甲状旁腺素、25-羟维生素D及血清钙磷与原发性骨质疏松症的相关性分析

目的分析原发性骨质疏松症患者血清钙、磷、甲状旁腺激素(PTH)和25-羟维生素D水平,分析原发性骨质疏松症发生的主要危险因素,并探讨PTH水平与性别和年龄的关系。方法将2017年1月至2018年12月就诊于该院并确诊为原发性骨质疏松症的患者254例纳入研究作为病例组。另外,将同期于该院体检中心体检合格的健康体检者254例纳入研究作为对照组。比较两组生化指标,并进行Logistic回归分析。将2018年6-7月于该院体检中心体检合格的健康体检者2551例纳入研究作为健康人群,分析PTH水平与性别、年龄的关系。结果健康人群PTH水平与性别、年龄均无关(P>0.05)。病例组PTH水平高于对照组(P<0.001),病例组血清钙、磷及25-羟维生素D水平均低于对照组(P<0.001)。Logistic回归分析显示,PTH升高是导致原发性骨质疏松症的主要危险因素,OR值为1.495(95%CI:1.310~1.707,P<0.001)。结论健康人群PTH水平与性别和年龄无关。PTH升高是导致原发性骨质疏松症发生的主要危险因素。     

Comparison of four current 25-hydroxyvitamin D assays

ObjectivesThe performance of recently developed vitamin D total assays (ADVIA Centaur and Elecsys) was compared to that of liquid chromatography–tandem mass spectrometry (LC–MS/MS) and LIASON 25-OH Vitamin D total assays.Design and methodsA total of 157 clinical samples and standard reference material (SRM) 972 were analyzed.ResultsThe correlations of LC–MS/MS with the three immunoassays were acceptable. However, compared to LC–MS/MS, LIAISON and ADVIA Centaur showed negative bias, and Elecsys showed positive bias. There was a lack of agreement among the four methods with only LC–MS/MS results close to the certified values of SRM 972. The prevalence of vitamin D insufficiency (< 50 nmol/L) was higher with ADVIA Centaur (51.6%) and LIAISON (52.2%) and lower with Elecsys (37.6%), compared with that of LC–MS/MS (44.6%).ConclusionThe new, automated total vitamin D assays show acceptable correlation with LC–MS/MS, and could be used in routine laboratories. However, standardization of vitamin D assays and consideration of assay-specific decision limits should be addressed.     

Two direct (nonchromatographic) assays for 25-hydroxyvitamin D

We describe direct, nonchromatographic assays for 25-hydroxyvitamin D3 in which we use either rat vitamin D-binding protein or rabbit antibodies to 25-hydroxyvitamin D3-3-hemisuccinate-bovine serum albumin as binding protein. Nonspecific interferences in serum could be eliminated by an appropriate extraction method or in obtaining data for the calibration curve, by using vitamin D-free human serum. The latter is prepared by affinity chromatography with use of immobilized antibodies against human vitamin D-binding protein. Values obtained by the direct assays and those obtained by two different chromatographic methods correlated well (r greater than 0.95). The direct competitive protein-binding assay overestimated the true 25-hydroxyvitamin D3 concentration by about 20%, but this percentage was constant from 5 to 600 micrograms/L. Overestimation by the direct radioimmunoassay was less than 10%. These two direct assays for 25-hydroxyvitamin D3 allow reliable, rapid, and simple screening for vitamin D deficiency or excess.     

An evaluation of automated methods for measurement of serum 25-hydroxyvitamin D

Objectives

To compare two new automated assays with the well-established reference method, DiaSorin radioimmunoassay (RIA), for quantitation of serum total 25-hydroxyvitamin D [25(OH)D].

Methods

25(OH)D from human sera (n = 158) was measured using DiaSorin RIA and two automated platforms, DiaSorin “LIAISON 25 OH Vitamin D TOTAL”, and Roche Modular “Vitamin D3 (25-OH)”. Methods were compared by regression and Bland-Altman analyses.

Results

DiaSorin LIAISON demonstrated a stronger correlation (r = 0.918) and better agreement (bias = − 0.88 nmol/L) with DiaSorin RIA than the Roche Modular assay (r = 0.871, bias = − 2.55 nmol/L). Precision ranges (CV%) for the RIA, LIAISON, and Roche Modular assays, respectively, were: within run (6.8-12.9%, 2.8-8.1%, and 1.9-5.5%), and total precision (7.4-14.5%, 7.3-17.5%, and 7.6-14.5%).

Conclusion

DiaSorin LIAISON displayed the best correlation and agreement with DiaSorin RIA. The DiaSorin LIAISON 25 OH Vitamin D TOTAL assay is an accurate and precise automated tool for serum total 25(OH)D determination.     

Seasonal and age-related differences in serum 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and parathyroid hormone in patients from Western Norway

Abstract

Objective. The aim of this study was to investigate the seasonal and age-related variation of vitamin D and PTH serum concentrations in a large general patient population in Western Norway. Design. A retrospective study was conducted at the Hormone laboratory, Haukeland University Hospital, Bergen, Norway. All analyses of 25-hydroxyvitamin D (25(OH)D) (n = 8325), 1,25-dihydroxyvitamin D (1,25(OH)₂D) (n = 4509) and PTH (n = 4203) requested from private practitioners from 2005 to 2008 were included. All three analytes were available in 1551 subjects. Subjects. Mean age of the study population was 49.8 years and 70.9% of the samples were from women. Results. The highest concentrations of 25(OH)D and 1,25(OH)₂D were observed in July–September. In April 43% of the studied population had 25(OH)D concentrations below 50 nmol/L. There was a positive correlation between 25(OH)D and 1,25(OH)₂D (p < 0.001). The levels of 25(OH)D and PTH were negatively correlated (p < 0.001) while 1,25(OH)₂D and PTH showed a weak positive correlation (p = 0.015). We observed higher concentrations of 25(OH)D (p = 0.003) and lower 1,25(OH)₂D levels (p < 0.001) in the older age groups. PTH increased throughout the whole age span (p < 0.001). Conclusion. We observed a seasonal variation in 25(OH)D and 1,25(OH)₂D with low serum concentrations during winter/early spring while PTH showed an inverse pattern. Higher levels of PTH in winter and the elderly may reflect an impaired vitamin D status that may affect calcium homeostasis and bone health.     

Vitamin D status regulates 25-hydroxyvitamin D3-1 alpha-hydroxylase and its responsiveness to parathyroid hormone in the chick.

We asked this question: Under normal or near-normal metabolic conditions, does the prevailing normal or near-normal vitamin D status dampen the activity of 25-hydroxyvitamin-D3-1 alpha-hydroxylase (1 alpha-hydroxylase) such that it determines not only its "basal" activity but also its responsiveness to stimulation by increased circulating concentrations of parathyroid hormone (PTH)? To answer this question, we measured the activity of 1 alpha-hydroxylase in chicks, with and without administration of PTH, immediately before and during deprivation of vitamin D. Before deprivation of vitamin D, 1 alpha-hydroxylase activity increased only slightly with administration of PTH. With deprivation of vitamin D for 5 and 10 d, while the plasma concentrations of calcium and phosphorus persisted normal and unchanged, 1 alpha-hydroxylase activity not only increased progressively but also became sharply and increasingly responsive to stimulation by administration of PTH. But after 15 d of vitamin D deprivation, and the supervention of hypocalcemia, 1 alpha-hydroxylase activity was not further increased by the administration of PTH. With deprivation of vitamin D, the progressive increase in 1 alpha-hydroxylase correlated inversely with circulating levels of 1,25-dihydroxyvitamin D (1,25-[OH]2D), and the decreasing calcemic response to PTH correlated inversely with the responsiveness of 1 alpha-hydroxylase to PTH (in chicks deprived of vitamin D for 1-10 d). These results demonstrate that: under normal metabolic conditions, the normal vitamin D status regulates the activity of 1 alpha-hydroxylase so as to dampen both its "basal" activity and its responsiveness to stimulation by PTH; and vitamin D deprivation insufficient to cause hypocalcemia enhances both the "basal" activity of 1 alpha-hydroxylase and its responsiveness to stimulation by PTH. The results suggest that the normal dampening of 1 alpha-hydroxylase and both of the demonstrated enhancements of its activity are mediated by normal and reduced levels of circulating 1,25-(OH)2D, respectively. The finding that PTH fails to further stimulate 1 alpha-hydroxylase when vitamin D deprivation is sufficient in duration to cause hypocalcemia confirms the findings of other investigators and again demonstrates that observations made during abnormal metabolic circumstances may bear little on the physiologic regulation of 1 alpha-hydroxylase under normal or near-normal metabolic circumstances.     

A comparison of automated methods for the quantitation of serum 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D

OBJECTIVE: To compare automated platforms with the routinely used methods in our clinical laboratory for the quantitation of 25-hydroxyvitamin D [25(OH)D] and 1,25-dihydroxyvitamin D [1,25(OH)2D]. METHOD: The NEXgen Four and Triturus ELISA platforms, utilizing the IDS enzyme immunoassay (EIA) kit for 25(OH)D, and the DiaSorin Liaison 25(OH)D methods were compared with the DiaSorin radio immunoassay (RIA) kit. The NEXgen Four and the Triturus, utilizing IDS EIA for 1,25(OH)2D, were compared with the thymus radioreceptor assay (RRA) for measurement of 1,25(OH)2D. RESULTS: NEXgen correlated best with DiaSorin RIA (r(2)=0.652). NEXgen correlated best with the thymus RRA method (r(2)=0.541). Imprecision CV values for NEXgen 1,25(OH)2D were 2.8-9.4% within-run and 10.2-13.9% between-run compared with a between-run precision of 14.0-16.9% with the thymus RRA method. CONCLUSION: NEXgen correlated best with DiaSorin RIA for measurement of 25(OH)D. NEXgen correlated best and demonstrated better precision than thymus RRA for quantitation of 1,25(OH)2D.     

Measurement of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in clinical settings

A quick and simple method for the selective measurement of 25-hydroxyvitamin D3 (25OHD3) and 25-hydroxyvitamin D2 (25OHD2) is described. It includes a rapid sample preparation technique and a combination of a selective radioimmunoassay for 25OHD3 and a competitive protein-binding assay using vitamin D-binding protein for the determination of total 25OHD, including 25OHD3 and 25OHD2. The method was compared with a procedure which include methanol/methylene chloride extraction and chromatography on Sephadex LH 20, and a procedure which includes HPLC and final quantification by u. v. detection. The methods were applied to three groups of patients in order to obtain information on how far assay procedures could be simplified for use in the clinical settings. It is concluded that the method described is applicable for following patients on vitamin D2 therapy. When groups of patients have to be compared, the mean values of the estimates are comparable, whether a simple method or a laborious method is used. Hence, the selection of assay method should take into account the clinical problem and the cost of the analysis.     

Measured free 25-hydroxyvitamin D in healthy children and relationship to total 25-hydroxyvitamin D,calculated free 25-hydroxyvitamin D and vitamin D binding protein

Backgroundvitamin D deficiency in children is still a global health problem. Measuring free 25-hydroxyvitamin D concentrations could provide a better estimate of the vitamin D status than total 25-hydroxyvitamin D (25(OH)D) levels.ObjectiveTo assess the relationship between measured free vitamin D (m-f25(OH)D) and calculated free 25(OH)D (c-f25(OH)D), total 25(OH)D, intact parathyroid hormone (iPTH) and other markers of phosphocalcic metabolism.To establish serum m-f25(OH)D concentrations corresponding to a total 25(OH)D > 50 nmol/L which is accepted as vitamin D-sufficiency status in children.DesignProspective cohort study.SettingJanuary and February 2017 in a Mediterranean population.Patientshealthy children.Measurementsm-f25(OH)D and vitamin D binding protein (VDBP) by ELISA. Free 25(OH)D was calculated using the formula described by Bikle.Resultsm-f25(OH)D directly correlated with total 25(OH)D (r:0.804,p < .001), serum calcium (r:0.26,p:0.035), and c-f25(OH)D (r:0.553,p:0.016); and inversely with iPTH (r:-0.374, p:0.002), alkaline phosphatase (r:-0.28, p:0.026), and age (r:-0.289, p:0.018). Total 25(OH)D correlated with the same parameters as m-f25(OH)D except for serum calcium. However, c-f25(OH)D correlated only with total 25(OH)D and VDBP, both included in the calculation formula.Multiple regression analysis showed that m-f25(OH)D variations were independently explained by calcium (β:0.156, p:0.026) and total 25(OH)D (β:0.043, p < .001).The optimal m-f25(OH)D cut-off for discriminating between insufficient and sufficient total 25(OH)D was >9.8 pmol/L (Area Under Curve (AUC): 0.897 (95% confidence interval (CI): (0.798–0.958); p < .001; sensitivity:72.7% (95%CI: 49.8–89.3); specificity: 95.5% (95%CI: 84.5–99.4)).ConclusionsDirectly measured free vitamin D correlated better with markers of phosphocalcic metabolism than total 25(OH)D and c-f25(OH)D in a population of healthy children.     

Performance evaluation of automated assays for beta-CrossLaps, N-MID-Osteocalcin and intact parathyroid hormone (BIOROSE Multicenter Study).

INTRODUCTION: Biochemical markers of bone metabolism have been mainly determined manually until now and the precision and accuracy of these methods have not always been satisfactory. This has been shown in several external quality assessment schemes (EQAS). OBJECTIVE AND STUDY DESIGN: A study named BIOROSE was undertaken to evaluate new automated assays for serum markers of bone metabolism. The main focus was to evaluate the assay performance in a multicenter setting with 20 laboratories participating in Germany. The evaluation consists of a familiarization phase to determine precision and accuracy and an EQAS to evaluate the comparability between laboratories. MATERIALS: The parameters beta-CrossLaps (CTX), N-MID-Osteocalcin (OC) and intact parathyroid hormone (PTH) were measured with reagents including calibrators and control sera obtained from Roche Diagnostics, Mannheim, Germany, with electrochemiluminescence immunoassays (ECLIA) on the automated analyzer Elecsys 2010. RESULTS: We calculated for the control samples, PCB 1-3, the mean and median values from the measured values of all participating laboratories and used these as target values. From these target values, a recovery range for the participating laboratories was calculated for beta-CrossLaps, OC and intact PTH of better than 80-126% for PCB 2 and PCB 3, and for PCB 1 (low concentration range) for beta-CrossLaps 79-129%, OC 90-120% and intact PTH 78-126%. The between-day imprecision was 2.4-7.2% for beta-CrossLaps, 1.1-5.9% for OC and 1.7-5.5% for intact PTH in the elevated range (sample PCB 2). In the EQAS, the inter-laboratory imprecision for beta-CrossLaps in the sample with a value of 0.8 ng/ml (above the upper limit of normal, which is 0.6 ng/ml) was 9.8% on day 1 and 9.7% on day 2. CONCLUSION: The performance evaluation of automated assays for beta-CrossLaps, N-MID-Osteocalcin and intact parathyroid hormone in the BIOROSE multicenter study showed that the participating laboratories had no problems in setting up these methods and they yielded results for precision and accuracy that are superior to results achieved in external quality assessment schemes for manually performed methods. In addition, at the clinically important decision level of the upper limit of the normal range, all three tested analytes gave precise results that improved medical decisions.     

液相色谱串联质谱法测定血清中25-羟基维生素D2及25-羟基维生素D3含量

目的建立液相色谱串联质谱法(LC-MS/MS)测定血清25-羟基维生素D2[25(OH)D2]及25-羟基维生素D3[25(OH)D3]含量的方法。方法采用乙醇沉淀蛋白,正己烷液液萃取目标组分,乙腈复溶。采用LCMS/MS[正离子电喷雾离子化(ESI+)的多反应监测模式(MRM)]氘代同位素内标法检测血清25(OH)D2及25(OH)D3含量并进行相关方法学验证。结果 LC-MS/MS检测血清25(OH)D2及25(OH)D3的批内精密度为0.88%~7.69%,批间精密度为1.56%~9.90%;25(OH)D2在0.5~10.0 ng/m L、25(OH)D3在5~100 ng/m L范围内线性良好,线性相关系数分别为0.999 1、0.999 9,校准品测试结果正确度为95.5%~101.2%。281名志愿者男、女之间25(OH)D2含量差异无统计学意义(P0.05),25(OH)D3含量差异有统计学意义(P0.05)。结论 LC-MS/MS检测血清25(OH)D2及25(OH)D3的敏感性高,结果准确、稳定,可应用于临床分析。     

Isotope dilution ultra performance liquid chromatography-tandem mass spectrometry method for simultaneous measurement of 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and 3-epi-25-hydroxyvitamin D3 in human serum

BackgroundAn ultra performance liquid chromatography-tandem mass spectrometry method with calibration traceable to NIST SRM was developed and validated to measure concentrations of 25-hydroxyvitamin D₂ (25OHD₂), 25-hydroxyvitamin D₃ (25OHD₃) and the C-3 epimer of 25OHD₃ (epi-25OHD₃) in human serum.MethodsTri- and hexa-deuterated internal standards were added to serum (100 μl) to monitor recovery. Liquid–liquid extraction was used to extract the hexane-soluble materials. Calibration solutions [8–100 nmol/L 25OHD_2, 12–150 nmol/L 25OHD₃, and 4–50 nmol/L epi-25OHD₃] prepared in phosphate-buffered saline containing 4% albumin were similarly processed. Using a pentafluorophenyl column (2.1 × 100 mm) and isocratic methanol/water (72/28, v/v) flowing at 0.4 ml/min, run time was 14 min per sample; 25OHD₃ and epi-25OHD₃ were baseline separated. Atmospheric pressure chemical ionization in the positive ion mode with selected reaction monitoring captured the following transitions: 25OHD₂, m/z 395.3 > 377.3 (209.1 qualifier); (epi-)25OHD₃, m/z 383.3 > 365.3 (105.1 qualifier); d₃-25OHD₂, m/z 398.3 > 380.3; and d₆-25OHD₃, m/z 389.3 > 371.3.ResultsRecovery averaged ≥ 98%. Total imprecision was ≤ 10% when concentrations were ≥ 20 nmol/l. Bias averaged < 5%. Detection limits were < 5 nmol/l. Median (nmol/l) 25OHD₂, 25OHD₃ and epi-25OHD₃ were quantitated in 98 blood donors (< LOD, 56.0, < LOD) and 35 pregnant women (< LOD, 87.6, 3.70).ConclusionsThis method is highly accurate, precise and specific.     

长骨骨折患者血清全段甲状旁腺激素联合25羟维生素D3诊断骨质疏松的价值

目的 探讨血清全段甲状旁腺激素(iPTH)联合25-羟维生素D3[25-(OH)-D3]诊断长骨骨折患者骨质疏松的价值.方法 选取2018年1月至2019年10月该院收治的137例长骨骨折患者作为研究对象,根据腰椎(L1～L4)和股骨颈任意部位的骨密度T值将其分为A组(骨量正常)45例、B组(骨量减少)40例、C组(骨质疏松)52例.检测3组血清iPTH、25-(OH)-D3、骨密度水平,并对其水平进行比较;分析血清iPTH、25-(OH)-D3水平、骨密度的相关性,绘制受试者工作特征曲线(ROC曲线)评估血清iPTH、25-(OH)-D3水平对骨质疏松的诊断价值.结果 B组、C组iPTH、25-(OH)-D3水平、腰椎(L1～L4)和股骨颈骨密度明显低于A组,C组明显低于B组,差异有统计学意义(P<0.05).血清iPTH、25-(OH)-D3水平与骨密度水平呈正相关(P<0.05).iPTH联合25-(OH)-D3诊断长骨骨折患者骨质疏松的曲线下面积(AUC)最大,为0.955,诊断效能最高.结论 长骨骨折骨质疏松患者血清iPTH、25-(OH)-D3水平显著降低,与骨密度呈正相关,在长骨骨折患者骨质疏松发生情况的诊断中具有重要临床意义,且两者联合检测的诊断效能更高.     

A simple micromethod for 25-hydroxyvitamin D estimation.

A quick and simple method for estimating 25-hydroxyvitamin D is described, It involves dichloromethane extraction followed by competitive protein-binding assay without chromatography. This assay can be performed on 100 microliter of serum and enables 50 samples to be estimated in one working day. The simplicity, speed, and sample size of this method make it very suitable for use in a routine clinical biochemistry laboratory. It is particularly useful when calcium disorders secondary to abnormalities in vitamin D intake and metabolism are suspected.