DiaSorin Radioimmunoassay Research Articles

Vitamin D, Cognitive Function, and Gait Speed in Older Adults: a NHANES Study

Vitamin D deficiency has been linked to poor cognition and neuromuscular impairment. We evaluated the relationships of vitamin D levels with cognitive function and gait speed in older adults. The study sample included 1076 individuals (age = > 60 years) from the 2001–2002 National Health Examination Survey (NHANES). The relationships between vitamin D and cognition and gait speed were studied. Cognitive function was measured as the number of questions correct on a digit-symbol test. Gait speed was measured as seconds to walk 20 ft. Serum 25(OH)D concentrations were measured via the DiaSorin radioimmunoassay. In our study, 32% were deficient in vitamin D (< 20 ng/ml) and 43% were insufficient in vitamin D (20–29 ng/ml). Only 25% had vitamin D values in the normal range (30–100 ng/ml). The mean vitamin D level, cognition score, and gait speed were found to be 24.71 ng/ml, 48.55 number correct, and 6.80 s, respectively. The relationship between vitamin D and cognitive function was an inverted U-shaped curve. Maximum cognition score was at a vitamin D value of 28.09 ng/ml. The relationship between vitamin D and gait speed was U-shaped. Minimum walking time was at a vitamin D level of 31.42 ng/ml. Optimal vitamin D levels were similar for both cognition and gait speed. Given the high prevalence of vitamin D deficiency in the elderly, we recommend that older individuals are tested and treated to achieve 25(OH) D values at least between 28 and 30 ng/ml.

The association of telomere length and serum 25-hydroxyvitamin D levels in US adults: the National Health and Nutrition Examination Survey.

IntroductionHigher vitamin D levels and longer telomere length (TL) have been associated with lower risk of several chronic diseases and all-cause mortality. However, direct relationships between 25-hydroxyvitamin D (25(OH)D) levels and TL are not well established. Vitamin D could influence TL through its anti-inflammatory properties. This study aimed to assess the relationship between vitamin D levels and TL in US adults.Material and methodsParticipants of the National Health and Nutrition Examination Survey (NHANES) with data available on 25(OH)D and TL measures from 2001 to 2002 were included. 25(OH)D levels were measured by the DiaSorin Radioimmunoassay. We used multivariable-adjusted linear regression models, accounting for the survey design and sample weights.ResultsOf the 4347 eligible participants, 47.0% (n = 2045) were men. The mean age was 42.7 years overall, 49.2 years in men and 42.5 years in women (p = 0.060). After adjustment for age, race, marital status, education, and C-reactive protein, each 1 ng/ml higher 25(OH)D level was associated with a 0.045 (95% confidence interval (CI): 0.032 to 0.059) longer telomere-to-single copy gene (T/S) ratio. This was driven by a significant association in women (0.054 (0.043 to 0.064)) and in men (0.036 (0.020 to 0.052)). However, after we further adjusted for smoking, body mass index, and physical activity, no significant relation was found in the overall sample (β coefficient –0.026, 95% CI: –3.16, 1.67), for men (–0.016 (–3.72, 2.64)), or for women (–0.052 (–6.85, 2.26)).ConclusionsOur findings support a possible positive association between 25(OH)D levels and telomere length. The implications of this association deserve further investigation.

Vitamin D deficiency is independently associated with greater prevalence of erectile dysfunction: The National Health and Nutrition Examination Survey (NHANES) 2001–2004

Background and aimsErectile dysfunction (ED) and atherosclerotic cardiovascular disease (ASCVD) share many common risk factors, and vascular ED is a marker for increased ASCVD risk. Low 25-hydroxyvitamin D [25(OH)D] concentrations have been associated with increased ASCVD risk, but less is known regarding the relationship of low 25(OH)D with ED. We determined whether 25(OH)D deficiency is associated with ED independent of ASCVD risk factors. MethodsWe performed cross-sectional analyses of 3390 men aged ≥20 years free of ASCVD who participated in NHANES 2001–2004. Serum 25(OH)D was measured by the DiaSorin radioimmunoassay; deficiency was defined as levels <20 ng/ml (<50 nmol/L). Self-reported ED, assessed by a single validated question, was defined as men who reported being “never” or “sometimes able” to maintain an erection. We assessed the relationship between 25(OH)D deficiency and ED prevalence using adjusted Poisson regression methods. ResultsAfter accounting for NHANES sampling, the weighted prevalence of 25(OH)D deficiency and of ED were 30% and 15.2%, respectively. 25(OH)D levels were lower in men with vs. those without ED (mean 22.8 vs 24.3 ng/mL, respectively; p = 0.0005). After adjusting for lifestyle variables, comorbidities, and medication use, men with 25(OH)D deficiency had a higher prevalence of ED compared to those with levels ≥30 ng/ml (Prevalence Ratio 1.30, 95% CI 1.08–1.57). ConclusionIn this cross-sectional analysis of a representative sample of U.S. men, vitamin D deficiency was associated with an increased prevalence of ED independent of ASCVD risk factors. Additional research is needed to evaluate whether treating vitamin D deficiency improves erectile function.

Abstract 15583: Serum Vitamin D Deficiency is Independently Associated With Greater Prevalence of Erectile Dysfunction: Results From the National Health and Nutrition Examination Survey (NHANES) 2001-2004

Background: Erectile dysfunction (ED) and cardiovascular disease (CVD) share common risk factors. Furthermore, up to 80% of ED is from vascular causes, and thus ED may be a marker for subclinical CVD. Low serum 25-hydroxyvitamin D [25(OH)D] levels have been associated with increased CVD risk, perhaps mediated through hypertension and diabetes, but it is unclear if low serum 25(OH)D is associated with ED, independent of CVD risk factors. Methods: We performed a cross-sectional analysis of 3,486 men aged ≥20 years free of CVD who participated in NHANES 2001-2004. Serum 25(OH)D was measured by the DiaSorin radioimmunoassay. We defined vitamin D deficiency as a serum level of 25(OH)D &lt;20 ng/mL. Self-reported erectile function was assessed by a single question: “How would you describe your ability to get and keep an erection adequate for satisfactory intercourse?” We defined ED as those who answered “sometimes able” or “never able.” We assessed the relationship between serum 25(OH)D deficiency and prevalence of ED using logistic regression after adjusting for potential confounders. Results: The prevalence of vitamin D deficiency and of ED was 30% and 16.4%, respectively. Serum 25(OH)D levels were statistically significantly lower in men with vs those without ED (mean 22.9 vs 24.3 ng/mL, respectively; p=0.0007). Vitamin D deficient men had a significantly higher prevalence of ED compared to those without deficiency (age- and race-adjusted OR 1.53, 95% CI 1.17-2.00). This association was materially unchanged after adjusting for multiple lifestyle variables, comorbidities, and medication use (OR 1.32, 95% CI 1.02-1.74) [ Figure ]. Conclusion: In this cross-sectional analysis of a representative sample of U.S. men, vitamin D deficiency was associated with an increased prevalence of ED independent of CVD risk factors. Additional research is needed to evaluate if treating vitamin D deficiency results in an improvement of erectile function or prevents vascular events.

AB0086 IL - 17 Levels with Vitamin D Repletion in Rheuamtoid Arthritis

BackgroundIncreasing scientific evidence implicates a pathogenic role of Th17 cells and associated cytokines in rheumatoid arthritis (RA). Vitamin D may have suppressive effect on Th17 cells. In vitro studies show...

Pretreatment vitamin D level and response to neoadjuvant chemotherapy in women with breast cancer on the I‐SPY trial (CALGB 150007/150015/ACRIN6657)

Laboratory studies suggest that vitamin D (vitD) enhances chemotherapy-induced cell death. The objective of this study was to determine whether pretreatment vitD levels were associated with response to neoadjuvant chemotherapy (NACT) in women with breast cancer. Study patients (n = 82) were enrolled on the I-SPY TRIAL, had HER2-negative tumors, and available pretreatment serum. VitD levels were measured via DiaSorin radioimmunoassay. The primary outcome was pathologic residual cancer burden (RCB; dichotomized 0/1 vs. 2/3). Secondary outcomes included biomarkers of proliferation, differentiation, and apoptosis (Ki67, grade, Bcl2, respectively) and 3-year relapse-free survival (RFS). Mean and median vitD values were 22.7 ng/mL (SD 11.9) and 23.1 ng/mL, respectively; 72% of patients had levels deemed “insufficient” (<30 ng/mL) by the Institute of Medicine (IOM). VitD level was not associated with attaining RCB 0/1 after NACT (univariate odds ratio [OR], 1.01; 95% CI, 0.96–1.05) even after adjustment for hormone receptor status (HR), grade, Ki67, or body mass index (BMI). Lower vitD levels were associated with higher tumor Ki67 adjusting for race (OR, 0.95; 95% CI, 0.90–0.99). VitD level was not associated with 3-year RFS, either alone (hazard ratio [HzR], 0.98; 95% CI, 0.95–1.02) or after adjustment for HR, grade, Ki-67, BMI, or response. VitD insufficiency was common at the time of breast cancer diagnosis among women who were candidates for NACT and was associated with a more proliferative phenotype. However, vitD levels had no impact on tumor response to NACT or short-term prognosis.

Vitamin D status, functional decline, and mortality in peripheral artery disease

Associations of vitamin D levels with prospectively measured functional decline and mortality in people with lower extremity peripheral artery disease (PAD) are unknown. We determined whether lower baseline vitamin D levels are associated with a faster decline in functional performance and higher mortality among people with and without PAD. A total of 658 participants (395 with PAD) underwent baseline measurement of 25-hydroxyvitamin D (DiaSorin radioimmunoassay), a 6-minute walk test, 4-meter walking velocity and the Short Physical Performance Battery (SPPB), and were followed annually for up to 4 years. Analyses were adjusted for age, sex, race, body mass index, comorbidities, the ankle-brachial index, and other confounders. Among participants with PAD, lower baseline vitamin D levels were associated with a faster decline in the 6-minute walk (vitamin D < 30 nmol/L: -70.0 feet/year; vitamin D 30 to < 50 nmol/L: -72.3 feet/year; vitamin D 50 to < 75 nmol/L: -35.5 feet/year; vitamin D 75 to < 120 nmol/L: -35.9 feet/year; p trend=0.012). PAD participants with vitamin D < 30 nmol/L had a faster decline in the SPPB and 6-minute walk compared to those with levels of 50 to < 75 (p=0.034 and p=0.04, respectively). Among participants without PAD, lower vitamin D was associated with a faster decline in the fast 4-meter walking velocity (p trend=0.003). There were no significant associations of baseline vitamin D levels with all-cause or cardiovascular disease mortality in PAD or non-PAD participants. In conclusion, among individuals with and without PAD, low vitamin D status was associated with a faster decline in some measures of functional performance but was not related to mortality.

Plasma Vitamin D Levels are Lower among Ethnic Indians in Matched Pairs of Male Acute Ischaemic Stroke Patients of Indian and Chinese Ethnicity

Introduction: Ethnic Indians have a high burden of vascular disease. Vitamin D deficiency is associated with an increased risk of vascular disease including ischaemic stroke. Vitamin D levels are lower among ethnic Indians compared to ethnic Chinese in healthy individuals. We compared the plasma 25-hydroxyvitamin D [25(OH)D] levels between ethnic Indians and Chinese male ischaemic stroke patients. Methods: We prospectively recruited consecutive ethnic Indian acute ischaemic stroke male patients within seven days of symptom onset, who were admitted to the Singapore General Hospital. The subsequent ethnic Chinese acute ischaemic stroke male patient matched for age and diabetes status was recruited. Plasma 25(OH)D was measured using DiaSorin radioimmunoassay. Results: We studied 22 matched pairs of ethnic Indian and Chinese ischaemic stroke male patients. Mean plasma 25(OH)D level was lower among ethnic Indians (16.7 ± 6.1 μg/L) than ethnic Chinese patients (19.7 ± 4.4 μg/L) ( p=0.04). The mean difference was 2.9 ± 6.46 μg/L corresponding to 12.9 ± 33.1%. Conclusion: Ethnic Indian male ischaemic stroke patients had lower vitamin D levels compared to matched ethnic Chinese counterparts. Thus it is important to consider ethnicity in the understanding of stroke risk due to vitamin D deficiency.

Calculated free and bioavailable vitamin D metabolite concentrations in vitamin D-deficient hip fracture patients after supplementation with cholecalciferol and ergocalciferol

We previously showed that oral cholecalciferol and ergocalciferol have comparable effects in decreasing circulating parathyroid hormone (PTH), despite a greater increase in total serum 25-hydroxyvitamin D (25OHD) concentration with cholecalciferol supplementation. However, the effects of cholecalciferol and ergocalciferol on total serum 1,25-dihydroxyvitamin D (1,25(OH)2D), vitamin D-binding protein (DBP), free 25OHD and free 1,25(OH)2D concentrations have not been previously studied.We randomized 95 hip fracture patients (aged 83±8years) with vitamin D deficiency (serum 25OHD <50nmol/L) to oral supplementation with either cholecalciferol 1000IU/day (n=47) or ergocalciferol 1000IU/day (n=48) for three months. All were given matching placebos of the alternative treatment to maintain blinding. We measured serum 25OHD (high-pressure liquid chromatography), 1,25(OH)2D (Diasorin radioimmunoassay), DBP (immunonephelometry), ionized calcium (Bayer 800 ion-selective electrode) and albumin (bromocresol green) concentrations before and after treatment. We calculated free and bioavailable concentrations of the vitamin D metabolites using albumin and DBP, and calculated free vitamin D metabolite indices as the ratios between the molar concentrations of the vitamin D metabolites and DBP.Seventy participants (74%) completed the study with paired samples for analysis. Total serum 1,25(OH)2D did not change significantly with either treatment (p>0.05, post-treatment vs baseline). Both treatments were associated with comparable increases in DBP (cholecalciferol: +18%, ergocalciferol: +16%, p=0.32 between groups), albumin (cholecalciferol: +31%, ergocalciferol: +21%, p=0.29 between groups) and calculated free 25OHD (cholecalciferol: +46%, ergocalciferol: +36%, p=0.08), with comparable decreases in free 1,25(OH)2D (cholecalciferol: −17%, ergocalciferol: −19%, p=0.32 between groups). In the treatment-adherent subgroup the increase in ionized calcium was marginally greater with cholecalciferol compared with ergocalciferol (cholecalciferol: +8%, ergocalciferol: +5%, p=0.03 between groups). There were no significant differences between the treatments in their effects on the calculated bioavailable concentrations or free indices of the vitamin D metabolites (p>0.05 between groups).In vitamin D-deficient hip fracture patients, oral supplementation with cholecalciferol and ergocalciferol had no effect on total serum 1,25(OH)2D, and comparable effects on DBP and free vitamin D metabolite concentrations. This is despite cholecalciferol having greater effects than ergocalciferol in increasing total 25OHD, and in increasing ionized calcium in treatment-adherent subjects. These findings may explain why cholecalciferol and ergocalciferol supplementation result in similar magnitudes of PTH reduction, but implicate potential differences in other vitamin D metabolites, such as 24,25(OH)2D, that could explain their different effects on ionized calcium.

Serum 25-hydroxyvitamin D status in individuals with psoriasis in the general population

There is a dearth of data assessing serum 25-hydroxyvitamin D (25[OH]D) status in psoriasis. This population-based study in the United States evaluated 25(OH)D status in psoriasis and examined the associations between 25(OH)D and psoriasis severity. The 2003-2006 National Health and Nutrition Examination Survey was analyzed. Participants aged 20-59 years self-reported psoriasis, psoriasis body surface area (BSA), and psoriasis life impairment (PLI). Serum 25(OH)D was assessed with the DiaSorin radioimmunoassay. General linear models were used to examine the associations between psoriasis and 25(OH)D while accounting for age, gender, race/ethnicity, season, and body mass index (BMI). Among the 5,841 participants with complete data, 148 reported a psoriasis diagnosis. Mean 25(OH)D levels and deficiency prevalences (<20 and <30 ng/mL) were not different between those with and without psoriasis. Among those with psoriasis, a multivariate model showed participants with BSA >10 hand palms trended towards lower 25(OH)D compared to those with minimal BSA (-4.98 ng/mL, P = 0.07). PLI was not associated with 25(OH)D, but BMI showed an inverse association with 25(OH)D (coefficient = -0.40, P < 0.001). In summary, dermatologists may consider measures of adiposity as better screening tools for vitamin D deficiency than BSA involvement among psoriatics with mild-to-moderate disease.

THU0421 Rheumatoid Versus Psoriatic Arthritis: Similar or Different Bone Loss?

BackgroundRheumatoid (RA) and psoriatic (PsA) arthritis are chronic diseases characterized by inflammation, often common therapies and prolonged joint immobilization are possible risk factors of bone loss and fragility fractures.1 Vitamin...

Abstract P3-06-10: Pretreatment Vitamin D Levels and Response to Neoadjuvant Chemotherapy in the I-SPY 1 TRIAL

Abstract Background: Vitamin D has been shown, in vitro, to promote differentiation and apoptosis while suppressing proliferation of breast cancer cells. Breast cancer cell lines show improved responsiveness to chemotherapy after pretreatment with vitamin D. This relationship has not been explored in breast cancer patients. The objective of this study was to determine whether pretreatment vitamin D levels were associated with response to neoadjuvant chemotherapy. Secondary aims examined relationships between serum vitamin D levels, tumor biomarkers and survival. Methods: Study patients were enrolled on the I-SPY TRIAL of neoadjuvant chemotherapy for locally-advanced breast cancer, had tumors that were Her2-negative and had pretreatment serum available. Vitamin D levels were measured using the Diasorin radioimmunoassay. Outcomes included pathologic complete response (pCR)/residual cancer burden (RCB) and 3-year recurrence-free survival (RFS). The study had 80% power to detect an increase in the odds of response of 9.8% or more for every one ng/mL increase in serum vitamin D with a two sided α=0.05. Statistical analyses were performed using STATA software (Version 12). Logistic regression was used for multivariate modeling. Results: 157 subjects with Her2-negative tumors were enrolled on I-SPY1; 82 of these patients had serum available for analysis. The mean vitamin D value was 22.7ng/mL (SD 11.9), the median was 23.1ng/mL (IQR 13.08 to 30.52ng/mL); only 28% had “sufficient” levels (&amp;gt;30ng/mL). Vitamin D level was significantly lower in those of non-Caucasian race (p = 0.0001), with blood collected in winter/spring (p = 0.009) and with increasing body mass index (BMI; p-trend=0.01). Higher vitamin D levels were also associated with lower tumor Ki67 (OR 0.95, 95%CI 0.91, 0.99, p = 0.017). Vitamin D level was not associated with achievement of pCR in univariable analysis (OR 1.01, 95% CI 0.96, 1.05) and after adjustment for hormone receptor status (HRS; OR 1.02, 95%CI 0.97, 1.07), tumor grade (OR 1.01, 95%CI 0.96, 1.06), Ki67 (OR 1.02, 95%CI 0.97, 1.07) or BMI (OR 1.00, 95%CI 0.96, 1.05). Vitamin D was independently associated with Ki67 (OR 0.95, 95%CI 0.91, 0.99). Vitamin D level was also not associated with 3 year RFS in univariable analysis (HR 0.98, 95% CI 0.95, 1.02), and after adjustment for HRS (HR 0.99, 95%CI 0.95, 1.03), tumor grade (HR 0.99, 95%CI 0.95, 1.02), Ki-67 (HR 0.99, 95%CI 0.95, 1.03), BMI (HR 0.96, 95% CI 0.92, 1.01) or pCR (HR 0.99, 0.95, 1.02); no interaction was seen when stratifying on HRS (p-interaction= 0.71). Conclusions: The majority of patients in this study had insufficient vitamin D levels (&amp;lt;30ng/mL) at the time of diagnosis of breast cancer. Pre-chemotherapy vitamin D level did not predict response to neoadjuvant chemotherapy in these Her2-negative patients, despite being significantly associated with tumor proliferation, as measured by Ki-67; nor was it independently associated with RFS. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-06-10.

Abstract P1-09-02: Pilot study of a 1-year intervention of high-dose vitamin D in women at high risk for breast cancer.

Abstract Background: Selective estrogen receptor modulators (SERMs) have been shown to decrease breast cancer incidence among high-risk women, but uptake for prevention has been poor. Observational studies have demonstrated that serum 25-hydroxyvitamin D (25-OHD) is inversely related to breast cancer risk, such that levels &amp;gt;40 ng/ml are associated with about a 40% reduction in breast cancer risk compared to women who are vitamin D deficient (25-OHD &amp;lt;20 ng/ml). Uncertainty remains about whether vitamin D supplementation will reduce breast cancer risk, the optimal dose of vitamin D, and the target level of serum 25-OHD. We examined the safety of high dose vitamin D and the effects on serum 25-OHD in women at high risk for breast cancer. Methods: Forty high-risk women (defined as a 5-year Gail risk ³1.67%, lobular or ductal carcinoma in situ [LCIS/DCIS], BRCA1/BRCA2 mutation carrier, or stage I/II invasive breast cancer in remission for &amp;gt;5 years) were assigned to a 1-year intervention of vitamin D3 20,000 or 30,000 IU weekly. Other eligibility criteria included baseline mammographic density (MD) ≥25%, serum 25-OHD ≤32 ng/ml, no current SERM use and no history of kidney stones. Women underwent a digital mammogram at baseline and 12 months, and serial blood draws every 3 months. In addition, random core breast biopsies were conducted in premenopausal women, whereas postmenopausal women underwent a breast MRI at baseline and 12 months. Participants were monitored for toxicity, particularly hypercalcemia and hypercalciuria, every 3 months. The primary objective is to determine the safety and feasibility of high-dose vitamin D in this study population. Secondary objectives are to determine changes in breast density and blood-based biomarkers (25-OHD, 1,25(OH)D, PTH, IGF-I, IGFBP-3). Serum 25-OHD was measured by Diasorin radioimmunoassay. Results: From November 2007 to January 2011, 292 women were screened and 142 were ineligible. Main reasons for ineligibility (%) included 25-OHD &amp;gt;32 ng/ml (27), opted for SERM (23), prior kidney stones (11), and MD &amp;lt;25% (9). Of the 40 enrolled participants: median age 50 years (range, 37–73); pre/postmenopausal: 20/20; white/hispanic/black/asian: 19/14/6/1; median body mass index 26.6 kg/m2 (20–39.6); elevated Gail risk/LCIS/DCIS/stage I or II breast cancer: 20/10/8/2; mean baseline serum 25-OHD 20.2 ng/ml (9–31). Currently, 1 participant is on-study, 31 completed the intervention, 6 were lost to follow-up, 1 withdrew due to hypercalciuria (spot urine Ca/Cr &amp;gt;0.37) and 1 withdrew due to dyspepsia. Mean serum 25-OHD rose to 47 ng/ml at 3 months, 49.1 ng/ml at 6 months, and 53.7 ng/ml (range, 26–77) at 12 months. No significant hypercalcemia (serum Ca &amp;gt;10.5 mg/dl) occurred at either dose level. Imaging and biomarker analyses are ongoing. Discussion: We have demonstrated that a 1-year intervention of high-dose vitamin D3 is well tolerated and can increase serum 25-OHD above a target level of 40 ng/ml. This preliminary data has informed an ongoing phase IIb randomized placebo-controlled trial (SWOG 0812) of high-dose vitamin D in 200 high-risk premenopausal women and highlights the importance of early phase breast cancer chemoprevention trials with intermediate biomarker endpoints to test novel agents. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-09-02.

Preanalytical evaluation of serum 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 measurements using LC–MS/MS

BackgroundVitamin D testing is increasing worldwide. Although immunoassays are still widely used in Japan for the measurement of serum 25-hydroxyvitamin D (25OHD) as an indicator of vitamin D status, development of a simple and high-throughput MS-based method is still needed for routine use in clinical laboratories. MethodsWe designed a method using a triple quadrupole mass spectrometer equipped with a two-step separation approach that used the Aria TLX-2 HPLC system in the selected reaction monitoring mode. Analytical performance of the system and effects of various preanalytical factors were tested. ResultsHigh-throughput quantitative analysis of 25OHD3 and D2 at 15samples/h was achieved using 25μl of serum/plasma. Intra- and inter-assay CVs for 25OHD3 were 5% and 7%, respectively. Limit of detection for 25OHD3 was 0.31ng/ml. No significant effects were seen for clotting time, repeated freeze–thaw cycles, anti-coagulants and possible interfering substances. A good correlation (r2=0.947) was found between the present system and the DiaSorin radioimmunoassay. Serum 25OHD3 levels in apparently healthy Japanese subjects were 25.5±9.8ng/ml for men and 20.9±7.1ng/ml for women. ConclusionsThis high-throughput LC–MS/MS 25-OHD assay has the potential to be used as a routine clinical laboratory assay for assessing vitamin D status.

Analytical methods and performance of the immunoassay methods for determination of vitamin d in comparison to mass spectrometry / Analitičke metode i izvođenje imunometrijskih određivanja vitamina d u poređenju sa masenom spektrometrijom

Summary Demand for vitamin D testing has been on a constant rise worldwide, partially due to mounting evidence linking vitamin D status to overall health and well-being. Currently available assays measure 25-hydroxy vitamin D (25-OHD), a major circulating form of vitamin D. Available methodologies include immunoassays and mass spectrometry based methods (LC-MS/MS). Until recently, the only immunoassays available for diagnostic use in the US have been DiaSorin radioimmunoassay (RIA) and an automated immunoassay on a LIAISON® platform. Within the last year, Siemens and Abbott successfully launched immuno - assays for determination of total vitamin D on their respective automated platforms, Centaur® and ARCHITECT®. Development of robust and precise Vitamin D immunoassays has historically been plagued with difficulty. One of the major challenges is development of specific antibodies against such a small antigen. Vitamin D is also highly hy - drophobic molecule predominantly bound to vitamin D binding protein (DBP). It is likely, therefore, that immuno - assays might be affected to varying extent by the DBP concentration. Adoption of LC-MS/MS into clinical laboratories has enabled development of accurate and almost fully automated methods that could handle increasing volume demands, especially in large volume reference laboratories. Smaller to mid-size hospital laboratories as well as physician offices have neither funds nor technical expertise to implement LC-MS/MS based testing. Our laboratory at the University of Chicago Medical Center has also seen in - crease in vitamin D volume and currently performs close to 20,000 25-OHD assays per year. We have recently deve - loped an LC-MS/MS method for quantitation of 25-OHD2 (obtained from plant sources) and 25-OHD3 (endogenous and animal sources). Prior to acquisition of LC-MS/MS instrument, we performed 25-OHD analysis by RIA. Du - ring the transition period, we encountered several challenges, including the necessity to streamline sample preparation as well as the bias introduced by calibration dif ferences. We chose to match our LC-MS/MS method to the RIA method in order to make this transition transparent to the clinician. Most immunoassays available today are acceptable for clinical use and might be method of choice for smaller laboratories. Larger clinical laboratories and aca demic institutions that possess technical expertise, particularly the ones with large pediatric population where assay sensitivity and specificity may be important, might find LC-MS/MS methodology a more suitable choice.

Vitamin D Deficiency in Critically Ill Children

Vitamin D influences cardiovascular and immune function. We aimed to establish the prevalence of vitamin D deficiency in critically ill children and identify factors influencing admission 25-hydroxy vitamin D (25(OH)D) levels. We hypothesized that levels would be lower with increased illness severity and in children with serious infections. Participants were 511 severely or critically ill children admitted to the PICU from November 2009 to November 2010. Blood was collected near PICU admission and analyzed for 25(OH)D concentration by using Diasorin radioimmunoassay. We enrolled 511 of 818 (62.5%) eligible children. The median 25(OH)D level was 22.5 ng/mL; 40.1% were 25(OH)D deficient (level <20 ng/mL). In multivariate analysis, age and race were associated with 25(OH)D deficiency; summer season, vitamin D supplementation, and formula intake were protective; 25(OH)D levels were not lower in the 238 children (46.6%) admitted with a life-threatening infection, unless they had septic shock (n = 51, 10.0%) (median 25(OH)D level 19.2 ng/mL; P = .0008). After adjusting for factors associated with deficiency, lower levels were associated with higher admission day illness severity (odds ratio 1.19 for a 1-quartile increase in Pediatric Risk of Mortality III score per 5 ng/mL decrease in 25(OH)D, 95% confidence interval 1.10-1.28; P < .0001). We found a high rate of vitamin D deficiency in critically ill children. Given the roles of vitamin D in bone development and immunity, we recommend screening of those critically ill children with risk factors for vitamin D deficiency and implementation of effective repletion strategies.

Vitamin D status and functional performance in peripheral artery disease

The clinical implications of low vitamin D in peripheral artery disease (PAD) are unknown. We hypothesized that among individuals with PAD, lower levels of 25-hydroxyvitamin D would be associated with poorer functional performance, more adverse calf muscle characteristics, and poorer peripheral nerve function. Participants were 402 men and women with PAD who underwent measurement of 25-hydroxyvitamin D (DiaSorin radioimmunoassay) along with 6-minute walk testing, measurement of walking velocity at usual and fastest pace, computed tomography-measured calf muscle density, and peripheral nerve conduction velocity (NCV). Among PAD participants, 20.4% had 25-hydroxyvitamin D levels < 30 nmol/L, consistent with deficient vitamin D status. Adjusting for age, sex, and race, lower 25-hydroxyvitamin D levels were associated with poorer 6-minute walk performance (p trend = 0.002), slower usual-paced 4-meter walking velocity (p trend = 0.031), slower fast-paced 4-meter walking velocity (p trend = 0.043), and lower calf muscle density (p trend = 0.031). After additional adjustment for body mass index (BMI) and diabetes, none of these associations remained statistically significant. However, lower levels of 25-hydroxyvitamin D were associated with poorer peroneal NCV (p trend = 0.013) and poorer sural NCV (p trend = 0.039), even after adjusting for age, sex, race, BMI, comorbidities, smoking, physical activity, and other confounders. In conclusion, vitamin D deficiency is common among people with PAD encountered in clinical settings. After adjusting for BMI and diabetes mellitus, we found no significant associations of lower levels of 25-hydroxyvitamin D with poorer functional performance or calf muscle characteristics. Associations of low vitamin D levels with poorer peripheral nerve function require further study.

Current 25-hydroxyvitamin D assays: Do they pass the test?

BackgroundVitamin D testing is becoming increasingly important with recent research demonstrating a correlation between vitamin D insufficiency and metabolic diseases, immunodeficiencies and other diseases. However, existing 25-hydroxyvitamin D (25OHD) assays lack comparability to the candidate reference method, causing difficulties in diagnosis and monitoring of vitamin D deficiency. MethodsWe looked at the accuracy of 3 automated assays (Roche Diagnostics Elecsys® Total 25OHD assay, Abbott Architect® Total vitamin D assay, Advia Centaur® vitamin D Total assay) and Diasorin® Radioimmunoassay (RIA) compared to a routine laboratory Liquid Chromatography–Tandem Mass Spectrometry (LC–MS/MS). ResultsThe correlation based on Passing Bablok regression was good with the slopes between 0.95 and 1.31 and the intercepts between −3.24 and 3.68. However, a significant positive bias was observed using the Abbott Architect and the Diasorin RIA. Using published analytical goals of coefficient of variation (CV) <10% and bias <5%, most methods did not meet these criteria. Using measurement of uncertainty of 9%, most methods were able to meet criteria using quality control materials but not patient samples. ConclusionInadequacies of these assay performances are contributed by differences in method of extraction of vitamin D from vitamin D binding protein, cross-reactivities to 25OHD2, 25OHD3 and other vitamin D metabolites, matrix interferences and a lack of standardization.

Estimation of Estradiol in Mouse Serum Samples: Evaluation of Commercial Estradiol Immunoassays

The University of Virginia Center for Research in Reproduction Ligand Core performed an evaluation of nine commercial estradiol (E2) immunoassays for use with mouse serum. The evaluation had two components. 1) Recovery Studies: a mouse pool was spiked with E2 concentrations across the assay range, and percent recovery and parallelism to the assay standard curve were determined. 2) Correlation Studies: serum pools were collected from intact females, ovariectomized (OVX) and OVX-E2 treated mice and E2 assayed, then measured by gas chromatography/tandem mass spectrometry (GC/MSMS) for comparison to a gold standard method. Recovery results showed that E2 recovery from spiked mouse pools varied greatly (from <18% to >640%) among kits tested. However, three kits (DiaSorin Radioimmunoassay, Siemens Double Antibody RIA, and CalBiotech Enzyme Immunoassay) showed reasonable recoveries and parallelism. Data collected from the Correlation Study showed that values from intact, OVX and OVX-E2-treated mouse pools varied by several fold vs. GC/MSMS for most of the kits tested. The DiaSorin RIA and CalBiotech Enzyme Immunoassay Kits showed the best correlation to GC/MSMS. Unfortunately, while this evaluation was ongoing, the DiaSorin Kit was discontinued. In summary, the CalBiotech Kit was the only available assay tested that demonstrated good E2 parallelism to the assay standard curve and accuracy vs. a gold standard method (i.e. GC/MSMS). Also of note, the CalBiotech assay is sensitive and requires minimal sample volume. Therefore, based on these findings the CalBiotech E2 assay has been implemented for use in mouse serum samples within the Ligand Core.

High serum 25-hydroxyvitamin D is associated with a low incidence of stress fractures

Low serum 25-hydroxyvitamin D [25(OH)D] concentrations are associated with hip fractures, but the dose-response relationship of serum 25(OH)D with risk of stress fractures in young women is unknown. This nested case-control study in a cohort of female Navy recruits was designed to determine whether those with low prediagnostic serum 25(OH)D concentrations had greater risk of stress fracture. Sera were drawn in 2002-2009 from 600 women who were diagnosed subsequently with stress fracture of the tibia or fibula and 600 matched controls who did not experience a stress fracture. The 25(OH)D concentration was measured using the DiaSorin radioimmunoassay method. Controls were individually matched to cases on race (white, black, or other), length of service (±30 days), and day blood was drawn (±2 days). There was approximately half the risk of stress fracture in the top compared with the bottom quintile of serum 25(OH)D concentration (odds ratio [OR] = 0.51, 95% CI 0.34-0.76, p ≤ 0.01). The range of serum 25(OH)D in the lowest quintile was 1.5 to 19.7 (mean 13.9) ng/mL, whereas in the highest it was 39.9 to 112 (mean 49.7) ng/mL. It is concluded that there was a monotonic inverse dose-response gradient between serum 25(OH)D and risk of stress fracture. There was double the risk of stress fractures of the tibia and fibula in women with serum 25(OH)D concentrations of less than 20 ng/mL compared to those with concentrations of 40 ng/mL or greater. A target for prevention of stress fractures would be a serum 25(OH)D concentration of 40 ng/mL or greater, achievable with 4000 IU/d of vitamin D(3) supplementation.