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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

This study examines the associations between endogenous sex steroids and bone mineral density (BMD), using data from a geographically defined cohort in Rancho Bernardo, California. Participants were community-dwelling women and men aged 50–89 years who took part in a study of endogenous sex steroid measurement between 1984–1987 and who had BMD measured in 1988–1991. Those taking corticosteroids or estrogen at the time of sex steroid determination were excluded. The main study outcomes were BMD of the ultradistal radius, midshaft radius, lumbar spine, and total hip by sex steroid level, adjusted for age, body mass index, cigarette smoking, alcohol consumption, leisure exercise, use of thiazides, thyroid hormones, and former estrogen use (women only). At the time of the hormone measurements, the mean age of the 457 women was 72.1 years and that of the 534 men was 68.6 years. A statistically significant positive relation was seen between bioavailable estradiol and BMD at all sites in women and men. Total estradiol was significantly associated with BMD at all sites in women and at all but the ultradistal radius in men. Estrone had a global effect on BMD in women and was not measured in men. Higher bioavailable (but not total) testosterone levels were associated with higher BMD of the ultradistal radius, spine, and hip in men and the ultradistal radius in women. Dehydroepiandrosterone was positively associated with BMD of the midradius, spine, and hip in women and was not associated with BMD at any site in men. Of the sex steroids tested, bioavailable estrogen was most strongly associated with BMD in both women and men. We conclude that endogenous sex steroid levels are significantly related to bone density in older women and men. Individual variation in age-related bone loss may be partially accounted for by alterations in sex steroid levels with aging. Further study to elucidate safe environmental and medical methods to maintain optimal sex steroid levels in old age is needed.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

RECENT EVIDENCE indicates that the rate of bone loss, measured at the hip and calcaneus, increases in the eighth and ninth decades of life.1 This newly described phenomenon invites consideration of the determinants of skeletal integrity in later years. Variation in endogenous sex steroid levels has been postulated as one factor underlying individual differences in age-related bone loss.2

Many studies have examined the associations between endogenous sex steroids and osteoporosis in women3–8 and men9–11 of various ages with contradictory results; few investigations have been performed in large, population-based samples. The present study expands upon prior information by exploring the relation between both total and bioavailable sex steroids in large numbers of community-dwelling elders, before and after adjustment for other major determinants of bone mineral density (BMD). Data gathered from participants in the Rancho Bernardo Heart and Chronic Disease Study were used to address the following question: What are the associations between endogenous sex steroids and BMD density in older women and men?

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The Rancho Bernardo Heart and Chronic Disease Study began as a survey of heart disease risk factors conducted between 1972 and 1974 in a geographically defined, upper-middle class community in Rancho Bernardo, California. Eighty-two percent of the eligible residents participated. Subsequently, subjects have been invited for studies of other chronic diseases. Data used in this analysis are from studies conducted in 1984–1987 and 1988–1991. A description of the eligibility criteria, participation rates, and measurements follows.

1984–1987 measurements

Eighty-two percent of the surviving cohort were examined between 1984 and 1987, when medical and behavioral histories were obtained using a standardized questionnaire. All medications were brought to the clinic for verification. Height and weight were measured with participants wearing light clothing and without shoes. Body mass index (BMI) was calculated as kilograms per square meter. Information regarding age, BMI, medication use (thyroid hormones, thiazides, and, in women, estrogens), alcohol (in grams during the 2 weeks prior to the visit), cigarette use (current, former, or never), and exercise (at least three times per week) were derived from this 1984–1987 study visit. Participants who were then between 50 and 89 years of age had blood drawn for the determination of sex steroid hormone levels. Specimens were frozen at −70°C in plastic cyrovials, and all assays were performed on previously unthawed plasma. Radioimmunoassay (RIA) methods were used to measure total estradiol (E2), total testosterone (T), dihydrotestosterone (DHT), estrone (E1), dehydroepiandrosterone (DHEA), and dehydroepiandrosterone sulfate (DHEAS).12 Non–sex hormone-binding globulin (SHBG) bound (bioavailable) testosterone and estradiol were measured using the method of Tremblay and Dube.13 The sensitivity, inter-assay and intra-assay coefficients of variation (respectively) for the hormone assays are as follows: estradiol 4 pg/ml, 8% and 12%; testosterone 25 pg/ml, 4% and 10%; DHT 34 pg/ml, 7.5% and 7.5%; estrone 7 pg/ml, 15% and 16%; DHEAS 0.02 ng/ml, 10% and 5.2%; DHEA 0.04 ng/ml, 6.7% and 6.1%; bioavailable estradiol 1 pg/ml, 3.7% and 4.2%; and bioavailable testosterone 8 pg/ml, 5.8% and 6%.

1988–1991 measurements

Between 1988 and 1991, all surviving, community-dwelling participants aged 55 or older were invited to participate in a study of osteoporosis; 80% of eligible participants did so. BMD of the nondominant arm was measured at the ultradistal radius and midshaft radius using single photon absorptiometry (Lunar Corporation, Madison, WI, U.S.A., model SP2B). BMD of the spine (L1–L4) and total hip was obtained using dual-energy X-ray absorptiometry (Hologic, Inc., Waltham, MA, U.S.A., model QDR 1,000). Bone densitometers were calibrated daily. At our bone density unit, in vivo precision for hip BMD, calculated from duplicate measures with repositioning is 1.9% and that for lumbar spine is 1.1%. We do not have in vivo precision data for the radial measures. Phantom calibration precision is 0.58% and 0.49% for the hip and spine, respectively. Participants gave written informed consent at each study visit, and each study was approved by the Institutional Review Board of the University of California, San Diego.

Data analysis

Because corticosteroid use influences sex steroid levels, participants using corticosteroids (n = 6 women) were excluded. The analysis was restricted to women who were not using exogenous hormones at the time of sex steroid determination (1984–1987 visit).

Analyses were performed using the Statistical Analysis System.14 Age, BMI, alcohol use, and cigarette use were regarded as necessary covariates, due to their prior reported associations with both sex steroid hormone levels and bone density.15–17 Additional covariates (thyroid hormone use, thiazide use, former estrogen use, and exercise) were added to the full models because of their known associations with BMD in this cohort.18–20

Linear regressions were conducted using each BMD site as the dependent variable and each sex steroid as the independent variable. All BMD and sex steroid values except total and bioavailable testosterone in women were normally distributed; log normal transformation of the testosterone values was performed for the analyses in women. Three models were run for each bone site and sex steroid: adjusted for age; adjusted for age and BMI; and finally adjusted for age, BMI, thyroid hormone use, thiazide use, cigarette smoking, alcohol consumption, exercise, and former estrogen use (women only). Continuous variables (BMD, age, BMI, grams of alcohol intake) were modeled continuously; thyroid use, thiazide use, exercise at least three times per week, and former estrogen use were dichotomous (yes/no); smoking was trichotomous (never/former/current). To examine the relative effect size of each sex steroid on BMD, standardized Beta coefficients were calculated. These Beta coefficients describe the increment in BMD per standard deviation increment in each sex steroid. (Note: There is no generally accepted method of comparing effect sizes across exposures with different units of measurement, as is the case here with sex steroids. The size of the standard deviation will necessarily influence the standardized regression coefficient.) Linearity across the range of measured values was confirmed by calculating age-adjusted BMD by quartile of each hormone value.

For all hormones except estradiol, models were run excluding those participants with measured hormone below the level of sensitivity of the assay. For each hormone, this represented fewer than 1.5% of the participants. Estradiol levels were below the level of detection in 61 women (13%). Therefore, for estradiol only, models were initially run excluding participants with missing values and then rerun substituting a value for estradiol that was one unit lower than the lowest measurable estradiol value. No other outlying hormone values were excluded.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

At the time blood was collected for hormone measurements (1984–1987), the mean age of the 457 women in the study was 72.1 years (±8.0 years), and the mean age of the 534 men was 68.6 years (±9.0 years). The age range was 51–89 years for women, with 91.2% age 60 years or older and 68.1% 70 years or older. Men ranged in age from 50 to 89 years with 79.2% age 60 years or older and 47.4% 70 years or older, respectively. All were Caucasian. The average BMI in women was 24.1 kg/m2 (±3.4 kg/m2) and for men 25.9 kg/m2 (±3.2 kg/m2). Current cigarette use was reported by 12.5% of women and 11.0% of men; 36.8% of women and 58.2% of men had smoked previously. Sixty-six percent of women and 78% of men reported current alcohol use. Among those who drank alcohol, the average alcohol consumption over the previous 2 weeks was 94.2 g/week for women and 135.1 g/week in men. Thiazide diuretics were taken by 26.9% of women and 20.2% of men. Thyroid hormone was taken by 14.4% of women and 3.0% of men. Seventy-nine percent of women and 87.8% of men said they engaged in regular exercise at least three times per week. Former estrogen use was reported by 66.3% of women.

Table 1 summarizes the average values of the sex steroid hormones and sex hormone binding globulin in the cohort. Estradiol levels were undetectable in 13% of women. The mean value of estradiol shown is that for the 396 women who had detectable levels. For the remainder of the sex steroids in women and men, a minimum of one and a maximum of seven participants had values below the level of detection of the assays. The average hormone values shown in Table 1 exclude the values that were missing for these participants. Estrone was not assayed in men, and DHT was not assayed in women. Also shown in Table 1 are the means and standard deviations of BMD values for women and men.

Table Table 1.. UNADJUSTED MEAN VALUES (STANDARD DEVIATIONS) OF SEX STEROID HORMONES AND BONE MINERAL DENSITY IN POSTMENOPAUSAL WOMEN AND MEN AGED 50 YEARS AND OLDER, RANCHO BERNARDO, CALIFORNIA, 1984–1987
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Sex-specific correlations between each of the measured steroids are summarized in Table 2. While most of the correlations were highly significant statistically (p < 0.001), the correlations between most sex steroids were modest to low. Bioavailable estradiol and estrone (measured on women only) were moderately correlated at r = 0.64. Bioavailable estradiol and bioavailable testosterone were also moderately correlated (r = 0.45 and r = 0.50 in men and women) as were total estradiol and total testosterone (r = 0.51 and r = 0.40 in men and women). As expected, correlations were higher between bioavailable and bound forms of estrogen and testosterone and between DHEA and DHEAS.

Table Table 2.. UNADJUSTED CORRELATIONS* BETWEEN SEX HORMONES IN POSTMENOPAUSAL WOMENAND MEN AGED 50 YEARS AND OLDER, RANCHO BERNARDO, CALIFORNIA
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The results of linear regression models that estimated the effect of DHEAS on BMD are displayed in Table 3. The age-adjusted model, the regression of DHEAS adjusted for age and BMI, and the fully adjusted model accounting for DHEAS, age, BMI, thiazide use, thyroid hormone use, alcohol consumption, cigarette smoking, leisure exercise, and former use of estrogen (women only) are shown. Among women, in all models, small but statistically significant positive associations between DHEAS and bone density were seen at the midradius, the lumbar spine, and the total hip. In multiply adjusted models across bone sites, BMD increased by 0.0097 to 0.0188 g/cm2 per each standard deviation increment in DHEAS. DHEAS was not associated with BMD at any measured bone site in men. DHEA was not related to BMD in women or men (data not shown).

Table Table 3.. BONE MINERAL DENSITY OF THE ULTRADISTAL RADIUS, MIDSHAFT RADIUS, SPINE, AND TOTAL HIP: CRUDE; ADJUSTED FOR AGE; AGE AND BMI; AND MULTIPLY ADJUSTED BY DHEAS
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Table 4 summarizes the relation between bioavailable testosterone and BMD. Among women, in fully adjusted models, an effect of log bioavailable testosterone was evident only at the ultradistal radius (p = 0.016). Among men, in fully adjusted models, bioavailable testosterone was positively associated with BMD at the ultradistal radius, the lumbar spine, and hip, with increments in BMD of between 0.0093 and 0.0189 g/cm2 evident across bone sites. Total testosterone levels were not associated with BMD in women or men (data not shown). DHT, measured only in men, was also not associated with BMD (data not shown).

Table Table 4.. BONE MINERAL DENSITY OF THE ULTRADISTAL RADIUS, MIDRADIUS, SPINE, AND TOTAL HIP: CRUDE; ADJUSTED FOR AGE; AGE AND BMI; AND MULTIPLY ADJUSTED, BY LOG BIOAVAILABLE TESTOSTERONE (WOMEN) AND BIOAVAILABLE TESTOSTERONE (MEN)
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Bioavailable estradiol was strongly and statistically significantly associated with BMD at all sites in both women and men (Table 5). For women, in multiply adjusted models, BMD of the ultradistal radius, midradius, lumbar spine, and total hip increased by between 0.0116 and 0.0264 g/cm2 for each standard deviation increment in bioavailable estradiol. The relation between bioavailable estradiol and BMD in men was similar. In fully adjusted models, increments in BMD of the ultradistal radius, midradius, lumbar spine, and total hip ranged between 0.0136 and 0.0292 g/cm2 for each standard deviation increment in bioavailable estradiol. Thus, per standard deviation unit, in both women and men, the increment in BMD was the largest for bioavailable estradiol compared with the other hormones measured. Total estradiol was also associated with BMD at all measured sites in women, and at the midradius, lumbar spine, and hip in men, but the effect sizes were less than those seen with bioavailable estradiol (data not shown). Results were unchanged when bioavailable and total estradiol models were re-run using the one-unit below lowest measurable value for the 61 women with undetectable levels of these hormones (data not shown).

Table Table 5.. BONE MINERAL DENSITY OF THE ULTRADISTAL RADIUS, MIDRADIUS, SPINE, AND TOTAL HIP: CRUDE ADJUSTED FOR AGE; AGE AND BMI; AND MULTIPLY ADJUSTED, BY BIOAVAILABLE ESTRADIOL
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The association between estrone and BMD was examined in women only. A global, statistically significant, positive relation was evident, but the overall effect size appeared somewhat lower than that of estradiol (Table 6).

Table Table 6.. BONE MINERAL DENSITY OF THE ULTRADISTAL RADIUS, MIDRADIUS, SPINE, AND TOTAL HIP: CRUDE; ADJUSTED FOR AGE; AGE AND BMI; AND MULTIPLY ADJUSTED BY ESTRONE IN WOMEN
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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

In this large, geographically defined cohort of older persons, the sex steroid that demonstrated the strongest positive association with BMD at all bone sites in both women and men was bioavailable estradiol. Total estradiol had similarly global effects on BMD in both genders, and estrone, measured in women only, was positively associated with BMD at all sites. In men, bioavailable testosterone was associated with higher BMD at three of four bone sites, while in women, a relation was found only for BMD of the ultradistal radius. No association between total testosterone and BMD was evident in either gender, nor was DHT related to BMD in men. DHEAS was positively associated with BMD at three sites in women but was unrelated to bone density in men.

Estrogen

Of the sex steroids tested, bioavailable estrogen was most strongly associated with BMD in both women and men (i.e., demonstrated the greatest increment in BMD per standard deviation unit of the hormone). The loss of bone mass after the menopause and the maintenance of postmenopausal bone mass by noncontraceptive estrogen use after the menopause infers a strong relation between estrogen and skeletal integrity in women. However, an effect of endogenous estradiol or estrone on BMD has not been consistently demonstrated: cross-sectional and longitudinal studies have reported both positive3–6,21 and negative7,22 findings. The present analysis revealed a strong association between bioavailable estradiol and estrone and BMD among women. There are several possible explanations for the robust relation seen in this group of largely septuagenarian women. First, endogenous estrogen may exert its predominant effect on bone maintenance later in life. Johnston and colleagues have proposed that women who produce more sex steroids when they are older may have a slower rate of later bone loss, thus enjoying relative protection from fractures.2 Alternatively, in the early peri- and postmenopause, fluctuating hormone levels due to unstable ovarian function may lead to single-sample measurement errors too large to show a relation between estrogen and bone mass in younger women.22 It appears that substantial variability in estrogen persists even into early postmenopause; in a reliability study of estrogen levels measured 4 weeks apart in nine early postmenopausal women, the intraclass correlation coefficients for estradiol and estrone were 0.19 and 0.51, respectively.23 Our large sample size would mitigate against this measurement error. Finally, some prior studies concluding no effect of estradiol on BMD may also have been hampered by the lower limits of detection of their estradiol assays.24 In the present study, 13% of the estradiol assays were undetectable.

There was a positive association between bioavailable estrogen and BMD at all sites in men. Limited prior data link endogenous estrogen and skeletal development and maintenance in males. Notably, Smith and colleagues25 recently reported the case of a young man with severe osteopenia and a mutation in the estrogen receptor. In this patient, skeletal development and density were quite abnormal, despite normal testosterone levels. In a recent open-label trial of intramuscular testosterone administration, Andersen et al. reported a 5% increase in BMD among eugonadal men with crush fractures.26 Serum estradiol increased by 45%, and change in BMD was significantly correlated with change in estradiol (but not with change in testosterone). These data support the hypothesis that estrogen benefits bone in men and are concordant with the present study's findings for endogenous estradiol and BMD in men. There are androgen and estrogen receptors on bone in both sexes,27 and it is plausible that estrogen is a functional intracellular receptor,28 even in men.

The absolute values of bioavailable and total estradiol levels of men were approximately four times higher than those of women. However, the effects of bioavailable and total estradiol on BMD were similar across the range of values measured in both men and women. The four-to-one ratio of male-to-female estradiol levels evident in the present study is higher than that reported by Pfeilschifter and colleagues.29 In a large population-based study of men and woman aged 50–80 years, they noted average estradiol levels of 23.9 pg/ml and 12.3 pg/ml in men and women, respectively. Other studies of exogenous sex steroids in men aged 50–80 years also report mean estradiol levels in the range of 22–26 pg/ml,30,31 while reported mean levels of estradiol in postmenopausal women vary between 4 and 13 pg/ml.24,29,32 The current study's female estradiol levels are on the low end of this range, with a mean value of 5.54 pg/ml. Prior results from the same cohort, measured when the women were approximately 12 years younger (1972–1974), were about two times higher.32 Estradiol levels are negatively associated with age24; in addition, the lower values seen here may be partially due to long-term storage in plastic vials. However, this storage effect would not be different in women and men. Importantly, a systematically lower absolute level in women and men would not produce the observed association between the hormones and BMD.

Testosterone

Few studies have examined the relation between endogenous testosterone and BMD in postmenopausal women.2,21,33 Johnston and colleagues reported a weak (r = 0.24) unadjusted correlation between testosterone and vertebral BMD in peri- and postmenopausal women.2 In another pilot study of 16 women, no association was found between trabecular bone at L3 and endogenous testosterone.33 Recently, Slemenda et al. reported correlations of 0.16 and 0.24 between total testosterone and non–SHBG-bound testosterone and longitudinal change in BMD in postmenopausal women; in multivariable models, unbound testosterone was related only to change in femoral neck BMD.21 In the present study, total testosterone was unrelated to BMD in women. An association between bioavailable testosterone and BMD was evident at all sites, but after accounting for BMI and other covariates, statistical significance remained only at the ultradistal radius. Our analyses differ from those of Slemenda and colleagues in two important ways: our outcome was not longitudinal change in BMD and our models included different covariates.

More studies of the relation between testosterone and BMD have been done in men, but a coherent conclusion has not emerged.10,11,34–36 In 112 male volunteers of average age 72 years, Drinka and colleagues were unable to show a relation between age-adjusted bioavailable testosterone levels and BMD at axial or appendicular sites.10 Studies of idiopathic hypogonotropic hypogonadism34 and hyperprolactinemic hypogonadism35 similarly failed to find an effect of male sex hormones on bone. In contrast, the free androgen index (FAI, calculated as testosterone/SHBG) was positively correlated with ultradistal radius BMD in one study of 30 men ranging in age from 21–79 years.36 A positive association was also reported between total testosterone, FAI, and BMD of the spine and hip in 134 British men.11 The current, larger study of older men directly measured bioavailable testosterone which was positively associated with BMD at the ultradistal radius, spine, and hip. It is striking that DHT, which in other organs is the tissue active form of testosterone, was unrelated to BMD in this study.

DHEAS

Compared with the other hormones, DHEAS was associated with a smaller, but statistically significant, effect on BMD of the forearm, spine, and hip among women only. Previous investigations of the potential bone-preserving effects of DHEAS in women have been discordant. In one study of early postmenopausal women, in multiply-adjusted models, DHEAS was positively associated with spine but not hip BMD.22 Another study that examined the effect of DHEAS on spine and radius BMD in women ranging in age from 40 to over 80 found a positive, age-adjusted effect at both sites.5 Others, including an earlier study in Rancho Bernardo, have not found a significant effect of DHEAS on radial or forearm BMD.6,32 Differences between this study and the former Rancho Bernardo analysis may be due to the substantially larger sample size of the current study (approximately 462 vs. approximately 965), the shorter interval between hormone and BMD measurement in the present study, the fact that women in the current study were 10 years older at the time of the BMD measurements, and that BMD was analyzed in tertiles previously versus continuously in the current study. As in the case of estrogen,2 the relation of endogenous levels of DHEAS to BMD may be more apparent with increasing age.

The role of DHEAS in the maintenance of male bone mass has received limited investigation. We37 and others11 have not been able to demonstrate an association previously; the present study is congruent with these results.

There are several limitations to this study. First, as in other reported studies, a single determination of sex steroid levels was made. This introduces a null bias, because sex steroids are not static, even in postmenopausal women.23 There are numerous interrelations between sex steroids. For example, testosterone is converted to estrogen, in both women and men. It is difficult to judge which hormone(s) are influencing bone directly and which are “upstream” sources of substrate to form the bone-active hormone(s). Further, if the steroid conversion occurs intracellularly,28 it would not be possible to detect which hormones are metabolic substrates by measuring circulating levels. Although others have used stepwise regression models,5 we do not believe that a mathematical model can be used to determine the complex relations among hormones; factors other than biology strongly influence what variables remain in a mathematical model (e.g., precision of measurement). Understanding the relative impact of different exposures is of clinical and biologic interest, but there remains no agreed-upon method for comparing effect sizes among several exposures, particularly when they have different units of measurement. With the recognition of its limitations,38 we gauged the relative strength of each hormone's effect by examining the increment in BMD per standard deviation unit of each hormone.

In sum, these data suggest that endogenous sex steroid levels influence bone density in old age. Rather than a gender difference in bone metabolism, we postulate that endogenous estrogen may be a potent bone trophic hormone in both older women and men. Finally, variation in age-related bone loss may be partially accounted for by variability in the production or clearance of sex steroids with aging.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

This work was supported by NIA 5RO1 AG07181 and NIDDK RO1 DK 31801. Dr. Greendale was also supported for this work by the Geriatrics Academy Program NIA K12 AG00489 and the Iris Cantor-UCLA Women's Center.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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