Annemieke C. Heijboer

Effects of Dietary Phosphate and Calcium Intake on Fibroblast Growth Factor-23

BACKGROUND AND OBJECTIVES Little is known about the influence of dietary phosphate intake on fibroblast growth factor-23 (FGF23) and its subsequent effects on vitamin D levels. This study addresses changes in intact FGF23 (iFGF23) and C-terminal FGF23 (cFGF23), phosphaturia, and levels of vitamin D on high and low phosphate and calcium intake. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Ten healthy subjects adhered to a diet low or high in phosphate and calcium content for 36 hours each with a 1-week interval during which subjects adhered to their usual diet. Serum phosphate, calcium, vitamin D metabolites, parathyroid hormone (PTH), and FGF23 levels (cFGF23 and iFGF23) were measured several times a day. Phosphate, calcium, and creatinine excretion was measured in 24-hour urine on all study days. RESULTS Serum phosphate levels and urinary phosphate increased during high dietary phosphate intake (from 1.11 to 1.32 mmol/L, P<0.0001 and 21.6 to 28.8 mmol/d, P=0.0005, respectively). FGF23 serum levels increased during high dietary phosphate/calcium intake (cFGF23 from 60 to 72 RU/ml, P<0.001; iFGF23 from 33 to 37 ng/L, P=0.003), whereas PTH declined. 1,25-dihydroxyvitamin D (1,25D) showed an inverse relation with FGF23. CONCLUSIONS Variation in dietary phosphate and calcium intake induces changes in FGF23 (on top of a circadian rhythm) and 1,25D blood levels as well as in urinary phosphate excretion. These changes are detectable the day after the change in the phosphate content of meals. Higher FGF23 levels are associated with phosphaturia and a decline in 1,25D levels.

Oral Nutritional Supplements Containing (n-3) Polyunsaturated Fatty Acids Affect the Nutritional Status of Patients with Stage III Non-Small Cell Lung Cancer during Multimodality Treatment

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), (n-3) fatty acids from fish oil, have immune-modulating effects and may improve nutritional status in cancer. The objective of this study was to investigate the effects of an oral nutritional supplement containing (n-3) fatty acids on nutritional status and inflammatory markers in patients with non-small cell lung cancer (NSCLC) undergoing multimodality treatment. In a double-blind experiment, 40 patients with stage III NSCLC were randomly assigned to receive 2 cans/d of a protein- and energy-dense oral nutritional supplement containing (n-3) fatty acids (2.0 g EPA + 0.9 g DHA/d) or an isocaloric control supplement. EPA in plasma phospholipids, energy intake, resting energy expenditure (REE), body weight, fat free mass (FFM), mid-upper arm circumference (MUAC), and inflammatory markers were assessed. Effects of intervention were analyzed by generalized estimating equations and expressed as regression coefficients (B). The intervention group (I) had a better weight maintenance than the control (C) group after 2 and 4 wk (B = 1.3 and 1.7 kg, respectively; P < 0.05), a better FFM maintenance after 3 and 5 wk (B = 1.5 and 1.9 kg, respectively; P < 0.05), a reduced REE (B = -16.7% of predicted; P = 0.01) after 3 wk, and a trend for a greater MUAC (B = 9.1; P = 0.06) and lower interleukin-6 production (B = -27.9; P = 0.08) after 5 wk. After 4 wk, the I group had a higher energy and protein intake than the C group (B = 2456 kJ/24 h, P = 0.03 and B = 25.0 g, P = 0.01, respectively). In conclusion, a protein- and energy-dense oral nutritional supplement containing (n-3) fatty acids beneficially affects nutritional status during multimodality treatment in patients with NSCLC.

Delay Discounting and Frontostriatal Fiber Tracts: A Combined DTI and MTR Study on Impulsive Choices in Healthy Young Adults

Delay discounting, a measure of impulsive choice, has been associated with decreased control of the prefrontal cortex over striatum responses. The anatomical connectivity between both brain regions in delaying gratification remains unknown. Here, we investigate whether the quality of frontostriatal (FS) white matter tracts can predict individual differences in delay-discounting behavior. We use tract-based diffusion tensor imaging and magnetization transfer imaging to measure the microstructural properties of FS fiber tracts in 40 healthy young adults (from 18 to 25 years). We additionally explored whether internal sex hormone levels affect the integrity of FS tracts, based on the hypothesis that sex hormones modulate axonal density within prefrontal dopaminergic circuits. We calculated fractional anisotropy (FA), mean diffusivity (MD), longitudinal diffusivity, radial diffusivity (RD), and magnetization transfer ratio (MTR), a putative measure of myelination, for the FS tract. Results showed that lower integrity within the FS tract (higher MD and RD and lower FA), predicts faster discounting in both sexes. MTR was unrelated to delay-discounting performance. In addition, testosterone levels in males were associated with a lower integrity (higher RD) within the FS tract. Our study provides support for the hypothesis that enhanced structural integrity of white matter fiber bundles between prefrontal and striatal brain areas is associated with better impulse control.

Dynamics of serum testosterone during the menstrual cycle evaluated by daily measurements with an ID-LC-MS/MS method and a 2nd generation automated immunoassay.

BACKGROUND Testosterone concentrations in normally cycling women are assumed to be elevated around the time of ovulation. The clinical relevance of changing testosterone concentrations during the menstrual cycle, however, is unclear. Poor performance of current direct immunoassays for testosterone at low concentrations confounds this issue. Therefore, our objective was to assess daily testosterone fluctuation during the menstrual cycle by a thoroughly validated isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method and to evaluate whether an ARCHITECT® 2nd Generation Testosterone fully automated immunoassay is equally suited for this purpose. METHODS Testosterone was measured in serum obtained daily during the menstrual cycle of 25 healthy women, characterized by biochemical and physical examination. RESULTS Performance of the ID-LC-MS/MS method was concordant with a published reference method (y=1.007x-0.056 nmol/L; r=0.9998). Comparison of the immunoassay to ID-LC-MS/MS yielded y=1.095x+0.104 nmol/L (r=0.9031). Overall, testosterone concentrations were higher mid-cycle, but a peak was not discernible in each individual. Apart from a persistent positive bias, the immunoassay measured the same testosterone profiles as the ID-LC-MS/MS method. The reference interval in women was 0.30-1.69 nmol/L (8.7-48.7 ng/dL) for ID-LC-MS/MS and 0.50-2.00 nmol/L (14.4-57.7 ng/dL) for the immunoassay. CONCLUSION The elevation of mid-cycle testosterone concentrations is statistically significant, although not clinically relevant since day-to-day variation is higher and independent of the menstrual cycle. In this light, a single testosterone measurement might not be reflective of the overall testosterone status in an individual. Measurements obtained using the 2nd generation immunoassay gave comparable results across the menstrual cycle.

Serum sarcosine is not a marker for prostate cancer

The recent finding by Sreekumar et al. that sarcosine might play a role in the progression of prostate cancer was warmly welcomed by professionals in the field. The assessment of sarosine concentration in body fluids was hypothesized to serve as a novel marker for prostate cancer and prostate cancer progression. Currently, the blood concentration of prostate-specific antigen (PSA), a protein produced by the prostate gland, is used as a biological marker, with the limitation that an increased concentration of PSA alone does not differentiate between benign prostate conditions and prostate cancer. Sarcosine, also known as N-methylglycine, is a small endogenous molecule present in low concentrations in blood. Careful interpretation of the data presented (Supplementary Figure 14) by Sreekumar et al. already showed a substantial overlap in the urinary sarcosine concentrations between men with positive and negative biopsies for cancer, implying that assessment of urinary sarcosine alone will not have clinical relevance on an individual basis. Potentially however, serum PSA concentrations in relation to serum sarcosine concentrations might have additional diagnostic value. We retrospectively determined serum sarcosine concentrations in samples obtained from three groups of men: controls (i.e. individuals who have been assessed for vitamin B12 status [n 1⁄4 15]), individuals with increased serum PSA (n 1⁄4 24) and individuals with prostate cancer metastases (n 1⁄4 18). Sarcosine was measured using liquid chromatographytandem mass spectrometry (LC-MS/MS) using stableisotope labelled [H3]sarcosine as internal standard. The LC-MS/MS method was evaluated with respect to linearity, recovery and reproducability. The structural analogues alanine and beta-alanine were physically separated from sarcosine by the LC column allowing proper quantification. The outcomes of the sarcosine determinations are depicted in Figure 1. Our results clearly show that the mean serum sarcosine concentration does not discriminate between the three groups of men. Moreover, on an individual basis the sarcosine concentration was uninformative with respect to increased PSA concentrations and to prostate cancer progression. Our serum sarcosine data in conjunction with the urinary sarcosine data presented by Sreekumar and colleagues shows that there are no major alterations in the extracellular concentrations of sarcosine, implying that the assessment of sarcosine in easily obtainable body fluids like urine and serum has limited potential in the diagnostic algorithm of prostate cancer. DECLARATIONS

Multicenter comparison study of current methods to measure 25-hydroxyvitamin D in serum

OBJECTIVES Measurement of serum 25-hydroxyvitamin D [25(OH)D] is generally considered to be a reliable indicator of vitamin D status. The recent increase in diversity of 25(OH)D assays prompted us to evaluate the performance of chromatographic methods (two in-house ID-LC-MS/MS and HPLC (ClinRep, Recipe)), a protein binding method (Cobas-25(OH)D-total, Roche) and immunochemical methods (Liaison and RIA (Diasorin), iSYS (IDS), ADVIA Centaur (Siemens), and Architect i1000 and i2000 (Abbott)). METHODS Blood was drawn from randomly selected outpatients (N=60) at one site after informed consent. DEQAS and SRM 972 samples were obtained from the scheme organizer and NIST, respectively. Serum aliquots were prepared, frozen and transported to participating centers. Method comparison was performed according to CLSI-EP9 specifications. RESULTS With these patient samples, and in comparison with ID-LC-MS/MS, Deming regression parameters slope, intercept and R were found to be within the ranges [0.57-1.07], [-1.7 to 6.9 nmol/L] and [0.88-0.98], respectively. 25(OH)D2 in DEQAS and SRM samples was fully recognized by chromatographic methods, but only partially by protein binding and immunochemical methods. Chromatographic methods, and to a lesser extent the protein binding assay, showed cross-reactivity with 3-epi-25(OH)D3. Agreement of 25(OH)D assays to ID-LC-MS/MS in sorting patients into distinct 25(OH)D categories varied between 53% and 88%. CONCLUSIONS Significant bias exists between ID-LC-MS/MS and many, but not all, other 25(OH)D assays. The variable response among different assays for 25(OH)D metabolites impedes the use of uniform cut-off values for defining vitamin D status. Our results indicate the need towards further standardizing assays for 25(OH)D measurement.

Determination of fibroblast growth factor 23

Background Fibroblast growth factor 23 (FGF-23) is a recently discovered hormone, which plays a key role in phosphate regulation. To investigate whether FGF-23 can be determined reliably, we validated the three available FGF-23 assays. Methods Currently, two intact FGF-23 assays (Kainos; Immutopics) and one C-terminal FGF-23 assay (Immutopics) are available. We determined intra- and inter-assay variation, linearity and matrix interference in these assays. Moreover, we compared assay results from healthy subjects with those of patient groups with expected high FGF-23 concentrations. Results Intra-assay variation was reasonably good in all three assays. Inter-assay variation and linearity were poor for the intact Immutopics assay but reasonable for both other assays. Immutopics assays gave best results in ethylenediaminetetraacetic acid (EDTA) plasma, while the Kainos assay showed comparable reproducibility in EDTA plasma and serum. Although the manual of the Kainos assay states that an automatic washing machine can be used, acceptable results were only found by manual washing. Patients with kidney disease and patients with hypophosphatemic osteomalacia had increased C-terminal FGF-23 concentrations compared with healthy controls. Conclusion Two assays of reasonable quality are available for FGF-23, the intact FGF-23 assay (Kainos) provided proper attention is paid to the washing procedure, and the C-terminal assay (Immutopics).

Bone mass in young adulthood following gonadotropin-releasing hormone analog treatment and cross-sex hormone treatment in adolescents with gender dysphoria.

CONTEXT Sex steroids are important for bone mass accrual. Adolescents with gender dysphoria (GD) treated with gonadotropin-releasing hormone analog (GnRHa) therapy are temporarily sex-steroid deprived until the addition of cross-sex hormones (CSH). The effect of this treatment on bone mineral density (BMD) in later life is not known. OBJECTIVE This study aimed to assess BMD development during GnRHa therapy and at age 22 years in young adults with GD who started sex reassignment (SR) during adolescence. DESIGN AND SETTING This was a longitudinal observational study at a tertiary referral center. PATIENTS Young adults diagnosed with gender identity disorder of adolescence (DSM IV-TR) who started SR in puberty and had undergone gonadectomy between June 1998 and August 2012 were included. In 34 subjects BMD development until the age of 22 years was analyzed. INTERVENTION GnRHa monotherapy (median duration in natal boys with GD [transwomen] and natal girls with GD [transmen] 1.3 and 1.5 y, respectively) followed by CSH (median duration in transwomen and transmen, 5.8 and 5.4 y, respectively) with discontinuation of GnRHa after gonadectomy. MAJOR OUTCOME MEASURES How BMD develops during SR until the age of 22 years. RESULTS AND CONCLUSION Between the start of GnRHa and age 22 years the lumbar areal BMD z score (for natal sex) in transwomen decreased significantly from -0.8 to -1.4 and in transmen there was a trend for decrease from 0.2 to -0.3. This suggests that the BMD was below their pretreatment potential and either attainment of peak bone mass has been delayed or peak bone mass itself is attenuated.

Laboratory aspects of circulating α-Klotho

BACKGROUND α-Klotho is a protein mainly produced in the kidney. Its circulating form has been suggested to link renal damage and distant tissue pathology. As three assays to measure α-Klotho became commercially available, we performed an evaluation of these commercially available Klotho assays. METHODS We studied within-run variation, between-run variation, matrix effects, linearity, and recovery of added recombinant human Klotho in the α-Klotho assays of IBL (IBL International GmbH, Hamburg, Germany), Cusabio (Cusabio Biotech, Wuhan, China) and USCN (USCN life Science, Inc., Wuhan, China) using both serum and ethylenediaminetetraacetic acid plasma. RESULTS Within run variation was 4, 13 and 32% for the IBL, Cusabio and USCN assay, respectively. Agreement between serum and EDTA plasma was good in the IBL assay, but poor in the USCN and Cusabio assays however improved after modifications in the Cusabio assay. Standardization and agreement between assays was poor. CONCLUSIONS The commercially available methods for the measurement of α-Klotho differ in quality. Some of the manufacturers should improve their assays in order to produce accurate results so that reliable conclusions can be drawn from studies in which these assays are used.

Vitamin D Deficiency in School-Age Children Is Associated with Sociodemographic and Lifestyle Factors

BACKGROUND There is concern about a reemergence of vitamin D deficiency in children in developed countries. OBJECTIVES The aims of this study were to describe vitamin D status in the Generation R study, a large multiethnic cohort of 6-y-old children in The Netherlands, and to examine sociodemographic, lifestyle, and dietary determinants of vitamin D deficiency. METHODS We measured serum 25-hydroxyvitamin D [25(OH)D] concentrations in 4167 children aged 6 y and defined deficiency following recommended cutoffs. We examined the associations between subject characteristics and vitamin D deficiency with the use of multivariable logistic regression analyses. RESULTS Serum 25(OH)D concentrations ranged from 4 to 211 nmol/L (median: 64 nmol/L), with 6.2% of the children having severely deficient (<25 nmol/L), 23.6% deficient (25 to <50 nmol/L), 36.5% sufficient (50 to <75 nmol/L), and 33.7% optimal (≥75 nmol/L) 25(OH)D concentrations. The prevalence of vitamin D deficiency [25(OH)D <50 nmol/L] was higher in winter (51.3%) than in summer (10.3%); and higher in African, Asian, Turkish, and Moroccan children (54.5%) than in those with a Dutch or other Western ethnic background (17.6%). In multivariable models, several factors were associated with vitamin D deficiency, including household income (OR: 1.74; 95% CI: 1.34, 2.27 for low vs. high income), child age (OR: 1.39; 95% CI: 1.20, 1.62 per year), child television watching (OR: 1.32; 95% CI: 1.06, 1.64 for ≥2 vs. <2 h/d), and playing outside (OR: 0.71; 95% CI: 0.57, 0.89 for ≥1 vs. <1 h/d). In a subgroup with dietary data (n = 1915), vitamin D deficiency was associated with a lower diet quality, but not with vitamin D intake or supplement use in early childhood. CONCLUSIONS Suboptimal vitamin D status is common among 6-y-old children in The Netherlands, especially among non-Western children and in winter and spring. Important modifiable factors associated with vitamin D deficiency were overall diet quality, sedentary behavior, and playing outside.