How the reference values for serum parathyroid hormone concentration are (or should be) established?

Well-validated reference values are necessary for a correct interpretation of a serum PTH concentration. Establishing PTH reference values needs recruiting a large reference population. Exclusion criteria for this population can be defined as any situation possibly inducing an increase or a decrease in PTH concentration. As recommended in the recent guidelines on the diagnosis and management of asymptomatic primary hyperparathyroidism, PTH reference values should be established in vitamin D-replete subjects with a normal renal function with possible stratification according to various factors such as age, gender, menopausal status, body mass index, and race. A consensus about analytical/pre-analytical aspects of PTH measurement is also needed with special emphasis on the nature of the sample (plasma or serum), the time and the fasting/non-fasting status of the blood sample. Our opinion is that blood sample for PTH measurement should be obtained in the morning after an overnight fast. Furthermore, despite longer stability of the PTH molecule in EDTA plasma, we prefer serum as it allows to measure calcium, a prerequisite for a correct interpretation of a PTH concentration, on the same sample. Once a consensus is reached, we believe an important international multicentre work should be performed to recruit a very extensive reference population of apparently healthy vitamin D-replete subjects with a normal renal function in order to establish the PTH normative data. Due to the huge inter-method variability in PTH measurement, a sufficient quantity of blood sample should be obtained to allow measurement with as many PTH kits as possible.

Individual site-specific bone mineral density gain in normocalcemic primary hyperparathyroidism.

UNLABELLED: In this study, we show that successful parathyroidectomy is followed at 1 year by a significant individual bone mineral density (BMD) gain in nearly half of normocalcemic PHPT patients with reduced bone mass. Alkaline phosphatase levels above median were identified as an independent predictor of individual BMD gain in normocalcemic PHPT patients. INTRODUCTION: The aims of this study were to assess bone mineral density (BMD) gains after parathyroidectomy (PTX) in normocalcemic primary hyperparathyroidism (PHPT) at the individual level and to identify predictors of BMD gain after PTX in this context. METHODS: Longitudinal cohort study of 55 PHPT patients referred for low bone mass and mild abnormalities of calcium/phosphorus metabolism, and successfully treated by PTX. BMD gain at 1 year was considered significant if ≥0.030 g/cm(2) at one site or more, without any equivalent BMD loss at another site. A logistic regression analysis was performed to identify predictive factors of individual BMD gain. RESULTS: Among the 55 PHPT patients included, 29 patients with hypercalcemia, 36 patients with normocalcemic PHPT, defined by normal pre-PTX serum total (albumin-corrected) calcium (tCa), including 15 patients with normal ionized calcium (iCa), were identified. At 1 year of PTX, an individual BMD gain was observed in 73.7 % of hypercalcemic, 44.4 % of normocalcemic, and 46 % of PHPT patients with both normal tCa and iCa. Site-specific BMD gains were most important at the spine and hip in all subgroups including patients with normal iCa. Alkaline phosphatase activity above median, which reflects high bone turnover, was predictive of individual BMD gain, both in the overall cohort (OR = 4.9, 95 % CI 1.3-18.9), and in the normocalcemic group: OR = 8.4, 95 % CI 1.4-56.6. CONCLUSIONS: Successful PTX is followed at 1 year by a significant individual BMD gain in nearly half of normocalcemic PHPT patients with osteoporosis. ALP levels above median could contribute to the therapeutic decision in this context.

Vitamin D status of schoolchildren in Northern Algeria, seasonal variations and determinants of vitamin D deficiency.

UNLABELLED: There are no published data on the vitamin D status of children living in North Africa. In 435 healthy Algerian children 5-15 years old, we found that vitamin D insufficiency (serum 25-hydroxyvitamin D (25OHD) <50 nmol/L) was frequent, especially in winter. Low vitamin D status was associated with increased parathyroid hormone (PTH) and leg deformation INTRODUCTION: As there are no published data on the vitamin D status of children living in North Africa, we evaluated the 25OHD concentration of healthy Algerian children at the end of summer and at the end of winter. As secondary objectives, we studied the various determinants of vitamin D status and the PTH-25OHD relationship in these subjects. METHODS: Four hundred thirty-five children 5-15 years old were examined and had a blood sample in September 2010. Of them, 408 were sampled again in March 2011. RESULTS: Median 25OHD concentration in the whole group was 71.4 nmol/L in September and 52.9 nmol/L in March. In September, 58.4, 29.9, and 8.1% had a 25OHD concentration below 75, 50, and 30 nmol/L respectively. In March, these percentages increased to 65.2, 41.4, and 17.4% for the 75, 50, and 30 nmol/L threshold, respectively. In multivariate analysis, older age, darker skin phototype, low daily vitamin D and calcium intake, poor socioeconomic status, and short daily sun exposure remained significantly associated with a 25OHD <50 nmol/L at both visits. In 72 (16.6%) children, genu varum/valgum was present. Compared to the 363 children without leg deformation, they presented more frequently with the risk factors of vitamin D insufficiency. They also had lower 25OHD concentrations and higher PTH and tALP. Serum PTH and 25OHD concentrations were negatively and significantly correlated (r = -0.43; p < 0.001) without a 25OHD threshold above which PTH does not decrease anymore. CONCLUSION: Despite a sunny environment, vitamin D insufficiency is frequent in healthy Algerian children.

Cost-effectiveness analysis of hip fracture prevention with vitamin D supplementation: a Markov micro-simulation model applied to the French population over 65 years old without previous hip fracture.

UNLABELLED: We performed a cost-effectiveness analysis of four vitamin D supplementation strategies for primary prevention of hip fracture among the elderly population and found that the most cost-effective strategy was screening for vitamin D insufficiency followed by adequate treatment to attain a minimum 25(OH) serum level. INTRODUCTION: Vitamin D supplementation has a demonstrated ability to reduce the incidence of hip fractures. The efficiency of lifetime supplementation has not yet been assessed in the population over 65 years without previous hip fracture. The objective was to analyze the efficiency of various vitamin D supplementation strategies for that population. METHODS: A Markov micro-simulation model was built with data extracted from published studies and from the French reimbursement schedule. Four vitamin D supplementation strategies were evaluated on our study population: (1) no treatment, (2) supplementation without any serum level check; (3) supplementation with a serum level check 3 months after initiation and subsequent treatment adaptation; (4) population screening for vitamin D insufficiency followed by treatment based on the vitamin D serum level. RESULTS: "Treat, then check" and "screen and treat" were two cost-effective strategies and dominated "treat without check" with incremental cost-effectiveness ratios of €5,219/quality-adjusted life-years (QALY) and €9,104/QALY, respectively. The acceptability curves showed that over €6,000/QALY, the "screen and treat" strategy had the greatest probability of being cost-effective, and the "no treatment" strategy would never be cost-effective if society were willing to spend over €8,000/QALY. The sensitivity analysis showed that among all parameters varying within realistic ranges, the cost of vitamin D treatment had the greatest effect and yet remained below the WHO cost-effectiveness thresholds. CONCLUSIONS: Population screening for vitamin D insufficiency followed by treatment based on the vitamin D serum level is the most cost-effective strategy for preventing hip fracture occurrence in the population over 65 years old.

Association of nutrition in early life with body fat and serum leptin at adult age.

BACKGROUND: There is overwhelming evidence that experiences during early life could have long-term health consequences. However, the role of early nutrition in programming obesity and leptin resistance is still poorly understood. OBJECTIVE: We aimed at determining whether nutritional intakes in early life are associated with body composition and hormonal status at 20 years. SUBJECTS: Healthy infants participating in the two-decade-long prospective ELANCE (Etude Longitudinale Alimentation Nutrition Croissance des Enfants) study were examined at 10 months and 2 years. At 20 years, weight, height, subscapular and triceps skinfold thicknesses, fat mass (FM) and fat-free mass (FFM) assessed via bioelectrical impedance analysis, and serum leptin concentration were recorded in 73 subjects still participating in the follow-up. RESULTS: In adjusted linear regression models, an increase by 100 kcal in energy intake at 2 years was associated with higher subscapular skinfold thickness (ß=6.4% SF, 95% confidence interval 2.53-10.30, P=0.002) and higher FFM (0.50 kg, 0.06-0.95, P=0.03) at 20 years. An increase by 1% energy from fat at 2 years was associated with lower subscapular skinfold thickness (-2.3% SF, -4.41 to -0.18, P=0.03), lower FM (-0.31 kg, -0.60 to -0.01, P=0.04) and lower serum leptin concentration (-0.21 µg l(-1), -0.39 to -0.03, P=0.02) at 20 years. CONCLUSIONS: Low-fat intake in early life was negatively associated with body fat (particularly at the trunk site) and serum leptin concentration at 20 years, suggesting that early low-fat intake could increase the susceptibility to develop overweight and leptin resistance at later ages. These findings substantiate current recommendations against restricting fat intake in early life and open new directions for investigating the origin of obesity.

Measurement uncertainty of 25-OH vitamin D determination with different commercially available kits: impact on the clinical cut offs.

SUMMARY: Due to "measurement uncertainty", the "true" 25-OH vitamin D (25(OH)D) of a patient (whatever the commercially available assay tested) will be >80 nmol/L if its measured concentration is >100 nmol/L. Thus, if a physician considers that a normal VTD status is a 25(OH)D level >or=80 nmol/L, he should ensure that the patient's results are >or=100 nmol/L. INTRODUCTION: Many experts recommend that serum levels of 25(OH)D should be above a lower normal limit of 75-80 nmol/L. However, the value delivered by laboratories is only an estimation of the "true" value due to "measurement uncertainty." When using a cut off, measurement uncertainty around the cut off is important because therapeutic actions may differ if the measured value is below or above the limit. We aimed to establish the "measurement uncertainty" at different levels of concentration for several commercially available 25(OH)D analytical techniques. METHODS: We constituted three pools of serum with different 25(OH)D concentrations. Each pool was assayed in triplicate during 5 days with the DiaSorin RIA, Liaison, Elecsys, and Chromsystems-HPLC assays. RESULTS: We report a relatively high "measurement uncertainty" for the measurement of 25(OH)D for the four different techniques: the mean relative uncertainties, all techniques confounded were 19.4%, 16.0%, and 11.3% for pool 1 (35.3 nmol/L), pool 2 (79.5 nmol/L), and pool 3 (126.1 nmol/L), respectively. CONCLUSIONS: Our results show that, whatever the assay, the "true" 25(OH)D of a patient will be >80 nmol/L if its measured concentration is >100 nmol/L. In other words, if a physician considers that a normal VTD status is defined by a 25(OH)D level >or=80 nmol/L, he should ensure that the patients present a 25(OH)D >or=100 nmol/L.

Osteopenia and vitamin D deficiency in children with sickle cell disease.

OBJECTIVES: To assess the prevalence in children with sickle cell disease of low bone mineral density (BMD), a feature found in up to 82% of adults but not well known in children. METHODS: In 53 children (45 SS, 4 SC, 4 Sbeta-thalassemia) with a mean age of 12.8 +/- 2.4 years, we assessed height; weight; sexual maturation; number of hospitalizations, painful crises, and transfusions in the last 3 years; calcium intake; steady-state hemoglobin and leukocyte count; calcaemia, phosphataemia, and calciuria/creatinuria; serum 25-(OH)D and PTH concentrations; and osteocalcin, urinary deoxypyridinoline, and the C-terminal component of pro-collagen type I. BMD was assessed using dual X-ray absorptiometry. RESULTS: Mean lumbar spine Z-score was -1.1 +/- 1.3 (-3.9 to +1.8). The Z score was significantly lower in girls than in boys in the prepubertal subgroup (-1.74 +/- 0.27 vs. -0.53 +/- 0.31) (P = 0.0169), but not in the pubertal group (-1.15 +/- 0.41 vs. -1.33 +/- 0.70). BMD was not associated with any of the disease-severity markers in girls but was unexpectedly associated with fewer vaso-occlusive crises and hospitalizations in boys. BMD did not correlate with hemoglobin or leukocyte counts. Vitamin D deficiency [25-(OH)D < 12 ng/mL] was found in 76% of patients and secondary hyperparathyroidism (PTH > 46 pg/mL) in 38%. BMD was not related to calcium intake, vitamin D status, osteocalcin, or bone resorption markers. CONCLUSION: A slight BMD decrease was found in SCD children, starting before puberty and being more marked in females. The decrease was unrelated to disease severity, vitamin D deficiency, or bone hyperresorption, suggesting abnormal bone formation as the underlying mechanism.

Pamidronate treatment of children with moderate-to-severe osteogenesis imperfecta: a note of caution.

BACKGROUND/AIMS: Bisphosphonates have been reported to decrease fractures related to osteogenesis imperfecta (OI). We assessed the efficacy and long-term safety of pamidronate therapy in patients with moderate-to-severe OI. METHODS: We conducted an open-label uncontrolled study in 14 boys and 13 girls whose mean age was 6.8 years at baseline. Intravenous pamidronate, 1 mg/kg/day, was given for 3 consecutive days every 4 months for 2-6 years, with physical therapy and orthopedic surgery as appropriate. Mobility score, fracture rate, height, bone mineral density (BMD) and bone healing were evaluated throughout follow-up. RESULTS: In 24 (89%) patients, the fracture rate decreased to 6 months) occurred in 8 (29.6%) patients; their BMD gains, baseline age and treatment duration were not significantly different from those in the other patients. Tolerance was good. CONCLUSION: Pamidronate with physiotherapy and orthopedic management improved outcomes without delaying fracture healing in 19 (70%) of 27 patients. Delayed fracture healing occurred in 8/27 patients. Pamidronate should be reserved for severe OI with multiple fractures and/or flattened vertebras.

[Vitamin D2 or vitamin D3?]. / Vitamine D2 ou vitamine D3?

PURPOSE: Nearly one billion people around the world are deficient in vitamin D and need to be supplemented. Vitamin D is available in medicines and fortified foods. It is available in two forms: vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). KEY POINTS: The pharmacopeiae consider these steroid hormones as equivalent and interchangeable. However, several studies have showed that serum level of 25(OH)D is increased more effectively with vitamin D3 than vitamin D2. Vitamin D2 has shorter plasma half-life and a lower affinity for the vitamin D binding protein, the hepatic vitamin D hydroxylase and the vitamin D receptor. CONCLUSION: Vitamin D2 should not be regarded anymore as suitable for supplementation or fortification. Currently though, it is still the most used in some countries such as Portugal and Australia.

[Recommendations for the management of bone demineralization in cystic fibrosis]. / Recommandations pour la prise en charge de la déminéralisation osseuse dans la mucoviscidose.

A high prevalence of low bone mineralization is documented in adult patients with cystic fibrosis (CF). Osteopenia is present in as much as 85% of adult patients and osteoporosis in 13 to 57% of them. In children, studies are discordant probably because of different control database. Denutrition, inflammation, vitamin D and vitamin K deficiency, altered sex hormone production, glucocorticoid therapy, and physical inactivity are well known risk factors for poor bone health. Puberty is a critical period and requires a careful follow-up for an optimal bone peak mass. This review is a consensus statement established by the national working group of the French Federation of CF Centers to develop practice guidelines for optimizing bone health in patients with CF. Recommendations for screening and for calcium, vitamin D and K supplementation are given. Further work is needed to define indications for treatment with biphosphonates and anabolic agents.

[Update on vitamin D and evaluation of vitamin D status]. / Actualité sur les effets de la vitamine D et l'évaluation du statut vitaminique D.

Knowledge about vitamin D has greatly improved during the last few years. Vitamin D cannot any more be considered as exclusively necessary to prevent ricket/osteomalacia. Its role in the prevention of some osteoporotic fractures in the elderly (in association with calcium nutrition) is now well demonstrated and many epidemiologic and laboratory data argue for a role in the prevention of several diseases or anomalies (cancer, auto-immune diseases, cardiovascular events, sarcopenia...). A few intervention studies confirming some of these effects also exist. Vitamin D status can easily be assessed by measuring serum 25 hydroxy vitamin D (25OHD) level. However, many experts have claimed that the population-based reference values for 25OHD are too low and that the cut-off value below which vitamin D insufficiency can be present is somewhere between 20 and 40 ng/mL with a clear tendency to target values above 30 ng/mL (75 nmol/L). The main consequences are that vitamin D insufficiency is highly frequent whereas the currently recommended supplementation doses are not sufficient.

Serum procollagen type 1 amino-terminal propeptide (P1NP) as an early predictor of the growth response to growth hormone treatment: Comparison of intrauterine growth retardation and idiopathic short stature.

UNLABELLED: There is no way to predict early the growth response to growth hormone (GH) treatment in short children with intrauterine growth retardation (IUGR) or idiopathic short stature (ISS). OBJECTIVE: To evaluate the capacity of the procollagen type 1 amino-terminal propeptide (P1NP), a new marker of bone formation, to help in this prediction. PATIENTS AND METHODS: Longitudinal study of 30 patients treated at 7.7 (range: 2.2-12.5) years for IUGR (n=16) or ISS (n=14) with GH (0.47 and 0.33 or 0.4mg/kg/week respectively). P1NP and insulin-like growth factor I (IGF I) were measured before and after 3-6 months of GH treatment. RESULTS: Before treatment, IUGR patients were younger and shorter than ISS patients, but their other characteristics were similar. IGF I Z-score (ZS) and P1NP concentrations were positively correlated in the whole population (Rho=0.48; P=0.01). After 3-6 months of treatment, both concentrations increased in IUGR and ISS (P<0.01). They remained correlated only in ISS (Rho=0.54; P<0.05). P1NP before treatment was negatively correlated (Rho=-0.67, P=0.015) with the growth rate (SD) during the first year of treatment in ISS but not in IUGR; IGF I ZS was not. The changes in P1NP for the whole population over 3-6 months, but not the changes in IGF I ZS, were positively correlated with the growth rate (Rho=0.41, P=0.03). CONCLUSIONS: Lower basal plasma P1NP concentrations predict better growth response to GH treatment during the first year in ISS children. Greater increases in its concentrations after 3-6 months of GH treatment may also predict a better growth response in both ISS and IUGR.

Methylmalonic and propionic acidurias: management without or with a few supplements of specific amino acid mixture.

In a series of 137 patients with methylmalonic acidaemia (MMA) and propionic acidaemia (PA) diagnosed since the early 1970s, we report in more detail 81 patients (51 MMA and 30 PA) diagnosed between 1988 and 2005. In this series, 14% of patients died at initial access revealing the disease before or despite treatment, 18% died later, and the remainder (68%) are still alive. All patients were treated with the same protocol of enteral feeds with a low-protein diet adjusted to individual tolerance, carnitine, antibiotics, and only occasional use of an amino acid (AA) mixture. There was intensive follow-up and monitoring using measurements of urinary urea. Thirty-nine patients with severe forms, followed for more than 3 years, are analysed in particular detail. Of the 17 PA patients, 6 had moderate disability (all neonatal-onset forms), whereas 11 were normal or slightly delayed in their mental development. Four presented with cardiomyopathy, of whom 2 died. Of the 22 MMA patients, 13 presented in the neonatal period, of whom 3 died later, 2 are in renal failure and only 5 are still alive and have a normal or slightly delayed mental development. In the 9 patients with late-onset forms, there were no deaths and all patients but one have normal mental development. Among the 39 patients, only 40% were given an AA supplement at 3 years, and 50% between 6 and 11 years. The actual intake of natural protein was 0.92, 0.78 and 0.77 g/kg per day at 3, 6 and 11 years, respectively, in patients without AA supplementation, whereas it was 0.75, 0.74 and 0.54 g/kg per day in the group who received small quantities of AA (0.4-0.6 g/kg per day). In both groups, feeding disorders were frequent: 55% at 3 years, 35% at 6 years and 12% at 11 years. Many patients were given a food supplement by tube overnight or were even exclusively tube fed: 60% at 3 years, 48% at 6 years and still 27% at 11 years. Growth velocity was near the normal values. Plasma valine and isoleucine were low to very low, as were leucine and phenylalanine but to a lesser extent. Albumin, vitamins, trace elements and markers of bone metabolism were within the normal values. IGF1, 24-hour urine calcium and body mass density were low. Body composition showed a normal to low lean mass and a normal to high fat mass.

[New definition of optimal vitamin D status and redefening serum parathyroid hormone reference range]. / Nouvelles définitions de l'insuffisance vitaminique D, retentissement sur les normes de PTH.

SCOPE: Knowledge concerning vitamin D has greatly improved during the past few years. Vitamin D can no longer be considered only as a preventive therapy for rickets-osteomalacia. Indeed, beside its role in the prevention of osteoporotic fractures in the elderly, many data suggest that it may be involved in the prevention of various diseases including cancers and auto-immune diseases. CURRENT SITUATION AND SALIENT POINTS: Vitamin D status may be easily assessed by the measurement of 25OHD serum concentration. However, many specialists in the field regard most 25OHD reference values as being too low, and believe that the 25OHD threshold below which vitamin D status can be considered as insufficient is somewhere between 50 and 100 nmol/L (20 to 40 ng/mL). It then appears that usually recommended amounts of supplemental vitamin D may be too low to reach these 25OHD concentrations, and thus need to be revised. We have proposed that PTH reference values should be established in healthy subjects with a normal vitamin D status. This supposes that 25OHD is measured in the reference population beforehand, and that the subjects with vitamin D insufficiency are eliminated from the reference group. PERSPECTIVES: Although more complicated than the usual way to establish normative data, we have shown that it decreases the upper limit of normal by 25-35%, enhancing thus the diagnostic sensitivity for hyperparathyroidism without a decrease in specificity.

Management of boys with short stature and delayed puberty.

OBJECTIVE: To review the management of boys with short stature and delayed puberty and the testosterone priming protocol. METHODS: In 148 boys aged > 14 years seen for height < -2 SDS and constitutional delayed puberty we evaluated growth hormone (GH) secretion and final height (80 boys). RESULTS: The GH peak was < 10 microg/l after arginine-insulin tests performed with testosterone heptylate priming in 8/32 (25%) and without in 62/153 (41%), including first and second evaluations. It was low in 7/11 boys given 2 x 100 mg testosterone (14.7 +/- 1.7 microg/l) and in 1/21 given 4 x 100 mg (21.3 +/- 2.0 microg/l, p = 0.04). It was low during sleep in 4/29 (14%) boys, all having basal plasma testosterone below 3.5 nmol/l. The basal insulin-like growth factor (IGF)-I concentration was below -2 SDS in 22% of the boys evaluated. Final height was -0.8 +/- 0.1 SDS. It was similar in those with low (n = 9) and normal (n = 71) GH peak, and in those treated (n = 22) or untreated (n = 58) with testosterone. It was over 1 SDS lower than the target height in 20% and than the predicted height at the initial evaluation in 14% of the boys. Pubertal growth was not correlated with the GH peak or plasma IGF-I. CONCLUSIONS: The GH peak during the sleep is more frequently normal than the peak after stimulation. The number of testosterone doses influences the quality of priming. The medical problems involved in treating boys with delayed puberty are excluding disease and deciding on testosterone treatment.

Measurement of circulating 25-hydroxyvitamin D: A historical review.

The constantly increasing requests for the measurement of serum 25-hydroxyvitamin D over the last years has led reagent manufacturers to market different automated and semi-automated methods, that being unfortunately not fully harmonized, yield different results. Liquid chromatography coupled to tandem mass spectrometry (LC/MS2) has more recently been introduced. This approach allows the distinction between the two forms of 25-hydroxyvitamin D and to measure other metabolites. This approach also requires harmonization to curtail the differences between the different analytical methods. To meet this requirement, the American National Institutes of Health (NIH), the Centre for Disease Control and Prevention (CDC) in Atlanta, the National Institute of Standards and Technology (NIST) and the vitamin D Reference laboratory of Ghent University have pooled their expertise to develop a standardization program. This article reviews the main elements and the difficulties of the automated and semi-automated methods for 25-hydroxyvitamin D, from sample preparation to the analytical phase, as well as those related to mass spectrometry. It also emphasizes the need for standardization to better define the clinical decision thresholds of vitamin D nutritional status.

CYP24A1 Mutations in a Cohort of Hypercalcemic Patients: Evidence for a Recessive Trait.

CONTEXT: Loss-of-function mutations of CYP24A1 (which encodes the 25-OH-D3-24-hydroxylase) have recently been reported to cause hypercalcemia. OBJECTIVES: The aims of this study were: 1) to evaluate the frequency of CYP24A1 mutations in patients with medical history of hypercalcemia; 2) to show the clinical utility of a simultaneous assay of serum 25-hydroxyvitamin D3 (25-OH-D3) and 24,25-dihydroxyvitamin D3 (24,25-[OH]2D3) by liquid chromatography tandem mass spectrometry (LC-MS/MS); and 3) to investigate biochemical parameters in heterozygous gene carriers with CYP24A1 mutations. PATIENTS AND METHODS: We screened for CYP24A1 mutations in 72 patients with serum calcium levels > 2.6 mmol/L and PTH levels < 20 pg/mL and recruited 24 relatives after genetic counseling for subsequent investigations. Vitamin D metabolite concentrations were assessed in a subset of patients by LC-MS/MS and results expressed as a ratio (R) of 25-OH-D3:24,25-(OH)2D3. RESULTS: Twenty-five patients with hypercalcemia (35%) harbored CYP24A1 variations. Twenty (28%) had biallelic variations, mostly found in subjects with nephrocalcinosis or renal stones (19/20). Five patients, all neonates, were heterozygous, without renal disease. We describe 15 new variations leading to loss-of-function according to pathogenicity prediction programs, and we functionally characterized 5 of them in vitro. A dramatic increase of R, usually >80, was found in patients harboring biallelic mutations providing evidence in vivo for the loss of CYP24A1 activity. In contrast, R value remains <25 in patients without CYP24A1 mutations. Subjects carrying one mutant allele, hypercalcemic individuals, as well as gene-carrier relatives, had a detectable 24,25-(OH)2D3 level and R < 25, indicating normal 24-hydroxylase activity. CONCLUSION: CYP24A1 biallelic mutations are frequently found in patients presenting with hypercalcemia, low PTH, and renal disease. We confirm the accuracy and effectiveness of a novel blood test estimating the ratio between relevant vitamin D metabolites as a useful screening tool for CYP24A1 mutations. Haploinsufficiency is not associated with CYP24A1 deficiency.

Persistence of Ca2+-sensing receptor expression in functionally active, long-term human parathyroid cell cultures.

An original human parathyroid cell culture model from uremic patients with IIo hyperparathyroidism has been developed, with its main feature being long-term functionally active viability up to 5 months, as assessed by persistent responsiveness to changes of extracellular Ca2+ concentrations ([Ca2+]e). In addition to the inhibitory effect of increasing [Ca2+]e, increasing extracellular phosphate exerted a biphasic effect on parathyroid hormone (PTH) secretion. The presence of the Ca2+-sensing receptor (CaR), on which depends the response to [Ca2+]e and its persistence, has been demonstrated in our culture system both by direct detection and by inhibition of its activity. CaR protein was detected by Western blot analysis with a specific anti-CaR antibody. CaR gene transcripts have been identified by reverse transcription-polymerase chain reaction analysis. mRNA (by in situ hybridization) and protein (by immunocytochemistry) expression were detected for both CaR and PTH. Adding a specific anti-CaR antibody to the medium induced a marked reduction of low [Ca2+]e-stimulated PTH release, which decreased to levels equivalent to those obtained in high [Ca2+]e medium. The described long-term functionality could be due to several factors, including the clustered cell type of culture yielded by our preparation procedure, the growth characteristics of hyperplastic uremic tissue, and the use of a phosphate-rich medium. The present model, because of its long-term functionality, is a unique tool for the exploration of PTH synthesis and secretion and for studies of parathyroid cell growth in vitro.

Serum pyridinoline as a specific marker of collagen breakdown and bone metabolism in hemodialysis patients.

Type I collagen represents more than 90% of bone matrix. Quantitative analysis of collagen cross-link molecules such as pyridinoline (PYD) provides valuable information on bone resorption rate. We have studied 37 hemodialysis patients who underwent a systematic transiliac bone biopsy for histomorphometry study. Eighteen of them had tetracycline double labeling, allowing to determine dynamic, in addition to static bone parameters. Measurement of serum-free PYD was performed using a new competitive enzyme immunoassay. Serum PYD values were compared with those of three other serum markers of bone metabolism, namely intact PTH (iPTH), bone-specific alkaline phosphatase (bAP), and osteocalcin, for the correlations with bone histomorphometric parameters. Serum PYD levels (mean +/- SD) were significantly higher in dialysis patients than in normal individuals, 90.6 +/- 99.6 nM versus 1.9 +/- 0.4 nM, respectively. Patients with high turnover bone disease had significantly higher serum PYD levels than patients with normal or low bone turnover, 108.8 +/- 108.0 nM versus 34.1 +/- 12.8 nM, respectively. Serum PYD levels were positively correlated with bone resorption parameters including osteoclast surface (r = 0.59, p < 0.0001) and osteoclast number/mm2 (r = 0.61, p < 0.0001), and also with bone formation parameters, osteoblast surface (r = 0.43, p < 0.008), double-labeled surface (r = 0.81, p < 0.001), and BFR (r = 0.91, p < 0.0001). The BFR was better correlated with serum PYD levels than with either serum iPTH or osteocalcin concentrations. However, correlation with serum bAP was comparable.(ABSTRACT TRUNCATED AT 250 WORDS)