1ß,25-Dihydroxyvitamin D3: A new vitamin D metabolite in human serum.

BACKGROUND: The measurement of 1α,25(OH)2D3 in human serum poses a true challenge as concentrations are very low and structurally similar metabolites can interfere. MATERIALS AND METHODS: During optimization of our in-house LC-MSMS method for serum 1α,25(OH)2D3 a previously co-eluting isobaric interference was separated. The isobar was identified as 1ß,25(OH)2D3 by comparing retention time and fragmentation spectra to standards (other isobaric dihydroxylated vitamin D3 analogs). 1ß,25(OH)2D3 showed specific cluster formation (water), not present in 1α,25(OH)2D3. 1ß,25(OH)2D3 was measured in serum of apparently healthy human volunteers (n=20), patients with high serum 25-hydroxyvitamin D [25(OH)D] concentrations (>50ng/mL) (n=33 among which 4 with very high levels (>150ng/mL)) and patients with kidney failure (n=68; 39 stage 1-3, 29 stage 4-5). Pearson's r was calculated for correlations and Mann-Whitney statistic to compare group medians. RESULTS: Median serum 1ß,25(OH)2D3 was 11pg/mL in apparently healthy volunteers and increased to 20pg/mL for serum 25(OH)D concentrations above 80ng/mL (n=22) (p<0.0001). 1ß,25(OH)2D3 concentrations were significantly correlated to serum 25(OH)D concentrations (r=0.85) for the combined results from healthy volunteers and patient sera (n=53) (p<0.0001). For patients with kidney failure, median serum 1ß,25(OH)2D3 was 7pg/mL and not different from the median level in healthy volunteers (p=0.06). The median concentration did not vary with different stages. CONCLUSIONS: We present evidence for the widespread presence of 1ß,25(OH)2D3, a new vitamin D metabolite, in human serum. The level increases with rising serum 25(OH)D concentrations and is particularly high in patients with very high 25(OH)D levels. We previously demonstrated that 1ß,25(OH)2D3 is a poor genomic agonist but a potent non-genomic antagonist of 1α,25(OH)2D3. The clinical implications of the presence of this analog therefore require further exploration.

Possibilities and limitations of signal summing for an immunosuppressant LC-MS/MS method.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the method of choice for quantifying small molecules in research and clinical setting. Although there is a large toolkit to increase quantification levels for LC-MS/MS, these techniques are sometimes insufficient to attain the needed limits of quantification (LOQs) or the method becomes too impractical for routine use. We examined the possibilities and limitations of signal summing, an under-utilized, easy-to-apply practice to increase LOQs for an immunosuppressant LC-MS/MS method. The limits of signal summing for everolimus were tested by running samples of everolimus at three concentrations in triplicate programming, increasing amounts of identical transitions in a constant cycle time up to the maximum number the software permitted to sum. The increase in peak area and the signal-to-noise ratio were determined. The effect on imprecision of peak areas and response ratios was evaluated by injection of a low concentration of everolimus tenfold using respectively one and five identical transitions, retaining an identical ion counting time. We compared the imprecision, LOQ, and recovery for our routine everolimus method (using one transition for everolimus and one for d3-everolimus) and an adapted method summing three identical transitions for everolimus (and one for d3-everolimus). The increase in signal was close to the theoretically expected one with a larger experimental spread for everolimus once more than five transitions were used. There was no clear beneficial effect of summing on imprecision. The adapted everolimus method showed a lower LOQ, but comparable imprecision and recovery as the routine method. Quantification levels can be improved by signal summing. No clear effect on imprecision was observed.

Associations of 25-Hydroxyvitamin D and 1,25-Dihydroxyvitamin D With Bone Mineral Density, Bone Mineral Density Change, and Incident Nonvertebral Fracture.

Relationships between 1,25-dihydroxyvitamin D (1,25(OH)2 D) and skeletal outcomes are uncertain. We examined the associations of 1,25(OH)2 D with bone mineral density (BMD), BMD change, and incident non-vertebral fractures in a cohort of older men and compared them with those of 25-hydroxyvitamin D (25OHD). The study population included 1000 men (aged 74.6 ± 6.2 years) in the Osteoporotic Fractures in Men (MrOS) study, of which 537 men had longitudinal dual-energy X-ray absorptiometry (DXA) data (4.5 years of follow-up). A case-cohort design and Cox proportional hazards models were used to test the association between vitamin D metabolite levels and incident nonvertebral and hip fractures. Linear regression models were used to estimate the association between vitamin D measures and baseline BMD and BMD change. Interactions between 25OHD and 1,25(OH)2 D were tested for each outcome. Over an average follow-up of 5.1 years, 432 men experienced incident nonvertebral fractures, including 81 hip fractures. Higher 25OHD was associated with higher baseline BMD, slower BMD loss, and lower hip fracture risk. Conversely, men with higher 1,25(OH)2 D had lower baseline BMD. 1,25(OH)2 D was not associated with BMD loss or nonvertebral fracture. Compared with higher levels of calcitriol, the risk of hip fracture was higher in men with the lowest 1,25(OH)2 D levels (8.70 to 51.60 pg/mL) after adjustment for baseline hip BMD (hazard ratio [HR] = 1.99, 95% confidence interval [CI] 1.19-3.33). Adjustment of 1,25(OH)2 D data for 25OHD (and vice versa) had little effect on the associations observed but did attenuate the hip fracture association of both vitamin D metabolites. In older men, higher 1,25(OH)2 D was associated with lower baseline BMD but was not related to the rate of bone loss or nonvertebral fracture risk. However, with BMD adjustment, a protective association for hip fracture was found with higher 1,25(OH)2 D. The associations of 25OHD with skeletal outcomes were generally stronger than those for 1,25(OH)2 D. These results do not support the hypothesis that measures of 1,25(OH)2 D improve the ability to predict adverse skeletal outcomes when 25OHD measures are available. © 2015 American Society for Bone and Mineral Research.

Arthralgia induced by endocrine treatment for breast cancer: A prospective study of serum levels of insulin like growth factor-I, its binding protein and oestrogens.

BACKGROUND: Aromatase inhibitors (AIs) frequently induce or enhance musculoskeletal problems (AI-induced musculoskeletal syndrome (AIMSS)) which sometimes are debilitating. Apart from low oestrogen levels, underlying mechanisms are unknown and likely multiple. We previously hypothesised a role for the growth hormone/insulin like growth factor-I (IGF-I) axis. Here, we report the effect of tamoxifen and AI on IGF-I, IGF binding protein-3 (IGFBP-3) and oestrogen levels from a prospective study. MATERIALS AND METHODS: Postmenopausal women with an early breast cancer scheduled to start adjuvant endocrine therapy with an AI or tamoxifen were recruited. A rheumatologic questionnaire was completed and serum was collected for assessment of IGF-I, IGFBP-3 and oestrogen levels. Re-evaluation was done after 3, 6 and 1 2months of therapy. RESULTS: 84 patients started on tamoxifen (n=42) or an AI (n=42). 66% of the latter group experienced worsening of pre-existing or de novo complaints in joint and/or muscle, compared to 29% of tamoxifen-treated patients. AI therapy resulted in elevated IGF-I levels with a statistically significant increase at 6months (p=0.0088), whereas tamoxifen users were characterised by a decrease in IGF-I levels at all follow-up times (p<0.0004). No effect on IGFBP-3 was seen in the latter group. AI-users, however, showed decreased IGFBP-3 levels at 12 months (p=0.0467). AIMSS was characterised by a decrease in IGFBP-3 levels (p=0.0007) and a trend towards increased IGF-I/IGFBP-3 ratio (p=0.0710). CONCLUSION: These findings provide preliminary evidence that AI-induced musculoskeletal symptoms are associated with changes in the growth hormone (GH)/IGF-I axis.

Higher 25(OH)D2 is associated with lower 25(OH)D3 and 1,25(OH)2D3.

CONTEXT: Despite common use of supplemental vitamin D2 in clinical practice, the associations of serum vitamin D2 concentrations with other vitamin D metabolites and total vitamin D are unclear. OBJECTIVE: The aim of the study was to measure vitamin D2 and D3 levels and examine their associations with each other and with total vitamin D. DESIGN: We performed a cross-sectional analysis of 679 randomly selected participants from the Osteoporotic Fractures in Men Study. 25-Hydroxyvitamin D2 [25(OH)D2], 25(OH)D3, 1,25-dihydroxyvitamin D2 [1,25(OH)2D2], and 1,25(OH)2D3 were measured using liquid chromatography-tandem mass spectrometry and were summed to obtain total 25(OH)D and 1,25(OH)2D. Associations between all metabolites (D2, D3, and total levels) were examined using Wilcoxon rank-sum tests and Spearman correlations. RESULTS: 25(OH)D2 and 1,25(OH)2D2 were detectable in 189 (27.8%) and 178 (26.2%) of the men, respectively. Higher 25(OH)D2 levels did not correlate with higher total 25(OH)D (r = 0.10; P = .17), although median total 25(OH)D was slightly higher in those with detectable vs undetectable 25(OH)D2 (25.8 vs 24.3 ng/mL; P < .001). 25(OH)D2 was not positively associated with total 1,25(OH)2D levels (r = -0.11; P = .13), and median 1,25(OH)2D level was not higher in those with detectable vs undetectable 25(OH)D2. Higher 25(OH)D2 was associated with lower 25(OH)D3 (r = -0.35; P < .001) and 1,25(OH)2D3 (r = -0.32; P < .001), with median levels of both D3 metabolites 18-35% higher when D2 metabolites were undetectable. CONCLUSIONS: In a cohort of older men, 25(OH)D2 is associated with lower levels of 25(OH)D3 and 1,25(OH)2D3, suggesting that vitamin D2 may decrease the availability of D3 and may not increase calcitriol levels.

Sensitive routine liquid chromatography-tandem mass spectrometry method for serum estradiol and estrone without derivatization.

The need for a routinely applicable assay to measure low estradiol levels in adult men, postmenopausal women, and young adolescents was recently discussed in an Endocrine Society position statement. Our aim was to develop a sensitive liquid chromatography-tandem mass spectrometry method for estradiol and estrone in human serum without the need for derivatization or extended extraction protocols. After protein precipitation of serum with a mixture of methanol/acetonitrile (85/15) (v/v) containing isotopic internal standards (17ß-estradiol-16,16,17-d 3 and estrone-2,3,4-(13)C), we quantified estradiol and estrone by two-dimensional liquid chromatography-tandem mass spectrometry with electrospray ionization in the negative mode monitoring 5 × 271.20→145.00 (17ß-estradiol) and 269.20→145.00 (estrone). Sensitivity was increased by using fluoride and summation of 5 identical transitions for estradiol. Our method was analytically validated, compared against direct immunoassays using serum of 25 adult men, and clinically tested by measuring samples of 3 men at baseline and after chemical castration, 30 postmenopausal women and 15 patients receiving aromatase inhibitors. Total imprecision was below 20% for the low quality controls. Limit of quantification was 1.3 ng/L (4.8 pmol/L) for estradiol and 1.2 ng/L (4.4 pmol/L) for estrone. Estradiol in Certified Reference Material BCR-576 was within specified uncertainty limits. No significant ion suppression or interference was observed. Our method showed modest correlation with direct immunoassay for estradiol (r(2) = 0.64) but no correlation for estrone (r(2) = 0.12). Patient sample results were within expected ranges. In conclusion, we developed a routinely applicable liquid chromatography-tandem mass spectrometry method for estradiol and estrone measurement which is sensitive enough for use in men, postmenopausal women, and young adolescents.

Active vitamin D (1,25-dihydroxyvitamin D) and bone health in middle-aged and elderly men: the European Male Aging Study (EMAS).

CONTEXT: There is little information on the potential impact of serum 1,25-dihydroxyvitamin D [1,25(OH)2D] on bone health including turnover. OBJECTIVE: The objective of the study was to determine the influence of 1,25(OH)2D and 25-hydroxyvitamin D [25(OH)D] on bone health in middle-aged and older European men. DESIGN, SETTING, AND PARTICIPANTS: Men aged 40-79 years were recruited from population registers in 8 European centers. Subjects completed questionnaires that included questions concerning lifestyle and were invited to attend for quantitative ultrasound (QUS) of the heel, assessment of height and weight, and a fasting blood sample from which 1,25(OH)2D, 25(OH)D, and PTH were measured. 1,25(OH)2D was measured using liquid chromatography tandem mass spectrometry. Bone markers serum N-terminal propeptide of type 1 procollagen (P1NP) and crosslinks (ß-cTX) were also measured. Dual-energy x-ray absorptiometry (DXA) of the hip and lumbar spine was performed in 2 centers. MAIN OUTCOME MEASURE(S): QUS of the heel, bone markers P1NP and ß-cTX, and DXA of the hip and lumbar spine were measured. RESULTS: A total of 2783 men, mean age 60.0 years (SD 11.0) were included in the analysis. After adjustment for age and center, 1,25(OH)2D was positively associated with 25(OH)D but not with PTH. 25(OH)D was negatively associated with PTH. After adjustment for age, center, height, weight, lifestyle factors, and season, 1,25(OH)2D was associated negatively with QUS and DXA parameters and associated positively with ß-cTX. 1,25(OH)2D was not correlated with P1NP. 25(OH)D was positively associated with the QUS and DXA parameters but not related to either bone turnover marker. Subjects with both high 1,25(OH)2D (upper tertile) and low 25(OH)D (lower tertile) had the lowest QUS and DXA parameters and the highest ß-cTX levels. CONCLUSIONS: Serum 1,25(OH)2D is associated with higher bone turnover and poorer bone health despite being positively related to 25(OH)D. A combination of high 1,25(OH)2D and low 25(OH)D is associated with the poorest bone health.

Oncogenic osteomalacia illustrating the effect of fibroblast growth factor 23 on phosphate homeostasis.

In oncogenic osteomalacia (OOM), fibroblast growth factor 23 (FGF23) induces renal phosphate wasting and inhibits the appropriate increase of calcitriol. A patient suffering from OOM is described. Serum calcium, phosphate, biointact parathyroid hormone and intact FGF23 as well as the calcitriol and 24,25-vitamin D levels were measured before and after tumour removal. The clinical approach to a patient with hypophosphataemia is discussed and the changes in mineral metabolism after removal of a FGF23-producing tumour are described.

Development of a method for the quantification of 1alpha,25(OH)2-vitamin D3 in serum by liquid chromatography tandem mass spectrometry without derivatization.

Quantification of 1alpha,25(OH)(2)-vitamin D(3) in serum is technically challenging because of its very low concentration. Even mass spectrometry faces a challenge due to the low sensitivity for this molecule, unless a specific derivatization is implemented. Therefore, there is still a need for a robust, simplified, sensitive and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantification of 1alpha,25(OH)(2)-vitamin D(3). Thanks to tandem mass- spectrometry associated to an on-line sample extraction and clean-up, sample preparation has been reduced to a simple protein precipitation step and derivatization has been avoided. Specimen volume has been kept to a minimum. The innovative aspects of the hereby-presented method are the use of stable lithium adducts and a highly sensitive tandem mass spectrometer. The achieved limit of quantification is at a level of 15 pg mL(-1), with a % CV of 5-15% at physiological concentration levels. The linearity is good and spiking experiments yielded a recovery of 87-102%. Comparison of selected samples with an established reference method, using an extensive purification procedure (high-performance liquid chromatography and radioimmunoassay), has shown a good correlation.

Cortisol response to critical illness: effect of intensive insulin therapy.

CONTEXT: Both excessive and insufficient activation of the hypothalamic-pituitary-adrenal axis in response to critical illness is associated with increased mortality. OBJECTIVE: The objective of the study was to study the effect of intensive insulin therapy, recently shown to reduce mortality and morbidity of critically ill patients, on the cortisol response to critical illness. DESIGN: This was a preplanned subanalysis of a large randomized, controlled study measuring serum total cortisol, cortisol-binding globulin, and albumin and calculating free cortisol levels. SETTING: The study was conducted at a university hospital surgical intensive care unit. PATIENTS: Four hundred fifty-one critically ill patients dependent on intensive care for more than 5 d and 45 control subjects matched for gender, age, height, and weight participated in this study. INTERVENTION: The intervention was strict blood glucose control to normoglycemia with insulin. RESULTS: Total and calculated free cortisol levels were equally elevated upon admission in both patient groups and thereafter were lower in intensive insulin-treated patients. Lower cortisol levels statistically related to the outcome benefit of intensive insulin therapy. Cortisol-binding globulin levels and structure were affected by critical illness but not insulin therapy, and neither were albumin levels. Administration of hydrocortisone in so-called replacement dose resulted in severalfold higher total and free cortisol levels, indicating that reevaluation of the doses used is warranted. CONCLUSIONS: Lower serum cortisol levels in critically ill patients receiving intensive insulin therapy statistically related to improved outcome with this intervention. The lower cortisol levels were not related to altered cortisol-binding capacity.