Dietary Protein Intake and Dynamic Aspects of Whole Body Nitrogen Metabolism in Adult Humans William

P. Steffee,

Ralph S. Goldsmith,

Nevin S. Scrimshaw, The constant isotope-infusion method of Picou and Taylor-Roberts was used to study rates of total body protein synthesis and breakdown in adult subjects following acute changes in the Ievol of dietary protein intake. Six healthy adults, four males and two females, were studied after adaptation to dietary protein intakes of 1.5 and 0.38 g of protein/kilogmm body weight/day. Dietary periods were from 7 to 15 days duration. “N-glycine was used as a tmcer, and was administered orally for 60 hr at 3-hr intervals, or by continuous intmvenous infusion for 48 hr. Results were similar for both routes of iwtope administration for the comparison conducted at the higher protein intake. At

and Vernon

Paul 6. Pencharz, R. Young

the 1.5-g protein level the mean N flux was 28.2 mg nitrogen/kg/hr, with total body protein (N x 6.25) synthesis and breakdown mtes being 3.0 g/kg/day and 2.7 g/kg/day, respectively.“Reducing the protein intake to 0.38 g/kg/day caused an 8% decreaw ( p < 0.05) in N flux, a 27% increaw (p < 0.005) iti’ the mte of total body protein breakdown, and a 15% increaw (p < 0.05) in the mte of protein synthesis. Endogenous amino acids were reutilized more efficiently under thew conditions. The findings are discussed in relation to the way in which adult subjects adapt to acute changes in dietary protein intake.

A

N IMPORTANT KEY to the survival of living organisms is the ability to maintain homeostatic regulation of major metabolic processes. For example, a favorable balance between the anabolic and catabolic phases of body nitrogen (N) metabolism is often achieved during marked fluctuations in the adequacy and level of dietary protein intake. However, the nature of this adaptive response in human subjects is poorly understood.’ The prompt reduction in urinary nitrogen excretion in response to lowered protein intake reflects adaptive metabolic changes in the protein and amino acid metabolism of various organs, which are integrated finally into the N economy of the whole body. In the past, the N-balance technique has been a principal method for investigating adaptive aspects of body protein metabolism at the whole body level in human subjects.*This technique measures changes in body N without revealing the individual processes responsible for such changes. Hence, reduced N excretion with decreased N intake might result from a reduc-

From the Department of Nutrition and Food Science. Massachusetts Institute of Technology. Cambridge, Mass. and the Clinical Research Center, Mayo Clinic, Rochester, Minn. Receivedfor publication July 1, 1975. Supported by Grant AM 15856 from the National Institutes of Health, and utilized the facilities of the MIT and Mayo Clinic Clinical Research Centers. which are supported bv Grants RR-88 and RR-585 from the Division of Research Resources, National Institutes of Health. Dr. Steffee was supported by NIH Training Grant 5-TOI-AM 0537, and Dr. Pencharz received a fellowship from the Canadian Medical Research Council. Reprint requests should be addressed to Dr. Vernon R. Young, Associate Professor of Nutritional Biochemistry, Massachusetts Institute of Technology. Department of Nutrition and Food Science, Cambridge, Mass. 02139. 0 1976 by Grune & Stratton. Inc. Mctobotism, Vol. 25, No. 3 (March), 1976

281

282

STEFFE

ET Al.

tion in the total body protein breakdown rate. Alternatively, a reduced protein intake may enhance the rate of body protein breakdown, which may then be coupled to compensatory mechanisms that increase the rate of total body protein synthesis and the efficient reutilization of amino acids. Waterlow’ has thoughtfully reviewed these aspects of the adaptive nature of body protein metabolism. According to the limited experimental data available, short-term adaptation to a low-protein diet is not accompanied by a decrease in total body N turnover in rats,* and comparable conclusions may be drawn from the work of Picou and Taylor-Roberts3 with infants given low- and high-protein diets. Studies of N turrrover and total body protein synthesis and breakdown rates are needed to explore the adaptive nature of protein metabolism in the human subject. Furthermore, they are necessary for understanding the ways in which the efficiency of dietary nitrogen utilization and the amount of body nitrogen are altered in response to physiologic and pathologic conditions, such as during growth and under stress brought about by physical injury or infectious disease. Studies of energy metabolism add further significance to investigations of dynamic aspects of total body nitrogen metabolism. Estimates of body N turnover in healthy adult subjects suggest values of approximately 3 g protein/ kg/day! Considerable energy is required for this extensive and continuous protein turnover, involving both the high energy requirement for polypeptide chain synthesis and the subsequent dissipation of the free energy during proteolysis. Calculations indicate that normal body protein turnover accounts for a significant portion of the basal energy expenditure (e.g., s), and that alterations in the turnover rate would be expected to have important influences on energy utilization and metabolism. Therefore, it would be of considerable interest to know whether acute alterations in the level of dietary protein intake cause significant changes in the rates of total body protein turnover, and consequently, in the utilization of major dietary energy constituents. Various approaches have been used to estimate total body protein turnover in man (’ [review]). The earlier methods relied upon a single administration of labeled amino acids, and the calculation of turnover rate based on the analysis of changes in the labeling of plasma amino acids or urinary urea. However, this general procedure is no longer considered adequate: and the continuous infusion of an amino acid, labeled with r5N or r4C, has been applied in studies of the dynamic aspects of body protein metabolism in human subjects.6*7 This more recent approach offers a number of advantages over those involving only a single dose of labeled amino acid. 4 However, it must be recognized that all models of whole body N turnover necessarily oversimplify the complex situation in vivo. Nevertheless, when the approach and methodology are carefully chosen and applied in comparative studies, estimations of whole body N turnover under various conditions should’offer new insight into the physiology of human protein metabolism. For these reasons we have investigated the adaptive nature of body protein metabolism by measuring total N flux (disposal rate) and total body protein synthesis and breakdown rates in adult subjects adapted to differing levels of dietary protein intake. We used the r5N-amino acid approach developed by Picou and Taylor-Roberts3 in their studies with malnourished children. Our

283

PROTEIN INTAKE

Table 1. Choractorirtics of Subits

Studied for the Ws

of Diotory Protein Intake

on Dynamic Aspects of Total Body Protein Metobolirm JF

HF

So

LF

NP

SP

Acw

20

21

20

21

25

23

Sex Initial Weight (kg)

M 70.7

M

M

F

M

F

63.7

07.5

52.3

76.8

54.0

Subject

Height (cm)

183

178

183

166

173

155

findings show that during short-term adaptation to a low:protein, but otherwise adequate diet in adult humans, there is a small decrease in N flux, a marked increase in the rate of total body protein breakdown, and a greater degree of endogenous amino acid reutilization. A preliminary account of some of the results reported here has appeared recently in a study of the effects of age on whole body N turnover.* MATERIALS

AND

METHODS

Subjects. Six adult volunteers, four males and two females, were studied in the Clinical Research Centers of either the Mayo Clinic (subjects LF, NP, and SP) or Massachusetts Institute of Technology (Table 1). All were in good health, as determined by physical examination, medical history, complete blood count, and a routine biochemical screen on serum blood samples. The purpose of the studies and the potential risks involved were fully explained to each subject, and written consent was obtained. The experimental protocols received the administrative approval of the MIT Committee on the Use of Humans as Experimental Subjects, the Executive and Policy Committees of the MIT Clinical Research Center, and the Human Studies Committee of the Mayo Clinic. Diers and experimenrol periods. The subjects were studied at two levels of dietary protein intake: 1.5 and 0.38 g protein/kg body weight/day. These dietary levels were chosen to reflect a usual and near maintenance intake of protein, respectively. The major dietary protein source at the 1.5 g level was either beef or hen’s egg, and at the 0.38 g level the protein source was egg. The compositions of the diets were essentially similar to those previously described,9 and total energy intake was maintained at 45 kcal/kg/day throughout the entire experimental period. MinFOOD 15N-GLYCINE

N

1

CATABOLISM

F/g. 1. The Picou ond Taylor-Roberts med.1 for studying dynamic ospoctr of whole body N metabolism by continuous infusion of “N-glycino. I, C, ond S ore intake, protein breakdown, ond synthesis, respectively (mg N/kg/day); E,, E,, ond E, ore urinary urea, urinoy nonuv, and total (urinary plus fecal) nitrogen lxuetions, respectively, (mg N/kgldoy), and Q is tho ftux (m@ N/kg/ day) of nitmgon for the metabolic pool, P. F is the fraction of the administered dose (d) of “N-glycine thot is excntod OS ‘IN-uroo (fraction of total N entering the pool thot is excreted or urea N) ond eu is the rate of excretion of ‘“N OSurea.

284

STEFFE

ET AL.

era1 and vitamin supplements have also been described.’ Four equal meals were consumed, at 8 a.m., noon, 5 p.m., and 9 p.m. Each subject was introduced into the study with the diet providing 1.5 g protein/kg body weight/ day, and maintained at this intake .Ievel for 5 days before the 15N-tracer studies were conducted, as described in the following. Thereafter, dietary protein intake was reduced to 0.38 g/ kg/day for I2 days before another 15N study was conducted. Three subjects (LF, NP, SP) continued to consume the latter diet for an additional 21 days, after which “N-tracer studies were repeated. The ex erimental model. We chose to modify slightly the approach taken by Picou and TaylorP Roberts, in which “N-labeled amino acid was administered at a constant rate to achieve a steady state of 15N-enrichment in urinary urea N. For purposes of clarity, Picou and TaylorRoberts” description of the model (Fig. 1) and the assumptions they discuss are repeated here: It is assumed that under steady state conditions, the rate(Q) at which amino-N enters the metabolic pool (P) equals the rate at which it leaves the pool: Q=I+C=S+E, where Q is the flux of N, or disposal rate;” I, the rate of N intake; C, the catabolic rate; S, the synthetic rate; and Et, the total N excretion rate (urine and fecal). With a constant administration of “N-labeled amino acid, the “N-enrichment of urinary urea will gradually increase towards a plateau (or quasi-plateau) level. When this isotopic steady state is achieved, the fraction (F) of the administered isotope that is excreted as “N-urea is assumed to be the same as the fraction of total amino-N entering the metabolic pool which is excreted as urea-N. Hence, F = e,d = E,/Q where e, is the rate of excretion of “N as urea (mg “N/kg/ hr), d is the dose of 15N administered, and E, is the total urinary urea N. Therefore, Q = EM/F. Since ey, d, and E, are measured, Q can be calculated. Because I and E, are known, S and C can be calculated. The assumptions made are: (1) 15N is handled in a manner similar to “N. Although there are differences between the behavior of “N and “N, as revealed by countercurrent techniques,” they presumably have little biologic significance in “N tracer studies. (2) The level of labeled amino acid administered is negligible in relation to the amino acid within the metabolic pool, and “N-glycine is a valid tracer for studies of total body amino N metabolism. (3) The size of the metabolic pool with respect to labeled and unlabeled N is constant during the period used for determining the rates of body N metabolism. Since all subjects were studied in a metabolic ward, were free of disease, and maintained under strict dietary conditions, this appears to be a valid assumption. (4) Dietary and endogenous nitrogen are treated in a similar manner. (5) There is no significant reentry of isotope into the metabolic pool during the period of isotope administration. Assumptions (2) and (4) were tested by Picou and Taylor-Roberts,3 and were found to be valid for their conditions. The assumptions and the problem of isotope reentry are also discussed below. fsotope administration. Either 95 or 99 atom-per cent excess 15N-glycine was used.* Enrichment of the purchased material was confirmed by mass spectrographic analysis. In all studies the 15N-glycine was administered at a rate of about 0.5 mg “N/kg body weight/24 hr. Picou and Taylor-Roberts’ used constant intravenous or intragastric infusion to administer the labeled amino acid. Since this method was not felt to be practical for studies with ambulatory subjects, we determined whether the provision of “N-glycine at frequent intervals would also achieve a steady state of “N-enrichment of urinary urea. With subjects consuming normal levels of dietary protein, we found that 15N-glycine given orally every 3 hr for 60 hr allowed a satisfactory plateau level of urinary “N-urea enrichment to be obtained. To evaluate the effects of the route of isotope administration, three subjects were studied with orally administered ‘sN-glycine, and subsequently with a constant intravenous infusion of the amino acid.

lStohler Isotope Chemical Corp., Waltham, Mass.

PROTEIN

285

INTAKE

For the oral studies, an amount of isotope sufficient to last a 60-hr tracer period was prepared in deionized water. Individual doses of IO ml each were administered quantitatively at precise 3-hr intervals. The tracer studies were begun at 8 p.m. and ended 60 hr later. For the intravenous studies, a stock 15N-glycine solution containing 2 mg glycine/ml was prepared with sterile isotonic saline. It was passed through a Millipore filter (0.22 r)* into clean 60-ml serologic vials, autoclaved, and stored at -20°C until used. Randomly selected vials were submitted for pyrogen and sterility testing.? For intravenous administration, 100 ml of the stock solution were added to I liter sterile isotonic saline. The glycine solution was infused into an antecubital vein with an infusion pump.i The rate of infusion (approximately 0.7 ml/min) was adjusted to supply 0.5 mg 15N/kg/24 hr. The “N-glycine concentration of each liter of solution was determined and the actual dose was calculated. Infusions were begun at 8 p.m. and continued for 48 hr. The subjects were free to move around the room during the infusion period, and normal activity was encouraged. Complete daily urine and fecal collections were made throughSample collections and analyses. out each dietary period. During the “N tracer periods, urinary collections were taken at precise 3-hr intervals. Urine was analyzed for total nitrogen, urea nitrogen, and creatinine by methods previously described.9 Blood urea N was also determined’ at the end of each dietary period. Fecal samples were pooled into either 4- or S-day collections, using fecal markers,12 and analyzed for total nitrogen. At the Mayo Clinic, 500 mg chromium sesquioxide were administered three times daily, and stools were analyzed for chromium.‘3 Urea N was isolated from urine for 15N analysis by one of two techniques. For the oral 15Nglycine studies conducted at the I.5 g protein intake level, the method used by Picou and Phillips’* was followed. For the remaining samples, we used the Conway diffusion methodI after pretreating urine with premutit.16 Several individual tracer studies were checked using both extraction methods to confirm that the “N-enrichment of urea N was the same for the two procedures. 15N-enrichment of urea N was determined following the reaction of urea (or ammonia) with hypobromite, “*” and the 29N/28N ratio was measured with a dual collector, isotope ratio mass spectrometer.4 Determinations, over a number of months, of purified tank nitrogen gave a value of 0.365 f 0.018 (n = 50) atom-per cent “N. Thus, each time standards and unknowns were analyzed, they were run between two determinations of tank nitrogen, and corrections made for variations in the calculated “N-abundance of natural N2 gas. This achieved good analytical reproducibility, since frequent determinations of “N-glycine standards calculated to provide 0.03 and 0.06 atom-per cent excess “N gave 0.0265 f 0.0008 and 0.0561 f 0.0024, respectively. The lower enrichment of the standards was consistent, and we assumed that the cause was a lower-than-expected enrichment level of the “N-glycine as purchased. Data analyses. “N-enrichment of urinary urea N was calculated as described by Rittenberg, Keston, Rosebury, and Schoenheimer.” Enrichment was corrected for background values determined on urine samples collected during a 3-hr period preceding initiation of each tracer study. For most of the results described below, the plateau level of “N-enrichment of urea was determined by visual inspection and averaging the data points for that segment of the “N-tracer period. In the studies at the 1.5 g level of protein intake, the increase in urea 15N-enrichment followed a smooth course. Therefore, we also estimated plateau values by using the least squares method to fit the individual data sets to an exponential equation: Y = C(1 - e-*1) The overall N flux was calculated by estimating the asymptote: Y(a) = C, as the plateau level. The fitting was carried out by means of the nonlinear program of the BMD computer program package.19 This program uses a modified Gauss-Newton techniquezuJ’ to search the parameter space until five successive iterations do not change the total error mean square by more than 0.001%. In addition, the program calculates the covariance matrix of the final parameter estimates.

*Millipore Corp., Bedford, Mass. tLeverco Laboratories, Roselle Park, N.J. tModel900 Harvard Apparatus Corp., Millis, Mass. $Model MS I I, Vacumetrics, Waltham, Mass.

286

STEFFE

ET Al.

The covariance matrix is used to calculate standard deviation of the estimates. Our purpose in using this approach, to the extent that the early urinary “N-urea values allowed, was to evaluate further the results of overall N flux obtained in the studies with intravenously administered ‘sN-glycine. These latter studies were of shorter duration (48 hr) than those involving oral administration of the isotope. Nitrogen balance data were based on urinary N output during the last three days of the initial dietary periods at the I.5 g protein/kg/day intakes, and on the last four days of the period providing the 0.38 g levels of protein intake. An approximation of the turnover rate of the urea pool was made for each subject at both levels of protein intake. The body urea pool was calculated from blood urea N concentration and total body water (TBW). For three subjects (LF, SP, and NP) TBW was determined using D20,22 for the others it was assumed to be 55% of total body weight.23 The turnover rate of the urea pool was estimated from the urinary urea output. Although this measurement underestimated the true turnover rate, since no account was taken of urea entry into the intestinal tract,24 it provided a satisfactory approximation for comparative evaluation of the “N urea results discussed below. Statistical evaluations were performed using Student’s t test2’ RESULTS

All subjects completed both phases of the study and tolerated the diets and procedures without incident. No illnesses or adverse influences were encountered. Initial and final blood screening tests were all normal and without significant change, except for a small decrease in hematocrit. No significant changes in body weight were observed in any of the studies. N Balances

Nitrogen balance data are summarized in Table 2. “True” N balance was estimated by assuming that 5 mg N/kg/day would approximate integumental and unmeasured losses.2* At the 1.5 g protein/kg/day level all subjects were in Table

2.

Nitrogen

8aloncer for Adult Subiects Consuming

Dieh Supplying

1.5 and 0.38 G Protein/Kg/Day Dietary Protein’

FecalN

EstimatedTrue Balancst

Diet N

Urine N

JF

19.11

15.30

1.70

SO

20.70

15.14

1.37

+3.75

HF

15.42

11.71

1.47

+1.92

LF

12.99

7.94

1.32

+3.45

NP

17.64

11.63

0.72

+4.91

SP

12.12

0.69

0.76

Subiact

g N/day 1.5 g

Mean f

SD

0.38 g

Mean f

SD

+1.72

+2.40 +3.03

JF

4.67

3.49

so

5.66

HF

3.77

LF

f

0.97

-0.18

3.44

0.84

+0.34

3.24

0.89

- 0.68

3.17

2.08

0.84

-10.67

NP

4.44

3.52

0.b9

-0.15

SP

2.94

1.91

0.60

1.23

+0.16 +0.03

f

0.47

*g protein/kg/day. tg N/Flay, arsumi~g an additional 5 mg N/kg/day for integumental and other unmeasured 10sses.~~

PROTEIN

287

INTAKE

distinct positive balance, whereas at the 0.38 g protein/kg/day level the mean N balance approached equilibrium. The distinctly positive balance at the higher protein intake level cannot be directly explained, although it has been observed by many others. Methodological errors in estimating N intake and urinary and fecal N outputs would not appear to account for these findings, which were made under carefully controlled metabolic balance conditions. Furthermore, nitrogen excretion was essentially steady throughout the balance period, suggesting that the values were not caused by insufficient adjustment time to the higher protein intake level. The validity of this finding is further supported by the fact that the N intake was close to the level consumed by the subjects during their usual living conditions. However, underestimation of N losses via the integument and other known minor routes may partially account for the positive balances. Another explanation is that a significant proportion of the “retention” may have been caused by nitrogen loss from the body in the form of molecular nitrogen. There is now increasing evidence to indicate that this route, hitherto regarded as unlikely in mammalian organisms, may be an important factor in nitrogen loss at generous intakes of protein in healthy subjects, and possibly under some pathologic conditions. The evidence, based on various approaches, has been reviewed,27*28and appears to provide a likely explanation for the positive balance values obtained at the high protein intake in our studies. We are currently exploring this problem in experimental animals, with the aid of ‘SN-tracer techniques, in an attempt to provide definitive support for this argument. ‘SN-Glycine Tracer Studies Time course of enrichment of urinary ‘jN-urea. Figure 2 depicts the change in “N-enrichment of urinary urea with time during a 60-hr period of oral ‘*Nglycine administration for a representative subject consuming 1.5 ,g protein/kg/ day. The actual values are also compared with those presented by regression analysis. lsN-urea enrichment increased during the first 36 hr, and reached a

0 = ACTUAL x = COMPUTED

P

0

I

I

I

I

I

I

I

I

I

I

6

12

I6

24

30

36

42

46

54

60

HOURS

OF GLYCINE

- 15N

ADMINISTRATION

Fig. 3. “N enrichment of urinary urea during frequent 3-hr oral administrations of ‘“Nglycine for subioct JF, when consuming a diet providing 1.5 9 prbtein/kg/day. Actual values (o) are compared with computed values (X) obtained by nonlinear rsgrerrion analysis.

288

STEFFE ET Al. foble 3. Ploteou Values for Urea ‘IN Enrichment, Obtained by Visual InsPection ot Beth Protein Intoke levels and Nonlineor Regression Analysis for Plateau Estimotion far Subjects Consuming 1.5 G Protein/Kg/Day

Nt

Subject’

Inspected

Predicted

Protein intake, 1.5 g/kg/day jz 0.0025t 0.0868 * 0.00 17

JF

6

0.0784

HF

7

0.0840 f

0.0035

0.0976 f 0.0049

so

7

0.0881 f 0.0027

0.0999 f 0.0044

LF

7

0.0986

0.1038

NP

7

0.0941 f

SP

8 Mean

f 0.0046 0.0051

f

0.0025

0.1010 z!G0.0040

0.1092 f

0.0078

0.1129 f

0.0916 f

0.0108

0.1003 zt 0.0085

0.0028$

0.0038

Protein intake, 0.38 g/kg/day JF

4

0.0747 f

HF

4

0.0953 f

0.0034

SO

4

0.1116 f

0.0072

LF

5

0.0986 f

0.0034

NP

5

0.1056 zt 0.0051

SP

4

0.1307 f

0.0037

o.c!O5o

LF§

6

0.1140 f

NP§

7

0.0899 f

0.0066

sP§

7

0.1168 f

0.0101

*All subjects received 15N-glycine orally. tNumber of 3-hr points used for calculation of inspected plateau. tMean values (& SD), expressed as 15N otom-per cent excess. 4Repeat study after a total of 33 days on diet.

relatively constant rSN-enrichment level, which was maintained for the remainder of the study period (Table 3). Visual determination of the r5N-urea plateau level for each subject studied at 1.5 g protein intake provided values for body N flux that compared reasonably well with those derived by regression analysis (Table 4), although the latter were significantly lower statistically than those derived by visual inspection. The small variance in the computed plateau values indicated that the change in urea-15N enrichment followed a consistent and progressive course of change during the early period of isotope administration at the higher protein intake level. Table 4. A Comparison of Total Body Nitrogen Flux Inspected Values for Ploteou “N-urea

(0)

Using Predicted and

Enrichment in Subiects

Consuming 1.5 G Pretein/Kg/Doy Inspected

Predicted

Subject’

mg N/kg body weight/hr JF

28.8

31.9

HF

25.6

29.8

SO

25.0

28.4

LF

25.0

26.8

NP

26.2

28.1

SP

23.4 Mean f

SD

25.8 f

Pt *All subjects received 15N-glycine via the or01 route. tp value determined by paired t test.

24.2 1.77

28.2 f 2.62 < 0.005

PROTEIN

289

INTAKE

I-

.os

zw .oa

0

0 0

0

0

0

0

0

0

0

O

0 0

; .07 w 8 .06

0 0

g .05 z.04 z *I

0 0

.03

2 n

.02

=

.Ol

0 0

0

1

0

I

I

I

I

I

I

I

I

I

I

6

12

18

24

30

36

42

48

54

60

HOURS

OF GLYCINE

-15N

ADMINISTRATION

15N-enrichment of urinary urea during frequent 3-hr oml odministmtions of ‘5N-glycine Fig. 3. for subject LF, when consuming a diet providing 0.38 g protein/kg/day.

Although a relatively constant level of ‘SN-urea enrichment appears to have been achieved during the final 15 hr of the 60-hr tracer period at the lower protein intake (Fig. 3, Table 3), the earlier portion of the “N-urea curve showed somewhat erratic behavior. Thus, the estimation of plateau values by least squares fitting would not be useful in this case. The reason for the variability of the r5N-urea enrichment curves during the first 24-30 hr of ‘SN-glycine administration, when subjects consumed the 0.38 g protein diet, is not clear. It may reflect metabolic instability at this barely adequate level of dietary protein intake, in contrast to a more stable metabolic situation in which either adequate or grossly deficient levels of protein intake are given. However, since visual inspection of the data clearly indicated a 12- to 15-hr period of steady state 15N-urea enrichment (Table 3), inspected values were used for calculating dynamic aspects of total body N metabolism at the 0.38 g protein intake level. Thus, estimates derived from plateau values obtained by visual inspection proTable 5. Total Body Nitmgen Flux (0) in Adult Subjects Adopted to Diets Providing 1.S and 0.38 G Protein/Kg/Dny ProteinIntake (g/kg/day) subjpct*

1.5

JF

31.9

33.5

HF

29.8

26.2

SO

28.4

22.4

LF

26.8

24.8

NP

28.1

25.0

SP

24.2

20.2

Mean f

SD

28.2 f 2.62

Dt

‘All subjects received‘“N-glycine by oral administration.

tp

0.38

mg N/kg body weight/hr

value was determined by poked t test.

25.7 f 4.56 < 0.05

290

STEFFE ET Al.

Table 6. Total Body Nitrogen

Flux (0)

in Three Subjects Studied after 12 and 35 Days

at the 0.38 G Protein/Kg/Day Subject’

Level of Intake

12 Days

35 Days mg N/kg/hr

LF

26.8

23.2

NP

25.0

26.5

SP

20.2 Mean f

SD

24.01 f

22.6 3.4

25.1 l 3.8 NS

Pt *Subjects received isotope by oral administration.

tp wasdetermined

by paired t test.

vided the basis for assessing the effects of protein intake on whole body nitrogen metabolism. N frux and rates of total body protein synthesis and breakdown (oral lsN-* glycine). After “N-urea plateau values were determined, N flux and rates of total body protein synthesis and catabolism were calculated. Table 5 summarizes the estimates of N flux obtained at the two levels of dietary protein intake. Mean values for N flux were slightly, although significantly (p < 0.05), reduced with the reduction in dietary protein intake. For the three subjects studied after 12 and 35 consecutive days at the 0.38 g dietary protein level, the mean values for N flux did not differ significantly (p > 0.1) (Table 6). Rates of total body protein synthesis and catabolism for each level of dietary protein intake are summarized in Table 7. The mean rate of total body protein catabolism increased (p < 0.005) by 27% when dietary protein was reduced from 1.5 g/kg/day to 0.38 g/kg/day. This rise in catabolic rate was accompanied by a smaller, although statistically significant (p < 0.05), increase (15%) in the rate of total body protein synthesis. At the higher protein intake, the estimated mean rate of synthesis was 3.0 g protein/kg/day, and catabolism, 2.7 g protein/kg/day. With reduced protein intake, the rates of synthesis and catabolism were essentially equal. The synthesis rate may overestimate the actual in vivo rate, if total N losses are underestimated. However, the estimate of the breakdown rate depends Table 7. Rates of Total Body Protein Synthesis (5) and Catabolism (C) in Adult Subjects Adapted to Diets Providing 1.5 and 0.38 G Protein/Kg/Day Synthesis(S) Subject’

1.5

Cotobolism(C) 0.38

1.5

0.38

mg N/kg body weight/hr JF

22.7

30.9

21.8

31.0

HF

20.9

23.3

19.7

23.0

SO

20.3

20.1

18.5

20.0

LF

19.2

24.3

16.5

24.3

NP

20.0

22.4

1a.5

22.6

SP

16.7

1a.0

1A.8

20.1 f 2.01

23.2 zt 4.42

Mean f Pt

SD

< 0.05

*All subiects received “N-glycina by oral administration.

tp

value was determined by paired t test.

18.3 f

17.9 2.4

23.3 + 4.5 < 0.005

291

PROTEIN INTAKE

Table 8. Effect of Protein intake on the Percentage of Nitrogen Entering the Metabolic Nitrogen Pool that Is Used for Protein Synthesis (S/O Derived from Catabolism (C/Q Protein

x 1DD) or

x 100)

Number

intake

of C/Q

x loo

x loo

Subjects’

S/Q

1.5

6

71.3*

l&q

64.7 + 2.0

0.38

6

90.1 *

1.2

90.4 *

(-a/k&W

1.3

*All subjects received 15N-glycine by oral route. tMeon

f SD.

upon the accurate assessment of N intake, which may be overestimated. Therefore, the measured differences between the synthesis and breakdown rates, particularly at the higher protein intake level, may have been slightly overestimated. The effect of reduced protein intake on total body N metabolism is also evident in the contributions of protein synthesis and catabolism to the N flux (Table 8). Significantly more of the N entering the pool was utilized for protein synthesis, and less was excreted as urea, when dietary protein intake was reduced. Similarly, the amino acids entering the pool through body protein breakdown accounted for a significantly greater proportion of the N flux when the level of dietary protein was decreased. Studies with intravenously administered glycine at diflering protein intakes. From the above observations a question arose as to whether the route of t5Nglycine administration might influence the estimates of body N flux and rates of protein synthesis and breakdown. Therefore, “N-tracer studies in which isotope was administered intravenously were conducted in three subjects receiving 1.5 g protein/kg/day. All had been previously studied at both protein intake levels with orally administered *‘N-glycine. The dietary and experimental protocols were otherwise identical. “N-glycine was infused intravenously for 48 hr, and for comparative purposes, plateau levels of urinary urea- “N enrichment were obtained by the method of least squares. Table 9. Comparison of Dynamic Parameters of Total Body Nitrogen Metabolism in Subiects Receiving “N-glycine

by Oml and Intravenous Routes

Route of

kotope Oral

Subject

0’

S/Q x loo

C/Q x 100

68.0

64.9

65.5

60.6

67.7

28.8

19.6

HF

25.6

16.8

18.7 15.5

so

25.0

16.9

15.2

26.5 zt 2.0 28.1

JF

Mean ri SD

C’

JF

Mean f SD IV

s*

17.8 f

1.6

16.5 zt 1.5

67.1 f

18.3

17.6

65.2

60.6 1.4

62.0 f

HF

20.2

10.9

10.3

58.3

51.1

so

24.6

15.8

15.2

64.2

61.7

24.3 f 4.0

Pt

NS

15.0 f NS

3.8

14.4 It 3.7 NS

62.6 f

2.5

62.9

3.7

< 0.05

*Values are mg N/kg body weight/hr. tp value obtained by paired t test. (Q = nitrogen flux, S = total body protein synthesis, C = total body protein catabolism.)

58.6 f 6.5 NS

292

STEFFE

ET Al.

Table 9 summarizes the estimates for various parameters of total body N metabolism in the three subjects consuming 1.5 g protein/kg/day. Estimates of nitrogen flux, and total body protein synthesis and catabolism did not differ significantly between the two routes of administration. Because the plateau r5N-urea enrichment was delayed at the lower protein intake, and because it was only practical to conduct the intravenous.studies for 48 hr, it was not possible to obtain similar comparisons of the effects of the route of isotope administration at the lower protein intake level. DISCUSSION

Various approaches have been taken in estimating rates of total body protein synthesis and breakdown in the human subject. Waterlow has reviewed this area critically, and pointed out the serious limitations inherent in most of the earlier studies in which a single dose of 15N-labeled material was used to enrich the body N pool. We have also conducted comparative nutritional studies* using the San Pietro and Rittenberg mode1,29 which we found to give highly variable and biologically unrealistic results. Our experience supports objections raised by Wu, Sendroy, and Bishop,m and Tschudy, Bacchus, Weissman, Watkin, Eubanks, and White3* of studies based on a single dose of isotope administration. Methods utilizing a constant infusion of labeled amino acids provide the most reliable measures of body N metabolism rates in man.4.6*7For these reasons, we chose to use the Picou and Taylor-Roberts model3 in the present studies. For adults consuming adequate dietary protein, the mean estimate for total body protein synthesis was determined to be approximately 3 g protein/kg/ day. This value agrees with those reported for adults by Waterlow, whose studies were based on the constant administration of 14C-labeled amino acids. Evaluation of tracer studies carried out at the whole body level involves certain simplifying assumptions. The major ones have been discussed earlier,3 and the present findings provide additional support for their general validity. When dietary protein intake was adequate, there was agreement between the inspected plateau of rsN-urea enrichment and the calculated asymptote. However, the reason for the failure to predict a reliable value for the asymptote after an acute reduction in dietary protein intake is not entirely clear. During the early part of the lSN-tracer period only small amounts of isotope would have entered the metabolic N pool. At this early time one would expect perturbations, such as those created by meal ingestion, to affect more markedly the pattern of “Nurea production. If sufficient time had elapsed to ensure that the j5N in the metabolic N pool had reached an isotopic steady state, these effects would tend to have less influence on the isotope excretory pattern. Nevertheless, it was possible to detect a consistently stable plateau by visual inspection at the 0.38 g protein level. The studies with orally administered ‘5N-glycine revealed significantly increased rates of body protein breakdown and synthesis with a reduced protein intake. We thought that these increases might be more apparent than real, *Steffee and Young, in preparation.

PROTEIN

293

INTAKE

particularly as they may have been influenced by the route of isotope administration. However, the good agreement between the absolute values for the parameters of total body N metabolism obtained with oral and intravenously administered i5N-glycine at the 1.5 g protein intake level suggests that the interpretation of the data is not confounded by the route of isotope entry into the metabolic N pool. This conclusion agrees with those of Picou and TaylorRoberts3 and Neale and Waterlow.” Two further points require consideration in the interpretation of these results. The first concerns the kinetics of the urea pool and the effect of changes in protein intake. If the turnover of the urea pool is reduced with a fall in protein intake, then the time required to reach a plateau level of i5N-urea enrichment with continuous administration of “N-glycine will be delayed, relative to the time required at a higher protein intake. Thus, the plateau enrichment might be underestimated, and values for N flux (Q) overestimated at the lower protein intake. We have attempted to assess this problem by approximating the fractional turnover rate of the urea pool at both protein levels, as discussed in the Methods. Table 10 shows that reducing protein intake to about 0.4 g/kg/day causes an approximate 50% fall in the turnover rate of the body urea pool. The 15Nurea data are in line with this observation (Table 3). They indicate that plateau was achieved after about 36-39 hr of 15N-glycine administration when subjects were consuming the higher protein diet, but not until about the 47th hr of isotope administration when the lower protein diet was consumed. Thus, although these data suggest that a satisfactory estimate of body N flux can be achieved at the lower protein intake, we cannot be entirely certain that the observed delay is solely caused by alterations in urea metabolism, or that the changes in urea flux caused a slight overestimation of the rates of protein synthesis and breakdown. This problem might be evaluated more directly by monitoring the level of lSN-enrichment of an accessible free amino acid compartment, such as Table 10.

An Estimate of the Fmctional Turnover Rate of the Rody Urea Pool in Subjects Consumina

Protein Intake WkelW 1.5

Mean

f

f

Rodv Weiaht/Dav

Total

Total

Daily

Fractional

Body

Body

Urine

TlJrWWer

water

BUN

Urea Pool

Urea N

Rate

Subject

(liters)

(e/l)

(e of N)

(g/day)

(day-‘)

LF

29.7

0.11

3.27

6.87

NP

41.4

0.22

9.11

12.17

1.34

SP

27.3

0.14

3.82

8.57

2.24

JF

43.3

0.18

7.79

14.21

1.82

I-IF

35.0

0.16

5.60

11.36

2.03

so

48.1

0.16

7.70

14.78

SD

0.38

Mean

1.5 and 0.38 G Protein/Ka

SD

2.10

1.92 1.91 l 0.31

LF

28.9

0.05

1.45

1.42

NP

46.6

0.07

3.26

2.87

0.98 0.88

SP

29.0

0.06

1.74

1.64

0.94

JF

43.3

0.09

3.90

2.50

0.64

HF

35.0

0.07

2.45

2.54

1.04

so

48.1

0.04

1.92

2.64

1.38

0.98

f

0.24

294

STEFFE ET AL.

the plasma free amino acid pool. Unfortunately,

with currently available techniques involving isotope-ratio mass spectrometry this type of evaluation cannot be done in the human subject because of the size of blood samples required. These considerations also point out a potential limitation in the use of the Picou and Taylor-Roberts model, as well as others that depend upon the analysis of urea for “N-enrichment. The use of i4C-amino acids for measuring amino acid flux6,’ presumably circumvents the problems that might arise from changes in the kinetic behavior of the urea pool. However, we could not use radioactive labels for this purpose in healthy young adults. The second point, which should be considered in interpreting our findings, concerns the reentry of isotope into the metabolic N pool. In rats, Aub and Waterlow” estimated a 3% reentry rate of labeled amino acid during 6 hr of intravenous infusion. This estimate has been taker3 to indicate that the problem of reentry is insignificant in humans because of their generally slower metabolic rates. The degree of isotope reentry in our study can be crudely approximated, assuming that the metabolically active protein content of the body is about 100 g/kg body weight. The estimated breakdown rate in the six subjects given the higher protein diet was about 3 g/kg body wt/day, or 3% of the protein pool/day. Over the infusion period, 19% of the administered “N-tracer dose appeared in the urine; thus, 8 1% was incorporated into body nitrogen. Thus, reentry would be only about 2.4% of the dose, and can be neglected in the evaluation of the effects of dietary protein in the present studies. Again, although a more precise evaluation would be possible using the multicompartmental analysis approach proposed by Aub and Waterlow,” it is not feasible in the human subject. There have been no satisfactory studies in normal adults on the dynamic aspects of total body protein metabolism at differing protein intakes. Tschudy et al.)’ studied one female patient with lymphosarcoma at various protein and energy intakes. However, because they used an inadequate experimental model,* unequivocal conclusions cannot be drawn from their work. Picou and Taylor-Roberts3 examined the effect of dietary protein level on three infants who had recovered from malnutrition, and observed that although the rate of total body protein breakdown increased with reduced protein intake, total body protein synthesis rate was unchanged. Hence, our observations on body protein breakdown are very similar to these findings in children, but differ with respect to whole body protein synthesis. Also, it needs to be pointed out that the effects of chronic dietary inadequacy may be different from those evident in short-term response. For example, in rats, chronic deficiency appears to result in reduced rates of body protein catabolism and a reduced body N fl~x.~ In contrast, short-term protein deprivation in rats does not markedly affect total body N fl~x,~ and in our human subjects only a small decrease was observed with an acute reduction in protein intake. Evidence that man may show similarly reduced rates of body protein catabolism after long-term nutritional deprivation was presented by Smith, Pozefsky, and Chhetri,35 who found re*Steffee and Young,

in preparation.

PROTEIN

295

INTAKE

duced arterio-venous amino acid differences-suggesting reduced protein breakdown-across the forearms of severely malnourished adult subjects. In contrast to the limited number of experiments using human subjects, published studies are more numerous for the effects of reduced protein intake on rates of whole body and tissue protein synthesis and catabolism in experimental animals (e.g.,3639). Although many of these studies may be used in partial support of our findings, caution must be exercised in extrapolating the data from animal models. A major problem is the applicability of the findings to adult man because of the far higher intensity of N metabolism in rats compared to that in children and adult human subjects.‘*8 Our findings indicate that acutely reduced protein intake in adult man causes only a small decrease in body N flux. However, there is a marked increase in the rate of total body protein breakdown, which is partially compensated for by an increased rate of total protein synthesis. A further feature of the adaptive response to acute changes in protein intake is a more efficient use of the N entering the pool for anabolic purposes, and less excreted as urea. This latter finding is consistent with reduced amino acid oxidation,40*4’ increased recycling of amino acids,37*39and a lowered enzymatic capacity for urea synthesis4*“’ when dietary protein intake is reduced. ACKNOWLEDGMENT We express our appreciation to the nursing staff of the Clinical Research Centers and to Dr. H. Zyas-Bazan, Misses C. Bilmazes, J. Allen, and M. Miller for their help in various phases of the studies. Our grateful thanks are offered to the subjects who willingly accepted the demands of the experiment. REFERENCES 1. Waterlow JC: Observations on the mechanism of adaptation to low protein intakes. Lancet ii:1091-1097,1968 2. Waterlow JC, Stephen JML: The effect of low protein diets on the turnover rates of serum, liver and muscle proteins in the rat, measured by continuous infusion of L-[ “C] lysine. Clin Sci 35287-305, 1968 3. Picou D, Taylor-Roberts T: The measurement of total protein synthesis and catabolism and nitrogen turnover in infants in different nutritional states and receiving different amounts of dietary protein. Clin Sci 36:283296, 1969 4. Waterlow JC: The assessment of protein nutrition and metabolism in the whole animal, with special reference to man, in Mammalian Protein Metabolism vol. III. Munro HN (ed): New York, Academic Press, 1969, pp 326-390 5. Blaxter KL: Methods of measuring the energy metabolism of animals and interpretation of results obtained. Fed Proc 30:1436-1443, 1971 6. Waterlow JC: Lysine turnover in man

measured by intravenous infusion of L-(U-‘4C) lysine. Clin Sci 33507-515, 1967 7. O’Keefe SJD Sender PM, and James WPT: “Catabolic” loss of body nitrogen in response to surgery. Lancet ii:103551037,1974 8. Young VR, Steffee WP, Pencharz PB, Winterer JC, Scrimshaw NS: Total human body protein synthesis in relation to protein requirements at various ages. Nature 253: 192194,1975 9. Young VR, Taylor YSM, Rand WM, Scrimshaw NS: Protein requirements of man: efficiency of egg protein utilization at maintenance and submaintenance levels in young men. J Nutr 103:11641174,1973 IO. Shipley RA, Clark RE: Tracer Methods for In Vivo Kinetics. New York, Academic Press, 1972 11. Matwiyoff NA, Ott DG: Stable isotope tracers in the life sciences and medicine. Science 181:1125-1133, 1973 12. Lutwak L, Burton BT: Fecal dye methods in metabolic balance studies. Am J Clin Nutr 14:109-111, 1964

296 13. Rose GA: Experience with the use of interrupted carmine red and continuous chromium sesquioxide marking of human feces with reference to calcium, phosphorus and magnesium. Gut 5:274-279, 1964 14. Picou D, Phillips M: Urea metabolism in malnourished and recovered children receiving a high or low protein diet. Am J Clin Nutr 25: 1261-1266, 1972 15. Hawk PB, Oser BL, Summerson NH: Practical Physiological Chemistry, 13th ed. New York, McGraw-Hill, 1954, p 886 16. Folin 0, Bell RD: Applications of a new reagent for the separation of ammonia. J Biol Chem 29:329-335, 1917 17. Sprinson DB, Rittenberg D: The rate of utilization of ammonia for protein synthesis. J Biol Chem 180:707-714, 1949 18. Rittenberg D, Keston AS, Rosebury F, Schoenheimer R: Studies in protein metabolism. II. The determination of nitrogen isotopes in organic compounds. J Biol Chem 127: 291-299, 1939 19. Dixon WJ: Biomedical Computer Programs, 3rd ed. Los Angeles, University of California Press, 1973, p 387 20. Hartley HO: The modified Gauss-Newton method for the fitting of nonlinear regression functions by least squares. Technometrics 3:269-280, 1961 21. Jennrich RJ, Sampson PF: Application of stepwise regression to nonlinear estimation. Technometrics 10:63-72, 1968 22. Soloman AK, Edelman JS, Soloway S: Use of the mass spectrometer to measure deuterium in body fluids. J Clin Invest 29:13111319, 1950 23. Moore FD: Metabolic Care of the Surgical Patient. 1959, pp 5-24 Philadelphia, W.B. Saunders 24. Walser M: Urea metabolism in chronic renal failure. J Clin Invest 53:1385-1392, 1974 25. Snedecor GW, Cochran WG: Statistical Methods 6th ed. Ames, Iowa, Iowa State University Press, 1967 26. Calloway DH, Ode11 ACF, Margen S: Sweat and miscellaneous nitrogen losses in human balance studies. J Nutr 101:775-786, 1971 27. Costa G, Kerins ME, Kantor F, Griffith K, Cummings WB: Conversion of Protein nitrogen into gaseous catabolites by the chick embryo. Proc Nat1 Acad Sci USA 71:451-454, 1974 28. Muysers K, Smidt U, von Nieding G, Krekeler H, Schaefer KE: Diffusional and metabolic components of nitrogen elimination

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41. Sketcher RD, James WPT: Branchedchain amino acid oxidation in relation to catabolic enzyme activities in rats given a proteinfree diet at different stages of development. Br J Nutr 32:615-623, 1974 42. Nuzum CT, Snodgrass PJ: Urea cycle enzyme adaptation to dietary protein in pri-

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