Maximal dose-response of vitamin-K2 (menaquinone-4) on undercarboxylated osteocalcin in women with osteoporosis.

Low concentrations of serum vitamin K accompany high concentrations of undercarboxylated osteocalcin (ucOC) and osteoporotic fractures. Although vitamin K2 (MK-4) is approved as a therapeutic agent for the treatment of osteoporosis in some countries, the dose-response is unknown. The objective of this study was to assess the improvement in carboxylation of osteocalcin (OC) in response to escalating doses of MK-4 supplementation. A nine-week, open-labeled, prospective cohort study was conducted in 29 postmenopausal women who suffered hip or vertebral compression fractures. Participants took low-dose MK-4 (0.5 mg) for 3 weeks (until the second visit), then medium-dose MK-4 (5 mg) for 3 weeks (until the third visit), then high-dose MK-4 (45 mg) for 3 weeks. The mean ± SD age of the participants was 69 ± 9 years. MK-4 dose (p < 0.0001), but neither age nor other relevant medications (e.g. bisphosphonates) correlated with improvement in %ucOC. As compared to baseline concentrations (geometric mean ± SD) of 16.8 ± 2.4, 0.5 mg supplementation halved %ucOC to 8.7 ± 2.2 (p < 0.0001) and the 5-mg dose halved %ucOC again (to 3.9 ± 2.2; p = 0.0002 compared to 0.5-mg dose). However, compared to 5 mg/day, there was no additional benefit of 45 mg/day (%ucOC 4.6; p = NS vs. 5-mg dose). MK-4 supplementation resulted in borderline increases in γ-carboxylated osteocalcin (glaOC; p = 0.07). There were no major side effects of MK-4 supplementation. In postmenopausal women with osteoporotic fractures, supplementation with either 5 or 45 mg/day of MK-4 reduces ucOC to concentrations typical of healthy, pre-menopausal women.

Fermented Oyster Extract Promotes Osteoblast Differentiation by Activating the Wnt/ß-Catenin Signaling Pathway, Leading to Bone Formation.

The Pacific oyster, Crassostrea gigas, is well-known as a nutritious food. Recently, we revealed that fermented extract of C. gigas (FO) inhibited ovariectomy-induced osteoporosis, resulting from suppression of osteoclastogenesis. However, since the beneficial effect of FO on osteogenesis is poorly understood, it was examined in mouse preosteoblast MC3T3-E1 cells, human osteosarcoma MG-63 osteoblast-like cells, and zebrafish larvae in this study. We found that FO increased mitochondrial activity from days 1 to 7; however, total cell number of MC3T3-E1 cells gradually decreased without any change in cell viability, which suggests that FO stimulates the differentiation of MC3T3-E1 cells. FO also promoted the expression of osteoblast marker genes, including runt-related transcription factor 2 (mRUNX2), alkaline phosphatase (mALP), collagen type I α1 (mCol1α1), osteocalcin (mOCN), osterix (mOSX), bone morphogenetic protein 2 (mBMP2), and mBMP4 in MC3T3-E1 cells accompanied by a significant increase in ALP activity. FO also increased nuclear translocation of RUNX2 and OSX transcription factors, ALP activity, and calcification in vitro along with the upregulated expression of osteoblast-specific marker proteins such as RUNX2, ALP, Col1α1, OCN, OSX, and BMP4. Additionally, FO enhanced bone mineralization (calcein intensity) in zebrafish larvae at 9 days post-fertilization comparable to that in the ß-glycerophosphate (GP)-treated group. All the tested osteoblast marker genes, including zRUNX2a, zRUNX2b, zALP, zCol1a1, zOCN, zBMP2, and zBMP4, were also remarkably upregulated in the zebrafish larvae in response to FO. It also promoted tail fin regeneration in adult zebrafish as same as the GP-treated groups. Furthermore, not only FO positively regulate ß-catenin expression and Wnt/ß-catenin luciferase activity, but pretreatment with a Wnt/ß-catenin inhibitor (FH535) also significantly decreased FO-mediated bone mineralization in zebrafish larvae, which indicates that FO-induced osteogenesis depends on the Wnt/ß-catenin pathway. Altogether, the current study suggests that the supplemental intake of FO has a beneficial effect on osteogenesis.

Vitamin K-induced effects on body fat and weight: results from a 3-year vitamin K2 intervention study.

BACKGROUND/OBJECTIVES: Vitamin K status has been linked to fat and glucose metabolism by several authors, but whether high vitamin K intake influences body weight or composition has remained unclear. Here we tested the hypothesis that increased vitamin K intake decreases body fat or fat distribution. SUBJECTS/METHODS: In a randomized placebo-controlled human intervention trial, 214 postmenopausal women, 55-65 years of age, received either 180 mcg/day of vitamin K2 (menaquinone-7, MK-7) or placebo for 3 years. Osteocalcin (OC) carboxylation was used as a marker for vitamin K status, and fat distribution was assessed by dual-energy X-ray absorptiometry total body scan. RESULTS: In the total cohort, MK-7 supplementation increased circulating carboxylated OC (cOC) but had no effect on body composition. In those with an above-median response in OC carboxylation ('good responders'), MK-7 treatment resulted in a significant increase in total and human molecular weight adiponectin and a decrease in abdominal fat mass and in the estimated visceral adipose tissue area compared with the placebo group and the poor responders. CONCLUSIONS: The fact that changes in body composition measures or markers for fat or glucose metabolism were not associated with changes in uncarboxylated OC (ucOC) does not support the assumption that ucOC stimulates fat metabolism in humans. Instead, high vitamin K2 intake may support reducing body weight, abdominal and visceral fat, notably in subjects showing a strong increase in cOC. A causal relation between the changes in cOC and body fat or distribution cannot be concluded from these data.

Exogenous and endogenous determinants of vitamin K status in cystic fibrosis.

Cystic fibrosis (CF) patients are at high risk for vitamin K deficiency. The effects of vitamin K supplementation are very ambiguous. Therefore, we aimed to define the determinants of vitamin K deficiency in a large cohort of supplemented - 146 (86.9%) and non-supplemented - 22 (13.1%) CF patients. Vitamin K status was assessed using prothrombin inducted by vitamin K absence (PIVKA-II) and undercarboxylated osteocalcin (u-OC). The pathological PIVKA-II concentration (≥ 2 ng/ml) and abnormal percentage of osteocalcin (≥ 20%) were found in 72 (42.8%) and 60 (35.7%) subjects, respectively. We found that liver involvement, diabetes, and glucocorticoid therapy were potential risk factors for vitamin K deficiency. Pathological concentrations of PIVKA-II occurred more frequently in patients with pancreatic insufficiency and those who have two severe mutations in both alleles of the CFTR gene. Pathological percentage of u-OC was found more frequently in adult CF patients and those not receiving vitamin K. However, it seems that there are no good predictive factors of vitamin K deficiency in CF patients in everyday clinical care. Early vitamin K supplementation in CF patients seems to be warranted. It is impossible to clearly determine the supplementation dose. Therefore, constant monitoring of vitamin K status seems to be justified.

Using the RosettaSurface algorithm to predict protein structure at mineral surfaces.

Determination of protein structure on mineral surfaces is necessary to understand biomineralization processes toward better treatment of biomineralization diseases and design of novel protein-synthesized materials. To date, limited atomic-resolution data have hindered experimental structure determination for proteins on mineral surfaces. Molecular simulation represents a complementary approach. In this chapter, we review RosettaSurface, a computational structure prediction-based algorithm designed to broadly sample conformational space to identify low-energy structures. We summarize the computational approaches, the published applications, and the new releases of the code in the Rosetta 3 framework. In addition, we provide a protocol capture to demonstrate the practical steps to employ RosettaSurface. As an example, we provide input files and output data analysis for a previously unstudied mineralization protein, osteocalcin. Finally, we summarize ongoing challenges in energy function optimization and conformational searching and suggest that the fusion between experiment and calculation is the best route forward.

Synthesis and preliminary biological evaluation of 20-epi-eldecalcitol [20-epi-1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D3: 20-epi-ED-71].

Eldecalcitol [1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D3, developing code: ED-71] is an analog of active vitamin D3, 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] that possesses a hydroxypropoxy substituent at the 2beta-position of 1,25(OH)2D3. Eldecalcitol has potent biological effects on bone and is now in preparation for approval as a promising medicine for the treatment of osteoporosis in Japan. To explore chemical structure-biological activity relationships between eldecalcitol and related analogs, we have already synthesized 1-epi-eldecalcitol, 3-epi-eldecalcitol, and 1,3-diepi-eldecalcitol with inherent biological interests of each targeted analog and evaluated their biological responses. It has been reported that 20-epi-1,25(OH)2D3, a diastereomer of 1,25(OH)2D3 that possesses an inverted methyl substituent at the 20-position of the side chain, shows remarkably enhanced biological activities compared to parental compound, 1,25(OH)2D3. As a continuation of our modification studies on eldecalcitol, we took great interest in 20-epi-eldecalcitol and its biological responses. In this paper, the synthesis of 20-epi-eldecalcitol by the Trost coupling reaction between the A-ring fragment and the C/D-ring fragment as well as in vitro preliminary biological evaluation of 20-epi-eldecalcitol are described. In the induction of human myeloid leukemia cell (HL-60) differentiation, inhibition of the human histiocytic lymphoma cell (U937) proliferation, and increase in osteocalcin concentration in the human osteosarcoma cell (MG-63), 20-epi-eldecalcitol showed significantly enhanced activity compared to eldecalcitol.

Influence of different modifications of a calcium phosphate bone cement on adhesion, proliferation, and osteogenic differentiation of human bone marrow stromal cells.

Collagen and noncollagenous proteins of the extracellular bone matrix are able to stimulate bone cell activities and bone healing. The modification of calcium phosphate bone cements used as temporary bone replacement materials with these proteins seems to be a promising approach to accelerate new bone formation. In this study, we investigated adhesion, proliferation, and osteogenic differentiation of human bone marrow stromal cells (hBMSC) on Biocement D/collagen composites which have been modified with osteocalcin and O-phospho-L-serine. Modification with osteocalcin was carried out by its addition to the cement precursor before setting as well as by functionalization of the cement samples after setting and sterilization. hBMSC were cultured on these samples for 28 days with and without osteogenic supplements. We found a positive impact especially of the phosphoserine-modifications but also of both osteocalcin-modifications on differentiation of hBMSC indicated by higher expression of the osteoblastic markers matrix metalloproteinase-13 and bone sialo protein II. For hBMSC cultured on phosphoserine-containing composites, an increased proliferation has been observed. However, in case of the osteocalcin-modified samples, only osteocalcin adsorbed after setting and sterilization of the cement samples was able to promote initial adhesion and proliferation of hBMSC. The addition of osteocalcin before setting results in a finer microstructure but the biological activity of osteocalcin might be impaired due to the sterilization process. Thus, our data indicate that the initial adhesion and proliferation of hBMSC is enhanced rather by the biological activity of osteocalcin than by the finer microstructure.

Effect of chemically modified titanium surfaces on protein adsorption and osteoblast precursor cell behavior.

PURPOSE: To investigate the effects of different chemically modified titanium surfaces on protein adsorption and the osteoblastic differentiation of human embryonic palatal mesenchymal (HEPM) cells. MATERIALS AND METHODS: Three different surfaces were evaluated. The first, a machined surface (Ti-M), was considered a control. The second surface was acid etched (Ti-AE). The third surface was prepared by exposing the Ti-AE samples to sodium hydroxide (NaOH) solution (Ti-AAE). The surface characteristics of chemically modified titanium were investigated by means of scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and profilometry. To evaluate the production of biomarkers, commercial kits were utilized. RESULTS: Surface composition and morphology affected the kinetics of protein adsorption. Ti-AE surfaces manifested a greater affinity for fibronectin adsorption compared to Ti-M or Ti-AAE surfaces. It was observed that Ti-AE and Ti-AAE surfaces promoted significantly greater cell attachment compared to Ti-M surfaces. Statistically significant differences were also observed in the expression of alkaline phosphatase (ALP) activity, osteocalcin, and osteopontin on all 3 titanium surfaces. ALP activity and osteocalcin production up to day 12 suggested that differentiation of the cells into osteoblasts had occurred and that cells were expressing a bone-forming phenotype. CONCLUSIONS: It was thus concluded from this study that surface morphology and composition play a critical role in enhancing HEPM cell proliferation and differentiation into osteoblast cells.

Two-year randomized controlled trial of vitamin K1 (phylloquinone) and vitamin D3 plus calcium on the bone health of older women.

UNLABELLED: Dietary supplementation with vitamin K(1), with vitamin D(3) and calcium or their combination, was examined in healthy older women during a 2-year, double-blind, placebo-controlled trial. Combined vitamin K with vitamin D plus calcium was associated with a modest but significant increase in BMC at the ultradistal radius but not at other sites in the hip or radius. INTRODUCTION: The putative beneficial role of high dietary vitamin K(1) (phylloquinone) on BMD and the possibility of interactive benefits with vitamin D were studied in a 2-year double-blind, placebo-controlled trial in healthy Scottish women > or =60 years of age. MATERIALS AND METHODS: Healthy, nonosteoporotic women (n = 244) were randomized to receive either (1) placebo, (2) 200 microg/day vitamin K(1), (3) 10 microg (400 IU) vitamin D(3) plus 1000 mg calcium/day, or (4) combined vitamins K(1) and D(3) plus calcium. Baseline and 6-month measurements included DXA bone mineral scans of the hip and wrist, markers of bone turnover, and vitamin status. Supplementation effects were tested using multivariate general linear modeling, with full adjustment for baseline and potential confounding variables. RESULTS: Significant bone mineral loss was seen only at the mid-distal radius but with no significant difference between groups. However, women who took combined vitamin K and vitamin D plus calcium showed a significant and sustained increase in both BMD and BMC at the site of the ultradistal radius. Serum status indicators responded significantly to respective supplementation with vitamins K and D. Over 2 years, serum vitamin K(1) increased by 157% (p < 0.001), the percentage of undercarboxylated osteocalcin (%GluOC) decreased by 51% (p < 0.001), serum 25-hydroxyvitamin D [25(OH)D] increased by 17% (p < 0.001), and PTH decreased by 11% (p = 0.049). CONCLUSIONS: These results provide evidence of a modest synergy in healthy older women from nutritionally relevant intakes of vitamin K(1) together with supplements of calcium plus moderate vitamin D(3) to enhance BMC at the ultradistal radius, a site consisting of principally trabecular bone. The substantial increase in gamma-carboxylation of osteocalcin by vitamin K may have long-term benefits and is potentially achievable by increased dietary intakes of vitamin K rather than by supplementation.

Effect of calcium, vitamins K1 and D3 on bone in galactosemia.

Classical galactosemia is an inherited disorder of galactose metabolism. Recently, diminished bone mineral content (BMC) in children and adolescents has been found. The aim of this study was to evaluate the effect of calcium, vitamins K(1) and D(3) supplementation on bone in children with galactosemia. A 2-year randomized, double-blind, placebo-controlled clinical trial was undertaken in which 40 children with classical galactosemia (13 males and 27 females, aged 3-17 years) were included to receive daily either 750 mg calcium, 1.0 mg vitamin K(1) and 10.0 microg vitamin D(3) or placebo. BMC of femoral neck, lumbar spine and total body and body composition data were determined by dual energy X-ray absorptiometry (DXA) at baseline and after 1 and 2 years. Diet was assessed using a food frequency questionnaire and a 3-day food diary. Biochemical measurements were determined at baseline and after 1 and 2 years. In the children receiving treatment, carboxylated osteocalcin (cOC) concentration significantly increased (P < 0.001) and undercarboxylated osteocalcin (ucOC) concentration significantly decreased (P = 0.001) when compared to the children receiving placebo. Furthermore, there was a statistically significant increase in BMC of lumbar spine (P = 0.001), lean tissue mass (LTM: P = 0.016) and fat mass (FM: P = 0.014) in the treatment group when compared to the placebo group. The significant increase in cOC and decrease in ucOC concentration in the treatment group were present in prepubertal (P < 0.001 and P = 0.006 respectively) and pubertal children (P = 0.004 and P = 0.042 respectively). The significant increase in BMC of lumbar spine in the treatment group was present only in the prepubertal children (P = 0.015). Supplementation of calcium, vitamins K(1) and D(3) given in this dose (750 mg, 1.0 mg and 10.0 mug respectively) is likely to have a role in the treatment of BMC abnormalities in galactosemia.

Identification of an osteocalcin isoform in fish with a large acidic prodomain.

Osteocalcin is a small, secreted bone protein whose gene consists of four exons. In the course of analyzing the structure of fish osteocalcin genes, we recently found that the spotted green pufferfish has two possible exon 2 structures, one of 15 bp and the other of 324 bp. Subsequent analysis of the pufferfish cDNA showed that only the transcript with a large exon 2 exists. Exon 2 codes for the osteocalcin propeptide, and exon 2 of pufferfish osteocalcin is approximately 3.4-fold larger than exon 2 previously found in other vertebrate species. We have termed this new pufferfish osteocalcin isoform OC2. Additional studies showed that the OC2 isoform is restricted to a unique fish taxonomic group, the Osteichthyes; OC2 is the only osteocalcin isoform found so far in six Osteichthyes species, whereas both OC1 and OC2 isoforms coexist in zebrafish and rainbow trout. The larger size of the OC2 propeptide is due to an acidic region that is likely to be highly phosphorylated and has no counterpart in the OC1 propeptide. We propose 1) that OC1 and OC2 are encoded by distinct genes that originated from a duplication event that probably occurred in the teleost fish lineage soon after divergence from tetrapods and 2) that the novel OC2 propeptide could be, if secreted, a phosphoprotein that participates in the regulation of biomineralization through its large acidic and phosphorylated propeptide.

The effect of incorporating RGD adhesive peptide in polyethylene glycol diacrylate hydrogel on osteogenesis of bone marrow stromal cells.

Advances in tissue engineering require biofunctional scaffolds that can not only provide cells with structural support, but also interact with cells in a biological manner. To achieve this goal, a frequently used cell adhesion peptide Arg-Gly-Asp (RGD) was covalently incorporated into poly(ethylene glycol) diacrylate (PEODA) hydrogel and its dosage effect (0.025, 1.25 and 2.5 mm) on osteogenesis of marrow stromal cells in a three-dimensional environment was examined. Expression of bone-related markers, osteocalcin (OCN) and Alkaline phosphatase (ALP), increased significantly as the RGD concentration increased. Compared with no RGD, 2.5 mm RGD group showed a 1344% increase in ALP production and a 277% increase in OCN accumulation in the medium. RGD helped MSCs maintain cbfa-1 expression when shifted from a two-dimensional environment to a three-dimensional environment. Soluble RGD was found to completely block the mineralization of marrow stromal cells, as manifested by quantitative calcium assay, phosphorus elemental analysis and Von Kossa staining. In conclusion, we have demonstrated that RGD-conjugated PEODA hydrogel promotes the osteogenesis of MSCs in a dosage-dependent manner, with 2.5 mm being optimal concentration.

Characterization of the cDNA encoding bullfrog, Rana catesbeiana, osteocalcin and two forms of the protein isolated from bone.

A full-length cDNA clone encoding osteocalcin from the bullfrog, Rana catesbeiana (bone Gla-protein, BGP) has been isolated, and the complete coding sequence for the 100-amino-acid pre-pro-osteocalcin protein was determined. The amino acid sequence of Rana catesbeiana osteocalcin, especially the mature 49-amino acid sequence, is closer to the mammalian than to the fish, Sparus osteocalcin. Rana mature osteocalcin has a similarity of 67% with human or 59% with rat osteocalcin, and only 42% with fish mature osteocalcin. The 51-amino-acid pre-pro-peptide contains the expected hydrophobic leader sequence and the dibasic Arg-Arg sequence preceding the NH2-terminal Ser of the mature 49-amino-acid Rana osteocalcin. The pro-peptide sequence also contains the expected motif of polar and hydrophobic residues, which targets vitamin K-dependent gamma-carboxylation of three specific Glu residues at positions 17, 21, and 24 in the mature protein. At the native protein expression levels, extraction from Rana cortical bone in the presence of protease inhibitor cocktail resulted in the isolation of two distinct forms of osteocalcin, P-1 and P-2, with a 3:2 distribution. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and amino acid sequence analysis of the N-terminal domain, we confirmed that P-1 is the intact 49-residue osteocalcin with N-terminal SNLRNAVFG., and that P-2 lacks four amino acids from the N-terminus, (NAVFG.). These results demonstrate the existence of a form of osteocalcin lacking four N-terminal amino acids in Rana bone, and that mature Rana osteocalcins remained highly conserved in their molecular evolution, especially with respect to the conservation of the C-terminal domain (residues 14-49).

Bone recognition mechanism of porcine osteocalcin from crystal structure.

Osteocalcin is the most abundant noncollagenous protein in bone, and its concentration in serum is closely linked to bone metabolism and serves as a biological marker for the clinical assessment of bone disease. Although its precise mechanism of action is unclear, osteocalcin influences bone mineralization, in part through its ability to bind with high affinity to the mineral component of bone, hydroxyapatite. In addition to binding to hydroxyapatite, osteocalcin functions in cell signalling and the recruitment of osteoclasts and osteoblasts, which have active roles in bone resorption and deposition, respectively. Here we present the X-ray crystal structure of porcine osteocalcin at 2.0 A resolution, which reveals a negatively charged protein surface that coordinates five calcium ions in a spatial orientation that is complementary to calcium ions in a hydroxyapatite crystal lattice. On the basis of our findings, we propose a model of osteocalcin binding to hydroxyapatite and draw parallels with other proteins that engage crystal lattices.

Carboxylation of osteocalcin in post-menopausal osteoporotic women following vitamin K and D supplementation.

The effect of vitamin supplements on bone metabolism indices in patients with osteoporosis has received scant attention in the literature. Over a 2-week period, vitamin supplements of K and K+D were given to 20 post-menopausal osteoporotic women with previous Colles fractures. Osteoporosis was confirmed by bone mass measurements that demonstrated that broadband ultrasound attenuation (os calcis) was almost as discriminatory as dual energy X-ray absorptiometry (spine and hip) in Colles fracture patients compared with matched controls. Vitamin K corrected the carboxylation defect in osteocalcin and while less marked 4 weeks later, the improvement was still detectable. The result after K+D was similar. The level of carboxylation became the same as in premenopausal women. Total osteocalcin level (bound) osteocalcin. While there was vitamin K correctable undercarboxylation of osteocalcin, simultaneously there was no evidence of undercarboxylation of prothrombin.

Characterization of structural sequences in the chicken osteocalcin gene: expression of osteocalcin by maturing osteoblasts and by hypertrophic chondrocytes in vitro.

Osteocalcin is one of the major noncollagenous proteins specific to mineralized connective tissues of vertebrates. A cDNA clone encoding the chicken osteocalcin gene was isolated, and the complete coding sequence for the 97-amino-acid pre-pro-osteocalcin was deduced. The 48-amino-acid pre-pro-peptide contains the expected hydrophobic leader sequence and the dibasic Lys-Arg sequence preceding the NH2-terminal His of the mature 49-amino-acid chicken osteocalcin, which is believed to be necessary for pro-peptide cleavage. The pro-peptide sequence also contains the expected motif of polar and hydrophobic residues, including Phe at -16, which targets vitamin K-dependent gamma-carboxylation of the three specific Glu residues at positions 17, 21, and 24 in the mature protein. Northern blots of total RNA were prepared from embryonic and adult chicken tissues (bone, brain, heart, intestine, kidney, muscle) and probed with chicken osteocalcin cDNA. The appearance of a single 0.5 kb mRNA species confirms that bone is the major site of osteocalcin expression in vivo. In primary osteoblasts isolated from 17-day embryonic chicken calvaria, an osteocalcin mRNA of similar size is expressed concurrently with culture mineralization in vitro. Hypertrophic chondrocytes from 12-day ventral vertebrae and from the cephalic half of 17-day caudal sternae also express osteocalcin mRNA, but nonhypertrophic chondrocytes from the caudal half of 17-day sternae do not express osteocalcin mRNA.

Rapid and sensitive method of quantitation of bone gla protein mRNA using competitive polymerase chain reaction.

A method for sensitive quantitation of bone gla protein (BGP, osteocalcin) mRNA has been developed using competitive polymerase chain reaction after reverse transcription (competitive RT-PCR). The complementary DNA (cDNA) were transcribed from sample RNA was co-amplified in a PCR with a known amount of mutant BGP cDNA (competitor) using the identical oligonucleotide primers. The mutant cDNA with its unique restriction site allowed quantitation of sample and mutant PCR products after densitometric analysis of ethidium bromide-stained agarose gels. A linear relationship between initial sample BGP amount and the ratio of BGP to mutant BGP band intensity was obtained and used to make a standard curve to determine the initial BGP mRNA of unknown samples. These standard curves were made with known amounts of recombinant BGP cDNA. The competitive RT-PCR for BGP allows measurement of twofold differences in 1 and 2 micrograms total RNA and requires at least 10 times less sample RNA than usual Northern blotting. Moreover, heteroduplexes with one BGP strand and one mutant BGP strand formed as a result of high PCR cycles were quantifiable. This provided the advantages of rapid quantitation from ethidium bromide-stained gels without blotting, hybridization, or autoradiography. Multiple samples could be assayed for greater confidence in the results. The sensitivity, accuracy, and ease of the assay will facilitate analysis of BGP mRNA from a small amount of sample. The assay has been used to confirm the BGP mRNA changes with hormonal treatment in cultured cells and the age-related changes in whole tibia in vivo.