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1.  Increasing Incidence of Nutritional Rickets: A Population-Based Study in Olmsted County, Minnesota 
Objective
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.
Results
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).
Conclusion
Nutritional rickets remains rare, but the incidence has dramatically increased after 2000. Not all cases can be attributed to vitamin D deficiency.
doi:10.1016/j.mayocp.2012.10.018
PMCID: PMC3612965  PMID: 23374621
bone diseases; metabolic; epidemiology; pediatric; vitamin D
2.  Comparison between Immunoturbidimetry, Size-Exclusion Chromatography, and LC-MS to Quantify Urinary Albumin 
Clinical chemistry  2008;54(9):1504-1510.
BACKGROUND
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.
METHODS
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.
RESULTS
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.
CONCLUSIONS
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.
doi:10.1373/clinchem.2008.107508
PMCID: PMC3903150  PMID: 18617580
3.  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.
doi:10.1159/000339149
PMCID: PMC3472801  PMID: 22759465
alveolar macrophage; sarcoidosis; cathelicidin; vitamin D; cytokines; steroid receptor co-activators
4.  Vitamin D (25OHD) Serum Seasonality in the United States 
PLoS ONE  2013;8(6):e65785.
Background
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.
Objectives
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.
Methods
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.
Results
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.
Conclusions
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).
doi:10.1371/journal.pone.0065785
PMCID: PMC3689782  PMID: 23805188
5.  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.
doi:10.1677/ERC-10-0004
PMCID: PMC3417306  PMID: 20418362
pheochromocytoma; paraganglioma; genetic testing
6.  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.
doi:10.1183/09031936.00146509
PMCID: PMC3070416  PMID: 20595151
Pulmonary Disease, Chronic Obstructive; Smoking; Spirometry; Vitamin D
7.  Comparison of Multiple Steroid Concentrations in Serum and Dried Blood Spots throughout the Day of Patients with Congenital Adrenal Hyperplasia 
Background/Aim
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.
Methods
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.
Results
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%).
Conclusions
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.
doi:10.1159/000315910
PMCID: PMC3202930  PMID: 20798478
Congenital adrenal hyperplasia; 17α-hydroxyprogesterone; Tandem mass spectrometry; Circadian rhythms of hormones; Glucocorticoid therapy
8.  Vitamin D Insufficiency and Prognosis in Non-Hodgkin's Lymphoma 
Journal of Clinical Oncology  2010;28(27):4191-4198.
Purpose
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.
Results
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.
Conclusion
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.
doi:10.1200/JCO.2010.28.6674
PMCID: PMC2953973  PMID: 20713849
9.  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.
doi:10.1002/jbmr.99
PMCID: PMC3153403  PMID: 20499377
metabolic bone; vitamin D; calcium; pediatric; nutrition
10.  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
11.  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.
doi:10.2500/ar.2011.2.0002
PMCID: PMC3390127  PMID: 22852112
Adherence; asthma; budesonide; drug monitoring; fluticasone propionate; methylprednisolone; prednisolone; prednisone; steroids; steroid dependent
12.  Comparison of Sex Steroid Measurements in Men by Immunoassay versus Mass Spectroscopy and Relationships with Cortical and Trabecular Volumetric Bone Mineral Density 
Introduction
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1007/s00198-008-0591-5
PMCID: PMC2636568  PMID: 18338096
Assays; Osteoporosis; Bone
13.  Atorvastatin Inhibits Hypercholesterolemia-Induced Cellular Proliferation and Bone Matrix Production in the Rabbit Aortic Valve 
Circulation  2002;105(22):2660-2665.
Background
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.
Conclusions
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

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