Lower Limb Metaphyseal Bone Is Lost in Men with Coeliac Disease and Does Not Relate to Parathyroid Status.

Aims. To investigate regional lower limb bone density and associations with weight, PTH, and bone breakdown in coeliac men. Methods. From whole body DXA scans bone mineral density (BMD) was measured in 28 coeliac men, in the lower limb (subdivided into 6 regions, 3 being metaphyseal (mainly trabecular) and 2 diaphyseal (mainly cortical)). BMD at femoral neck (FN) and lumbar spine L2-4, body weight, height, serum calcium, alkaline phosphatase, parathyroid hormone (PTH), and urinary calcium and NTx/Cr, a measure of bone breakdown, were also measured. Age matched healthy men provided values for BMD calculation of z and T scores and for biochemical measurements. Results. Low BMD z scores were found at metaphyseal regions in the leg (p < 0.001) and in the FN (p < 0.05). The distal metaphyseal region BMD in the leg was lower than spine or FN (p < 0.05). PTH, urinary calcium/creatinine, and urinary NTx/Cr were similar to controls. Both metaphyseal and diaphyseal BMD z scores were associated with body weight (p < 0.02), but not with either PTH or urinary NTx/Cr. Conclusions. Low BMD lower limb regions comprising mostly trabecular bone occur early in CD and in the absence of elevated PTH or increased bone resorption. Low BMD is associated with low body weight.

Health-related quality of life in women referred for bone density assessment: relationships with bone mineral density, fracture and co-morbidity.

PURPOSE: To identify variables that might influence health-related quality of life (HRQoL) in patients with and without a history of fracture, attending bone mineral density (BMD) assessment prior to diagnosis of osteoporosis. METHODS: This cross-sectional study included 312 newly referred postmenopausal women attending for a DXA scan, without a diagnosis of osteoporosis. Data were obtained from the medical history and the General Practitioner's letter. HRQoL, using SF36 was scored using published algorithms with reference to an age-related population from England. Regression analyses were used to determine relationships between HRQoL and BMD, age, fracture status and co-morbidities. RESULTS: For all patients, the age-related physical component summary (PCS) and mental component summary (MCS) scores were 46 ± 10 and 47 ± 10, respectively. Controlling for confounding variables, low BMD at the femoral neck was associated with worse PCS scores (p = 0.010) and MCS scores (p = 0.034) in patients without fracture. In patients with a history of fracture, this relationship was less evident, and younger age (p < 0.00), increasing BMI (p = 0.016) and number of co-morbidities (p = 0.042) were associated with reductions in PCS scores. CONCLUSIONS: Patients referred for BMD assessment before a diagnosis of osteoporosis had reduced PCS scores. In patients without fracture, low BMD contributed to this reduction in health-related quality of life. Low PCS scores in patients with fracture were seen only in younger subjects with osteoporosis.

Application of sub-regional analysis to bone mineral density of the lower limb from whole body DXA scans.

Bone mineral density at spine and hip is widely used to diagnose osteoporosis. Certain conditions cause changes in bone density at other sites, particularly in the lower limb, with fractures occurring in non-classical locations. Bone density changes at these sites would be of interest for diagnosis and treatment. We describe an application, based on an existing software option for Hologic scanners, which allows reproducible measurement of bone density at six lower limb sites (upper femur, mid-femur, lower femur; upper leg, mid-leg, lower leg). In 30 unselected subjects, referred for bone density, precision (CV%) measured on 2 occasions, separated by repositioning, ranged from 1.7% (mid-femur) to 4.5% at the lowest leg site. Intra-operator precision, measured by three operators on ten subjects on three occasions, was between 1.0% and 2.9%, whilst inter-operator precision was between 1.0% and 3.6%, according to region. These values compare well with those at the spine and upper femur, and in the literature. There was no evidence that this operator agreement improved between occasions 1 and 3. This technique promises to be useful for assessing bone changes at vulnerable sites in the lower limb, in diverse pathological states and in assessing response to treatment.

Matched-cohort study of body composition, physical function, and quality of life in men with idiopathic vertebral fracture.

OBJECTIVE: To determine the effect of 6 years of routine management on body composition, physical functioning, and quality of life, and their interrelationships, in men with idiopathic vertebral fracture. METHODS: Twenty men with idiopathic vertebral fracture (patients: mean ± SD age 58 ± 6 years) were age and height matched to 28 healthy controls with no known disease. The primary outcome was skeletal muscle mass (appendicular lean mass by dual x-ray absorptiometry) assessed at 2 visits (0 and 6 years). Physical functioning and quality of life domains were assessed by the Senior Fitness Test and Short Form 36 (SF-36) questionnaire at visit 2 only. Data were analyzed by repeated-measures analysis of variance, independent t-tests, and correlation. RESULTS: At visit 1, appendicular lean mass was 9% lower in patients than controls. Although patients better maintained appendicular lean mass between visits (interaction P = 0.016), at visit 2 appendicular lean mass remained 5% lower in patients than controls. Furthermore, patients' appendicular lean mass change was correlated with femoral neck bone density change (r = 0.507, P = 0.023). Physical function tests were 13-27% lower in patients compared with controls (P = 0.056 to 0.003), as were SF-36 quality of life physical domains (13-26% lower; P = 0.028 to <0.001). CONCLUSION: Despite an association between changes in muscle mass and bone density, routine management of men with idiopathic vertebral fracture does not address muscle loss. Combined with the observation of reduced physical functioning and quality of life, this study identifies novel targets for intervention in men with idiopathic vertebral fracture.

Circulating and tissue-derived isoforms of bone alkaline phosphatase in Paget's disease of bone.

BACKGROUND: Alkaline phosphatase (ALP) is routinely used in the assessment of Paget's disease of bone (PDB); however, the individual bone ALP isoforms (B/I, B1, and B2) have not been investigated in this disorder. METHODS: Subjects comprised 37 patients (mean age 74 years) with symptomatic PDB confirmed by radiograph and stratified into high and low total ALP activity groups and 66 healthy individuals (mean age 64 years). Extracts of human cancellous and cortical bone tissues were also investigated. The bone ALP isoforms, measured by HPLC, were compared with two bone ALP immunoassays (Metra and Ostase), and the bone formation marker intact amino-terminal procollagen type I propeptide (iPINP). RESULTS: All bone ALP isoforms were increased in high ALP activity PDB compared with the low ALP activity and control groups (p < 0.0001). The B2 isoform had the greater relative activity representing 36%, 50%, and 71%, of the total ALP activity in the control, low and high ALP activity groups, respectively. Compared with controls, B2 was increased in the low ALP activity PDB group (p < 0.05). ROC analysis showed a validity of approximately 80% for B2 to discriminate patients with PDB. CONCLUSION: All bone ALP isoforms were increased in patients with high ALP activity PDB and the B2 isoform was even elevated in the low ALP activity PDB group. The bone ALP isoform B2 may be of use in the management of PDB but that has to be further elucidated in subsequent studies.

Disc cell clusters in pathological human intervertebral discs are associated with increased stress protein immunostaining.

Intervertebral disc (IVD) cells within the annulus fibrosus (AF) and nucleus pulposus (NP) maintain distinct functional extracellular matrices and operate within a potentially noxious and stressful environment. How disc cells respond to stress and whether stress is responsible for triggering degeneration is unknown. Disc cell proliferation and cluster formation are most marked in degenerate IVDs, possibly indicating attempts at matrix repair. In other tissues, stress proteins increase rapidly after stress protecting cell function and, although implicated in degeneration of articular cartilage, have received little attention in degenerative IVD pathologies. We have compared the distribution of stress protein immunolocalization in pathological and control IVDs. Disc tissues were obtained at surgery from 43 patients with degenerative disc disease (DDD) and herniation, and 12 controls at postmortem. Tissues were immunostained with a polyclonal antibody for heat shock factor 1 (HSF-1) and monoclonal antibodies for the heat shock proteins, Hsp27 and Hsp72, using an indirect immunoperoxidase method. Positively stained cells were expressed as a percentage of the total. Cell cluster formation was also assessed. The proportion of cells in clusters was similar in the AF (both 2%) and NP (8 and 9%) of control and DDD samples, whereas in herniated tissues this was increased (AF 12%, NP 14%). Stress antigen staining tended to be more frequent in clustered rather than in single/doublet cells, and this was significant (P < 0.005) in both the AF and NP of herniated discs. Clustered cells, which are most common in herniated discs, may be mounting a protective response to abnormal environmental factors associated with disc degeneration. A better understanding of the stress response in IVD cells may allow its utilization in disc cell therapies.

Increased circulating Dickkopf-1 in Paget's disease of bone.

UNLABELLED: Dickkopf-1 (Dkk-1) is a secreted inhibitor of Wnt signaling which in adults regulates bone turnover. Dkk-1 over-production is implicated in osteolytic disease where it inhibits bone formation and stimulates bone breakdown. Recently it was reported that osteoblastic cells from Paget's disease of bone (PDB) over-expressed Dkk-1. OBJECTIVE: To see if increased Dkk-1 was detected in serum from patients with PDB. RESULTS: Dkk-1 and total serum alkaline phosphatase activity (tsAP) were significantly elevated in sera from PDB patients. Patients with polyostotic PDB had significantly higher levels of tsAP but not Dkk-1, than monostotic patients. TsAP but not Dkk-1, was significantly lower in sera from bisphosphonate treated versus untreated PDB patients. Dkk-1 and tsAP were not significantly correlated. CONCLUSIONS: Dkk-1 may be a useful biomarker of PDB and we speculate that Dkk-1 may play a central role in the etiology of PDB.

Analysis of human bone alkaline phosphatase isoforms: comparison of isoelectric focusing and ion-exchange high-performance liquid chromatography.

BACKGROUND: Several isoforms of alkaline phosphatase (ALP) can be identified in human tissues and serum after separation by anion-exchange HPLC and isoelectric focusing (IEF). METHODS: We purified four soluble bone ALP (BALP) isoforms (B/I, B1x, B1 and B2) from human SaOS-2 cells, determined their specific pI values by broad range IEF (pH 3.5-9.5), compared these with commercial preparations of bone, intestinal and liver ALPs and established the effects of neuraminidase and wheat germ lectin (WGA) on enzyme activity. RESULTS: Whilst the isoforms B1x (pI=4.48), B1 (pI=4.32) and B2 (pI=4.12) resolved as well-defined bands, B/I resolved as a complex (pI=4.85-6.84). Neuraminidase altered the migration of all BALP isoforms to pI=6.84 and abolished their binding to the anion-exchange matrix, but increased their enzymatic activities by 11-20%. WGA precipitated the BALP isoforms in IEF gels and the HPLC column and attenuated their enzymatic activities by 54-73%. IEF resolved the commercial BALP into 2 major bands (pI=4.41 and 4.55). CONCLUSIONS: Migration of BALP isoforms is similar in IEF and anion-exchange HPLC and dependent on sialic acid content. HPLC is preferable in smaller scale research applications where samples containing mixtures of BALP isoforms are analysed. Circulating liver ALP (pI=3.85) can be resolved from BALP by either method. IEF represents a simpler approach for routine purposes even though some overlapping of the isoforms may occur.

Knee angles and rickets in nigerian children.

Knee angles of 2,036 normal Nigerian children up to 12 years old were measured directly or from photographs. The knees were bowed (varus) in the first 6 months. At 21 to 23 months, the distribution of angles became strongly bimodal: about half were varus and half were valgus (knock-kneed), with few in between. After this they were all valgus, with few exceptions. Hence, the change from varus to valgus in individual infants must be sudden (a few weeks), although the changeover of the whole population appears smooth and gradual. They became maximally and uniformly knock-kneed (-7.1 degrees +/- 1.4 degrees) between 3 and 3.5 years, with little change thereafter. On the other hand, 120 patients with rickets discovered during screening had large knee angles, in either sense, with a bimodal distribution and frequency maxima at +10 degrees (varus) and -12 degrees (valgus). Varus knee is uncommon after 2 years. Large knee angles between 2 and 5 years suggest rickets.

Differently cross-linked and uncross-linked carboxy-terminal telopeptides of type I collagen in human mineralised bone.

In bone matrix, type I collagen is stabilised by covalent cross-links formed between adjacent collagen molecules; the majority of which is believed to be immature, divalent bonds. For studying these immature forms in detail, we have developed an immunoassay for a synthetic peptide SP 4 that is analogous with and detects a linear epitope within the C-telopeptide of alpha1-chain of type I collagen. The SP 4 assay, together with the ICTP assay, which is specific for the trivalently cross-linked C-telopeptide, was used for the isolation of the differently cross-linked C-telopeptide structures of the alpha1-chain of type I collagen present in mineralised human bone. Amino acid analysis, peptide sequencing and MALDI-TOF mass spectrometry were used to identify and characterise each of the isolated structures. The cross-link content of each isolated peptide was identified. In the trivalent ICTP peptide, only 40% was cross-linked with pyridinoline, the remainder of the cross-links being currently uncharacterized. The divalent peptides contained only previously characterised cross-linking structures. Most of the divalent cross-links were dihydroxylysinonorleucine (DHLNL), with minor amounts of hydroxylysinonorleucine (HLNL). The relative proportion of the HLNL cross-link was slightly higher in the divalent alpha1Calpha2H peptide. A substantial amount of uncross-linked telopeptide structures was also found. Previous studies, where direct chemical cross-link analyses have been used to assess the maturity of cross-linking, have inferred that bone contains more divalently than trivalently cross-linked C-telopeptides. The immunochemical peptide approach used in this study may help to detect presently uncharacterized, trivalent cross-links, the presence of which is strongly suggested in this study. It also provides additional information regarding the extent and maturity of tissue type I collagen cross-linking in health and disease.

Different distributions of human bone alkaline phosphatase isoforms in serum and bone tissue extracts.

BACKGROUND: In vitro, bone alkaline phosphatase (BALP) is released from the osteoblast membrane with its glycosylphosphatidylinositol (GPI) anchor still attached (i.e., in an anchor-intact form); however, in vivo, BALP circulates as a variable mixture of anchorless isoforms, which can be identified by high-performance liquid chromatography (HPLC). Previous studies have shown that the relative abundance of these BALP isoforms in serum may be clinically useful for the diagnosis and management of metabolic bone disease. METHODS: In the current studies, we describe a method for the determination of anchorless BALP isoforms in extracts of bone and we present novel data on the conversion of anchor-intact to anchorless BALP by incubation with endogenous circulating GPI-specific phospholipase D (GPI-PLD). RESULTS: A 72-h extraction with 0.1% Triton X-100 released approximately 90% of the BALP activity from powdered bone. An average of 19% of this activity was anchorless, but essentially all of the activity could be converted to the anchorless form by incubation with partially purified GPI-PLD from human serum. Using HPLC, we detected four BALP isoforms (B/I, B1x, B1, and B2) in these GPI-PLD-treated extracts of bone. An additional BALP fraction was also detected in the samples during the initial phase of GPI-PLD treatment. CONCLUSIONS: The abundance of the BALP isoforms differed between bone and serum, particularly for the B/I isoform, which comprised, on average, 18% of the BALP in GPI-PLD-treated extracts of healthy bone tissue, but only 6% of the total BALP activity in serum from healthy individuals. Based on our recent finding of differences in the number of sialic acid residues between the BALP isoforms, we hypothesize that this difference between BALP isoforms in serum and extracts of bone is due to the different patterns of glycosylation, which results in different biological half-lives in the circulation. A preliminary application of our method to the extraction of BALP isoforms from a small number of human bone samples suggests that the method should be useful for studies of human skeletal site-specific and metabolic bone disease-specific differences in the amounts and distributions of the BALP isoforms in bone.