As an alternative to measuring the turnover of the whole amino-N pool per se, the kinetics of an individual amino acid can be followed from the dilution of an infused tracer of that amino acid. The simplest models consider only essential amino acids that have no de novo synthesis. The kinetics of essential amino acids mimic the kinetics of protein turnover as shown in Figure...2..10. The same type of model can be constructed but cast specifically in terms of a single essential amino acid, and the same steady-state balance equation can be defined:
Qaa = 4a + Baa = Caa + ^aa where Qaa is the turnover rate (or flux) of the essential amino acid, Iaa is the rate at which the amino acid is entering the free pool from dietary intake, Baa is the rate of amino acid entry from protein breakdown, Caa is the rate of amino acid oxidation, and Saa is the rate of amino acid uptake for protein synthesis. The most common method for defining amino acid kinetics has been a primed infusion of an amino acid tracer until isotopic steady state (constant dilution) is reached in blood. The flux for the amino acid is measured from the dilution of the tracer in the free pool. Knowing the tracer enrichment and infusion rate and measuring the tracer dilution in blood samples taken at plateau, the rate of unlabeled metabolite appearance is determined ( 64, 74, 75):
where iaa is the infusion rate of tracer with enrichment E. (mole % excess) and Ep is the blood amino acid enrichment.
For a carbon-labeled tracer, the amino acid oxidation rate can be measured from the rate of 13CO2 or 14CO2 excretion (42, 64, 74). The choice of a carbon label that is quantitatively oxidized is critical. For example, the 13C of an L-[1-13C]leucine tracer is quantitatively released at the first irreversible step of leucine catabolism. In contrast, a 13C-label in the leucine tail will end up in acetoacetate or acetyl-CoA, which may or may not be quantitatively oxidized. Other amino acids, such as lysine, have even more nebulous oxidation pathways.
Before the oxidized carbon-label is recovered in exhaled air, it must pass through the body bicarbonate pool. Therefore, information about body bicarbonate kinetics is required (76). To complete the oxidation rate calculation based upon the measured recovery of the administered carbon-label as CO 2, we must know what fraction of bicarbonate pool turnover is the release of CO 2 into exhaled air versus retention for alternative fates in the body. In general only about 80% of the bicarbonate produced is released immediately as expired CO2, as determined from infusion of labeled bicarbonate and measurement of the fraction infused that is recovered in exhaled CO2 (77). The other approximately 20% is retained in bone and metabolic pathways that "fix" carbon. The amount of bicarbonate retained is somewhat variable (ranging from 0 to 40% of its production) and needs to be determined when different metabolic situations are investigated. In cases in which the retention of bicarbonate in the body may change with metabolic perturbation, parallel studies measuring the recovery of an administered dose of 13C- or 14C-labeled bicarbonate are essential to interpretation of the oxidation results ( 78, 79).
The rate of amino acid release from protein breakdown and uptake for protein synthesis is calculated by subtracting dietary intake and oxidation from the flux of an essential amino acid—just as is done with the end-product method. The primary distinction is that the measurements are specific to a single amino acid's kinetics (pmol of amino acid per unit time) rather than in terms of N per se. Flux components can be extrapolated to whole-body protein kinetics by dividing the amino acid rates by the assumed concentration of the amino acid in body protein (as shown in TabJe...2...3).
The principal advantages to measuring the kinetics of an individual metabolite are that (a) the results are specific to that metabolite, improving the confidence of the measurement, and (b) the measurements can be performed quickly because turnover time of the free pool is usually rapid (a tracer infusion study can be completed in less than 4 hours using a priming dose to reduce the time required to come to isotopic steady state). Drawbacks to measuring the kinetics of an individual amino acid are that (a) an appropriately labeled tracer may not be available to follow the pathways of the amino acid being studied, especially with regard to amino acid oxidation, and (b) metabolism of amino acids occurs within cells, but the tracers are typically administered into and sampled from the blood outside cells. Amino acids do not freely pass through cells; they are transported. For the neutral amino acids (leucine, isoleucine, valine, phenylalanine, and tyrosine), transport in and out of cells may be rapid, and only a small concentration gradient between plasma and intracellular milieus exists ( Iable 2.4). However, even that small gradient limits exchange of intracellular and extracellular amino acids. For leucine, this phenomenon can be defined using a-ketoisocaproate (KIC), which is formed from leucine inside cells by transamination. Some of the KIC formed is then decarboxylated, but most of it is either reaminated to reform leucine (80) or released from cells into plasma. Ihus, plasma KIC enrichment can be used as a marker of intracellular leucine enrichment from which it came (81).
Previous workers have shown that generally, plasma KIC enrichment is about 25% lower than plasma leucine enrichment ( 75, 81, 82). If plasma KIC enrichment is substituted for the plasma leucine tracer enrichment in the calculation of leucine kinetics, then the measured leucine flux and oxidation and, likewise, estimates of protein breakdown and synthesis are increased by about 25%. However, when protein metabolism is studied under two different conditions and the resulting leucine kinetics are compared, the same relative response is obtained regardless of whether leucine or KIC enrichment is used for the calculation of kinetics ( 81). The prudent approach is to measure both species and to note occasions when the KIC/leucine enrichment ratio has changed, to signal a possible change in the partitioning of amino acids between intracellular and extracellular spaces (83).
Use of KIC to represent intracellular leucine is an application of a concept that adds definition to the model shown in Figure,2J..2 but does not require a more rigorous model to describe leucine kinetics. Because of confusion over a suitable model to describe leucine kinetics, a series of experiments were performed to develop a true multicompartmental model for the leucine-KIC system (84). Four leucine and three KIC pools were required to account for leucine kinetics. Clearly the kinetics of individual metabolites are far more complex than one- or two-compartment models. However, the conventional model using KIC as the precursor enrichment for calculating leucine kinetics as shown in Hgure,2.,12. agreed well with the multi-compartmental model, which means that under many metabolic circumstances, the simpler approaches should accurately follow directional changes without requiring introduction of complicated compartmental models. These and intermediate models have been reviewed (75, 85), and the various assumptions, limitations, strengths, and weaknesses have been discussed. The leucine/KIC tracer system remains the single most applied measure of whole-body amino acid kinetics used to reflect changes in protein metabolism ( 83).
Figure 2.12. Two-pool model of leucine kinetics. The leucine tracer is administered to the plasma pool (large arrow) and sampled from plasma and/or from exhaled CO2 (circles with sticks). Plasma leucine exchanges with intracellular leucine where metabolism occurs: uptake for protein synthesis (S) or conversion to a-ketoisocaproate (KIC). Oxidation (C) occurs from KIC. Unlabeled leucine enters into the free pool via dietary intake (I) or protein breakdown (B) into intracellular pools.
Most amino acids do not have a convenient metabolite that can be readily measured in plasma to define aspects of their intracellular metabolism, but an intracellular marker for leucine does not necessarily authenticate leucine as the tracer for defining whole-body protein metabolism. A variety of investigators have measured the turnover rate of many of the amino acids, both essential and nonessential, in humans, to define aspects of the metabolism of these amino acids. The general trend of these amino acid kinetic data has been reviewed by Bier (75). The fluxes of essential amino acids should represent their release rates from whole-body protein breakdown for postabsorptive humans in whom there is no dietary intake. Therefore, if the Waterlow model of Figure,2.10 is a reasonable representation of whole-body protein turnover, the individual rates of essential amino acid turnover should be proportional to each amino acid's content in body protein, and a linear relationship of amino acid flux and amino acid abundance in body protein should exist. That relationship is shown in Figure.,2.13. for data gleaned from a variety of studies in humans measured in the postabsorptive state (without dietary intake during the infusion studies) previously consuming diets of adequate N and energy intake. Amino acid flux is correlated with amino acid composition in protein across a variety of amino acid tracers and studies. This correlation suggests that even if there are problems in defining intracellular/extracellular concentration gradients of tracers to assess true intracellular events, changes in fluxes measured for the various essential amino acids reflect changes in breakdown in general.
Figure 2.13. Fluxes of individual amino acids measured in postabsorptive humans are plotted against amino acid concentration in protein. Closed circles represent nonessential amino acids, and open circles represent essential amino acids. The regression line is for the flux of the essential amino acids versus their content in protein. Error bars represent the range of reported values that were taken from various reports in the literature of studies of amino acid kinetics in healthy humans eating adequate diets of N and energy intake studied in the postabsorptive state. The amino acid content of protein data are taken for muscle values from TabJie,2.:3. The regression line slope of 4.1 g proteiN/kg/day is similar to other estimates of whole body protein turnover. (Redrawn from Bier DM. Diabetes Metab Rev 1989;5:111-32, with additional data added.)
Because nonessential amino acids are synthesized in the body, their fluxes are expected to exceed their expected flux based upon the regression line in Figure,2.13 by the amount of de novo synthesis that occurs. Because de novo synthesis and disposal of the nonessential amino acids would be expected to be based upon the metabolic pathways of individual amino acids, the degree to which individual nonessential amino acids lie above the line should also vary. For example, tyrosine is a nonessential amino acid because it is made by hydroxylation of phenylalanine, which is also the pathway of phenylalanine disposal. The rate of tyrosine de novo synthesis is the rate of phenylalanine disposal. In the postabsorptive state, 10 to 20% of an essential amino acid's turnover goes to oxidative disposal. For phenylalanine, with a flux of about 40 pmol/kg/h, phenylalanine disposal produces about 6 pmol/kg/h of tyrosine. We would predict from the tyrosine content of body protein that tyrosine release from protein breakdown would be 21 pmol/kg/h and that the flux of tyrosine (tyrosine release from protein breakdown plus tyrosine production from phenylalanine) would be 21 + 6 = 27 pmol/kg/h. The measured tyrosine flux approximates this prediction ( Fig 2.13) (.8.6).
Compared with tyrosine, which has a de novo synthesis component limited by phenylalanine oxidation, most nonessential amino acids have very large de novo synthesis components because of the metabolic pathways they are involved in. For example, arginine is at the center of the urea cycle ( Fig 2.3). Normal synthesis for urea is 8-12 g of N per day. That amount of urea production translates into an arginine de novo synthesis of approximately 250 pmoL/kg/h, which is four times the expected 60 pmoL/kg/h of arginine released from protein breakdown. As can be seen in Figure,2.13, however, the measured arginine flux approximates the arginine release from protein breakdown (87). The large de novo synthesis component does not exist in the measured flux. The explanation for this low flux is that the arginine involved in urea synthesis is very highly compartmentalized in the liver, and this arginine does not exchange with the tracer arginine infused intravenously.
Similar disparities are seen between the measured fluxes of glutamine and glutamate determined with intravenously infused tracers and their anticipated fluxes from their expected de novo synthesis components. The predicted flux for glutamate should include transamination with the BCAAs, alanine, and aspartate, as well as glutamate's contribution to the production and degradation of glutamine. However, the glutamate flux measured in postabsorptive adult subjects infused with [15N]glutamate is 80 pmol/kg/h, barely above the anticipated rate of glutamate release from protein breakdown (Fig... .. 2 1.3). The size of the free glutamate pool was also determined in this study from the tracer dilution. The tracer-determined pool of glutamate was very small and approximated only the pool size predicted for extracellular water. The much larger intracellular pool that exists in muscle ( Ta.b.!§...2.:.4) was not seen with the intravenously administered tracer. The flux measured for glutamine is considerably larger (350 pmol/kg/h), reflecting a large de novo synthesis component ( Fig 2.13.). However, the pool size determined with the [15N]glutamine tracer also was small—not much larger than glutamine in extracellular water. The large intracellular-muscle free pool of glutamine was not found ( 88). The results of this study showed that glutamine and glutamate tracers administered intravenously define pools of glutamine and glutamate that reflect primarily extracellular free glutamine and glutamate. The large intracellular pools (especially those in muscle) are tightly compartmentalized and do not readily mix with extracellular glutamine and glutamate. Intracellular events such as glutamate transamination are not detected by the glutamate tracer. The same is true of the glutamine tracer. However, the prominent role of glutamine in the body is interorgan transport, i.e., production by muscle and release for use by other tissues (89, 90), and that event is measured by the glutamine tracer (as is obvious from Fjg...2.13. in which the tracer-determined glutamine flux shows the highest measured flux of any amino acid).
The model in Flgyr§ 2.:10. does not consider the potential first-pass effect that the splanchnic bed (gut and liver) has on regulating the delivery of nutrients from the oral route. Under normal circumstances, the amino acid tracer is infused intravenously to measure whole-body systemic kinetics. However, enterally delivered amino acids pass through the gut and liver before entering the systemic circulation. Any metabolism of these amino acids by gut or liver on the first pass during absorption will not be "seen" by an intravenously infused tracer in terms of systemic kinetics. Therefore, another pool with a second arrow showing the first-pass removal by gut and liver should precede the input arrow for I (Fig 2..14) to indicate the role of the splanchnic bed. A fraction f of the dietary intake (I • f) is sequestered on the first-pass, and only I • (1 - f) enters the systemic circulation.
Figure 2.14. Model of whole-body protein metabolism for the fed state when first-pass uptake of dietary intake is considered. A labeled amino acid tracer is administered by the gastrointestinal route (ig) to follow dietary amino acid intake (I). The fraction of dietary amino acid sequestered on first pass by the splanchnic bed (f) can be determined by administering the tracer by both the gastrointestinal and the intravenous routes (iiv) and comparing the enrichments in blood for the two tracers (Eg. and Eiv, respectively).
There are two ways to address this problem. The first does not evaluate the fraction sequestered explicitly but builds the tracer administration scheme into the first-pass losses. One simply adds the amino acid tracer to the dietary intake so that the tracer administration is the oral route (Ig) and enrichments in blood (Eg) come after any first-pass metabolism by the splanchnic bed (91, 92). This approach is especially useful for studying the effect of varying levels of amino acid intake, but it does not evaluate per se the amount of material sequestered by the splanchnic bed.
The second approach applies the tracer by both the intravenous route and the enteral route. The intravenous tracer infusion (Iiv) and plasma enrichment (Eiv) are used to determine systemic kinetics, and the enteral tracer infusion and its plasma enrichment determine systemic kinetics plus the effect of the first pass. By difference, the fraction, f, is readily calculated (93). This approach can be applied even in the postabsorptive state to determine basal uptake of amino acid tracers by the splanchnic bed. As shown in Table.2,.11, a number of amino acids have been studied, and first-pass fractional uptake values for these different amino acids have been determined (93, 94, 95, 96, 97, 98, 99, 100 and 101). In general, the splanchnic bed removes less of the essential amino acids but more than half of the nonessential amino acids on the first-pass—especially glutamate, which is almost entirely removed.
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Table 2.11 First-Pass Sequestration of Enteral Amino and Keto Acid Tracers by the Gut and Liver in Humans
The above methods deal with measurements at the whole-body level but do not address specific proteins and their rates of synthesis and degradation. To do so requires obtaining samples of the proteins for purification. Some proteins are readily sampled (e.g., proteins in blood such as the lipoproteins, albumin, fibrinogen, and other secreted proteins). Other proteins require tissue sampling (e.g., muscle biopsy). If a protein (or group of proteins) can be sampled and purified, then its (their) synthetic rate can be determined directly from the rate of tracer incorporation. Proteins that turn over slowly (e.g., muscle protein or albumin) incorporate only a small amount of tracer during a tracer infusion. Because the incorporation rate of tracer is approximately linear during this time, protein synthesis can be measured by obtaining only two samples. This technique has been especially useful for evaluating protein synthesis of myofibrillar protein with a limited number of muscle biopsies (102, 103). For proteins that turn over at a faster rate, the tracer concentration rises exponentially in the protein toward a plateau value of enrichment that matches that of the precursor amino acids used for its synthesis (i.e., the intracellular amino acid enrichment). The types of protein that have been measured under these conditions have been the lipoproteins, especially apolipoprotein-B (apo-B) in very low density lipoprotein (VLDL) ( 104, 105 and 106).
Determination of the protein fractional synthetic rate is a "precursor-product" method that requires knowledge of both the rate of tracer incorporation into the protein being synthesized and the enrichment of the amino acid precursor used for synthesis. For muscle, L-[1-13C]leucine is often used as the tracer, and plasma KIC 13C enrichment is used to approximate the intracellular muscle leucine enrichment (102). Various other schemes have been used to estimate intracellular liver amino acid tracer enrichment. Hippuric acid is excreted in urine after formation in the liver by conjugation of benzoic acid and glycine. Therefore, urinary hippuric acid can be used as an index of hepatic intracellular glycine 15N enrichment (107, 108). Although the evidence is not specific, suggestions have been made that the hippuric acid does not accurately reflect the glycine precursor pool from which the export proteins are synthesized. However, in the absence of better approach, using the hippuric acid 15N enrichment is clearly better than using another tracer where the hepatic intracellular enrichment is completely unknown. For proteins such as VLDL apo-B that turn over quickly (typically 4-8 hours), tracer incorporation into the protein will approach a plateau within the period of tracer infusion. If the tracer enrichment in the protein does not reach a plateau during the time course of the tracer infusion, curve fitting can usually predict the plateau. When the standard precursor-product relationship holds (i.e., the product is made only from the precursor), the protein plateau amino acid enrichment will reflect the precursor enrichment, simplifying the kinetic calculation (106). Cryer et al. (104) were able to use the plateau in VLDL apo-B to measure the precursor enrichment in normal subjects but still had to use urinary hippurate in hyperlipemic subjects who had large, slow-turnover VLDL pools that did not approach plateau during the course of the 8-h infusion.
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