Adaptation of Protein Metabolism in Relation to Limits to High Dietary Protein Intake

Adaptation of protein metabolism in relation to limits to high dietary protein intake

PJ Garlick1*, MA McNurlan1 and CS Patlak1

1Department of Surgery, Health Sciences Center, T19-020, State University of New York at Stony Brook, Stony Brook, NY 11794-8191, USA

Studies of the effects of dietary protein level on human metabolism have usually concentrated on the effects of protein deprivation and on establishing a minimum dietary requirement. By contrast, less is known about the effects of very high protein diets, although general levels of protein intake in the developed world are increasing, and high protein diets have been advocated for maintaining or increasing muscle mass in certain groups of the population. This article, therefore, examines the response of protein metabolism to high dietary protein, studied in adults by nitrogen balance and isotopic tracer techniques, and concentrating on the evidence for increased lean body mass. It is concluded that high protein feeding initially results in protein retention, with greater cycling of body protein in response to meals, but that neither N-balance nor isotopic tracer methods possess suf®cient sensitivity to detect whether a long term increase in functional lean tissue ensues. Improved methods of body composition measurement will be needed to establish this. Moreover, the absence of strong evidence that high protein diets confer any advantage in terms of strength or health must be weighed against potentially injurious consequences. Descriptors: protein metabolism; dietary protein; nitrogen balance; human; stable isotopes; high protein; lean body mass

Introduction notable study male volunteers consumed 8 g=kg=d( 600 g protein=d) for 9 d (Calloway et al, 1971). Protein intakes as The ability of the human being to adapt to diets with a wide high as this can be achieved with diets based almost range of protein levels has been extensively studied and exclusively on meat or ®sh. For example, there is evidence guidelines have been agreed on the minimum levels needed that the men in the famous Lewis and Clark expedition to maintain health in most of the population (FAO= across America early last century might have eaten 9 lbs WHO=UNU, 1985). By contrast, the effects of diets high (4 kg) of meat daily when buffalos were abundant (Duncan in protein, their possible bene®ts and drawbacks, have been & Burns, 1997). It is dif®cult to see how this amount of much less studied. It is clear that adult humans in different meat could have contained less than 600 g protein, even if a cultures survive on a wide range of protein intakes, from high fat content, to provide energy for very high work rates, marginal levels of only 0.6 g=kg=d to perhaps ten times this is assumed. Moreover, it has been estimated that paleolithic amount. In developed countries, most people will be con- diets might have contained much more protein than is suming diets with protein contents in the range 1 ± 2 g=kg=d presently customary, in the range from 25% to more than (approx. 10 ± 20% of energy intake), but athletes, particu- 50% of dietary energy (Eaton & Konnor, 1985). However, larly when undergoing strength training, frequently take in this century, it seems unlikely that very many of the more (for example, 2.8 g=kg=d (Tarnopolsky et al, 1988; world's populations are consuming that much protein, see Table 1). Athletes and others with very high energy except possibly some Eskimos and gauchos of the expenditures can also consume large amounts of protein Pampas consuming traditional diets comprising mostly because of high total intakes, even when the proportion of ®sh or meat. protein in the food is moderate (Table 1). The purpose of this article is to describe what is known of the adaptations in protein metabolism that are associated with sustained high protein intake. In experimental studies, Table 1 `Habitual' dietary protein intake in sedentary and exercise- investigators have used the term `high protein' for diets training young men ranging from 1.8 g=kg=d±3.3g=kg=d (Fisher et al, 1967; Dietary protein intake Allen et al, 1979; Fern et al, 1991; Price et al, 1994; Quevedo et al, 1994; Yang et al, 1986). However, in one Sedentary Endurance training Body-building

g=d 84 124 222 g=kg=d 1.1 1.7 2.8 *Correspondence: Dr PJ Garlick, Department of Surgery, Health Sciences % of energy 11 12 19 Center, T19-020, State University of New York at Stony Brook, Stony Brook, NY 11794-8191, USA Data from Tarnopolsky et al, 1988. Adaptation of protein metabolism PJ Garlick et al S35 Amino acid oxidation protein, and have until recently been the basis of determi- nations of protein and amino acid requirements (reviewed The major changes in protein and amino acid metabolism by Fuller & Garlick, 1994). The long-term effects of a resulting from an increase in dietary protein intake can be change in protein intake are well illustrated by the study of separated into acute and chronic responses. An early and Oddoye & Margen (1979) who very carefully measured N sustained response to an increase in protein intake is an intake and excretion in six subjects over a period of 100 d. elevation of amino acid oxidation, with consequent In three subjects, the diet initially contained 12 gN=d increases in nitrogen excretion and the proportion of total (75 g protein) for 40 d, and was then increased to 36 gN=d energy expenditure derived from oxidation of amino acids. (225 g protein) for the remaining period, whereas the other There is also an initial net retention of amino acids in the three subjects took the diets in reverse order. In those body. These points are illustrated by the study of Robinson starting on the 12 gN=d diet, the N excretion remained et al (1990), who gave meals containing 70% protein at steady over the 40 d period and the balance was not hourly intervals for 9 h, and compared results with those signi®cantly different from zero. However, those who obtained with 70% carbohydrate meals (12% protein), in began on 36 gN=d consistently excreted less nitrogen than healthy subjects who had previously received a normal they consumed, so were in positive balance by about protein intake. The amount of protein oxidized during the 1gN=d. After the change of diet from 12 to 36 gN=d, N 9 h was estimated by measurements of urinary nitrogen output increased over the ®rst few days, so that over a excretion, corrected for changes in the size of the body urea period of 10 to 20 d the subjects retained about 16 g N. pool, on the assumption that all urinary nitrogen is derived After this the N excretion stabilized, but always remained ultimately from amino acid oxidation. Over the 9 h period, less than intake, so positive N balances of about 1 gN=d 290 g protein were consumed with the high protein diet, persisted over the remaining 40 d. Conversely, in those who compared with 50 g for the high carbohydrate diet. In both changed from high to lower protein, the N balance was cases the nitrogen excretion rose from the prefeeding level negative by about 30 gN over 20 d following the change, to rates corresponding to about 50% of the protein intake but the balance became zero for the remaining period on over the 9 h. Therefore, amino acid oxidation was rapidly the 12 gN=d diet. able to respond to the intake of a high protein meal with an The important points to be gained from this study are almost 3-fold increase. The regulation of oxidation for a ®rstly that after a change in dietary protein there is a variety of amino acids has been studied widely and has transient retention or loss of body N over 10 to 20 d. This been shown to operate partly through increases in amino is a very well recognized phenomenon, which has been acid concentration. There are also increases in the activity extensively discussed (Munro, 1964). It has been attributed of the enzymes for transamination, for disposal of the to a labile pool of body protein which expands or contracts carbon skeletons in intermediary metabolism and disposal in response to changes in dietary protein intake, but its of nitrogen mostly by the urea cycle. These processes have actual identity has not been proven in humans. A part of the been reviewed comprehensively (Harper 1983, 1994; retained N must result from an expansion of the body urea Harper et al, 1984). Changes in amino acid oxidation also pool. Urea is uniformly distributed in body water, which lead to alterations in the pathways used to derive energy. normally contains about 9 gN as urea in subjects on normal Oxidation of protein (amino acids) contributed as much as diets. When the dietary protein intake is increased the urea 57% of total energy expenditure with the high protein concentration also increases, about 2-fold with a change in meals, compared with 21% with high carbohydrate. diet from 12 to 36 gN=d (Oddoye & Margen, 1979; Callo- These data show that amino acid oxidation is very way et al, 1971). Therefore, about 9 gN, corresponding to ¯exible and can very quickly alter over a wide range. 30 ± 50% of the retained N could be contained in the urea However, despite the 3-fold increase in oxidation, only pool. A smaller amount of N might also result from an 50% of amino acids from the high protein meal were expansion of the pools of free amino acids such as gluta- oxidized, and the remaining 50% must have been retained mine, which has a very high concentration in some tissues, by the body. These could not have been retained as free particularly muscle (Vinnars et al, 1975). Whether the rest amino acids, as their total content in tissues is too small, corresponds to an increase in protein in muscle or other and amino acids must therefore have been retained as tissue tissues is not known, as an increase of 20 gN represents too protein. As will be discussed later, all of the retained small a proportion of total body N ( 2 kg) to be detect- protein from meals containing normal levels of protein able. would be expected to be lost again during a subsequent The second point to be gained from the study of Oddoye overnight fasting period, with no net gain over a full day. & Margen (1979) is that when subjects receive high intakes However, with high protein intakes, there are important of protein, the measured N balance remains positive for an questions of whether the net gain continues, for how long extended period of time, and shows no sign of returning to and by what mechanisms? These issues are the main emphasis of the current review, as the answers have a signi®cant impact on whether high protein diets may be of Table 2 Some published results of N-balance in healthy subjects given any value in promoting increased lean body and muscle high protein diets mass in athletes or body builders, or in preventing protein Protein Dietary loss in subjects who are at risk of body wasting (for intake period N-balance example, elderly subjects or patients with AIDS). Authors (g=kg=d) (d) (gN=d)

Oddoye & Margen, 1978 3.0 50 1.6 Fisher et al, 1967 2.8 21 3.1 Nitrogen balance Tarnopolski et al, 1988 2.8 12 13.4 Price et al, 1994 2.3 12 3.0 Measurements of nitrogen balance have been widely used Cheng et al, 1978 2.3 11 3.0 to study the response to changes in the dietary level of Adaptation of protein metabolism PJ Garlick et al S36 Table 3 Routes of nitrogen loss from the body signi®cant changes in urinary creatinine excretion, fat-free mass, or other measures of protein deposition (Fisher et al, mgN=d 1967; Cheng et al, 1978; Tarnapolsky et al, 1992). Even Major during 12 weeks of resistance training in older subjects urine (urea, ammonia, creatinine, etc.) 10,000 (Campbell et al, 1995) or 1 month of strength training in feces (diet residue, endogenous) 1,500 young adults (Lemon et al, 1992), no differences due to Minor dermal (skin debris, sweat) 200 higher dietary protein could be detected in fat-free mass, hair, nails 30 urinary creatinine, and muscle size (by computerized tomo- saliva (tooth brushing) 30 graphy) and composition. Hence, the weight of evidence breath (ammonia) 50 suggests that the positive N balances do not correspond to miscellaneous (nasal mucous, semen) each < 20 increased lean tissue, and particularly not skeletal muscle, Estimates of amounts of nitrogen taken from Calloway et al, 1971. so the observation of increased creatinine excretion by Oddoye & Margen (1979) remains to be con®rmed. The alternative conclusion, therefore, is that the N balance zero. This phenomenon has been demonstrated in almost all technique has an intrinsic error which leads to apparently studies of high protein intake, some examples being shown positive balances at high protein intake. Very careful in Table 2. Even if the very high positive balance of measurements have been made in an attempt to identify 13.4 gN=d (Tarnopolsky et al, 1988) is regarded as unrea- the source of such errors, and although none has been listic, positive balances in the region of 1 ± 3 gN=d appear detected (Calloway et al, 1971), it is commonly believed to be a consistent ®nding. This has been discussed pre- that the N balance technique is inadequate for assessing viously by Hegsted (1978), who noted a positive correlation changes in body protein content (Oddoye & Margen, 1979; between N intake and N balance in data taken from a range Yoshimura, 1972), especially in relation to high protein of studies, regardless of whether the subjects were adult, or intake. This lack of con®dence in N balance has led some were growing, and therefore likely to be depositing nitro- investigators to explore the use of isotopic tracer methods, gen as protein. His conclusion was therefore that the N as these can give information not only on net protein balance technique has some unrecognized error that leads balance, but also on the dynamics of protein and amino to excessively positive balances at higher protein intakes. acid metabolism that determine protein retention or loss. The suggested rationalization of this is that the two main sources of experimental error, failure to collect all N losses Stable isotopic measurements of protein and amino acid and failure to measure all uneaten food, both result in dynamics erroneously positive balances (Hegsted, 1978). Oddoye & Margen (1979) discussed this possibility with regard to Most early studies of the in¯uence of diet on protein their own data, and also in relation to a previous study from dynamics were based on infusions of the stable isotope, the same laboratory which very carefully assessed the 15N, followed by measurement of the excretion of the errors encountered with measurement of N intake and isotope in urinary end products of nitrogen metabolism output (Calloway et al, 1971). The routes and correspond- (Waterlow et al, 1978a, b). The ®rst systematic investiga- ing amounts of N loss for subjects consuming 12 gN=d are tion of dietary protein intake employed constant infusion of summarized in Table 3. The major loss ( 85%) is in the [15N]glycine for 48 h periods to measure whole-body pro- urine, and this together with fecal N makes up 97% of the tein synthesis and degradation in volunteers given adequate total. Other losses are less than 3%. Most of these other protein (1.5 g=kg=d) for 5 d then low protein (0.38 g=kg=d) losses were directly measured by Oddoye & Margen for a further 12 d (Steffee et al, 1976). Surprisingly, both (1979), and they argued that those that were not measured the synthesis and degradation of protein were higher when (breath ammonia) were far too small to have accounted for the low-protein diet was given. The importance of dietary the observed positive balance. protein for maintaining whole-body protein turnover was The critical question of whether a high protein diet can identi®ed in obese subjects given a low-energy, protein-free result in sustained retention of nitrogen, and therefore a diet for three weeks. When the low-energy diet contained functional expansion of lean body mass, remains unan- no protein, there were large decreases in protein synthesis, swered by the N balance studies. A retention of 1 gN=d for degradation and oxidation compared with rates on a normal 50 d, as reported by Oddoye & Margen (1979), corresponds diet, whereas rates were maintained when a low-energy, to a total increase of about 1.5 kg of fat-free body mass, protein containing diet was given (Garlick et al, 1980a). It assuming a factor of 34 gN=kg fat-free tissue (Widdowson was also shown that these changes took place very quickly, & Dickerson, 1964). If all the retained N were as muscle already being apparent on the ®rst day after the change in protein, it would comprise about 0.1% of muscle protein diet, with smaller changes thereafter (Garlick et al, 1980a). mass per day (assuming the body contains about 1 kg of The rapidity of these changes, and a growing awareness muscle protein), which is reasonable in relation to the that rates of protein turnover are not constant even within a knowledge that skeletal muscle protein is turned over single day, undergoing cycles of protein deposition during very slowly at a rate of 1.5 ± 2%=d, corresponding to a feeding and loss during fasting (Garlick et al, 1973, 1980b; half-life of 35 ± 47 d (Garlick et al, 1994). It seems thus Clugston & Garlick, 1982), emphasized the need for possible that an expansion of muscle protein mass would methods of measuring protein dynamics over short periods occur very slowly and might continue for periods in excess of hours, rather than the days required with the [15N]glycine of 50 d. Fat-free mass was not reported, but urinary crea- method. Although a more rapid technique with [15N]gly- tinine, as a measure of muscle mass, increased signi®cantly cine was developed (Fern et al, 1981), the primed constant by 4% (Oddoye & Margen,1979), which is consistent with infusion of [1-13C]leucine (Matthews et al, 1980; Motil et the 1%=d increase in muscle protein for 50 d predicted from al, 1981) has proved the most useful. Only 4 h are required the N balance. However, other studies have not detected to make measurements (Matthews et al, 1980; Motil et al, Adaptation of protein metabolism PJ Garlick et al S37 1981), or alternatively, an 8 h infusion can be divided into into the pool, and also equals the total rate of exit or two separate dietary periods of 4 h each (Garlick et al, disposal (Rd) from the pool. Leucine has very limited 1987). With this method it was shown that protein turnover metabolic pathways, so appearance is the sum of leucine and oxidation respond immediately to the intake of food. intake (from the diet plus infused tracer) and leucine When food containing a normal amount of protein was derived from whole-body protein degradation (Figure 1). consumed, there was an increase in amino acid oxidation The latter can then be calculated, as appearance and dietary and a decrease in protein degradation within the ®rst 2 ± 4 h intake of leucine are known. Similarly, disposal is the sum (Melville et al, 1989). The change in protein balance, from of oxidation, which is determined from the appearance of 13 negative during fasting to positive during feeding, resulted CO2 in the breath, and utilization by whole-body protein from a depression in whole-body protein degradation, with synthesis, permitting protein synthesis to be calculated. The little, if any, change in synthesis (Melville et al, 1989). The details of the technique have been described in previous importance of the above results is that they demonstrate the articles (Waterlow et al, 1978a; Bier et al, 1984; McNurlan requirement that any investigation of the metabolic & Garlick, 1989). Its validity depends on the following effects of dietary protein should take account of both the assumptions: immediate responses to meals of differing composition, and subsequent adaptations to the longer term intake of a 1. The metabolism of the single amino acid leucine accu- different diet. rately represents that of body protein: Leucine appearance occurs only from the diet, infused tracer and body protein degradation. 13 Assumptions and validity of the [1- C]leucine infusion Leucine disposal is only by oxidation to CO2 and into technique protein synthesis. The leucine concentration in body proteins is known. Before examining the use of isotopic methods to study the The free leucine pool is in a steady state. responses to high-protein diets, it is important to consider the basis and assumptions of the methods. Although there 2. Whole body leucine ¯ux rate can be determined accu- have been some studies with 15N, this technique also rately from the plateau enrichment of plasma KIC. depends on N balance, whereas methods employing 13C The enrichment of leucine at the sites of leucine are independent of N balance. Moreover, 13C has also been oxidation and protein synthesis is the same as that of the most widely used, particularly with [1-13C]leucine, so plasma KIC. the following sections will focus on studies with this tracer. There is no recycling of labeled leucine. The model for whole body leucine metabolism used to determine leucine and protein kinetics is shown in Figure 1. The basis of the method is that the labeled amino acid is The assumptions listed under (1) are in general well used to determine the turnover rate of the free amino acid accepted, with two reservations. The leucine composition pool, which has been termed the ¯ux rate. If [1-13C]leucine of these proteins in the body that are being synthesized is is given as a continuous infusion, and the body free leucine not, and cannot be known accurately. Hence, the rates of pool remains constant in size (steady state), the isotopic leucine metabolism cannot be converted into equivalent enrichment of the leucine in plasma will reach a constant rates for protein with complete con®dence. Of potentially (plateau) value from which the ¯ux rate is calculated from more signi®cance is the possibility that leucine might be the expression: synthesized by bacteria in the colon and made available to the host (Jackson 1994, 1995). In this case, leucine synth- infusion rate of 1-13Cleucine esis would have to be known in order to calculate rates of leucine flux : enrichment of plasma free leucine whole body protein degradation or leucine balance. The quantitative importance of bacterial synthesis of essential In practice, the enrichment of plasma ketoisocaproate amno acids in the colon is currently under investigation in (KIC) is usually measured rather than that of leucine, as it various centers. The assumptions under (2) above have is considered to be a better measure of leucine enrichment been studied in some detail. Recycling is known to be a in cells, where protein synthesis is actually occurring problem with long infusions, for example, 24 h (Melville et (Matthews et al, 1982). In the steady state, the ¯ux rate al, 1989; Schwenk et al, 1985), but for infusions of not is equal to the total entry or appearance rate (Ra) of leucine more than 8 h, it is not considered to be a serious source of error. In any case, the error would only apply to rates of protein synthesis and degradation, and would not in¯uence the estimate of leucine oxidation. Similarly, the use of KIC enrichment as a measure of intracellular leucine enrichment is unlikely to introduce a serious error for leucine oxidation, as KIC is on the direct pathway of leucine oxidation. However, it might have more serious consequences for determining rates of protein synthesis, as the main site of production of plasma KIC is thought to be skeletal muscle, whereas protein is being synthesized in all body tissues, each of which might have a different leucine enrichment. It has been shown that the enrichment of leucyl tRNA in muscle is different from that of plasma KIC, and that the Figure 1 The model representing whole-body leucine metabolism, used difference between the two enrichments varies with food to derive rates of protein turnover from leucine oxidation tracer kinetic intake (Ljungqvist et al, 1997). The present evidence, studies. therefore, suggests that leucine oxidation rates can be Adaptation of protein metabolism PJ Garlick et al S38 measured accurately by infusion of [1-13C]leucine, but plateau is reestablished. This differs from the situation with there is less information regarding the accuracy of mea- leucine oxidation, where all the relevant pools turn over sured protein turnover rates. relatively quickly. Because, the pool size of urea and the There is no truly independent method against which to rate of synthesis of urea are constantly changing, as a result validate the rates of whole-body protein turnover obtained of the discontinuous dietary intake, the plasma urea enrich- with [1-13C]leucine, except the methods employing ment never has time to reach a true plateau. This situation [15N]glycine, which rely on many of the same assumptions. has been modeled, by assuming that urea synthesis follows However, in general the methods with [15N]glycine show a course that can be described by a sine function with phase reasonable consistency with [1-13C]leucine (Garlick et al, and amplitude chosen to represent the measured curve for 1980a; Garlick & Fern, 1985). However, leucine oxidation leucine oxidation (Figure 3). It was then assumed that urea rates can be compared with nitrogen excretion, as subjects turnover is measured by infusion of labeled urea and that who are in N balance should also be in leucine balance. the system follows ®rst order kinetics. The changes with Moreover, the ratio of leucine to total N in the diet should time of the enrichment of urea and apparent values for urea be the same as the ratio of leucine oxidation to total N production rate calculated from successive values for urea excretion. This approach must be taken over periods long enrichment were then simulated (additional details of the enough that the discontinuous intake of food during the day simulation are available from the authors). The result is does not interfere, namely 24 h or more. One such study has illustrated in Figure 3, showing that the apparent rate of been performed by El Khoury et al (1994), who made urea production has a smaller amplitude and a delay of frequent measurements during a 24 h infusion of about 5 h in comparison with the curve for leucine oxida- [1-13C]leucine in subjects who had been adapted to a diet tion rate. Moreover, the simulated curves are very similar to of known leucine content. The comparison of measured the experimentally derived curves shown in Figure 2. This rates of N excretion, with N excretion calculated from simulation, therefore, shows that the differences between leucine oxidation, on the assumption that the leucine to N the measured curves for leucine oxidation and urea produc- ratio was the same as that for the diet, showed very good tion could have arisen because of the delay in the large, agreement. In a group of seven subjects, the estimate from slowly turning over urea pool, and do not necessarily leucine oxidation was 160 mgN=kg=d (s.d. 6), no more indicate a dissociation between leucine oxidation and than 5% different from the estimate from total N excretion urea synthesis. On this basis, it is reasonable to conclude of 153 mgN=kg=d (s.d. 18, P NS). Despite the agreement of leucine oxidation and N excretion over 24 h, it has been suggested that the time course of leucine oxidation differs substantially from that of urea production, indicating inconsistencies between the two methods (Jackson, 1994, 1995). However, in our opinion, these discrepancies may be more apparent than real. The data are illustrated in Figure 2, which shows the 24 h time courses of leucine oxidation and urea excretion, compared with urea production measured simultaneously by infusion of [15N]urea. The ®rst point is that the curve labeled as `urea excretion' is in reality more an indication of net urea production, since the excreted amounts of urea during 3 h intervals were adjusted for changes in the body urea pool. The time course of this curve agrees quite well with that for leucine oxidation, which would be expected if leucine oxidation was a ®xed proportion of protein oxidation and hence of urea synthesis. However, the curve for total urea production measured with [15N]urea differs appreciably from the other two. Although, like the other two curves, it displays a periodicity related to the diurnal feeding pattern, the phase is different, with a delay of about 5 h, and the amplitude is less. There are two reasons why this curve should be different. Firstly, the isotopic method measures total urea production, which includes a proportion, about 20%, which is degraded in the gut and the nitrogen recycled into new urea synthesis. It is the possibility that this recycling might include leucine synthesized in the gut that has alerted Jackson (1994, 1995) to point out the discrepancies, as de novo synthesis of leucine would invalidate the [1-13C]leucine method. However, there is a second reason for the discrepancies between these curves. When urea production is measured by infusion of Figure 2 Hourly values for leucine oxidation (upper curve) and total [15N]urea, the plateau in plasma urea enrichment is only urea production (middle curve) measured by simultaneous infusion of reached very slowly, unless a large priming dose is given, [1-13C]leucine and [15N]urea over a 24 h period. Nitrogen excretion was because of the slow turnover rate of the urea pool measured in three-hourly urine collections and adjusted for changes in plasma urea concentration (lower curve). Subjects were fasted or given (T1 10 h). This also means that any disturbance of the 2 small hourly meals as shown on the time axis. Data are taken from El plateau will require an extended period of time before a Khouri et al (1994), with permission. Adaptation of protein metabolism PJ Garlick et al S39 last 4 h of fasting and the ®rst 4 h of feeding. In addition, urine collections were made over the two times 12 periods of feeding and fasting, and nitrogen excretion, corrected for changes in the body urea pool, was determined. In the study by Price et al (1994), four levels of protein were given to volunteers for two weeks of adaptation before measurements were made. Figure 4 illustrates the relationship of nitrogen intake to nitrogen excretion during the fasting and fed periods. At the lowest intake, N excretion was not altered by feeding, but at higher levels of intake, N output was higher in the fed state. Figure 5 shows the N balance values calculated from the same original data. This illustrates the magnitude of the `diurnal cycling' of body nitrogen resulting from the discontinuous intake of food and extends the knowledge gained in earlier studies. Sev- Figure 3 Simulated curves for leucine oxidation and net urea production rates, illustrating damping of the apparent rate of urea production by the eral points are worthy of note. During feeding, as described body urea pool. Urea production rate, as derived from [15N]urea infusion, previously, the balance is strongly dependent on the protein is predicted from the curve for leucine oxidation. For details, see text. intake. Moreover, positive balance is possible during feeding even with the lowest protein intake, which is below the accepted requirement level. However, N balance even that the [1-13C]leucine infusion method is valid, at least during fasting is also strongly dependent on previous when judged in relation to N excretion. level of intake, as a result of adaptation. Figure 5 also shows the daily balance, which is negative at the lowest, sub-requirement intake. However, the daily balance is also Protein turnover in relation to dietary protein level negative at an intake of 0.77 g=kg=d (approximately equal The ®rst systematic study of protein turnover in relation to to the accepted requirement level), but becomes positive at dietary protein intake to take account of variations induced higher intakes. The notable feature is the almost linear by the diurnal pattern of food intake employed [1-13C]leu- relationship between N intake and balance, with substan- cine infusion in volunteers with three levels of protein tially positive balances at high protein intakes, consistent intake in the range from very low to generous (0.1 ± with the N-balance data discussed earlier. 1.5 g=kg=d, Motil et al, 1981). Subjects were given the The rates of leucine oxidation determined by Price et al diets for adaptation periods of 6 ± 8 d before measurements (1994) in the fed and fasting states show a similar pattern to of leucine kinetics were performed with 4 h infusions of the N excretion shown in Figure 4. However, when leucine [1-13C]leucine in the postabsorptive period or while ingest- oxidation rates were converted to equivalent rates of N ing the appropriate diet as small hourly meals, each excretion, by the same method as that employed by El comprising one twelfth of the total day's intake. At the Khoury et al (1994), described in the previous Section, the higher levels of protein intake, protein synthesis was values are in all cases lower than expected; between 61% elevated in both the fed and fasting states, whereas protein degradation was elevated only during fasting. The net result of these changes on protein balance was an increasing protein loss during the fasting period at higher protein intakes during fasting, and increasing protein retention during feeding. Therefore, the effects of protein intake are not only apparent during the absorptive phase, but also during a subsequent period of fasting. Responses can therefore be characterized either as immediate consequences of food absorption or as persistent adaptations to differing protein intake. More recently, there has been a variety of studies of Figure 4 Rates of leucine oxidation and dietary intake, measured by infusion of [1-13C]leucine, after fasting overnight or while eating small whole-body protein turnover at low to moderate levels of hourly meals. For details refer to the text and Price et al (1994). protein intake in humans (Robinson et al, 1990; Garlick et al, 1991; Campbell et al, 1995; Pannemans et al, 1995; Gibson et al, 1996) and also at high intake (Yang et al, 1986; Fern et al, 1991). However, a recent and systematic series of studies with levels of protein intake extending from low to high (0.36 ± 2.31 g=kg=d) has further developed the concepts of immediate effects of food intake and adaptive responses (Pacy et al, 1994; Price et al, 1994; Quevedo et al, 1994). As in earlier studies described above (Motil et al, 1981; Clugston & Garlick, 1982; Melville et al, 1989), the experimental protocol consisted of a day divided into two notional periods, 12 h of fasting (overnight) and 12 h of feeding, involving hourly small meals. Figure 5 Leucine balance afer fasting overnight or while eating small Measurements of leucine kinetics were made during a hourly meals, calculated from the data shown in Figure 4 (Price et al, single 8 h infusion of [1-13C]leucine which spanned the 1994). Adaptation of protein metabolism PJ Garlick et al S40 feeding might have led to an underestimate of the total daily oxidation rate. The ®nal question, therefore, is whether the rates of protein synthesis and degradation calculated from the leucine kinetic data are valid. As leucine oxidation rates and balance are generally small in comparison to rates of protein turnover, there is no reason to suppose that the trends in protein synthesis and degradation will not be predicted correctly by this technique. This is especially true of the trend in degradation, since its value does not depend on measurements of leucine oxidation. The rates of whole-body protein turnover at the four Figure 6 Rates of leucine oxidation, measured by infusion of [1-13C]leu- different levels of dietary protein were calculated from the cine, after fasting overnight or while eating small hourly meals for 4 h, 8 h data in the above study by Pacy et al (1994) (Figure 7). In or 12 h (Melville et al, 1989). the fasting state, the effects of protein intake are small, with no change in protein synthesis at higher intakes, but a small increase in protein degradation, which is responsible for the increase in protein loss (Figure 5). The changes in the fed state are more pronounced, involving a progressive decline in protein degradation with increasing intake and a lesser increase in protein synthesis. These two changes combine to give a large increase in protein retention at higher intakes. Overall, it is apparent that protein balance is far more dependent on changes in degradation than changes in synthesis, when considered in terms of both immediate and adaptive responses. The transition from fasting to Figure 7 Rates of whole-body protein synthesis and degradation, mea- feeding, resulting in a rapid switch from negative to sured by infusion of [1-13C]leucine, after fasting overnight or while eating positive balance, involves a depression in degradation, small hourly meals. For details refer to the text and Pacy et al (1994). which is greater at higher intakes. Synthesis, by comparison, is not responsive to feeding at the lower intakes and is only slightly stimulated at high intake. There is also a and 75% of the N excretion during fasting and between higher rate of protein degradation in the fasting state, 84% and 99% during feeding. The impact of these dis- resulting from two weeks of adaptation to the high protein crepancies on N balance estimated from leucine balance is diet. However, as pointed out by the authors (Pacy et al, substantial, leading to more positive daily balances at all 1994), when the average daily rates of synthesis and intakes. Therefore, only the lowest intake resulted in a degradation are calculated, there is little difference over negative value for balance (720 mgN=kg=d), whereas the full range of protein intake. the highest intake showed a very large positive The period of time after a change in dietary protein for balance (133 mgN=kg=d). These values compare with adaptations to occur has also been examined by the same 738 mg=kg=d and 40 mg=kg=d obtained by direct mea- group, employing a similar experimental protocol (Que- surement of N intake and excretion (Figure 5). vedo et al, 1994). The subjects were initially adapted to a These data raise three important questions. Firstly, can protein intake of 1.82 g=kg=d for 14 d, followed by a further leucine kinetics allay the misgivings expressed previously 14 d with 0.77 g=kg=d. N excretion after the diet change about the validity of the N retention seen in adults on high was progressively reduced during both fasting and feeding, protein intakes? Clearly, the present data do not help with with the most rapid changes occurring during the ®rst 3 d. interpreting the N balance data, as they lead to even more Further reductions occurred up to day 9, with little change extreme positive balances. The second question is why does thereafter. N balance was initially positive, became highly N excretion measured directly differ from that calculated negative during the 3 d after the change, and then from leucine oxidation? As discussed before, when mea- approached, but did not reach, zero balance, as has been surements are made over a complete 24 h period, good shown previously. Measurements of leucine kinetics in the agreement between these two measurements has been fasting and fed states were made as described above (Price demonstrated. Therefore, it seems likely that the discre- et al, 1994; Pacy et al, 1994), ®rstly when subjects were on pancy observed by Price et al (1994) relates to the speci®c the higher protein diet, and then on three additional occa- experimental protocol. The main differences between pro- sions, at 3, 7 and 14 d after the change to the lower protein tocols are that Price et al (1994) estimated, but did not diet. Rates of whole-body protein synthesis, degradation measure non-urinary N losses, and they also measured and balance derived from leucine kinetics during fasting leucine kinetics during 8 h rather than the whole day. The and feeding are shown in Figure 7. Although there were latter difference is likely to be the more important explana- changes in balance after the change in diet, it is not possible tion of the discrepancy. As can be seen from Figure 6, in a to attribute these to protein synthesis or degradation, as the study with the same experimental protocol as that changes were too small. However, in the fed state, the employed by Price et al (1994), the rate of leucine oxida- changes in balance were much greater, and it is clear that tion increases gradually from the beginning to the end of the adaptation resulted mainly from a diminished suppres- the feeding period (Melville et al, 1989). Therefore, mea- sion of protein degradation by food intake. Moreover, this surement of leucine kinetics during only the ®rst 4 h of response occured quite quickly, being almost complete at 3 d. Adaptation of protein metabolism PJ Garlick et al S41 A number of conclusions can be drawn concerning the training. Studies with more sensitive methods over longer adaptation of whole-body protein turnover to high protein periods of dietary treatment may be necessary to detect diets. slow rates of accumulation of protein of 1 gN=d, which is equivalent to only 0.05% of total body N per day. Adaptive changes are apparent on both the fasting and The second question relates to possible sites of deposi- fed states. tion of protein, both during the ®rst few days after a change Most of the adaptation takes place within the ®rst 3 d of diet, or in the longer term. This knowledge is potentially after an increase in protein intake, and small subsequent important in relation to the likely bene®t. Skeletal muscle is changes are not detectable by measurements of leucine thought to be particularly important as a `reserve' of protein kinetics. that can be drawn upon during critical illness or other stress The most conspicuous adaptation is an increase in amino (Garlick & Wernerman, 1997). Amino acids from the acid oxidation and subsequent nitrogen excretion, which breakdown of muscle protein are used to synthesize gluta- is especially pronounced in the fed state. mine, which is an important substrate for maintaining the Alterations in protein balance result largely from an function of the gut and the cells of the immune system enhanced inhibition of protein degradation by feeding, (Newsholme, 1990; Souba et al, 1990). Measurements of whereas protein synthesis is relatively unaffected. the balance of amino acids across limbs have shown that The amplitude of diurnal cycling of body protein skeletal muscle takes up amino acids during food absorp- increases at high protein intake, with no change in tion and releases amino acids during fasting (Halliday et al, mean daily protein turnover rates. 1988). This has been attributed to variations in the rate of muscle protein synthesis (Halliday et al, 1988), although other studies have demonstrated no change in muscle protein synthesis in fed compared with fasting subjects Conclusions and implications (McNurlan et al, 1993), suggesting that changes in protein It is clear from the foregoing discussion that healthy degradation occur. However, differences in amino acid humans have a wide capacity to adapt to differences in uptake and release by muscle due to variations in dietary protein intake. After an increase in dietary protein intake, protein level do not appear to have been studied. Similarly, various phases of response and adaptation can be discerned. there appear to have been no studies of the effects of Initially, the acute response to a meal with a higher protein dietary protein intake on rates of muscle protein synthesis content is modi®ed, so that amino acid oxidation is or degradation in adult humans or animals. increased and more nitrogen is retained in the body, Although skeletal muscle has been the tissue most possibly as protein. Over the following few days, oxidation studied in humans, work in animals has shown that the adapts further, so that the net balance of protein approaches liver protein content is highly variable, depending on the zero. This phase involves adaptation, especially of rates of time after feeding (Millward et al, 1974) and dietary protein degradation and oxidation, not only during absorp- protein content (Munro, 1964). These changes in protein tion of meals, but also during the postabsorptive state. After content are brought about mainly by changes in the rate of 10 ± 20 d, no further changes in protein balance can be liver protein degradation rather than synthesis (Garlick et detected, but in a wide variety of studies, high protein al, 1973, 1975; Conde & Scornik, 1976). There appears to intake has been shown to result in continued positive N- have been only one study of liver protein synthesis in balance of 1 gN=d or more. It is not known whether this relation to nutrition in humans, showing no difference apprent retention of protein is real, and if so, for how long it between patients who were fasting and those who were persists. given total parenteral nutrition prior to surgery (Barle et al, The conclusions raise a number of pivotal questions. It is 1997), consistent with the data from animals. However, of considerable importance that the question of whether or there have been a number of investigations of the synthesis not lean body mass can be increased by high protein diets of serum albumin by the liver, showing higher rates of can be resolved. This question is not only salient to synthesis at higher dietary protein intakes (Gersowitz et al, athletes, who wish to improve their total body and 1980; Kaysen et al, 1986). muscle protein mass and performance. The decline in A third question is whether high protein diets cause a muscle mass as age progresses, with consequent loss of generalized increase in the turnover of proteins. It has been mobility, is well characterized. Similarly, various chronic hypothesized that proteins of the body are continually illnesses, such as rheumatoid arthritis and AIDS, cause synthesized and degraded to confer adaptability (Waterlow substantial lean tissue wasting. Moreover, acute critical et al, 1978). Therefore, amino acids would always be illness can result in substantial losses of body protein available, regardless of feeding status, to meet the (Garlick & Wernerman, 1997). Whether high protein demand for amino acids as substrates for the synthesis of diets can have any role to play in reversing or preventing a new protein and other products (Young & Marchini, these losses is therefore a very important question. It seems 1990). Adaptability might also be enhanced if turnover unlikely that the current uncertainty regarding the positive were more rapid. Pacy et al (1994) have suggested that N-balances at high intake can be resolved by additional their data (Figure 7) do not support the idea of improved studies of balance of either nitrogen or a single amino acid adaptability, since there was no increase in the average (leucine), as these will always be hampered by the inac- daily rates of protein turnover with high protein intake. curacies of calculating small differences between large However, during the crucial fasting period, when there was numbers. If the apparent N retention is real, then it must no other source of amino acids for new protein synthesis, ultimately be detectable by measurement of body composi- the supply from protein degradation was indeed somewhat tion, if it is of any physiological signi®cance. So far, body higher when subjects were given high protein diets (Figure composition measurement has, in general, failed to detect 7), which is consistent with the concept of enhanced changes due to dietary protein, even during exercise adaptability. Adaptation of protein metabolism PJ Garlick et al S42 So far, effects of high protein intake on protein the absence of strong evidence that these adaptations confer metabolism have been considered only in terms of any advantage in terms of strength or health, must be potential bene®ts, but it should be pointed out that weighed against possibly injurious consequences. there are documented drawbacks. In certain speci®c groups there have been clear recommendations regarding the upper level of protein intake. For example, at a References symposium to discuss the nutrient content of infant Allen LH, Oddoye AE & Margen S (1979): Protein-induced hypercal- formulas (Fomon & Ziegler, 1989) it was suggested ciuria: a longer term study. Am. J. Clin. Nutr. 32, 741 ± 749. that the recommended maximum level of protein in Barle H, Nyberg B, Andersson K, EsseÂn P, McNurlan MA, Wernerman J & Garlick PJ (1997): The effects of short term parenteral nutrition on formulas for healthy term infants should be revised human liver protein and amino acid metabolism. JPEN 21, 330 ± 335. downwards from 4.5 g protein=100 kcal to 3.5 g pro- Bier DM, Matthews DE & Young VR (1984): Interpretation of amino acid tein=100 kcal. The rationale was based on the abnormal kinetic studies in the context of whole-body protein metabolism. In plasma amino acid pro®le of high-protein formula-fed Substrate and Energy Metabolism in Man, eds. JS Garrow & D Halli- day. London: John Libbey & Co. Limited, pp 27 ± 37. babies compared with those fed breast milk, and con- Calloway DH, Odell ACF & Margen S (1971): Sweat and miscellaneous cerns for acid-base balance and renal solute load (Young nitrogen losses in human balance studies. J. Nutr. 101, 775 ± 786. & Pelletier, 1989). Similarly, it is quite well accepted Campbell WW, Crim MC, Young VR, Joseph LJ & Evans WJ (1995): that patients suffering from chronic renal failure should Effects of resistance training and dietary protein intake on protein restrict their dietary protein intake (Pedrini et al, 1996; metabolism in older adults. Am. J. Physiol. 268, E1143 ± E1153. Cheng AHR, Gomez A, Bergan JG, Lee TC, Monckeberg F & Chichester Maroni & Mitch, 1997; Mitch & Maroni, 1998). This is CO (1978): Comparative nitrogen balance study between young and partly because the symptoms are ameliorated, through the aged adults using three levels of protein intake from a combination decreased build-up of end products of protein metabolism wheat-soy-milk mixture. Am. J. Clin. Nutr. 31, 12 ± 22. in the blood (urea, creatinine, phosphate), and partly Clugston GA & Garlick PJ (1982): The response of protein and energy metabolism to food intake in lean and obese man. Hum. Nutr.: Clin. because a low-protein diet seems to slow the progressive Nutr. 36C, 57 ± 70. loss of renal function (Mitch & Maroni, 1998). High Conde RD & Scornik OA (1976): Role of protein degradation in the protein intake is also known to interfere with calcium growth of livers after a nutritional shift. Biochem. J. 158, 385 ± 390. homeostasis. At ®xed intake of phosphorus, a doubling of Duncan D & Burns K (1997): Lewis and ClarkÐThe Journey of the Corps protein intake can cause a 50% increase in urinary of Discovery, Chap. 9. New York: Alfred A Knopf Inc., pp 154. Eaton SB & Konner M (1985): Paleolithic nutrition. N. Engl. J. Med. 312, calcium excretion (Schuette & Linkswiler, 1982). More- 283 ± 289. over, this increased excretion continues for at least two El-Khoury AE, Fukagawa NK, Sanchez M, Tsay RH, Gleason RE, Chap- months after the change in diet (Allen et al, 1979). In man TE & Young VR (1994): Validation of the tracer-balance concept the absence of an increase in calcium absorption from with reference to leucine: 24-h intravenous tracer studies with L- [1-13C]leucine and [15N-15N]urea. Am. J. Clin. Nutr. 59, 1000 ± 1011. the intestine, this would lead to bone resorption and FAO=WHO=UNO Committee (1985): Energy and Protein Requirements. potentially to osteoporosis. The intake of phosphorus, Geneva: World Health Organization. (WHO technical report series # which is generally related to the intake of protein, 724.) reduces the urinary loss of calcium. However, phosphorus Fern EB, Garlick PJ, McNurlan MA & Waterlow JC (1981): The excretion also increases intestinal losses, by a similar amount to the of isotope in urea and ammonia for estimating protein turnover in man with [15N]glycine. Clin. Sci. 61, 217 ± 228. decrease in urinary loss (Heaney, 1993). However, the effects Fern EB, Bielinski RN & Schutz Y (1991): Effects of exaggerated amino of a high protein diet can be negated by increasing the calcium acid and protein supply in man. Experientia 47, 168 ± 172. intake (Heaney, 1993). It would therefore be important for Fisher H, Brush MK, Griminger P & Sostman ER (1967): Nitrogen subjects taking high protein diets to ensure that intakes of retention in adult man: a possible factor in protein requirement. Am. J. Clin. Nutr. 20, 927 ± 934. calcium were also adequate. Finally, in a wide range of studies Fomon SJ & Ziegler EE (1989): Nutrients in infant formulas: introduction. in animals, it has been shown that dietary intake has a J. Nutr. 119, 1763 ± 1764. pronounced effect on longevity. Therefore, a restriction of Fuller MF & Garlick PJ (1994): Human amino acid requirements: can the total intake of food by 25 ± 65% from a young age resulted in controversy be resolved? Ann. Rev. Nutr. 14, 217 ± 241. increases in lifespan of mice and rats ranging from 20 ± 65% Gajjar A, Johnson BC & Good RA (1987): In¯uence of extremes of protein and energy intake on survival of b=w mice. J. Nutr. 117, (Weindruch et al, 1986; Shimokawa et al, 1993). However, 1136 ± 1140. much of this effect has been attributed to reduced total energy Garlick PJ & Fern EB (1985): Whole-body protein turnover: theoretical intake (Gajjar et al, 1987). Although large differences in considerations. In Substrate and Energy Metabolism in Man, eds. JS longevity have been shown in studies comparing diets with Garrow & D Halliday. London: John Libbey, pp 7 ± 15. Garlick PJ & Wernerman J (1997): Protein metabolism in injury. In 4% and 26% protein (Goodrick, 1978; Leto et al, 1976), no Scienti®c Foundations of Trauma, eds. GJ Cooper, HAF Dudley, DS controls for total energy intake were included. In one study Gann, RA Little & RL Maynard. Chap 48. Oxford: Butterworth with isoenergetic diets, it was suggested that protein might Heineman, pp 690 ± 727. have some effect on life span, but this effect was small in Garlick PJ, Millward DJ & James WPT (1973): The diurnal response of comparison to that induced by changes in dietary energy muscle and liver protein synthesis in vivo in meal-fed rats. Biochem. J. 136, 935 ± 946. (Shimokawa et al, 1993). On the other hand, it has been Garlick PJ, Millward DJ, James WPT & Waterlow JC (1975): The effect of pointed out that despite correlations between dietary protein protein deprivation and starvation on the rate of protein synthesis in and the incidence of various cancers, the big improvements in tissues of the rat. Biochim. Biophys. Acta 414, 71 ± 84. longevity of humans during the last few decades correlate Garlick PJ, Clugston GA & Waterlow JC (1980a): In¯uence of low-energy diets on whole-body protein turnover in obese subjects. Am. J. 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