Preparation of CO2 from blood and protein-bound amino acid carboxyl groups for quantification and 13C-isotope measurements.

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).

Sources of ammonia for mammalian urea synthesis.

The initial rate of incorporation of [15N]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 McGivan & Chappell [(1975) FEBS Lett. 52, 1--7], the experiments support the view that hepatic glutamate dehydrogenase can supply the required ammonia.

Abnormality in metabolism of 15N-labeled nitrate in toxemia.

The urinary excretion of 15N-labeled ammonium nitrate was studied during the last few weeks in patients with normal pregnancy and in those with toxemia. Marked diminution of the 15N label was found consistently in patients with toxemia in the urinary fraction consisting of nitrogenous compounds other than urea and ammonia. The possible significance of this finding is discussed.

Weight/length classification of nutritional status.

The use of somatic measurements for the assessment of nutritional status is briefly reviewed. Two classifications using weight and length being advocated at present--those of McLaren and Read (1972) and Waterlow and Rutishauser (1974)--were applied to data from three sets of children surveyed in hospital and field studies. The results obtained differed considerably. The system of Waterlow and Rutishauser tended to underestimate the problem. A simple chart has been devised to aid the practical application of the classification of McLaren and Read.

15N studies of endogenous faecal nitrogen in infants.

(15)N from labelled yeast protein, fed to marasmic and recovered infants, appeared in the stool shortly after administration and continued to be excreted for 100 hours. Unabsorbed dietary (15)N, which appeared with carmine-marked stools, formed only part of this excretion. Calculations of the endogenous nitrogen content of infant stool agreed closely with those of previous workers. The use of intravenously injected (15)N showed that part of the stool nitrogen was derived from this source. It was excreted as urea, ammonia, and other compounds, and totalled about 2% of the administered urea. The persistence of the (15)N in the stool suggests that it had in part been incorporated into cellular material.

Studies with 15N-labeled ammonia and urea in the malnourished child.

Investigations using ammonium citrate-(15)N and urea-(15)N showed that children in the acute stage of kwashiorkor and marasmus receiving a diet of adequate protein content retained a considerable percentage of the label from both compounds. Excretion of both total (15)N and urea-(15)N was subnormal and elimination was virtually completed 36 hr after administration of the isotope. During recovery from kwashiorkor total (15)N excretion had approached normal a month after commencement of rehabilitation. Urea-(15)N excretion was still slightly subnormal after 3 months. In marasmus urea-(15)N formed a normal proportion of total (15)N excretion after 1 month, although total (15)N excretion then was still low. Ammonia nitrogen was retained to a greater extent than urea nitrogen in all cases. As it is known that a considerable amount of urea is degraded to ammonia in the gastrointestinal tract, it seems probable that urea nitrogen became available for use after this degradation. Examination of blood from one marasmic child after feeding ammonia-(15)N and from another after intravenous injection of urea-(15)N showed incorporation of the label into blood cells and plasma proteins. This did not occur in well nourished controls. It is concluded that ammonia and urea as sources of nonessential nitrogen may play an important part in protein metabolism in the malnourished child.