Isotope Tracers in Metabolic Research: Principles and Practice of Kinetic Analysis / Edition 2

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Overview

In the past few years, the number of applications of tracers for in vivo biomedical studies has greatly increased. New analytical tools at the genetic and protein levels have spurred this growth, opening the door for a deeper understanding of metabolic events. This in turn promises to yield significant advances in the understanding and treatment of human disease.

Now fully revised and expanded, Isotope Tracers in Metabolic Research, Second Edition is the established definitive text on stable and radioactive isotope tracers. In unique, multidisciplinary fashion, it presents comprehensive coverage of new methodological, mathematical, and theoretical approaches.

This new Second Edition includes:

  • All-new chapters on nuclear magnetic resonance, mass isotopomer analysis, and methods of protein metabolism analysis
  • A completely updated categorized list of over 750 references
  • Major advances in the development of mass isotopomer and positional isotopomer techniques, noninvasive isotope techniques for studying metabolic pathways, hyphenated techniques, and new tracer techniques
  • The latest developments in quantification of DNA synthesis and mass spectrometry spurred by genome sequencing and proteomics
  • New coverage of mathematical modeling
  • Expanded coverage of microdialysis probes, laboratory procedures, and regulatory issues related to human studies

In this complete guide to performing tracer studies, the authors systematically cover tracer selection, modeling considerations, sample derivitization, mass spectrometry analysis, and data interpretation. Problems and discussion questions highlight key points in each chapter. Isotope Tracers in Metabolic Research, Second Edition offers students and researchers a comprehensive, practical resource for utilizing the latest tracer methodologies.

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Editorial Reviews

From the Publisher
"...is supported by a wealth of illustrations, tables, and exemplary calculations that will aid in comprehension...[an] excellent and much needed textbook." (The Quarterly Review of Biology, March 2007)

"...should be required for anyone who desires to know more about metabolic tracer kinetics." (Journal of the American Society for Mass Spectrometry, September 2005)

"...an effective integration of theory and practical implementation...It effectively serves as a teaching textbook as well as a research item." (E-STREAMS, August 2005)

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Product Details

  • ISBN-13: 9780471462095
  • Publisher: Wiley
  • Publication date: 10/8/2004
  • Edition description: REV
  • Edition number: 2
  • Pages: 488
  • Product dimensions: 7.19 (w) x 10.00 (h) x 1.12 (d)

Read an Excerpt

Isotope Tracers in Metabolic Research

Principles and Practice of Kinetic Analysis
By Robert R. Wolfe David L. Chinkes

John Wiley & Sons

Copyright © 2005 John Wiley & Sons, Inc.
All right reserved.

ISBN: 0-471-46209-8


Chapter One

BASIC CHARACTERISTICS OF ISOTOPIC TRACERS

1.1 WHAT IS A TRACER?

A tracer is a compound that is chemically and functionally identical to the naturally occurring compound of interest (the tracee) but is distinct in some way that enables detection. By following the fate of a tracer in the body, information can be obtained regarding the metabolism of the tracee.

There are three general ways in which tracers are used in metabolic research. In one case, the tracer is bound to a compound that is injected into the body, and the fate of the compound, and possibly its metabolites, is followed by virtue of the convenience of the distinct property of the tracer. Injection of a pharmacological compound labeled with a tracer is a common application of this approach. For example, if a pharmaceutical compound is labeled with radioactive iodine, the kinetics of the compound can be determined by simply counting the radioactivity, rather than performing the potentially tedious task of measuring the concentration of the compound. The second use of a tracer is to measure its rate of incorporation into another compound. This can be used to calculate therate of synthesis of a product (e.g., protein), or the rate of oxidation of a compound (by measuring the rate of incorporation of labeled carbon into C[O.sub.2]). Also certain aspects of metabolism can be measured using the incorporation principle, such as measuring the rate of gluconeogenesis from a particular precursor or quantifying specific pathways of degradation. The third general use of a tracer is called the "tracer dilution" technique. The most common use of this method is the measurement of the rate of appearance of a substrate into the plasma.

An ideal tracer can be detected with sufficient precision so that it can be given in such a small dose that the metabolism of the tracee produced in the body (the endogenous substrate) is not affected. Alternatively, a tracer can be used in a larger dose, provided that account can be taken of the effect of the tracer on the endogenous kinetics of the tracee. Also the metabolism of tracer must reflect the metabolism of the tracee. In other words, the labeling of the tracer cannot make it so distinct from the tracee that the two compounds are metabolized at different rates.

1.2 TYPES OF TRACERS

A tracer is made by labeling a molecule otherwise identical to the tracee with one or more atoms that are distinct from the most abundant form of that atom. Tracers are conventionally labeled with either radioactive or stable isotopes of one or more atoms in a molecule. Atoms are composed of a dense core of positively charged protons and uncharged neutrons. In the lighter elements, such as carbon, hydrogen, oxygen, and nitrogen, there are approximately equal numbers of neutrons and protons. The number of protons plus neutrons is called the mass number. The mass number is traditionally denoted by the symbol A. The number of protons is the atomic number (symbol Z). Given that the mass units of protons and neutrons are each equal to one, early students of the atom questioned why atomic weights were not whole numbers. In 1911 Soddy used equipment developed the previous year by Thompson that accurately determined relative nuclear charges and masses, and he demonstrated experimentally that certain elements were composed of atoms that were chemically identical but that differed slightly in weight. He proposed the term isotopes for such atoms. Mass differences of isotopes are due to different numbers of nuclear neutrons. The number of neutrons in the atom does not affect the chemical properties of the atom, which are determined by the electronic configuration. Thus a commonly used radioactive isotope of carbon ([sup.14]C) is the same mass as the most abundant isotope of nitrogen ([sup.14]N), yet these two atoms are chemically distinct as carbon and nitrogen.

1.3 NUMBERING OF MOLECULES

The exact position in which a tracer molecule is labeled and the specific isotope label are denoted by the numbering of the molecule. Molecules are numbered according to a system approved by the International Union of Pure and Applied Chemistry (IUPAC). In general, the longest continuous chain of C atoms is considered to be the "parent" hydrocarbon, and any branching alkyls (carbons and hydrogens) or functional groups (e.g., carboxyl, amino groups) attached to it are considered to be substituents of that parent. The parent chain is numbered so as to give the lowest possible set of numbers to the carbons bearing the alkyl or functional groups. For substrates involved in metabolic studies, the functional group is often a carboxyl group (COOH), and consequently the carboxyl carbon is generally the only carbon of the molecule. The hydrogens and oxygen atoms are not specifically numbered but are referred to in relation to the number of the carbon atom to which they are attached. The specifically enriched atom is referred to by identification of the weight of the atom by a superscript prior to the letter. Thus [sup.12]C, [sup.13]C, and [sup.14]C refer to carbon atoms of atomic masses 12, 13, and 14, respectively. [sup.12]C is the most abundant mass, approximately 99%. [sup.13]C is the naturally occurring stable isotope, and [sup.14]C is the radioactive isotope. The position within a molecule of an enriched carbon is referred to by the appropriate carbon number, preceding the abbreviation of the description of the isotope used as a tracer. Thus 1-[sup.13][C.sub.1]-glucose refers to a molecule of glucose in which the 1 position is labeled with carbons enriched with the stable isotope of mass 13. The subscript following the C refers to the number of specifically enriched atoms of carbon (in this example, one) in the molecule. If the 1 and 2 positions were both specifically enriched with [sup.13]C, then the compound would be called 1,2-[sup.13][C.sub.2]-glucose. If all carbons are labeled, it is considered to be uniformly labeled, which is abbreviated by U. Thus a glucose molecule with all positions containing [sup.13]C-enriched atoms would be U-[sup.14][C.sub.6]-glucose, since there are 6 carbons in glucose. If hydrogen is used as a tracer, it is denoted as [sup.2]H for deuterium, the stable isotope of hydrogen of mass 2, and [sup.3]H for tritium, the radioactive isotope of hydrogen of mass 3. The number of carbon to which the hydrogen is attached is denoted by the first number. Thus 2-[sup.3]H-glucose refers to glucose labeled with one hydrogen atom, of mass 3, which is the radioactive isotope (also called tritium), attached to the 2 carbon of the molecule. In the case of hydrogen it is possible for more than one labeled atom to be attached to the same carbon. The commonly used tracer [6,6,-[sup.2][H.sub.2]]-glucose is an example, in which 2 hydrogens attached to the 6 carbons are specifically enriched with atoms of mass 2 (deuterium). The abbreviation d is sometimes used for deuterium, so that 6,6,-[d.sub.2]-glucose might also be used to describe the same tracer.

1.4 RADIONUCLIDES

Radionuclides have been the most commonly used tracers for metabolic studies. A radioactive nuclide spontaneously disintegrates to form an atom of another element, with radiation being emitted in the process.

There are three distinct types of radiation: alpha, beta, and gamma rays. [sup.3]H and [sup.14]C are the most commonly used radionuclides for metabolic studies. Both [sup.3]H and [sup.14]C emit beta rays. Beta rays are actually comprised of electrons with a mass of about 1/1800 that of the hydrogen atom and travel at about the speed of light. In spontaneous decay, one of the neutrons in the nucleus becomes a proton and an electron, the latter of which is emitted from the nucleus. Energy accompanies the stream of emitted electrons from an atom undergoing decay. For example, [sup.14.sub.6]C has 8 neutrons and 6 protons. The superscript 14 refers to the sum of the neutrons and protons, and the 6 refers to the number of protons. When undergoing decay, a neutron becomes a proton. In the case of [sup.14.sub.6]C, a neutron becoming a proton when an electron is released resulting in the formation of nitrogen, which has 7 neutrons and 7 protons (denoted). Thus

[sup.14.sub.6]C [right arrow] [sup.14.sub.7]N + [sup.0.sub.-1]e

The atomic mass units (amu) of reactants and products are equal to the masses of neutral atoms of C and N:

Reactant. Mass of [sup.14.sub.6]C atom: 14.003242 amu

Product. Mass of [sup.14.sub.7]N atom: 14.003074 amu

Loss of mass in reaction. 0.000168 amu

The energy (E) equivalent to this loss of mass is

E = 0.000 16 8 amu · 931 MeV [amu.sup.-1] = 0.156 MeV,

where MeV is millielectron volts. Thus the emitted electrons have an energy of 0.156 MeV.

The rate at which the atoms undergo transformation is directly proportional to the number of radioactive atoms present. As the number of radioactive atoms decreases due to the radioactive transformations, the rate of emission of beta particles decreases. The half-life of a radionuclide is the time required for half of the atoms to be transformed through radioactive decay. The half-life of [sup.14]C is 5730 years, and therefore the decay of stored [sup.14]C, for example, need not be considered as an important factor in terms of loss of tracer. It also means that any [sup.14]C infused into a subject as part of a substrate will be lost entirely as a function of the metabolic turnover of the infused tracer, as opposed to spontaneous decay. The half-life of [sup.3]H is 12.3 years, which is still lengthy in relation to physiological turnover of molecules, including water, in the body. However, the half-life could be pertinent if a tritiated nuclide is stored for a few years and then used, since, in that example, 10% of the radioactivity would be lost after 2 years as a result of spontaneous decay. Such spontaneous decay can easily be accounted for by determining the decays per minute per milliliter of a stock solution of the tracer before mixing a solution for infusion.

1.5 STABLE ISOTOPES

In contrast to radioactive isotopes, there is no spontaneous decay of stable isotopes (hence the name stable isotope). Stable isotopes of an atom contain variable numbers of neutrons in the nucleus. This alters the mass of the atom but not its chemical nature. The most commonly occurring isotope has the lowest mass in the case of carbon, hydrogen, oxygen, and nitrogen. However, this is not true for all atoms. Table 1.1 shows a partial list of stable isotopes. In the case of selenium, for example, there are six naturally occurring stable isotopes, with none comprising more than 50% of the total. Further the lowest atomic weight (74) is the least common isotope, and the most common atomic weight (80) is almost the heaviest isotope. In the discussion of calculation of isotopoic enrichment by mass spectrometry, it is therefore important to note that the assumption that the lowest atomic weight of the tracer atom is its most abundant isotope is implicit in the calculation of enrichment by the technique described most extensively in this book. It is also assumed that the natural abundance of other isotopes is infrequent.

The use of stable isotopes as metabolic tracers in vivo actually predates the use of radioactive isotopes by almost 20 years. In early studies at Columbia University, Schoenheimer and Rittenberg used the stable isotope of hydrogen ([sup.2]H, deuterium) to study fat metabolism in mice. They soon extended their isotopic studies by using [sup.15]N-labeled glycine to demonstrate the dynamic nature of the protein pool of the body. Experiments were also performed as early as the 1930s with [sup.13]C and [sup.18]O. With the advent of scintillation counting and the availability of a wide variety of radioactive tracers, most studies in which metabolic rates were determined (i.e., kinetic studies) in the 1950s and 1960s used radioactive tracers. In the 1970s a resurgence of the use of stable, nonradioactive isotopes began. The most important reasons for this were probably availability of tracers labeled with stable isotopes and the improved ease of analysis due to the availability of the quadruple mass spectrometer interfaced with the gas chromatograph (GCMS), which made possible the convenient use of selective ion monitoring for the quantitation of isotopic enrichment. An increased awareness of the health hazards of radioactivity for human investigations also stimulated the use of stable isotopes.

1.6 ADVANTAGES OF STABLE ISOTOPES

1.6.1 Biological Effects

The most obvious advantage of stable isotopes is that they are nonradioactive and present little or no risk to human subjects. Carbon 13 is a naturally occurring isotope present to the extent of approximately 1.1% of the major isotopic species, carbon 12 (Table 1.1). Since carbon 13 naturally contributes 1.1% of the carbon pool, and since it has not been possible to demonstrate more than trivial in vitro isotopic effects on chemical reactions with carbon 13-labeled substrates, significant side effects in vivo are not expected from administration of "tracer" doses of carbon 13. In fact Gregg et al. raised mice on [sup.13]C enriched algae, thereby raising the carbon 13 content of the total body pool from 60% to 70% of total carbon without discernible effect on the animals. Replacement of [H.sub.2]O with [D.sub.2]O, however, can affect the growth of microorganisms. To see an effect, however, the deuterated water must be in excess of 20% of the total water. Similarly [H.sup.18.sub.2]O, carbon 13, and [sup.15]N can be shown to affect certain parameters of cell function at extremely high levels of enrichment that would never be attained in an in vivo study in humans. There is no evidence that stable isotope tracers present an identifiable risk to human subjects at the highest levels of enrichment that might reasonably be achieved, with the possible exception of [sup.2][H.sub.2]O given at a dose sufficient to raise the enrichment of the total body pool by about 5%.

1.6.2 Enzymatic Effects

The use of a labeled tracer requires the assumption that the labeled molecule will not be discriminated from the unlabeled molecule and that the labeled molecule will trace the movement of the unlabeled molecules. However, certain enzymatic effects of stable isotopes have been reported, such that the isotope will be selectively fractioned from its more abundantly occurring counterpart. Potential isotope effects when carbon, nitrogen, and oxygen are used as tracers are rarely of concern. However, it is possible that sufficient isotope effects might occur with [sup.3]H or [sup.2]H to be of physiological significance. For example, an isotope effect was claimed in the clearance of 3-[sup.3]H-glucose from blood, as well as for 6-[sup.3]H-glucose and 6,6-[sup.2][H.sub.2]-glucose.

Continues...


Excerpted from Isotope Tracers in Metabolic Research by Robert R. Wolfe David L. Chinkes Copyright © 2005 by John Wiley & Sons, Inc.. Excerpted by permission.
All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
Excerpts are provided by Dial-A-Book Inc. solely for the personal use of visitors to this web site.

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Table of Contents

Preface.

Chapter 1. Basic Characteristics of Isotopic Tracers.

Chapter 2. Calculating Specific Activity and Radiation Dosages.

Chapter 3. Calculation of Substance Kinetics: Single-Pool Model.

Chapter 4. Calculation of Substrate Kinetics: Multiple-Pool Model.

Chapter 5. Mass Spectrometry: Instrumentation.

Chapter 6. Determination of Isotopic Enrichment.

Chapter 7. Measurement of Substrate Oxidation.

Chapter 8. Measurement of Total Energy Expenditure Using the Doubly Labeled Water Method.

Chapter 9. Mass Isotopomer Distribution Analysis.

Chapter 10. Glucose Metabolism.

Chapter 11. Lipid Kinetics.

Chapter 12. Whole Body Protein Synthesis and Breakdown.

Chapter 13. Measurement of the Synthesis of Specific Proteins.

Chapter 14. Measurement of Regional or Tissue Protein Breakdown.

Chapter 15. Arterial-Venous Balance Technique to Measure Amino Acid Kinetics.

Chapter 16. Nuclear Magnetic Resonance.

Glossary.

Abbreviations.

Index.

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