Nitrogen balance studies in aging rats

Experimental Gerontology, Vol. 20, pp. 29-34, 1985 Printed in the USA. All rights reserved.

NITROGEN

BALANCE

0531-5565/85 $3.00 + .00 Copyright ©1985Pergamon Press Ltd

STUDIES

IN AGING

RATS

J.K. BALDWINand P. GRIMINGER Department of Nutrition, Rutgers, The State University, New Brunswick, New Jersey 08903

A b s t r a c t - Nitrogen balance studies were conducted to compare the protein requirements of aged

(24-month-old) and mature (12-month-old) rats for possible age-related differences. Four diets with protein supplied as amino acid mixtures simulating casein protein at 4.5%, 4.5°70 plus 1.5°70 indispensable amino acids of casein, 4.5% plus 1.5% dispensable amino acids of casein or 6.0% casein protein were offered for one week each during a four-week counter-balanced dietary sequence. Lower food and nitrogen intake of aged rats resulted in a lower mean nitrogen balance compared to that of the mature animals. Calculations of the level of N intake necessary to provide for N equilibrium in the aged rats indicated an apparent increase in the actual net need for nitrogen and indispensable amino acids when compared with the needs of the mature animals.

INTRODUCTION STUDIES OF possible age-related changes in the protein requirements of aged subjects have provided contradictory results (Watkin, 1964; Swendseid, 1963). However, the importance of determining the amount and direction of such changes regardless of the probable multiplicity of underlying physiological and biochemical mechanisms remains undisputed. Although the population of the United States has shown an increase in the number of individuals aged 65 and older (Tauber, 1972), the recommended levels of intake do not make provision for possible differences in requirements for older individuals (Nat'l Res. Council, 1980). It is assumed that any changes in protein requirement with the onset of senescence in healthy individuals are minimal and that the requirement may be slightly, but insignificantly, lower than the recommended level (Watkin, 1964). However, since the elderly comprise approximately 10070 of the national population and incur at least 30070 of national health costs, further investigation of the actual requirements of the aged is justified. If differences between the requirement of the mature and the aged have not been clearly demonstrated, this may, at least in part, be the consequence of the extreme difficulty of obtaining sufficiently comparable subjects, and the inherent problems in the methodology of assessment.

The present address for J.K. Baldwin is: Exxon Corp., P.O. Box 235, E. Millstone, NJ 08873 New Jersey Agricultural Experiment Station Publication No. D-14406-1-82. Supported by state funds and by NIH Grant PHS AG00160 from the National Institute on Aging. (Received 26 April 1983, Revised 5 March 1984) 29

30

J,K. BALDWIN AND P. GRIMINGER

A number of preliminary studies were carried out to investigate the protein requirements of older rats using the nitrogen balance method (Baldwin, 1978). Based on the resuits obtained from these preliminary investigations, an experiment was devised to determine whether there was an age-dependent increase in the requirement or a change in the response to nitrogen (N) intake near the maintenance requirement level. M E T H O D S AND P R O C E D U R E Barrier-reared Fischer 344 male rats were used; they were obtained from the National Institute of Aging colony maintained by the Charles River Breeding Laboratories, Wilmington, MA. For the purpose of this study, the term "mature" was assigned to the 12-month-old animals, and the 24-month-old animals were referred to as "aged." Each age group consisted of eight rats. The assignments to treatments and diet offerings were made without regard to initial body weight. Four test diets (A,B,C,D) were given during each of the five trial weeks in a counter-balanced sequence providing duplicated Latin square blocks for each of the two age groups, thus minimizing the effect of dietary sequencing on the nitrogen responses (Keppel, 1973). Differences in protein requirement of aged and mature rats were asssessed by the effect of the four test diets (Table 1) on nitrogen balance. N balance was calculated as nitrogen intake minus nitrogen excreted in urine and feces, with integumental losses assumed minimal and essentially equal among all animals. Each experimental diet was given ad libitum for seven consecutive days (Table 2). Daily individual N balances were determined for all rats for the last four days of each trial week. This arrangement of diet presentation and measurement of N balance allowed for the collection of data concerning the effects of age, diet, week and week/day as factors of the protein requirement and provided a total of 32 observations for each diet within each age group. Diets A (4.5% simulated casein protein, methionine supplemented) and D (6% simulated casein protein, methionine supplemented) were chosen to study the quantitative aspect of the protein requirement of aged vs. mature animals. Diet A had been shown in preliminary tests to provide the minimum protein requirement to maintain older (mature) rats in N equilibrium. Diet D was chosen to provide protein at a level near that requirement, but sufficiently higher to produce a positive N balance as demonstrated in preliminary experiments. Diets B and C (4.5% simulated casein protein (SC), methionine supplemented, plus 1.5% essential amino acid (EAA) mix, and 4.5% SC, methionine supplemented, plus 1.5% non-essential amino acid (NEAA) mix, respectively) were chosen for evaluation of qualitative aspects of possible differences in the protein requirement of the two age groups. All diets were analyzed for N content by the Kjeldahl method prior to use. When necessary, they were then adjusted with EAA and/or NEAA mix to provide, as closely as possible, the desired N levels. Animals were housed individually in suspended stainless steel wire bottom metabolism cages. During the entire study, animals were maintained in an isolated controlled environment. Light was provided on a 12 hour off/on cycle. Temperature was maintained at 72-74 F. TABLE 1. SIMULATED PROTEIN CONTENT OF EXPERIMENTAL DIETS.

Diet

A m i n o Acids Simulating Casein

A m i n o Acid Mixtures

Basal c

A B C D

4.5 4.5 4.5 6.0

-1.5 EAAa 1.5 NEAAb -

94 94 94 94

aMixture of indispensable (essential) amino acids simulating those found in Casein Protein. OMixture of dispensable (non-essential) amino acids simulating those found in Casein Protein. cSucrose 40.0%, Fiber (Solka-floc, Brown Co.) 5.0%, Corn oil 10.0%, Vitamin Mix 0.2% (Bauer and Griminger 1980), Biotin 0.006%, Choline Chloride 0.1%, Salt Mix 4.0% (Draper et al., 1964), ZnSOd-H20 0.0068%, CuSO4 0.0038%, Corn starch 34.7, DL methionine, 0.1%. Corn starch added to make up to 100%.

NITROGEN BALANCE STUDIES

31

TABLE 2. EXPERIMENTAL DESlON.

Week on Experiment 1

2

Experimental Animal Pairs a

3

4

5

Experimental Diets b

1,5 2,6 3,7 4,8

A B C D

B D A C

C A D B

D C B A

A B C D

aEach o f the two pairs on each diet sequence (for example, pair 1 and pair 5) consisted o f a mature (12-month old) and an aged (24m o n t h old) rat. bExperimental diets, see Table 1. Individual food intakes were recorded dally. During the collection period (last four days o f each week), urine and feces were collected for each day. While urinary N excretion was measured on a daily basis, feces were pooled for the four-day collection period o f each animal and dried to a constant weight prior to N analysis.

RESULTS AND DISCUSSION Nitrogen balance responses to the 4.5°7o protein diet (A) were considerably different for the two age groups of rats. The average N balance of the mature animals (9.5 mg/rat/day) on diet A indicates that these animals were close to or in N equilibrium; in contrast, the average N balance of the aged rats was definitely negative ( - 14.4 mg/rat/day) (Table 3). TABLE 3. AVERAGE ( ± SE) DAILY N INTAKE, N EXCRETION, AND N BALANCE OF MATURE AND AGED RATS ON FOUR DIFFERENT DIETS.

Diet a

A A B B C C D D M (A,B,C,D) A(A,B,C,D)

Age b

M A M A M A M A

Nitrogen Intake

Urinary Nitrogen

Nitrogen Balancec

mg

mg

mg

102 ± 11 79±12 145 ± 10 1164-12 153 ± 12 129 ± 17 149 ± 15 132 ± 13

58 ± 5 70± 7 65 ± 5 76± 8 87 ± 7 100 ± 11 70 ± 6 87 ± 7

137 ± 14 114 ± 15

70 ± 10 83 ± 9

10 ± -14± 47 ± 12± 31 ± -2 ± 45 ± 15 ±

5 6 5 6 6 9 7 8

33 ± 9 3 ± 10

aDiet A, 4 . 5 0 simulated casein; diet B, 4.5070 simulated casein plus 1.5070 E A A mixture; diet C, 4.5070 simulated casein plus 1.5070 N E A A mixture; diet D, 6.0070 simulated casein. bM = mature (12-month old); A = aged (24-month old) male Fischer 344 rats. CN balance = N intake - (urinary N + fecal N), average of weeks 1-4. Fecal N (not shown) = N intake - (urinary N + N balance).

32

J.K. BALDWIN AND P. GRIMINGER

The difference in N balance is approximately equal and probably directly attributable to the difference in N intake. The lower N balance on diet A observed in the aged rats compared to that of the mature was fairly consistent during each of the four test weeks. The results indicate that the 4.5% protein diet (diet A) produced a N balance close enough to N equilibrium to be considered as the requirement level for mature rats. However, this diet was insufficient for the maintenance of N equilibrium in the aged rats. The N balance responses to each of the three 6.0% protein diets (diets B,C,D) of the aged animals during each of the five test weeks indicated in all instances considerably less N retention than those of the mature animals. The average daily N balances of mature animals on diets B,C, and D showed a greater N retention than those seen on diet A and were all strongly positive; the average retentions were 47.3, 31.1 and 44.8 mg/rat/day, respectively, versus 9.5 mg for diet A. The average daily N balances of the aged animals on diets B (12.5 mg/rat/day), C ( - 1.6 mg/rat/day) and D (15.1 mg/rat/day), were more positive than on diet A ( - 14.4 mg/rat/day), but were still lower than the N balances observed in the mature animals. Comparing the combined experimental diets B,C, and D for the two age groups, one obtains N balances of 41.1 m g / r a t / d a y for the mature, and 8.7 mg/rat/day for the aged; this difference of 32.4 mg is slightly more than the difference observed in mean N intake between the two age groups (23.5 mg). Although it was noted that the major factor contributing to the more negative N balance of the aged animals was the lower food intake and, therefore, lower N intake, the urinary excretions of the aged animals were in almost all instances greater than those of the mature. As previously noted, diets B and D produced more positive N balances than did diet C for both ages (46 and 30 mg vs. 15 mg). Although in both ages the N intake and urinary N were not significantly different among diets, the urinary N per mg of N intake of diet C was higher than that of diets B and D (Table 4). The resultant lower balance on diet C suggests that some difference in N response to the amino acid composition of this diet occurs at both ages. As diet C contains 4.5% simulated casein protein plus 1.5% NEAA, this difference may be the result of a dilution of the effective level of the indispensable amino acids, or of a particular indispensable amino acid.

TAn~ 4. RATXO OF URINARY NXTROOEN TO NITROGEN INTAKE IN MATURE AND

AGED RATS FED Tm~E APPROXIMATELY ISONITROGENOUS DIETS. Dietb Age a

B

C

D

Average Urinary Nitrogen/ Average Nitrogen Intake M A

.45 .66

aSee Table 3, footnote b bSee Table 3, footnote a

.57 .78

.47 .66

NITROGEN BALANCE STUDIES

33

The apparent digestibility coefficients of the three 6.0°70 protein diets B, C, and D, which were isocaloric and approximately isonitrogenous, were equal for both age groups, suggesting that the differences noted in N balance were not the result of diet- or agerelated differences in the absorption of dietary N. Since the differences in N balance were seen to be mainly the result of differences in the N consumption by the rats of the two age groups on each of the four diets, a further examination of the differences in response of the mature and aged animals to the test diets was made by a comparison of the amount of N intake necessary to produce equivalent responses. This evaluation was made by calculating a regression line (N balance/N intake) for each age group on each of the four diets (Table 5). The N intake at N equilibrium (NB = 0) was estimated from these lines. As indicated by the R-square value, about twothirds of all observations of each age-diet group are explained by the regression equation. Using a computerized statistical analysis system (SAS) to generate analysis of variance and least significant differences, it was determined that the amount of N required for N equilibrium for the aged rats on diet B (105.0 mg) was significantly greater (p < 0.05) than the amount required by the mature (78.6 mg) for an equivalent response (Table 5). The N intake necessary for the aged animals to achieve N equilibrium on each of the remaining diets A (98.0 mg), C (136.2 mg), and D (111.2 mg) was larger than the N intake required by the mature on diets A (90.0 mg), C (112.6 mg), and D (83.3 mg). However, these differences did not reach the 0.05 level of significance. The N intake required by the aged to produce N equilibrium on diet C was significantly greater than that on diets A and

TABLE 5. REGRESSIONCOEFFICIENTS FOR NITROGEN BALANCE REGRESSEDON NITROGEN INTAKE AND STATISTIC OF FIT OF NITROGENBALANCEOBSERVEDIN MATURE AND AGED RATS ON FOUR DIFFERENT DIETS.a Diet & Age b

Intercept d

NB c

Slope e

R Square f

N I at N B = Og

mg A,M A,A B,M B,A C,M C,A D,M D,A

9.1 -14.4 47.2 12.5 27.6 -4.4 43.7 15.1

± ± ± ± ± ± ± ±

15.1 18.1 13.4 16.5 16.7 24.3 20.0 21.7

-70.4 -80.3 -55.8 -95.5 -77.7 -83.1 -55.8 -80.1

± ± ± ± ± ± ± ±

9.2 9.1 13.1 11.2 14.1 12.7 13.6 14.8

0.78 0.86 0.71 0.91 0.69 0.61 0.67 0.72

± ± ± ± ± ± ± ±

0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

0.73 0.70 0.67 0.77 0.67 0.60 0.66 0.60

90.0 98.0 78.6 105.0 112.6 136.2 83.3 111.2

± ± ± ± ± ± ± ±

13.0 4.2 i** 10.0 h* 3.3 i** 18.6 7.2 18.0 5.0 i*

an = 32, 4 (24 hr) N balances of 2 rats of each age group on each diet for weeks 1-4. bLetters denote diet and age (see Table 3, footnotes a and b). cNB = average daily nitrogen balance (~) ± standard deviation. dEstimated NB at zero nitrogen intake (a) ± standard error. eSlope NB/N1 (8) ± standard error fStatistic of fit, percent of observations which are included in estimate. gAverage daily NI when NB = 0 ± standard error, calculated from equation of line when NB is regressed against NI, standard error of NI where var = standard error of a or 8,

a 2 (var a)~

(var 8)2

(when NB = 0) = / 8 (a)-----F - 4- ~

(var 8 2)

4- 2 (NI[a) (~)

hSignificant difference between ages on this diet. *p < 0.05 **p < 0.01 iSignificantly different from C, A value.

34

J.K. BALDWIN AND P. GRIMINGER

B (p < 0.01), and D (p < 0.05). While the differences in the mature rats were in the same direction, they were not significant. The finding that the N intake for aged rats to achieve N equilibrium is more than the amount necessary for the same response in the mature is demonstrated by additional observations. The level of N intake (98.0 to 136.2 mg) required by the aged on all four diets for N equilibrium was higher than that o f the mature (78.6 to 112.6 mg). On the three 6.0°7o protein diets the N intake (mg/kg BW) for N equilibrium of the aged rats was 37°7o greater than that o f the mature. On the 6.007o protein diet with a high indispensable amino acid content (diet B) aged rats required significantly greater N intake (a difference o f 83 m g / k g BW) than the mature to maintain N equilibrium. These results tend to confirm studies conducted in older men which also reported an increase in indispensable amino acid requirements with age (Tuttle et al., 1965). The results obtained on diets C and D in aged animals are also in agreement with the findings that the indispensable amino acids may be used more effectively as the supplemental N source (Tuttle et al., 1965). The dietary protein level necessary for aged rats to maintain adequate N balance is apparently higher than that o f mature animals. This is a result o f the lower food intake and, therefore, N intake o f the aged animals. However, in addition, comparisons o f N balance studies in mature and aged rats have provided evidence for an apparent increase in the actual net needs for N and indispensable amino acids o f the aged. Similarity o f results obtained in the aged animals o f this study and reported studies in elderly humans indicate that this animal model may be useful in providing experimentally derived information concerning age-related changes in N requirements of aged humans. REFERENCES BALDWIN,J.K. (1978) Investigation of Age-Related Changes in the Protein Needs of Aged and Mature Rats,

Ph.D. Thesis, Rutgers University, New Brunswick, NJ. BAttER, K.D., and G~n~OER, P. (1980) Poultry Sci. 59, 1493. Dlt~a,ER, H.H., B~RGAN,J.G., MEI CHro, CS~LANY,A.S. and BOARO,A.V. (1964) J. Nutrition 84, 395. KEI'I'EL, G. (1973) Design and analysis, a researcher's handbook, 397 Prentice Hall Inc., Englewood Cliffs, New Jersey. NATIONALI~S~Crl Cour~crt (1980) Recommended dietary allowances, 9th ed. National Academy of Sciences, Washington, D.C. SwEz,nasE~, M.E. (1963) Syrup. protein nutrition and metabolism, University of Illinois Special Publication No. 4. TAUBER,C. (1972) Science 176, 773. TtJTrt~, S.G., BASS~Tr,S.H., GRirrrrH, W.H., M t r u c ~ , D.B., and SWENDS~ID,M.E. (1965)Am. J. Clin. Nutr. 16, 225. WAa~n~, D.M. (1964)In: Mammalian protein metabolism Vol. 2 (Edited by H.N. Monro and J.B. Allison), pp. 247-261, Academic Press, Inc., New York.