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6.1. Nitrogen Balance
In most studies nitrogen balance will be the main indicator. In children each treatment should last for seven days, divided into four days for adaptation to the new level of intake and three days for measurement of N balance. Stools should be collected between markers and may be pooled over these three days. In adults each treatment should last a minimum of eight to ten days. preceded by one day on a nitrogen-free diet; five days should be allowed for adaptation to the test level and three to five days for urine collection. Faecal collections should be pooled for at least the last six days of each test period, because of the greater difficulty of obtaining accurate estimates of faecal output in adults than in children. Procedures for the conduct of nitrogen balance studies in adults have been described in detail (33).
No special methods were recommended for determining nitrogen losses through the skin and other minor routes. Skin losses may, however, be important when studies are being made in tropical climates, particularly if the subjects are fairly active physically.
Nitrogen measurements should be made in duplicate.
The energy content of the diets and stools should be measured in duplicate by bomb calorimetry. If this method is not available. the dry weight and fat content of diet and stools should be determined.
Wherever possible quality control should be maintained by exchange of specimens with a reference laboratory.
6.2. Growth and Body Composition
In infants and young children reasonable estimates of the rate of weight gain can be obtained from the change over a period as short as one week, provided that the weight is measured under carefully standardized conditions. Rates of gain should be compared with those expected for a normal child of the same biological age, i.e. the same height.
In adults it will take a much longer time to observe a significant change in weight. Body weight should, however, be measured at frequent intervals during the study, in order to assess the size of change that will be statistically significant.
Measurements of skinfold thickness are too insensitive and inaccurate to be of much value in short-term studies, except for assessing the initial nutritional state.
Creatinine output is still considered a useful measure of muscle mass, as well as a check on the completeness of urine collections. The duration of the collection period (usually about 24 hours) must be accurately timed. Creatinine excretion has been found to be erratic if the daily urine volume is very low (less than 150 ml in pre-school children). Children also excrete considerable amounts of creatine, which is converted to creatinine at acid pH, even at ambient temperatures. Urine should therefore not be collected in acid, as is often done to prevent ammonia formation. It is preferable to collect the urine on ice, or to use bacteriostatic agents such as toluene. The creatine content of the diet should be checked, because there is some creatine even in milk.
In laboratories where the facilities are available, changes in body composition have been estimated from measurements of total body potassium (6, 24). Total body water could also be used. Neither method is precise enough to be of much use except perhaps in long-term studies.
In general, all these measurements will be more useful when repeated measurements are made on the same subject, so that interindividual variability is reduced.
6.3. Energy Expenditure and Physical Fitness
The basal metabolic rate should be measured wherever possible, although this may not be feasible in young children. Rectal temperature should be taken daily.
Mention was made in section 5 of the need for standardizing physical activity. It will obviously be an advantage if energy expenditure can actually be measured. If the subjects are carrying out a standard task, the energy cost of this could be measured by indirect calorimetry, and the cost of other activities (sitting, lying, etc.) could be estimated from standard tables. There was some difference of opinion about the reliability of the integrated heart-rate method for determining 24-hour activity. In any case, this method has to be individually calibrated by indirect calorimetry.
When physical fitness is one of the functional indicators that are assessed in longerterm studies, tests of it should take into account the function that it is supposed to represent in the context of the population under study. For example, in non-anaemic adults whose occupation involves long-continued physical activity (e.g. nonmechanized agricultural tasks or walking long distances while carrying heavy loads), the use of physical fitness tests of short duration (e.g. step tests) or of muscle strength may be inadequate, whereas endurance or maximal tests will be more appropriate. Changes in fitness of children of pre-school age may be tested by determining the regression equation of changes in oxygen consumption and heart-rate induced by exercise.
6.4. Biochemical Measurements
The ideal is to take blood samples at the beginning and end of each test period (i.e. at intervals of seven to nine days), but it is important not to disturb the subjects by too frequent sampling. Fingertip samples should be used wherever possible.
Measurements that may be useful are: haemoglobin or haematocrit, total protein, albumin, and perhaps other circulating proteins that are considered to be indicators of protein nutrition, e.g. transferrin (29), retinol-binding protein (30). There is some evidence that the effect of protein intake on albumin concentration depends on whether the dietary protein is of animal or vegetable origin (Graham, unpublished data) (31); free amino acid concentrations, and ratio of non-essential to essential amino acids; aminotransferases, since increases have been noted in long-term experiments (6).
The amount of blood taken in metabolic studies should be recorded, and account take of it in computing nitrogen balances.
It was not considered that tests of immune response, challenging with skin-test antigens etc. are likely to be of much value in the studies on normal, healthy subjects proposed in the first phase of the programme.
The following measurements are proposed as a basic set to be included in each study. Weight/height and weight change; basal or resting metabolic rate; nitrogen balance; creatinine (plus creatine) excretion; faecal dry weight, amount and composition of dietary intake; and estimate of the level and pattern of physical activity.
Different groups of investigators are encouraged to add on special studies that are of particular interest to them, such as measurements of protein turnover. These will increase our understanding of the basic interrelationships between protein and energy metabolism and nutrition, and help to develop functional measures of nutritional adequacy.
Formulation of test diets based on habitual foods is more difficult than when the diets are composed entirely of commercial products of uniform composition. As far as possible the components of the diet should be purchased in bulk, but this will not, of course, be possible with perishable items such as vegetables unless frozen storage is feasible.
Preliminary analyses of the foodstuffs will have to be made to determine the proportions to be used in each particular test. The mixture of foodstuffs that make up the diet will then have to be analysed at intervals. This requires great care in homogenization and sampling. It is recommended that standardized methods be used, and that duplicate samples be sent for analysis (N determination and bomb calorimetry) in a reference laboratory. Amino acid analysis will also be necessary for calculating the protein score.
The most useful outcome of the meeting was that there was general agreement on the priority objective for the next phase of metabolic studies: to examine nitrogen balance at a fixed level of protein intake (either the safe or habitual level) over a range of energy intakes, varying from the habitual to the recommended, account being taken of the degree of physical activity.
Nevertheless, changes in nitrogen retention will indicate the effect of dietary energy on an important aspect of protein metabolism. Furthermore, the investigations proposed will allow comparisons with the results of earlier metabolic studies in which nitrogen balance was often the only indicator of protein requirements.
It is not to be expected that such studies will lead to the definition of a single level of protein requirement for any particular age group. Rather, they will enlarge our knowledge of the relationships between protein requirement and energy intake, and may lead to the concept of a zone of requirements, depending on the intake and output of energy.
It was not found feasible to define a standardized protocol, largely because the different scientific groups represented at the meeting are interested in different age and sex groups - young infants, older pre-school children, and adults. This may be regarded as a source of strength to the programme, since there are gaps in our knowledge of all age groups.
There was also some difference between the groups in the emphasis given to short-term and longerterm studies. This diversification will also be an advantage to the programme. The pros and cons of different experimental designs were thoroughly discussed, and all members were agreed on the principles involved.
The emphasis on habitual diets in the experiments now being planned is the first step in a move away from the rather artificial conditions of most previous metabolic studies. However, it was accepted that in the first phase of the programme it is necessary, as a baseline, to study subjects who are well nourished and healthy. In a second phase comparisons could be made with subjects whose nutritional and health status is more representative of poor communities. This will provide information that is badly needed on the extent and nature of the adaptation that occurs under these conditions.
The Group fully recognized the limitations of short-term nitrogen balance as an indicator, and reaffirmed the need to develop functional criteria of nutritional status.
In the end, the conclusions from strictly controlled laboratory investigations will have to be tested by studies in the field. This might well form a third phase of the overall programme.
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19. V R. Young, Y.S.M. Taylor, W.M. Rand, and N.S. Scrimshaw, "Protein Requirements of Man: Efficiency of Egg Protein Utilization at Maintenance and Submaintenance Levels in Young Men, J. Nutrition, 103: 1164 (1973).
20. C. Garza, N.S. Scrimshaw, and V.R. Young, "Human Protein Requirements: The Effect of Variations in Energy Intake Within the Maintenance Range," Amer J. Clin. Nutr., 29: 280 (1976).
21. P.C. Huang, H.E. Chang, and W.M. Rand, "Obligatory Urinary and Fecal Nitrogen Losses in Young Chinese Men," J. Nutn6On, 102: 1605 (1972).
22. C.N Rao, A.N. Naidu, and B.S. Rao, "Influence of Varying Energy Intake on Nitrogen Balance in Men on Two Levels of Protein Intake," Amer J, Clin Nutr., 28: 1116 (1975).
23. B.M. Nicol, and P,G, Phillips, "The Utilization of Dietary Protein by Nigerian Men," Brit J. Nutrition, 36: 337 (1976)
24. N,S, Scrimshaw, W.D.A. Perera. and V.R Young. "Protein Requirements of Man: Obligatory Urinary and Fecal Nitrogen Losses in Elderly Women." J. Nutrition. 106: 665 (1976).
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29. P.J. Reeds, and A.A.O. Laditan, "Serum Albumin and Transferrin in Protein-Energy Malnutrition," Brit. J. Nutr, 36: 255 (1976).
30. Y Ingenbleek, H.G. van den Schrieck, P. de Nayer, and M. de Visscher, "Albumin, Transferrin and the Thyroxine-binding Prealbumin/Retinol-binding Protein (TBPA-RBP) Complex in Assessment of Malnutrition," Clinica Chimica Acta, 63: 61 (1975).
31. G.H. Whipple, "Protein Production and Exchange in the Body including Hemoglobin Plasma Protein and Cell Protein," Amer J. Med. Sci., 196: 609 (1938).
32. H. Chan, and J.C Waterlow, "The Protein Requirements of Infants at the Age of About 1 Year," Brit. J. Nutrition, 20: 775 (1966).
33. R. Uauy, N.S. Scrimshaw, W.M. Rand, and V.R Young, "Human Protein Requirements: Obligatory Urinary and Fecal Nitrogen Losses and the Factorial Estimation of Protein Needs in Elderly Males," J. Nutrition, 108: 97 (1978)
Dr. G.H. Beaton, Professor and Head, Department of Nutrition, School of Hygiene, University of Toronto, Toronto 5, Ontario, Canada.
Dr. D.H. Calloway, Professor of Nutrition and Chairman, Department of Nutrition Sciences, University of California, Berkeley, California 94720, USA.
Dr. Kraisid Tontisirin, Department of Pediatrics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.
Dr. D. Picou, Director, Tropical Metabolism Research Unit, University of the West Indies, Kingston, Jamaica.
Dr. B. Torún, Chief, Programme of Physiology and Clinical Nutrition, Institute of Nutrition for Central America and Panama. Guatemala City, Guatemala.
Dr. Vinodini Reddy, Assistant Director, Clinical Research, National Institute of Nutrition, Hyderabad, 500007, India.
Dr. J. Waterlow, (chairman) Professor of Human Nutrition, London School of Hygiene and Tropical Medicine, Keppel Street (Gower Street), London WC1E 7HT, United Kingdom.
Representatives of Other Organizations
Dr. V.R. Young, Professor Nutritional Biochemistry, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Dr. E.M. De Maeyer, Medical Officer, Nutrition, WHO, Geneva, Switzerland.
Dr. P. Lunven, Chief, Food and Nutrition Assessment Service, Food Policy and Nutrition Division, FAO, Rome, Italy.
Dr. J. Períssé, Senior Officer, Food and Nutrition Assessment Service. Food Policy and Nutrition Division, FAO, Rome, Italy.
Dr. N. Rao Maturu (Secretary), Nutrition Officer, Food and Nutrition Assessment Service, Food Policy and Nutrition Division, FAO, Rome, Italy.
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