Fifty years of human space travel: implications for bone and calcium research.

Calcium and bone metabolism remain key concerns for space travelers, and ground-based models of space flight have provided a vast literature to complement the smaller set of reports from flight studies. Increased bone resorption and largely unchanged bone formation result in the loss of calcium and bone mineral during space flight, which alters the endocrine regulation of calcium metabolism. Physical, pharmacologic, and nutritional means have been used to counteract these changes. In 2012, heavy resistance exercise plus good nutritional and vitamin D status were demonstrated to reduce loss of bone mineral density on long-duration International Space Station missions. Uncertainty continues to exist, however, as to whether the bone is as strong after flight as it was before flight and whether nutritional and exercise prescriptions can be optimized during space flight. Findings from these studies not only will help future space explorers but also will broaden our understanding of the regulation of bone and calcium homeostasis on Earth.

Calcium kinetics during bed rest with artificial gravity and exercise countermeasures.

UNLABELLED: We assessed the potential for countermeasures to lessen the loss of bone calcium during bed rest. Subjects ingested less calcium during bed rest, and with artificial gravity, they also absorbed less calcium. With exercise, they excreted less calcium. To retain bone during bed rest, calcium intake needs to be maintained. INTRODUCTION: This study aims to assess the potential for artificial gravity (AG) and exercise (EX) to mitigate loss of bone calcium during space flight. METHODS: We performed two studies: (1) a 21-day bed rest (BR) study with subjects receiving 1 h/day AG (n = 8) or no AG (n = 7) and (2) a 28-day BR study with 1 h/day resistance EX (n = 10) or no EX (n = 3). In both studies, stable isotopes of Ca were administered orally and intravenously, at baseline and after 10 days of BR, and blood, urine, and feces were sampled for up to 14 days post dosing. Tracers were measured using thermal ionization mass spectrometry. Data were analyzed by compartmental modeling. RESULTS: Less Ca was absorbed during BR, resulting in lower Ca balance in BR+AG (-6.04 ± 3.38 mmol/day, P = 0.023). However, Ca balance did not change with BR+EX, even though absorbed Ca decreased and urinary Ca excretion increased, because endogenous excretion decreased, and there was a trend for increased bone deposition (P = 0.06). Urinary N-telopeptide excretion increased in controls during BR, but not in the EX group. Markers of bone formation were not different between treatment groups for either study. Ca intake decreased during BR (by 5.4 mmol/day in the AG study and 2.8 mmol/day in the EX study), resulting in lower absorbed Ca. CONCLUSIONS: During BR (or space flight), Ca intake needs to be maintained or even increased with countermeasures such as exercise, to enable maintenance of bone Ca.

Evaluation of a whole-farm model for pasture-based dairy systems.

In the temperate climate of New Zealand, animals can be grazed outdoors all year round. The pasture is supplemented with conserved feed, with the amount being determined by seasonal pasture growth, genetics of the herd, and stocking rate. The large number of factors that affect production makes it impractical and expensive to use field trials to explore all the farm system options. A model of an in situ-grazed pasture system has been developed to provide a tool for developing and testing novel farm systems; for example, different levels of bought-in supplements and different levels of nitrogen fertilizer application, to maintain sustainability or environmental integrity and profitability. It consists of a software framework that links climate information, on a daily basis, with dynamic, mechanistic component-models for pasture growth and animal metabolism, as well as management policies. A unique feature is that the component models were developed and published by other groups, and are retained in their original software language. The aim of this study was to compare the model, called the whole-farm model (WFM) with a farm trial that was conducted over 3 yr and in which data were collected specifically for evaluating the WFM. Data were used from the first year to develop the WFM and data from the second and third year to evaluate the model. The model predicted annual pasture production, end-of-season cow liveweight, cow body condition score, and pasture cover across season with relative prediction error <20%. Milk yield and milksolids (fat + protein) were overpredicted by approximately 30% even though both annual and monthly pasture and supplement intake were predicted with acceptable accuracy, suggesting that the metabolic conversion of feed to fat, protein, and lactose in the mammary gland needs to be refined. Because feed growth and intake predictions were acceptable, economic predictions can be made using the WFM, with an adjustment for milk yield, to test different management policies, alterations in climate, or the use of genetically improved animals, pastures, or crops.

Quantification of biochemical markers of bone turnover by kinetic measures of bone formation and resorption in young healthy females.

The quantification of biochemical markers of bone formation and resorption with kinetic measures of bone turnover is an essential step in their validation. Some biochemical markers have been validated by quantification against formation and resorption rates measured by calcium kinetics in adults with bone disease. However, none has been validated in healthy individuals who are undergoing skeletal growth and bone consolidation. Therefore, we have measured biochemical markers of bone formation (serum osteocalcin [OC], bone-specific alkaline phosphatase [BAP], and total alkaline phosphatase [ALP]) and resorption (serum tartrate resistant acid phosphatase [TRAP], urinary cross-linked N teleopeptides of type I collagen/creatinine [NTx/Cr], and hydroxyproline/creatinine [OHP/Cr]) in healthy females aged 11-32 years (n = 31) after an overnight fast to determine their relationship with bone formation (Vo+) and bone resorption (Vo-) as measured by calcium kinetics and balance. All biochemical markers were highly intercorrelated (r > 0.6, p < 0.001) as were Vo+ and Vo- (r = 0.91, p < 0.001). Highly significant correlations were present between bone formation measured by calcium kinetics (Vo+) and serum levels of bone biochemical markers (OC, r = 0.82, p = 0.001; ALP, r = 0.92, p = 0.001; and BAP, r = 0.90, p = 0.001) and between bone resorption measured by calcium kinetics (Vo-) and fasting serum levels and urine creatinine ratios of biochemical markers (TRAP, r = 0.77, p < 0.001; OHP/Cr, r = 0.79, p < 0.001; and NTx/Cr, r = 0.70, p < 0.001). Thus, biochemical markers of bone formation and resorption can be used to predict calcium kinetic rates during skeletal growth and the early years of formation of peak bone mass, ages at which strategies to build peak bone mass are important. Biochemical markers of formation and resorption are equally useful in predicting either the bone formation rate or the resorption rate.

Changes in calcium kinetics in adolescent girls induced by high calcium intake.

To identify the mechanism/s whereby calcium retention is increased by calcium intake in adolescent girls, kinetic studies were performed using stable calcium isotope tracers. Girls (n = 10; 12 +/- 1 yr old, mean +/- SD) were studied while on a controlled diet containing a low (21.2 mmol/day) and a high (47.4 mmol/day) calcium intake, in randomized order, using a cross-over design. Studies were separated by 1 month. Calcium tracers were administered after 1 week on the study diet, orally and iv; and serum, urine, and feces were collected for the following 14 days. Tracers were measured using fast atom bombardment mass spectrometry, and kinetic data were analyzed by compartmental modeling. Biochemical markers of bone turnover were measured in serum and urine samples. On high (compared with low) calcium intake, fractional absorption did not differ, absorbed calcium increased (19.6 +/- 7.5 vs. 8.0 +/- 2.5 mmol/day, mean +/- SD, P: < 0.001), calcium excreted in urine increased (2.8 +/- 1.7 vs. 2.1 +/- 1.1 mmol/day, P: < 0.01), calcium retained in bone increased (14.5 +/- 8.9 vs. 3.2 +/- 3.6 mmol/day, P: < 0.001), bone formation did not change, and bone resorption decreased by 32%. These changes, measured by kinetics, were corroborated by changes in markers of bone turnover. We conclude that increased bone retention of calcium, with high calcium intake in adolescent girls, is attributable to an increase in absorption and a decrease in bone resorption.

Racial differences in bone turnover and calcium metabolism in adolescent females.

Blacks develop a higher peak bone mass than whites which is associated with a reduced risk for bone fracture. The physiological basis for the difference in bone mass was investigated by metabolic balance and calcium kinetic studies in adolescent black and white girls. The hypothesis that the greater peak bone mass in blacks compared with whites is due to suppressed bone resorption was tested. Subjects were housed in a supervised environment for 3 wk during which time they consumed a controlled diet and collected all excreta. Subjects were given stable calcium isotopes orally and intravenously after 1 wk adaptation. Blacks have greater calcium retention (mean +/- SD, 11.5 +/- 6.1 vs. 7.3 +/- 4.1 mmol/d, P < 0.05) consistent with greater bone formation rates (49.4 +/- 13.5 vs. 36.5 +/- 13.6 mmol/d, P < 0.05) relative to bone resorption rates (37.4 +/- 13.2 vs. 29.4 +/- 10.9 mmol/d, P = 0.07), increased calcium absorption efficiency (54 +/- 19 vs. 38 +/- 18%, P < 0.05) and decreased urinary calcium (1.15 +/- 0.95 vs. 2.50 +/- 1.35 mmol/d, P < 0.001), compared with whites. The racial differences in calcium retention in adolescence can account for the racial differences in bone mass of adults.

Differences in calcium metabolism between adolescent and adult females.

A 3-wk metabolic study measured calcium balance in 14 white adolescent girls and 11 young adult women. Subjects were housed in a sorority to simulate a free-living environment. A 6-d menu cycle consisted of foods typically eaten by teenagers and averaged 1332 mg Ca/d. Adolescents had a significantly higher calcium balance of 326 +/- 107 mg/d (mean +/- SD) than adults, who averaged 73 +/- 104 mg/d (P < 0.001). No adult > age 21 y was in positive calcium balance. Adolescents had lower urinary calcium excretion values (P < 0.001), lower fecal calcium excretion (P < 0.01), and greater net absorption (P < 0.001) than adults. Calcium balance was negatively correlated with years postmenarche (r = -0.788, P = 0.0001) and height (r = -0.650, P = 0.001). Net calcium absorption was positively correlated with parathyroid hormone concentrations (r = 0.537, P = < 0.01). Thus, the growth demands of adolescents are met by more efficient net absorption and retention of calcium compared with young adults.

Using models to explore whole-body metabolism and accessing models through a model library.

A model is a mathematical representation of a system that can be used to explore the system in a number of ways: to determine the system's internal connections, to calculate properties of the system such as flow rates and pool sizes, and to make predictions about the system's behavior under different conditions. The use of modeling to explore whole-body metabolism is demonstrated using a compartmental model of zinc kinetics as an example. Because models are useful tools for exploring systems, a facility called a "model library" is being established on the Internet to provide access to working versions of published models.

Calcium kinetics with microgram stable isotope doses and saliva sampling.

Studies of calcium kinetics require administration of tracer doses of calcium and subsequent repeated sampling of biological fluids. This study was designed to develop techniques that would allow estimation of calcium kinetics by using small (micrograms) doses of isotopes instead of the more common large (mg) doses to minimize tracer perturbation of the system and reduce cost, and to explore the use of saliva sampling as an alternative to blood sampling. Subjects received an oral dose (133 micrograms) of 43Ca and an i.v. dose (7.7 micrograms) of 46Ca. Isotopic enrichment in blood, urine, saliva and feces was well above thermal ionization mass spectrometry measurement precision up to 170 h after dosing. Fractional calcium absorptions determined from isotopic ratios in blood, urine and saliva were similar. Compartmental modeling revealed that kinetic parameters determined from serum or saliva data were similar, decreasing the necessity for blood samples. It is concluded from these results that calcium kinetics can be assessed with micrograms doses of stable isotopes, thereby reducing tracer costs and with saliva samples, thereby reducing the amount of blood needed.

Distributing working versions of published mathematical models for biological systems via the Internet.

Mathematical models are useful tools for investigating complex systems. By representing physiological systems as models, theories can be tested quantitatively against data from the system. Models can be used to explore new theories prior to experimentation and to design studies to optimize experimental resources. They can also be used as teaching tools to illustrate physiochemical principles. In spite of their usefulness and the time invested in developing models, published models are often underused due to the difficulty in obtaining working versions of the model. To address this problem we have designed a library for mathematical models of biological systems on the Internet. The library contains published models of biological systems in formats compatible with several modeling packages, from the fields of physiology, metabolism, endocrinology, biochemistry, and chemistry. The models can be viewed graphically, model solutions can be viewed as plots against data, and models can be downloaded to be run with software on the user's own system. The address of the library is: http://biomodel.georgetown.edu/model/ Investigators are invited to submit working versions of published models to the library. Models can be submitted electronically at the time a manuscript is accepted for publication. As journals go online, articles containing models can be linked to working versions of the models in the library. By increasing access to working versions of models, more of the investment in kinetic studies and model development can be realized.

Compartmental analysis of zinc kinetics in mature male rats.

A compartmental model of zinc kinetics in mature male rats was developed. The model was based on zinc and radioisotopic zinc measured in samples of plasma, skeletal muscle, kidneys, testes, spleen, bone, and intestinal segments collected at various times for up to 4 days after 65Zn was injected intravenously. Zinc intake, excretion of zinc and 65Zn in urine and feces, and the whole body retention of 65Zn were also determined. Other data used to develop the model included published information on zinc concentration and zinc kinetics in tissues that were not sampled. In the model, the intestinal tract was represented by five compartments. Plasma, spleen, kidneys, and testes were each represented by an individual compartment. In contrast, two compartments each were used to represent exchangeable zinc in liver, bone, skeletal muscle, skin, and red blood cells. The present model extends earlier models of zinc kinetics, describes the distribution of zinc in the whole body, and may provide a means to evaluate the influence of either pathophysiological conditions or dietary extremes on the metabolism of zinc.

Development and application of a model for zinc metabolism in humans.

A model has been developed to describe zinc metabolism in humans. The model was developed from a series of studies in normal volunteers and patients with various clinical disorders. Zinc metabolism was studied with radiotracers for 9-12 mo before and after a daily oral zinc load of 100 mg. Data were analysed by compartmental analysis and a model was developed that consists of compartments for zinc in the gut, plasma, red blood cells, liver, muscle, bone and other tissues with excretion via urine and feces. Using the model parameters of zinc metabolism including absorption, tissue exchange, secretion and excretion have been determined, together with mass of zinc in tissues and in the whole body. The model has been used to identify five sites of long-term regulation of zinc metabolism in humans. These sites are absorption, excretion, exchange with red blood cells, release of zinc from muscle and secretion of zinc into gut. The model is currently being applied to several areas of nutrition including the effects of dietary fiber on zinc stores, the effects of aging on zinc metabolism and the zinc requirements of neonates.

Calculation of zinc absorption in humans using tracers by fecal monitoring and a compartmental approach.

Zinc absorption is often determined following oral administration of tracer by fecal monitoring as dose minus tracer excreted in feces. The value obtained for absorption with this method is influenced by excretion of absorbed tracer into feces during the fecal collection period and by any incomplete elimination of unabsorbed tracer. In the present study a published, physiologically based compartmental model of zinc metabolism has been used to calculate, after oral tracer administration, the fecal appearance of unabsorbed and absorbed tracer and the appearance when fractional rates of endogenous excretion and elimination were varied. Absorption was determined by fecal monitoring and compared to the value determined using parameter values of the compartmental model. The value calculated for absorption using fecal monitoring varied with length of fecal collection, rate of excretion of absorbed tracer (secretion) and rate of elimination of unabsorbed tracer. Absorption was determined correctly by fecal monitoring only when the amount of absorbed tracer excreted was equivalent to the amount of unabsorbed tracer remaining in the gut. Fecal monitoring determines a parameter representing the combined processes of absorption, endogenous excretion (or secretion) and fecal elimination.

Glucose turnover and hepatocyte glucose production of starved and toxaemic pregnant sheep.

Ewes bearing twins were starved for 10 days during the last month of gestation to induce ovine pregnancy toxaemia (OPT). Glucose turnover was measured by a primed continuous infusion of [U-14C]- and [6-3H]glucose at the end of 10 days of starvation (non-susceptible), or earlier when ewes became recumbent with OPT (susceptible). All ewes were slaughtered at the end of the infusion and hepatocytes were prepared in order to measure glucose production from different substrates. Many of the ewes had dead foetuses when slaughtered. Glucose production rates by hepatocytes with the substrates propionate, lactate or alanine were significantly less from the susceptible ewes than were those from non-susceptible ewes. These low rates were not stimulated by incubation with glucagon (10(-8) M), glutamine or glycerol. Rates of glucose turnover and of hepatic glucose production from all substrates were higher for ewes with dead than with live foetuses. The data support the hypothesis that pathogenesis of OPT is related to an impairment of hepatic gluconeogenesis, and further suggest that, in starved pregnant ewes, maternal glucose production may be restrained in the presence of a live foetus.

Ketone body kinetics in humans: a mathematical model.

A model has been developed to account for ketone body kinetics in man based on data following bolus injections of [14C]acetoacetate (A) and [14C]beta-OH butyrate (B) into normal humans in the postabsorptive state. The model consists of separate compartments for blood A and B that are linked by a tissue compartment in which rapid interconversion of the ketone bodies occurs. The probability of movement from blood into this compartment was assumed to be the same for both ketone bodies. Two slowly equilibrating tissue compartments are required to account for the slow components in the tracer data, and thus a five-compartment model is proposed. By modeling the transient tracer data with the tracee in a steady state, ketone body kinetics were defined in terms of the rapid interconversions of A and B, and the slow exchanges of carbon within the tissues. The rates of release of new A and B into blood, (UA and UB) were calculated. These rates were less than the apparent production rates, PRA and PRB, as the PR's included carbon atoms first released as the other ketone body. The exchange constants between the compartments were determined in addition to the fractional catabolic rates (FCR) and metabolic clearance rates (MCR) of A and B. The initial space of distribution was 10 L and the mean values +/- SD (n = 11), normalized to this volume, were UA = 6.4 +/- 5.0, UB = 8.8 +/- 8.0 (mumol L-1 min-1), FCRA = 0.226 +/- 0.142, FCRB = 0.188 +/- 0.124 (min-1), MCRA = 2.26 +/- 1.42, MCRB = 1.87 +/- 1.23 (L min-1) and PRA = 11.1 +/- 7.6, PRB = 12.7 +/- 10.0 (mumol L-1 min-1).

Changes in regulation of human zinc metabolism with age.

To assess changes in zinc metabolism with age, kinetic studies were performed in healthy adults (26 men, 21 women) aged 20-84 yr after a single oral or intravenous bolus of 65Zn. Studies covered two consecutive 9-mo periods while subjects were on a basal dietary intake of approximately 10 mg Zn/day and while taking an additional 100 mg Zn/day orally. Zinc metabolism was analyzed by compartmental analysis using data from plasma, red blood cells, urine, feces, liver, thigh, and whole body [M. E. Wastney, R. L. Aamodt, W. F. Rumble, and R. I. Henkin. Am. J. Physiol. 251 (Regulatory Integrative Comp. Physiol. 20): R398-R408, 1986]. Changes in observed and model calculated values of zinc metabolism were assessed on age by regression. During basal state, zinc release from red blood cells decreased with age. During zinc loading, response (defined as change from basal state) of plasma zinc concentration, urinary zinc excretion, and liver zinc increased with age, while response of fraction of zinc taken up by red blood cells decreased with age. In men, response of amount of zinc absorbed increased with age and in women response of fraction of endogenous zinc excreted decreased with age. Four responses that changed with age (urinary excretion, red blood cell exchange, absorption, and endogenous excretion) occurred at previously defined sites of regulation of zinc metabolism. Results show that regulation of zinc metabolism changes with age.

Mechanisms of the transfer of aminoacyl-tRNA from aminoacyl-tRNA synthetase to the elongation factor 1 alpha.

Aspartylation of mammalian tRNAAsp by bacteria-expressed human aspartyl-tRNA synthetase (hDRS) was examined. The kinetics of the aspartylation of tRNA was consistent with the following reaction pathway, [formula: see text] where E, represents aspartyl-tRNA synthetase. A set of rate constants was obtained which fit single turnover time courses at varying concentrations of the enzyme, tRNA, and AMP using the SAAM program. The dissociation of Asp-tRNA (k3) was found to be rate limiting. The elongation factor 1 alpha (EF1 alpha) and GTP stimulated the hDRS aspartylation. The stimulation depended on the presence of both EF1 alpha and GTP. Kinetic analysis indicated that EF1 alpha formed a complex with the hDRS-Asp-tRNA complex and stimulated the dissociation of Asp-tRNA. In the presence of 0.5 M NH4Cl, which enhances the binding of Asp-tRNA by EF1 alpha, hDRS-bound Asp-tRNA can be transferred directly to EF1 alpha. The implications of these results on the function of the multi-tRNA synthetase complex will be discussed.

DNA structural polymorphism modulates the kinetics of superhelical DNA cleavage by BamHI restriction endonuclease.

A compartmental model developed by Hensley (Hensley, P., Nardone, G., Chirikjian, J.G., and Wastney, M. E., (1990) J. Biol. Chem. 265, 15300-15307) for analysis of the time courses of the cleavage of superhelical DNA substrates by the restriction endonuclease, BamHI, has been used to quantify the effects of changes in temperature, ionic strength, superhelical density, and the DNA substrate on the binding and strand cleavage processes. Studies reported here indicate that changes in topology may be introduced into the DNA substrate solely as a result of the plasmid preparation process and in the absence of covalent bond cleavage and ligation. These changes in topology have qualitatively different effects on the kinetics than those promoted by changes in the superhelical density. The former are removed by briefly warming the DNA prior to assay, suggesting that they are only kinetically stable, while the latter changes are not affected by heating. Increasing the [NaCl] from 0.01 M to 0.1 M increases the overall rate of plasmid cleavage by increasing both the rates of cleavage and enzyme DNA association. To describe the decrease in the overall cleavage rate observed in 0.15 M NaCl, an ionic strength-dependent rate-determining structural transition in the DNA substrate was incorporated into the model. The largest changes in the rate of the cleavage process resulted from changes in the DNA substrate. For the SV40 substrate compared to pBR322, the rate constants describing the two association processes and the first bond cleavage event were increased 6- to 7-fold. The rate of the second bond cleavage process was not affected. These changes may be due to differences in the flanking sequences.