Measurements of total nitrogen intake and output can be used to calculate protein nutritional status (Leverton & Gram 1949; Hegsted 1978). This approach is based on principles of chemical mass-balance, and although practical matters concerning collecting total nitrogen waste make this method difficult to implement, measurements of nitrogen balance generally permit a reliable determination of protein homeostasis. Use of the method requires special consideration since urinary nitrogen may occur in the forms of urea and ammonia (derived from amino acid and protein catabolism) and uric acid and creatinine (derived from nonprotein sources), and since nitrogen may be lost in faeces, sweat and other products (Figure 10.1). To facilitate studies, investigators have proposed correction factors that account for the relationship between total urinary nitrogen and urinary urea nitrogen (Mackenzie et al. 1985) and for miscellaneous non-urinary nitrogen losses (Calloway et al. 1971).
While measurements of whole-body nitrogen balance can be used to address questions related to the wasting of lean mass and to determine the efficiency of nitrogen retention, nitrogen balance does not yield a measure of the turnover of specific proteins or the remodeling rate(s) of an individual tissue(s) within the body, i.e. one cannot determine whether changes in nitrogen balance reflect changes in protein synthesis and/or protein breakdown. To quantify endogenous protein turnover, and to examine interactions between dietary intake and stored nitrogen, investigators have proposed the use of 'end-product' methods (Figure 10.2). Briefly, these methods consider two pools of endogenous nitrogen, a pool of bound nitrogen (amino acids in existing proteins) and a pool of free nitrogen (amino acids circulating in plasma). The flux of nitrogen in the free pool is described using Equation 10.4, where we let Q represent nitrogen flux.
Investigators have demonstrated that the metabolism of a 15N-labeled precursor affects the excretion of 15N (Fern et al. 1985). To quantify nitrogen flux, investigators typically administer [15N]glycine (San Pietro & Rittenberg 1953; Matthews et al. 1981; Stein et al. 1980) and then measure the 15N-labeling of urea and/or ammonia. The rate of nitrogen flux is calculated using the equation:
nitrogen flux in the free pool = dose of the administered isotope/labeling of the end-product (10.5)
Rates of protein synthesis (S) and protein breakdown (B) are calculated by measuring the quantity of the excreted end-products (urea and ammonia) and using equations:
S = nitrogen flux — quantity of excreted end-products (10.6)
B = nitrogen flux — dietary nitrogen intake (10.7)
where the conversion factor of 1 g nitrogen is equal to 6.25 g protein. As noted earlier, if subjects are studied during a post-absorptive state, dietary nitrogen intake is equal to 0 and the rate of protein breakdown is equal to nitrogen flux in the free pool.
Duggleby & Waterlow (2005) present a thorough critique of the studies that have used the [15N]glycine method. They discuss the implications of measuring the 15N-labeling in the end-products of ammonia or urea and the effect of using a single bolus or constant administration of [15N]glycine. We comment on the elaborate experiments done by Gougeon et al. (1994, 1997, 1998, 2000), who used the [15N]glycine method to examine protein metabolism in obese patients with type 2 diabetes. Studies were typically performed over the course of several weeks, during which time subjects lived in a clinical research centre. [15N]glycine was administered over ~60 hour intervals several times during the study. Using this method, the investigators made several important overall observations including that: 1) there is an increased nitrogen flux in poorly controlled patients, i.e. those with glycated hemoglobins of ~12 % and fasting glucose of ~12 mM; and 2) normalisation of glycemia (either via insulin or oral hypoglycemic agents) leads to a reduction in protein breakdown thereby decreasing nitrogen flux and promoting nitrogen retention.
To further dissect the nature of endogenous protein turnover, Gougeon et al. (2000) estimated the contribution of muscle protein breakdown to whole-body nitrogen flux by measuring the excretion of methylhistidine. Briefly, the excretion of a modified amino acid (e.g. methylhistidine, hydroxyproline) can be used to measure protein breakdown, i.e. since post-translational modifications render amino acids unsuitable for use during the synthesis of new proteins, the excretion of modified amino acids reflects protein breakdown (Bilmazes et al. 1978; Elia et al. 1980; Marchesini et al. 1982; Selby et al. 1995).
Was this article helpful?
Find out why long exhausting workouts may do more harm than good. Most of the body-building workout and diet regimens out there are designed for the guys that gain muscle and fat easily. They focus on eating less and working out more in order to cut the excess fat from their bodies while adding needed muscle tone.