A conserved role of the insulin-like signaling pathway in diet-dependent uric acid pathologies in Drosophila melanogaster.

Elevated uric acid (UA) is a key risk factor for many disorders, including metabolic syndrome, gout and kidney stones. Despite frequent occurrence of these disorders, the genetic pathways influencing UA metabolism and the association with disease remain poorly understood. In humans, elevated UA levels resulted from the loss of the of the urate oxidase (Uro) gene around 15 million years ago. Therefore, we established a Drosophila melanogaster model with reduced expression of the orthologous Uro gene to study the pathogenesis arising from elevated UA. Reduced Uro expression in Drosophila resulted in elevated UA levels, accumulation of concretions in the excretory system, and shortening of lifespan when reared on diets containing high levels of yeast extract. Furthermore, high levels of dietary purines, but not protein or sugar, were sufficient to produce the same effects of shortened lifespan and concretion formation in the Drosophila model. The insulin-like signaling (ILS) pathway has been shown to respond to changes in nutrient status in several species. We observed that genetic suppression of ILS genes reduced both UA levels and concretion load in flies fed high levels of yeast extract. Further support for the role of the ILS pathway in modulating UA metabolism stems from a human candidate gene study identifying SNPs in the ILS genes AKT2 and FOXO3 being associated with serum UA levels or gout. Additionally, inhibition of the NADPH oxidase (NOX) gene rescued the reduced lifespan and concretion phenotypes in Uro knockdown flies. Thus, components of the ILS pathway and the downstream protein NOX represent potential therapeutic targets for treating UA associated pathologies, including gout and kidney stones, as well as extending human healthspan.

Coronary Artery Calcium Score and Association with Recurrent Nephrolithiasis: The Multi-Ethnic Study of Atherosclerosis.

PURPOSE: Subclinical coronary artery calcification is an established predictor of cardiovascular events. While a history of kidney stones has been linked to subclinical carotid atherosclerosis, to our knowledge no study has examined its relationship with coronary artery calcification. We studied the association between kidney stone history and prevalent coronary artery calcification in MESA (Multi-Ethnic Study of Atherosclerosis). MATERIALS AND METHODS: MESA is a multisite cohort study of participants 45 to 84 years old without known cardiovascular disease at baseline from 2000 to 2002. Computerized tomography was done in 3,282 participants at followup in 2010 to 2012 to determine coronary artery calcification and kidney stone history was assessed by self-report. Coronary artery calcification scores were categorized as none-0, mild-1 to 99, moderate-100 to 399 or severe-400 or greater. Cross-sectional analysis was performed adjusting for demographic and dietary factors related to kidney stones. RESULTS: The prevalence of kidney stone disease history was approximately 9%, mean ± SD participant age was 69.5 ± 9.3 years, 39% of participants were Caucasian, 47% were men and 69% had detectable coronary artery calcification (score greater than 0). No difference in the score was seen between single stone formers and nonstone formers. Recurrent kidney stone formation was associated with moderate or severe calcification on multivariable logistic regression vs none or mild calcification (OR 1.80, 95% CI 1.22-2.67). When coronary artery calcification scores were separated into none, mild, moderate and severe calcification, recurrent stone formation was associated with a higher score category on multivariable ordinal logistic regression (OR 1.44 per category, 95% CI 1.04-2.01). CONCLUSIONS: Recurrent kidney stone formation is associated with subclinical coronary atherosclerosis. This association appeared stronger with coronary artery calcification severity than with coronary artery calcification presence.

Activity, energy intake, obesity, and the risk of incident kidney stones in postmenopausal women: a report from the Women's Health Initiative.

Obesity is a strong risk factor for nephrolithiasis, but the role of physical activity and caloric intake remains poorly understood. We evaluated this relationship in 84,225 women with no history of stones as part of the Women's Health Initiative Observational Study, a longitudinal, prospective cohort of postmenopausal women enrolled from 1993 to 1998 with 8 years' median follow-up. The independent association of physical activity (metabolic equivalents [METs]/wk), calibrated dietary energy intake, and body mass index (BMI) with incident kidney stone development was evaluated after adjustment for nephrolithiasis risk factors. Activity intensity was evaluated in stratified analyses. Compared with the risk in inactive women, the risk of incident stones decreased by 16% in women with the lowest physical activity level (adjusted hazard ratio [aHR], 0.84; 95% confidence interval [95% CI], 0.74 to 0.97). As activity increased, the risk of incident stones continued to decline until plateauing at a decrease of approximately 31% for activity levels ≥10 METs/wk (aHR, 0.69; 95% CI, 0.60 to 0.79). Intensity of activity was not associated with stone formation. As dietary energy intake increased, the risk of incident stones increased by up to 42% (aHR, 1.42; 95% CI, 1.02 to 1.98). However, intake <1800 kcal/d did not protect against stone formation. Higher BMI category was associated with increased risk of incident stones. In summary, physical activity may reduce the risk of incident kidney stones in postmenopausal women independent of caloric intake and BMI, primarily because of the amount of activity rather than exercise intensity. Higher caloric intake further increases the risk of incident stones.

Dietary intake of fiber, fruit and vegetables decreases the risk of incident kidney stones in women: a Women's Health Initiative report.

PURPOSE: We evaluated the relationship between dietary fiber, fruit and vegetable intake, and the risk of kidney stone formation. MATERIALS AND METHODS: Overall 83,922 postmenopausal women from the Women's Health Initiative observational study were included in the analysis and followed prospectively. Cox proportional hazards regression analyses were used to evaluate the associations between total dietary fiber, fruit and vegetable intake, and the risk of incident kidney stone formation, adjusting for nephrolithiasis risk factors (age, race/ethnicity, geographic region, diabetes mellitus, calcium supplementation, hormone therapy use, body mass index and calibrated caloric intake; and dietary water, sodium, animal protein and calcium intake). Women with a history of kidney stones (3,471) were analyzed separately. RESULTS: Mean age of the women was 64±7 years, 85% were white and 2,937 (3.5%) experienced a kidney stone in a median followup of 8 years. In women with no history of kidney stones higher total dietary fiber (6% to 26% decreased risk, p <0.001), greater fruit intake (12% to 25% decreased risk, p <0.001) and greater vegetable intake (9% to 22% decreased risk, p=0.002) were associated with a decreased risk of incident kidney stone formation in separate adjusted models. In women with a history of stones there were no significant protective effects of fiber, fruit or vegetable intake on the risk of kidney stone recurrence. CONCLUSIONS: Greater dietary intake of fiber, fruits and vegetables was associated with a reduced risk of incident kidney stones in postmenopausal women. The protective effects were independent of other known risk factors for kidney stones. In contrast, there was no reduction in risk in women with a history of stones.

Drosophila melanogaster as an emerging translational model of human nephrolithiasis.

PURPOSE: The limitations imposed by human clinical studies and mammalian models of nephrolithiasis have hampered the development of effective medical treatments and preventive measures for decades. The simple but elegant Drosophila melanogaster is emerging as a powerful translational model of human disease, including nephrolithiasis. It may provide important information essential to our understanding of stone formation. We present the current state of research using D. melanogaster as a model of human nephrolithiasis. MATERIALS AND METHODS: We comprehensively reviewed the English language literature using PubMed®. When necessary, authoritative texts on relevant subtopics were consulted. RESULTS: The genetic composition, anatomical structure and physiological function of Drosophila malpighian tubules are remarkably similar to those of the human nephron. The direct effects of dietary manipulation, environmental alteration and genetic variation on stone formation can be observed and quantified in a matter of days. Several Drosophila models of human nephrolithiasis have been developed, including genetically linked and environmentally induced stones. A model of calcium oxalate stone formation is among the most recent fly models of human nephrolithiasis. CONCLUSIONS: The ability to readily manipulate and quantify stone formation in D. melanogaster models of human nephrolithiasis presents the urological community with a unique opportunity to increase our understanding of this enigmatic disease.

Strontium substitution for calcium in lithogenesis.

PURPOSE: Strontium has chemical similarity to calcium, which enables the replacement of calcium by strontium in biomineralization processes. Incorporating strontium into human bone and teeth has been studied extensively but little research has been performed of the incorporation of strontium into urinary calculi. We used synchrotron based x-ray fluorescence and x-ray absorption techniques to examine the presence of strontium in different types of human kidney stones. MATERIALS AND METHODS: Multiple unique human stone samples were obtained via consecutive percutaneous nephrolithotomies/ureteroscopies. A portion of each stone was sent for standard laboratory analysis and a portion was retained for x-ray fluorescence and x-ray absorption measurements. X-ray fluorescence and x-ray absorption measurements determined the presence, spatial distribution and speciation of strontium in each stone sample. RESULTS: Traditional kidney stone analyses identified calcium oxalate, calcium phosphate, uric acid and cystine stones. X-ray fluorescence measurements identified strontium in all stone types except pure cystine. X-ray fluorescence elemental mapping of the samples revealed co-localization of calcium and strontium. X-ray absorption measurements of the calcium phosphate stone showed strontium predominately present as strontium apatite. CONCLUSIONS: Advanced x-ray fluorescence imaging identified strontium in all calcium based stones, present as strontium apatite. This finding may be critical since apatite is thought to be the initial nidus for calcium stone formation. Strontium is not identified by standard laboratory stone analyses. Its substitution for calcium can be reliably identified in stones from multiple calcium based stone formers, which may offer opportunities to gain insight into early events in lithogenesis.

Microcomposition of human urinary calculi using advanced imaging techniques.

PURPOSE: Common methods of commercial urolithiasis analysis, such as light microscopy and Fourier transform infrared spectroscopy, provide limited or no information on the molecular composition of stones, which is vital when studying early stone pathogenesis. We used synchrotron radiation based microfocused x-ray fluorescence, x-ray absorption and x-ray diffraction advanced imaging techniques to identify and map the elemental composition, including trace elements, of urinary calculi on a µm (0.0001 cm) scale. MATERIALS AND METHODS: Human stone samples were obtained during serial percutaneous nephrolithotomy and ureteroscopy procedures. A portion of each sample was sent for commercial stone analysis and a portion was retained for synchrotron radiation based advanced imaging analysis. RESULTS: Synchrotron radiation based methods of stone analysis correctly identified stone composition and provided additional molecular detail on elemental components and spatial distribution in uroliths. Resolution was on the order of a few µm. CONCLUSIONS: Knowledge of all elements present in lithogenesis at this detail allows for better understanding of early stone formation events, which may provide additional insight to prevent and treat stone formation.

Impact of calcium intake and intestinal calcium absorption on kidney stones in older women: the study of osteoporotic fractures.

PURPOSE: Intestinal calcium absorption is thought to have a critical role in nephrolithiasis. However, to our knowledge no study has directly assessed this association. Therefore, we explored the relationship among intestinal fractional calcium absorption, calcium intake and nephrolithiasis. MATERIALS AND METHODS: The Study of Osteoporotic Fractures is a prospective cohort of 9,704 postmenopausal women recruited from population based listings in 1986 and followed for more than 20 years. Secondary analyses were performed of 7,982 women who reported their history of nephrolithiasis, of which 5,452 (68%) underwent an oral radioactive calcium assay (45Ca). The impact of dietary and supplemental calcium on intestinal fractional calcium absorption was evaluated, and factors independently associated with nephrolithiasis were determined. RESULTS: Fractional calcium absorption decreased with increased calcium intake, with no difference between dietary and supplemental calcium. Fractional calcium absorption was higher in women with a nephrolithiasis history among all calcium intake groups. Increased dietary calcium intake reduced the likelihood of nephrolithiasis by 45% to 54% (p=0.03). Women with a history of nephrolithiasis were less likely to supplement calcium (p<0.001). In adjusted analyses women who supplemented calcium were 21% to 38% less likely to have a nephrolithiasis history (p=0.007) and there was a 24% increased risk of kidney stones for each 10% increase in fractional calcium absorption (p=0.008). CONCLUSIONS: Fractional calcium absorption is higher in women with a history of nephrolithiasis. Higher intestinal fractional calcium absorption is associated with a greater risk of historical nephrolithiasis. Dietary and supplemental calcium decrease fractional calcium absorption, and may protect against nephrolithiasis.

Impact of nutritional factors on incident kidney stone formation: a report from the WHI OS.

PURPOSE: Increased fluid intake, and decreased dietary sodium and animal protein intake are thought to reduce the risk of kidney stones but the role of calcium intake is controversial. We evaluated the relationship between dietary factors and incident kidney stone formation. MATERIALS AND METHODS: Secondary analysis was done of 78,293 women from the prospective WHI OS (Women's Health Initiative Observational Study) with no history of nephrolithiasis who completed the validated food frequency questionnaire. Multivariate logistic regression was used to determine demographic and dietary factors, and supplement use independently associated with incident kidney stones. RESULTS: Overall 1,952 women (2.5%) reported an incident kidney stone in 573,575 person-years of followup. The risk of incident kidney stones was decreased by 5% to 28% (p = 0.01) with higher dietary calcium intake and by 13% to 31% (p = 0.002) with higher water intake after adjusting for nephrolithiasis risk factors. Conversely higher dietary sodium intake increased the risk of nephrolithiasis by 11% to 61% (p <0.001) after adjustment with the most pronounced effect in women with the highest intake. Higher body mass index independently increased the risk of incident nephrolithiasis (adjusted OR 1.19-2.01, p <0.001). Animal protein intake was not associated with nephrolithiasis on multivariate analysis. CONCLUSIONS: This study adds to the growing evidence underscoring the importance of maintaining adequate fluid and dietary calcium intake. Greater dietary calcium intake significantly decreased the risk of incident kidney stones. In contrast, excess sodium intake increased the risk of incident nephrolithiasis, especially in women with the highest intake. Animal protein intake was not independently associated with nephrolithiasis.

Kidney stones and subclinical atherosclerosis in young adults: the CARDIA study.

PURPOSE: Recent reports suggest that nephrolithiasis and atherosclerosis share a number of risk factors. To our knowledge there has been no previous examination of the relationship between kidney stones and subclinical atherosclerotic disease. We studied the relationship between nephrolithiasis, and carotid wall thickness and carotid stenosis assessed by B-mode ultrasound in the general community using data from the CARDIA study. MATERIALS AND METHODS: The CARDIA study is a United States, population based, observational study of 5,115 white and African-American men and women between the ages of 18 and 30 years at recruitment in 1985 to 1986. RESULTS: By the year 20 examination 200 (3.9%) CARDIA participants had reported ever having kidney stones. Symptomatic kidney stones were associated with greater carotid wall thickness measured at the year 20 examination, particularly of the internal carotid/bulb region. Using a composite dichotomous end point of carotid stenosis and/or the upper quartile of internal carotid/bulb wall thickness, the association of kidney stones with carotid atherosclerosis was significant (OR 1.6, 95% CI 1.1-2.3, p=0.01), even after adjusting for major atherosclerotic risk factors. CONCLUSIONS: The association between a history of kidney stones and subclinical carotid atherosclerosis in young adults adds further support to the notion that nephrolithiasis and atherosclerosis share common systemic risk factors and/or pathophysiology.

Attributions of blame and responsibility in sexual harassment: reexamining a psychological model.

Kelley's (Nebr Symp Motiv 15:192-238, 1967) attribution theory can inform sexual harassment research by identifying how observers use consensus, consistency, and distinctiveness information in determining whether a target or perpetrator is responsible for a sexual harassment situation. In this study, Kelley's theory is applied to a scenario in which a male perpetrator sexually harasses a female target in a university setting. Results from 314 predominantly female college students indicate that consistency and consensus information significantly affect participants' judgments of blame and responsibility for the situation. The authors discuss the importance of the reference groups used to derive consensus and distinctiveness information, and reintroduce Kelley's attribution theory as a means of understanding observers' perceptions of sexual harassment.

The Role of Advanced Glycation End Products in Aging and Metabolic Diseases: Bridging Association and Causality.

Accumulation of advanced glycation end products (AGEs) on nucleotides, lipids, and peptides/proteins are an inevitable component of the aging process in all eukaryotic organisms, including humans. To date, a substantial body of evidence shows that AGEs and their functionally compromised adducts are linked to and perhaps responsible for changes seen during aging and for the development of many age-related morbidities. However, much remains to be learned about the biology of AGE formation, causal nature of these associations, and whether new interventions might be developed that will prevent or reduce the negative impact of AGEs-related damage. To facilitate achieving these latter ends, we show how invertebrate models, notably Drosophila melanogaster and Caenorhabditis elegans, can be used to explore AGE-related pathways in depth and to identify and assess drugs that will mitigate against the detrimental effects of AGE-adduct development.

α-Lipoic acid treatment prevents cystine urolithiasis in a mouse model of cystinuria.

Cystinuria is an incompletely dominant disorder characterized by defective urinary cystine reabsorption that results in the formation of cystine-based urinary stones. Current treatment options are limited in their effectiveness at preventing stone recurrence and are often poorly tolerated. We report that the nutritional supplement α-lipoic acid inhibits cystine stone formation in the Slc3a1-/- mouse model of cystinuria by increasing the solubility of urinary cystine. These findings identify a novel therapeutic strategy for the clinical treatment of cystinuria.

In silico modeling and simulation of bone biology: a proposal.

Contemporary, computer-based mathematical modeling techniques make it possible to represent complex biological mechanisms in a manner that permits hypothesis testing in silico. This perspective shows how such approaches might be applied to bone remodeling and therapeutic research. Currently, the dominant conceptual model applied in bone research involves the dynamic balance between the continual build-up and breakdown of bone matrix by two cell types, the osteoblasts and osteoclasts, acting together as a coordinated, remodeling unit. This conceptualization has served extraordinarily well as a focal point for understanding how mutations, chemical mediators, and mechanical force, as well as external influences (e.g., drugs, diet) affect bone structure and function. However, the need remains to better understand and predict the consequences of manipulating any single factor, or combination of factors, within the context of this complex system's multiple interacting pathways. Mathematical models are a natural extension of conceptual models, providing dynamic, quantitative descriptions of the relationships among interacting components. This formalization creates the ability to simulate the natural behavior of a system, as well as its modulation by therapeutic or dietetic interventions. A number of mathematical models have been developed to study complex bone functions, but most include only a limited set of biological components needed to address a few specific questions. However, it is possible to develop larger, multiscale models that capture the dynamic interactions of many biological components and relate them to important physiological or pathological outcomes that allow broader study. Examples of such models include entelos' physiolab platforms. These models simulate the dynamic, quantitative interactions among a biological system's biochemicals, cells, tissues, and organs and how they give rise to key physiologic and pathophysiologic outcomes. We propose that a similar predictive, dynamical, multiscale mathematical model of bone remodeling and metabolism would provide a better understanding of the mechanisms governing these phenomena as well as serve as an in silico platform for testing pharmaceutical and clinical interventions on metabolic bone disease.

Regaining lost youth: the controversial and colorful beginnings of hormone replacement therapy in aging.

The quest for regaining lost youth seems to have existed since the beginning of recorded history and has taken many forms. One strategy that began in earnest in the latter part of the 19th century and continues to have enormous momentum today is based on the notion that by replacing internally secreted substances, that is, hormones, that decline with age, the vitality and physical attributes associated with youth can be regained. Although the approach remains highly controversial as, for example, in "anti-aging medicine," it is no more controversial than it was many years ago when the work of three high profile investigators, Charles Eduoard Brown-Séquard, Eugen Steinach, and Serge Voronoff set the basis for using this strategy. In the case of all three individuals, the therapies they developed received widespread attention (including ridicule) in the popular press, were spread rapidly by practitioners of questionable training and ethical motivation, and finally and relatively quickly disappeared from common use. However, and ultimately more importantly, in the process of developing and promoting their therapies, these individuals made important contributions to the origins of endocrinology, the biology of sex, and establishment of hormone replacement therapy. It remains to be seen whether contemporary efforts using hormone replacement therapy to blunt and reverse aging have the same fate as their predecessors and make comparable important contributions to biology and medicine.

FOXO3 and related transcription factors in development, aging, and exceptional longevity.

In June 2013, a workshop was convened in San Francisco to explore, in depth, the role of the Forkhead transcription factor FOXO3 (and related FOXOs) in development, aging, and, in particular, exceptional longevity. The presentations covered results derived from model systems, computational analysis and bioinformatics, and genomics and genome-wide association studies of a number of cohorts. Although the data collectively strongly reinforce FOXO3 and the FOXO/FOXO3 pathway as very important determinants in aging and life span, much of the detail of how the latter is achieved still remains unknown, in part, because of the very large number of genes (~2,200 in Caenorhabditis elegans) the transcription factor is involved in helping regulate. Particularly challenging at the present time is understanding the association of apparently nonfunctional specific variants (single nucleotide polymorphisms) of FOXO3 and exceptional longevity in humans, a finding replicated in a number of studies. Nonetheless, as summarized in this report, valuable information and insights were presented at the workshop on the transcription factor including but not limited to its role in determining longevity in C elegans and Drosophila (in flies, eg, an important interaction in aging occurs between dFOXO and the transforming growth factor-ß/activin pathway), stem cell function and aging (notably in hematopoiesis), downstream regulatory activity (eg, by binding near sites of RNAse occupancy and altering chromatin structure), and as a potential target for the development a healthy aging drug (in this example, using compounds developed and screened to effect FOXO function in cancer cells).

The elementome of calcium-based urinary stones and its role in urolithiasis.

Urolithiasis affects around 10% of the US population with an increasing rate of prevalence, recurrence and penetrance. The causes for the formation of most urinary calculi remain poorly understood, but obtaining the chemical composition of these stones might help identify key aspects of this process and new targets for treatment. The majority of urinary stones are composed of calcium that is complexed in a crystalline matrix with organic and inorganic components. Surprisingly, mitigation of urolithiasis risk by altering calcium homeostasis has not been very effective. Thus, studies to identify other therapeutic stone-specific targets, using proteomics, metabolomics and microscopy techniques, have been conducted, revealing a high level of complexity. The data suggest that numerous metals other than calcium and many nonmetals are present within calculi at measurable levels and several have distinct distribution patterns. Manipulation of the levels of some of these elemental components of calcium-based stones has resulted in clinically beneficial changes in stone chemistry and rate of stone formation. The elementome--the full spectrum of elemental content--of calcium-based urinary calculi is emerging as a new concept in stone research that continues to provide important insights for improved understanding and prevention of urinary stone disease.

Elemental Content of Calcium Oxalate Stones from a Canine Model of Urinary Stone Disease.

One of the most common types of urinary stones formed in humans and some other mammals is composed of calcium oxalate in ordered hydrated crystals. Many studies have reported a range of metals other than calcium in human stones, but few have looked at stones from animal models such as the dog. Therefore, we determined the elemental profile of canine calcium oxalate urinary stones and compared it to reported values from human stones. The content of 19 elements spanning 7-orders of magnitude was quantified in calcium oxalate stones from 53 dogs. The elemental profile of the canine stones was highly overlapping with human stones, indicating similar inorganic composition. Correlation and cluster analysis was then performed on the elemental profile from canine stones to evaluate associations between the elements and test for potential subgrouping based on elemental content. No correlations were observed with the most abundant metal calcium. However, magnesium and sulfur content correlated with the mineral hydration form, while phosphorous and zinc content correlated with the neuter status of the dog. Inter-elemental correlation analysis indicated strong associations between barium, phosphorous, and zinc content. Additionally, cluster analysis revealed subgroups within the stones that were also based primarily on barium, phosphorous, and zinc. These data support the use of the dog as a model to study the effects of trace metal homeostasis in urinary stone disease.

A Drosophila model identifies a critical role for zinc in mineralization for kidney stone disease.

Ectopic calcification is a driving force for a variety of diseases, including kidney stones and atherosclerosis, but initiating factors remain largely unknown. Given its importance in seemingly divergent disease processes, identifying fundamental principal actors for ectopic calcification may have broad translational significance. Here we establish a Drosophila melanogaster model for ectopic calcification by inhibiting xanthine dehydrogenase whose deficiency leads to kidney stones in humans and dogs. Micro X-ray absorption near edge spectroscopy (µXANES) synchrotron analyses revealed high enrichment of zinc in the Drosophila equivalent of kidney stones, which was also observed in human kidney stones and Randall's plaques (early calcifications seen in human kidneys thought to be the precursor for renal stones). To further test the role of zinc in driving mineralization, we inhibited zinc transporter genes in the ZnT family and observed suppression of Drosophila stone formation. Taken together, genetic, dietary, and pharmacologic interventions to lower zinc confirm a critical role for zinc in driving the process of heterogeneous nucleation that eventually leads to stone formation. Our findings open a novel perspective on the etiology of urinary stones and related diseases, which may lead to the identification of new preventive and therapeutic approaches.

Dehydroepiandrosterone supplementation and bone turnover in middle-aged to elderly men.

In the present placebo-controlled, double-blind study, we assessed the effect of dehydroepiandrosterone (DHEA) supplementation (90 mg orally/d) on bone turnover in 43 healthy men, 56-80 yr old. Placebo or steroid was given for 6 months, followed by a 1-month washout period and then a further 6 months of the opposite agent. Serum samples were collected at baseline 3, 6, 7, and 13 months and assayed for procollagen peptide, bone-specific alkaline phosphatase, and osteocalcin, all markers of bone formation. Measurements were also made of serum cortisol, DHEA/DHEA-S, E2 and free and total T. First void, fasting urine was collected at baseline, 6, 7, and 13 months and assessed for deoxypyridinoline, a marker of bone resorption. Mean serum DHEA and DHEA-S levels in treated men were increased approximately 3-fold ( approximately 2.2 ng/ml to approximately 6 ng/ml) and 4.5-fold ( approximately 1000 ng/ml to approximately 4500 ng/ml), respectively, after 6 months and returned to baseline after washout. Similarly, circulating E2 concentrations were also increased 1.4-fold (from approximately 16-23 pg/ml; P < 0.001), a finding not observed with any other measured hormone. Bone marker levels remained remarkably constant at each sampling interval; procollagen peptide at approximately 8.0 ng/ml; bone-specific alkaline phosphatase at approximately 21.0 U/liter; deoxypyridinoline at approximately 4.5 nmol/mmol Cr. Osteocalcin showed a transient reduction from approximately 10.2- 6.2 ng/ml, P < 0.005 to P < 0.001, at 3 months, but this decline was observed in both treated and controls. Stratifying the marker levels by age or baseline DHEA/DHEA-S levels did not affect the findings. We conclude that oral DHEA does not affect bone turnover in middle-aged to elderly men when used for a 6-month period at doses targeted to restore circulating levels of the steroid to that seen in young adults.