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The gut microbiome affects the inflammatory environment through effects on T-cells, which influence the production of immune mediators and inflammatory cytokines that stimulate osteoclastogenesis and bone loss in mice. However, there are few large human studies of the gut microbiome and skeletal health. We investigated the association between the human gut microbiome and high resolution peripheral quantitative computed tomography (HR-pQCT) scans of the radius and tibia in two large cohorts; Framingham Heart Study (FHS [n=1227, age range: 32 - 89]), and the Osteoporosis in Men Study (MrOS [n=836, age range: 78 - 98]). Stool samples from study participants underwent amplification and sequencing of the V4 hypervariable region of the 16S rRNA gene. The resulting 16S rRNA sequencing data were processed separately for each cohort, with the DADA2 pipeline incorporated in the16S bioBakery workflow. Resulting amplicon sequence variants were assigned taxonomies using the SILVA reference database. Controlling for multiple covariates, we tested for associations between microbial taxa abundances and HR-pQCT measures using general linear models as implemented in microbiome multivariable association with linear model (MaAslin2). Abundance of 37 microbial genera in FHS, and 4 genera in MrOS, were associated with various skeletal measures (false discovery rate [FDR] ≤ 0.1) including the association of DTU089 with bone measures, which was independently replicated in the two cohorts. A meta-analysis of the taxa-bone associations further revealed (FDR ≤ 0.25) that greater abundances of the genera; Akkermansia and DTU089 , were associated with lower radius total vBMD, and tibia cortical vBMD respectively. Conversely, higher abundances of the genera; Lachnospiraceae NK4A136 group , and Faecalibacterium were associated with greater tibia cortical vBMD. We also investigated functional capabilities of microbial taxa by testing for associations between predicted (based on 16S rRNA amplicon sequence data) metabolic pathways abundance and bone phenotypes in each cohort. While there were no concordant functional associations observed in both cohorts, a meta-analysis revealed 8 pathways including the super-pathway of histidine, purine, and pyrimidine biosynthesis, associated with bone measures of the tibia cortical compartment. In conclusion, our findings suggest that there is a link between the gut microbiome and skeletal metabolism., Competing Interests: ESO: Consultation for Amgen, Bayer, BioCon, Radius; research support from Mereo; travel funding from the OI Foundation. DPK has received grants to his institution from Amgen, Inc, and Radius Health; he has received consulting fees from Solarea Bio, Radius Health, and Pfizer for serving on Scientific Advisory Boards, fees from Agnovos for serving on a Data and Safety Monitoring Committee, and royalties for publication in UpToDate by Wolters Kluwer. SS reports institutional grants from Dairy Management Inc. ended September 2022 and Solarea Bio Inc. ended March 2022, has reviewed grants for the American Egg Board’s Egg Nutrition Center and National Dairy Council. DMK has consulting fees from Amgen and Ultragenyx for serving on Scientific Advisory Boards, royalties for publication in UpToDate by Wolters Kluwer, and grant funding from Starkey. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest, (Copyright © 2023 Okoro, Orwoll, Huttenhower, Morgan, Kuntz, McIver, Dufour, Bouxsein, Langsetmo, Farsijani, Kado, Pacifici, Sahni and Kiel.)

Background & Aims: There is a considerable degree of variation in bone mineral density (BMD) within populations. Use of plasma metabolomics may provide insight into established and novel determinants of BMD variance, such as nutrition and gut microbiome composition, to inform future prevention and treatment strategies for loss of BMD. Using high-resolution metabolomics (HRM), we examined low-molecular weight plasma metabolites and nutrition-related metabolic pathways associated with BMD., Methods: This cross-sectional study included 179 adults (mean age 49.5 ± 10.3 yr, 64% female). Fasting plasma was analyzed using ultra-high-resolution mass spectrometry with liquid chromatography. Whole body and spine BMD were assessed by dual energy X-ray absorptiometry and expressed as BMD (g/cm 2 ) or Z-scores. Multiple linear regression, pathway enrichment, and module analyses were used to determine key plasma metabolic features associated with bone density., Results: Of 10,210 total detected metabolic features, whole body BMD Z-score was associated with 710 metabolites, which were significantly enriched in seven metabolic pathways, including linoleic acid, fatty acid activation and biosynthesis, and glycerophospholipid metabolism. Spine BMD was associated with 970 metabolites, significantly enriched in pro-inflammatory pathways involved in prostaglandin formation and linoleic acid metabolism. In module analyses, tryptophan- and polyamine-derived metabolites formed a network that was significantly associated with spine BMD, supporting a link with the gut microbiome., Conclusions: Plasma HRM provides comprehensive information relevant to nutrition and components of the microbiome that influence bone health. This data supports pro-inflammatory fatty acids and the gut microbiome as novel regulators of postnatal bone remodeling., (Copyright © 2020 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

The sensitivity of bone mineral density (BMD) as a predictor of fracture risk is limited by the fact that this index does not take into account the geometrical and material characteristics of bone. In contrast, both BMD and bone architecture influence the quality factor (QF), the fraction of the inverse of the energy lost in one cycle of deformation. In this study we have compared the sensitivity of a QF analyzer and dual-energy X-ray absorptiometry (DXA) in detecting the changes induced by ovariectomy (OVX) on the QF, impact strength, and BMD of the femur of mature rats. QF and BMD were measured noninvasively before and 4 weeks after OVX or sham operation using a QF analyzer developed in our laboratory and a Hologic QDR 2000 bone densitometry, respectively. Impact strength was measured in excised femurs at the end of the study. The in vivo short-term precision (coefficient of variation) of the QF analyzer was 1.9%. BMD and QF measurements were highly correlated (r = 0.80,p <0.0001). At baseline, QF and BMD were similar in OVX and sham-operated rats. At 4 weeks, BMD was 14.7 + or - 0.9% lower than at baseline (p < 0.001) in OVX rats and 5.3 + or - 1.3% lower in sham-operated rats (p <0.05). QF decreased 36.0 + or - 2.8% (p <0.0001) in OVX and 10.6 + or - 3.6% in sham rats (p <0.01). As a result, at 4 weeks the difference between sham-operated and OVX rats was larger (p < 0.05) by QF than by BMD. Moreover, QF correlated better than BMD with impact strength and the difference in impact strength between sham and OVX mice was closer to that in QF than that in BMD. These data demonstrate that QF analysis is a precise technique that is more sensitive than DXA in detecting the changes in bone density and strength induced by OVX. QF analysis may represent a new, simple, and economic technique for predicting fracture risk.

The recent finding that treatment with the interleukin-1 (IL-1) inhibitor, interleukin-1 receptor antagonist (IL-1ra) decreases bone loss and bone resorption in ovariectomized rats, strongly suggested that IL-1 mediates, at least in part, the effects of estrogen deficiency on bone resorption. Although in vitro studies have shown that IL-1 activates mature osteoclasts and stimulates osteoclastogenesis, the two main mechanisms by which estrogen deficiency stimulates bone resorption, it is still unclear whether IL-1 mediates both effects of estrogen deficiency in vivo. To investigate this matter, we have examined the changes in bone mineral density (BMD) which occur in ovariectomized rats after completion of 1 month of estrogen or IL-1ra treatment begun at the time of ovariectomy. Ovariectomy caused a marked decreased in BMD which was blocked by 17 beta estradiol and decreased by IL-1ra. Cessation of estrogen therapy was followed by a rapid induction of bone loss, indicating that estrogen blocks the activation and utilization of mature osteoclasts without depleting the bone microenvironment of osteoclast precursors and mature, inactive osteoclasts. In contrast, ovariectomized rats treated with IL-1ra maintained a stable bone density for the first 4 weeks after completion of the treatment. In these rats, bone loss resumed not earlier than 6 weeks after discontinuation of the IL-1ra treatment. Estrogen deficiency was necessary to unveil the bone-sparing effect of IL-1ra because in a control experiment in which rats were treated with IL-1ra for the 4 weeks before ovariectomy, BMD began to decrease immediately after ovariectomy.(ABSTRACT TRUNCATED AT 250 WORDS)

Pregnancy-associated osteoporosis is a rare and poorly characterized clinical entity. We describe two women, aged 28 and 31 yr, who developed vertebral osteopaenia with non-traumatic compression fractures within a few months after delivery. In both women secondary causes of osteoporosis were excluded and routine laboratory data including indices of calcium metabolism were normal. In one patient (Patient 1) spontaneous secretion of IL-1 by cultured blood monocytes was elevated 6 months after delivery and a bone biopsy revealed histomorphometric indices consistent with accelerated bone remodelling. BMD was measured by quantitative computed tomography (QCT) and/or dual energy X-ray absorptiometry (DXA). In Patient 1, BMD, measured with QCT, 6 months after delivery was 3.32 Z-score units below the predicted value for age and sex-matched subjects. At that time treatment (phosphate followed by sodium etidronate) was instituted, and QCT measurements obtained 24 and 30 months after delivery revealed a BMD of -2.98 and -2.67 Z-score units, respectively. The treatment was discontinued just before she became pregnant again, and 3 months after the second delivery QCT showed a BMD of -3.06 Z-score units. Further BMD assessments were obtained with DXA at 15 and 18 months after the second delivery, and the BMD values were -1.41 and -1.08, respectively. In Patient 2, spine BMD, measured with QCT, 4 and 10 months after delivery were -1.68 and -1.59 Z-score units, respectively. At 10, 16 and 22 months after delivery, BMD was assessed with DXA. The initial measurement with this technique disclosed a BMD of 0.52 Z-score units.(ABSTRACT TRUNCATED AT 250 WORDS)

Dual-energy x-ray absorptiometry (DEXA) is now an established method of measuring bone mineral density (BMD) in humans. We have applied the technique to measuring BMD in the rat. The short-term precision of the technique was assessed by calculating the BMD coefficient of variation at the rat spine (1.20%), total femur (0.52%), proximal femur (1.16%), midfemur (1.00%), distal femur (0.96%), and proximal tibia (2.2%). The long-term precision of femoral measurements in rat cadavers over a 4 week period was 0.72% for the total femur and 1.2% for the distal femur. The accuracy of the technique was assessed by comparing DEXA-measured bone mineral content (BMC) in vitro and in vivo with ashed BMC. Results indicated a highly significant positive correlation between in vitro DEXA measurements and ash BMC (r = 0.99) and between in vivo DEXA measurements and ash BMC (r = 0.89). The ability of the technique to detect bone loss was assessed at the femoral site by comparing the BMD of ovariectomized (OVX) and sham-operated rats at baseline and at a 1 month follow-up. There was no significant difference in BMD between the groups at baseline. However, at 1 month follow-up the OVX group showed a significant (p < 0.001) decline in BMD at the distal femur (-8.6%) and in the total femur (-4.8%) compared with sham-operated rats. A comparison of retired breeder female rats with age-matched nulliparous rats indicated that the BMD of retired breeder rats was significantly lower than that of virgin females at all femoral sites (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogen and calcitonin increase bone density of osteoporotic women, particularly on the axial skeleton. To verify whether the effect of these drugs might in part be biased by spurious increases in bone density due to radiologically irrelevant microfractures, and consequent subtle decreases in vertebral height, we followed the changes in vertebral bone density (VBD), assessed by quantitative computed tomography, in relation to vertebral height (VH) in 60 osteoporotic women. VH was measured as the sum of anterior, central and posterior heights of L1-L3 vertebral bodies on lateral radiographs. Patients received either salmon calcitonin (sCT: 50 IU subcutaneously three times per week, n = 18), hormonal replacement therapy (HRT: conjugated estrogen 0.625 mg/day, days 1-25, plus medroxyprogesterone acetate 10 mg/day, days 16-25 of each month; n = 21) or calcium alone (Ca: 1000 mg/day, n = 21). After 1 year, VBD increased in the HRT group (+5.0 +/- 1.9%, p = 0.010), did not change significantly in the sCT group (+3.3 +/- 2.3%, p = 0.167), and decreased by 6.1 +/- 1.0% (p less than 0.001) in the Ca group. By analysis of variance, the changes induced by HRT and sCT were significantly different from those observed in the Ca group (F = 7.982, p less than 0.001). VH decreased slightly in all three subsets of patients (-0.9 +/- 0.5% in sCT, -1.5 +/- 0.3% in HRT, -0.1 +/- 0.5% in Ca), but these changes were not significantly different between groups (F = 2.545, p = 0.081).(ABSTRACT TRUNCATED AT 250 WORDS)

A lateral projection of the lumbar spine with dual-energy radiography (DER) may be a more sensitive tool for the detection of vertebral demineralization than the standard anterior-posterior (AP) DER measurement. The lateral view allows measurement of the vertebral body and selective measurement of the central trabecular compartment, excluding the majority of the cortical envelope and posterior vertebral elements. The lateral DER typically contains L2-4 in the region of interest. The present study was designed to investigate the frequency and bone mineral content (BMC) contribution of rib to the L2 vertebral body BMC measurement in a lateral view DER scan. In addition, the frequency of pelvic overlap of L4 was evaluated. All patients were scanned in the supine position. Abdominal CT images of L2 were examined in 20 women aged 28-82 years to determine the frequency of rib and pelvic interference. QCT images of L2 also were examined in an additional 35 women aged 20-82 years to further investigate the frequency of rib overlap and to calculate the potential contribution of rib superimposition to the L2 BMC in a lateral projection. Pelvic overlap of L4 occurred in 15% of the 20 cases reviewed. Rib overlapped the L2 vertebral body in 100% of the L2 images. The expected contribution of rib BMC to L2 BMC in a lateral DER scan was 10.4% on average. There was no relationship between the percentage of rib BMC contribution and age and relatively poor correlations between rib BMC and that of either the vertebral body or the central trabecular compartment of L2. We conclude that supine positioning allows routine inclusion of L3 and L4 in a lateral lumbar scan. Results also suggest that L2 should be excluded from DER scans on patients scanned in either the lateral decubitus or supine position.

Previous studies comparing axial and appendicular skeleton have shown that trabecular bone loss is greater than cortical bone loss. However, whether the same difference exists between the trabecular and the cortical compartments of the vertebral body remains to be determined. In this study, we used quantitative computer tomography (QCT) to simultaneously measure the cortical rim of the vertebral body as well as trabecular bone. In 99 Caucasian women (mean age 53.8 +/- 13.0 years, range 26-79 years) we found a significant correlation between cortical mineral content (BMCC) and both single (SE) and dual energy (DE) trabecular mineral content (BMCT) (r = 0.62, P less than 0.0001 for both regressions). The cross-sectional rates of bone loss per year were 1.32%, 1.16%, and 0.59% for SE-BMCT, DE-BMCT, and BMCC, respectively. BMCC decreased at a rate that was 45-51% that of SE-BMCT and DE-BMCT, respectively. Our results indicate that (1) QCT may provide a useful means to selectively measure cortical density in vertebral bodies; (2) the decrease of cortical density over time in the spine appears to have been underestimated previously by extrapolation from appendicular bone measurements; (3) because measurements of the entire vertebral body (exclusive of the posterior elements) may provide information that is more representative of spine changes with age, a measurement that includes both areas might be more useful than one measuring only the trabecular region.

Idiopathic hypercalciuria (IH) is a heterogeneous disorder frequently observed in patients with nephrolithiasis. At one extreme of its clinical spectrum is fasting hypercalciuria (FH), a condition characterized by increased bone resorption and turnover. In previous studies we have shown that monocytes from patients with high turnover osteoporosis and from women in early postmenopause elaborate increased amounts of interleukin-1 (IL-1), a cytokine that stimulates bone resorption in vitro and in vivo. Since IL-1 could also mediate the resorptive mechanism of FH and cause a clinically significant bone loss, we have studied the relationship of IH, vertebral mineral density, bone turnover, and monocyte IL-1 activity in 47 patients with absorptive hypercalciuria (AH), 23 with FH, and 38 nonhypercalciuric subjects with recurrent nephrolithiasis (controls). Vertebral mineral density, as measured by quantitative computer tomography, was decreased in each of the three patient groups, but was significantly lower in FH patients than in AH patients or control subjects. Twenty-four-hour total urinary hydroxyproline excretion was increased in FH patients compared to that in AH patients or controls, but blood levels of osteocalcin were not. Monocytes from FH subjects yielded significantly more IL-1 (alpha + beta) activity than those from AH patients or controls; levels of IL-1 activity in monocytes of AH and control patients were similar. In IH subjects, significant correlations were found between IL-1 and hydroxyproline (r = 0.70; P less than 0.0001), IL-1 and quantitative computer tomography values (r = -0.49; P less than 0.005), and IL-1 and urinary calcium (r = -0.36; P less than 0.05). Serum PTH levels were within normal limits in all subjects and were similar in the three study groups, 1,25-Dihydroxyvitamin D3 levels, although higher in IH patients than in controls, were not significantly different in FH and AH subjects. Increased IL-1 activity and decreased vertebral mineral density are features of a subset of patients with IH. Although a cause-effect relationship remains to be established, increased monocytic IL-1 activity, rather than elevated PTH or 1,25-dihydroxyvitamin D3 levels, could underlie the resorptive component of FH.

Dual energy radiography (DER) is a new technique for non-invasive measurement of vertebral bone mineral density, which because of its high precision (1%), is particularly suitable for prospective analyses of time-related changes in bone mass. However, we recently found that DER is less sensitive to demineralization of trabecular bone than quantitative computed tomography (QCT). Since the compact bone of the posterior vertebral elements are included in the standard anterior-posterior DER (APDER), but not in QCT measurements, changes in trabecular bone mass attending the early phases of the osteoporotic process go undetected with the APDER technique due to the preponderance of cortical mass. Lateral-view DER (LDER), however, would minimize the contribution of vertebral compact bone as well as intra-abdominal calcifications to the trabecular component. To determine the in vivo short-term precision of the LDER method, we obtained three measurements in 18 healthy women, aged 23 to 76, during a four-week period. In addition, both APDER and LDER measurements were obtained in 53 healthy women, aged 23 to 80. The coefficient of variation for LDER was 2.8%. LDER density values accounted for a higher percentage of the variance of bone mass measurements with age (47%) than APDER (23%). Moreover, LDER declined significantly with age in both premenopausal (r = -0.42, p less than 0.05) and post-menopausal women (r = -0.50, p less than 0.005), whereas the age-related changes in APDER values failed to reach statistical significance in either group. The rate of bone loss with age in the premenopausal group was 0.45% per year with LDER, and 0.10% per year with APDER. Postmenopausal women showed a 0.78% loss of bone mass per year with LDER and 0.47% per year with APDER. The correlation between APDER and LDER was significant (r = 0.78, p less than 0.01). The accumulated data indicate that LDER may be a more appropriate technique than APDER for detecting the initial loss of vertebral trabecular bone that characterizes early subclinical osteoporotic syndrome.

Interleukin-1 (IL-1), a cytokine best known for its ability to stimulate lymphocyte proliferation, has recently been shown to stimulate bone resorption and modulate bone formation in vivo. Consequently, the authors have devised a series of studies to investigate the relationship between bone remodeling, menopause, and monocyte IL-1-secretion. In a first study, monocytes from osteoporotic patients were found to produce more IL-1 than monocytes from control subjects. IL-1 activity was also found to reflect histomorphometric indices of bone formation, but not of bone resorption. In a second study, devised to assess the effect of menopause on the relationship between IL-1 and bone turnover, a significant correlation was found between IL-1 and BGP in premenopausal osteoporotic women and osteoporotic men, but not in both postmenopausal osteoporotic subjects and normal subjects of either sex. In a third study, IL-1 from untreated postmenopausal women was found to be higher than in either untreated premenopausal or estrogen/progesterone-treated postmenopausal women. A significant negative correlation was found between IL-1 and years since menopause in both the healthy and osteoporotic postmenopausal women. Premenopausal IL-1 levels were achieved within eight years of menopause in the healthy but not in the osteoporotic subjects. In osteoporotic women, high IL-1 levels were evident as long as 15 years after menopause. IL-1 also correlated inversely with mineral density as measured by quantitative computer tomography. In prospective study, treatment with estrogen/progesterone caused a significant increase in IL-1 activity. This data indicates that monocyte IL-1 production mirrors the rate of bone turnover in both the healthy and osteoporotic patient, and that alteration in IL-1 production may underlie the postmenopausal acceleration of bone loss and its inhibition by ovarian steroids.

Distal radius photodensitometric and second metacarpal radiogrammetric measurements were obtained from computerized analyses of standard hand X-Ray films of 296 Caucasian subjects (189 women and 107 men). This sample included 134 subjects > or = 65 years old (75 women and 59 men). Distal radius bone density and metacarpal index showed a significant linear decrease with age in both sexes. Rates of bone loss, calculated from the regression curves, were -0.7% per year in women and -0.5% per year in men by distal radial photodensitometry, and -0.49% per year in women and -0.33% per year in men by metacarpal radiogrammetry. In the elderly subgroup, women > or = 65 years of age showed an even faster bone loss, with an annual decrease of -1.4% by distal radial photodensitometry. Conversely, men > or = 65 years of age had no significant bone loss, not even by metacarpal radiogrammetry. In conclusion, these data suggest that appendicular cortical bone loss occurs at a higher rate in elderly females than in the elderly males, both at the distal radial and at the metacarpal site. [ABSTRACT FROM AUTHOR]