Nitrogen children1’
with
normal
2
E. Ziegler,3
Steven
studies
M.D.,
E. Nelson,6
B.S.,
Alejandro Leon
M. O’Donnell,4
F. Burmeister,7
M.D.,
Ph.D.,
Genevieve
and
Samuel
Stearns,5
Ph.D.,
J. Fomon,8
M.D.
ABSTRACT A total of 1 148 nitrogen balance studies were conducted in 100 boys and 23 girls 1 to 1 1 years old. Mixed, customary diets supplied between 8.9 and 21 .4% of calories from protein . Duration of balance studies ranged from 72 hr in the youngest subjects to 1 20 hr in older children . Regressions and 95 % confidence intervals of nitrogen retention on nitrogen intake are presented . The slope of the regression was significantly greater for children 12 to 1 8 months old than for children of other age groups. Correlation coefficients between various parameters of nitrogen balance and energy intake are presented. Although variability in results of nitrogen balances within subjects appears to be rather large , a significant difference between subjects was observed. Am. I. Clin. Nut,’. 30: 939-946, 1977.
Few satisfactory reference data from studies of nitrogen balance with normal children of preschool and school age are available in the literature Moreover, as pointed out by Irwin and Hegsted (1), results of published studies lack agreement. The present report presents data from 1 148 nitrogen balance studies with 1 23 normal children from 1 to 1 1 years of age These studies were conducted between 1930 and 1953 under relatively uniform conditions in the Pediatric Metabolic Unit, University of Iowa, under the direction of one of the authors (G . S.) and Dr. P. C. Jeans. Macy and associates (2) have presented data from a large number of nitrogen balance studies with normal children. These studies involved 33 children, none of whom was less than 4 years old. The range of nitrogen intakes was relatively small compared with that in the present series. Data from about 80% of these studies were reported by Stearns et al. (3) in their discussion of protein requirements of children The present report includes results of further studies by the same investigators and presents regressions and confidence intervals for the entire data. .
.
Iowa, children awaiting adoption, or normal children of mothers being treated for tuberculosis. These children had been living in the Pediatric Metabolic Unit since early infancy. Most of the children over 2 years old came from orphanages in the State of Iowa and lived for at least several months in the Pediatric Metabolic Unit. In analyzing the data, we have elected to exclude from consideration the small percentage of the children who at the time of admission demonstrated height and/ or weight less than the 10th percentile values of widely used reference data (4) . The children were all judged to be adequately nourished. No child lost weight while the studies were being conducted and most children gained weight over the interval of study. Subjects have been divided into the following age groups: 1 2 to 18 months, 18 to 36 months, 3 to 6 years and 6 to 1 1 years. The last age category, for example, included all children who had reached their sixth birthday and had not yet reached their eleventh birthday. The youngest age group was identified on the basis of preliminary analysis of the data which showed that the slope of the regression of nitrogen retention on nitrogen intake changed little after 18 months of age. The other age divisions were arbitrary.
Procedures Those subjects who were children staff of the University of Iowa were
.
Subjects,
and
procedures
In most instances subjects between 1 and 2 years old were children of students or staff of the University of
American
Journal
of
Clinical
Nutrition
30: JUNE
From
the
Department
7578 . ‘ Associate low. Present address: mento
methods
Subjects
The
1
Medicine, University 2 Supported in
de
PediatrIa,
of students permitted
of Pediatrics,
Iowa, Iowa City, part by U.S.P.H.S. of
Professor. Unidad Hospital
or to
go
College
of
Iowa 52242. Grant HD
Postdoctoral
Fel-
Metabdlica, DepartaRam#{243}n Sard#{225},Universi-
dad Nacional de Buenos Aires, Buenos Aires, Argentina . Professor Emeritus. #{176} Research Assistant . Assistant Professor, Department of Preventive Medicine and Environmental Health . Professor. 1977,
pp. 939-946.
Printed
in U.S.A.
939
Downloaded from https://academic.oup.com/ajcn/article-abstract/30/6/939/4650377 by East Carolina University user on 11 March 2019
Ekhard
balance
940
ZIEGLER
ET
home on Sundays; otherwise the children lived in the Pediatric Metabolic Unit continuously for 6 or more months. Children from orphanages were studied no sconer than 4 weeks after admission to the Pediatric Metabolic Unit to permit adjustment to the new living conditions
and
diet.
An
effort
was
made
to provide
a
playground
located
just
outside
the
ward
unit,
so they
could be watched at all times. Toys, books, and games were provided. The difficulty of collecting excreta quantitatively from very young children is evidenced by the fact that few data other than these are available in the literature. Excreta of subjects less than approximately 2 years old were collected with the use of a metabolic bed , similar in design to, but larger than, that subsequently described (5). These children were permitted short periods of activity off the metabolic bed during waking hours. As the children became more dependable in their toilet training, activity periods were lengthened until the metabolic beds were used only for sleeping. Separate collections of urine and feces were rarely attempted with girls less than 3 years old. No attempt was made with any age group to measure dermal losses or losses from external secretions. Fecal collections were made between carmine markers. Urine was collected under toluene. With the youngest subjects, one 72-hr study was usually performed every week or every other week. Occasionally, two consecutive 72-hr studies were performed. As the children became more ambulatory, study periods were frequently lengthened to 96 hr. In a few cases, as many as four consecutive 96-hr studies were carried out, but more commonly one study was performed per week. After a change in diet, an adjustment period of at least 1 0 days was allowed before resumption of studies. Children above approximately 4 years old were studied for periods of 120 hr. Usually, three such studies were performed consecutively. The diet was then altered and a 2-week adjustment period allowed before resumption of studies. Diet
A mixed diet appropriate for age was fed, resembling to a large extent home-prepared American diets. Bananas were generally included during all seasons. These were a great treat to orphanage children and encouraged them to accept the entire diet . Inclusion of other fruits and vegetables varied with the season. Proteins of animal origin accounted for approximately 90% of protein intake, with milk, meat, and eggs serving as the major sources. Changes in protein intake were brought about in younger children by altering chiefly the amount of milk products, but also of meat and eggs. Diets offered to subjects 1 to 3 years old contributed an average of 1 6 .5 % (range: 1 1 .9 to 21 .4%) of calories from protein and diets of subjects 6 to 1 1 years old contributed an average of 13.8% (range: 8.9 to 19.2%) of calories from protein. During adjustment periods subjects consumed as much food as they pleased and records were made of
amounts consumed. During subsequent balance periods, constant amounts of food were offered which slightly exceeded the mean amount consumed during the preceding adjustment period. Very likely this caused variation of fcod intake within a given subject to be less than would otherwise have been the case. In the early years of the study, energy content of the diets offered to the subjects was calculated from unpublished locally assembled tables of food composition and, after 1950, from USDA published tables (6). Duplicates or, in the case of older children, aliquots of all food offered in the course of a balance study were pooled, homogenized and analyzed for nitrogen. In the case of milk, however, each new supply was analyzed only once and the amount of nitrogen offered from this source calculated separately. If any portion of a meal was not eaten, it was analyzed for nitrogen. From the nitrogen offered minus the nitrogen in uneaten portions, intake of nitrogen was calculated. Although uneaten portions of diet were not measured for energy content, food refusals were few and of small amount. Therefore , energy offered is referred to subsequently in this report as “energy intake.” Methods Aliquots of food and complete fecal contents were digested with 20% hydrochloric acid and made up to a convenient volume with water. Nitrogen content of these digests and of urine was determined in duplicate by the Kjeldahl method as described by Hawk and Bergeim (7). Later in the study a selenium catalyst was employed as described by Campbell and Hanna (8). Net absorption of nitrogen was estimated from the quantity consumed minus the amount excreted in feces. Similarly, intake of nitrogen minus amounts excreted in feces and urine yielded apparent net retention of nitrogen. Standard statistical methods, including regression analysis, calculation of correlation coefficients, analysis of variance and of covariance, were used (9). Normality of distribution was assessed by calculating Kolmogorov-Smirnov statistics (1 0). In most instances parameters of nitrogen balance were found not to follow a normal distribution. Therefore, nonparametric correlations using Kendall’s r (1 1) were also computed. However, confidence intervals and standard deviations were retained simply because they describe samples in familiar and thus convenient terms.
Results Among the children in Iowa orphanages at the time of these studies, there were many more males than females. Males were much more often available for study Of the total number of 1 148 metabolic balance studies, 986 were carried out with 100 males and 1 62 were carried out with 23 females (Table 1). Preliminary statistical analysis failed to reveal significant sex-related differences in regressions of nitrogen retention on nitro.
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home-like atmosphere. The older children were taken on walks, trips to the City Park, and shopping trips. Those of school age attended both the hospital school and its craft shop. The young children had their own
AL.
NITROGEN
gen intake Therefore female subjects have
BALANCE
.
,
been
Intake, absorption, and nitrogen (Table 1)
data of pooled.
urinary
STUDIES
male
WITH
and
,
excretion
of
,
of nitrogen
balance
No.
of subjects’
3 years
old,
data
on absorption
No.
of studies
excretion
Nitrogen
.
Nitrogen
NiO
in-
Fecal
en retention
12-l8months
thesis.
urinary
M, male;
excretions
TABLE Relations
2 among
F, female.
115 71d
235 199” 302 496 in more (I
Subset
581” (113) 529 55 (137) (24) 546 (84) 543 63 (81) (21) 507 51 (100) (15) 447 44 (84) (15) than one age group.
Energy
Urinaiy
mg/kg/day
21 (12M, 9F)C 13” (12M, iF) 18-36 months 20 (14M, 6F) 19” (14M, SF) 3-.6years 32 (30M, 2F) 63 6.-il years (54M, 9F) a Several subjects were included
and
studies .
Age
than
% ofirnake
364 (63)
b
390 (68) 374 (82) 339 (63) Mean;
of first line in this age group,
149 23.7 (105) (12.2) 19.4 110 (81) (10.6) 95 17.0 (51) (8.4) 90 16.2 (48) (8.0) 82 15.8 (43) (7.8) 64 13.7 (50) (9.7) standard deviation given
representing
studies
in which
kcal/kg/day
100 (18) 93 (14) 91 (14) 91 (14) 90 (12) 81 (14) in parenfecal and
were measured. parameters
of nitrogen
balance
and energy
intake Age
Parameter,’
Correlation N intake N absorption
12-18
18-36
3-6
6-11
months
months
years
years
vs energy intake vs N intake
0.804 0.979
0.561 0.966
0.266 0.990
0.672 0.985
vs N intake vs N intake
0.628 0.839
0.764
0.882
0.780
vs N intake vs N absorption
0.661 0.805
0.554 0.311 0.541
0.524 0.231 0.525
0.581 0.374 0.606 0.503 0.891
coefficient”
Urinary N excretion N retention % N retention N retention
Kendall’s N intake
.
Mb
vs energy
intake
0.617
0.443
0.246
N absorption
vs N intake
0.839
0.850
0.897
Urinary
vs N intake vs N intake
0.427 0.61 1
vs N intake vs N absorption
0.447 0.536
0.579 0.379 0.194
0.670 0.364 0.130 0.375
N excretion
N retention % N retention N retention a
Expressed
significant
per kilogram
(P < 0.001).
of body
weight
except
where
indicated
0.369
as percent
of intake.
“
0.57 1 0.405 0.259 0.425 All values are highly
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less
urinary excretion of nitrogen in the age groups 1 2 to 1 8 months and 1 8 to 36 months were, with few exceptions, available for male subjects only. Nevertheless, fecal and urinary excretion could be determined
,
1 of results
941
CHILDREN
not vary independently and were, as mdicated in Table 2 highly correlated in each age group. In this case, as in the case of all other correlations listed in Table 2 Kendall’s i- reached the same high level of significance (P < 0.001) as the correlation coefficient. Because urine and feces were generally not collected separately in studies of girls
Mean nitrogen intake per kilogram of body weight was 581 mg/day in the age group 1 2 to 1 8 months and decreased progressively in subsequent age groups. As already mentioned, protein generally accounted for 10 to 20% of energy intake. With the same diet, a child who consumed a greater quantity of food achieved a greater intake both of energy and protein. Therefore nitrogen intake and energy intake did TABLE Summary
NORMAL
942
ZIEGLER
ET
AL.
4
12
I NTAKE
U
URINE z
..
w
./
06
4
RETENTION 2
#..-
.
--.-...
._______S___
.._.. -
FECES I
2
3
4
5 AGE
FIG. 1 . Summary of nitrogen balance studies months and 1 year are used in this graph only.
6
7
8
9
tO
II
(years)
in children
from
age 1 to 1 1 years.
Note
that age intervals
,
of 6
‘Ps
E z
0 Iz Ui
00
IUi
300
600
INTAKE
00
0o
(mg/kg)
2. Daily nitrogen retention in relation to daily nitrogen intake in 21 children 12 to 18 months old. Each the result of one balance study. Y = 0.635X 219.6 (Y = retention of nitrogen, and X = intake of nitrogen, both in mg/kg/day). N = 1 15 . Solid lines indicate regression line and 95% confidence intervals. Points off the scale are indicated by arrows. FIG.
dot
represents
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I0
NITROGEN
BALANCE
STUDIES
,
,
.
,
.
,
Energy
intake
As already tween quantity
mentioned, oL food
the difference offered and that
becon-
NORMAL
CHILDREN
943
sumed was probably small and may thus be ignored Mean energy intake (estimated from diet offered) averaged 100 kcal/kg of body weight at age 1 2 to 1 8 months and decreased progressively to 81 kcal/kg at age 6 to 1 1 years (Table 1). Since no attempt was made at controlling energy intakes at predetermined levels, the observed energy intakes represent essentially ad libitum intakes. .
Retention
of nitrogen
Mean retention of nitrogen per kilogram of body weight was greatest in the age group 12 to 18 months (149 mg/kg/day) and decreased progressively at older ages Similarly, retention of nitrogen, expressed as percentage of intake decreased from 23 .7 % at age 1 2 to 1 8 months, to 1 3 .7 % at age 6 to 1 1 years (Table 1). The relationship between intake and retention of nitrogen in the various age groups is illustrated in Figures 2 to 5 . As indicated in Table 2, correlation coefficients and Kendali’s T were highly significant in all age groups. Analysis of covariance in conjunction with t-tests revealed that the slope of the regression for the age group 1 2 to 18 months (Fig. 2) was significantly greater (P < 0.05) than the slopes for the three other age groups (Figs. 3 to 5); slopes of the latter .
,
300
200
E z
0 I-
00
z
Ui I-. Ui
0
200
FIG.
3. Daily
dot represents
the
nitrogen result
300
retention in relation of one balance study.
400
500
INTAKE to daily nitrogen Y 0.330X -
600
JO0
00
(mg/kg) intake in 20 children 18 to 36 months 84.4 (see legend to Fig. 1). N = 235.
old. Each
Downloaded from https://academic.oup.com/ajcn/article-abstract/30/6/939/4650377 by East Carolina University user on 11 March 2019
in 71 and 199 balance studies, respectively, in the two age groups. Net absorption of nitrogen was highly correlated with intake of nitrogen at all ages (Table 2). Regardless of age fecal excretion of nitrogen averaged 10 to 1 2 % and, therefore net absorption of nitrogen amounted to 88 to 90% of intake (Table 1) Mean urinary excretion, expressed as percent of intake increased slightly from 69 % at age 1 2 to 1 8 months to 76 % at age 6 to 1 1 years. In each age group, urinary nitrogen excretion, expressed in milligrams per kilogram of body weight, was highly correlated with nitrogen intake expressed in the same terms (Table 2). A summary of results of nitrogen balance studies is presented in Figure 1 In this figure (but not in the other figures or tables) age intervals of 6 months during the first 3 years of life and intervals of 1 year for older children have been used. Expressed in grams per 24 hr (rather than grams per kilogram of body weight), urinary nitrogen excretion increased as intake rose with advancing age whereas fecal excretion and retention of nitrogen showed little change.
WITH
944
ZIEGLER
ET
400
500
AL.
300
.*
z
0
I.z Ui
100
IUi
0
00 200
300
INTAKE FIG. 4. Daily nitrogen retention represents the result of one balance
in relation study. Y
=
to daily nitrogen 0.228X 33.8
600
700
800
(mg/kg)
intake (see
in 32 children legend
to Fig.
3 to #{246} years 1).
N
=
old.
Each
dot
302.
400
300
.
E 200
z
0
I-. z
00
Ui Ui
0
-100 200
300
500
400
INTAKE FIG. 5 . Daily nitrogen retention represents the result of one balance scale is indicated by arrow.
in relation study. Y
=
to daily 0.341X
did not differ significantly from one another. To facilitate comparison, Figure 6 includes the regression of apparent nitrogen
700
600
800
(mg/kg)
nitrogen intake in 63 children 6 to 1 1 years old. Each dot 88.1 (see legend to Fig. 1). N 496. One point off the
retention on nitrogen intake for infants 90 to 1 20 days of age receiving similar intakes of nitrogen - i .e more than 1 1 % of cabries from protein (1 1). As may be seen, the . ,
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E
NITROGEN
STUDIES
BALANCE
WITh
NORMAL
945
CHILDREN
400
90-120
days
2 E
200
6-Ilysors
18-36
z
months
0 Iz Ui
IUi
3-6
ysors
00
0
-100
400
FIG. Included calories (15).
6. Daily also
from
800
600
INTAKE
00
(mg/kg)
nitrogen
is regression
protein
,
retention in relation to daily nitrogen intake in the various age groups (solid lines). line for infants 90 to 1 20 days of age (interrupted line) receiving more than 1 1 % of from Fomon et al . (1 2) based on data of Nelson (1 3), Jeans and Stearns (14), and et al
regression of apparent nitrogen nitrogen intake closely parallels 1 2- to 1 8-month-old children.
retention on that for the
Reproducibility subjects
between
to 0 .274
the and
differences
The 95 % confidence limits of the regressions of retention of nitrogen on intake of nitrogen (Figs. 2 to 5) and the standard deviations listed in Table 1 provide some measure of reproducibility with respect to the total number of studies. Reproducibility of nitrogen balance was further estimated by calculating intraclass (intrasubject) correlation coefficients. Since nitrogen retention was strongly correlated with nitrogen intake analysis of covariance was utilized to correct nitrogen retention for nitrogen intake Intraclass correlation coefficients of nitrogen retention corrected for nitrogen intake ranged from 0 1 36 (1 2 to 18 months) to 0.275 (6 to 1 1 years) when retention was expressed as milligrams per kilograms and from 0 1 1 5 (1 2 to 1 8 months) ,
.
.
.
(6 to 1 1 years)
when
expressed as percent of Thus, variability in results same
subject
appears
retention
was
nitrogen intake. of studies with to be
rather
large.
On the other hand, the F-test for betweensubject variation was significant for age 12 to 18 months (P < 0.03) and for all other ages (P < 0.001). Thus, nitrogen retention corrected for nitrogen intake differs signifi.cantly between subjects. Comment The present series of 1 148 nitrogen balance studies probably represents the largest available body of such data concerning normal children from 1 through 3 years old and one of the largest bodies of data concerning children from 4 through 1 1 years old. Although the majority of the data have already been summarized (3), we believed the usefulness of the data would be enhanced by a more detailed description of the studies and by further statistical analyses. Irwin and Hegsted (1) have reviewed the
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300
ZIEGLER
946
.
,
References 1 . IRwIN, M . I . , AND D . M . HEGSTED. of research on protein requirements Nuts. 101: 385, 1971.
A conspectus of man. J.
AL.
2. MACY, I. G. Nutrition and Chemical Growth in Childhood. Vol II. Original data. Springfield, illinois: Charles C Thomas, 1946. 3. Smzns, G., K. J. NEWMAN, J. B. McKnz AND P. C. Jws. The protein requirements of children from one to ten years of age. Ann. N.Y. Acad. Sci. 69: 857, 1958. 4. VAUGHAN, V. C., III, AND R. J. MCKAY. Nelson Textbook of Pediatrics (10th ed.). Philadelphia: w. B. Saunders Co., 1975, pp. 40-43. 5. F0M0N, S. J., L. N. THOMAS, R. L. JENSEN AND C. D. MAY. Determination of nitrogen balance of infants less than 6 months of age . Pediatrics 22: 94, 1958 6. WAn, B. K., AND A. L. MERRILL. Composition of Foods Raw , Processed, Prepared . Washington, D.C.: USDA Agriculture Handbook No. 8. Agri-
-
cultural HAWK,
Research
Administration,
1950.
P. B., AND 0. BERGEIM. Practical Physiological Chemistry (10th ed.). Philadelphia: Blakiston, 1931, p. 814. 8. CAMPBELL, W. R., AND M. I. HANNA. The determination of nitrogen by modified Kjeldahl methods. J. Biol. Chem. 119: 1, 1937. 9. SNEDECOR, G. W., AND W. G. CocHaN. Statistical Methods. 6th edition. Ames, Iowa: Iowa State University Press, 1967. 10. Osri., B., AND R. W. MENSING. Statistics in Research (3rd ed.). Ames, Iowa: Iowa State Uni7.
Press, 1975, p. 352. W. J. Practical Nonparametric StatisYork: John Wiley & Sons, 1971. 12. F0M0N, S. J., A. M. O’DONNELL AND E. E. ZizGLER. Bilancio azotato nei primi 3 anni di eta. In: Metabolismo Proteico in Infanzia, edited by E. Schwarz-Tiene. Milan: Minerva Medica. In press. versity
1 1 . CoNovna, tics. New
13.
M. V. K. The growth and nitrogen metabolism of infants receiving undiluted milk. Am. J. Diseases Children 39: 701, 1930. 14. Jr.s, P. C., AND G. Smi.asis. Growth and retentions of calcium, phosphorus and nitrogen of infants fed evaporated milk. Am. J. Diseases Children 46: 69, 1933. 15. JEANS, P. C., G. Sm.aris, J. B. MCKINLEY, E. A. GoFi’ AND D. STINGER. Factors possibly influencing the retention of calcium, phosphorus, and nitrogen by infants given whole milk feedings. I. The curding agent. J. Pediat. 8: 403, 1936. 16. Joint FAO/WHO Ad Hoc Expert Committee: Energy and Protein Requirements. WHO Technical Report Series No. 522. Geneva: World Health Organization, 1973. 17. YOUNG, V. R., AND N. S. Scamisnw. Human protein and amino acid metabolism and requirements in relation to protein quality. In: Evaluation of Protein for Humans, edited by C. E. Bodwell. Westport, Connecticut: Avi Publishing Company, 1977. NELSON,
Downloaded from https://academic.oup.com/ajcn/article-abstract/30/6/939/4650377 by East Carolina University user on 11 March 2019
voluminous literature on the subject of nitrogen balance studies in children The lack of agreement among investigators noted by Irwin and Hegsted (1) prompted us to prepare the present summary of studies which, although carried out some time ago, were conducted under standardized conditions in a uniform manner. It was our intention to establish relationships between intake and retention of nitrogen in normal children of various ages receiving protein intakes similar to those commonly consumed by children in the United States. Extremely high or low protein intakes were not included and thus even the lowest protein intakes used about 1 2 % of calories from protein in children less than 3 years old and about 9% in older subjectswere in excess of presumed requirements (16). Statistical evaluation was complicated by the fact that in the majority of instances parameters of nitrogen balance did not follow normal distributions. Relationships between these parameters were therefore examined by Kendall’s r, a nonparametric technic. At least for those relationships summarized in Table 2 these calculations yielded correlations which were as significant as the conventional correlation coefficients. Although it is well recognized that nitrogen retention is influenced by the availability of dietary energy (17), the present studies, in which energy and protein intakes were highly correlated (Table 2), were not designed to provide additional insight into the nature of the relationship. Our results suggest that the predictability of results of nitrogen balance studies, even with the same subject, is rather low. In spite of the wide variability in individuals, significant differences were shown to exist between individuals.
ET
with
normal
2
E. Ziegler,3
Steven
studies
M.D.,
E. Nelson,6
B.S.,
Alejandro Leon
M. O’Donnell,4
F. Burmeister,7
M.D.,
Ph.D.,
Genevieve
and
Samuel
Stearns,5
Ph.D.,
J. Fomon,8
M.D.
ABSTRACT A total of 1 148 nitrogen balance studies were conducted in 100 boys and 23 girls 1 to 1 1 years old. Mixed, customary diets supplied between 8.9 and 21 .4% of calories from protein . Duration of balance studies ranged from 72 hr in the youngest subjects to 1 20 hr in older children . Regressions and 95 % confidence intervals of nitrogen retention on nitrogen intake are presented . The slope of the regression was significantly greater for children 12 to 1 8 months old than for children of other age groups. Correlation coefficients between various parameters of nitrogen balance and energy intake are presented. Although variability in results of nitrogen balances within subjects appears to be rather large , a significant difference between subjects was observed. Am. I. Clin. Nut,’. 30: 939-946, 1977.
Few satisfactory reference data from studies of nitrogen balance with normal children of preschool and school age are available in the literature Moreover, as pointed out by Irwin and Hegsted (1), results of published studies lack agreement. The present report presents data from 1 148 nitrogen balance studies with 1 23 normal children from 1 to 1 1 years of age These studies were conducted between 1930 and 1953 under relatively uniform conditions in the Pediatric Metabolic Unit, University of Iowa, under the direction of one of the authors (G . S.) and Dr. P. C. Jeans. Macy and associates (2) have presented data from a large number of nitrogen balance studies with normal children. These studies involved 33 children, none of whom was less than 4 years old. The range of nitrogen intakes was relatively small compared with that in the present series. Data from about 80% of these studies were reported by Stearns et al. (3) in their discussion of protein requirements of children The present report includes results of further studies by the same investigators and presents regressions and confidence intervals for the entire data. .
.
Iowa, children awaiting adoption, or normal children of mothers being treated for tuberculosis. These children had been living in the Pediatric Metabolic Unit since early infancy. Most of the children over 2 years old came from orphanages in the State of Iowa and lived for at least several months in the Pediatric Metabolic Unit. In analyzing the data, we have elected to exclude from consideration the small percentage of the children who at the time of admission demonstrated height and/ or weight less than the 10th percentile values of widely used reference data (4) . The children were all judged to be adequately nourished. No child lost weight while the studies were being conducted and most children gained weight over the interval of study. Subjects have been divided into the following age groups: 1 2 to 18 months, 18 to 36 months, 3 to 6 years and 6 to 1 1 years. The last age category, for example, included all children who had reached their sixth birthday and had not yet reached their eleventh birthday. The youngest age group was identified on the basis of preliminary analysis of the data which showed that the slope of the regression of nitrogen retention on nitrogen intake changed little after 18 months of age. The other age divisions were arbitrary.
Procedures Those subjects who were children staff of the University of Iowa were
.
Subjects,
and
procedures
In most instances subjects between 1 and 2 years old were children of students or staff of the University of
American
Journal
of
Clinical
Nutrition
30: JUNE
From
the
Department
7578 . ‘ Associate low. Present address: mento
methods
Subjects
The
1
Medicine, University 2 Supported in
de
PediatrIa,
of students permitted
of Pediatrics,
Iowa, Iowa City, part by U.S.P.H.S. of
Professor. Unidad Hospital
or to
go
College
of
Iowa 52242. Grant HD
Postdoctoral
Fel-
Metabdlica, DepartaRam#{243}n Sard#{225},Universi-
dad Nacional de Buenos Aires, Buenos Aires, Argentina . Professor Emeritus. #{176} Research Assistant . Assistant Professor, Department of Preventive Medicine and Environmental Health . Professor. 1977,
pp. 939-946.
Printed
in U.S.A.
939
Downloaded from https://academic.oup.com/ajcn/article-abstract/30/6/939/4650377 by East Carolina University user on 11 March 2019
Ekhard
balance
940
ZIEGLER
ET
home on Sundays; otherwise the children lived in the Pediatric Metabolic Unit continuously for 6 or more months. Children from orphanages were studied no sconer than 4 weeks after admission to the Pediatric Metabolic Unit to permit adjustment to the new living conditions
and
diet.
An
effort
was
made
to provide
a
playground
located
just
outside
the
ward
unit,
so they
could be watched at all times. Toys, books, and games were provided. The difficulty of collecting excreta quantitatively from very young children is evidenced by the fact that few data other than these are available in the literature. Excreta of subjects less than approximately 2 years old were collected with the use of a metabolic bed , similar in design to, but larger than, that subsequently described (5). These children were permitted short periods of activity off the metabolic bed during waking hours. As the children became more dependable in their toilet training, activity periods were lengthened until the metabolic beds were used only for sleeping. Separate collections of urine and feces were rarely attempted with girls less than 3 years old. No attempt was made with any age group to measure dermal losses or losses from external secretions. Fecal collections were made between carmine markers. Urine was collected under toluene. With the youngest subjects, one 72-hr study was usually performed every week or every other week. Occasionally, two consecutive 72-hr studies were performed. As the children became more ambulatory, study periods were frequently lengthened to 96 hr. In a few cases, as many as four consecutive 96-hr studies were carried out, but more commonly one study was performed per week. After a change in diet, an adjustment period of at least 1 0 days was allowed before resumption of studies. Children above approximately 4 years old were studied for periods of 120 hr. Usually, three such studies were performed consecutively. The diet was then altered and a 2-week adjustment period allowed before resumption of studies. Diet
A mixed diet appropriate for age was fed, resembling to a large extent home-prepared American diets. Bananas were generally included during all seasons. These were a great treat to orphanage children and encouraged them to accept the entire diet . Inclusion of other fruits and vegetables varied with the season. Proteins of animal origin accounted for approximately 90% of protein intake, with milk, meat, and eggs serving as the major sources. Changes in protein intake were brought about in younger children by altering chiefly the amount of milk products, but also of meat and eggs. Diets offered to subjects 1 to 3 years old contributed an average of 1 6 .5 % (range: 1 1 .9 to 21 .4%) of calories from protein and diets of subjects 6 to 1 1 years old contributed an average of 13.8% (range: 8.9 to 19.2%) of calories from protein. During adjustment periods subjects consumed as much food as they pleased and records were made of
amounts consumed. During subsequent balance periods, constant amounts of food were offered which slightly exceeded the mean amount consumed during the preceding adjustment period. Very likely this caused variation of fcod intake within a given subject to be less than would otherwise have been the case. In the early years of the study, energy content of the diets offered to the subjects was calculated from unpublished locally assembled tables of food composition and, after 1950, from USDA published tables (6). Duplicates or, in the case of older children, aliquots of all food offered in the course of a balance study were pooled, homogenized and analyzed for nitrogen. In the case of milk, however, each new supply was analyzed only once and the amount of nitrogen offered from this source calculated separately. If any portion of a meal was not eaten, it was analyzed for nitrogen. From the nitrogen offered minus the nitrogen in uneaten portions, intake of nitrogen was calculated. Although uneaten portions of diet were not measured for energy content, food refusals were few and of small amount. Therefore , energy offered is referred to subsequently in this report as “energy intake.” Methods Aliquots of food and complete fecal contents were digested with 20% hydrochloric acid and made up to a convenient volume with water. Nitrogen content of these digests and of urine was determined in duplicate by the Kjeldahl method as described by Hawk and Bergeim (7). Later in the study a selenium catalyst was employed as described by Campbell and Hanna (8). Net absorption of nitrogen was estimated from the quantity consumed minus the amount excreted in feces. Similarly, intake of nitrogen minus amounts excreted in feces and urine yielded apparent net retention of nitrogen. Standard statistical methods, including regression analysis, calculation of correlation coefficients, analysis of variance and of covariance, were used (9). Normality of distribution was assessed by calculating Kolmogorov-Smirnov statistics (1 0). In most instances parameters of nitrogen balance were found not to follow a normal distribution. Therefore, nonparametric correlations using Kendall’s r (1 1) were also computed. However, confidence intervals and standard deviations were retained simply because they describe samples in familiar and thus convenient terms.
Results Among the children in Iowa orphanages at the time of these studies, there were many more males than females. Males were much more often available for study Of the total number of 1 148 metabolic balance studies, 986 were carried out with 100 males and 1 62 were carried out with 23 females (Table 1). Preliminary statistical analysis failed to reveal significant sex-related differences in regressions of nitrogen retention on nitro.
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home-like atmosphere. The older children were taken on walks, trips to the City Park, and shopping trips. Those of school age attended both the hospital school and its craft shop. The young children had their own
AL.
NITROGEN
gen intake Therefore female subjects have
BALANCE
.
,
been
Intake, absorption, and nitrogen (Table 1)
data of pooled.
urinary
STUDIES
male
WITH
and
,
excretion
of
,
of nitrogen
balance
No.
of subjects’
3 years
old,
data
on absorption
No.
of studies
excretion
Nitrogen
.
Nitrogen
NiO
in-
Fecal
en retention
12-l8months
thesis.
urinary
M, male;
excretions
TABLE Relations
2 among
F, female.
115 71d
235 199” 302 496 in more (I
Subset
581” (113) 529 55 (137) (24) 546 (84) 543 63 (81) (21) 507 51 (100) (15) 447 44 (84) (15) than one age group.
Energy
Urinaiy
mg/kg/day
21 (12M, 9F)C 13” (12M, iF) 18-36 months 20 (14M, 6F) 19” (14M, SF) 3-.6years 32 (30M, 2F) 63 6.-il years (54M, 9F) a Several subjects were included
and
studies .
Age
than
% ofirnake
364 (63)
b
390 (68) 374 (82) 339 (63) Mean;
of first line in this age group,
149 23.7 (105) (12.2) 19.4 110 (81) (10.6) 95 17.0 (51) (8.4) 90 16.2 (48) (8.0) 82 15.8 (43) (7.8) 64 13.7 (50) (9.7) standard deviation given
representing
studies
in which
kcal/kg/day
100 (18) 93 (14) 91 (14) 91 (14) 90 (12) 81 (14) in parenfecal and
were measured. parameters
of nitrogen
balance
and energy
intake Age
Parameter,’
Correlation N intake N absorption
12-18
18-36
3-6
6-11
months
months
years
years
vs energy intake vs N intake
0.804 0.979
0.561 0.966
0.266 0.990
0.672 0.985
vs N intake vs N intake
0.628 0.839
0.764
0.882
0.780
vs N intake vs N absorption
0.661 0.805
0.554 0.311 0.541
0.524 0.231 0.525
0.581 0.374 0.606 0.503 0.891
coefficient”
Urinary N excretion N retention % N retention N retention
Kendall’s N intake
.
Mb
vs energy
intake
0.617
0.443
0.246
N absorption
vs N intake
0.839
0.850
0.897
Urinary
vs N intake vs N intake
0.427 0.61 1
vs N intake vs N absorption
0.447 0.536
0.579 0.379 0.194
0.670 0.364 0.130 0.375
N excretion
N retention % N retention N retention a
Expressed
significant
per kilogram
(P < 0.001).
of body
weight
except
where
indicated
0.369
as percent
of intake.
“
0.57 1 0.405 0.259 0.425 All values are highly
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less
urinary excretion of nitrogen in the age groups 1 2 to 1 8 months and 1 8 to 36 months were, with few exceptions, available for male subjects only. Nevertheless, fecal and urinary excretion could be determined
,
1 of results
941
CHILDREN
not vary independently and were, as mdicated in Table 2 highly correlated in each age group. In this case, as in the case of all other correlations listed in Table 2 Kendall’s i- reached the same high level of significance (P < 0.001) as the correlation coefficient. Because urine and feces were generally not collected separately in studies of girls
Mean nitrogen intake per kilogram of body weight was 581 mg/day in the age group 1 2 to 1 8 months and decreased progressively in subsequent age groups. As already mentioned, protein generally accounted for 10 to 20% of energy intake. With the same diet, a child who consumed a greater quantity of food achieved a greater intake both of energy and protein. Therefore nitrogen intake and energy intake did TABLE Summary
NORMAL
942
ZIEGLER
ET
AL.
4
12
I NTAKE
U
URINE z
..
w
./
06
4
RETENTION 2
#..-
.
--.-...
._______S___
.._.. -
FECES I
2
3
4
5 AGE
FIG. 1 . Summary of nitrogen balance studies months and 1 year are used in this graph only.
6
7
8
9
tO
II
(years)
in children
from
age 1 to 1 1 years.
Note
that age intervals
,
of 6
‘Ps
E z
0 Iz Ui
00
IUi
300
600
INTAKE
00
0o
(mg/kg)
2. Daily nitrogen retention in relation to daily nitrogen intake in 21 children 12 to 18 months old. Each the result of one balance study. Y = 0.635X 219.6 (Y = retention of nitrogen, and X = intake of nitrogen, both in mg/kg/day). N = 1 15 . Solid lines indicate regression line and 95% confidence intervals. Points off the scale are indicated by arrows. FIG.
dot
represents
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I0
NITROGEN
BALANCE
STUDIES
,
,
.
,
.
,
Energy
intake
As already tween quantity
mentioned, oL food
the difference offered and that
becon-
NORMAL
CHILDREN
943
sumed was probably small and may thus be ignored Mean energy intake (estimated from diet offered) averaged 100 kcal/kg of body weight at age 1 2 to 1 8 months and decreased progressively to 81 kcal/kg at age 6 to 1 1 years (Table 1). Since no attempt was made at controlling energy intakes at predetermined levels, the observed energy intakes represent essentially ad libitum intakes. .
Retention
of nitrogen
Mean retention of nitrogen per kilogram of body weight was greatest in the age group 12 to 18 months (149 mg/kg/day) and decreased progressively at older ages Similarly, retention of nitrogen, expressed as percentage of intake decreased from 23 .7 % at age 1 2 to 1 8 months, to 1 3 .7 % at age 6 to 1 1 years (Table 1). The relationship between intake and retention of nitrogen in the various age groups is illustrated in Figures 2 to 5 . As indicated in Table 2, correlation coefficients and Kendali’s T were highly significant in all age groups. Analysis of covariance in conjunction with t-tests revealed that the slope of the regression for the age group 1 2 to 18 months (Fig. 2) was significantly greater (P < 0.05) than the slopes for the three other age groups (Figs. 3 to 5); slopes of the latter .
,
300
200
E z
0 I-
00
z
Ui I-. Ui
0
200
FIG.
3. Daily
dot represents
the
nitrogen result
300
retention in relation of one balance study.
400
500
INTAKE to daily nitrogen Y 0.330X -
600
JO0
00
(mg/kg) intake in 20 children 18 to 36 months 84.4 (see legend to Fig. 1). N = 235.
old. Each
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in 71 and 199 balance studies, respectively, in the two age groups. Net absorption of nitrogen was highly correlated with intake of nitrogen at all ages (Table 2). Regardless of age fecal excretion of nitrogen averaged 10 to 1 2 % and, therefore net absorption of nitrogen amounted to 88 to 90% of intake (Table 1) Mean urinary excretion, expressed as percent of intake increased slightly from 69 % at age 1 2 to 1 8 months to 76 % at age 6 to 1 1 years. In each age group, urinary nitrogen excretion, expressed in milligrams per kilogram of body weight, was highly correlated with nitrogen intake expressed in the same terms (Table 2). A summary of results of nitrogen balance studies is presented in Figure 1 In this figure (but not in the other figures or tables) age intervals of 6 months during the first 3 years of life and intervals of 1 year for older children have been used. Expressed in grams per 24 hr (rather than grams per kilogram of body weight), urinary nitrogen excretion increased as intake rose with advancing age whereas fecal excretion and retention of nitrogen showed little change.
WITH
944
ZIEGLER
ET
400
500
AL.
300
.*
z
0
I.z Ui
100
IUi
0
00 200
300
INTAKE FIG. 4. Daily nitrogen retention represents the result of one balance
in relation study. Y
=
to daily nitrogen 0.228X 33.8
600
700
800
(mg/kg)
intake (see
in 32 children legend
to Fig.
3 to #{246} years 1).
N
=
old.
Each
dot
302.
400
300
.
E 200
z
0
I-. z
00
Ui Ui
0
-100 200
300
500
400
INTAKE FIG. 5 . Daily nitrogen retention represents the result of one balance scale is indicated by arrow.
in relation study. Y
=
to daily 0.341X
did not differ significantly from one another. To facilitate comparison, Figure 6 includes the regression of apparent nitrogen
700
600
800
(mg/kg)
nitrogen intake in 63 children 6 to 1 1 years old. Each dot 88.1 (see legend to Fig. 1). N 496. One point off the
retention on nitrogen intake for infants 90 to 1 20 days of age receiving similar intakes of nitrogen - i .e more than 1 1 % of cabries from protein (1 1). As may be seen, the . ,
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E
NITROGEN
STUDIES
BALANCE
WITh
NORMAL
945
CHILDREN
400
90-120
days
2 E
200
6-Ilysors
18-36
z
months
0 Iz Ui
IUi
3-6
ysors
00
0
-100
400
FIG. Included calories (15).
6. Daily also
from
800
600
INTAKE
00
(mg/kg)
nitrogen
is regression
protein
,
retention in relation to daily nitrogen intake in the various age groups (solid lines). line for infants 90 to 1 20 days of age (interrupted line) receiving more than 1 1 % of from Fomon et al . (1 2) based on data of Nelson (1 3), Jeans and Stearns (14), and et al
regression of apparent nitrogen nitrogen intake closely parallels 1 2- to 1 8-month-old children.
retention on that for the
Reproducibility subjects
between
to 0 .274
the and
differences
The 95 % confidence limits of the regressions of retention of nitrogen on intake of nitrogen (Figs. 2 to 5) and the standard deviations listed in Table 1 provide some measure of reproducibility with respect to the total number of studies. Reproducibility of nitrogen balance was further estimated by calculating intraclass (intrasubject) correlation coefficients. Since nitrogen retention was strongly correlated with nitrogen intake analysis of covariance was utilized to correct nitrogen retention for nitrogen intake Intraclass correlation coefficients of nitrogen retention corrected for nitrogen intake ranged from 0 1 36 (1 2 to 18 months) to 0.275 (6 to 1 1 years) when retention was expressed as milligrams per kilograms and from 0 1 1 5 (1 2 to 1 8 months) ,
.
.
.
(6 to 1 1 years)
when
expressed as percent of Thus, variability in results same
subject
appears
retention
was
nitrogen intake. of studies with to be
rather
large.
On the other hand, the F-test for betweensubject variation was significant for age 12 to 18 months (P < 0.03) and for all other ages (P < 0.001). Thus, nitrogen retention corrected for nitrogen intake differs signifi.cantly between subjects. Comment The present series of 1 148 nitrogen balance studies probably represents the largest available body of such data concerning normal children from 1 through 3 years old and one of the largest bodies of data concerning children from 4 through 1 1 years old. Although the majority of the data have already been summarized (3), we believed the usefulness of the data would be enhanced by a more detailed description of the studies and by further statistical analyses. Irwin and Hegsted (1) have reviewed the
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300
ZIEGLER
946
.
,
References 1 . IRwIN, M . I . , AND D . M . HEGSTED. of research on protein requirements Nuts. 101: 385, 1971.
A conspectus of man. J.
AL.
2. MACY, I. G. Nutrition and Chemical Growth in Childhood. Vol II. Original data. Springfield, illinois: Charles C Thomas, 1946. 3. Smzns, G., K. J. NEWMAN, J. B. McKnz AND P. C. Jws. The protein requirements of children from one to ten years of age. Ann. N.Y. Acad. Sci. 69: 857, 1958. 4. VAUGHAN, V. C., III, AND R. J. MCKAY. Nelson Textbook of Pediatrics (10th ed.). Philadelphia: w. B. Saunders Co., 1975, pp. 40-43. 5. F0M0N, S. J., L. N. THOMAS, R. L. JENSEN AND C. D. MAY. Determination of nitrogen balance of infants less than 6 months of age . Pediatrics 22: 94, 1958 6. WAn, B. K., AND A. L. MERRILL. Composition of Foods Raw , Processed, Prepared . Washington, D.C.: USDA Agriculture Handbook No. 8. Agri-
-
cultural HAWK,
Research
Administration,
1950.
P. B., AND 0. BERGEIM. Practical Physiological Chemistry (10th ed.). Philadelphia: Blakiston, 1931, p. 814. 8. CAMPBELL, W. R., AND M. I. HANNA. The determination of nitrogen by modified Kjeldahl methods. J. Biol. Chem. 119: 1, 1937. 9. SNEDECOR, G. W., AND W. G. CocHaN. Statistical Methods. 6th edition. Ames, Iowa: Iowa State University Press, 1967. 10. Osri., B., AND R. W. MENSING. Statistics in Research (3rd ed.). Ames, Iowa: Iowa State Uni7.
Press, 1975, p. 352. W. J. Practical Nonparametric StatisYork: John Wiley & Sons, 1971. 12. F0M0N, S. J., A. M. O’DONNELL AND E. E. ZizGLER. Bilancio azotato nei primi 3 anni di eta. In: Metabolismo Proteico in Infanzia, edited by E. Schwarz-Tiene. Milan: Minerva Medica. In press. versity
1 1 . CoNovna, tics. New
13.
M. V. K. The growth and nitrogen metabolism of infants receiving undiluted milk. Am. J. Diseases Children 39: 701, 1930. 14. Jr.s, P. C., AND G. Smi.asis. Growth and retentions of calcium, phosphorus and nitrogen of infants fed evaporated milk. Am. J. Diseases Children 46: 69, 1933. 15. JEANS, P. C., G. Sm.aris, J. B. MCKINLEY, E. A. GoFi’ AND D. STINGER. Factors possibly influencing the retention of calcium, phosphorus, and nitrogen by infants given whole milk feedings. I. The curding agent. J. Pediat. 8: 403, 1936. 16. Joint FAO/WHO Ad Hoc Expert Committee: Energy and Protein Requirements. WHO Technical Report Series No. 522. Geneva: World Health Organization, 1973. 17. YOUNG, V. R., AND N. S. Scamisnw. Human protein and amino acid metabolism and requirements in relation to protein quality. In: Evaluation of Protein for Humans, edited by C. E. Bodwell. Westport, Connecticut: Avi Publishing Company, 1977. NELSON,
Downloaded from https://academic.oup.com/ajcn/article-abstract/30/6/939/4650377 by East Carolina University user on 11 March 2019
voluminous literature on the subject of nitrogen balance studies in children The lack of agreement among investigators noted by Irwin and Hegsted (1) prompted us to prepare the present summary of studies which, although carried out some time ago, were conducted under standardized conditions in a uniform manner. It was our intention to establish relationships between intake and retention of nitrogen in normal children of various ages receiving protein intakes similar to those commonly consumed by children in the United States. Extremely high or low protein intakes were not included and thus even the lowest protein intakes used about 1 2 % of calories from protein in children less than 3 years old and about 9% in older subjectswere in excess of presumed requirements (16). Statistical evaluation was complicated by the fact that in the majority of instances parameters of nitrogen balance did not follow normal distributions. Relationships between these parameters were therefore examined by Kendall’s r, a nonparametric technic. At least for those relationships summarized in Table 2 these calculations yielded correlations which were as significant as the conventional correlation coefficients. Although it is well recognized that nitrogen retention is influenced by the availability of dietary energy (17), the present studies, in which energy and protein intakes were highly correlated (Table 2), were not designed to provide additional insight into the nature of the relationship. Our results suggest that the predictability of results of nitrogen balance studies, even with the same subject, is rather low. In spite of the wide variability in individuals, significant differences were shown to exist between individuals.
ET
