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The methods to measure stable isotope tracers and their applications to clinical nutrition problems keep getting better . . .

Matthews, Dwight E.

Current Opinion in Clinical Nutrition and Metabolic Care: November 1999 - Volume 2 - Issue 6 - p 439-440
Editorial Comment
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Correspondence to Dwight E. Matthews, PhD, Department of Medicine, University of Vermont, Given Building-B217, Burlington, VT 05405, USA. Tel: +1 802 656 8114; fax: +1 802 656 1229

Stable isotopically labelled tracers were first used to measure metabolic processes in the 1930s by Schoenheimer and Rittenberg [1]. At that time man-made radioactive materials were not available, and only non-radioactive stable isotopes were being separated in limited amounts. The work of Harold Urey in identifying and isolating stable isotopes, of which his discovery of deuterium (2H) led to his being awarded the Nobel Prize in 1934, made it possible for Schoenheimer and Rittenberg to conduct their studies. These scientists were true pioneers. Before their work, people thought that many biological compounds were static in the body, i.e. proteins were synthesized for the life of the cell. Schoenheimer and Rittenberg used stable isotopes incorporated into a variety of compounds from amino acids to fatty acids to prove that compounds such as lipids and proteins were dynamic; that they were both simultaneously synthesized and degraded [2]. Schoenheimer and Rittenberg would surely have been awarded the Nobel Prize for their work had it not been for the untimely death of Schoenheimer in 1941.

A fundamental key to the work of Schoenheimer and Rittenberg was the mass spectrometer. The early mass spectrometers were used to measure the abundance of different isotopes contained in simple gas molecules, such as carbon dioxide or nitrogen. These first mass spectrometers became the basis of the isotope ratio mass spectrometer (IRMS), which although greatly modernized, exists in substantially the same form as used by Rittenberg. However, it would not be until after the 1940s and into the 1950-1960s that mass spectrometers would be designed for measuring organic compounds [3]. These mass spectrometers would be used to produce the classical mass spectra for identification associated with every college textbook of organic chemistry. Although the focus of mass spectrometry has shifted from applications of organic compound identification to studies of complex biological compounds, modern mass spectrometry remains primarily devoted to the identification and detection of specific compounds. Stable isotope ratio measurements have taken a back seat. In fact, IRMS is barely mentioned in a 50 year review of mass spectrometry by one of the major producers of mass spectrometers [4].

Stable isotopes and their measurement are, however, very important to scientists in nutrition. The only way to follow endogenous compounds in the body is by using isotopically labelled tags. As Meier-Augenstein points out in his article in this issue (pp. 465-470), stable isotopically labelled tracers have significant advantages over the use of radioactively labelled compounds. These advantages go well beyond the advantage of no radioactive exposure with stable isotopically labelled compounds. Gas chromatography-mass spectrometry (GCMS) allows for the very specific measurement of different stable isotopically labelled tracers of the same compound. The differently labelled leucines of [1-13C]leucine, [1,2-13C]leucine, [5,5,5-2H3]leucine, [15N]leucine, . . . can all be measured and defined simultaneously. With radioisotopes we only have a choice of ‘the chicken or the beef’, i.e. the use of 14C or 3H. The advantages of using stable isotopes in the 1990s has been enormous. The problem has been in their measurement.

GCMS revolutionized stable isotope use in nutrition starting in the 1980s [5,6]. A textbook written by Wolfe [7] provides extensive information on the subject of stable isotope use in nutrition and metabolism. IRMS has also been extensively used for stable isotope tracer measurement, but for different purposes. The reason for selecting one technique or the other is also defined in the article by Meier-Augenstein.

In the late 1970s we built a modified device, neither GCMS nor IRMS [8]. We gave the device a name and demonstrated its ability to measure small enrichments of both 13C and 15N. However, this instrument was not at the right time and place. Further development of other technologies were required to make this device commercially feasible. The development of modern low-flow fused silica capillary gas chromatographic columns revolutionized gas chromatography, but this development also made it possible to design low-flow oxidation and reduction reactors that could be coupled to an IRMS. New developments in how the ion source produces ions in the IRMS and how gas is removed from it by turbomolecular pumps also made it possible to have continuous flow IRMS devices. These developments gave rise to what is now commercially available, commonly called gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). GC-C-IRMS is truly a hybrid between GCMS and IRMS. It offers distinct advantages over the other methods. Meier-Augenstein, in his review, discusses the latest advancements and applications of GC-C-IRMS. He describes in detail how it can and has been applied in nutrition research. As we enter the next century, we find ourselves armed with multiple, different mass spectrometry techniques for measuring stable isotopically labelled tracers. With these techniques there are relatively few if any metabolic questions that cannot be answered.

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References

1 Keston AS, Rittenberg D, Schoenheimer R. Determination of deuterium in organic compounds. J Biol Chem 1937; 122:227-237.
2 Guggenheim KY. Rudolf Schoenheimer and the concept of the dynamic state of body constituents. J Nutr 1991; 121:1701-1704.
3 Gohlke RS, McLafferty FW. Early gas chromatography/mass spectrometry. J Am Soc Mass Spectrom 1993; 4:367-371.
4 Brunnée C. 50 Years of MAT in Bremen. Rapid Commun Mass Spectrom 1997; 11:694-707.
5 Matthews DE, Bier DM. Stable isotope methods for nutritional investigation. Annu Rev Nutr 1983; 3:309-339.
6 Matthews DE. Proteins and amino acids. In: Modern nutrition in health and disease, 9th ed. Shils ME, Olson JA, Shike M, Ross AC (editors). Baltimore: Williams and Wilkins; 1999. pp. 11-48.
7 Wolfe RR. Radioactive and stable isotope tracers in biomedicine: principles and practice of kinetic analysis. New York: Wiley Liss; 1992.
8 Matthews DE, Hayes JM. Isotope-ratio-monitoring gas chromatography-mass spectrometry. Anal Chem 1978; 50:1465-1473.
© 1999 Lippincott Williams & Wilkins, Inc.