Phosphate Metabolism in Health and Disease.

Phosphorus, a 5A element with atomic weight of 31, comprises just over 0.6% of the composition by weight of plants and animals. Three isotopes are available for studying phosphorus metabolism and kinetics. 31P is stable, whereas the radioactive isotope 33P has a half-life of 25 days and 32P has a half-life of 14 days. Phosphate ester and phosphoanhydride are common chemical linkages and phosphorus is a key element in organic molecules involved in a wide variety of essential cellular functions. These include biochemical energy transfer via adenosine triphosphate (ATP), maintenance of genetic information with nucleotides DNA and RNA, intracellular signaling via cyclic adenosine monophosphate (cAMP), and membrane structural integrity via glycerophospholipids. However, this review focuses on the metabolism of inorganic phosphorus (Pi) acting as a weak acid. Phosphoric acid has all three hydrogens attached to oxygen and is a weak diprotic acid. It has 3 pKa values: pH 2.2, pH 7.2, and pH 12.7. At physiological pH of 7.4, Pi exists as both H2PO4(-) and HPO4(2-) and acts as an extracellular fluid (ECF) buffer. Pi is the form transported across tissue compartments and cells. Measurement of Pi in biological fluids is based on its reaction with ammonium molybdate which does not measure organic phosphorus. In humans, 80% of the body phosphorus is present in the form of calcium phosphate crystals (apatite) that confer hardness to bone and teeth, and function as the major phosphorus reservoir (Fig. 1). The remainder is present in soft tissues and ECF. Dietary phosphorus, comprising both inorganic and organic forms, is digested in the upper gastrointestinal tract. Absorbed Pi is transported to and from bone, skeletal muscle and soft tissues, and kidney at rates determined by ECF Pi concentration, rate of blood flow, and activity of cell Pi transporters (Fig. 2). During growth, there is net accretion of phosphorus, and with aging, net loss of phosphorus occurs. The bone phosphorus reservoir is depleted and repleted by overall phosphorus requirement. Skeletal muscle is rich in phosphorus used in essential biochemical energy transfer. Kidney is the main regulator of ECF Pi concentration by virtue of having a tubular maximum reabsorptive capacity for Pi (TmPi) that is under close endocrine control. It is also the main excretory pathway for Pi surplus which is passed in urine. Transcellular and paracellular Pi transports are performed by a number of transport mechanisms widely distributed in tissues, and particularly important in gut, bone, and kidney. Pi transporters are regulated by a hormonal axis comprising fibroblast growth factor 23 (FGF23), parathyroid hormone (PTH), and 1,25 dihydroxy vitamin D (1,25D). Pi and calcium (Ca) metabolism are intimately interrelated, and clinically neither can be considered in isolation. Diseases of Pi metabolism affect bone as osteomalacia/rickets, soft tissues as ectopic mineralization, skeletal muscle as myopathy, and kidney as nephrocalcinosis and urinary stone formation. Fig. 1 Content of phosphorus in human adult: skeleton, soft tissue, and extracellular fluid (grams, log scale). Corresponding data for calcium are shown for comparison Fig. 2 Phosphate (Pi) transport to and from tissue compartments in mg/24 h. At a dietary phosphorus of 1400 mg, 1120 mg is absorbed in upper intestine to the ECF, 210 mg returned to intestine by endogenous secretion, resulting in 910 mg net Pi absorption and 490 mg fecal excretion. At bone, 180 mg is deposited by bone formation and 180 mg return to the ECF by bone resorption. At kidney, 5040 mg is filtered at the glomerulus and 4130 mg return to the ECF by tubular reabsorption with 910 mg excreted in the urine. In soft tissue, Pi is exchanged between ECF and cells.

Effects of Iron Isomaltoside vs Ferric Carboxymaltose on Hypophosphatemia in Iron-Deficiency Anemia: Two Randomized Clinical Trials.

Importance: Intravenous iron enables rapid correction of iron-deficiency anemia, but certain formulations induce fibroblast growth factor 23-mediated hypophosphatemia. Objective: To compare risks of hypophosphatemia and effects on biomarkers of mineral and bone homeostasis of intravenous iron isomaltoside (now known as ferric derisomaltose) vs ferric carboxymaltose. Design, Setting, and Participants: Between October 2017 and June 2018, 245 patients aged 18 years and older with iron-deficiency anemia (hemoglobin level ≤11 g/dL; serum ferritin level ≤100 ng/mL) and intolerance or unresponsiveness to 1 month or more of oral iron were recruited from 30 outpatient clinic sites in the United States into 2 identically designed, open-label, randomized clinical trials. Patients with reduced kidney function were excluded. Serum phosphate and 12 additional biomarkers of mineral and bone homeostasis were measured on days 0, 1, 7, 8, 14, 21, and 35. The date of final follow-up was June 19, 2018, for trial A and May 29, 2018, for trial B. Interventions: Intravenous administration of iron isomaltoside, 1000 mg, on day 0 or ferric carboxymaltose, 750 mg, infused on days 0 and 7. Main Outcomes and Measures: The primary end point was the incidence of hypophosphatemia (serum phosphate level <2.0 mg/dL) between baseline and day 35. Results: In trial A, 123 patients were randomized (mean [SD] age, 45.1 [11.0] years; 95.9% women), including 62 to iron isomaltoside and 61 to ferric carboxymaltose; 95.1% completed the trial. In trial B, 122 patients were randomized (mean [SD] age, 42.6 [12.2] years; 94.1% women), including 61 to iron isomaltoside and 61 to ferric carboxymaltose; 93.4% completed the trial. The incidence of hypophosphatemia was significantly lower following iron isomaltoside vs ferric carboxymaltose (trial A: 7.9% vs 75.0% [adjusted rate difference, -67.0% {95% CI, -77.4% to -51.5%}], P < .001; trial B: 8.1% vs 73.7% [adjusted rate difference, -65.8% {95% CI, -76.6% to -49.8%}], P < .001). Beyond hypophosphatemia and increased parathyroid hormone, the most common adverse drug reactions (No./total No.) were nausea (iron isomaltoside: 1/125; ferric carboxymaltose: 8/117) and headache (iron isomaltoside: 4/125; ferric carboxymaltose: 5/117). Conclusions and Relevance: In 2 randomized trials of patients with iron-deficiency anemia who were intolerant of or unresponsive to oral iron, iron isomaltoside (now called ferric derisomaltose), compared with ferric carboxymaltose, resulted in lower incidence of hypophosphatemia over 35 days. However, further research is needed to determine the clinical importance of this difference. Trial Registration: ClinicalTrials.gov Identifiers: NCT03238911 and NCT03237065.

Genetic Variants Associated with Circulating Fibroblast Growth Factor 23.

BACKGROUND: Fibroblast growth factor 23 (FGF23), a bone-derived hormone that regulates phosphorus and vitamin D metabolism, contributes to the pathogenesis of mineral and bone disorders in CKD and is an emerging cardiovascular risk factor. Central elements of FGF23 regulation remain incompletely understood; genetic variation may help explain interindividual differences. METHODS: We performed a meta-analysis of genome-wide association studies of circulating FGF23 concentrations among 16,624 participants of European ancestry from seven cohort studies, excluding participants with eGFR<30 ml/min per 1.73 m2 to focus on FGF23 under normal conditions. We evaluated the association of single-nucleotide polymorphisms (SNPs) with natural log-transformed FGF23 concentration, adjusted for age, sex, study site, and principal components of ancestry. A second model additionally adjusted for BMI and eGFR. RESULTS: We discovered 154 SNPs from five independent regions associated with FGF23 concentration. The SNP with the strongest association, rs17216707 (P=3.0×10-24), lies upstream of CYP24A1, which encodes the primary catabolic enzyme for 1,25-dihydroxyvitamin D and 25-hydroxyvitamin D. Each additional copy of the T allele at this locus is associated with 5% higher FGF23 concentration. Another locus strongly associated with variations in FGF23 concentration is rs11741640, within RGS14 and upstream of SLC34A1 (a gene involved in renal phosphate transport). Additional adjustment for BMI and eGFR did not materially alter the magnitude of these associations. Another top locus (within ABO, the ABO blood group transferase gene) was no longer statistically significant at the genome-wide level. CONCLUSIONS: Common genetic variants located near genes involved in vitamin D metabolism and renal phosphate transport are associated with differences in circulating FGF23 concentrations.

Genetic Variants Associated with Circulating Parathyroid Hormone.

Parathyroid hormone (PTH) is a primary calcium regulatory hormone. Elevated serum PTH concentrations in primary and secondary hyperparathyroidism have been associated with bone disease, hypertension, and in some studies, cardiovascular mortality. Genetic causes of variation in circulating PTH concentrations are incompletely understood. We performed a genome-wide association study of serum PTH concentrations among 29,155 participants of European ancestry from 13 cohort studies (n=22,653 and n=6502 in discovery and replication analyses, respectively). We evaluated the association of single nucleotide polymorphisms (SNPs) with natural log-transformed PTH concentration adjusted for age, sex, season, study site, and principal components of ancestry. We discovered associations of SNPs from five independent regions with serum PTH concentration, including the strongest association with rs6127099 upstream of CYP24A1 (P=4.2 × 10-53), a gene that encodes the primary catabolic enzyme for 1,25-dihydroxyvitamin D and 25-dihydroxyvitamin D. Each additional copy of the minor allele at this SNP associated with 7% higher serum PTH concentration. The other SNPs associated with serum PTH concentration included rs4074995 within RGS14 (P=6.6 × 10-17), rs219779 adjacent to CLDN14 (P=3.5 × 10-16), rs4443100 near RTDR1 (P=8.7 × 10-9), and rs73186030 near CASR (P=4.8 × 10-8). Of these five SNPs, rs6127099, rs4074995, and rs219779 replicated. Thus, common genetic variants located near genes involved in vitamin D metabolism and calcium and renal phosphate transport associated with differences in circulating PTH concentrations. Future studies could identify the causal variants at these loci, and the clinical and functional relevance of these variants should be pursued.

GLUCOCORTICOID EXCESS IN BONE AND MUSCLE.

Glucocorticoids (GC), produced and released by the adrenal glands, regulate numerous physiological processes in a wide range of tissues. Because of their profound immunosuppressive and anti-inflammatory actions, GC are extensively used for the treatment of immune and inflammatory conditions, the management of organ transplantation, and as a component of chemotherapy regimens for cancers. However, both pathologic endogenous elevation and long-term use of exogenous GC are associated with severe adverse effects. In particular, excess GC has devastating effects on the musculoskeletal system. GC increase bone resorption and decrease formation leading to bone loss, microarchitectural deterioration and fracture. GC also induce loss of muscle mass and strength leading to an increased incidence of falls. The combined effects on bone and muscle account for the increased fracture risk with GC. This review summarizes the advance in knowledge in the last two decades about the mechanisms of action of GC in bone and muscle and the attempts to interfere with the damaging actions of GC in these tissues with the goal of developing more effective therapeutic strategies.

Genome-wide association study of serum iron phenotypes in premenopausal women of European descent.

A genome-wide association study was performed on 1130 premenopausal women to detect common variants associated with three serum iron-related phenotypes. Total iron binding capacity was strongly associated (p=10(-14)) with variants in and near the TF gene (transferrin), the serum iron transporting protein, and with variants in HFE (p=4×10(-7)), which encodes the human hemochromatosis gene. Association was also detected between percent iron saturation (p=10(-8)) and variants in the chromosome 6 region containing both HFE and SLC17A2, which encodes a phosphate transport protein. No significant associations were detected with serum iron, but variants in HFE were suggestive (p=10(-6)). Our results corroborate prior studies in older subjects and demonstrate that the association of these genetic variants with iron phenotypes can be detected in premenopausal women.

Associations between genetic variants and the effect of letrozole and exemestane on bone mass and bone turnover.

Adjuvant therapy for hormone receptor (HR) positive postmenopausal breast cancer patients includes aromatase inhibitors (AI). While both the non-steroidal AI letrozole and the steroidal AI exemestane decrease serum estrogen concentrations, there is evidence that exemestane may be less detrimental to bone. We hypothesized that single nucleotide polymorphisms (SNP) predict effects of AIs on bone turnover. Early stage HR-positive breast cancer patients were enrolled in a randomized trial of exemestane versus letrozole. Effects of AI on bone mineral density (BMD) and bone turnover markers (BTM), and associations between SNPs in 24 candidate genes and changes in BMD or BTM were determined. Of the 503 enrolled patients, paired BMD data were available for 123 and 101 patients treated with letrozole and exemestane, respectively, and paired BTM data were available for 175 and 173 patients, respectively. The mean change in lumbar spine BMD was significantly greater for letrozole-treated (-3.2 %) compared to exemestane-treated patients (-1.0 %) (p = 0.0016). Urine N-telopeptide was significantly increased in patients treated with exemestane (p = 0.001) but not letrozole. Two SNPs (rs4870061 and rs9322335) in ESR1 and one SNP (rs10140457) in ESR2 were associated with decreased BMD in letrozole-treated patients. In the exemestane-treated patients, SNPs in ESR1 (Rs2813543) and CYP19A1 (Rs6493497) were associated with decreased bone density. Exemestane had a less negative impact on bone density compared to letrozole, and the effects of AI therapy on bone may be impacted by genetic variants in the ER pathway.

The Apparent Relation between Plasma 25-Hydroxyvitamin D and Insulin Resistance is Largely Attributable to Central Adiposity in Overweight and Obese Adults.

BACKGROUND: Research indicates that plasma 25-hydroxyvitamin D [25(OH)D] is associated with insulin resistance, but whether regional adiposity confounds this association is unclear. OBJECTIVE: This study assessed the potential influence of adiposity and its anatomical distribution on the relation between plasma 25(OH)D and insulin resistance. METHODS: A secondary analysis of data from middle-aged overweight and obese healthy adults [n = 336: 213 women and 123 men; mean ± SD (range); age: 48 ± 8 y (35-65 y); body mass index (BMI; in kg/m2): 30.3 ± 2.7 (26-35)] from West Lafayette, Indiana (40.4 °N), were used for this cross-sectional analysis. Multiple linear regression analyses that controlled for multiple covariates were used as the primary statistical model. RESULTS: Of all participants, 8.6% and 20.5% displayed moderate [20.1-37.5 nmol/L plasma 25(OH)D] to mild (37.6-49.9 nmol/L) vitamin D insufficiency, respectively. A regression analysis controlling for age, sex, race, plasma parathyroid hormone concentration, season of year, and supplement use showed that 25(OH)D was negatively associated with fasting insulin (P = 0.021). Additional regression analyses showed that total and central adiposity but not peripheral adiposity predicted low plasma 25(OH)D [total fat mass index (FMI): P = 0.018; android FMI: P = 0.052; gynoid FMI: P = 0.15; appendicular FMI: P = 0.07) and insulin resistance (homeostasis model assessment of insulin resistance: total and android FMI, P <0.0001; gynoid FMI, P = 0.94; appendicular FMI, P = 0.86). The associations of total and central adiposity with insulin resistance remained significant after adjusting for plasma 25(OH)D. However, adjusting for central adiposity but not other anatomical measures of fat distribution eliminated the association between plasma 25(OH)D and insulin resistance. CONCLUSION: Central adiposity drives the association between plasma 25(OH)D and insulin resistance in overweight and obese adults. The trial was registered at clinicaltrials.gov as NCT00812409.

Dietary calcium requirements do not differ between Mexican-American boys and girls.

Mexican Americans are an understudied ethnic group for determinants of bone health, although the risk of age-related osteoporosis is high in this rapidly growing sector of the U.S. population. Thus, the objective of the present study was to establish the dietary calcium requirements for bone health in Mexican-American adolescents by measuring calcium retention calculated from balance in response to a range of dietary calcium intakes and to determine predictors of skeletal calcium retention. Adolescents aged 12-15 y were studied twice on paired calcium intakes ranging from 600 to 2300 mg/d using randomized-order, crossover 3-wk balance studies. Skeletal calcium retention was calculated as dietary calcium intake minus calcium excreted in feces and urine over the last 2 wk of balance. A linear model was developed to explain the variation in calcium retention. Boys (n = 20) were taller and had higher lean mass, usual dietary calcium intake, bone mineral content, and serum alkaline phosphatase compared with girls, whereas girls (n = 20) had higher Tanner scores and greater fat mass. Calcium retention increased with calcium intake (P < 0.0001) and did not differ by sex (P = 0.66). In boys and girls considered together, calcium intake explained 33% of the variation in calcium retention. Serum alkaline phosphatase explained an additional 11% of the variation in calcium retention. Other variables measured, including the urine N-telopeptide of type I collagen/creatinine ratio, Tanner score, serum parathyroid hormone and 25-hydroxyvitamin D, weight, height, and body mass index, did not contribute to the variance in calcium retention. In adolescence, calcium retention in both Mexican-American boys and girls was higher than determined previously in adolescent nonHispanic white girls. This trial was registered at clinicaltrials.gov as NCT01277185.

Velcalcetide (AMG 416), a novel peptide agonist of the calcium-sensing receptor, reduces serum parathyroid hormone and FGF23 levels in healthy male subjects.

CONTEXT: Velcalcetide, also known as AMG 416, is a novel, long-acting selective peptide agonist of the calcium sensing receptor. It is being developed as an intravenous treatment of secondary hyperparathyroidism (SHPT) in hemodialysis patients with chronic kidney disease-mineral and bone disorder. OBJECTIVE: To assess the safety, tolerability, pharmacokinetics and pharmacodynamics of velcalcetide in healthy male volunteers. METHODS: The study was a double-blind, randomized, placebo-controlled, single-dose, dose-escalation study in healthy males aged 18-45 years conducted at a single center. Each cohort included eight subjects randomized 6:2 to velcalcetide or placebo. INTERVENTION: Velcalcetide at 0.5, 2, 5 and 10 mg or placebo was administered intravenously. OUTCOMES: Measurements included plasma ionized calcium (iCa), serum total calcium, intact parathyroid hormone (iPTH), phosphorus and fibroblast growth factor-23 (FGF23), 1,25-dihydroxyvitamin D, calcitonin and urine creatinine, calcium and phosphorus and plasma pharmacokinetics for velcalcetide. Vital signs, safety biochemical and hematological indices, and adverse events were monitored throughout the study. RESULTS: Intravenous administration of velcalcetide was well tolerated with no adverse reaction of nausea, vomiting or diarrhea reported. Velcalcetide mediated dose-dependent decreases in serum iPTH at 30 min, FGF23 at 24 h and iCa at 12 h post dose (P<0.05) and in urine fractional excretion of phosphorus and increases in tubular reabsorption of phosphorus. Velcalcetide plasma exposure increased in a dose-related manner and the terminal elimination of half-life was comparable across the dose range evaluated and ranged from 18.4 to 20.0 h. CONCLUSION: Single IV doses of velcalcetide were well tolerated and associated with rapid, sustained, dose-dependent reductions in serum PTH. The results support further evaluation of velcalcetide as a treatment for SHPT in hemodialysis patients.

Soluble maize fibre affects short-term calcium absorption in adolescent boys and girls: a randomised controlled trial using dual stable isotopic tracers.

Soluble maize fibre (SCF) has been found to significantly improve bone mineral density and strength in growing rats compared with several other novel prebiotic fibres. The objective of the present study was to investigate the effect of SCF on Ca absorption and retention in pubertal children by studying the potential absorption mechanisms of the intestinal microbiota. A total of twenty-four adolescent boys and girls (12-15 years) participated in two 3-week metabolic balance studies testing 0 g/d SCF (control (CON) treatment) and 12 g/d SCF (SCF treatment) in a random order by inclusion in a low-Ca diet (600 mg/d). Fractional Ca absorption was measured at the end of the two intervention periods using a dual-stable isotope method. Diet composites and faecal and urine samples were collected daily and analysed for Ca content. Ca retention was calculated as dietary Ca intake minus Ca excretion in faeces and urine over the last 2 weeks. Microbial community composition in the faecal samples collected at the beginning and end of each session was determined by 454 pyrosequencing of the PCR-amplified 16S ribosomal RNA gene. Fractional Ca absorption was 12 % higher (41 mg/d) after the SCF treatment compared with that after the CON treatment (0·664 (sd 0·129) and 0·595 (sd 0·142), respectively; P= 0·02), but Ca retention was unaffected. The average proportion of bacteria of the phylum Bacteroidetes was significantly greater in the participants after the SCF treatment than after the CON treatment. These results suggest that moderate daily intake of SCF, a well-tolerated prebiotic fibre, increases short-term Ca absorption in adolescents consuming less than the recommended amounts of Ca.

Iron deficiency drives an autosomal dominant hypophosphatemic rickets (ADHR) phenotype in fibroblast growth factor-23 (Fgf23) knock-in mice.

Autosomal dominant hypophosphatemic rickets (ADHR) is unique among the disorders involving Fibroblast growth factor 23 (FGF23) because individuals with R176Q/W and R179Q/W mutations in the FGF23 (176)RXXR(179)/S(180) proteolytic cleavage motif can cycle from unaffected status to delayed onset of disease. This onset may occur in physiological states associated with iron deficiency, including puberty and pregnancy. To test the role of iron status in development of the ADHR phenotype, WT and R176Q-Fgf23 knock-in (ADHR) mice were placed on control or low-iron diets. Both the WT and ADHR mice receiving low-iron diet had significantly elevated bone Fgf23 mRNA. WT mice on a low-iron diet maintained normal serum intact Fgf23 and phosphate metabolism, with elevated serum C-terminal Fgf23 fragments. In contrast, the ADHR mice on the low-iron diet had elevated intact and C-terminal Fgf23 with hypophosphatemic osteomalacia. We used in vitro iron chelation to isolate the effects of iron deficiency on Fgf23 expression. We found that iron chelation in vitro resulted in a significant increase in Fgf23 mRNA that was dependent upon Mapk. Thus, unlike other syndromes of elevated FGF23, our findings support the concept that late-onset ADHR is the product of gene-environment interactions whereby the combined presence of an Fgf23-stabilizing mutation and iron deficiency can lead to ADHR.

Oral calcium carbonate affects calcium but not phosphorus balance in stage 3-4 chronic kidney disease.

Patients with chronic kidney disease (CKD) are given calcium carbonate to bind dietary phosphorus, reduce phosphorus retention, and prevent negative calcium balance; however, data are limited on calcium and phosphorus balance during CKD to support this. Here, we studied eight patients with stage 3 or 4 CKD (mean estimated glomerular filtration rate 36 ml/min) who received a controlled diet with or without a calcium carbonate supplement (1500 mg/day calcium) during two 3-week balance periods in a randomized placebo-controlled cross-over design. All feces and urine were collected during weeks 2 and 3 of each balance period and fasting blood, and urine was collected at baseline and at the end of each week. Calcium kinetics were determined using oral and intravenous (45)calcium. Patients were found to be in neutral calcium and phosphorus balance while on the placebo. Calcium carbonate supplementation produced positive calcium balance, did not affect phosphorus balance, and produced only a modest reduction in urine phosphorus excretion compared with placebo. Calcium kinetics demonstrated positive net bone balance but less than overall calcium balance, suggesting soft-tissue deposition. Fasting blood and urine biochemistries of calcium and phosphate homeostasis were unaffected by calcium carbonate. Thus, the positive calcium balance produced by calcium carbonate treatment within 3 weeks cautions against its use as a phosphate binder in patients with stage 3 or 4 CKD, if these findings can be extrapolated to long-term therapy.

Long-Term Safety and Efficacy of Recombinant Human Parathyroid Hormone (1-84) in Adults With Chronic Hypoparathyroidism.

Context: Chronic hypoparathyroidism is conventionally treated with oral calcium and active vitamin D to reach and maintain targeted serum calcium and phosphorus levels, but some patients remain inadequately controlled. Objective: To assess long-term safety and efficacy of recombinant human parathyroid hormone (1-84) (rhPTH(1-84)) treatment. Methods: This was an open-label extension study at 12 US centers. Adults (n = 49) with chronic hypoparathyroidism were included. The intervention was rhPTH(1-84) for 6 years. The main outcome measures were safety, biochemical measures, oral supplement doses, bone indices. Results: Thirty-eight patients (77.6%) completed the study. Throughout 72 months, mean albumin-adjusted serum calcium was within 2.00 to 2.25 mmol/L (8.0-9.0 mg/dL). At baseline, 65% of patients with measurements (n = 24/37) were hypercalciuric; of these, 54% (n = 13/24) were normocalciuric at month 72. Mean serum phosphorus declined from 1.6 ± 0.19 mmol/L at baseline (n = 49) to 1.3 ± 0.20 mmol/L at month 72 (n = 36). Mean estimated glomerular filtration rate was stable. rhPTH(1-84)-related adverse events were reported in 51.0% of patients (n = 25/49); all but 1 event were mild/moderate in severity. Mean oral calcium supplementation reduced by 45% ± 113.6% and calcitriol by 74% ± 39.3%. Bone turnover markers declined by month 32 to a plateau above pretreatment values; only aminoterminal propeptide of type 1 collagen remained outside the reference range. Mean bone mineral density z score fell at one-third radius and was stable at other sites. Conclusion: 6 years of rhPTH(1-84) treatment was associated with sustained improvements in biochemical parameters, a reduction in the percentage of patients with hypercalciuria, stable renal function, and decreased supplement requirements. rhPTH(1-84) was well tolerated; no new safety signals were identified.

Moderate consumption of freeze-dried blueberry powder increased net bone calcium retention compared with no treatment in healthy postmenopausal women: a randomized crossover trial.

BACKGROUND: Preclinical studies suggest that blueberry consumption is associated with improved bone health. OBJECTIVES: We conducted a blueberry dose-response study in ovariectomized (OVX)-rats that informed a study in postmenopausal women using the urinary appearance of calcium (Ca) tracers from prelabeled bone to reflect changes in bone balance. We hypothesized that blueberry consumption would reduce bone loss in a dose-dependent manner compared with no treatment. METHODS: OVX rats were fed 4 doses of blueberry powder (2.5%, 5%, 10%, and 15%) in randomized order to determine bone 45Ca retention. Fourteen healthy, nonosteoporotic women ≥4 y past menopause were dosed with 50 nCi of 41Ca, a long-lived radioisotope, and equilibrated for 5 mo to allow 41Ca deposition in bone. Following a 6-wk baseline period, participants were assigned to a random sequence of 3 6-wk interventions, a low (17.5 g/d), medium (35 g/d), or high (70 g/d) dose of freeze-dried blueberry powder equivalent to 0.75, 1.5, or 3 cups of fresh blueberries incorporated into food and beverage products. Urinary 41Ca:Ca ratio was measured by accelerator mass spectrometry. Serum bone resorption biomarkers and urinary polyphenols were measured at the end of each control and intervention period. Data were analyzed using a linear mixed model and repeated measures analysis of variance. RESULTS: In both OVX rats and postmenopausal women, blueberry interventions benefited net bone calcium balance at lower but not at higher doses. In women, net bone calcium retention increased by 6% with the low (95% CI: 2.50, 8.60; P < 0.01) and 4% with the medium (95% CI: 0.96, 7.90; P < 0.05) dose compared with no treatment. Urinary excretion of hippuric acid increased dose-dependently with blueberry consumption. No significant relationships were found between bone resorption biomarkers, 25-hydroxyvitamin D, and interventions. CONCLUSIONS: Moderate consumption (<1 cup/d) of blueberries may be an effective strategy to attenuate bone loss in healthy postmenopausal women. This trial was registered at clinicaltrials.gov as NCT02630797.

Long-term Burosumab Administration Is Safe and Effective in Adults With X-linked Hypophosphatemia.

CONTEXT: Burosumab was developed as a treatment option for patients with the rare, lifelong, chronically debilitating, genetic bone disease X-linked hypophosphatemia (XLH). OBJECTIVE: Collect additional information on the safety, immunogenicity, and clinical response to long-term administration of burosumab. METHODS: UX023-CL203 (NCT02312687) was a Phase 2b, open-label, single-arm, long-term extension study of adult subjects with XLH who participated in KRN23-INT-001 or KRN23-INT-002 studies. The long-term UX023-CL203 study (January 5, 2015 through November 30, 2018) provided data up to 184 weeks. Participants in UX023-CL203 received burosumab based on the last dose in the prior KRN23-INT-001 or KRN23-INT-002 studies (0.3, 0.6, or 1.0 mg/kg given by subcutaneous injection every 4 weeks). At Week 12, burosumab could be titrated upward/downward to achieve fasting serum phosphate levels within the normal range. Primary objectives included long-term safety, the proportion of subjects achieving fasting serum phosphate in the normal range, changes in bone turnover markers, patient-reported outcomes for pain and stiffness, and measures of mobility. RESULTS: Fasting serum phosphate levels at the midpoint of the dosing interval (2 weeks postdose, the time of peak effect) were within the normal range in 85% to 100% of subjects. Measures of phosphate metabolism and bone biomarkers generally improved with burosumab therapy, approaching or reaching their respective normal ranges by study end. Improvements in patient-reported outcomes and mobility were sustained throughout the observation period. No new safety findings emerged with longer-term burosumab treatment. CONCLUSION: These data support the conclusion that burosumab therapy may be a safe and effective long-term treatment option for adult patients with XLH.

Classical and Nonclassical Manifestations of Primary Hyperparathyroidism.

This narrative review summarizes data on classical and nonclassical manifestations of primary hyperparathyroidism (PHPT). It is based on a rigorous literature search, inclusive of a Medline search for systematic reviews from 1940 to December 2020, coupled with a targeted search for original publications, covering four databases, from January 2013-December 2020, and relevant articles from authors' libraries. We present the most recent information, identify knowledge gaps, and suggest a research agenda. The shift in the presentation of PHPT from a predominantly symptomatic to an asymptomatic disease, with its varied manifestations, has presented several challenges. Subclinical nephrolithiasis and vertebral fractures are common in patients with asymptomatic disease. The natural history of asymptomatic PHPT with no end organ damage at diagnosis is unclear. Some observational and cross-sectional studies continue to show associations between PHPT and cardiovascular and neuropsychological abnormalities, among the different disease phenotypes. Their causal relationship is uncertain. Limited new data are available on the natural history of skeletal, renal, cardiovascular, neuropsychological, and neuromuscular manifestations and quality of life. Normocalcemic PHPT (NPHPT) is often diagnosed without the fulfillment of rigorous criteria. Randomized clinical trials have not demonstrated a consistent long-term benefit of parathyroidectomy (PTX) versus observation on nonclassical manifestations. We propose further refining the definition of asymptomatic disease, into two phenotypes: one without and one with evidence of target organ involvement, upon the standard evaluation detailed in our recommendations. Each of these phenotypes can present with or without non-classical manifestations. We propose multiple albumin-adjusted serum calcium determinations (albumin-adjusted and ionized) and exclusion of all secondary causes of high parathyroid hormone (PTH) when establishing the diagnosis of NPHPT. Refining the definition of asymptomatic disease into the phenotypes proposed will afford insights into their natural history and response to interventions. This would also pave the way for the development of evidence-based guidance and recommendations. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Common genetic determinants of vitamin D insufficiency: a genome-wide association study.

BACKGROUND: Vitamin D is crucial for maintenance of musculoskeletal health, and might also have a role in extraskeletal tissues. Determinants of circulating 25-hydroxyvitamin D concentrations include sun exposure and diet, but high heritability suggests that genetic factors could also play a part. We aimed to identify common genetic variants affecting vitamin D concentrations and risk of insufficiency. METHODS: We undertook a genome-wide association study of 25-hydroxyvitamin D concentrations in 33 996 individuals of European descent from 15 cohorts. Five epidemiological cohorts were designated as discovery cohorts (n=16 125), five as in-silico replication cohorts (n=9367), and five as de-novo replication cohorts (n=8504). 25-hydroxyvitamin D concentrations were measured by radioimmunoassay, chemiluminescent assay, ELISA, or mass spectrometry. Vitamin D insufficiency was defined as concentrations lower than 75 nmol/L or 50 nmol/L. We combined results of genome-wide analyses across cohorts using Z-score-weighted meta-analysis. Genotype scores were constructed for confirmed variants. FINDINGS: Variants at three loci reached genome-wide significance in discovery cohorts for association with 25-hydroxyvitamin D concentrations, and were confirmed in replication cohorts: 4p12 (overall p=1.9x10(-109) for rs2282679, in GC); 11q12 (p=2.1x10(-27) for rs12785878, near DHCR7); and 11p15 (p=3.3x10(-20) for rs10741657, near CYP2R1). Variants at an additional locus (20q13, CYP24A1) were genome-wide significant in the pooled sample (p=6.0x10(-10) for rs6013897). Participants with a genotype score (combining the three confirmed variants) in the highest quartile were at increased risk of having 25-hydroxyvitamin D concentrations lower than 75 nmol/L (OR 2.47, 95% CI 2.20-2.78, p=2.3x10(-48)) or lower than 50 nmol/L (1.92, 1.70-2.16, p=1.0x10(-26)) compared with those in the lowest quartile. INTERPRETATION: Variants near genes involved in cholesterol synthesis, hydroxylation, and vitamin D transport affect vitamin D status. Genetic variation at these loci identifies individuals who have substantially raised risk of vitamin D insufficiency. FUNDING: Full funding sources listed at end of paper (see Acknowledgments).

Effect of calcium carbonate particle size on calcium absorption and retention in adolescent girls.

OBJECTIVE: Increasing calcium bioavailability by decreasing calcium salt particle size in the supplement may be one way to increase calcium absorption. The aim of the study was to compare (1) large versus small particle size CaCO(3) supplements and (2) small particle size CaCO(3) supplement versus placebo on calcium absorption and retention in adolescent girls. METHODS: Thirty-one adolescent girls, aged 11 to 14 years, participated in two 3-week calcium balance periods separated by a 1-week washout period. During both balance periods, the subjects consumed a controlled diet containing 804 mg/d calcium. Using a crossover design, one group (n = 19) received an additional ∼600 mg/d calcium of two ∼300-mg calcium doses as either large particle (18 µm; i.e., standard commercial form) or small particle (13.5 µm) CaCO(3). A second group (n = 12) received ∼600 mg/d calcium from small-particle CaCO(3) or placebo. RESULTS: The parathyroid hormone suppression curve, following a challenge, from the first arm of the study indicated that calcium absorption from the small particle size CaCO(3) was less than that from the large particle size CaCO(3). The parathyroid hormone suppression curve from the small particle versus placebo arm indicated that calcium absorption from small particle size CaCO(3) was greater than placebo. Calcium balance (Ca intake - [urine Ca + fecal Ca]) demonstrated that the small particle size CaCO(3) supplement increased Ca retention nearly 2-fold compared with placebo (p < 0.05; 496 ± 213 and 256 ± 94 mg/d, respectively). However, there was no significant difference in Ca retention due to small versus large particle size of CaCO(3) (p > 0.05; 349.1 ± 131.6 and 322.0 ± 194.2 mg/d, respectively). CONCLUSIONS: Dietary supplementation with CaCO(3) is effective in increasing calcium absorption and retention compared with placebo. But there is no advantage of small compared with large particle size CaCO(3) on calcium absorption and retention.

Development and optimization of an LC-MS/MS-based method for simultaneous quantification of vitamin D2 , vitamin D3 , 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3.

Simultaneous and accurate measurement of vitamin D and 25-hydroxyvitamin D in biological samples is a barrier limiting our ability to define "optimal" vitamin D status. Thus, our goal was to optimize conditions and evaluate an LC-MS method for simultaneous detection and quantification of vitamin D(2) , vitamin D(3) , 25-hydroxyvitamin D(2) and 25-hydroxyvitamin D(3) in serum. Extraction and separation of vitamin D forms were achieved using acetone liquid-liquid extraction and by a reversed phase C8 column, respectively. Detection was performed on a triple quadrupole tandem mass spectrometer (QQQ-MS/MS) equipped with atmospheric pressure photo ionization source. The LOQs for all analytes tested were 1 ng/mL for hydroxylated molecules and 2 ng/mL for the parent vitamin Ds. RSD at lower LOQ (2 ng/mL) and in medium (80 ng/mL) and high (200 ng/mL) quality control samples did not exceed 20 and 15% CV, respectively. Accuracy of the method for determination of hydroxylated molecules was also validated using National Institutes of Standards and Technology standard samples and found to be in the range of 90.9-111.2%. In summary, a sensitive and reproducible method is reported for simultaneous quantification of vitamin D(2) , vitamin D(3) , 25-hydroxyvitamin D(2) and 25-hydroxyvitamin D(3) molecules in biological samples.