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1.  Altered Calcium and Vitamin D Homeostasis in First-Time Calcium Kidney Stone-Formers 
PLoS ONE  2015;10(9):e0137350.
Elevated serum 1,25-dihydroxyvitamin D (1,25(OH)2D) concentrations have been reported among cohorts of recurrent calcium (Ca) kidney stone-formers and implicated in the pathogenesis of hypercalciuria. Variations in Ca and vitamin D metabolism, and excretion of urinary solutes among first-time male and female Ca stone-formers in the community, however, have not been defined.
In a 4-year community-based study we measured serum Ca, phosphorus (P), 25-hydroxyvitamin D (25(OH)D), 1,25(OH)2D, 24,25-dihydroxyvitamin D (24,25(OH)2D), parathyroid hormone (PTH), and fibroblast growth factor-23 (FGF-23) concentrations in first-time Ca stone-formers and age- and gender frequency-matched controls.
Serum Ca and 1,25(OH)2D were increased in Ca stone-formers compared to controls (P = 0.01 and P = 0.001). Stone-formers had a lower serum 24,25(OH)2D/25(OH)D ratio compared to controls (P = 0.008). Serum PTH and FGF-23 concentrations were similar in the groups. Urine Ca excretion was similar in the two groups (P = 0.82). In controls, positive associations between serum 25(OH)D and 24,25(OH)2D, FGF-23 and fractional phosphate excretion, and negative associations between serum Ca and PTH, and FGF-23 and 1,25(OH)2D were observed. In SF associations between FGF-23 and fractional phosphate excretion, and FGF-23 and 1,25(OH)2D, were not observed. 1,25(OH)2D concentrations associated more weakly with FGF-23 in SF compared with C (P <0.05).
Quantitative differences in serum Ca and 1,25(OH)2D and reductions in 24-hydroxylation of vitamin D metabolites are present in first-time SF and might contribute to first-time stone risk.
PMCID: PMC4558059  PMID: 26332888
2.  Myostatin as a mediator of sarcopenia versus homeostatic regulator of muscle mass: insights using a new mass spectrometry-based assay 
Skeletal Muscle  2015;5:21.
Myostatin is a protein synthesized and secreted by skeletal muscle that negatively regulates muscle mass. The extent to which circulating myostatin levels change in the context of aging is controversial, largely due to methodological barriers.
We developed a specific and sensitive liquid chromatography with tandem mass spectrometry (LC-MS/MS) assay to measure concentrations of myostatin and two of its key inhibitors, follistatin-related gene (FLRG) protein and growth and serum protein-1 (GASP-1) in 80 younger (<40 years), 80 older (>65 years), and 80 sarcopenic older women and men.
Older women had 34 % higher circulating concentrations of myostatin than younger women. Per unit of lean mass, both older and sarcopenic older women had >23 % higher myostatin levels than younger women. By contrast, younger men had higher myostatin concentrations than older men with and without sarcopenia. Younger men had approximately twofold higher concentrations of myostatin than younger women; however, older women and sarcopenic older women had significantly higher relative myostatin levels than the corresponding groups of men. In both sexes, sarcopenic older subjects had the highest concentrations of FLRG. Circulating concentrations of myostatin exhibited positive, but not robust, correlations with relative muscle mass in both sexes.
Our data suggest that myostatin may contribute to the higher prevalence of sarcopenia in women but acts as a homeostatic regulator of muscle mass in men. Moreover, this new LC-MS/MS-based approach offers a means to determine the extent to which myostatin serves as a biomarker of muscle health in diverse conditions of muscle loss and deterioration.
Electronic supplementary material
The online version of this article (doi:10.1186/s13395-015-0047-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4502935  PMID: 26180626
Myostatin; Aging; Sarcopenia; Skeletal muscle mass; Strength; Body composition
3.  Immunologic and mass-spectrometric estimates of SHBG concentrations in healthy women 
Sex-hormone binding globulin (SHBG) concentrations across the adult female lifespan are not well defined. To address this knowledge gap, SHBG was quantified by both immunological and criterion methods, viz, mass spectrometry (MS).
Setting: Center for Translational Science Activities (CTSA). Participants: Healthy nonpregnant women (N = 120) ages 21 to 79 years. Outcomes: SHBG, testosterone (T), estradiol (E2) and estrone (E1) each determined by MS. Uni- and multivariate regression of SHBG concentrations on age, body mass index (BMI), total and visceral abdominal fat (TAF, AVF), albumin, glucose, insulin, sex steroids, selected cytokines, blood pressure, and lipids.
By univariate regression, MS-estimated SHBG correlated negatively with BMI, TAF, AVF, insulin, free T and bioavailable T (bio T) (each P ≤ 10−4), but not with blood pressure or lipids. By stepwise multivariate regression analysis, free and total T (both positive) and bio T (negative) were correlated with SHBG in all 4 assays (each P < 10−15, R2 ≥ 0.481). In addition, TAF and BMI were negatively associated with SHBG (P ≤ 0.0066) in 2 SHBG assays, and estrone and IL-8 with SHBG weakly (P ≤ 0.035) in one SHBG assay each. When nonsignificant cytokines were excluded, SHBG was jointly associated with AVF, total T and HDL (P < 10−9, R2 = 0.358).
According to MS, three metabolic factors, T, AVF and HDL, together explain more than one-third of the interindividual variation in SHBG levels. We speculate that these measures reflect insulin action.
PMCID: PMC4066221  PMID: 24746136
Female; Sex steroid; Body composition; Obesity; Insulin
4.  Pharmacokinetics of daily versus monthly vitamin D3 supplementation in non-lactating women 
This study compared serum cholecalciferol and 25-hydroxyvitamin D [25(OH)D] concentrations over four weeks in healthy, non-pregnant, non-lactating females aged 18-40 years, who were randomized to oral cholecalciferol 5,000 international units (IU) daily for 28 days or a single dose of 150,000 IU. The study was conducted in Rochester, MN in March and April of 2010. We found no difference in mean 25(OH)D between treatment groups on study day 0 or day 28 (p = 0.14 and 0.28, respectively). The daily group had 11 more days of detectable serum cholecalciferol than the single-dose group (p < 0.001). There was no difference observed in cholecalciferol area under the curve (AUC28) between groups (p = 0.49). However, The single dose group had a significantly greater mean 25(OH)D AUC28 compared with the daily group (p < 0.001).
PMCID: PMC4266550  PMID: 24424073
nutrition; women; metabolism; Vitamin D; pharmacokinetics
5.  Maternal vitamin D supplementation to improve the vitamin D status of breastfed infants: a randomized control trial 
Mayo Clinic proceedings  2013;88(12):1378-1387.
To determine if a single monthly supplement was as effective as a daily maternal supplement in increasing breast milk vitamin D to achieve vitamin D sufficiency in their infants.
Patients and Methods
Forty mothers with exclusively breastfed infants were randomized to receive oral cholecalciferol (vitamin D3) 5000 IU/d for 28 days or 150,000 IU once. Maternal serum, breast milk, and urine were collected on days 0, 1, 3, 7, 14 and 28; infant serum was obtained on days 0 and 28. Enrollment occurred between January 7 and July 29, 2011.
In mothers given daily cholecalciferol, concentrations of serum and breast milk cholecalciferol attained steady levels of 18 and 8 ng/mL, respectively, from day 3 through 28. In mothers given the single dose, serum and breast milk cholecalciferol peaked at 160 and 40 ng/mL, respectively at day 1, before rapidly declining. Maternal milk and serum cholecalciferol concentrations were related (r=0.87). Infant mean serum 25(OH)D (±SD) increased from 17±13 to 39±6 ng/mL in the single dose group and from 16±12 to 39±12 ng/mL in the daily dose group (P=.88). All infants achieved serum 25(OH)D concentrations >20 ng/mL).
Either single dose or daily cholecalciferol supplementation of mothers provided breast milk concentrations that result in vitamin D sufficiency in breastfeeding infants.
PMCID: PMC3923377  PMID: 24290111
6.  Atorvastatin Inhibits Hypercholesterolemia-Induced Cellular Proliferation and Bone Matrix Production in the Rabbit Aortic Valve 
Circulation  2002;105(22):2660-2665.
Despite the common occurrence of aortic stenosis, the cellular causes of the disorder are unknown, in part because of the absence of experimental models. We hypothesized that atherosclerosis and early bone matrix expression in the aortic valve occurs secondary to experimental hypercholesterolemia and that treatment with atorvastatin modifies this transformation.
Methods and Results
To test this hypothesis, we developed an experimental hypercholesterolemic rabbit model. New Zealand White rabbits (n=48) were studied: group 1 (n=16), normal diet; group 2 (n=16), 1% (wt/wt) cholesterol diet; and group 3 (n=16), 1% (wt/wt) cholesterol diet plus atorvastatin (3 mg/kg per day). The aortic valves were examined with hematoxylin and eosin stain, Masson trichrome, macrophage (RAM 11), proliferation cell nuclear antigen (PCNA), and osteopontin immunostains. Cholesterol and highly sensitive C-reactive protein (hsCRP) serum levels were obtained by standard assays. Computerized morphometry and digital image analysis were performed for quantifying PCNA (% area). Electron microscopy and immunogold labeling were performed for osteopontin. Semiquantitative RT-PCR was performed for the osteoblast bone markers [alkaline phosphatase, osteopontin, and osteoblast lineage-specific transcription factor (Cbfa-1)]. There was an increase in cholesterol, hsCRP, PCNA, RAM 11, and osteopontin and osteoblast gene markers (alkaline phosphatase, osteopontin, and Cbfa-1) in the cholesterol-fed rabbits compared with control rabbits. All markers except hsCRP were reduced by atorvastatin.
These findings of increased macrophages, PCNA levels, and bone matrix proteins in the aortic valve during experimental hypercholesterolemia provide evidence of a proliferative atherosclerosis–like process in the aortic valve associated with the transformation to an osteoblast-like phenotype that is inhibited by atorvastatin. (Circulation. 2002; 105:2660-2665.)
PMCID: PMC3951862  PMID: 12045173
valves; cardiovascular diseases; lipids; atherosclerosis; physiology
7.  Increasing Incidence of Nutritional Rickets: A Population-Based Study in Olmsted County, Minnesota 
To determine temporal trends in incidence and risk factors of nutritional rickets in a community-based population.
Patients and Methods
Rochester Epidemiology Project (REP) data were used to identify all children (<18 years) residing in Olmsted County, Minnesota between January 1, 1970 and December 31, 2009 with diagnostic codes corresponding to rickets, vitamin D deficiency, hypovitaminosis D, rachitis, osteomalacia, genu varum, genu valgum, craniotabes, hypocalcemia, hypocalcemic seizure, and tetany. Record abstraction was performed to select subjects with radiographic confirmation of rickets. Age- and gender-matched controls were identified for evaluation of risk factors. The main outcome measure was radiographic rickets without identifiable inherited, genetic, or non-nutritional causes. Incidence rates were calculated using REP census data.
Of 768 children with eligible diagnostic codes, 23 had radiographic evidence of rickets; of these, 17 children had nutritional rickets. All were younger than 3 years and 13 (76%) were nonwhite. Clinical presentation included poor growth (n=12), leg deformity (n=8), motor delay (n=5), leg pain (n=3), weakness (n=3), and hypocalcemia or tetany (n=2). The incidence of nutritional rickets in children under 3 years was 0, 2.2, 3.7, and 24.1 per 100,000 for the decades beginning in 1970, 1980, 1990, and 2000, respectively (P=.003 for incidence trend). Nutritional rickets was associated with black race, breastfeeding, low birth weight, and stunted growth (P<.05 for all). Four of 13 (31%) who underwent 25-hydroxyvitamin D testing had values less than 10 ng/mL (25 nmol/L).
Nutritional rickets remains rare, but the incidence has dramatically increased after 2000. Not all cases can be attributed to vitamin D deficiency.
PMCID: PMC3612965  PMID: 23374621
bone diseases; metabolic; epidemiology; pediatric; vitamin D
8.  Comparison between Immunoturbidimetry, Size-Exclusion Chromatography, and LC-MS to Quantify Urinary Albumin 
Clinical chemistry  2008;54(9):1504-1510.
The accurate and precise measurement of urinary albumin is critical, since even minor increases are diagnostically sensitive indicators of renal disease, cardiovascular events, and risk for death. To gain insights into potential measurement biases, we systematically compared urine albumin measurements performed by LC-MS, a clinically available immunoturbidimetric assay, and size-exclusion HPLC.
We obtained unused clinical urine samples from 150 patients who were stratified by degrees of albuminuria (<20 mg/L, 20–250 mg/L, >250 mg/L) as determined by the immunoturbidimetric assay used in our clinical laboratory (Roche Hitachi 912). Urine albumin was then remeasured via LC-MS and HPLC (Accumin™) assays.
The immunoturbidimetric assay, calibrated using manufacturer-supplied serum-derived calibrators (Diasorin), underestimated albumin compared with LC-MS. After calibration with purified HSA, this immunoturbidimetric assay correlated well with LC-MS. HPLC overestimated albumin compared with both LC-MS and immunoturbidimetry. The current LC-MS and HPLC assays both performed poorly at concentrations <20 mg/L.
Efforts are needed to establish gold-standard traceable calibrators for clinical assays. LC-MS is a specific method to quantify albumin in native urine when concentrations exceed 20 mg/L, and therefore could be employed for standardization among assays.
PMCID: PMC3903150  PMID: 18617580
9.  Alveolar Macrophage Cathelicidin Deficiency in Severe Sarcoidosis 
Journal of innate immunity  2012;4(5-6):569-578.
Dysfunctional immune responses characterize sarcoidosis but the status of cathelicidin, a potent immunoregulatory and anti-microbial molecule has not been established in clinical disease activity. Alveolar macrophage cathelicidin expression was determined in biopsy-proven sarcoidosis patients classified clinically as “severe” (requiring systemic treatment) or “non-severe” (never requiring treatment). Sarcoidosis and healthy control bronchoalveolar lavage (BAL) cells were analyzed for mRNA expression of cathelicidin, vitamin D receptor (VDR), and the VDR co-activator, steroid receptor co-activator-3 (SRC3) by quantitative PCR. Cathelicidin-derived peptide LL-37 was determined by immunocytochemistry. Serum calcidiol [25(OH)vitD2] (vitD2) and calcitriol [1,25(OH)2vitD3] (vitD3) were quantified. Results indicated reduced BAL cell expression of cathelicidin and SRC3 in severe but not non-severe sarcoidosis compared to controls. Serum levels of biologically active vitD3 in both severe and non-severe patients were within control range even though vitD2 levels in both groups were below recommended level (30ng/ml). Sarcoidosis and control alveolar macrophages were studied in vitro to determine cathelicidin responses to vitD3 and tumor necrosis factor-alpha (TNFα), a vitD3 antagonist elevated in active sarcoidosis. Alveolar macrophage cathelicidin was stimulated by vitD3 but repressed by TNFα which also repressed SRC3. Findings suggest that TNFα-mediated repression of SRC3 contributes to alveolar macrophage cathelicidin deficiency in severe sarcoidosis despite healthy vitD3 levels. Deficiency of cathelicidin, a multifunctional regulator of immune cells and pro-inflammatory cytokines, may impede resolution of inflammation in severe sarcoidosis lung.
PMCID: PMC3472801  PMID: 22759465
alveolar macrophage; sarcoidosis; cathelicidin; vitamin D; cytokines; steroid receptor co-activators
10.  Vitamin D (25OHD) Serum Seasonality in the United States 
PLoS ONE  2013;8(6):e65785.
Vitamin D is an important micronutrient for health. Hypovitaminosis D is thought to play a role in the seasonality of a number of diseases and adverse health conditions. To refine hypotheses about the links between vitamin D and seasonal diseases, good estimates of the cyclicality of serum vitamin D are necessary.
The objective of this study is to describe quantitatively the cyclicality of 25-hydroxyvitamin D (25OHD) in the United States. We provide a statistical analysis with weekly time resolution, in comparison to the quarterly (winter/spring/summer/fall) estimates already in the literature.
We analyzed time series data on 25OHD, spanning 287 consecutive weeks. The pooled data set comes from 3.44 million serum samples from the United States. We statistically analyzed the proportion of sera that were vitamin D sufficient, defined as 25OHD ng/mL, as a function of date.
In the United States, serum 25OHD follows a lagged pattern relative to the astronomical seasons, peaking in late summer (August) and troughing in late winter (February). Airmass, which is a function of solar altitude, fits the 25OHD data very well when lagged by 8 weeks.
Serum vitamin D levels can be modeled as a function of date, working through a double-log transformation of minimal solar airmass (easily calculated from solar altitude, retrievable from an online solar altitude/azimuth table).
PMCID: PMC3689782  PMID: 23805188
11.  Succinate Dehydrogenase Gene Mutations are Strongly Associated with Paraganglioma of the Organ of Zuckerkandl 
Endocrine-related cancer  2010;17(3):581-588.
Organ of Zuckerkandl paragangliomas (PGLs) are rare neuroendocrine tumors that are derived from chromaffin cells located around the origin of the inferior mesenteric artery extending to the level of the aortic bifurcation. Mutations in the genes encoding succinate dehydrogenase subunits (SDH) B, C, and D (SDHx) have been associated with PGLs, but their contribution to PGLs of the organ of Zuckerkandl PGLs is not known. We aimed to describe the clinical presentation of patients with PGLs of the organ of Zuckerkandl and investigate the prevalence of SDHx mutations and other genetic defects among them. The clinical characteristics of 14 patients with PGL of the organ of Zuckerkandl were analyzed retrospectively; their DNA was tested for SDHx mutations and deletions. Eleven out of 14 (79%) of patients with PGLs of the organ of Zuckerkandl were found to have mutations of the SDHB (9), or SDHD (2) genes; one patient was found to have the Carney-Stratakis syndrome (CSS) and his PGL was discovered during surgery for gastrointestinal stromal tumor (GIST). Our results show that SDHx mutations are prevalent in pediatric and adult PGLs of the organ of Zuckerkandl. Patients with PGLs of the organ of Zuckerkandl should be screened for SDHx mutations and the CSS; in addition asymptomatic carriers of an SDHx mutation among the relatives of affected patients may benefit from tumor screening for early PGL detection.
PMCID: PMC3417306  PMID: 20418362
pheochromocytoma; paraganglioma; genetic testing
12.  Vitamin D Status and Longitudinal Lung Function Decline in the Lung Health Study 
Low vitamin D blood levels are postulated to be a risk factor for worse lung function, largely based on cross-sectional data. We sought to use longitudinal data to test the hypothesis that baseline plasma 25-hydroxyvitamin D [25(OH)D] is lower in subjects with more rapid lung function decline, compared to those with slow lung function decline.
We conducted a nested, matched case-control study in the Lung Health Study 3 cohort. Cases and controls were continuous smokers with rapid and slow lung function decline, respectively, over approximately 6 years of follow-up. We compared baseline 25(OH)D levels between cases and controls, matching on date of blood draw and clinical center.
Among 196 subjects, despite rapid and slow decliners experiencing strikingly and significantly different rates of decline of forced expiratory volume in one second (−152 vs. −0.3 mL/year; p<0.001), there was no significant difference in baseline 25(OH)D levels (25.0 vs. 25.9 ng/mL; p=0.54). There was a high prevalence of vitamin D insufficiency (35%) and deficiency (31%); only 4% had a normal 25(OH)D level in the winter.
Although vitamin D insufficiency and deficiency are common among continuous smokers with established mild to moderate COPD, baseline 25(OH)D levels are not predictive of subsequent lung function decline.
PMCID: PMC3070416  PMID: 20595151
Pulmonary Disease, Chronic Obstructive; Smoking; Spirometry; Vitamin D
13.  Comparison of Multiple Steroid Concentrations in Serum and Dried Blood Spots throughout the Day of Patients with Congenital Adrenal Hyperplasia 
Periodic measurement of plasma concentrations of cortisol precursors on a clinic visit may be of limited value in patients with congenital adrenal hyperplasia because it does not reflect a patient's circadian patterns of adrenal steroid secretion. Steroid profiling in dried blood spots (DBS) may allow for more frequent and sensitive monitoring.
We compared the agreement between 17α-hydroxyprogesterone (17-OHP) and androstenedione (D4A) levels determined from DBS samples and concurrently collected serum samples. Blood was drawn from 9 congenital adrenal hyperplasia patients every 4 h over a 24-hour period. Serum and DBS steroid levels were measured by liquid chromatography tandem mass spectrometry.
DBS determinations of 17-OHP overestimated corresponding serum levels (mean difference 1.67 ng/ml), and underestimated D4A serum levels (mean difference 0.84 ng/ml). However, the DBS assay yielded excellent agreement (97%) with serum 17-OHP, but did considerably poorer for D4A (31%).
Our results indicate an excellent agreement between DBS and serum 17-OHP measurements to identify the peaks and troughs associated with an individual's circadian pattern. Larger-scale studies are required to evaluate the utility of DBS for home monitoring and to determine if more frequent monitoring leads to improved clinical outcomes.
PMCID: PMC3202930  PMID: 20798478
Congenital adrenal hyperplasia; 17α-hydroxyprogesterone; Tandem mass spectrometry; Circadian rhythms of hormones; Glucocorticoid therapy
14.  Vitamin D Insufficiency and Prognosis in Non-Hodgkin's Lymphoma 
Journal of Clinical Oncology  2010;28(27):4191-4198.
Vitamin D insufficiency is common in the United States, with low levels linked in some studies to higher cancer incidence, including non-Hodgkin's lymphoma (NHL). Recent data also suggest that vitamin D insufficiency is related to inferior prognosis in some cancers, although there are no data for NHL.
Patients and Methods
We tested the hypothesis that circulating 25-hydroxyvitamin D [25(OH)D] levels are predictive of event-free survival (EFS) and overall survival (OS) in a prospective cohort of 983 newly diagnosed patients with NHL. 25(OH)D and 1,25-dihydroxyvitamin D [1,25(OH)2D] levels were measured by liquid chromatography-tandem mass spectrometry.
Mean age at diagnosis was 62 years (range, 19 to 94 years); 44% of patients had insufficient 25(OH)D levels (< 25 ng/mL) within 120 days of diagnosis. Median follow-up was 34.8 months; 404 events and 193 deaths (168 from lymphoma) occurred. After adjusting for known prognostic factors and treatment, 25(OH)D insufficient patients with diffuse large B-cell lymphoma (DLBCL) had inferior EFS (hazard ratio [HR], 1.41; 95% CI, 0.98 to 2.04) and OS (HR, 1.99; 95% CI, 1.27 to 3.13); 25(OH)D insufficient patients with T-cell lymphoma also had inferior EFS (HR, 1.94; 95% CI, 1.04 to 3.61) and OS (HR, 2.38; 95% CI, 1.04 to 5.41). There were no associations with EFS for the other NHL subtypes. Among patients with DLBCL and T-cell lymphoma, higher 1,25(OH)2D levels were associated with better EFS and OS, suggesting that any putative tumor 1-α-hydroxylase activity did not explain the 25(OH)D associations.
25(OH)D insufficiency was associated with inferior EFS and OS in DLBCL and T-cell lymphoma. Whether normalizing vitamin D levels in these patients improves outcomes will require testing in future trials.
PMCID: PMC2953973  PMID: 20713849
15.  Comparison of Metabolism of Vitamins D2 and D3 in Children With Nutritional Rickets 
Journal of Bone and Mineral Research  2010;25(9):1988-1995.
Children with calcium-deficiency rickets may have increased vitamin D requirements and respond differently to vitamin D2 and vitamin D3. Our objective was to compare the metabolism of vitamins D2 and D3 in rachitic and control children. We administered an oral single dose of vitamin D2 or D3 of 1.25 mg to 49 Nigerian children—28 with active rickets and 21 healthy controls. The primary outcome measure was the incremental change in vitamin D metabolites. Baseline serum 25-hydroxyvitamin D [25(OH)D] concentrations ranged from 7 to 24 and 15 to 34 ng/mL in rachitic and control children, respectively (p < .001), whereas baseline 1,25-dihydroxyvitamin D [1,25(OH)2D] values (mean ± SD) were 224 ± 72 and 121 ± 34 pg/mL, respectively (p < .001), and baseline 24,25-dihydroxyvitamin D [24,25(OH)2D] values were 1.13 ± 0.59 and 4.03 ± 1.33 ng/mL, respectively (p < .001). The peak increment in 25(OH)D was on day 3 and was similar with vitamins D2 and D3 in children with rickets (29 ± 17 and 25 ± 11 ng/mL, respectively) and in control children (33 ± 13 and 31 ± 16 ng/mL, respectively). 1,25(OH)2D rose significantly (p < .001) and similarly (p = .18) on day 3 by 166 ± 80 and 209 ± 83 pg/mL after vitamin D2 and D3 administration, respectively, in children with rickets. By contrast, control children had no significant increase in 1,25(OH)2D (19 ± 28 and 16 ± 38 pg/mL after vitamin D2 and D3 administration, respectively). We conclude that in the short term, vitamins D2 and D3 similarly increase serum 25(OH)D concentrations in rachitic and healthy children. A marked increase in 1,25(OH)2D in response to vitamin D distinguishes children with putative dietary calcium-deficiency rickets from healthy children, consistent with increased vitamin D requirements in children with calcium-deficiency rickets. © 2010 American Society for Bone and Mineral Research.
PMCID: PMC3153403  PMID: 20499377
metabolic bone; vitamin D; calcium; pediatric; nutrition
16.  LC-MS/MS in the Clinical Laboratory – Where to From Here? 
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has seen enormous growth in clinical laboratories during the last 10–15 years. It offers analytical specificity superior to that of immunoassays or conventional high performance/pressure liquid chromatography (HPLC) for low molecular weight analytes and has higher throughput than gas chromatography-mass spectrometry (GC-MS). Drug/Toxicology and Biochemical Genetics/Newborn Screening laboratories were at the vanguard of clinical LC-MS/MS use, but have been eclipsed by Endocrine laboratories. In USA reference/referral laboratories, most steroids and biogenic amines are now assayed by LC-MS/MS, and the technology has started to penetrate into smaller laboratories. Assays for mineralo- and gluco-corticoids and their precursors, sex steroids, metanephrines and 25-hydroxy vitamin D highlight the advantages of LC-MS/MS.
However, several limitations of LC-MS/MS have become apparent, centring on the interacting triangle of sensitivity – specificity – throughput. While sample throughput is higher than for conventional HPLC or GC-MS, it lags behind automated immunoassays. Techniques which improve throughput include direct sample injection, LC-multiplexing and samplemultiplexing. Measures to improve specificity and sensitivity include sample clean-up and optimising chromatography to avoid interferences and ion suppression due to sample-matrix components. Next generation instrumentation may offer additional benefits.
The next challenge for clinical LC-MS/MS is peptide/protein analysis. The quest for multi-biomarker profiles for various diseases has largely failed, but targeted peptide and protein testing by LC-MS/MS, directed at analytical and clinical questions that need to be answered, is proving highly successful. We anticipate that this will result in similar growth of clinical protein/peptide LC-MS/MS as has been seen for low molecular weight applications.
PMCID: PMC3052391  PMID: 21451775
17.  Assessment of synthetic glucocorticoids in asthmatic sputum 
Allergy & Rhinology  2011;2(1):33-35.
Nonadherence with anti-inflammatory treatment is a frequent cause of continued symptoms in asthmatic patients. Clinical assessments including patient-reported medication administration may provide the asthma specialist incomplete information regarding actual adherence to anti-inflammatory medications. The objective of this report was to describe the first case where adherence to inhaled asthma therapy was assessed by direct analysis of glucocorticoids in induced sputum. The patient's blood, urine, and sputum were tested for synthetic corticosteroids using mass spectrometry. To evaluate a clinical suspicion of poor adherence, sputum, urine, and blood were used to assess for current compliance to medication use. We report a case where asthma specialists attributed poorly controlled asthma to nonadherence to medical therapy. After modification of the medical regimen, adherence with oral and inhaled steroids was assessed—via examination of the urine, blood, and sputum. Direct analysis of glucocorticoids in sputum is feasible and in theory could provide a novel tool to document current medication adherence. Concomitant assessment of glucocorticoids and eosinophils in the same induced sputum specimen could provide insight into possible steroid resistance in select referral patients with difficult asthma.
PMCID: PMC3390127  PMID: 22852112
Adherence; asthma; budesonide; drug monitoring; fluticasone propionate; methylprednisolone; prednisolone; prednisone; steroids; steroid dependent
18.  Comparison of Sex Steroid Measurements in Men by Immunoassay versus Mass Spectroscopy and Relationships with Cortical and Trabecular Volumetric Bone Mineral Density 
While immunoassays have been used extensively for measurement of serum testosterone (T) and estradiol (E2) levels, there is concern about their specificity, particularly at low E2 levels as present in men.
We compared T and E2 measured by mass spectroscopy to levels measured by immunoassay in men (n = 313, age 22 to 91 years) and related these to volumetric bone mineral density (vBMD) at various skeletal sites.
Serum T and non-SHBG bound (or bioavailable) T levels by immunoassay correlated well with the corresponding mass spectroscopy measurements (R = 0.90 and 0.95, respectively, P < 0.001); the correlations for serum E2 measured using the two techniques were less robust (R = 0.63 for total E2 and 0.84 for bioavailable E2, P < 0.001). Overall relationships between serum bioavailable T and E2 levels with vBMD at various skeletal sites were similar for the immunoassay and mass spectroscopic measures.
Although E2 levels with immunoassay correlate less well with the mass spectroscopic measurements than do the T measurements in men, our findings indicate that the fundamental relationships observed previously between vBMD and the sex steroids by immunoassay are also present with the mass spectroscopic measurements.
PMCID: PMC2636568  PMID: 18338096
Assays; Osteoporosis; Bone

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