Accelerating clinical insights: how to use accelerator mass spectrometry to make better early development decisions

This paper is an overview of the applications of the technique of Accelerator Mass Spectrometry (AMS) in the biomedical drug development field. The work described here has been carried out at Xceleron (York, UK and Germantown, MD, USA), and it aims to apply AMS to provide better information about the human pharmacokinetic/metabolic behaviour of drugs or drug candidates as early as possible. It is hoped that the use of this technique will contribute to the delivery of better, more effective drugs onto the market sooner, which will be good news for all concerned.     

Detection of Adriamycin–DNA adducts by accelerator mass spectrometry at clinically relevant Adriamycin concentrations

Limited sensitivity of existing assays has prevented investigation of whether Adriamycin–DNA adducts are involved in the anti-tumour potential of Adriamycin. Previous detection has achieved a sensitivity of a few Adriamycin–DNA adducts/10⁴ bp DNA, but has required the use of supra-clinical drug concentrations. This work sought to measure Adriamycin–DNA adducts at sub-micromolar doses using accelerator mass spectrometry (AMS), a technique with origins in geochemistry for radiocarbon dating. We have used conditions previously validated (by less sensitive decay counting) to extract [¹⁴C]Adriamycin–DNA adducts from cells and adapted the methodology to AMS detection. Here we show the first direct evidence of Adriamycin–DNA adducts at clinically-relevant Adriamycin concentrations. [¹⁴C]Adriamycin treatment (25 nM) resulted in 4.4 ± 1.0 adducts/10⁷ bp (~1300 adducts/cell) in MCF-7 breast cancer cells, representing the best sensitivity and precision reported to date for the covalent binding of Adriamycin to DNA. The exceedingly sensitive nature of AMS has enabled over three orders of magnitude increased sensitivity of Adriamycin–DNA adduct detection and revealed adduct formation within an hour of drug treatment. This method has been shown to be highly reproducible for the measurement of Adriamycin–DNA adducts in tumour cells in culture and can now be applied to the detection of these adducts in human tissues.     

NMR analysis of plant nitrogen metabolism

The analysis of primary and secondary nitrogen metabolism in plants by nuclear magnetic resonance (NMR) spectroscopy is comprehensively reviewed. NMR is a versatile analytical tool, and the combined use of ¹H, ²H, ¹³C, ¹⁴N and ¹⁵N NMR allows detailed investigation of the acquisition, assimilation and metabolism of nitrogen. The analysis of tissue extracts can be complemented by the in vivo NMR analysis of functioning tissues and cell suspensions, and by the application of solid state NMR techniques. Moreover stable isotope labelling with ²H-, ¹³C- and ¹⁵N-labelled precursors provides direct insight into specific pathways, with the option of both time-course and steady state analysis increasing the potential value of the approach. The scope of the NMR method, and its contribution to studies of plant nitrogen metabolism, are illustrated with a wide range of examples. These include studies of the GS/GOGAT pathway of ammonium assimilation, investigations of the metabolism of glutamate, glycine and other amino acids, and applications to tropane alkaloid metabolism. The continuing development of the NMR technique, together with potential applications in the emerging fields of metabolomics and metabolic flux analysis, leads to the conclusion that NMR will play an increasingly valuable role in the analysis of plant nitrogen metabolism.     

SIMS MICROSCOPY: METHODOLOGY,PROBLEMS AND PERSPECTIVES IN MAPPING DRUGS AND NUCLEAR MEDICINE COMPOUNDS

Secondary ion mass spectrometry (SIMS) microscopy, a mass spectrometry method designed in the 1960s, offers new analytical capabilities, high sensitivity (ppm to ppb region), high specificity and improved lateral resolution, thus facilitating insight into many physiological and biomedical questions. Apart from the sample preparation and the physical characteristics of the detection, the biological model must also be considered. SIMS analysis of diffusible ions and molecules requires strict cryogenic procedures which always begin by a flash-freeze fixation. Cellular integrity can be checked by mapping the major element distributions since intra and extracellular ions are redistributed only in damaged cells. Cryofixing may be followed either by a freeze-fracture methodology or by cryoembedding and dry-cutting. Chemical sample preparation is only used for ions or molecules bound to fixed cell structures. The use of scanning procedures ameliorates the lateral resolution and chromosome imaging has been reported with probe size of below 50nm. Absolute quantification can be derived for embedded specimen by using internal references included in tissue equivalent resins. The sensitivity is limited by the ionization yield of the tag element and may be further impaired when working at high mass resolution (≥5000) to eliminate interfering cluster ions. SIMS drug mapping is usually performed after in vitro administration of a molecule to cell culture systems. Drug detection is accomplished indirectly by detecting a tag isotope naturally present or introduced by labelling, mainly with halogens,¹⁵N and¹⁴C. Molecular imaging with TOF-SIMS is an appealing alternative especially for heavier compounds. We stress some biological problems through a critical review of published SIMS drug studies. SIMS proved useful in assessing the targeting specificity of nuclear medicine pharmaceutics, even after in vivo administration. The first microscopic evidence of a thionamide induced follicular blockade of the iodine organification process is presented in a human sample.     

Clinical cardiac magnetic resonance spectroscopy - present state and future directions

MR spectroscopy opens a window to the non-invasive evaluation of various aspects of cardiac metabolism. Experimentally, the method has extensively been used since 1970's. ³¹P-MR allows the registration of cardiac high-energy phosphate metabolism to non-invasively estimate the energetic state of the heart: ATP, phosphocreatine, inorganic phosphate, monophosphate esters and intracellular pH can all be quantitated. In conjunction with extracellular shift reagents such as [DyTTHA]^3- or [TmDOTP]^5-5-, ²³Na- and ³⁹K-MR allow the measurement of intra- and extra-cellular cation pools. ¹H-MR spectroscopy allows the detection of a large number of metabolites such as, e.g. creatine, lactate, or carnitine.Human cardiac spectrocsopy has so far been confined to the³¹ P nucleus. Localization techniques (DRESS, ISIS, 3D-CSI etc.) are required to confine the acquired signal to the heart region. Relative quantification is straightforward (phosphocreatine/ATP ratio), absolute quantification (mM) is under development. Cardiac³¹ P-MR spectroscopy has research application in at least three clinical areas: (1) Coronary artery disease: A biochemical stress test for non-invasive ischemia detection (decrease of phosphocreatine with exercise) and viability assessment via quantification of ATP may become feasible. (2) Heart failure: The phosphocreatine /ATP ratio may provide an independent index for grading of heart failure, allow to monitor the longterm effects of different forms of drug therapy on cardiac energy metabolism in heart failure, and may also hold prognostic information on survival. (3) Valve disease: It is possible that the decrease of phosphocreatine/ATP can be used to guide the timing for the valve replacement.At the present time, no routine clinical applications can be defined for the use of human cardiac spectroscopy in patients with cardiac disease. However, the technique holds great potential for the future as a non-invasive approach to cardiac metabolism, and in coming years routine applications may become reality.     

Radiocarbon dating: The continuing revolution

Radiocarbon (¹⁴C) dating, now in its fifth decade of general use, continues to be the most widely employed method of inferring chronometric age for late Pleistocene and Holocene age materials recovered from archeological contexts. Over the last decade, several technical advances in ¹⁴C studies have provided contexts for a number of significant applications in archeology that were previously either not possible or not practical. These include the extension of the calibrated ¹⁴C time scale into the late Pleistocene and the development of accelerator mass spectrometry (AMS). The contribution of AMS-based ¹⁴C values to the critical evaluation of archeological data is illustrated by considering the problems of dating early plant domestication in the Near East and Mesoamerica, New World Paleoindian human skeletal materials, and European Upper Paleolithic and Mesolithic materials.     

The chick embryo: hatching a model for contemporary biomedical research

Animal models play a crucial role in fundamental and medical research. Progress in the fields of drug discovery, regenerative medicine and cancer research among others are heavily dependent on in vivo models to validate in vitro observations, and develop new therapeutic approaches. However, conventional rodent and large animal experiments face ethical, practical and technical issues that limit their usage. The chick embryo represents an accessible and economical in vivo model, which has long been used in developmental biology, gene expression analysis and loss/gain of function experiments. It is also an established model for tissue/cell transplantation, and because of its lack of immune system in early development, the chick embryo is increasingly recognised as a model of choice for mammalian biology with new applications for stem cell and cancer research. Here, we review novel applications of the chick embryo model, and discuss future developments of this in vivo model for biomedical research.     

Stable isotope analysis of modern human hair collected from Asia (China,India, Mongolia,and Pakistan)

We report isotopic data (δ²H, δ¹⁸O n = 196; δ¹³C, δ¹⁵N n = 142; δ³⁴S n = 85) from human hair and drinking water (δ²H, δ¹⁸O n = 67) collected across China, India, Mongolia, and Pakistan. Hair isotope ratios reflected the large environmental isotopic gradients and dietary differences. Geographic information was recorded in H and O and to a lesser extent, S isotopes. H and O data were entered into a recently developed model describing the relationship between the H and O isotope composition of human hair and drinking water in modern USA and pre‐globalized populations. This has anthropological and forensic applications including reconstructing environment and diet in modern and ancient human hair. However, it has not been applied to a modern population outside of the USA, where we expect different diet. Relationships between H and O isotope ratios in drinking water and hair of modern human populations in Asia were different to both modern USA and pre‐globalized populations. However, the Asian dataset was closer to the modern USA than to pre‐globalized populations. Model parameters suggested slightly higher consumption of locally producedfoods in our sampled population than modern USA residents, but lower than pre‐globalized populations. The degree of in vivo amino acid synthesis was comparable to both the modern USA and pre‐globalized populations. C isotope ratios reflected the predominantly C₃‐based regional agriculture and C₄ consumption in northernChina. C, N, and S isotope ratios supported marine food consumption in some coastal locales. N isotope ratios suggested a relatively low consumption of animal‐derived products compared to western populations. Am J Phys Anthropol 2010. © 2009 Wiley‐Liss, Inc.     

基于微流控芯片的秀丽隐杆线虫的研究进展

微流控芯片技术作为近年来最前沿的分析技术之一,已经在化学、生物学、医药学等研究领域取得了突破性的进展.微流控芯片具有高通量、微型化和多功能集成化等独特优势,已经成为生物医学研究的新平台之一,被越来越多地应用于秀丽隐杆线虫的研究.综述了基于微流控芯片上的秀丽隐杆线虫在生物医学领域中的研究进展,侧重介绍了微流控芯片在线虫的自动化固定、行为学、衰老与发育学、神经学、药物筛选及基因筛选等六大方面所取得的最新进展,并展望了微流控芯片的应用前景.     

Dating archaeobotanical remains: a cautionary tale from Port au Choix,Newfoundland

In this paper we report on the first ¹⁴C dated archaeological seeds from the island of Newfoundland, Canada. Ninety-three archaeobotanical specimens were recovered from a midden deposit adjacent to a small dwelling at Point Riche (EeBi-20), a large Dorset Palaeoeskimo site near Port au Choix, northwestern Newfoundland. These remains were collected from a seemingly secure context within the midden, but AMS ¹⁴C testing of a sample of specimens produced modern ¹⁴C dates, indicating that the remains are intrusive to the Dorset occupation. While the majority of Newfoundland-based research assumes antiquity of archaeobotanical remains, we recommend using AMS ¹⁴C dating and other proxy data in future archaeobotanical studies to confirm antiquity prior to making interpretations regarding human–plant interactions.     

Dual‐tracer‐based isotope turnover rates in a highly invasive mysid Limnomysis benedeni from Lake Constance

Understanding the ecological patterns of invasive species and their habitats require an understanding of the species’ foraging ecology. Stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope values provide useful information into the study of animal ecology and evolution, since the isotope ratios of consumers reflect consumer's dietary patterns. Nevertheless, the lack of species‐ and element‐specific laboratory‐derived turnover rates could limit their application. Using a laboratory‐based dual stable isotope tracer approach (Na¹⁵NO₃ and NaH¹³CO₃), we evaluated the δ¹⁵N and δ¹³C isotope turnover rates in full‐grown adult invasive Limnomysis benedeni from Lake Constance. We provide δ¹⁵N and δ¹³C turnover rates based on nonlinear least‐squares regression and posterior linear regression models. Model precisions and fit were evaluated using Akaike's information criterion. Within a couple of days, the δ¹⁵N and δ¹³C of mysids began to change. Nevertheless, after about 14 days, L. benedeni did not reach equilibrium with their new isotope values. Since the experiment was conducted on adult subjects, it is evident that turnover was mainly influenced by metabolism (in contrast to growth). Unlike traditional dietary shifts, our laboratory‐based dual stable isotope tracer approach does not shift the experimental organisms into a new diet and avoids dietary effects on isotope values. Results confirm the application of isotopic tracers to label mysid subpopulations and could be used to reflect assimilation and turnover from the labeled dietary sources. Field‐based stable isotope studies often use isotopic mixing models commonly assuming diet‐tissue steady state. Unfortunately, in cases where the isotopic composition of the animal is not in equilibrium with its diet, this can lead to highly misleading conclusions. Thus, our laboratory‐based isotopic incorporation rates assist interpretation of the isotopic values from the field and provide a foundation for future research into using isotopic tracers to investigate invasion ecology.     

15N自然丰度法在生态系统氮素循环研究中的应用

稳定性同位素技术是现代生态学研究中的一门新兴技术,在生态学研究的诸多领域都展示了广阔的应用前景。其中,稳定性同位素     

Metabolic origin of δ15N values in nitrogenous compounds from Brassica napus L. leaves

Nitrogen isotope composition (δ¹⁵N) in plant organic matter is currently used as a natural tracer of nitrogen acquisition efficiency. However, the δ¹⁵N value of whole leaf material does not properly reflect the way in which N is assimilated because isotope fractionations along metabolic reactions may cause substantial differences among leaf compounds. In other words, any change in metabolic composition or allocation pattern may cause undesirable variability in leaf δ¹⁵N. Here, we investigated the δ¹⁵N in different leaf fractions and individual metabolites from rapeseed (Brassica napus) leaves. We show that there were substantial differences in δ¹⁵N between nitrogenous compounds (up to 30‰) and the content in (¹⁵N enriched) nitrate had a clear influence on leaf δ¹⁵N. Using a simple steady‐state model of day metabolism, we suggest that the δ¹⁵N value in major amino acids was mostly explained by isotope fractionation associated with isotope effects on enzyme‐catalysed reactions in primary nitrogen metabolism. δ¹⁵N values were further influenced by light versus dark conditions and the probable occurrence of alternative biosynthetic pathways. We conclude that both biochemical pathways (that fractionate between isotopes) and nitrogen sources (used for amino acid production) should be considered when interpreting the δ¹⁵N value of leaf nitrogenous compounds.     

Xenon for NMR biosensing – Inert but alert

NMR studies with hyperpolarized xenon as functionalized sensor or contrast agent recently made notable progress in developing a new approach for detecting molecular markers and parameters of biomedical interest. Combining spin polarization enhancement with novel indirect detection schemes easily enables a 10⁷-fold signal gain, thus having promising potential to solve the NMR sensitivity problem in many applications. Though an inert element, ¹²⁹Xe has exquisite NMR properties to sense molecular environments. This review summarizes recent developments in the production of hyperpolarized xenon and the design and detection schemes of xenon biosensors.     

基于单分子量子相干调制的细胞温度成像技术

目的 细胞温度成像可以帮助科学家研究和理解细胞内部的温度分布,揭示细胞代谢和生物化学过程的关键信息。目前,基于荧光温度探针的细胞温度成像技术存在低温度分辨率和有限测量范围等限制。本文旨在利用单分子量子相干过程依赖温度的特性,开发一种单细胞温度成像和实时检测技术。方法 基于飞秒脉冲激光制备延时和相位可调的飞秒脉冲对,调制的脉冲对通过显微系统激发细胞内标记的荧光单分子,之后收集并记录每个荧光光子的到达时间。利用单分子相干过程与周围环境温度的关系,定义单分子量子相干可视度（V）,建立V与环境温度的对应关系。通过调制解调荧光光子的到达时间,获取单分子周围环境温度,结合扫描成像,实现细胞的温度成像和实时检测。结果 该方法可以实现高精度（温度分辨率<0.1℃）和大范围温度（10～50℃）的温度成像和测量,并观测到了单个细胞代谢相关的温度变化。结论 该研究有助于深入了解细胞代谢、蛋白质功能和疾病机制,为生物医学研究提供重要工具。     

Aberrant Water Homeostasis Detected by Stable Isotope Analysis

While isotopes are frequently used as tracers in investigations of disease physiology (i.e., ¹⁴C labeled glucose), few studies have examined the impact that disease, and disease-related alterations in metabolism, may have on stable isotope ratios at natural abundance levels. The isotopic composition of body water is heavily influenced by water metabolism and dietary patterns and may provide a platform for disease detection. By utilizing a model of streptozotocin (STZ)-induced diabetes as an index case of aberrant water homeostasis, we demonstrate that untreated diabetes mellitus results in distinct combinations, or signatures, of the hydrogen (δ²H) and oxygen (δ¹⁸O) isotope ratios in body water. Additionally, we show that the δ²H and δ¹⁸O values of body water are correlated with increased water flux, suggesting altered blood osmolality, due to hyperglycemia, as the mechanism behind this correlation. Further, we present a mathematical model describing the impact of water flux on the isotopic composition of body water and compare model predicted values with actual values. These data highlight the importance of factors such as water flux and energy expenditure on predictive models of body water and additionally provide a framework for using naturally occurring stable isotope ratios to monitor diseases that impact water homeostasis.     

Kinetic isotope effects significantly influence intracellular metabolite 13C labeling patterns and flux determination

Rigorous mathematical modeling of carbon-labeling experiments allows estimation of fluxes through the pathways of central carbon metabolism, yielding powerful information for basic scientific studies as well as for a wide range of applications. However, the mathematical models that have been developed for flux determination from ¹³C labeling data have commonly neglected the influence of kinetic isotope effects on the distribution of ¹³C label in intracellular metabolites, as these effects have often been assumed to be inconsequential. We have used measurements of the ¹³C isotope effects on the pyruvate dehydrogenase enzyme from the literature to model isotopic fractionation at the pyruvate node and quantify the modeling errors expected to result from the assumption that isotope effects are negligible. We show that under some conditions kinetic isotope effects have a significant impact on the ¹³C labeling patterns of intracellular metabolites, and the errors associated with neglecting isotope effects in ¹³C-metabolic flux analysis models can be comparable in size to measurement errors associated with GC–MS. Thus, kinetic isotope effects must be considered in any rigorous assessment of errors in ¹³C labeling data, goodness-of-fit between model and data, confidence intervals of estimated metabolic fluxes, and statistical significance of differences between estimated metabolic flux distributions.     

In Vivo NMR Studies of Neurodegenerative Diseases in Transgenic and Rodent Models

In vivo magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) provide unique quality to attain neurochemical, physiological, anatomical, and functional information noninvasively. These techniques have been increasingly applied to biomedical research and clinical usage in diagnosis and prognosis of diseases. The ability of MRS to detect early yet subtle changes of neurochemicals in vivo permits the use of this technology for the study of cerebral metabolism in physiological and pathological conditions. Recent advances in MR technology have further extended its use to assess the etiology and progression of neurodegeneration. This review focuses on the current technical advances and the applications of MRS and MRI in the study of neurodegenerative disease animal models including amyotrophic lateral sclerosis, Alzheimer's, Huntington's, and Parkinson's diseases. Enhanced MR measurable neurochemical parameters in vivo are described in regard to their importance in neurodegenerative disorders and their investigation into the metabolic alterations accompanying the pathogenesis of neurodegeneration.     

Quantitative detection of engineered nanoparticles in tissues and organs: An investigation of efficacy and linear dynamic ranges using ICP-AES

The absence of effective non-isotopic quantification methods to determine in vivo nanoparticle kinetics and distribution is a key obstacle to the development of various biomedical nanotechnologies. This paper presents a novel adaptation of the established technology of Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES) to a simple technique intended to address this obstacle. Applicability to three varieties of nanoparticles, (CdSe/ZnS quantum dots (QD), gold nanoparticles, and Fe₃O₄ nanoparticles) was investigated, and particle detection sensitivity was shown in moles of particles per gram of tissue. Using gold nanoparticles, increased particle size corresponded with lower molar detection thresholds. Minimum linear detection ranges of 2.5 orders of magnitude for QDs and 1.5 orders of magnitude for all three sizes of gold were demonstrated. The detection of the Fe₃O₄ particles was hampered by the natural presence of Fe²⁺ in tissues, showing that the technique is not suitable for measuring nanoparticles composed of endogenous elements. These detection levels and ranges demonstrate that this technique is useful for quantifying nanoparticles in excised organs, after in vivo dosing.