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This is the first study to examine clinical human bone specimens by three-dimensional imaging to characterize osteocyte lacunar properties as a function of macroanatomic bone type and estrogen loss. We applied laboratory-based instrumentation [3D X-ray microscope (3DXRM), MicroXCT-200; Carl Zeiss/Xradia, Inc.] that reaches the same resolution as synchrotron microscopy. We used serial transiliac bone biopsy specimens to examine the effect of macroanatomic bone type and estrogen status on osteocyte lacunar properties. These properties include lacunar size (volume, axes lengths of the ellipsoidal lacunar voids), distribution (density, average near-neighbor lacunar distance), and shape factors (sphericity ratio, average eigenvalues, degree of equancy, elongation, and flatness) in both cortical and trabecular bone tissue. The lacunar properties (volume, surface area, density, near-neighbor distance, etc.) and the shape factors (E1, L1, L2, degree of equancy, degree of elongation) were different between cortical and trabecular bone regardless of estrogen status. In cortical bone and trabecular nodes, the lacunar void volume and surface area were either smaller or tended to be smaller in postmenopausal as compared to premenopausal women. The void volume-to-bone volume ratio of cortical bone showed declining trends with estrogen loss. While there were differences between trabecular and cortical bone tissue, the lacunar void sphericity ratio for trabecular struts shows decreasing trends in postmenopausal women. These data suggest that using 3DXRM can provide new insight into osteocyte lacunar properties in transiliac bone biopsies from patients with various skeletal disease/conditions and pharmaceutical treatments.

Bone mineralization density distribution (BMDD) is an important determinant of bone mechanical properties. The most available skeletal site for access to the BMDD is the iliac crest. Compared to cancellous bone much less information on BMDD is available for cortical bone. Hence, we analyzed complete transiliac crest bone biopsy samples from premenopausal women (n = 73) aged 25-48 years, clinically classified as healthy, by quantitative backscattered electron imaging for cortical (Ct.) and cancellous (Cn.) BMDD. The Ct.BMDD was characterized by the arithmetic mean of the BMDD of the cortical plates. We found correlations between Ct. and Cn. BMDD variables with correlation coefficients r between 0.42 and 0.73 (all p < 0.001). Additionally to this synchronous behavior of cortical and cancellous compartments, we found that the heterogeneity of mineralization densities (Ct.Ca(Width)), as well as the cortical porosity (Ct.Po) was larger for a lower average degree of mineralization (Ct.Ca(Mean)). Moreover, Ct.Po correlated negatively with the percentage of highly mineralized bone areas (Ct.Ca(High)) and positively with the percentage of lowly mineralized bone areas (Ct.Ca(Low)). In conclusion, the correlation of cortical with cancellous BMDD in the iliac crest of the study cohort suggests coordinated regulation of bone turnover between both bone compartments. Only in a few cases, there was a difference in the degree of mineralization of >1wt % between both cortices suggesting a possible modeling situation. This normative dataset of healthy premenopausal women will provide a reference standard by which disease- and treatment-specific effects can be assessed at the level of cortical bone BMDD.

This 2-year trial evaluated the efficacy and tolerability of a monthly oral regimen of risedronate. Postmenopausal women with osteoporosis were randomly assigned to double-blind treatment with risedronate 75 mg on 2 consecutive days each month (2CDM) or 5 mg daily. The primary end point was the percentage change from baseline in lumbar spine bone mineral density (BMD) at 12 months. Secondary end points included the change in BMD of the lumbar spine and proximal femur and in bone turnover markers as well as the number of subjects with at least one new vertebral fracture over 24 months. Among 1,229 patients who were randomized and received at least one dose of risedronate, lumbar spine BMD was increased in both treatment groups: mean percentage change from baseline was 4.2 ± 0.19 and 4.3 ± 0.19 % in the 75 mg 2CDM and 5 mg daily groups, respectively, at month 24. The treatment difference was 0.17 (95 % confidence interval -0.35 to 0.68). There were no statistically significant differences between treatment groups on any secondary efficacy parameters. Both treatment regimens were well tolerated. Risedronate 75 mg 2CDM was noninferior in BMD efficacy and did not show a difference in tolerability compared to 5 mg daily after 24 months of treatment in women with postmenopausal osteoporosis. This monthly regimen may provide a more convenient dosing schedule to some patients with postmenopausal osteoporosis.

Vitamin D deficiency is a common public health problem in the US. It is related to the high risk of rickets, osteoporosis and other diseases. Currently, serum 25-hydroxy vitamin D [25(OH)D] concentration is the best indicator of vitamin D status, and determination of its deficiency or sufficiency. This level has high heritability (28-80%). However, genes contributing to the wide variation in serum 25(OH)D are generally unknown. In this study, we screened nine important genes in vitamin D metabolic pathways using 49 single nucleotide polymorphism (SNP) markers in a group of 156 unrelated healthy Caucasian subjects. Significant confounding factors that may affect serum 25(OH)D variations were used as covariates for the association analyses. An association test for quantitative trait was performed to evaluate the association between candidate genes and serum 25(OH)D levels. Permutation was conducted for correcting multiple testing problems. Evidence of association was observed at SNPs in the CYP2R1 (cytochrome P450, family 2, subfamily R, polypeptide 1) and the GC (vitamin D binding protein) gene. Next, we performed a replication study for six promising SNPs in the gene CYP2R1 and GC, using another group of 340 unrelated healthy Caucasian subjects. Association analyses were conducted in the replication cohort (n = 340) and the pooled cohort (n = 496). The CYP2R1 gene and the GC gene remain significant in the pooled cohort. The results suggest that the CYP2R1 and GC genes may contribute to the variation of serum 25(OH)D levels in healthy populations.

Treatment of monkeys and humans with parathyroid hormone (PTH) 1-84 stimulates skeletal remodeling, which increases trabecular (Tb) bone mineral density (BMD) but decreases cortical (Ct) BMD at locations where these bone types predominate. We report the effects of daily PTH treatment (5, 10, or 25 microg/kg) of ovariectomized (OVX) rhesus monkeys for 16 months on bone structure and biomechanical properties at the proximal femur, a mixed trabecular and cortical bone site. PTH reversed the OVX-induced decrease in BMD measured by dual-energy X-ray absorptiometry at the proximal femur, femoral neck, and distal femur. Peripheral quantitative computed tomography confirmed a significant decrease in Ct.BMD and an increase in Tb.BMD at the total proximal femur and at the proximal and distal femoral metaphyses. The decrease in Ct.BMD resulted primarily from increased area because cortical bone mineral content was unaffected by PTH. Histomorphometry revealed that PTH significantly increased the trabecular bone formation rate (BFR) as well as trabecular bone volume and number. PTH did not affect periosteal or haversian BFR at the femoral neck, but cortical porosity was increased slightly. PTH had no effects on stiffness or peak load measured using a shear test, whereas work-to-failure, the energy required to fracture, was increased significantly. Thus, PTH treatment induced changes in trabecular and cortical bone at the proximal femur that were similar to those occurring at sites where these bone types predominate. Together, the changes had no effect on stiffness or peak load but increased the energy required to break the proximal femur, thereby making it more resistant to fracture.

Most studies that have investigated the anabolic effects of parathyroid hormone (1-84) (PTH) or PTH fragments on the skeleton of ovariectomized (OVX) rats have evaluated the short-term effects of high-dose PTH(1-34) in young animals. This study used densitometry, histomorphometry, and biomechanical testing to evaluate the effects of 12-month daily treatment with low-dose PTH (15 or 30 microg/kg) in rats that were 10 months old at baseline, 4 months after OVX. Bone mineral density (BMD) and bone strength were reduced substantially in control OVX rats. The 15 microg/kg dose of PTH restored BMD to levels similar to those in sham animals within 6 months at the lumbar spine, distal and central femur, and whole body and maintained the BMD gain from 6 to 12 months. The 30 microg/kg dose produced greater effects. Both PTH doses normalized the trabecular bone volume-to-total volume ratio (BV/TV) at lumbar vertebra 3 but not at the proximal tibia (where baseline BV/TV was very low), solely by increasing trabecular thickness. PTH dose-dependently increased bone formation by increasing the mineralizing surface, but only the 30 microg/kg dose increased resorption. PTH increased cortical BMD, area, and thickness, primarily by increasing endocortical bone formation, and restored all measures of bone strength to levels similar to those in sham animals at all skeletal sites. PTH increased bone mass safely; there was no osteoid accumulation, mineralization defect, or marrow fibrosis and there were no abnormal cells. Thus, long-term PTH therapy normalized bone strength in the aged OVX rat, a model of postmenopausal osteoporosis, through increased bone turnover and enhanced formation of both trabecular and cortical bone.

Bone mineral density (BMD) is a complex trait having genetic and environmental determination. There are gender-specific differences in BMD measurements, and the rate of BMD changes with age and lifestyle. Previous studies have shown that the genetic loci underlying BMD variation are gender-specific in mice and humans. Our study aimed to investigate correlations between BMD at the spine, hip, and ultradistal radius (UD) and degree of shared genetic and environmental factors among them in females and males, separately. For a large sample of 4,489 subjects containing 2,667 females and 1,822 males from 512 Caucasian pedigrees, we performed bivariate variance decomposition analyses. Our results showed that the genetic correlations (rhoG), environmental correlations (rhoE), and phenotypical correlations (rhoP) were all significant and positive. Strong genetic correlations were observed in both female and male groups, ranging 0.590-0.738 and 0.583-0.773, respectively. Genetic correlations of BMD at the spine, hip, and UD were generally higher than environmental correlations. In summary, we are the first to test the genetic and environmental correlations in females and males, separately. It is suggested that the phenotypic correlations of BMDs at the three different sites may have more genetic than environmental components. BMDs at the spine and hip may share more environmental components in females than males. We did not detect gender-specific difference in spine/UD and hip/UD. It is also indicated that the environmental factors that preserve or increase BMD at one skeletal site may have similar beneficial effects on some other skeletal sites and vice versa.

Evidence from several studies suggests that soy protein and/or its isoflavones may have beneficial effects on bone in postmenopausal women and animal models who have osteoporosis. The present study examined the dose-dependent effects of soy isoflavones in the context of soy protein or casein on the male skeleton. Thirteen-month-old male Fisher 344 rats were orchidectomized (ORX; 5 groups) or sham-operated (Sham; 1 group) and immediately placed on dietary treatments for 180 days. Diets were semi-purified and the protein source was either casein (Sham and ORX; controls), casein with two added doses of isoflavones (Iso1; 600 mg/kg diet and Iso2; 1200 mg/kg diet), soy protein with normal isoflavones content (Soy; 600 mg/kg diet), or soy protein with added isoflavones (Soy+; 1200 mg/kg diet). A 7% loss of whole body bone mineral density (BMD) was observed due to orchidectomy; however, the ORX induced BMD loss was significantly reduced to 4.3 and 4.7 % with the Soy and Soy+, respectively. Both doses of isoflavones in conjunction with casein also reduced the loss of whole body BMD, albeit not significantly different from ORX control animals. Trabecular bone histomorphometric analysis of the proximal tibia further supported the bone-sparing role of soy isoflavones as indicated by higher percent bone volume and trabecular number, and lower trabecular separation. We conclude that isoflavones exert modest beneficial effects on the male skeleton whether provided with casein or a soy protein.

Low bone mineral density (BMD) is a major risk factor for osteoporotic fracture. To identify genomic regions harboring quantitative trait loci (QTLs) contributing to BMD variation, we performed a two-stage genome screen. The first stage involved genotyping of a sample of 53 pedigrees with 630 individuals using 400 microsatellite markers spaced at approximately 10-cM intervals throughout the genome. Ten genomic regions with multi- and/or two-point LOD scores greater than 1.5 were observed. In the present second-stage study, 60 microsatellite markers, with a mean spacing of about 5 cM, were genotyped in these regions in an expanded sample of 79 pedigrees that contained 1816 subjects. Each pedigree was ascertained through a proband with extreme BMD at the hip or spine. BMD at the spine (L1-4), hip (the femoral neck, trochanter, and intertrochanteric region), and wrist (the ultradistal region) was measured by dual-energy X-ray absorptiometry (DXA) and was adjusted for age, sex, height, and weight. Two-point and multipoint linkage analyses were performed for each BMD site using statistical genetic methods that are implemented in the computer package SOLAR. Several regions (7q11, 10q26, 12q13, and 12q24) achieved LOD scores in excess of 1 in the second-stage followup study. The current results replicate some of our previous linkage findings and also highlight some of the difficulties facing microsatellite linkage mapping for complex human diseases.

The purpose of this study was to assess breed-related differences in bone histomorphometry, bone biomechanics, and serum biochemistry in three mouse breeds shown to differ in bone mineral density (BMD) (as measured by DXA) and bone mineral content (BMC). Femurs, tibiae, and sera were collected from 16-week-old C3H/HeJ C3H, C57BL/6J BL6, and DBA/2J DBAmice (n = 12/breed). Data collected included BMC and BMD (femora), histomorphometry of cancellous (distal femur) and cortical bone (diaphyseal tibiae and femora), bone strength (femora), and serum alkaline phosphatase (ALP). Consistent with previous reports, BMC and BMD were higher in C3H than in BL6 or DBA mice. The higher BMD in the C3H breed was associated with greater cancellous bone volume, cortical bone area, periosteal bone formation rate, biomechanical strength, and serum ALP. However, mid-diaphyseal total femoral and tibial cross-sectional area and moment of inertia were greatest in BL6, intermediate in C3H, and lowest in DBA mice. The specific distribution of cortical bone in C3H, BL6, DBA mice represents a difference in adaptive response to similar mechanical loads in these breeds. This difference in adaptive response may be intrinsic to the adaptive mechanism, or may be intrinsic to the bone tissue material properties. In either case, the bone-adaptive response to ordinary mechanical loads in the BL6 mice yields bones of lower mechanical efficiency (less stiffness per unit mass of bone tissue) and does not adapt as well as that of the C3H mice where the final product is a bone with greater resistance to bending under load. We suggest that the size, shape, and BMD of the bone are a result of breed-specific genetically regulated cellular mechanisms. Compared with the C3H mice, the lower BMD in BL6 mice is associated with long bones that are weaker because the larger cross-sectional area fails to compensate completely for their lower BMD and BMC.

Bone, being sensitive to mechanical stimulus, adapts to mechanical loads in response to bending or deformation. Although the signal/receptor mechanism for bone adaptation to deformation is still under investigation, the mechanical signal is related to the amount of bone deformation or strain. Adaptation to changes in physical activity depends on both the magnitude of increase in strain above average daily levels for maintaining current bone density and the Minimum Effective Strain (MES) for initiating adaptive bone formation. Given the variation of peak bone density that exists in any human population, it is likely that variation in levels for MES is, to a considerable degree, inherited and varies among animal species and breeds. This study showed a dose-related periosteal response to loading in C3H/HeJ mice. The extent of active formation surface, the rate of periosteal bone formation, and area of bone formation increased with increasing peak periosteal strain. In these mice, the loaded tibia consistently showed lower endocortical formation surface and mineral apposition rate than the nonloaded bones at every load level. Although periosteal expansion is the most efficient means of increasing moment of inertia in adaptation to bending, a dose response increase in endocortical formation would have been predicted. Our characterization of the mouse bone formation response to increasing bending loads will be useful in the design of experiments to study the tibial adaptive response to known loads in different mouse breeds.

Our purpose is to assess whether genotypes of the vitamin D receptor (VDR) and estrogen receptor (ER) and their interaction influence changes in bone mass in postmenopausal Caucasian women with and without hormone replacement therapy (HRT). A population of 108 US Mid-West women who participated in a study of low-dose continuous estrogen/progestin was genotyped at the VDR BsmI site and the ER XbaI and PvuII sites. Adequate vitamin D and calcium nutritional intakes were assured in all the study subjects. For the 3.5-year duration of the study, we analyzed changes in bone mineral density (BMD) at the spine, femoral neck, distal radius, and the total body (total body bone mineral content, tbBMC). We adjusted for confounding factors, such as age and weight, in the analysis. We found that VDR and/or ER genotypes and/or their interaction generally had significant effects on the changes in the bone mass measurements in both the placebo and HRT groups. When a significant gene-by-gene interaction exists between VDR and ER genotypes, failure to account for them in analyses may yield nonsignificant results, even if significant genotypic effects exist. The amount of variation in changes in bone mass measurements explained by the total genotypic effects of the VDR and ER loci varies from approximately 1.0% (for the tbBMC changes in combined placebo and HRT groups) to approximately 18.7% (for the spine BMD changes in the HRT group). These results suggest that individual genotypes are important factors in determining changes in bone mass in the elderly with and without HRT and thus may need to be considered with respect to the treatment to preserve bone mass in elderly Caucasian women.

We investigated the bone response to external loading in C57BL/6J and C3H/HeJ mice, both breeds with low and high bone density, respectively. An in vivo tibial four-point bending device previously used for application of measured external loads in rats was adapted for mice. It delivered a uniform medio-lateral bending moment to the region of the tibia located 1-5.5 mm proximal to the tibio-fibula junction. The right legs of six C57BL/6J [low bone density (LBD)] and C3H/HeJ [high bone density (HBD)] mice were externally loaded in the device for 36 cycles/day at 2 Hz, 6 days/week for 2 weeks at 9.3 +/- 0.9 N force, inducing estimated lateral periosteal surface compressive strains of 5121 +/- 1128 mu epsilon in C3H/HeJ (HBD) mice (n = 6), significantly higher than the estimated 3988 +/- 820 mu epsilon in C57BL/6J mice (n = 6) (mean +/- SD). In addition, C3H/HeJ HBD mice (n = 11) were externally sham (pad pressure or no bending) loaded in the device for 36 cycles/day at 2 Hz, 3 days/week for 3 weeks at 9.3 +/- 0.9 N force. Calcein injections for bone labeling were given at the 10th and 3rd days before sacrifice. At the end of the experiment, all mice were killed and both tibiae were removed, fixed, embedded, and cross-sectioned through the loaded region. Both tibiae were measured for marrow area (Ma.Ar), cortical area (Ct.Ar), total area (Tt.Ar), cross-sectional moment of inertia (CSMI), and periosteal and endocortical woven bone surface (Wo.B/BS), single-labeled surface (sLS), double-labeled surface (dLS), and total formation surface (FS/BS). Differences in all variables due to breed and loading (both bending and sham-bending) were tested by two-way analysis of variance (ANOVA) (P < 0.05). Ma.Ar, Tt.Ar, and CSMI were greater in C57BL/6J (LBD) than in C3H/HeJ (HBD) mice. Periosteal and endocortical woven bone and formation surface were increased significantly more by loading (bending) in C57BL/6J than in C3H/HeJ mice. Periosteal woven bone response due to sham-bending or sham-loading was significantly lower than due to bending loads in the C3H/HeJ mice. We conclude that the bone response to external loading is greater in LBD mice than in HBD mice. The high bone density of C3H/HeJ (HBD) mice is related to breed-specific factors other than the response to loading.

The Saunders Bone Quality Study was designed to determine the feasibility of ultrasonic bone measurement, at the patella, as a predictor of low-trauma fractures in a rural population-based study. At the first visit of this 4-year longitudinal study, anthropometric and clinical measurements and medical, surgical and fracture histories were obtained for the 1428 participants 9899 women and 529 men). Explored risk factors for low-trauma fractures included age, sex, calcium intake, alcohol and caffeine ingestion, tobacco use, body mass and grip strength, age of menopause, estrogen replacement therapy, propensity to fall, distal radius and ulna bone mineral content, and bone density. Forward multivariate logistic regression analysis showed that lower ultrasound values are more consistently associated with reported low-trauma appendicular fractures than the commonly reported forearm absorptiometry measures of radius mineral content and density. When ultrasound, age, and the extra skeletal risk factors were included in an additional multivariate model, only age and ultrasound were significantly associated with appendicular fracture history in women (P = 0.0003), whereas only ultrasound was associated in the men (P = 0.001). We conclude that ultrasound is a better measure as association with reported low-trauma fractures than the commonly reported forearm SPA measures. Even after adjustment for many of the extra skeletal risk factors, low AVU is highly associated with low-trauma fracture status for both men and women.

Although mechanical forces regulate bone mass and morphology, little is known about the signals involved in that regulation. External force application increases periosteal bone formation by increasing surface activation and formation rate. In this study, the early tibial periosteal response to external loads was compared between loaded and nonloaded contralateral tibia by examining the results of blot hybridization analyses of total RNA. To study the impact of external load on gene expression, RNA blots were sequentially hybridized to cDNAs encoding the protooncogene c-fos, cytoskeletal protein beta-actin, bone matrix proteins alkaline phosphatase (ALP), osteopontin (Op), and osteocalcin (Oc), and growth factors insulin-like growth factor I (IGF-I) and transforming growth factor-beta (TGF-beta). The rapid yet transient increase in levels of c-fos mRNA seen within 2 hours after load application indirectly suggests that the initial periosteal response to mechanical loading is cell proliferation. This is also supported by the concomitant decline in levels of mRNAs encoding bone matrix proteins ALP, Op, and Oc, which are typically produced by mature osteoblasts. Another early periosteal response to mechanical load appeared to be the rapid induction of growth factor synthesis as TGF-beta and IGF-I mRNA levels were increased in the loaded limb with peak levels being observed 4 hours after loading. These data indicate that the acute periosteal response to external mechanical loading was a change in the pattern of gene expression which may signal cell proliferation. The altered pattern of gene expression observed in the present study supports previous evidence of increased periosteal cell proliferation seen both in vivo and in vitro following mechanical loading.

Bone strength is determined by mass density, structural arrangement, and the presence of microdamage. This presentation focuses on trabecular microstructure as an element of bone strength, particularly in the syndrome of vertebral compression fracture. The base material comes from transilial biopsies from 90 patients with the crush fracture syndrome and from 34 normal volunteers who underwent transilial biopsy. Trabecular connectivity was compared in these two groups. Patients and normals were then matched for trabecular bone volume (BV/TV). To highlight measures of trabecular connectivity, three general methods for examining trabecular connectivity were applied to the 23 patient-control pairs matched for BV/TV. The conventional method of Parfitt showed that trabecular number was decreased by 11%, trabecular separation was increased by 9%, and trabecular thickness was increased by 9% in patients compared with controls. The method of counting trabecular nodes and trabecular free ends showed that in the patients, free ends were decreased by 37% and nodes were decreased by 37%. The marrow space star volume (the average volume of marrow measured from radii placed at random points within the marrow and extending to trabecular surface) was 36% greater in the patients. All three methods are measures of trabecular connectivity (the density of connections between trabeculae). All three demonstrated less trabecular connectivity in the patients versus controls for identical BV/TV. Patients with vertebral fractures had fewer trabecular elements rather than global thinning of trabeculae. Differences in measures or connectivity were not accompanied by significant differences in any of the measures of bone remodeling dynamics, as examined by tetracycline labels.(ABSTRACT TRUNCATED AT 250 WORDS)

Fractional absorption of seven chemically defined calcium sources was measured in normal adult women under standardized load conditions. Solubility of the sources in water at neutral pH ranged from a low of 0.04 mM to a high of 1500 mM. The relationship of solubility to absorbability was weak. In the range from 0.1 to 10 mM, within which most calcium supplement sources fall, there was no detectable effect of solubility on absorption. Data from four food sources are presented for comparison. Absorbability of food calcium was not clearly related to absorbability of the dominant chemical form in the food concerned. These findings suggests that (1) even under controlled, chemically defined conditions, solubility of a source has very little influence on its absorbability; and (2) absorbability of calcium from food sources is determined mainly by other food components.

We examined the time course of calcium absorption (CaAbs) in 155 studies, using a double isotope technique. The subjects were 118 healthy peri-menopausal women (mean age 53.3 years), studied as inpatients under metabolic balance conditions. We measured the ratio of radiolabeled calcium (oral:IV) in serum and urine for 144 hours after the oral dose, and generated a composite CaAbs curve for all 155 studies using normalized data. Although CaAbs was 80.9% complete at 3 hours, it was still only 95.8% complete at 7 hours; the remaining 4.2% was absorbed in a slower late component, and did not reach completion until about 26 hours. The rapid initial component probably represents mainly small intestinal absorption and the late component, colonic. At the dietary intakes of our subjects, we estimate the size of the late component at about 6.8 mg/day. For fully accurate measurements of CaAbs, it is necessary to allow for this small late component.

Eighteen postmenopausal osteoporotic (PMO) women on placebo therapy were followed for 1 year. Serial measurements of calcium balance and kinetics, bone histomorphometry, photon absorptiometry, and radiogrammetry were obtained. Few significant changes in balance and kinetics or bone histology were seen in these women over time. Photon absorptiometry and radiogrammetry of the femoral cortex showed a significant annual bone loss however. When compared with premenopausal and healthy postmenopausal women, the annual bone loss in those with PMO was significantly greater. The ability of balance studies, photon absorptiometry, and radiogrammetry to predict the magnitude of this bone loss was similar. Finally, there was an illustration of the use of the estimated variance provided by this study for estimating sample size for future studies.