Measurements of protein and amino acid metabolism in man using stable isotopes and selected ion monitoring gas chromatographic/mass spectrometric techniques are limited by the requirement of relatively high levels of labelling for adequate precision (greater than 0.05 at % excess). We describe here a means of extending the scope of such studies by measurement of lower levels of enrichment achieved in gaseous CO2 derived from whole blood or protein-bound amino acids following the administration of tracer amounts of appropriately labelled substrates. Construction and operation of a novel glass vacuum line required for this work are described in detail and specific applications relevant to clinical investigations are outlined. Measurements of both the total amount of CO2 and its 13C enrichment are performed in an isotope-ratio mass spectrometer which provides acceptable levels of accuracy and reproducibility for both measurements (+/- 0.1% and +/- 0.0001 at % excess respectively).

The initial rate of incorporation of ['5N]alanine into the 6-amino group of the adenine nucleotides in rat hepatocytes was about one-eighteenth of the rate of incorporation into urea. Thus the purine nucleotide cycle cannot provide most of the ammonia needed in urea synthesis for the carbamoyl phosphate synthase reaction (EC 2.7.2.5). On the other hand, contrary to the view expressed by , because experiments on isolated mitochondria suggested that the activity of the glutamate dehydrogenase system in situ is too low because of the slow entry of glutamate into the mitochondrial matrix. These workers also noted that leucine accelerates glutamate dehydrogenase in sonicated mitochondria, whereas it inhibits urea synthesis in intact hepatocytes. These observations led to the tentative conclusion that glutamate dehydrogenase plays no major role in connection with the synthesis of urea. As for an alternative source of ammonia, they expressed the view that the deamination of AMP and the purine nucleotide cycle (Raggi et al., 1969; Lowenstein, 1972) For the measurement of the incorporation of 15N into the 6-amino group of the adenine nucleotides and into urea it is necessary to isolate a derivative from which gaseous nitrogen can readily be prepared for direct isotopic analysis in a mass spectrometer. A suitable derivative is ammonia. This was obtained from urea with urease (EC 3.5.1.5) and from adenine nucleotides by the combined action of potato apyrase (EC 3.6.1.5) and AMP deaminase (EC 3.5.4.6). In the early attempts to isolate ammonia irregular results were obtained, which were traced to the contamination of the samples with external ammonia. Such contamination dilutes adenine nucleotide N to a much greater extent than urea N (on a percentage basis) because the amounts of urea N are much greater under the test conditions. The results became consistent only when all sources of ammonia contamination had been minimized. The distilled water used was freshly prepared and then passed through a mixed-bed-resin deionizer. The HCI solutions used in the Conway units were prepared immediately before use from a BDH volumetric solution ampoule. This solution contains HgCl2 and therefore could be used only in the Conway dishes. An alternative source of low-ammonia HCI, used for neutralization of solutions to which enzymes had to be added, was the 5 M-HCI analytical volumetric

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The object of this communication is to focus attention on the design and analytical capabilities of isotope ratio and organic mass spectrometers and their potential areas of application to the study of amino acid and protein metabolism in man using stable isotopes.The isotope ratio mass spectrometer Dual inlet double collector gas isotope ratio mass spectrometers by inherent design, possess a low resolving power but extremely high sensitivity. These specialized instruments are capable of precise estimates of the heavy isotopic content of pure, low molecular weight permanent gases such as H,, CO,, N, and

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