Measurement of protein turnover in normal man using the end-product method with oral [¹⁵N]glycine: comparison of single-dose and intermittent-dose regimens

Abstract

The 'single-dose end-product' approach for the measurement of protein turnover is the only method suited for application to free-living individuals and for field studies. However, the method has never been widely accepted because the results obtained appear to vary from one situation to another. There is the need for a formal comparison of the different approaches that have been used and the assumptions on which they are based in order to be able to understand the extent to which the variation in the results obtained is real or might be attributed to methodological differences. The present study used the 'primelintermittent-dose end-product' approach over 18 h as a reference with which to compare the 'singledose end-product' approach, with urine being collected for periods of 9 to 48 h. N flux was derived in a total of thirteen normal men using oral ['%]glycine and measurement of l3N enrichment in urinary NH, or urea, with isotope being given either as prime/intermittent doses or a single dose in separate studies. The pattern of results was similar to those reported in the literature. In all studies the rates of N flux derived from urea were higher than those derived from NH,, under equivalent conditions, by about 2530%. The single-dose approach, with urine collection over 9 h, gave results which were consisteutly higher than the prime/intermittentdose approach. The extent of the difference was influenced by the duration of time over which the cumulative excretion of isotope in urine was determined, and with NH, as the end-product the most consistent estimates of N flux could be obtained with a urine collection of at least 12 h and no greater than 24 h. With urea as the end-product, correction for the label retained in the body pool at 9 h gave similar results for N flux to those derived from the total excretion of label in urea over 24 h. The derivation of values for protein synthesis and protein degradation from measures of flux requires care to ensure that the time periods over which N intake and excretion are measured accord with those for which the measurement of flux apply. It is concluded that measurements of protein turnover similar to those obtained with the prime/intermittent-dose approach can be obtained with the single-dose approach in the fed state during the daytime, either from the excretion of label in urinary NH, over a period of 12 h or with the excretion of label in urinary urea over a period of 24 h. The suitability of the method for use in the fasted state or at night time remains to be determined. Urea: Ammonia: Nitrogen fluxThe measurement of protein turnover has provided invaluable information on the dynamic changes in the relative rates of protein synthesis and degradation which take place in a range of physiological or pathological states (Waterlow et al. 1978a;Waterlow, 1984). For studies in adults there has been a tendency to use the relatively invasive precursor methods with the intravenous administration of isotope, either as a prime/continuous infusion or as a flooding dose of isot...

Postexercise protein ingestion increases whole body net protein balance in healthy children

“…Alternatively, the sensitivity of oral [ 15 N]glycine may have precluded our ability to detect differences in protein turnover with the relatively small acute differences in protein intake between conditions that other methodology, such as intravenous infusions, may have permitted; however, discrepancies between measured rates of whole body protein kinetics by oral [ 15 N]glycine and [ 13 C]leucine infusion are more pronounced with measurements made in the fasted rather than fed state (14). Nevertheless, in general, whole body protein turnover (i.e., both synthesis and breakdown) is higher in children than adults and can be less sensitive to even large fluctuations in protein intake (19), which opens the possibility that even subtle changes in whole body protein synthesis and/or protein breakdown with exercise or nutrition could translate into physiologically more meaningful differences in WBPB because of these rapid rates of tissue remodeling.…”

“…The potential limitation to oral tracers, such as [ 15 N]glycine, is they represent the net sum of all nitrogen metabolism in the body (e.g., within muscle, splanchnic bed, etc.) and generally have a lower time resolution given the need to adequately collect the metabolic end product, which based on differences in pool size is ϳ9 h and ϳ24 h for urinary [ 15 N]ammonia and [ 15 N]urea, respectively (19). In addition, the noninvasive study design precluded our ability to measure other potential biological effectors of protein metabolism such as circulating insulin, which can suppress whole body protein breakdown (Arslanian) and, provided there was a differential response between conditions, may have also contributed to the changes in WBPB observed herein.…”

“…Our decision to utilize [ 15 N]glycine methodology to measure whole body protein metabolism, which has a long history of use in humans of all ages (14), was related to the ease with which protein kinetics can be measured over relatively long time frames (e.g., up to 24 h) (19), the relatively low withinsubject variability (16), and, because of the oral ingestion and urinary endpoint analysis, its feasible and ethical application in healthy children. In addition, although urinary nitrogen excretion can account for ϳ80 -85% of all nitrogen loss in active adults (39,40), we also utilized the measured exercise-induced change in body mass with average sweat nitrogen content (1) to estimate sweat nitrogen excretion, which can contribute ϳ10% of nitrogen loss in active adults (39) and, if not accounted for, would subsequently increase the apparent net protein balance (1); this approach, in contrast to previous studies in active children that only considered urinary nitrogen excretion (7,8,33), would result in a conservative estimate of WBPB in our study in the absence of the technically and logistically challenging determination of all potential routes of nitrogen loss (e.g., fecal, integumentary, hair, etc.).…”

“…The concentration of the major nitrogen-containing metabolites urea and creatinine were determined colormetrically by commercially available kits (Quantichrom, Bioassay Systems) as an estimate of urinary nitrogen excretion. The [ 15 N] enrichments (i.e., ratio of tracer-trace, t/Tr) of urinary ammonia (at baseline, 9 h, and 24 h) and urea (at baseline and 24 h) were determined in duplicate by isotope ratio mass spectrometry by Metabolic Solutions Incorporated (Nashua, NH) to determine whole body nitrogen turnover and protein metabolism (19 N] enrichment of urinary ammonia, t is the time (i.e., 9 or 24 h), and BM is the participant's body mass. Q using [ 15 N]urea as the end product was calculated in an identical manner except no correction of time was used (i.e., rates were expressed in units of g N·kg Ϫ1 ·d Ϫ1 ).…”

A critical review of recommendations to increase dietary protein requirements in the habitually active

“…Glycine tracer methodology can be useful because a shorter experimental time will be necessary as NH 3 is the endproduct sampled. This procedure has been standardised and validated (49) and collection periods of 12 h have been found to be suitable to study whole-body protein metabolism (50,51) . A urea tracer, [ 15 N]urea, has also been studied but it is difficult to determine a suitable collection period because of the slow turnover rate of the urea pool.…”