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Serum total calcium (tCa) is routinely measured for diagnosing calcium disorders but may not reflect levels of biologically active ionized calcium (iCa) in disease or detect all cases of primary hyperparathyroidism.We investigated the utility of measuring iCa and tCa for diagnosing primary hyperparathyroidism.This was an observational, retrospective, cross-sectional study.We studied a biochemistry cohort of consecutive ambulatory outpatients with suspected bone or calcium metabolism disorders referred for calcium metabolism biochemistry panels and a surgical cohort of consecutive tertiary hospital patients whose parathyroid specimens were submitted to a single center, and consecutive parathyroidectomy patients of a single surgeon with specimens submitted to a different center.In 5490 biochemistry cohort patients, discordance between iCa and tCa in classifying calcium status occurred in 12.6% of cases overall but was worse in hypercalcemic (whether defined by tCa and/or iCa) cases (49%) and hypocalcemic cases (92%). Reliance on tCa alone would miss 45% with ionized hypercalcemia. In 315 biochemistry cohort cases with PTH-dependent hypercalcemia, 130 (41%) had isolated ionized hypercalcemia at diagnosis. In 143 patients with histologically proven parathyroid disease, 24% had isolated ionized hypercalcemia at diagnosis. These patients were younger (P = 0.022) with milder ionized hypercalcemia and better renal function (both P ≤ 0.001) than patients presenting with concurrently elevated iCa and tCa.In abnormal calcium states, tCa frequently disagrees with iCa in classifying calcium status. Histologically proven parathyroid disease can present with isolated ionized hypercalcemia. Measurement of iCa is required to accurately assess calcium status and improve diagnostic accuracy.

Previously reported Sequestosome 1(SQSTM1)/p62 gene mutations associated with Paget's disease of bone (PDB) cluster in, or cause deletion of, the ubiquitin-associated (UBA) domain. The aims of this study were to examine the prevalence of SQSTM1 mutations in Australian patients, genotype/phenotype correlations and the functional consequences of a novel point mutation (P364S) located upstream of the UBA. Mutation screening of the SQSTM1 gene was conducted on 49 kindreds with PDB. In addition, 194 subjects with apparently sporadic PDB were screened for the common P392L mutation by restriction enzyme digestion. HEK293 cells stably expressing RANK were co-transfected with expression plasmids for SQSTM1 (wildtype or mutant) or empty vector and a NF-kappaB luciferase reporter gene. GST-SQSTM1 (wildtype and mutant) proteins were used in pull-down assays to compare monoubiquitin-binding ability. We identified SQSTM1 mutations in 12 of 49 families screened (24.5%), comprising 9 families with the P392L mutation and 1 family each with the following mutations: K378X, 390X, and a novel P364S mutation in exon 7, upstream of the UBA. The P392L mutation was found in 9 of 194 (4.6%) patients with sporadic disease. Subjects with SQSTM1 mutations had more extensive disease, but not earlier onset, compared with subjects without mutations. In functional studies, the P364S mutation increased NF-kappaB activation compared with wildtype SQSTM1 but did not reduce ubiquitin binding. This suggests that increased NF-kappaB signaling, but not the impairment of ubiquitin binding, may be essential in the pathogenesis of PDB associated with SQSTM1 mutations.

Sequestosome 1/p62 (p62) mutations are associated with PDB; however, there are limited data regarding functional consequences. We report a novel mutation in exon 7 (K378X) in a patient with polyostotic Paget's disease of bone. p62 mutants increased NF-κB activation and significantly potentiated osteoclast formation and bone resorption in human primary cell cultures. Introduction:Sequestosome 1/p62 (p62) mutations are associated with Paget's disease of bone (PDB); however, there are limited data regarding functional consequences. One report has linked the common P392L mutation in the p62 ubiquitin binding associated (UBA) domain with increases in NF-κB activity, a transcription factor essential for osteoclastogenesis. To further clarify the functional impact of p62 mutations associated with PDB, we assessed the effect of p62 mutation (a novel mutation: K378X, and previously reported mutations: P392L and E396X) on RANK-induced NF-κB activation and compared this with the effect of wildtype p62. In addition, we studied the effect of p62 mutation on osteoclast formation and bone resorption. Materials and Methods: We performed co-transfection experiments with expression plasmids for p62 (wildtype or mutated) and RANK and an NF-κB luciferase reporter gene. Luciferase activities were recorded after addition of luciferin to cellular lysates. RAW264.7 cells stably expressing enhanced green fluorescent protein (EGFP)-tagged p62 (wildtype, K378X, or P392L) or EGFP alone were assessed for changes in cell proliferation. Additionally, these cells were stimulated with RANKL to produce osteoclast-like cells (OLCs). Primary human monocytes collected from the K378X-affected patient and a control subject were stimulated to form OLCs and bone resorption data were obtained. Results: The novel mutation introduces a premature stop codon in place of Lys-378 and thereby eliminates the entire p62 UBA domain; this and two additional natural mutations (P392L, E396X) increased NF-κB activation compared with wildtype p62. Wildtype p62 consistently inhibited NF-κB activation compared with empty vector. UBA mutations (K378X and P392L) significantly increased the number of OLCs formed in response to RANKL and also the number of nuclei of the OLCs. K378X-affected human monocytes formed more OLCs with more nuclei and increased bone resorption compared with control monocytes. Conclusions: Our data show that mutation of the p62 UBA domain results in increased activation of NF-κB and osteoclast formation and function compared with wildtype p62. These results may partially explain the mechanism by which p62 mutation contributes to the pathogenesis of PDB.

Human thoracic discs were analyzed for collagen and collagen cross-links to determine the distribution due to segmental, age, and gender influences. Thoracic discs from 26 cadaveric spines (1 to 90 years old) were graded macroscopically, then separated into anular and nuclear samples. Only grade I (i.e., normal) disc samples were selected (n ¼ 209). Pyridinoline and deoxypyridinoline cross-links were initially separated by column chromatography and analyzed by reverse-phase high-pressure liquid chromatography. The collagen content was lower and the extent of pyridinoline and deoxypyridinoline were significantly higher in the nucleus compared with the anulus (p , .001). The collagen content and extent of pyridinoline were significantly lower with increasing age in the anulus and nucleus (p , .001). Young male discs had a significantly higher extent of pyridinoline compared with females (p , .001). Age, gender, and disc region differences were found to have a significant influence on the biochemical composition of the normal disc extracellular matrix.

Oral (44Ca: 0.13-0.20 mmol) and intravenous (42Ca: 0.02-0.037 mmol) isotopically enriched stable calcium (Ca) tracers were given together with an oral dose of 2.5 mmol of natural Ca to normal subjects. Blood and urine samples were collected up to 24 h after the tracer doses and atom fractions (AFs) of these tracers (relative to natural Ca) were measured by high-precision thermal ionization mass spectrometry (TIMS). The time-dependent fractional rate of oral dose absorbed and true fractional intestinal Ca absorption (alpha) were derived from the Afs by mathematical deconvolution. After 6 h, the ratio AF oral tracer/AF intravenous tracer in blood equalled that in urine and did not change thereafter. Reproducibility of the combination of chemical precipitation of Ca (from a urine standard) and subsequent TIMS measurements, in nine runs over 13 months, was 1.2% (coefficient of variation). This was in accord with the within-run reproducibility. An estimate of alpha derived from a single blood or urine measurement was 6-10% higher than the reference value obtained by deconvolution. This discrepancy could be explained by a correction factor depending, in part, on the elapsed time for peak Ca intestinal absorption rate. Instrumentally induced mass fractionation, as well as contributions from radiogenic Ca, had a significant effect on the accuracy and reproducibility of the ratio of AFs of tracers in blood and urine.

Background: Current evidence indicates that increased desmosine excretion reflects the active inflammatory status of some connective tissue diseases. Our goal was to establish a reliable method of detection and to investigate the normal distribution of urinary desmosine excretion in a healthy pre-pubertal population. Method: Urine was collected from healthy volunteers aged four weeks to 12 years old. We modified a published high-performance liquid chromatography (HPLC) method by (a) increasing hydrolysis time and temperature and (b) increasing cellulose column size. Results: Our modified method had small inter- and intra-assay variability, with coefficients of variation of 2 = 0.91). There was no significant diurnal or day-to-day variability in total desmosine levels. A reference range for healthy pre-pubertal children aged four weeks to 12 years was established. Conclusion: The modified HPLC method is reliable with low variability. The technique can now be applied as a non-invasive research or diagnostic tool for children with chronic lung disease.

Intravenous disodium 3-amino-1-hydroxypropylidene-1,1-bisphosphonate pentahydrate (pamidronate disodium) was used to treat 39 patients (22 males and 17 females, age range 48–85 years) with symptomatic Paget's disease. Patients were stratified into three groups based on the biochemical severity of the disease as assessed by fasting urinary hydroxyproline excretion (HypE, μmol/liter GF, glomerular filtrate): group I (n = 23), HypE 10.0, 240 mg over 4 or 8 days. Bone mineral density (BMD) was measured before and 3 and 6 months following treatment in the spine (LI-4) using dual-energy x-ray absorptiometry and in the forearm at an ultradistal and a shaft site using single-photon absorptiometry. When groups I-III were combined, nonpagetic and pagetic lumbar spinal BMD had both risen significantly at 3 months compared with the pretreatment values (p < 0.001). In each group, lumbar spinal BMD in pagetic vertebrae rose markedly by 3 months, with no further significant change at 6 months. The percentage rises in the three groups were not different from each other at 3 or 6 months. Nonpagetic lumbar spinal BMD followed a similar and significant trend but with a significantly smaller rise than for pagetic bone. (For the combined groups, nonpagetic BMD rose 5.1 ± 1.1% SEM, above pretreatment at 6 months; pagetic BMD rose 17.8 ± 1.6%: significance of comparison = p < 0.0001). In contrast, forearm BMD in group III had fallen at 6 months by 8.3 ± 2.5% (p < 0.01) and 7.0 ± 1.2% (p < 0.001) in the ultradistal and shaft sites, respectively. There were no significant changes in forearm BMD in groups I and II. When groups I-III were combined, the maximum observed changes within each individual in ultradistal forearm BMD (seen posttreatment) were correlated inversely with maximum intraindividual changes (seen posttreatment) in intact parathyroid hormone (Spearman's Q = −0.53, p < 0.001). A group of 18 control subjects with untreated Paget's disease were studied for 3–6 months. No changes were seen in nonpagetic or pagetic lumbar spinal BMD or in forearm BMD. Three mechanisms are proposed to explain these findings: (1) in nonpagetic bone, persistence of bone formation following acute reduction in resorption, with a more marked effect in the axial than in the appendicular skeleton; (2) in pagetic bone, magnification of mechanism 1, caused by the presence of abnormally active osteoclasts, leading to a marked divergence between bone resorption and formation, and (3) in cancellous and cortical distal forearm bone, the onset of acute secondary hyperparathyroidism following treatment, leading to resorption. These findings have implications for treatment of Paget's disease using pamidronate or other bisphosphonates in patients with preexisting appendicular osteopenia, particularly if the treatment-induced appendicular deficit is sustained.

The calcium (Ca) metabolism of established human lactation was studied in 40 adult women (mean age 32.4 years) who had been breast-feeding for 6 months (Lac) and in 40 age-matched controls (Con) using fasting urine and blood biochemistry and forearm single-photon bone mineral densitometry (BMD). Serial studies were performed up to 6 months after weaning in Lac women and repeated once in Con women. During lactation the significant findings were (1) a selective reduction (7.1%, P < 0.03) in BMD at the ultradistal site containing 60% trabecular bone, but not at two more proximal, chiefly cortical bone sites; (2) increased bone turnover affecting bone resorption [fasting hydroxyproline excretion, Lac 2.22 + 0.12 μmol/liter GF (mean + SEM), Con 1.19 + 0.04, P < 0.001] and affecting bone formation (plasma alkaline phosphatase, Lac 81.9 + 2.5 IU/liter, Con 53.5 + 2.7, P < 0.001, and serum osteocalcin, Lac 14.0 + 0.7 μg/liter, Con 7.3 + 0.4, P < 0.001); and (3) renal conservation in the fasting state of both Ca and inorganic phosphate (Pi) with a resultant moderate increase in plasma Pi but not in plasma Ca (total or ionized). There were no differences between the groups in serum parathyroid hormone (PTH, intact and midmolecule assays), 25-hydroxy- and 1,25-dihydroxyvitamin D, nephrogenous cyclic AMP production, or plasma creatinine. In 25 of these Lac women restudied at one or more of the times 2, 4, or 6 months after weaning, the findings were (1) an early (2 months) normalization of bone resorption and renal Pi handling with continuing increased bone formation and renal Ca conservation, associated with the onset of increased intact PTH levels; and (2) a recovery, within 4-6 months in ultradistal BMD associated with normalization of bone formation but with persisting renal Ca conservation and elevated intact PTH levels. We conclude that in established adult human lactation there is increased bone turnover with an accompanying loss of trabecular bone, despite renal conservation of Ca and Pi. After weaning, the deficit in trabecular bone is made up during a period of imbalance between a normal bone resorption rate and an elevated bone formation rate. The moderately elevated PTH after weaning may play a role in the recovery of bone mass by maintaining renal Ca conservation and by an anabolic action on bone.

Calcitriol deficiency with retained synthetic reserve in chronic renal failure. Serum calcitriol and the free calcitriol index together with factors considered to regulate calcitriol production were measured in eleven patients with moderate chronic renal failure (MCRF) and eleven age- and sex-matched normal subjects. Although the serum dialysable calcium levels were similar in the two groups, there was depression of calcitriol levels and an elevation of PTH and nephrogenous cyclic AMP (NcAMP) levels in the MCRF patients. Furthermore, plasma phosphate levels were higher and the renal phosphate threshold was depressed in this patient group. When all subjects were grouped together calcitriol was positively correlated with GFR. When calcitriol levels were factored for GFR, to permit an assessment of calcitriol production per unit functioning renal mass, there was no significant difference between normal and MCRF subjects. To determine whether reserve for calcitriol production existed, six of the MCRF patients and six of the age- and sex-matched normal subjects received a low calcium diet for one week supplemented by cellulose phosphate to bind calcium within the gut. In both groups there was a significant rise in calcitriol, although the absolute levels were much lower in the MCRF patients than the normal subjects. These results suggest that calcitriol deficiency is a major feature of MCRF despite marked hyperparathyroidism. The rise in calcitriol levels in MCRF suggests persistent reserve secretory capacity in this condition. Therefore, the low serum calcitriol concentration may be due not only to structural renal damage, but also to suppression of calcitriol formation perhaps due to altered renal phosphate handling.

A comparison of the performance of a two-site immunochemiluminometric assay for intact parathyroid hormone with that of an in-house radioimmunoassay for carboxy terminal parathyroid hormone has been performed on samples from unselected patients being investigated for hypercalcaemia. The intact parathyroid hormone assay was found to be a simple and robust technique with a broad working assay range (CV less than 10% between 1.8-212 pmol/l) and a detection limit of 0.2 pmol/l. Clinically it is superior to the carboxy terminal assay in its ability to distinguish between patients with hyperparathyroidism from those with other causes of hypercalcaemia especially in the presence of impaired renal function.

The theoretical tubular maximum for calcium reabsorption was calculated and its usefulness assessed in the diagnosis and differential diagnosis of primary hyperparathyroidism and familial hypocalciuric hypercalcaemia. The sensitivity of the test in the diagnosis of primary hyperparathyroidism was only 12%. The theoretical tubular maximum for calcium reabsorption was recalculated after correction of calcium concentration in plasma for albumin concentration and for urinary sodium excretion. Despite these corrections, the sensitivity improved to only 44%. This contrasts with a sensitivity of 80% for the plot of fasting calcium excretion against calcium concentration in plasma in primary hyperparathyroidism. The calculation of theoretical tubular maximum for calcium reabsorption cannot be recommended as a useful test for distinguishing between primary hyperparathyroidism and familial hypocalciuric hypercalcaemia. The simple calculation of fractional excretion of calcium was a better test in distinguishing familial hypocalciuric hypercalcaemia from primary hyperparathyroidism.