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D.A. Navarrete, R. Bressani, and R. Siberián
Division of Food and Agricultural Chemistry Institute of Nutrition of Central America and Panama (INCAP) Guatemala City, Guatemala
1. To determine the effect of energy intake on nitrogen intake for nitrogen equilibrium.
2. To determine the nitrogen needs for nitrogen equilibrium in three rural diets of different protein quality.
A total of 24 healthy young adult male subjects were used in two studies. In study 1, 18 subjects were involved, while 10 subjects participated in study 2. All subjects performed their usual chores. Their characteristics are described in table 1.
The subjects lived in their homes in Guatemala City and worked at INCAP. All of their meals were eaten in the Metabolic Unit of the Division of Food and Agricultural Chemistry. The daily ambient temperature ranged from 21° to 25° C. Relative humidity ranged from 72 to 85 per cent. Guatemala City is 1,510 m above sea-level.
Based on actual dietary intakes of rural adult Guatemalan populations, a series of basal diets were formulated for each level of protein intake, as shown in table 2. These diets provided from 2,217 kcal/day when protein intake was practically 0 kg/day to 720 kcal/day when protein intake was equivalent to 0.7 9 P/kg/day. Calculations of actual dietary intakes in rural areas showed caloric intake to be equivalent to 36 kcal/kg/day. Therefore, the protein intake and the differences in energy intake were made up from diets based on lime-treated common corn, lime-treated opaque-2 corn, beans, and poultry meat, as shown in table 3. It was necessary to feed a few individuals who had a higher weight additional calories. In study 2, the level of calories was 45 kcal/kg/day and only the commoncorn diet was assayed.
TABLE 1. Physical Characteristics of the Subjects (36 kcal/kg/day)
Protein Quality Assay. Short-term Nitrogen Balance Method
After a four-day adaptation period with a high-quality diet fed at 0.6 9 protein/kg/day and 36 kcal/kg/day, the subjects were fed a low-nitrogen diet for three days. The low-nitrogen diet is that shown in table 2, but all protein sources were replaced with cassava. During the last two days, quantitative faecal and urine collections were made. The low-N diet was followed by feeding the experimental diets in study 1 to provide 0.2, 0.4, 0.6, and 0.7 9 P/kg/day. In study 2 the highest protein level fed was 0.6 9 P/kg/day. These levels of protein were given for two days each, with quantitative faecal and urine collections every 48 and 24 hours, respectively.
TABLE 2. Food Intake. Basal Diets (g/day)
|Artificial flavoured drink (glasses)||3||4||3||2|
|Analysed nitrogen (g/day)||1.575||1.866||1.639||1.730||1.603|
a. Made with corn starch. herbs. margarine
b Adjusted to 36 kcal/subject with the experimental diets (table 3) and with N free food items (candies. soft drinks)
TABLE 3. Average Food Intake (g/day). Protein Sources
|Common corn plus chicken||Tortilla||103.8||206.9||311.4||363.3|
a Served fried with vegetable oil and salt. added before trying b Boiled chicken deboned and chopped
TABLE 4. Summary of Nitrogen Retention at Various Levels of Nitrogen Intake of Different Dietary Treatments
|Energy Intake (kcal/kg/day)||Subjects||Average N Intake (mg/kg/day)|
|Common corn||36||6||26.5 ± 2 4a||64.1 ± 3.8||93.0 ± 3.2||127.1 ± 3.4||141.3 ± 3.2|
|- 50.3 ± 9.4a||- 2.8 ± 6.1||3.6 ± 11.1||19.0 ± 18.7||28.8 ± 8.0|
|Opaque-2 corn||36||6||26.5 ± 2.1||64.4 ± 03.7||93.4 ± 3.5||127.5 ± 3.5||141.3 ± 3.0|
|- 58.7 ± 12.4||- 13.7 ± 12.5||0.1 ± 10.9||17.7 ± 20.6||29.4 ± 27.2|
|Common corn||36||6||27.7 ± 1.6||64.8 ± 4.5||93.7 ± 4.1||128.5 ± 4.7||143.2 ± 5.2|
|plus chicken||- 47.2 ± 14.0||- 5.6 ± 15.2||6.1 ± 16.3||21.4 ± 20.0||23.0 ± 22.5|
|Common corn||45||6||22.8 ± 1.8||47.0 ± 0.9||88.6 ± 1.4||127.6 ± 1.4||-|
|- 74.3 ± 14.1||- 44.6 ± 11.3||9.8 ± 7.9||14.8 ± 20.1||-|
a. Standard deviation
TABLE 5. Regressions between Nitrogen Intake
and Nitrogen Retained of Rural Diets made with Common Corn
Opaque-2 Corn and Common Corn with Chicken
|Rural Diet||Calorie Intake (kcal/kg/day)||Number of Subjects||Number of Observations||Regression Equations||r2 (%)|
|Common corn||36||6||30||NR = - 58.07 + 0.64 Ni|
|P (B) <= 0.001||70.9|
|Opaque-2 corn||36||6||30||NR = - 70.54 + 0.72 Ni|
|P (B) <= 0.001||70.1|
|Common corn and opaque-2 corn||NR = - 64.29 + 0.68 Ni|
|36||12||60||P (B) <= 0.001||73.0|
|Common corn plus chicken||NR = - 79.84 + 1.38 Ni - 0.0047 Ni2|
|36||6||30||P (B) <= 0.003 P (B) <= 0.05||70.4|
|Common corn||45||6||24||NR = - 123.30 + 2.23 Ni - 0.0089 Ni˛|
|P (B) <= 0.000091 P(B) <= 0.003||87.1|
TABLE 6. Regressions between Nitrogen Retained
and Weight Losses of Subjects Fed Diets Made with Common Corn.
Opaque-2 Corn and Common Corn with Chicken at 36 kcal/kg/day
|Common corn||High||3||15||NR = - 61.550339 + 0.67957 Ni|
|P (B) <= 0.00044||65.6|
|Low||3||15||NR = - 84.354209 + 1.544372 Ni - 0.005529 Ni2|
|P (B) <= 0.001945 P (B2) <= 0.027418||86.6|
|Opaque-2 corn||High||3||15||NR = - 89.596578 + 1.458469 Ni - 0.003984 Ni˛|
|P (B) <= 0.000921 P (B2) <= 0.051018||93.1|
|Low||2||10||NR = - 59.838841 + 0.504027 Ni|
|P (B) <= 0.00316||69.2|
|High||3||14||NR = - 108.646796 + 2.379523 Ni - 0.011026 Ni˛|
|P (B) <= 0.001811 P (B2) <= 0.007086||75.6|
|Low||2||10||NR = - 60.158831 + 0.664204 Ni|
|P (B) <= 0.00027||86.0|
TABLE 7. Regressions between Nitrogen Retained and Weight Losses of Subjects Fed Diets Made with Common Corn at Two Different Calorie Intakes
|Gain||2||8||NR = - 126 80957 + 2.581358 Ni - 0.011596 N i˛|
|P (B) <= 0.001391 P (B2) <= 0.004855||97.2|
|Loss||2||8||NR = - 162.147021 + 3.197631 Ni0.014768 Ni2|
|P (B) <= 0.013716 P (B2) <= 0 041228||89.6|
|3||15||NR = - 61.660339 + 0.67957 Ni|
|P (B) <= 0.0004||65.6|
|Low||3||15||NR = - 84.354209 + 1 544372 Ni - 0 005529 Ni˛|
|P(B) <= 0.001945 P (B2) <= 0.027418||86.6|
At the end of each experiment, the samples of food, faeces, and urine were analysed for nitrogen. The results were analysed statistically by linear regression to estimate protein needs for nitrogen equilibrium and estimation of protein requirement.
Summary of Main Results
Table 4 summarizes the average nitrogen balance in diets fed at 36 kcal/kg/day and the common corn/bean diet at 45 kcal/kg/day.
The statistical analysis carried out for the three diets made with common corn, opaque-2 corn, and common corn plus chicken at an intake of 36 kcal/kg/day indicated no difference between the opaque-2corn and common-corn diets, but the common corn plus chicken was different from the other two because it showed a significant quadratic component, as indicated in table 5. As calorie intake was low, all subjects lost weight. The diet with chicken at 0.7 9 P/kg/day was no longer giving a linear response, while the other two at this level did show a linear response, suggesting that protein was being used as a calorie source.
Statistical analyses were made between the weight changes of individuals, and the relationship between nitrogen intake and nitrogen retention for each dietary treatment. These are shown in tables 6 and 7.
Within each dietary treatment, the individuals were divided into two groups: those who lost or gained more weight and those who gained or lost less weight. At a calorie intake of 36 kcal/kg/day all individuals with all diets lost weight; however, those eating common corn with a higher weight loss showed a linear regression, while those with a lower weight loss showed a significant quadratic component. The opposite was true according to the statistical analysis for subjects with greater weight loss as compared with those with lower weight loss on diets of higher protein quality, that is, that of opaque-2 corn and that with common corn plus chicken. With respect to the common-corn diet fed at 45 kcal/kg/day, some subjects gained weight while some lost weight. In this case, with "adequate calories" both groups showed a significant quadratic component.
The interpretation of the regression equations suggests different methods of adaptation to the experimental conditions imposed with respect to calorie intake and its interaction with protein quantity and quality in each diet.
A.A. Jackson, M.H.N. Golden, R. Byfield, F. Jahoor, J. Royes, L. Soutter
Tropical Metabolism Research Unit. University of the West Indies, Mona, Kingston 7. Jamaica
The study was designed to measure nitrogen balance and whole body nitrogen kinetics in subjects consuming intakes of protein and energy at or around the safe level.
Nine male children were studied in all. Seven came from a local orphanage and two had recently recovered from protein-energy malnutrition in the metabolic ward. Two children had been given intensive therapy for malnutrition in the past, but had been on a normal diet for at least four weeks. Each child was screened for anaemia and infection and had received appropriate therapy.
All studies were carried out in the metabolic ward of the Tropical Metabolism Research Unit at ambient temperature and humidity.
There was no restriction on activity between balances. Each day the child had a play activity session in the morning. A special nanny was used to attend to the personal needs of the child. During the study there was minimal restriction of activity, as the method of Golden et al. (11 was used to ensure near-normal activity levels.
Duration of the Study
The complete study time was four periods of seven days, a total of 28 days.
Five children were given a diet of 1.7 9 milk protein/kg/day with energy intakes of 80, 90, or 100 kcal/kg/day. Two children received this diet at the level of 100 kcal/kg/day only. Two children received a diet containing 0.7 9 milk protein/kg/day with varying energy intakes of 80, 90, or 100 kcal/kg/day. Each child was first maintained for one week on the diet containing 100 kcal/kg/day.
Over the next three weeks the child received the three diets for seven days each. The order of diets was varied according to a Latin square design. The diets were made up by weight, with added vitamin and mineral supplements. Over days 5, 6, and 7 of each diet period a tracer dose of 15N-glycine was added to the feed, and a balance study was conducted.
Total nitrogen was measured in the food, stool, and urine by the micro-Kjeldahl method. The energy content of the diet and stool was measured by ballistic bomb calorimetry (2). Protein turnover was calculated by the method of Picou and Taylor-Roberts (3) and Waterlow et al. (4) from the plateau enrichment of ammonia in urine, the samples being prepared as previously described (5).
The subjects were between 7 and 22 months old. Their heights were between 58 and 81 cm, or 85 to 99 per cent of that expected for their age. The weights ranged from 5 to 11.6 kg: 57 to 100 per cent of the weight expected for age, or 78 to 104 per cent of the expected weight-for-height (table 1).
The results from children recently recovered from malnutrition were not different from those of the other children.
TABLE 1. Clinical Characteristics of the Patients in the Study
(kg ± SD)
|D.H A||9||69 5||8.24 ± 0.02||98||91||96||100||1.7||Steady|
|B||10||70||881 ± 0.03||97||94||101||90||Steady|
|C||70||9.04 ± 0.04||97||96||104||100||Growing|
|D||71||9.27 ± 0.05||98||99||103||80||Growing|
|S.B. A||18||81||11.09 ± 0.06||99||97||99||80||Steady|
|B||11.25 ± 0.04||99||98||100||90||Growing|
|C||19||11.63 ± 0.06||100||103||100||Growing|
|N.J. A||12||70||7.52 ± 0.01||93||75||86||100||Steady|
|B||7.39 ± 0.02||73||85||80||Losing|
|C||7.32 ± 0.03||73||84||100||Steady|
|D||7.40 ± 0.02||73||85||90||Steady|
|J.B. A||9||70||6.72 ± 0.03||98||74||77||90||Steady|
|B||6.74 ± 0.01||74||77||80||Steady|
|C||6.86 ± 0.06||76||79||100||Growing|
|K.S. A||7||58||4.96 ± 0.04||85||62||97||100||Losing|
|G.J. A||9||67||7.26 ± 0.03||99||80||94||100||Steady|
|R.K. A||18||77||9.14 ± 0.02||94||80||88||100||Steady|
|B||9.05 ± 0.02||79||87||80||Steady|
|C||9.06 ± 0.04||79||87||90||Steady|
|A.W. A||22||66.5||7.03 ± 0.02||78||58||92||90||0.7||Steady|
|B||6.90 ± 0.01||57||91||80||Losing|
|C||6.99 ± 0.02||57||92||100||Gaining|
|S.W. A||11||71.5||7.21 ± 0.01||97||74||79||100||Steady|
|B||7.14 ± 0.02||73||78||80||Steady|
|C||7.15 ± 0.02||73||78||90||Steady|
a Expected height for age
b Expected weight for age
c Expected weight for height
In all balances it was assumed that unmeasured losses of nitrogen were 5 mg/kg/day, and this correction was made for all retentions. Only one diet was unable to maintain nitrogen balance, 0.7 9 protein and 80 kcal/kg/day (table 2). Three patterns of nitrogen retention emerged.
1. On 0.7 9 protein/kg/day: positive balance of 10 mg N/kg/day was maintained at an intake of 100 and 90 kcal/kg/day, but both subjects were in negative balance, -28 mg N/kg/day, on 80 kcal/kg/day.
TABLE 2. Energy Intakes, Nitrogen Balance and Whole Body Nitrogen Flux (Values are mean + SD)
|No.||Energy (kcal/kg/day)||Nitrogen (mg/kg/d)|
|7||108||6 ± 2||102 ± 2||288||36 ± 14||149 ± 34||98 ± 30||920 ± 180|
|(n = 5)|
|5||98||7 ± 3||91 ± 3||288||41 ± 09||170 ± 29||72 ± 36||960 ± 230|
|5||87||6 ± 1||82 ± 1||288||33 ± 11||188 ± 20||64 ± 15||1,175 ± 230|
|(n = 4)|
a. Includes an allowance of 5 mg/kg/d for
2. On 1.7 9 protein/kg/day: the five children showed two distinctive patterns. In three, the balance became progressively less positive as the energy intake fell: 116 mg N/kg/day at 100, 97 at 90, and 66 mg N/kg/day at 80 kcal/kg/day. The positive nitrogen balance of the other two children was 65 mg N/kg/day at 100 and fell to 34 mg N/kg/day at 90 kcal/kg/day. However, further reduction of the energy intake led to improved nitrogen balance of 57 mg N/kg/day.
The latter two children appeared to utilize energy less efficiently as they did not gain weight at any level of energy intake, and one lost weight on 80 kcal/kg/day. The other three maintained weight at 80 kcal/kg/day and started to gain at the higher energy intakes.
The energy content of the individual components of the formula diet was measured by bomb calorimetry. Table 2 gives the dietary energy intake, stool energy losses, and absorbed energy on the six diets. Stool energy was 6 to 7 per cent of the dietary intake.
There is a marked difference in flux between the two levels of protein intake. On a dietary intake of 1.7 9 protein/kg/day, flux is about twice that on an intake of 0.7 9 protein/kg/day. For each level of dietary protein there is greater inter-individual variation on the same diet than intra-individual variation for different diets.
The two subjects on 0.7 9 protein/kg/day showed an increased flux, on average, as the energy intake fell (470, 490, 530 mg N/kg/day). The subjects on 1.7 9 protein/kg/day showed a slight increase, on average, between 100 and 90 kcal/kg/day, and a much more marked increase as energy was reduced further to 80 kcal/kg/day (920, 960, 1,175 mg N/kg/day). None of these differences is statistically significant because of the small numbers in each group and the wide individual variations.
All the children had mild intercurrent infections initially, requiring therapeutic intervention. This was almost invariably associated with some weight loss on the 100 kcal/kg/day.
The diets were ingested without any difficulties. There were occasional refusals of small amounts. In the later studies, the total refusal for each 24-hour period was re-offered, and taken, at the end of each day. The diets were made up by weight on a Mettler top-loading balance, and the volume of feed ingested calculated as the difference between the weight of the cup before and after the feed, weighed on an electronic balance. During the study period the dietary intake was known exactly, as the constant infusion pump delivering the feed was carefully calibrated, and the vessel containing the feed weighed at the start and end of each delivery period.
Urine collection was almost complete in most studies. Those periods in which significant losses occurred have been excluded from the calculations. Therefore, the errors are much less than 5 per cent. In general, the stool collection was complete. Small losses did occur on occasion, but were unlikely to have exceeded 5 per cent of the total.
It is clear that the energy intake required for weight maintenance lies between 80 and 100 kcal/kg/day for all the children, whether they were taking 0.7 or 1.7 9 protein/kg/day. However, this is not a single value for energy intake either for the group as a whole, or for any individual child. Some children adapted over the entire range, and maintained a steady weight. Others gained at the higher energy intake, while some lost weight at the lower energy intake. In this study the children taking 1.7 9 protein/kg/day can be differentiated into two groups, one having a lower requirement for energy for maintenance and the other a somewhat higher requirement. This difference is reflected in the nitrogen balance and nitrogen kinetic data.
This difference in maintenance requirement could not be accounted for by the degree of stunting, wasting, chronological age, or antecedent infections, nor the elapse of time since recovery from PEM.
As all other nutrients had been provided in adequate amounts, it was presumed that energy and protein would be the first limiting nutrients. Possible explanations are:
1. Some children had total body deficits of specific nutrients that had not been repleted fully at the time of the study.
2. The inter-individual variation is real, representing metabolic diversity in, for example, the pattern of proteins being synthesized.
3. The metabolic requirements in relation to the dietary energy intake varied.
4. Variable psychosomatic responses to clinical investigation.
Urinary nitrogen excretion followed the expected pattern. The highest value was found in a child with fever. It is of interest that stool nitrogen fell to very low values in this child. The methods for collecting stools makes it difficult to pick up a clear pattern; however, stool nitrogen did vary in this study in relation to diet. Low nitrogen intake is associated with a low faecal nitrogen. When the contribution of faecal nitrogen to total nitrogen excretion is considered, then it has an important determinant effect on the direction in which nitrogen balance changes as energy intake varies. Hence, in terms of total nitrogen excretion or nitrogen retention, three clear patterns emerge:
1. On 0.7 9 protein/kg/day, nitrogen balance is maintained at equilibrium on 100 and 90 kcal/kg/day but becomes negative on 80 kcal/kg/day.
2. On 1.7 9 protein/kg/day nitrogen balance is positive at 116 mg N/kg/day on 100 kcal/kg/day; 97 mg N on 90 kcal/day, and 66 mg N on 80 kcal/kg/day.
3. On 1.7 9 protein/kg/day nitrogen balance is positive at about 65 mg N/kg/day on 100 kcal/kg/day, falling to 34 mg N on 90 kcal, but increasing to 57 mg N on 80 kcal.
In group 1, nitrogen flux was about half that in groups 2 and 3. About 80 per cent of the total was due to synthesis and breakdown, both of which were reduced relative to groups 2 and 3.
In group 2, the decreased nitrogen balance between 100 and 90 kcal/kg/day was brought about with very little change in nitrogen flux. The further reduction in nitrogen balance at 80 kcal/kg/day was associated with an increased flux. Breakdown and synthesis both increased, with the increase in breakdown greater.
In group 3, the changes in flux at 100, 90, and 80 kcal/kg/day were less marked, but there was a tendency towards an inverse relation. There was a greater relative increase in synthesis between 90 and 80 kcal/kg/day, which was associated with an improved nitrogen balance.
The pattern of excretion of 3-methyl-histidine and creatinine showed no consistent change. Therefore it is not possible to attribute the changes in turnover to a change in any particular tissue.
These results indicate the extent to which the body endeavours to maintain nitrogen balance. The exact mechanism invoked depends upon the particular circumstance of the individual in relation to his environment.
1. M.H.N. Golden, B.E. Golden, and A.A. Jackson, "Method of Trace Metal Balance Studies in Young Children." in J. McC. Howell. J.M Gawthorne. and C.L. White, eds., Trace Element Metabolism in Man (Australian Academy of Science, Canberra. 1981), vol. 4, pp. 69-72.
2. D.S. Miller and P.R. Payne, "A Ballistic Bomb Calorimeter," Brit. J. Nutr. 13 501-508.
3. D. Picou and T. Taylor-Roberts, "The Measurement of Total Protein Synthesis and Catabolism and Nitrogen Turnover in Infants in Different Nutritional Status and Receiving Different Amounts of Dietary Protein," Clin. Sci., 36: 283-296.
4. J.C. Waterlow, M.H.N. Golden and P.J. Garlick, "Protein Turnover in Man Measured with 15N: Comparison of End Products and Dose Regimes," Amer. J. Physiol, 235: E165-174.
5. A.A. Jackson, M.H.N. Golden, P. Jahoor and J. Landman, "The isolation of Urea Nitrogen and Ammonia Nitrogen from Biological Samples for Mass Spectrometry." Analyt Biochem 105: 14-17.
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