Amino acid isotope dilution

The isotope dilution principle, first employed by Hevesy and Hobbie (133) in 1932 for the determination of lead in ores, was applied by Schoenheimer et al. (241) to the determination of amino acids. [Shemin and Foster (248) have reviewed this topic.] An N15-amino acid derivative was added to a protein hydrolyzate, a sample of the amino acid to be determined was isolated and purified, the excess N15 in this product was estimated with the mass spectrograph, and the grams of amino acid originally present were calculated from Equation 2. 

Coupling constants are routinely used to determine the side-chain conformation of amino acids in peptides and proteins. Whereas proteins nowadays are almost exclusively studied as C- and N-labeled isotopomers, peptides usually have these isotopes in natural abundance, i.e. the magnetically active heteronuclei are highly diluted. Most amino acids contain a methylene group at the ji-position for which the X angle is determined by the conformation of the Ca—Cp bond. Two vicinal Jhh coupling constants can be measured Ha to and H to Usually... 

Preston et al. [698,699] have described novel approaches to 15N-isotope dilution determination of ammonium and of free amino acids in natural waters, incorporating chemical derivatization and conventional GC-MS analysis. 

Calder, A.G. Garden, K.E. Anderson, S.E. Lobley, G.E. Quantitation of Blood and Plasma Amino Acids Using Isotope Dilution GC-EI-MS with U- C Amino Acids As Internal Standards. Rapid Com-mun. Mass Spectrom. 1999, 13, 2080-2083. 

TABLE 2 Selected Free Amino Acid Concentrations (pM) in Milk Formula and Specimens Measured By Isotope Dilution MS/MS... 

Rittenberg, D., and Foster, G. L. (1940). A new procedure for quantitative analysis by isotope dilution, with application to the determination of amino acids and fatty acids. J. Biol. Chem. 133, 737. 

W. R. Wolf, H. Zainal, Methylseleno-amino acid content of food materials by stable isotope dilution mass spectrometry, Food Nutr. Bull., 23 (2002), 120D123. 

Determination of oxidized amino acids in urine is usually performed by isotope dilution gas chromatography-mass spectrometry (L9). DOPA is estimated by HPLC separation of acid protein hydrolysates with fluorescence detection (excitation 280 nm, emission at 320 nm) (A15). Other methods are based on borate-hydrochloric acid difference spectroscopy (this method suffers interference from tyrosine and tryptophan) (W2), derivatization of DOPA with nitrite and subsequent coulometric determination (W3), and fluorometric detection after derivatization with ethylenediamine (A15). 3-Hydroxylysine is quantitated by HPLC with 9-fluorenylmethyl chloroformate precolumn derivatization (M25) of amino acids obtained by gas-phase hydrolysis of proteins (F21). Other general methods to detect amino acid damage are mass spectometry methods applied to protein hydrolysates, such as tandem mass spectrometry (F6). 

Fuhrman, J. (1987). Close coupling between release and uptake of dissolved free amino acids in seawater studied by an isotope dilution approach. Mar. Ecol. Prog. Ser. 37, 45—52. 

Different species of macroalgae would be expected to have different N release rates. For example, some macroalgae are known to have large intracellular inorganic N storage pools (e.g., McGlathery et ah, 1996). Release of NH4", as measured with isotope dilution, was observed in Ulva fenestrata (0.08-11.27 pmol N g h ) and Gracilaria pacifica (0.12-0.77 pmol N g h ) when NH4+ was added to the medium. However, no net NH4+ release, over uptake, was observed. Small pulses of free amino acids were released and then rapidly reincorporated (Naldi and Wheeler, 2002), but no protein release was detectable. 

It is worth noting that the term isotope dilution has also been applied to apphcations where the concentration of a compound is determined isotopicaUy rather than by direct chemical determination. The most famihar appHcation is the determination of the magnitude of a blank (pre-existing in an analysis) by adding a known amount of to a sample and determining the decrease from the expected isotopic value of the tracer addition. This concept can also be used in field samples, such as for determining the concentrations of dissolved and combined amino acids. 

Preston, T., Bury, S., McMeekin, B., and Slater, C. (1996a). Isotope dilution analysis of combined nitrogen in natural waters. II. Amino acids. Rapid Commun. Mass Spectrom. 10, 965—968. 

Shemin, G., and G. L. Foster The Isotope Dilution Method of Amino Acid... 

This amino acid which is synthesized by muscle and excreted into urine without recycling has been methylated with [Djj-methyl iodide exclusively at N site and applied as tracer in vivo and as an isotopic dilution standard in vitro, by protecting the N and N positions in a cyclic urea derivative 34. The latter was obtained by treating the (5)-histidine methyl ester 35 with carbonyl diimidazole and hydrolysing the product 36 in boiling dilute hydrochloric acid to give isomerically and isotopically pure title compound 37 retaining at least 95% of its optical rotation (equation 5). 37 has been applied in clinical research. 

G( — M) values were determined by isotopic dilution using amino acids recrystallized as above, as well as with an automatic amino acid analyzer. 

The importance of the isotope-dilution technique is that it can be used for the quantitative determination of substances present in such small amounts that other methods are difficult to apply. For example, if a protein is hydrolyzed, some of the amino acids are present in very small proportions. In the isotope-dilution technique, we add to such a mixture a pure radioactively-Iabeled sample of the same compound, and isolate a sample of the substance in pure form regardless of yield. We then measure the specific radioactivity of the product, and from the specific activity of the added material we calculate the amount of unlabeled compound originally present. The method of calculation is best explained by means of an example. 

Explain the method of isotope dilution, with particular reference to the determination of the proportions of the various amino acids in a protein. 

The isotope dilution technique has been employed for Ihe determination of about thirty elements in a variety of matrix materials, Isotopic dilution procedures have also been most widely used for the determination of compounds of interest in organic chemistry and biochemistry, Thus, methods have been developed for the determination of such diverse substances as vitamin D, vitamin B12, sucrose, insulin, penicillin, various amino acids, corticosterone, various alcohols, and thyroxine. Isotope dilution analysis has experienced less widespread application since the advent of activation methods. Continued use of the procedure can be expected, however, because of the relative simplicity of the equipment required. In addition, isotope dilution is often applicable where activation analysis is not. 

In some cases the measurement of the final sample utilizes a technique other than weighing, but the principle remains the same. Isotope dilution is used, for example, in the determination of naphthalene in tar, of fatty acids in mixtures of natural fat, of amino acids in biological material, etc. 

D. M., Rasmussen, J. E., and Turk, J., Detecting oxidative modification of biomolecules with isotope dilution mass spectrometry Sensitive and quantitative assays for oxidized amino acids in proteins and tissues. Methods Enzymol. 300, 124-144 (1998). 

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