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.

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.

Bone-seeking labels as markers for bone turnover: validation of urinary excretion in rats.

UNLABELLED: Urinary excretion of tritiated tetracycline ((3)H-TC) and (41)Ca tracers was validated as reflecting skeletal disappearance of these bone-seeking tracers as a direct measure of bone turnover following ovariectomy in rats. INTRODUCTION: Tritiated tetracycline ((3)H-TC) and Ca tracers have been used to measure bone resorption in animal models, but urinary excretion of these labels has not been directly compared to skeletal turnover. We aimed to evaluate the use of bone-seeking labels by comparing label release into urine with label in the skeleton when bone turnover was perturbed following ovariectomy. METHODS: Sixty-four 6-month-old ovariectomized (OVX) rats were randomized to one of eight groups in a 2 × 4 design that differed in time of (3)H-TC and (41)Ca administration following ovariectomy (1 month, when bone turnover would be accelerated following estrogen depletion or 3 months when bone loss due to OVX had slowed down) and time of euthanasia (1 week, 1 month, 3 months, and 6 months post-dose). Twenty-four-hour urine pools over two to four consecutive days and total skeleton were collected and recovered for the assessment of (3)H-TC and (41)Ca. RESULTS: Urinary (3)H-TC levels reflected skeletal (3)H-TC levels (r = 0.93; p < 0.0001) over a wide range of bone turnover rates in response to an intervention. Urinary (41)Ca and (3)H-TC excretion were highly correlated (r = 0.95, p < 0.0001). CONCLUSION: This study confirms that bone-seeking label excretion into the urine directly measures bone turnover.

Antiresorptive effects of phytoestrogen supplements compared with estradiol or risedronate in postmenopausal women using (41)Ca methodology.

INTRODUCTION: Reduction of ovarian estrogen secretion at menopause increases net bone resorption and leads to bone loss. Isoflavones have been reported to protect bone from estrogen deficiency, but their modest effects on bone resorption have been difficult to measure with traditional analytical methods. METHODS: In this randomized-order, crossover, blinded trial in 11 healthy postmenopausal women, we compared four commercial sources of isoflavones from soy cotyledon, soy germ, kudzu, and red clover and a positive control of oral 1 mg estradiol combined with 2.5 mg medroxyprogesterone or 5 mg/d oral risedronate (Actonel) for their antiresorptive effects on bone using novel (41)Ca methodology. RESULTS: Risedronate and estrogen plus progesterone decreased net bone resorption measured by urinary (41)Ca by 22 and 24%, respectively (P < 0.0001). Despite serum isoflavone profiles indicating bioavailability of the phytoestrogens, only soy isoflavones from the cotyledon and germ significantly decreased net bone resorption by 9% (P = 0.0002) and 5% (P = 0.03), respectively. Calcium absorption and biochemical markers of bone turnover were not influenced by interventions. CONCLUSIONS: Dietary supplements containing genistein-like isoflavones demonstrated a significant but modest ability to suppress net bone resorption in postmenopausal women at the doses supplied in this study over a 50-d intervention period.

Crystal structure of human prion protein bound to a therapeutic antibody.

Prion infection is characterized by the conversion of host cellular prion protein (PrP(C)) into disease-related conformers (PrP(Sc)) and can be arrested in vivo by passive immunization with anti-PrP monoclonal antibodies. Here, we show that the ability of an antibody to cure prion-infected cells correlates with its binding affinity for PrP(C) rather than PrP(Sc). We have visualized this interaction at the molecular level by determining the crystal structure of human PrP bound to the Fab fragment of monoclonal antibody ICSM 18, which has the highest affinity for PrP(C) and the highest therapeutic potency in vitro and in vivo. In this crystal structure, human PrP is observed in its native PrP(C) conformation. Interactions between neighboring PrP molecules in the crystal structure are mediated by close homotypic contacts between residues at position 129 that lead to the formation of a 4-strand intermolecular beta-sheet. The importance of this residue in mediating protein-protein contact could explain the genetic susceptibility and prion strain selection determined by polymorphic residue 129 in human prion disease, one of the strongest common susceptibility polymorphisms known in any human disease.

Soy isoflavones do not affect bone resorption in postmenopausal women: a dose-response study using a novel approach with 41Ca.

INTRODUCTION: The purpose of this 3-way crossover study was to identify the effective dose of soy protein isolate enriched with isoflavones for suppressing bone resorption in postmenopausal women using a novel, rapid assessment of antibone resorbing treatments. METHODS: Thirteen postmenopausal women (>or=6 yr since menopause) were predosed with 41Ca iv. After a 200-d baseline period, subjects were given 43 g soy protein/d that contained 0, 97.5, or 135.5 mg total isoflavones in randomized order. The soy protein isolate powder was incorporated into baked products and beverages. Each 50-d intervention phase was preceded by a 50-d pretreatment phase for comparison. Serum isoflavone levels and biochemical markers were measured at the end of each phase. Twenty-four-hour urine samples were collected approximately every 10 d during each phase for 41Ca/Ca analysis by accelerator mass spectrometry. RESULTS: Serum isoflavone levels reflected the amount of isoflavones consumed in a dose-dependent manner. None of the isoflavone levels had a significant effect on biochemical markers of bone turnover, urinary cross-linked N teleopeptides of type I collagen and serum osteocalcin, or bone turnover as assessed by urinary 41Ca/Ca ratios. CONCLUSIONS: Soy protein with isoflavone doses of up to 135.5 mg/d did not suppress bone resorption in postmenopausal women. This is the first efficacy trial using the novel technique of urinary 41Ca excretion from prelabeled bone.

The role of the cellular prion protein in the immune system.

Prion protein (PrP) plays a key role in the pathogenesis of prion diseases. However, the normal function of the protein remains unclear. The cellular isoform (PrP(C)) is expressed widely in the immune system, in haematopoietic stem cells and mature lymphoid and myeloid compartments in addition to cells of the central nervous system. It is up-regulated in T cell activation and may be expressed at higher levels by specialized classes of lymphocyte. Furthermore, antibody cross-linking of surface PrP modulates T cell activation and leads to rearrangements of lipid raft constituents and increased phosphorylation of signalling proteins. These findings appear to indicate an important but, as yet, ill-defined role in T cell function. Although PrP(-/-) mice have been reported to have only minor alterations in immune function, recent work has suggested that PrP is required for self-renewal of haematopoietic stem cells. Here, we consider the evidence for a distinctive role for PrP(C) in the immune system and what the effects of anti-prion therapeutics may be on immune function.

The molecular pathology of CJD: old and new variants.

The study of prion disease has become an area of intense interest since experimental evidence emerged for the transmission of phenotypic variation without the involvement of a nucleic acid component. Additional impetus has come from the widespread concern that exposure to bovine spongiform encephalopathy contaminated material poses a distinct and, conceivably, a severe threat to public health in the UK and other countries. The occurrence of new variant Creutzfeldt-Jakob disease has dramatically highlighted the need for a precise understanding of the molecular basis of prion propagation. The molecular basis of prion strain diversity, previously a major challenge to the "protein only" model, can now be reconciled with propagation of infectious protein topologies. The conformational change known to be central to prion propagation, from a predominantly alpha-helical fold to one predominantly comprising beta-structure, can now be reproduced in vitro, and the ability of beta-PrP to form fibrillar aggregates provides a plausible molecular mechanism for prion propagation. Concomitantly, advances in the fundamental biology of prion disease have done much to reinforce the protein only hypothesis of prion replication.

Spontaneous conformational change within the prion protein--implications for disease pathogenesis?

A recent paper by Leclerc et al(1) describes how recombinant hamster prion protein can undergo a spontaneous change in conformation to a structure that has features in common with PrP(Sc). Structural change in the host prion protein, PrP(C) to an insoluble and aggregated form with increased beta-sheet content (PrP(Sc)) is central to the pathology of prion diseases.(2) A detailed understanding of the nature of these conformational changes will increase our knowledge of the molecular basis of prion pathology. These findings may have implications for how the disease is initiated and provide a format for further investigation.

XAFS study of the high-affinity copper-binding site of human PrP(91-231) and its low-resolution structure in solution.

Here, we describe the structure of a C-terminal high-affinity copper-binding site within a truncated recombinant human PrP containing residues 91-231, which lacks the octapeptide repeat region. We show that at least two extra co-ordinating groups are involved in binding this copper(II) ion in conjunction with histidine residues 96 and 111 in a region of the molecule known to be critical in conferring strain type. In addition, using X-ray solution scattering, a low-resolution shape of PrP(91-231) is provided. The restored molecular envelope is consistent with the picture where the N-terminal segment, residues 91-120, extends out from the previously known globular domain containing residues 121-231.

Location and properties of metal-binding sites on the human prion protein.

Although a functional role in copper binding has been suggested for the prion protein, evidence for binding at affinities characteristic of authentic metal-binding proteins has been lacking. By presentation of copper(II) ions in the presence of the weak chelator glycine, we have now characterized two high-affinity binding sites for divalent transition metals within the human prion protein. One is in the N-terminal octapeptide-repeat segment and has a K(d) for copper(II) of 10(-14) M, with other metals (Ni(2+), Zn(2+), and Mn(2+)) binding three or more orders of magnitude more weakly. However, NMR and fluorescence data reveal a previously unreported second site around histidines 96 and 111, a region of the molecule known to be crucial for prion propagation. The K(d) for copper(II) at this site is 4 x 10(-14) M, whereas nickel(II), zinc(II), and manganese(II) bind 6, 7, and 10 orders of magnitude more weakly, respectively, regardless of whether the protein is in its oxidized alpha-helical (alpha-PrP) or reduced beta-sheet (beta-PrP) conformation. A role for prion protein (PrP) in copper metabolism or transport seems likely and disturbance of this function may be involved in prion-related neurotoxicity.

The molecular biology of prion propagation.

Prion diseases such as Creutzfeldt-Jakob disease (CJD) in humans and scrapie and bovine spongiform encephalopathy (BSE) in animals are associated with the accumulation in affected brains of a conformational isomer (PrP(Sc)) of host-derived prion protein (PrP(C)). According to the protein-only hypothesis, PrP(Sc) is the principal or sole component of transmissible prions. The conformational change known to be central to prion propagation, from a predominantly alpha-helical fold to one predominantly comprising beta structure, can now be reproduced in vitro, and the ability of beta-PrP to form fibrillar aggregates provides a plausible molecular mechanism for prion propagation. The existence of multiple prion strains has been difficult to explain in terms of a protein-only infectious agent but recent studies of human prion diseases suggest that strain-specific phenotypes can be encoded by different PrP conformations and glycosylation patterns. The experimental confirmation that a novel form of human prion disease, variant CJD, is caused by the same prion strain as cattle BSE, has highlighted the pressing need to understand the molecular basis of prion propagation and the transmission barriers that limit their passage between mammalian species. These and other advances in the fundamental biology of prion propagation are leading to strategies for the development of rational therapeutics.

Use of accelerator mass spectrometry for studies in nutrition.

Accelerator mass spectrometry (AMS) is an ultrasensitive analytical technique for measuring rare nuclides such as 14C, 26Al and 41Ca. The low detection limit and wide dynamic range of AMS allow long-term and highly sensitive tracer studies in nutrition that cannot be performed with other methods. The present paper is intended to provide a description of AMS to the interested nutritionist and present proven applications. AMS is compared to liquid scintillation counting and stable isotope MS. A description of common AMS methodology is presented that consists of determining the dose, preparing the sample, diluting the sample (if necessary), and measuring the sample. Applications include Ca metabolism, Al uptake from the environment, dietary intake of carcinogens, fat meta-bolism and folate metabolism. Throughout this discussion the experimental advantages (small doses that pose no health risk, extremely long experimental lifetime, small sample sizes and high sensitivity) made possible by the unique analytical capabilities of AMS are emphasized. The future of AMS is discussed. As the number of AMS centres, instruments, and studies increases, the number of nutritional applications that employ AMS will continue to grow. The coupling of AMS with other analytical techniques (e.g. high performance liquid chromatography) will be developed as access to AMS improves.

Prion disease--the propagation of infectious protein topologies.

Prion propagation is associated with accumulation of a conformational isomer of host encoded cellular prion protein, PrP(C). Solution structures of several mammalian PrPs have now been reported and they have stimulated a significant advance in our understanding of the folding dynamics of PrP. Studies on recombinant PrP have shown the polypeptide chain is able to adopt different topologies in different solvent conditions. Concomitantly, advances in the analysis of the abnormal isoform, PrP(Sc), have expanded our knowledge on the molecular basis of prion strains and have done much to reinforce the protein-only hypothesis of prion replication.

Importance of gas-phase proton affinities in determining the electrospray ionization response for analytes and solvents.

The effect of gas-phase proton transfer reactions on the mass spectral response of solvents and analytes with known gas-phase proton affinities was evaluated. Methanol, ethanol, propanol and water mixtures were employed to probe the effect of gas-phase proton transfer reactions on the abundance of protonated solvent ions. Ion-molecule reactions were carried out either in an atmospheric pressure electrospray ionization source or in the central quadrupole of a triple-quadrupole mass spectrometer. The introduction of solvent vapor with higher gas-phase proton affinity than the solvent being electrosprayed caused protons to transfer to the gas-phase solvent molecules. In mixed solvents, protonated solvent clusters of the solvent with higher gas-phase proton affinity dominated the resulting mass spectra. The effect of solvent gas-phase proton affinity on analyte response was also investigated, and the analyte response was suppressed or eliminated in solvents with gas-phase proton affinities higher than that of the analyte.

Mammalian prion proteins.

The past two years have seen the extension of our knowledge on the cellular prion protein structure with new NMR data on both the hamster and human proteins. In addition, the folding dynamics of two cellular prion proteins have been elucidated. There are now several examples of recombinant prion proteins that are able to adopt different conformations in solution and recent work on the molecular basis of prion strains has done much to consolidate the protein-only hypothesis. Important advances in relating disease to structure have also been made through the identification of the minimal prion protein fragment that is capable of conferring susceptibility to and propagation of the scrapie agent.

Strain-specific prion-protein conformation determined by metal ions.

In animals infected with a transmissible spongiform encephalopathy, or prion disease, conformational isomers (known as PrPSc proteins) of the wild-type, host-encoded cellular prion protein (PrPc) accumulate. The infectious agents, prions, are composed mainly of these conformational isomers, with distinct prion isolates or strains being associated with different PrPSc conformations and patterns of glycosylation. Here we show that two different human PrPSc types, seen in clinically distinct subtypes of classical Creutzfeldt-Jakob disease, can be interconverted in vitro by altering their metal-ion occupancy. The dependence of PrPSc conformation on the binding of copper and zinc represents a new mechanism for post-translational modification of PrP and for the generation of multiple prion strains, with widespread implications for both the molecular classification and the pathogenesis of prion diseases in humans and animals.

Electrical equivalence of electrospray ionization with conducting and nonconducting needles.

An electrical equivalent circuit is derived for the electrospray process. It is a series circuit which consists of the power supply, the electrochemical contact to the solution, the solution resistance (R(s)), a constant-current regulator which represents the processes of charge separation and charge transport in the gap between the spray needle aperture and the counter electrode, and charge neutralization at the counter electrode. A current i, established by the constant-current regulator flows throughout the entire circuit. Current-voltage curves are developed for each element in the circuit. From these it is shown that in the case where R(s) is negligible (the power supply is connected directly to a conducting needle) the shape of the current-voltage curve is dictated by the constant-current regulator established by the charge separation process, the gap, and the counter electrode. The solution resistance may be significant if a nonconducting needle is used so that the electrochemical contact to the solution is remote from the tip. Experiments with a nonconducting spray needle quantify the effect of the solution resistance on the current-voltage curve. Subtracting the iRs voltage from Vapp (power supply voltage) yields the current-voltage curve for the constant-current regulator. When iRs drop is a significant fraction of Vapp, the current-voltage curve of the constant-current regulator is changed substantially from the case when the solution resistance is negligible.

Structural mobility of the human prion protein probed by backbone hydrogen exchange.

Prions, the causative agents of Creutzfeldt-Jacob Disease (CJD) in humans and bovine spongiform encephalopathy (BSE) and scrapie in animals, are principally composed of PrPSc, a conformational isomer of cellular prion protein (PrPC). The propensity of PrPC to adopt alternative folds suggests that there may be an unusually high proportion of alternative conformations in dynamic equilibrium with the native state. However, the rates of hydrogen/deuterium exchange demonstrate that the conformation of human PrPC is not abnormally plastic. The stable core of PrPC has extensive contributions from all three alpha-helices and shows protection factors equal to the equilibrium constant for the major unfolding transition. A residual, hyper-stable region is retained upon unfolding, and exchange analysis identifies this as a small nucleus of approximately 10 residues around the disulfide bond. These results show that the most likely route for the conversion of PrPC to PrPSc is through a highly unfolded state that retains, at most, only this small nucleus of structure, rather than through a highly organized folding intermediate.