Controversy and consensus regarding vitamin D: Recent methodological changes and the risks and benefits of vitamin D supplementation.

A dramatic and sustained surge in vitamin D test numbers has been attributed to the extraskeletal and probable intra/paracrine effects of vitamin D and not the important role of vitamin D in the regulation of extracellular calcium homeostasis and bone metabolism. This review summarizes recent data regarding the skeletal and extraskeletal effects of vitamin D, provides an overview of current methods of 25-hydroxyvitamin D measurement and includes the beneficial and adverse effects of vitamin D replacement. The role of 1,25-dihydroxyvitamin D, 24,25-dihydroxyvitamin D, vitamin D binding protein and free hormone levels are explored and potential future developments in this area are discussed. The adoption of a reference method for the measurement of 25-hydroxyvitamin D, certified reference standards and an independent certification program administered by the Centre of Disease Control is expected to improve routine analytical performance and is a major, crucial step forward. Improvements in accuracy, precision and sensitivity of 25-hydroxyvitamin D measurement is an important prelude to accurately defining the desirable level of 25-hydroxyvitamin D that is associated with the lowest risk for falls and fractures. Finally, the results of ongoing large, prospective, randomized clinical trials such as the Australian D-Health study should clarify the role of vitamin D supplementation in the prevention and management of skeletal and nonskeletal disorders, including vitamin D effects on mortality risk.

Measuring vitamin D.

When assessing vitamin D status, measure serum 25-hydroxyvitamin D concentration as this reflects total body vitamin D reserves. Recent Australasian guidelines outline who should be tested for vitamin D deficiency, who should be treated and when repeat testing should be performed. A 25-hydroxyvitamin D threshold of at least 50 nanomol/L at the end of winter is a suitable treatment target. Measurement can be repeated after three months of repletion, and thereafter less frequently unless new risk factors for vitamin D deficiency arise. When interpreting vitamin D pathology reports, practitioners should be aware that some laboratories quote reference limits which are based on overseas rather than Australian guidelines.

The Adrenal Vein Sampling Outcomes Study (AVOS): success rates following adrenalectomy for unilateral primary aldosteronism.

The objective was to determine the clinical and biochemical success rates and assess the nature of follow-up after adrenalectomy in patients with unilateral primary aldosteronism (PA), subtyped by adrenal vein sampling (AVS) in West Australia (WA) using the Primary Aldosteronism Surgical Outcome (PASO) criteria. Clinical and biochemical outcomes were retrospectively evaluated in patients with unilateral PA who underwent adrenalectomy according to AVS between September 2017 and September 2020. Pre- and post-surgical data were collected using a standardised questionnaire, review of clinic letters and examination of private and public pathology results and radiological reports. Follow-up data were available for 47 patients post-adrenalectomy; biochemical outcome data were available for 37 patients, clinical outcome data for 40 patients, with 30 patients having both outcomes available. Final assessment was performed between 0 to 3 months in 23/37 (62.2%) patients with biochemical outcomes, 15/40 (37.5%) with clinical outcomes, and 17/30 (56.7%) with both clinical and biochemical outcomes. Complete biochemical success was achieved in 83.8% (31/37) of patients, with 26.7% (8/30) obtaining both complete clinical and biochemical success. Complete clinical success was achieved in 35.0% (14/40) of patients, with 47.5% (19/40) obtaining partial clinical success. Overall, 93.6% (44/47) of patients derived benefit from adrenalectomy. The outcomes of adrenalectomy for unilateral PA in Western Australian using standardised PASO criteria demonstrate highly comparable clinical and biochemical success rates to international data. However, further standardisation of post-operative follow-up care needs to be implemented to ensure the recommended repeat follow-up assessment criteria are collected.

Endotoxin induced TNF and IL-10 mRNA production is higher in male than female donors: correlation with elevated expression of TLR4.

Modification of cytokine production by gender hormones has been postulated to affect disease susceptibility and outcome. Here we investigate the effect of gender and the menstrual cycle on production of cytokines. Mononuclear cells were isolated every week for 10 consecutive weeks from healthy pre-menopausal women and men. TNF and IL-10 mRNA and protein levels were measured as well as membrane CD14 and intracellular TLR4 protein. Endotoxin stimulation of mononuclear cells from men produced more TNF and IL10 mRNA than cells from women. TLR4 expression was also significantly higher in cells from men. These gender differences in the immune response may help to elucidate the sexual dimorphism observed in infectious diseases.

Clinical utility of bone turnover markers in the management of common metabolic bone diseases in adults.

Bone turnover marker (BTMs) concentrations in blood and urine reflect bone-remodelling activity, and may be useful adjuncts in the diagnosis and management of metabolic bone diseases. Newer biomarkers, mainly bone regulatory proteins, are currently being investigated to elucidate their role in bone metabolism and disease and may in future be useful in clinical diagnosis and management of metabolic bone disease. BTM concentrations increase around menopause in women, and at a population level the degree of increase in BTMs reflect bone loss. However, lack of adequate data precludes their use in individual patients for fracture risk assessment in clinical practice. The rapid and large changes in BTMs following anti-resorptive and anabolic therapies for osteoporosis treatment indicate they may be useful for monitoring therapy in clinical practice. The offset of drug effect on BTMs could be helpful for adjudicating the duration of bisphosphonate drug holidays. BTMs may offer useful additional data in skeletal diseases that are typically characterised by increased bone remodelling: chronic kidney disease (CKD), primary hyperparathyroidism (PHPT) and Paget's disease. In CKD, bone specific alkaline phosphatase (bAP) is currently endorsed for use for the assessment of mineral bone disease. The role of BTMsin predicting the bone mineral density response to successful parathyroidectomy in PHPT shows some utility but the data are not consistent and studies are limited in size and/or duration. In Paget's disease of bone, BTMs are used to confirm diagnosis, evaluate extent of disease or degree of activity and for monitoring the response to bisphosphonate treatment. Whilst BTMs are currently used in specific clinical practice instances when investigating or managing metabolic bone disease, further data are needed to consolidate their clinical use where evidence of utility is limited.

Improved technical success and radiation safety of adrenal vein sampling using rapid, semi-quantitative point-of-care cortisol measurement.

Objective Primary aldosteronism is a curable cause of hypertension which can be treated surgically or medically depending on the findings of adrenal vein sampling studies. Adrenal vein sampling studies are technically demanding with a high failure rate in many centres. The use of intraprocedural cortisol measurement could improve the success rates of adrenal vein sampling but may be impracticable due to cost and effects on procedural duration. Design Retrospective review of the results of adrenal vein sampling procedures since commencement of point-of-care cortisol measurement using a novel single-use semi-quantitative measuring device for cortisol, the adrenal vein sampling Accuracy Kit. MEASUREMENTS: Success rate and complications of adrenal vein sampling procedures before and after use of the adrenal vein sampling Accuracy Kit. Routine use of the adrenal vein sampling Accuracy Kit device for intraprocedural measurement of cortisol commenced in 2016. Results Technical success rate of adrenal vein sampling increased from 63% of 99 procedures to 90% of 48 procedures ( P = 0.0007) after implementation of the adrenal vein sampling Accuracy Kit. Failure of right adrenal vein cannulation was the main reason for an unsuccessful study. Radiation dose decreased from 34.2 Gy.cm2 (interquartile range, 15.8-85.9) to 15.7 Gy.cm2 (6.9-47.3) ( P = 0.009). No complications were noted, and implementation costs were minimal. Conclusions Point-of-care cortisol measurement during adrenal vein sampling improved cannulation success rates and reduced radiation exposure. The use of the adrenal vein sampling Accuracy Kit is now standard practice at our centre.

The challenge of improving the diagnostic yield from metanephrine testing in suspected phaeochromocytoma and paraganglioma.

Background Plasma-free metanephrines (PFM) or urinary fractionated metanephrines (UFM) are the preferred biochemical tests for the diagnosis of phaeochromocytoma and paraganglioma (PPGL). Borderline increased results should be followed up to either exclude or confirm diagnosis. Methods We extracted all PFM and UFM results reported by our laboratory over a six-month period from the laboratory information system. We categorized patients with borderline increased results according to whether follow-up testing had been performed as suggested in the initial laboratory report. Questionnaires were then sent to all requesting doctors and medical notes reviewed where available. Results Two hundred and four patients with borderline increased PFM or UFM were identified. Sixty-five (38.5%) of 169 patients with borderline increased PFM had a repeat test out of which 36 were normal and 29 did not normalize. Of 35 patients with borderline increased UFM, 17 (48.6%) had subsequent PFM measurement, out of which 15 were normal. Questionnaires were returned to 106 (52%) patients. Of these, the most frequent indication for testing was hypertension ( n = 50); 15 patients had an incidental adrenal mass and two of these patients were diagnosed with a phaeochromocytoma. Conclusion Only 38% of patients with borderline increased PFM had a repeat PFM measurement. This was not significantly higher when compared with the 28% in a previous audit that we reported in 2010 ( P = 0.10). Forty-nine per cent of patients with a borderline increased UFM had a repeat UFM or PFM measurement. There remains a substantial possibility of missed detection of PPGL.

Isolated brachydactyly type E and idiopathic pancreatitis in a patient presenting to a lipid disorders clinic.

An 18-year-old female tertiary student was referred to a lipid clinic with hypertriglyceridaemia discovered after presentation with acute pancreatitis. The patient's only medication was l-thyroxine for treatment of hypothyroidism. She was overweight, normotensive, with unremarkable facies. However, she had hypermobile hand joints and brachydactyly resulting in loss of left 3-5 and right 4 and 5 knuckle definitions. Radiography revealed shortening of metacarpals 3-5 on the left and 4 and 5 on the right. Her mother had similar skeletal changes, consistent with a dominant mode of inheritance. Abnormally short digits involving the metacarpals, classified as brachydactyly type E, can be isolated or occur as part of a syndrome. Turner syndrome, Albright hereditary osteodystrophy, hypertension with brachydactyly, chromosome 2q37 microdeletion and PTHLH mutations were excluded following clinical, biochemical and genetic testing. No specific treatment was required. Genetic testing for isolated and syndromic forms of brachydactyly facilitates family screening and prepregnancy counselling.

25-Hydroxyvitamin D C3-epimer is universally present in neonatal Western Australian samples but is unlikely to contribute to diagnostic misclassification.

BACKGROUND: The presence of C3-epimer (C-3-epi-25-hydroxyvitamin D) in infant serum may complicate 25-hydroxyvitamin D (25(OH)D) measurement when using liquid chromatography tandem mass spectrometry assays that do not separately measure the epimer. We measured the concentration of C3-epi-25(OH)D in neonatal samples in Western Australian using umbilical cord blood samples and a liquid chromatography tandem mass spectrometry assay that separately quantifies 25(OH)D and C3-epi-25(OH)D. METHODS: A total of 120 anonymized cord blood samples were analysed using a liquid chromatography tandem mass spectrometry assay that utilizes two CSH fluoro-phenyl columns in series. Chromatography was performed on a Waters Acquity Ultra Performance Liquid system, and quantification was using a Waters Quattro Premier XE mass spectrometer. RESULTS: C3-epi-25(OH)D3 was detected in all umbilical cord blood samples (median 5.2 nmol/L, IQR 3.7-6.6 nmol/L) and contributed 6.6% (SD 2.6, 95% CI [6.1, 7.1]) of the total 25(OH)D concentration. Mean 25(OH)D3 measured in cord blood was 79.1 nmol/L (SD 22.7 nmol/L). A positive relationship (R(2 )= 0.35, P < 0.0005) between 25(OH)D3 levels and C3-epi-25(OH)D3 was noted in this cohort. No samples contained 25(OH)D2 or C3-epi-25(OH)D2. CONCLUSION: C3-epi-25(OH)D3 is present in all neonatal samples but contributes <10% of the total 25(OH)D concentration which is unlikely to be clinically significant. Liquid chromatography tandem mass spectrometry assays that do not separately quantify C3-epi-25(OH)D3 from other vitamin D metabolites may potentially overestimate neonatal 25(OH)D levels, but diagnostic misclassification in neonates is unlikely.

Is vitamin D testing at a tertiary referral hospital consistent with guideline recommendations?

To determine if 25 hydroxyvitamin D (25OHD) testing at our tertiary referral hospital is consistent with guideline recommendations concerning the clinical indications for testing, the timing of repeat testing and utilisation of the test result, we conducted a retrospective audit of electronic laboratory and patient case records. We included adult inpatients and outpatients who had serum 25OHD measured during a randomly selected one-week audit period and who had patient case records available for detailed review. The audit sample comprised 184 serum 25OHD measurements (134 initial and 50 repeat tests). There were 81 (60%) initial and 15 (30%) repeat tests [96 (52%) overall] that were consistent with guideline recommendations concerning clinical indication, timing of repeat testing and utilisation of result. Almost half the 25 hydroxyvitamin D tests audited were potentially unnecessary and/or not utilised clinically. Improved adherence to guideline recommendations for 25 hydroxyvitamin D testing, utilisation of test results and enforcement of new indications for testing due to be introduced by Medicare Australia could result in significant cost savings without adversely affecting patient outcomes.

Long-term efficacy, safety, and patient acceptability of ibandronate in the treatment of postmenopausal osteoporosis.

Several second-generation bisphosphonates (BPs) are approved in osteoporosis treatment. Efficacy and safety depends on potency of farnesyl pyrophosphate synthase (FPPS) inhibition, hydroxyapatite affinity, compliance and adherence. The latter may be influenced by frequency and route of administration. A literature search using "ibandronate", "postmenopausal osteoporosis", "fracture", and "bone mineral density" (BMD) revealed 168 publications. The Phase III BONE study, using low dose 2.5 mg daily oral ibandronate demonstrated 49% relative risk reduction (RRR) in clinical vertebral fracture after 3 years. Non-vertebral fracture (NVF) reduction was demonstrated in a subgroup (pretreatment T-score ≤ -3.0; RRR 69%) and a meta-analysis of high annual doses (150 mg oral monthly or intravenous equivalent of ibandronate; RRR 38%). Hip fracture reduction was not demonstrated. Long-term treatment efficacy has been confirmed over 5 years. Long term safety is comparable to placebo over 3 years apart from flu-like symptoms which are more common with oral monthly and intravenous treatments. No cases of atypical femoral fracture or osteonecrosis of the jaw have been reported in randomized controlled trial studies. Ibandronate inhibits FPPS more than alendronate but less than other BPs which could explain rate of action onset. Ibandronate has a higher affinity for hydroxyapatite compared with risedronate but less than other BPs which could affect skeletal distribution and rate of action offset. High doses (150 mg oral monthly or intravenous equivalent) were superior to low doses (oral 2.5 mg daily) according to 1 year BMD change. Data are limited by patient selection, statistical power, under-dosing, and absence of placebo groups in high dose studies. Ibandronate treatment offers different doses and modalities of administration which could translate into higher adherence rates, an important factor when the two main limitations of BP treatment are initiation and adherence rates. However, lack of consistency in NVF reduction and absence of hip fracture data limits more generalized use of this agent.

Estrogen and androgen regulation of plasma membrane calcium pump activity in immortalized distal tubule kidney cells.

The ATP dependent plasma membrane calcium pump (PMCA) is a regulator of renal calcium reabsorption. The effect of estrogen and dihydrotestosterone to increase the activity of the PMCA in membrane vesicle preparations from a distal tubule cell line was investigated. 17beta Estradiol (10(-10)M) increased PMCA activity (1.5 +/- 0.2-fold increase compared to control) with 24 h, but not 1 or 5 h, of exposure, an effect that was blocked by the addition of the estrogen antagonist ICI 164384. alpha Estradiol did not increase PMCA activity. Dihydrotestosterone (10(-11)M ) resulted in a dose dependent increase in PMCA activity (1.5+/-0.1-fold increase compared to control) with 24h, but not 1 or 5h, of exposure, an effect that was blocked by the androgen receptor agonist flutamide. Testosterone (10(-5)M) also increased PMCA activity (1.9+/-0.3-fold increase compared to control). Neither estrogen nor dihydrotestosterone increased PMCA protein expression in MDBK cells, indicating that these hormones increase PMCA activity by regulating PMCA activity rather than PMCA expression. These results demonstrate receptor dependent stimulatory effects of both estrogen and dihydrotestosterone to increase PMCA activity. and have significance for our understanding of estrogen and androgen deficient states on calcium transport.

Issues of methodology, standardization and metabolite recognition for 25-hydroxyvitamin D when comparing the DiaSorin radioimmunoassay and the Nichols Advantage automated chemiluminescence protein-binding assay in hip fracture cases.

BACKGROUND: Deficiency of vitamin D is commonly associated with hip fracture and treatment with vitamin D reduces hip fracture rates. Consequently, the demand for assays to measure 25-hydroxyvitamin D (25-OHD) has increased. The Nichols Advantage chemiluminescence protein-binding assay (CLPBA) for 25-OHD is a first-generation automated immunoassay with decreased turnaround time, reduced manual handling and non-radioactive label. METHODS: We compared the CLPBA to the DiaSorin radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC) for the measurement of 25-OHD using 161 samples from hip fracture patients and samples before and after institution of ergocalciferol (vitamin D(2)) therapy. RESULTS: A negative bias for the CLPBA at concentrations below 30 nmol/L and a positive bias at 25-OHD values above 30 nmol/L compared with the RIA resulted in diagnostic discordance for one in three samples when using 30 and 50 nmol/L as decision limits. HPLC analysis confirmed the presence of a negative bias for the CLPBA at low values. Both immunoassays under-estimate 25-hydroxyvitamin D(2). CONCLUSIONS: The discordance between 25-OHD values may be due to differences in standardization of each assay relative to HPLC. Our results emphasize the need for assay-specific clinical decision limits.

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±8 years) with vitamin D deficiency (serum 25OHD <50 nmol/L) to oral supplementation with either cholecalciferol 1000 IU/day (n=47) or ergocalciferol 1000 IU/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.

A diagnostic conundrum: heterophilic antibody interference in an adrenocorticotropic hormone immunoassay not detectable using a proprietary heterophile blocking reagent.

CONTEXT: Heterophilic antibodies are a well-described interferent but poorly appreciated and are often not a recognized problem affecting most immunoassays. We describe for the first time heterophilic antibodies interference affecting an adrenocorticotropic hormone (ACTH) assay in a patient with Cushing's syndrome due to bilateral nodular adrenal hyperplasia. CASE: A 60-year-old retired female nurse underwent extensive invasive investigations, which were ultimately unnecessary, as a result of initial analytical interference in the ACTH assay, which could not be resolved using a proprietary heterophilic binding reagent. RESULTS: This case highlights the inherent difficulty of diagnosing Cushing's syndrome and the large emphasis placed on laboratory tests. The consequence of not initially identifying interference in this patient's laboratory test results led to unnecessary and costly investigations with potentially adverse outcomes. CONCLUSIONS: Clinicians and the laboratory community need to be continuously vigilant and view laboratory results with caution when they are inconsistent with the clinical picture. This approach is paramount, especially at a time of increasing automation and ever-diminishing scientist involvement in sample processing.

Screening for vitamin D deficiency: defining vitamin D deficiency, target thresholds of treatment and estimating the benefits of treatment.

Vitamin D sufficiency has been associated with improved health outcomes but cost benefit analyses of published data adopt a number of assumptions. Firstly, definitions of vitamin D deficiency vary. Secondly, available methods used for the analysis of 25-hydroxyvitamin D (25OHD) have significant limitations which could affect the adoption of specific target thresholds for treatment. Thirdly, although a variety of diseases are associated with vitamin D deficiency, randomised clinical trial data demonstrating the benefit of vitamin D supplementation only exist for the prevention of falls or fractures. This review will summarise the current evidence regarding an appropriate target threshold of 25OHD and review proposed therapeutic target thresholds of treatment. The limitations of current methods will be reviewed and objective data relating to the costs of diagnosis, the costs of treatment, and the level of evidence that screening could lessen disease burden in the community, will be provided. Finally, information needed by governments and health organisations to help justify population screening and what strategies could be adopted to make screening more cost-effective will be explored.

Vitamin D: methods of 25 hydroxyvitamin D analysis, targeting at risk populations and selecting thresholds of treatment.

Interest in vitamin D has intensified with the association of vitamin D deficiency (VDD) with many diseases. This review will outline the limitations of current 25 hydroxyvitamin D (25OHD) methods, the target treatment threshold, and review the classical (endocrine/bone) and non-classical (paracrine/non-bone) actions of vitamin D. Recent standardisation by the National Institutes of Standards and Technology and use of LC tandem mass methodology has reduced inter-method bias but insensitivity and imprecision of automated methods have challenged assay performance. Many diseases are associated with VDD but randomised clinical trial data demonstrating the benefit of un-activated sterol supplementation only exists for the prevention of falls and fractures. Consequently, 25OHD measurement should be restricted to high falls or fracture risk patients. Controversy regarding the 25OHD target of therapy requires consensus. Until resolved, widespread adoption of screening programmes and measurement of 25OHD in patients at risk of non-musculoskeletal disease is premature, costly and not supported by evidence.

Effects of three-monthly oral 150,000 IU cholecalciferol supplementation on falls, mobility, and muscle strength in older postmenopausal women: a randomized controlled trial.

Daily vitamin D in addition to calcium supplementation reduces falls and fractures in older women. However, poor adherence to therapy is a common clinical problem. To examine the effects of supervised oral 3-monthly vitamin D therapy on falls, muscle strength, and mobility, we conducted a 9-month randomized, double-blind, placebo-controlled trial in 686 community-dwelling ambulant women aged over 70 years. Participants received either oral cholecalciferol 150,000 IU every 3 months (n = 353) or an identical placebo (n = 333). All participants were advised to increase dietary calcium intake. Falls data were collected 3-monthly. At baseline, 3, 6, and 9 months, muscle strength was measured by a handheld dynamometer and mobility by the Timed Up and Go (TUG) test. Serum 25 hydroxyvitamin D (25OHD) was measured in a subgroup of 40 subjects. Mean age at baseline was 76.7 ± 4.1 years. The average serum 25OHD value at baseline was 65.8 ± 22.7 nmol/L. By 3, 6, and 9 months after supplementation, 25OHD levels of the vitamin D group were approximately 15 nmol/L higher than the placebo group. Calcium intake did not change significantly between baseline (864 ± 412 mg/day) and 9 months (855 ± 357 mg/day). Faller rates in the two groups did not differ: vitamin D group, 102 of 353 (29%); placebo group, 89 of 333 (27%). At 9 months, compared to placebo or baseline, muscle strength, and TUG were not altered by vitamin D. In conclusion, oral cholecalciferol 150,000 IU therapy administered 3-monthly had neither beneficial nor adverse effects on falls or physical function. These data together with previous findings confirm that intermittent large doses of vitamin D are ineffective or have a deleterious effect on falls. Thus despite adherence issues with daily vitamin D replacement, an intermittent, high-dose vitamin D regimen cannot be supported as a strategy to reduce falls and fractures.

Improved plasma free metadrenaline analysis requires mixed mode cation exchange solid-phase extraction prior to liquid chromatography tandem mass spectrometry detection.

BACKGROUND: The investigation and effective management of phaeochromocytoma involves biochemical measurement of either conjugated total urine or plasma free metadrenalines. Current analytical methods include enzyme-linked immunosorbent assays, high-performance liquid chromatography (HPLC) with electrochemical detection (ECD) or liquid chromatography tandem mass spectrometry (LCMS/MS). Since the first two methods are either extremely laborious, necessitate low sample run numbers, result in slow turnaround times or are subject to analytical interference, a robust, routine clinical method is not achievable. We established a novel sample preparation method to measure plasma free metadrenalines using LCMS/MS. METHODS: Three different solid-phase extraction (SPE) methods were compared: hydrophilic-lipophilic balance sorbent (HLB), weak cation exchange (WCX) and mixed mode cation exchange (MCX) and their ability to remove interfering compounds prior to LCMS/MS analysis. Maximum recovery of plasma free metadrenaline and plasma free normetadrenaline were achieved by positively charging compounds prior to SPE application. RESULTS: Compared with HLB and WCX cartridges, MCX extraction resulted in chromatography without co-eluting interference with superior assay precision and accuracy. Additionally, samples that could not be quantified because of interference using HPLC/ECD could be readily assayed using this new method. CONCLUSIONS: The use of the MCX SPE method with LCMS/MS detection provides an improved assay to measure plasma free metadrenalines in comparison to many available alternative methods.