Calcium-41: a technology for monitoring changes in bone mineral.

The rare, long-lived radiotracer, 41Ca, measured by accelerator mass spectrometry in the urine or serum following incorporation into the bone provides an ultra-sensitive tool to assess changes in bone calcium balance in response to an intervention. Changes in bone balance can be followed for years with one small dose that is both radiologically and biologically non-invasive. Sequential interventions can be compared, with greater precision than they can with biochemical markers of bone turnover and with greater power than with bone densitometry. This method is especially useful to screen interventions over a period of weeks. The development and validation of this tool and its applications are reviewed. Mini abstract: Use of 41Ca measured in the urine or blood by accelerator mass spectrometry to assess bone balance provides a tool to compare the relative efficacy of multiple interventions. This perspective provides insights in the use of this novel method and comparisons with more traditional methods for evaluating the efficacy of interventions.

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.

Soft tissue calcification in the Ossabaw miniature pig: experimental and kinetic modeling studies.

UNLABELLED: Calcium (Ca) deposition into vascular tissue was measured in Ossabaw miniature pigs with and without metabolic syndrome (MetS) using Ca tracer kinetics and coronary atherosclerosis measured with intravascular ultrasound. Pigs with MetS had higher Ca uptake into coronary arteries than lean pigs. INTRODUCTION: Ca deposition into arteries is a common disease in humans. The Ossabaw pig develops MetS when fed an atherogenic diet. The aim of this study was to measure Ca deposition into arteries of lean vs. MetS pigs. METHODS: Male pigs were fed for 5 months with chow diet (healthy, lean; n = 7) or atherogenic diet (n = 8) consisting of chow supplemented with 2 % cholesterol, 43 % kcal from fat, and 20 % kcal from fructose. Pigs were verified to have MetS by obesity, insulin resistance, impaired glucose tolerance, dyslipidemia, and hypertension. Two pigs received 50 nCi of (41)Ca i.v. and blood was drawn frequently for 24 h, and 2, 3, 6, 8, 10, 15, 20, and at sacrifice at 28 days after injection. Peripheral arteries were biopsied four times per pig over the 28th day and coronary artery sampled at sacrifice. Tissues were analyzed for (41)Ca:Ca. A compartmental model was used to estimate rates of Ca deposition into the arteries. RESULTS: The MetS swine had higher (41)Ca and atherosclerosis in coronary arteries than lean pigs. CONCLUSIONS: This pig model is a suitable model for studying vascular calcification in humans.

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.

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.

Kinetics of zinc metabolism: variation with diet, genetics and disease.

Kinetic studies are used to investigate metabolic processes. By adding an isotope to a system and measuring its movement in the system over time, pool sizes and transport rates can be determined by mathematically modeling the data. This approach enables rate differences to be determined in conditions that have been modified by diet, environment, genetics or disease. Kinetic studies in humans have shown that there are multiple pools of zinc that turnover from minutes to years and that processes, including zinc absorption and excretion, are regulated to maintain tissue levels when zinc intake varies. Animal studies allow for greater understanding of kinetics because more tissues can be sampled and environmental and genetic factors can be controlled. Kinetic studies in animals will provide information on the overexpression or the deletion of genes coding for specific proteins involved in zinc transport and metabolism. The advances that have been made in our understanding of the role of zinc in metabolism have been aided by the development of techniques for measuring isotopes in biological materials. In the future, the kinetics of zinc bound to different compounds will be measured. Modeling will enable this information, at the molecular level, to be integrated with knowledge of zinc metabolism at the cellular, organ and whole body level. To understand more fully the role of zinc in human health, kinetic studies are needed in healthy and disease states to identify differences in metabolic processes. This knowledge can be used as a basis for dietary and therapeutic recommendations.

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.

Calcium metabolism before, during, and after a 3-mo spaceflight: kinetic and biochemical changes.

The loss of bone during spaceflight is considered a physiological obstacle for the exploration of other planets. This report of calcium metabolism before, during, and after long-duration spaceflight extends results from Skylab missions in the 1970s. Biochemical and endocrine indexes of calcium and bone metabolism were measured together with calcium absorption, excretion, and bone turnover using stable isotopes. Studies were conducted before, during, and after flight in three male subjects. Subjects varied in physical activity, yet all lost weight during flight. During flight, calcium intake and absorption decreased up to 50%, urinary calcium excretion increased up to 50%, and bone resorption (determined by kinetics or bone markers) increased by over 50%. Osteocalcin and bone-specific alkaline phosphatase, markers of bone formation, increased after flight. Subjects lost approximately 250 mg bone calcium per day during flight and regained bone calcium at a slower rate of approximately 100 mg/day for up to 3 mo after landing. Further studies are required to determine the time course of changes in calcium homeostasis during flight to develop and assess countermeasures against flight-induced bone loss.

The Niemann-Pick C1 protein resides in a vesicular compartment linked to retrograde transport of multiple lysosomal cargo.

Niemann-Pick C disease (NP-C) is a neurovisceral lysosomal storage disorder. A variety of studies have highlighted defective sterol trafficking from lysosomes in NP-C cells. However, the heterogeneous nature of additional accumulating metabolites suggests that the cellular lesion may involve a more generalized block in retrograde lysosomal trafficking. Immunocytochemical studies in fibroblasts reveal that the NPC1 gene product resides in a novel set of lysosome-associated membrane protein-2 (LAMP2)(+)/mannose 6-phosphate receptor(-) vesicles that can be distinguished from cholesterol-enriched LAMP2(+) lysosomes. Drugs that block sterol transport out of lysosomes also redistribute NPC1 to cholesterol-laden lysosomes. Sterol relocation from lysosomes in cultured human fibroblasts can be blocked at 21 degrees C, consistent with vesicle-mediated transfer. These findings suggest that NPC1(+) vesicles may transiently interact with lysosomes to facilitate sterol relocation. Independent of defective sterol trafficking, NP-C fibroblasts are also deficient in vesicle-mediated clearance of endocytosed [14C]sucrose. Compartmental modeling of the observed [14C]sucrose clearance data targets the trafficking defect caused by mutations in NPC1 to an endocytic compartment proximal to lysosomes. Low density lipoprotein uptake by normal cells retards retrograde transport of [14C]sucrose through this same kinetic compartment, further suggesting that it may contain the sterol-sensing NPC1 protein. We conclude that a distinctive organelle containing NPC1 mediates retrograde lysosomal transport of endocytosed cargo that is not restricted to sterol.

Zinc absorption, distribution, excretion, and retention by healthy preterm infants.

Zinc (Zn) is an essential nutrient for growth, but little is known about Zn absorption, distribution, excretion, and retention in preterm infants. Nine infants with gestational age 32+/-1 wk (mean+/-SE), birth weight 1.44+/-0.08 kg, postnatal age 14+/-3 d, on Zn intake of 23+/-3 micromol/kg per d via enteral feeding of preterm formula were studied. A stable Zn isotope (70Zn) was administered orally or i.v., and plasma, red blood cells, urine, and feces were sampled for up to 30 d. Samples were analyzed for Zn by inductively coupled plasma atomic emission spectrometry and for isotope enrichment by inductively coupled plasma mass spectrometry. Data were analyzed by compartmental analysis using the Simulation Analysis and Modeling program, and absorption, distribution, excretion, and retention were calculated. Absorption was 36+/-5% or 7+/-1 micromol/kg per d; distribution in plasma was 15+/-1 micromol Zn/L and in RBC was 41+/-4 micromol Zn/L; excretion in urine was 0.55+/-0.03 micromol Zn/kg per d and in feces was 17+/-3 micromol Zn/kg per d and retention was 5+/-1 microl/kg per d. Results show that healthy preterm infants with Zn intake of 23 micromol/kg per d and expected growth rates (> 15 g/kg per d) absorb and retain Zn at rates comparable to in utero accretion. The values for absorption, distribution, and excretion by this population of healthy preterm infants provide a normal range for future studies, although further studies are required to determine endogenous excretion rates in healthy preterm infants. We speculate that these values can be used to determine whether Zn kinetics are abnormal in sick infants or in infants with slow growth.

Calcium and oxalic acid kinetics differ in rats.

Small molecular weight calcium salts, if absorbed intact, could provide a nutritional source of calcium in subjects with impaired absorption of calcium by the saturable pathway. An understanding of the mechanism of absorption of calcium oxalate (as a representative salt) may be important nutritionally and therapeutically. The aim of the present study was to develop models to study absorption, distribution and retention of calcium and oxalate in rats as a basis for studying calcium oxalate absorption. Labeled compounds (45Ca and [14C]-oxalic acid) were administered to separate groups of rats orally (n = 8-11) or intravenously (n = 3-5) and blood was sampled for up to 240 min. Data were analyzed using SAAM/CONSAM. Calcium kinetics were fitted by a model with three compartments in the body and one absorption pathway from the intestine. By contrast, oxalic acid kinetics were fitted by two pools in the body and two absorption pathways from the intestine. Calcium and oxalic acid, therefore, demonstrate different absorption and distribution kinetics in rats.

Absorption of calcium oxalate does not require dissociation in rats.

Calcium absorption is thought to occur only if calcium is in a soluble or dissociated form, although experimental evidence is lacking. The intestinal absorption of calcium oxalate, a small, neutral and virtually insoluble calcium salt, was elucidated in the whole body of awake rats. Suspensions of 45Ca ascorbate, 14C-oxalic acid and doubly labeled 45Ca-[14C]-oxalate were given by gavage to separate groups of rats. Following dosing, blood samples were drawn for up to 240 min through a previously inserted intravenous catheter. Serum was assayed for radioactive tracers, and data were then plotted as fraction of dose over time. Calcium absorption was 15% [with a loading of 0.3 mmol (15 mg) calcium], oxalic acid absorption was 22% and Ca-oxalate absorption was <2%. Appearance of 45Ca from calcium ascorbate and 14C from oxalic acid differed, whereas 45Ca and 14C from doubly labeled Ca-oxalate had identical serum appearance profiles. Therefore, we conclude that calcium oxalate was absorbed intact. Addition of excess, unlabeled calcium to the doubly-labeled calcium oxalate did not alter the relationship of the serum level of the two tracers, confirming absorption of calcium oxalate as the intact salt. Thus, calcium bound as a small, neutral, calcium salt such as calcium oxalate does not have to be dissociated prior to absorption. Possibly other small compounds would be similarly absorbed. These results alter our current understanding of calcium bioavailability from foods and therapeutic agents.

Balancing needs, efficiency, and functionality in the provision of modeling software: a perspective of the NIH WinSAAM Project.

The development of new software or the refinement of existing software for new operating environments each calls for judicious balancing. On the one hand, we strive for simplicity, predictability, and operational protection as it is well recognized that software with these attributes will attract an audience of satisfied users. But, on the other hand, these attributes do not conjure a sense of power, efficiency, or flexibility, and these other properties are also appreciated by users, albeit a somewhat different group of users. The goal is to achieve a blend which isolates critical functionality, flexible control, and user support while meeting the needs of the broadest collection of serious users. In this chapter, we discuss the issues impacting the migration of SAAM to the Windows environment, the NIH WinSAAM Project, and we outline the steps taken to ensure its feasibility. In addition, we describe a new paradigm for software development and use which ensures the durability of the software for modeling.

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.

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.

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.

Magnesium kinetics in adolescent girls determined using stable isotopes: effects of high and low calcium intake.

Magnesium kinetics were measured in five adolescent girls who were participating in a calcium balance study. Two calcium levels were fed in a randomized crossover design. After an acclimation period, 26Mg was consumed orally and 25Mg was given intravenously, and then blood, urine, and feces were collected for 14 days. Total magnesium and percent enrichment were determined, and data were fitted to a eight-compartment model. There was no significant difference between high and low calcium intakes for any of the parameters examined. Mean values for control (800 mg/day) and high (1,800 mg/day) calcium intake were as follows: Mg intake, 305 +/- 30 and 286 +/- 9 mg/day; absorption (percent), 44 +/- 7 and 39 +/- 9; absorption (mg/day), 134 +/- 35 and 110 +/- 28; urinary excretion, 96 +/- 22 and 101 +/- 31 mg/day; fecal excretion, 175 +/- 32 and 200 +/- 11 mg/day; and magnesium balance, 13 +/- 35 and -34 +/- 48 mg/day, respectively. In conclusion, high calcium intake did not alter magnesium kinetics or balance in adolescent girls.

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.