C) concentrations, yet men with low serum testosterone have elevated rates of cardiovascular disease (CVD). HDL function may better predict CVD risk than absolute HDL-C quantity. We evaluated the acute effects of medical castration in men on HDL-C, cholesterol efflux capacity and HDL protein composition. Twenty-one healthy men, ages 18–55, received the GnRH antagonist acyline and one of the following for 28 days: Group 1: placebo, Group 2: transdermal testosterone gel and placebo, Group 3: transdermal testosterone gel and an aromatase inhibitor. Sex steroids, fasting lipids, and cholesterol efflux to apoB-depleted serum were measured in all subjects. The HDL proteome was assessed in Group 1 subjects only. In Group 1, serum testosterone concentrations were reduced by >95%, and HDL-C and cholesterol efflux capacity increased (p=0.02 and p=0.04 vs. baseline, respectively). HDL-associated clusterin increased significantly with sex steroid withdrawal (p=0.007 vs. baseline). Testosterone withdrawal in young, healthy men increases HDL-C and cholesterol efflux capacity. Moreover, sex steroid deprivation changes HDL protein composition. Further investigation of the effects of sex steroids on HDL composition and function may help resolve the apparently conflicting data regarding testosterone, HDL-C, and CVD risk.
doi:10.1016/j.steroids.2012.01.002
PMCID: PMC3304018  PMID: 22266332
testosterone; estradiol; HDL cholesterol; cardiovascular disease; apolipoproteins; atherosclerosis
Kidney international  2012;82(6):693-700.
Chronic kidney disease is characterized, in part, as a state of decreased production of 1,25-dihydroxyvitamin D (1,25(OH)2D); however, this paradigm overlooks the role of vitamin D catabolism. We developed a mass spectrometric assay to quantify serum concentration of 24,25-dihydroxyvitamin D (24,25(OH)2D), the first metabolic product of 25-hydroxyvitamin D (25(OH)D) by CYP24A1, and determined its clinical correlates and associated outcomes among 278 participants with chronic kidney disease in the Seattle Kidney Study. For eGFRs of 60 or more, 45–59, 30–44, 15–29, and under 15 ml/min/1.73m2, the mean serum 24,25(OH)2D concentrations significantly trended lower from 3.6, 3.2, 2.6, 2.6, to 1.7 ng/ml, respectively. Non-Hispanic Black race, diabetes, albuminuria, and lower serum bicarbonate were also independently and significantly associated with lower 24,25(OH)2D concentrations. The 24,25(OH)2D concentration was more strongly correlated with that of parathyroid hormone than was 25(OH)D or 1,25(OH)2D. A 24,25(OH)2D concentration below the median was associated with increased risk of mortality in unadjusted analysis, but this was attenuated with adjustment for potential confounding variables. Thus, chronic kidney disease is a state of stagnant vitamin D metabolism characterized by decreases in both 1,25(OH)2D production and vitamin D catabolism.
doi:10.1038/ki.2012.193
PMCID: PMC3434313  PMID: 22648296
American Journal of Epidemiology  2011;174(12):1363-1372.
Low circulating concentrations of 25-hydroxyvitamin D (25(OH)D) are associated with adverse health outcomes in diverse populations. However, 25(OH)D concentrations vary seasonally with varying exposure to sunlight, so single measurements may poorly reflect long-term 25(OH)D exposure. The authors investigated cyclical trends in average serum 25(OH)D concentrations among 2,298 individuals enrolled in the Cardiovascular Health Study of community-based older adults (1992–1993). A sinusoidal model closely approximated observed 25(OH)D concentrations and fit the data significantly better than did a mean model (P < 0.0001). The mean annual 25(OH)D concentration was 25.1 ng/mL (95% confidence interval: 24.7, 25.5), and the mean peak-trough difference was 9.6 ng/mL (95% confidence interval: 8.5, 10.7). Male sex, higher latitude of study site, and greater physical activity levels were associated with larger peak-trough difference in 25(OH)D concentration (each P < 0.05). Serum concentrations of intact parathyroid hormone and bone-specific alkaline phosphatase also varied in a sinusoidal fashion (P < 0.0001), inversely to 25(OH)D. In conclusion, serum 25(OH)D varies in a sinusoidal manner, with large seasonal differences relative to mean concentration and laboratory evidence of biologic sequelae. Single 25(OH)D measurements might not capture overall vitamin D status, and the extent of misclassification could vary by demographic and behavioral factors. Accounting for collection time may reduce bias in research studies and improve decision-making in clinical care.
doi:10.1093/aje/kwr258
PMCID: PMC3276302  PMID: 22112344
alkaline phosphatase; parathyroid hormone; seasons; vitamin D
Radiation Research  2010;175(3):266-281.
The structural maintenance of chromosome 1 (Smc1) protein is a member of the highly conserved cohesin complex and is involved in sister chromatid cohesion. In response to ionizing radiation, Smc1 is phosphorylated at two sites, Ser-957 and Ser-966, and these phosphorylation events are dependent on the ATM protein kinase. In this study, we describe the generation of two novel ELISAs for quantifying phospho-Smc1Ser-957 and phospho-Smc1Ser-966. Using these novel assays, we quantify the kinetic and biodosimetric responses of human cells of hematological origin, including immortalized cells, as well as both quiescent and cycling primary human PBMC. Additionally, we demonstrate a robust in vivo response for phospho-Smc1Ser-957 and phospho-Smc1Ser-966 in lymphocytes of human patients after therapeutic exposure to ionizing radiation, including total-body irradiation, partial-body irradiation, and internal exposure to 131I. These assays are useful for quantifying the DNA damage response in experimental systems and potentially for the identification of individuals exposed to radiation after a radiological incident.
doi:10.1667/RR2402.1
PMCID: PMC3123689  PMID: 21388270
Clinical chemistry  2011;57(5):701-709.
Background
Macrophages and related cells are important cellular mediators of the innate system and play an important role in wound healing and fibrosis. Flux through different L-arginine metabolic pathways partially defines the functional behavior of macrophages. Methods to measure metabolites within the nitric oxide synthase/arginase pathways could potentially reveal insights into local and systemic inflammatory processes.
Methods
A targeted metabolomics approach was developed using HILIC chromatography and electrospray ionization-tandem mass spectrometry to simultaneously measure L-arginine, asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), L-citrulline, L-ornithine, and L-proline in plasma from humans and mice.
Results
All analytes were quantifiable in human plasma and mouse plasma and serum with a small volume (25µl), minimal sample preparation, and no derivatization. Patients with high plasma concentrations of C-reactive protein and mice with acute inflammation induced by lipopolysaccharide had significant reductions of arginine metabolites in plasma compared with normal controls.
Conclusions
This new assay uses plasma metabolomic measurements to help provide new insights into metabolic changes coupled to the innate immune response. We identified significant changes in arginine metabolism in both humans and mice following an inflammatory stimulus. These changes were associated with decreased plasma arginine metabolite concentrations and increased methylated arginine concentrations.
doi:10.1373/clinchem.2010.155895
PMCID: PMC3199374  PMID: 21406573
arginine; citrulline; ornithine; proline; ADMA; SDMA; metabolomics; HILIC; LC-MS/MS; hsCRP; plasma; serum
Journal of Obesity  2011;2011:495101.
Low-serum concentrations of 25-hydroxyvitamin D [25(OH)D] are associated with insulin resistance in adults. Less data are available in pediatric populations. Serum 25(OH)D serum concentrations were assessed in 125 obese and 31 nonobese children (age 11.9 ± 2.7 y, range 6–16 y, 49% male) living in Bonn, Germany. The relationship between 25(OH)D, measured by liquid chromatography-tandem mass spectrometry, and measures of insulin sensitivity and adipokines adiponectin and resistin were analyzed. Seventy-six % of subjects were 25(OH)D deficient (<20 ng/mL). Higher insulin, homeostasis model assessment-insulin resistance (HOMA-IR r = −0.269, P = 0.023), and hemoglobin A1c (HbA1c) as well as lower quantitative insulin-sensitivity check index (QUICKI r = 0.264, P = 0.030) values were found in obese children with lower 25(OH)D concentrations even after adjustment for gender, age, and body mass index. Furthermore, 25(OH)D correlated significantly with adiponectin, but not with resistin. Our results suggest that hypovitaminosis D is a risk factor for developing insulin resistance independent of adiposity.
doi:10.1155/2011/495101
PMCID: PMC3255292  PMID: 22254134

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