Effects of dietary lysine levels on the concentrations of selected nutrient metabolites in blood plasma of late-stage finishing pigs.

Lysine is the first-limiting amino acid (AA) in typical swine diets and plays very important roles in promoting growth performance of pigs. This research was conducted to study the effects of dietary lysine on blood plasma concentrations of protein, carbohydrate, and lipid metabolites of pigs. Eighteen crossbred finishing pigs (nine barrows and nine gilts; initial BW 92.3 ± 6.9 kg) were individually penned in an environment controlled barn. Pigs were assigned to three dietary treatments according to a randomized complete block design with gender as block and pig as experimental unit (6 pigs/treatment). Three corn and soybean meal-based diets were formulated to contain total lysine at 0.43%, 0.71%, and 0.98% (as-fed basis) for Diets I (lysine deficient), II (lysine adequate), and III (lysine excess) respectively. After 4 weeks on trial, jugular vein blood was collected and plasma was separated. The plasma concentrations of total protein, albumin, urea nitrogen (UN), triglyceride, total cholesterol, and glucose were determined using an ACE Clinical Chemistry System (Alfa Wassermann, Inc., West Caldwell, NJ, USA). Data were analysed using the GLM Procedure with PDIFF (adjust = T) option of SAS. No differences (p > 0.10) were found between barrows and gilts for any of the metabolites measured. While there were no differences (p > 0.10) between pigs fed Diets II and III in plasma concentrations of UN, albumin, and total cholesterol, the concentration of albumin in these pigs was higher (p < .05) than that of pigs fed Diet I, and the concentrations of UN and total cholesterol in these pigs were lower (p < .05) than that of pigs fed Diet I. There were no differences (p > 0.10) among the three dietary treatments in plasma concentrations of total protein, triglycerides, and glucose. These findings indicated that the plasma metabolite profile can be affected by changing dietary lysine content only. Thorough understanding how the plasma metabolite profile is alternated by dietary lysine will facilitate nutrient management for more sustainable swine production.

Spectroscopic imaging of mineral maturation in bovine dentin.

Dentin is a useful model for the study of mineral maturation. Using Fourier Transform Infrared Imaging (FTIRI), we characterized distinct regions in developing dentin at 7- micro m spatial resolution. Mineral-to-matrix ratio and crystallinity in bovine dentin from cervical and incisal parts of 3rd-trimester fetal compared with one-year-old incisor crowns showed that virtually all maturation stages in dentin could be spectroscopically isolated and analyzed. In the fetal incisors, mantle and circumpulpal dentin presented distinct patterns of mineral maturation. Gradients in both mineral properties examined were observed at the mineralization front and at the dentino-enamel junction.

In vitro apatite induction by phosphophoryn immobilized on modified collagen fibrils.

Noncollagenous phosphoproteins that interact with type I collagen are thought to nucleate the mineral phase to collagen network of mineralized tissues. Previously, we found that phosphophoryn cross-linked to type I collagen was an effective nucleator of apatite. Here, we investigated the potential role of collagen telopeptide structure on this nucleation. We used pepsin and sodium borohydride (NaBH4) to modify the telopeptide region and reducible cross-links in the collagen fibrils and determined the effect on mineral induction by phosphophoryn cross-linked to it. The amount of phosphophoryn cross-linked to NaBH4-reduced collagen fibrils was higher than that to intact (unmodified) collagen fibrils. However, the amount of phosphophoryn cross-linked to collagen that lacked the telopeptides (atelocollagen) was 25% of that cross-linked to intact collagen fibrils. Each preparation was incubated at 37 degrees C in metastable calcium phosphate solutions that did not spontaneously precipitate. Apatite was induced by phosphophoryn cross-linked to intact collagen fibrils at 15.0 h whereas phosphophoryn cross-linked to reduced collagen fibrils induced apatite formation after 10.9 h. Enough phosphophoryn was cross-linked to atelocollagen to induce mineral formation, but it did not. The failure of the phosphophoryn-atelocollagen complex to nucleate mineral might have been caused by a cross-linking pattern in the helical portion of the collagen molecule that did not promote the growth of the calcium-phosphate clusters into nuclei. The present study indicates that the telopeptide domains of type I collagen play a role in the interaction with phosphophoryn, which is critical for the nucleation process.

Apatite induction by insoluble dentin collagen.

Phosphoproteins are thought to play a role in mineral formation in dentin. A portion of this phosphoprotein is bound to collagen. We have investigated the requirement for bound phosphate in mineral induction by isolated dentin collagen. Insoluble bovine dentin collagen obtained by ethylene-diamino-tetra-acetic acid (EDTA) demineralization had 19.5 mol of P/mol of collagen that could not be extracted with 0.5 M EDTA in 4 M guanidine HCl. When this collagen was incubated in supersaturated solutions that did not spontaneously precipitate, apatite was induced. With progressive enzymatic dephosphorylation, induction times for mineral formation became progressively longer. The dentin did not induce mineral formation when 90% of the ester phosphate was removed. Insoluble bone collagen, which had even less phosphate, also did not induce mineral formation. Mineral induction times by dentin collagen increased with decreasing solution saturations. Using these data, the interfacial tension for mineral induction was determined to be 90 ergs/cm2. This value approximated that of phosphatidic acid liposomes and of phosvitin cross-linked to agarose beads, and it might reflect the energetics of heterogeneous nucleation on a highly phosphorylated surface. Sequestering of calcium-phosphate clusters on the phosphoprotein probably accounts for the observed calcium binding by dentin collagen in excess of that required to neutralize the phosphate esters of the collagen. Because the phosphoprotein is immobilized at a low density on the collagen, it cannot self-associate in calcium-phosphate solutions as it does when it is free in solution. This immobilized phosphoprotein allows the mineral clusters formed on its surface to grow into a crystalline order.

Mineral induction by immobilized phosphoproteins.

Dentin phosphoproteins are thought to have a primary role in the deposition of mineral on the collagen of dentin. In this study we determined the type of binding between collagen and phosphoproteins necessary for mineral formation onto collagen fibrils and whether the phosphate esters are required. Bovine dentin phosphophoryn or phosvitin from egg yolk were immobilized on reconstituted skin type I collagen fibrils by adsorption or by covalent cross-linking. In some samples the ester phosphate was removed from the covalently cross-linked phosphoproteins by treatment with acid phosphatase. All samples were incubated at 37 degrees C in metastable solutions that do not spontaneously precipitate. Reconstituted collagen fibrils alone did not induce mineral formation. The phosphoproteins adsorbed to the collagen fibrils desorbed when the mineralization medium was added, and mineral was not induced. The mineral induced by the cross-linked phosphoproteins was apatite, and the crystals were confined to the surface of the collagen fibrils. With decreasing medium saturation the time required for mineral induction increased. The interfacial tensions calculated for apatite formation by either phosphoprotein cross-linked to collagen were about the same as that for phosphatidic acid liposomes and hydroxyapatite. This similarity in values indicates that the nucleation potential of these highly phosphorylated surfaces is about the same. It is concluded that phosphoproteins must be irreversibly bound to collagen fibrils for the mineralization of the collagen network in solutions that do not spontaneously precipitate. The phosphate esters of phosphoproteins are required for mineral induction, and the carboxylate groups are not sufficient.

Three-dimensional analysis of mineralizing turkey leg tendon: matrix vesicle-collagen relationships.

The spatial and temporal relationships of mineral deposition between matrix vesicles and type I collagen fibrils have been studied in the turkey leg tendon by electron microscopy of cross sections and serial longitudinal thin sections and by electron tomography of longitudinal thick sections. Serial sectioning and electron tomography allow three-dimensional analysis of spatial relationships, overcoming the problems of missing depth information and over-projection of adjacent structures which exist for two-dimensional projections of isolated sections. These techniques reveal that while mineral deposits within matrix vesicles are found remote from calcifying collagen fibrils, the reverse relationship does not occur; all collagen-associated mineral can ultimately be linked to mineral-laden vesicles. These results suggest a temporal sequence of calcification beginning in matrix vesicles and spreading to adjacent collagen fibrils.

Calcium movement in vivo and in vitro in secretory-stage enamel of rat incisors.

The lower incisors of young rats were dissected, immersed in physiological saline containing 45Ca under various conditions, and processed for autoradiography. The data were compared with those from in vivo 45Ca autoradiography. In secretory-stage enamel, wiped free of the enamel organ and immediately immersed in radioactive saline, there was intense labelling in the surface layers. The labelled area expanded only gradually into the deeper layers at a rate similar to that observed in vivo. Labelling in the enamel was similar in pattern but much weaker in intensity when the incisor was identically treated in vitro with the enamel organ attached. Glutaraldehyde pretreatment of the exposed enamel abolished expansion of the labelled area, whereas a hypochlorite pretreatment allowed a rapid diffusion of the isotope into the deeper layers of the secretory-stage enamel. The findings confirm the role of the enamel organ as a diffusion barrier to the penetration of calcium from the extracellular fluid to the secretory-stage enamel, and suggest an intimate correlation between physicochemical properties of the organic enamel matrix and the rate of surface-to-interior diffusion of calcium within the secretory-stage enamel of rat incisors.

Mineral induction by immobilized polyanionic proteins.

The purpose of this study was to investigate the mineral induction capacity in vitro of polyanionic proteins covalently bound to a surface. Rat dentin gamma-carboxyglutamate-containing protein of the osteocalcin type (Gla-protein), proteoglycan (PG), and phosphoprotein (PP-H), as well as phosvitin (PhV) and bovine serum albumin (BSA), were covalently linked to agarose beads. There were incubated at 37 degrees C in solutions with a Ca/P molar ratio of 1.67, [Ca][P] molar products in the range 1.0-1.8 mM2, and an ionic strength of 0.165. The incubations were performed at constant pH and composition conditions; no spontaneous precipitation occurred under these conditions. Mineral formation, as monitored by scanning electron microscopy (SEM), was induced by all immobilized polyanions, including enzymatically dephosphorylated PP-H and PhV. No mineral was induced by BSA. The mineral inductive capacity of immobilized polyanionic proteins, as judged by the SEM after identical incubations, was found to differ between the different ligands. The mineral induced by PP-H and PG was shown by X-ray diffraction to be apatitic. It was concluded that, although polyanionic proteins in solution may inhibit mineral induction and growth, very minute quantities of such molecules, when immobilized on a surface, induce mineral at physiological concentrations of calcium and phosphate ions. The data presented may be taken to suggest that PP-H and PG, and perhaps other polyanions, may possibly be responsible for mineral nucleation in dentin and bone. The results, however, also point to the rather limited specificity in this type of reaction.

The Ca, Pi, F, and proline content of developing bovine enamel under GBHA-stained and unstained bands.

The surface enamel of fetal bovine teeth was stained with GBHA to indicate the position of bands of smooth-ended and ruffle-ended ameloblasts relative to the developing enamel. The boundaries of the bands were scored, under a dissecting microscope, and the bulk enamel under each band was collected. The enamel samples were assayed for Ca, Pi, F, and proline. The amount of Ca and Pi in the enamel increased in successive bands and seemed unrelated to the overlying ameloblast cell type. The loss of proline seemed unrelated to cell type. The fluoride content of enamel increased by approximately 50% in the first stained band immediately adjacent to the secretory zone. The F level returned to secretory values in the succeeding unstained band. Thus, only changes in the F level of developing enamel appeared to be related to GBHA staining patterns.

The in vitro uptake of fluoride by secretory and maturation stage bovine enamel.

The objectives of this study were to determine the specific surface area of secretory-stage and of maturation-stage enamel, to compare the fluoride uptake by isolated enamel at these two stages on a surface-area basis, and to examine the effect of the organic matrix on the fluoride uptake by whole enamel. Fetal bovine secretory and maturation stage enamel samples were collected, and a portion of the enamel at each developmental stage was treated with hydrazine for removal of the organic matrix. The specific surface areas of the enamel mineral, as determined by the multi-point BET method, were 59.3 m2/g in the secretory stage and 37.9 m2/g in the maturation stage. Whole and deproteinated enamel samples were equilibrated in buffered solutions containing 10(-5) to 10(-3) mol/L fluoride, and the uptake was measured with a fluoride specific electrode. The results indicate that the in vitro fluoride uptake was controlled solely by the surface area of the apatitic mineral and that the organic matrix did not contribute to the fluoride uptake.

Absence of in-vitro fluoride-binding by the organic matrix of developing bovine enamel.

The in-vitro binding of fluoride to isolated organic matrix of secretory bovine enamel was studied by direct fluoride measurement and equilibrium dialysis. Over a wide range of protein and fluoride concentrations there was no indication of fluoride binding by the matrix in contrast to earlier reports.

Induction and inhibition of hydroxyapatite formation by rat dentine phosphoprotein in vitro.

Highly phosphorylated rat incisor phosphoprotein (PP-H) was purified and covalently attached to agarose beads. The beads were incubated for 24 h in solutions having an ionic strength of 0.165, a molar Ca/P ratio of 1.67, and a pH of 7.4. The calcium-phosphate concentration products [( Ca][P]) in the stable incubation solutions ranged from 1.0 to 1.8 mM2, from which no spontaneous precipitation occurred. In a timed series mineral formation was monitored by SEM and X-ray diffraction. The inhibitory capacity of PP-H, free solution, was also studied. The first mineral appeared after 10 min at a [Ca][P] product as low as 1.2 mM2; X-ray diffraction showed that the mineral was (hydroxy)apatite. Thus small amounts of PP-H attached to a surface are capable of inducing mineral formation in vitro at comparatively low supersaturation, whereas PP-H is a mineral inhibitor when free in solution.

The short-term uptake and retention of fluoride in developing enamel and bone.

Eight- and 12-day-old rat pups were injected intraperitoneally with fluoride. Plasma, molar enamel, and bone samples were collected at observation times up to six hr after injection. In a second series, adult rats maintained for six weeks on water containing 5 ppm F were injected with fluoride. Plasma, incisor enamel, and bone samples were collected at the same observation times as those used in the first series. Fluoride assays were conducted by means of the microdiffusion, ion-selective-electrode method. In the suckling rats, plasma [F] levels peaked at 15 min and returned nearly to baseline in one hr. Significant increases in the [F] of developing enamel and bone were observed. No significant decline from the peak [F] seen in the hard tissues was observed over the six-hour period. Similar results were seen in the developing enamel of the adult rats. The data gave no evidence of a short-term reversible component of fluoride uptake in developing enamel. Apparent increases in F uptake in enamel and bone beyond peak plasma values suggest the presence of a diffusion-limiting membrane for fluoride from the extracellular fluids into the mineralizing matrix.

Diffusion of fluoride through the rat enamel organ in vitro.

This study investigated the diffusion of fluoride through the enamel organ in vitro. The rat molar explants used were entirely in the secretory stage or predominantly in the maturation stage of enamel formation. The removal of the enamel organ or metabolic inhibition with iodoacetate caused significant increases in enamel fluoride uptake at both stages of enamel formation. Inhibition with dinitrophenol caused a significant increase only in the maturation phase. Uptake of fluoride in enamel was related to the fluoride concentration in the medium, except in the maturation stage explants, where increasing the medium fluoride concentration from 0.05 ppm to 0.08 ppm did not significantly increase fluoride uptake at any of the three observation times. The findings indicate that the enamel organ exists as a diffusion-limiting membrane to the movement of fluoride from the extracellular fluid compartment to the developing enamel.

Ca-ATPase and ALPase activities at the initial calcification sites of dentin and enamel in the rat incisor.

Enzymatic activities of calcium-magnesium dependent adenosine triphosphatase (Ca-ATPase) and nonspecific alkaline phosphatase (ALPase) were localized at the initial calcification sites of dentin and enamel of rat incisor teeth using electron-microscopic cytochemistry. Ca-ATPase was localized in the Golgi cisternae, cytoplasmic vesicles and along the outer surface of the presecretory and secretory ameloblasts, whereas it was totally absent from the odontoblasts in the pulp. Inversely, ALPase reaction was localized along the outer surface of the odontoblasts, but almost completely absent from the ameloblasts. Diffuse extracellular reactions of both enzymes were distributed throughout the unmineralized fibrous matrix of mantle dentin in which a large number of matrix vesicles were scattered. Both Ca-ATPase and ALPase reactions, which appeared in the matrix vesicles in the process of formation of mantle dentin, became most conspicuous at the site of initial dentin calcification. At this stage, an intense Ca-ATPase reaction also appeared along some of the collagen fibrils adjacent to the reactive matrix vesicles. No ALPase reaction was localized along these Ca-ATPase reactive collagen fibrils. Our observations suggest strongly that Ca-ATPase in the matrix vesicles originates from the inner enamel epithelium and/or preameloblasts whereas ALPase originates from the odontoblasts in the pulp. The importance of the coexistence of both enzymes for the control of initial calcification of dental hard tissues is suggested.

Changes in the fluoride-induced modulation of maturation stage ameloblasts of rats.

The maturation stage of enamel development is characterized by a cyclic modulation of the ameloblasts between bands of smooth-ended cells and longer bands of ruffle-ended cells. There are cyclic patterns of calcein staining of and 45Ca uptake in the enamel associated with this cellular modulation. Rats were given 0, 75, 100, or 150 ppm fluoride in their drinking water. Fluoride disrupted the cyclic patterns of the maturation stage, resulting in fewer bands of smooth-ended ameloblasts, fewer calcein-stained stripes, and fewer cycles of 45Ca uptake. When animals were given water containing 0 ppm fluoride following ingestion of water containing 100 ppm fluoride, the pattern of calcein staining returned to that of the control enamel. The disruption of the cyclic patterns in the maturation stage and the increased protein content of maturation enamel seem to be among the early events in the development of fluorosis.

Evaluation of dietary practices of college women based on expressed concern for one's dietary habits.

On the basis of expressions of concern for their dietary practices, those noting much concern were more likely to practice nutrient supplementation as a margin of safety against possible dietary deficiencies and to rate their diets as good. Although those subjects were more likely to view their body size with satisfaction than subjects in other groups, the majority in each group tended to believe that they were overweight. Diets of those expressing much concern were more likely to meet 100% of the RDAs than diets of other groups.

The role of osteocytes in bone resorption during lactation: morphometric observations.

It has been suggested that osteocytes may resorb bone during the calcium mobilization that occurs during lactation. To test this hypothesis morphometric methods were used to evaluate changes in mature nonmated female rats, 6-day postpartum nonlactating rats, 21-day postpartum nonlactating rats, 6-day lactating rats, 15-day lactating rats, and 21-day lactating rats using femur cross sections in the diaphyseal region. Osteoclast resorption per unit length of periosteal surface, as well as other measures of resorption activity, demonstrated that by 15 days of lactation a significant mobilization of calcium was occurring. The volume density of osteocyte lacunae and individual lacunar volumes from serial section reconstruction showed no increase in the lactating groups. Indeed, the only significant change in lacunae volume was a decrease apparently due to pregnancy. It is concluded from this study that osteocytes do not resorb bone during lactation.