Reducing fluxes of faecal indicator compliance parameters to bathing waters from diffuse agricultural sources: the Brighouse Bay study, Scotland.

The European Water Framework Directive requires the integrated management of point and diffuse pollution to achieve 'good' water quality in 'protected areas'. These include bathing waters, which are regulated using faecal indicator organisms as compliance parameters. Thus, for the first time, European regulators are faced with the control of faecal indicator fluxes from agricultural sources where these impact on bathing water compliance locations. Concurrently, reforms to the European Union (EU) Common Agricultural Policy offer scope for supporting on-farm measures producing environmental benefits through the new 'single farm payments' and the concept of 'cross-compliance'. This paper reports the first UK study involving remedial measures, principally stream bank fencing, designed to reduce faecal indicator fluxes at the catchment scale. Considerable reduction in faecal indicator flux was observed, but this was insufficient to ensure bathing water compliance with either Directive 76/160/EEC standards or new health-evidence-based criteria proposed by WHO and the European Commission.

Influence of vitamin D status on hyaluronan localization in bone.

The distribution of hyaluronan was investigated in the proximal tibiotarsal bones of normal (vitamin D-treated) chicks, and chicks with rachitic lesions induced by vitamin D deficiency. Localization studies using a biotinylated hyaluronan-binding probe revealed that in vitamin D-treated chicks, a high level of hyaluronan staining was present in upper proliferative zone cartilage and upper hypertrophic zone cartilage. Hyaluronan staining was greatly reduced in the zone of provisional calcification. In the metaphyses and diaphyses of normal chicks hyaluronan was predominantly localized to the non-bone-forming surfaces of osteoblasts but was also present on the basolateral surfaces of lining cells and osteoclasts. Marked changes in hyaluronan distribution were observed in vitamin D-deficient chicks. The amount of hyaluronan present in proliferative zone growth cartilage was similar to control chicks, although with a more widespread distribution, extending into lower proliferative zone cartilage. In the zone of hypertrophy/calcification, biochemical analyses revealed that hyaluronan levels in rachitic chicks were about 3.6 times greater than in vitamin D-treated chicks; localization studies demonstrated that this increase was associated with the presence of hyaluronan-positive spindle-shaped cells in the metaphyseal vascular spaces. Intense hyaluronan staining was also associated with abundant spindle-shaped cells occupying the marrow spaces of rachitic diaphyseal bone. The distribution of hyaluronan in vitamin D-treated chick bone, and the alterations observed in rachitic tissue suggests a role for hyaluronan in endochondral bone formation.

Histomorphometric and biochemical characterization of bone following acute severe burns in children.

Severe burns in adults is associated with an uncoupling of normal remodeling, low bone formation without reduced resorption. The risk of osteopenia that may occur under such circumstances is heightened by our detection in a cross-sectional study of low bone mass in severely burned children. We report here the acute histomorphometric and biochemical response of bone to severe burn injury, as well as bone mass in severely burned children. We enrolled 24 patients ages 5.8 to 17.5 years following burns of 63 +/- 16% (SD) body surface area. Serum and urine were collected weekly until iliac crest bone biopsy was obtained 26 +/- 10 days postburn. Seventeen of 18 patients, including 5 patients receiving growth hormone treatment to accelerate wound healing, failed to take up doxycycline in trabecular bone, and had no detectable osteoblasts at the osteoid seam, while eroded surface was normal and osteoblasts were documented by staining. Thus, bone formation was virtually absent. There was an eightfold elevation in urinary free cortisol excretion and high serum levels of acute phase reactants and interleukin-1 beta and -6. Biochemical markers of bone formation, osteocalcin, and type I procollagen propeptide were low, as were resorptive markers urinary pyridinoline and deoxypyridinoline. However, there was no correlation with resorptive surface. Mean age-related z-score for bone mass was -1.06 +/- 1.05, 40 days postburn. Immobilization and endogenous corticosteroid production may be the main factors responsible for acutely reduced bone formation while inflammatory cytokines may mediate resorption.

Lumbar vertebral and femoral neck bone mineral density are higher in postmenopausal women with the alpha 2HS-glycoprotein 2 phenotype.

alpha 2HS-glycoprotein (AHSG) is a plasma protein which becomes concentrated in the organic matrix of bone. The two most common alleles, AHSG*1 and AHSG*2, give rise to three common phenotypes. A recent report showed that a group of postmenopausal white North American women with different AHSG phenotypes differed significantly with respect to their oestrogen status. We have studied variations in bone mineral density, measured by DEXA, and levels of sex hormones and biochemical markers of bone metabolism in a group of 88 post-menopausal women unselected as to their health status. Lumbar vertebral and femoral neck bone mineral density (BMD), and the free oestradiol index were all significantly higher (P < 0.05) in women with the AHSG 2 phenotype. Values of these three parameters were lowest in the AHSG 1 phenotype and intermediate in the AHSG 2-1 phenotype. Because the differences in BMD between the AHSG 2 and 1 phenotypes represent at least a 40% difference in fracture risk, the AHSG phenotype may be of some clinical relevance as a risk factor for osteoporosis.

The effects of vitamin D deficiency on proteoglycan and hyaluronate constituents of chick bone.

The effect of vitamin D deficiency on proteoglycan and hyaluronate constituents of cortical diaphyseal chick bone was studied. Proteoglycans in rachitic bone showed no significant change with respect to their size, composition, or amount relative to other extractable macromolecular components. In contrast, bone hyaluronate levels were raised in chicks fed on diets that were either vitamin D-deficient or depleted in calcium or phosphate, a 7-fold increase being seen in hypocalcaemic vitamin D-deficient chicks. This increase in hyaluronate was not directly related either to the absence of vitamin D or to abnormal levels of blood calcium or phosphate per se; hyaluronate levels are probably regulated by another factor, not yet identified, that is responsive to changes in vitamin D and mineral metabolism.

Changes with age in the urinary excretion of lysyl- and hydroxylysylpyridinoline, two new markers of bone collagen turnover.

Two intermolecular cross-linking amino acids, hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP), are promising markers in urine of collagen resorption because their levels in urine should reflect only collagen resorption and, unlike hydroxyproline, should not be influenced by degradation of either newly synthesized collagen molecules or noncollagenous proteins. Changes with age in the urinary excretion of HP and LP were studied in 24 h collections of urine from a group of 22 male and 27 female healthy subjects aged from 2 to 70 years. The pyridinolines were quantitated, utilizing their natural fluorescence, after resolution by reversed-phase HPLC. Levels of both pyridinolines were higher in children than in adults, but in adults no evidence of age or sex variations were observed except in the 20-30 year age group. Mean values of HP/Cr and LP/Cr in 37 adults (21-70 years) were 27.2 +/- 1.9 and 8.8 +/- 0.8 mumols/mol, respectively; in the 12 children (2-15 years) the mean values were 14.4 and 12.4 times higher than the respective adult values. Making certain assumptions, the mean amount of bone resorbed in normal adults was tentatively estimated at 1.9 g per 24 h. The finding that differences between children and adults in these relatively specific markers were greater than with hydroxyproline suggests that hydroxyproline values may considerably underestimate the actual amount of bone turnover occurring in growing children or overestimate the adult turnover rate.

Comparative studies of type X collagen expression in normal and rachitic chicken epiphyseal cartilage.

The levels of type X collagen in mineralizing normal chicken epiphyses and nonmineralizing rachitic chicken tibial epiphyses were measured and compared. Qualitative immunoperoxidase studies with anti-chick type X collagen monoclonal antibodies on sections from normal and rachitic cartilage demonstrated that the type X collagen levels in rachitic growth plates are reduced. Northern hybridization of mRNA and biosynthetic studies have confirmed that type X collagen synthesis in rickets is also decreased. In hypocalcemic rickets, the level of type X collagen mRNA is reduced by 80% whereas the level of type X collagen mRNA is only reduced by 50% in normocalcemic rickets. These observations provide additional evidence that type X collagen is involved in the process of cartilage mineralization and also suggest that the partial recovery of type X collagen synthesis in normocalcemic rickets may be related to the elevated plasma concentration of calcium. Calcium concentration may therefore play an important role in the control of type X collagen synthesis.

Vitamin A and bone formation. Different responses to retinol and retinoic acid of chick bone cells in organ culture.

The response of bone cells in organ culture to retinol and retinoic acid was studied. Both stimulated incorporation of [3H]thymidine into DNA by 16-day embryonic chick calvaria, but the time-course of the responses differed; the peak responses to retinol and retinoic acid occurred at about 18 h and 48 h, respectively. Although retinol inhibited chick bone collagen synthesis retinoic acid had no effect, but it did stimulate non-collagenous protein synthesis, whereas the effect on the latter of retinol was, if anything, inhibitory. When present with retinol, retinoic acid was able to attenuate the inhibitory effect of the former on chick bone collagen synthesis, but preincubation with retinoic acid had no such effect. In neonatal murine calvarial cultures, retinoic acid inhibited collagen synthesis selectively in the same manner as did retinol. The ability of chick osteoblasts to respond differently to retinol and retinoic acid suggests that both forms of the vitamin may have a role in bone formation and that their intracellular models of action may differ although the attenuation response indicates there may be some interaction between the two.

The carbonate environment in bone mineral: a resolution-enhanced Fourier Transform Infrared Spectroscopy Study.

The environment of carbonate ions in bones of different species (rat, rabbit, chicken, cow, human) was investigated by Fourier Transform Infrared Spectroscopy (FTIR) associated with a self-deconvolution technique. The carbonate bands in the v2 CO3(2-) domain show three components which were identified by using synthetic standards and different properties of the apatitic structure (ionic affinity for crystallographic locations, ionic exchange). The major component at 871 cm-1 is due to carbonate ions located in PO4(3-) sites (type B carbonate). A band at 878 cm-1 was exclusively assigned to carbonate ions substituting for OH-ions in the apatitic structure (type A carbonate). A band at 866 cm-1 not previously observed was shown to correspond to a labile carbonate environment. The intensity ratio of type A to type B carbonate appears remarkably constant in all bone samples. The 866 cm-1 carbonate band varies in its relative intensity in different species.

Regulation of new osteoclast formation by a bone cell-derived macromolecular factor.

Medium conditioned by incubation with embryonic chick calvarial bones, which contain osteoblasts but not osteoclasts, stimulated new osteoclast formation in foetal long bone cultures and in adult bone marrow cultures formation of tartrate-resistant acid phosphatase (TRAP) positive cells was greatly stimulated. We have termed the factor responsible for this activity osteoclast growth/inducing factor (OGF). OGF was soluble, heat-stable and of size greater than 10kda. OGF activity was present also in mouse bone conditioned medium and in extracts of demineralized cortical diaphyseal bone of five-week-old chickens. OGF appeared to differ from the osteoblast-derived bone-resorbing factors previously observed as well as from macrophage colony stimulating factor (CSF-1). It is therefore probable that different locally secreted factors independently regulate the formation of osteoclasts and their activity.

Influence of vitamin D status on the content of complexed acidic phospholipids in chick diaphyseal bone.

Calcium acidic phospholipid phosphate complexes cause hydroxyapatite formation in vitro and in vivo. Previous studies have shown that the amount of these complexed acidic phospholipids is altered in the bones of animals depleted of vitamin D and phosphate. To help differentiate the effects of vitamin D from those due to altered mineral metabolism, the amount of complexed acidic phospholipids present in diaphyseal bone was determined in five groups of chicks varying in calcium, phosphate and vitamin D status. Highest levels of complexed acidic phospholipids were found in chicks receiving vitamin D and having normal serum levels of Ca and P. Lowest levels were found in vitamin D-deficient chicks; levels were not altered in vitamin D-deficient chicks when their serum Ca was normalized. Intermediate levels were found in vitamin D-treated chicks fed a low Ca diet. Levels of the complexed acidic phospholipids were low in bones of hypophosphatemic, vitamin D-treated chicks but, when related to total lipid phosphorus content, values were similar to those of hypocalcemic chicks. These data suggest that while disturbances in calcium and phosphate metabolism which are present in rickets influence the bone content of these complexed acidic phospholipids, vitamin D may also play a direct role in their formation. Further, the ability of vitamin D to increase bone complexed acidic phospholipid content does not seem to be related to its hypercalcemic effect.

Collagen cross-linking in human bone and articular cartilage. Age-related changes in the content of mature hydroxypyridinium residues.

The concentration in collagen of hydroxypyridinium cross-linking amino acids was measured in samples of bone and cartilage from human subjects aged from 1 month to 80 years. Cortical and cancellous bone samples were dissected and analysed separately. In both bone and cartilage, the content of this mature form of cross-link reached a maximum by 10-15 years of age (the amount in cartilage being 5-10 times that in bone), then stayed essentially in the same range throughout adult life. In bone the ratio of the two chemical variants of the mature cross-link, hydroxylysylpyridinoline to lysylpyridinoline, was constant throughout adult life at 3.5:1, whereas in cartilage it was always greater than 10:1. The ratio of hydroxypyridinium cross-links to borohydride-reducible keto-amine cross-links also changed with age. The reducible cross-links in bone collagen decreased steeply in content between birth and 25 years, but persisted in significant amounts throughout adult life. Reducible cross-links had virtually disappeared from cartilage by 10-15 years of age, being replaced by hydroxypyridinium residues, their maturation products. Cancellous and cortical bone collagens showed similar trends with age in their content of mature cross-links, though for each subject the concentration in cancellous bone was always lower than in cortical bone, presumably reflecting the higher turnover rate and hence the more immature state of cancellous bone.

Studies on the stability of VIII:C during the manufacture of a factor VIII concentrate for clinical use.

The stability of VIII:C was investigated by monitoring samples taken at different points from a routine process for the manufacture of factor VIII concentrate and by examining the stabilising influence of a number of product formulations. Loss of VIII:C over process-finishing procedures (formulation, 0.22 micron filtration, dispensing) was associated with a citrate-induced inactivation which could be prevented by controlling the ionised calcium concentration of the solution. These results were obtained using a one-stage clotting assay but were not observed using a two-stage assay. No evidence for activation was found in vitro (e.g. by FPA generation and VIII:C stability) and the yield increase suggested by the one-stage assay was supported by results from a controlled clinical evaluation.

"Glycated" osteocalcin in human and bovine bone. The effect of age.

Purified osteocalcin from cow and calf bone was analyzed for nonenzymatic glycosylation (glycation) by sodium [3H]borohydride reduction. Calf bone was found to be approximately 5% glycated, while bone from mature cows was 10% glycated. These results were confirmed by a second method which utilizes periodate oxidation followed by formaldehyde fluorescence. Osteocalcin in human bone was also found to be glycated. The content of glycated osteocalcin from the bones of 47 nondiabetic individuals, aged 0.6-97, was dependent upon age. The extent of glycation was lowest in children, was constant through the adult years, and increased linearly in bone taken from individuals aged 60-97. Glycated osteocalcin was purified by boronate affinity chromatography and subjected to one-step Edman degradation. It was established that the site of glycation was the amino-terminal tyrosine. Increases in the amount of glycated osteocalcin in the bones of older individuals may play a role in the pathogenesis of senile osteoporosis and in the osteopenia which may accompany diabetes mellitus.

A comparison of proteoglycan from chick cartilage of different types and a study of the effect of vitamin D on proteoglycan structure.

Normal chick growth cartilage contains mainly large proteoglycans that interact well with hyaluronic acid. In contrast normal articular and sternal cartilages contain mainly smaller molecules that interact poorly with hyaluronic acid. Similar smaller proteoglycans are present in all the cartilages in the rachitic state. The smaller proteoglycans all appear to be synthesized as such, and not to arise from degradation of larger molecules unless such degradation occurs extremely rapidly after synthesis. Supplementation of the vitamin D-deplete animals with calcium resulted in the production of large proteoglycans by the growth cartilage, but did not affect the size of the proteoglycan produced by the articular or sternal cartilages. It would appear that in these chicks the chondrocytes of the growth cartilage may be unique in their ability to produce large aggregating proteoglycans, and in their response to plasma calcium levels.

The influence of vitamin D metabolites on collagen synthesis by chick cartilage in organ culture.

When growth cartilage from rachitic chicks was cultured in the presence of the calcium-regulating hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), collagen resorption was increased and collagen synthesis decreased compared to control cultures containing no hormone. The minimum concentration of the hormone that caused a statistically significant inhibition of collagen synthesis was 10(-8) mol/l. Collagen synthesis by growth cartilage from normal chicks was also reduced by 1,25-(OH)2D3, showing that it was not an abnormal response of vitamin D-depleted tissue. 25-Hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 also inhibited collagen synthesis by cultures of growth cartilage but only at higher metabolite concentrations. 1,25-Dihydroxyvitamin D3 (10(-7) mol/l) did not significantly inhibit collagen synthesis by cultures of articular fibrocartilage and of sternal cartilage, tissues that do not calcify physiologically. The minimum concentration of 1,25-(OH)2D3 (10(-9) mol/l) necessary to cause decreased collagen synthesis by embryonic chick calvaria was lower than the value obtained with growth cartilage; this suggests that bone cells may be more sensitive to the hormone in this respect than are growth cartilage chondrocytes. These findings provide evidence of a direct role of 1,25-(OH)2D3 in the control of endochondral bone formation which is consistent with its primary role in the maintenance of plasma calcium homeostasis.

Vitamin A and bone formation. Effect of an excess of retinol on bone collagen synthesis in vitro.

The influence of an excess of retinol on bone formation was studied by using cultures of embryonic-chick calvaria. Retinol decreased collagen synthesis in a dose-dependent manner, non-collagenous protein synthesis being relatively unaffected. Collagen synthesis was significantly inhibited after 24 h of culture with retinol and was progressively decreased, compared with control cultures containing no retinol, as the period of culture was increased. The effect of retinol on collagen synthesis could be reversed by incubation of calvaria for further periods in retinol-free medium. Incorporation of [3H]thymidine and [3H]uridine into DNA and RNA respectively was not altered by culturing calvaria with retinol for 22 h. These latter findings, and the selectivity for collagen synthesis, all suggested that the effect observed was not a cell-toxicity phenomenon. The effect of retinol on collagen synthesis by chick calvarial osteoblasts was probably direct and not mediated by osteoclasts, since a negligible number of the latter cells is present in chick calvaria. In cultures of neonatal murine calvaria, which contain many osteoclasts, retinol similarly inhibited synthesis of collagen, but not of non-collagenous protein; the concentrations of retinol necessary to produce the response were similar to those required to stimulate bone resorption in vitro.

The effects of parathyroid hormone on osteoblast-like cells from embryonic chick calvaria.

A bone cell fraction was isolated from 16 day old embryonic chick calvaria using a sequential enzymatic digestion procedure. The fraction contained cells, of an osteoblast-like character, which responded to parathyroid hormone (PTH) and prostaglandin E2, but not to calcitonin, in terms of increased production of cyclic AMP. Primary cultures of cells maintained their responsiveness to PTH for at least 2 weeks after reaching confluence. Production of alkaline phosphatase by the bone cells was inhibited when 1,25(OH)2 vitamin D3 was added to cultures at concentrations of 10(-8)M or greater. When cells were cultured in the presence of PTH a biphasic effect was observed; alkaline phosphatase levels were stimulated at low concentrations of this hormone but were decreased at higher concentrations. The latter finding appears consistent with observations that PTH can in vivo exert either anabolic or catabolic effects on bone, depending upon the circulating level of hormone present.

Changes with age in the non-collagenous proteins of human bone.

Little is known about the function of the non-collagenous proteins in the organic matrix of bone. Information about these proteins was obtained from studies of human cortical bone. Analysis of fractions of bone differing in density showed that 96% of the alpha 2HS-glycoprotein and 58% of the albumin was associated with the mineralized phase. A study of age-related changes in the composition of bone showed that the amounts present of alpha 2HS-glycoprotein, albumin, sialoprotein, soluble collagen and of EDTA-soluble protein were all higher in bone from children than in adults. In subjects aged over 60 y the content in bone of albumin, sialoprotein and of total protein was similar to that of children. These differences could not be correlated with age-related changes in the calcium/hydroxyproline ratio nor with previous reports of alterations in the crystal structure and the physical properties of human cortical bone. This suggests that these proteins are unlikely to be involved in stabilization of the mineralized matrix or in influencing the physical properties of bone; their function appears more likely to be related to bone formation and turnover.