|FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS||ESN:FAO/WHO/UNU|
|WORLD HEALTH ORGANIZATION|
|THE UNITED NATIONS UNIVERSITY|
Provisional Agenda Item 3.3.4
Joint FAO/WHO/UNU Expert Consultation on Energy and Protein Requirements
Rome, 5 to 17 October 1981
PROTEIN REQUIREMENTS FOR THE ELDERLY
Hamish N. Munro
The significance of nutrition for aging of man can be considered under three headings.First, many physiological functions decline progressively throughout adult life.The significance of nutrition in altering the progress of these changes is largely unknown.Second, aging is associated with the emergence of chronic diseases, some of which probably include nutritional factors in their etiology.Finally, it is well recognized that food intake in general diminishes with age, but there is little conclusive evidence on which to base recommendations for optimum intakes of nutrient intakes by the elderly.
Since many age-related body changes appear to occur continuously throughout adult life, protein allowances for adults should ideally be those which best preserve bodily functions from early adulthood through into old age.Protein needs may well change progressively with age as body composition, physiological functional capacity, physical activity, total food intake and frequency of disease alter with age.However, we are presently lacking the necessary information to construct recommendations based on such a continuum.Nevertheless, the elderly represent an important class for whom some estimates of protein allowances should be specifically developed as a public health measure, even although they are only the tail-end of a continuous process beginning in early adult life.
This background paper will therefore deal briefly with the changes in body composition, physiological functions and metabolism that characterize the aging process, followed by estimates of the protein and amino acid requirements of the elderly, and will finally make recommendations of allowances for those over 65 years of age. More extensive descriptions of these areas will be found in reviews by Munro (1981), Munro and Young (1980) and Young, Gersovitz and Munro (1981).
Age-related Changes in Body Composition, Physiological Function and Metabolism
Using whole-body potassium measured by 40K as an index of body protein content, both cross-sectional (Allen et al., 1960; Forbes and Reina, 1970; Parizkova et al., 1971; Zanni et al., 1979; /Cohn et al., 1980) and longitudinal studies (Forbes and Reina, 1970; Forbes, 1976; Steen et al., 1979), agree in showing a reduction in body protein content (lean body mass) with advancing age.For example, the data of Forbes and Reina (1970) show a continuous decline in lean body mass, tending to be greater for men and to accelerate in later life, and to be compensated by an increase in body fat. Using a combination of in vivo neutron: activation to determine body N content and 40K to measure body K content, Cohn et al. (1980) have not only confirmed the loss of body protein but have further calculated from comparison of the loss of N and of K (the latter richer in muscle) that a reduction in muscle mass accounts for most or all of the age-related change in lean body mass.This emphasis on muscle loss agrees with the autopsy data of Korenchevsky (1961), who observed that subjects over 70 years of age had 40% less muscle mass than young adults, liver 18%, kidney 9% and lung 11%.Finally, the preferential loss of muscle with aging is reflected in the diminishing output per unit body weight of creatinine (Uauy et al., 1978) and of 3-methylhistidine (Young and Munro, 1978).
In addition to reduction in lean body mass, there is also a diminution in the functional capacity of many tissues in aging.Shock (1972) has assembled data showing very strikingly that men between the ages of 30 and 80 years of age undergo a 30% loss in cardiac output and a 50% reduction in renal blood flow.Individual systems show other evidence of functional loss.Thus cellular immunity is lost progressively from adolescence onwards (Makinoden, 1978).
Metabolism of protein has been investigated for age-related changes in man. Using precursor amino acids labeled with stable isotopes, we (Uauy et al., 1978) have shown that whole-body protein synthesis diminishes per kg body weight but not per unit of lean body mass as the subject ages.Since loss of muscle accounts for most of the loss of lean body mass, the reduced protein synthesis of the elderly must be accounted for mainly by less muscle protein synthesis.Output of 3-methylhistidine confirms this (Gersovitz, Munro and Young, 1981).Using this as a measure of muscle protein breakdown, it was shown that muscle protein turnover accounts for 30% of whole body protein turnover in young men and 20% in old men.
While these observations on age-related changes in body composition and in protein metabolism, especially relating to muscle, would suggest that utilization of dietary protein and of essential amino acids may differ in the young and old adult, studies relevant to this are only beginning to be performed.A preliminary investigation into fasting young and old subjects produced no striking differences in leucine flux, oxidation or incorporation into protein in the post-absorption state (Robert, Bier, Schoeller, Matthews, Munro and Young, 1981). These studies need to be extended to young and old subjects while absorbing a meal of protein.
The Protein Requirements of the Elderly
The minimum physiological needs for total protein in adult humans have been determined using one of two N balance methods.These are: (a) the factorial approach and (b) the N balance response curve method in order to determin directly the intake required to just maintain body N balance.By the former approach the losses of obligatory N via urine and feces are measured while the subject is receiving a protein-free diet, and summated with additional corrections for N losses via the integument and other minor routes.This allows calculation of the amount of dietary protein needed to replace obligatory losses.
Table 1.Obligatory N Losses of Young and Old Subjects
|Young:||Men||37||9||46||51||Scrimshaw et al. (1972)|
|Men||38||14||52||57||Calloway and Margen (1971)|
|Women||25||8||33||38||Bricker and Smith (1951)|
|Old:||Men||35||12||47||52||Uauy et al. (1978)|
|Men||27||10||37||42||Zanni et al. (1979)|
|Women||24||10||34||39||Scrimshaw et al. (1976)|
(1) Assuming 5 mg N/kg via skin and other minor routes.
Table 1 summarizes published studies.It shows similar values for obligatory output per kg body weight for young and old men studied at M.I.T., but lower values for old versus young men studied at Berkeley, while young and old women appear to show the same output.Fecal N outputs per kg body weight follow the same pattern, so that the data for total obligatory N shows no difference for males of different age studied at M.I.T., but a much lower output in the Berkeley series, while women of different ages appear not to differ.Thus the data provide no reason for increasing protein allowances for elderly men and women, if we apply the factorial method to existing obligatory N output data.However, such calculations assume that utilization of dietary protein is similar in young and elderly.
A more direct assessment of requirements is based on determining the minimum amount of protein needed to bring the subject into N equilibrium.The difficulties of using this procedure are discussed by Munro and Young (1981), who provide literature references.First, N balance represents a relatively small difference (± 2 g N daily in most cases) between the amounts of nitrogen consumed and excreted (possibly 10 g or more).It has been pointed out frequently that errors in measurement of intake and of urinary output will generally favor an apparent N balance that is more favorable than the true N balance.A second, though minor, source of error favoring positive balances is the conversion of organic nitrogen to nitrite and nitrates by the body and the excretion of nitrate in the urine as a compound not reacting as N under usual reaction conditions involving the Kjeldahl method.Last, most investigators do not directly estimate cutaneous and other N losses from the body, although these losses may vary considerably.
In addition to these uncertainties of techniques, the state of the subject affects the N balance obtained.Factors influencing this include previous nutritional status; subjects who are protein-depleted respond to an increase in protein intake with a larger N retention than do subjects whose body protein content is adequate.In addition, stressful stimuli affect N balance, and finally the energy intake of the subject is a major factor in determining a positive or negative N balance.Since energy balance and requirement is not easily determined, the amount of protein needed to achieve N equilibrium for a given subject remains uncertain.
|Estimate and Conclusion||Remarks||Authors|
|N equilibrium in 7 of 8 women at 0.7 and 1.0 g protein/kg Dietary standards adequate||Healthy women, 52–74 years||Roberts et al. (1948)|
|Good nutritional state maintained at 54 ± 5 g protein||N balance assessed from diet records 20 women, 68–88 yrs.||Albanese et al. (1957)|
|Protein needs not different from younger adults||Review of studies with older men and women in a mental hospital||Horwitt, M.K. (1953)|
|No evidence of qualitative or quantitative changes with age||Balance studies in healthy old men||Watkin, D.M. (1957-58)|
|Elderly require 0.7 g protein/kg/day||4 men, 69–76 yrs., poorly nourished subjects||Kountz et al. (1952)|
|Protein requirement for elderly women may be 20–30% less than for young women||9 women, 66–94 yrs. old.Maintained health at self chosen intakes||Albanese et al. (1952)|
Published studies on the minimum protein needs to achieve N balance-equilibrium can be divided into early and recent investigations.Table 2 shows representative early studies which led to the conclusion that the protein needs of the elderly are either similar to or greater than those of young adults of the same sex.However, these studies were generally not carried out with the care and attention which has resulted from more recent re-assessment of the limitations of N balance such as those presented earlier in this text, and which stem from the impetus given to N balance studies following the 1965 FAO/WHO Report on Protein Requirements.Four recent studies form the basis on which to arrive at a decision on the protein allowances for people over 60 years of age (Table 3).
|Cheng et al.||NRC allowance of 0.8g/kg considered adequate||7 men; 61–72 yrs; 62 kg; 11-day periods;|
Energy intake; 40 kcal/kg Balance in 4/7 subjects.
|Zanni et al.||FAO/WHO 1973 safe level of 0.57g egg protein/kg considered adequate||6 men; 63–77 yrs; 83 kg;|
Test protein followed 17-day protein-free period.
15-day balance Energy intake; 31 kcal/kg
|Uauy et al.||Mean requirement 0.8g/kg, women|
|7 women, 71–78 yrs; 69 kg|
Energy intake, 28 Kcal/kg.
7 males; 68–74 yrs; 74 kg
Energy intake, 32 Kcal/kg
|Gersovitz et al. (unpublished)||0.8g egg protein/kg not adequate for long-term balance||7 women; 71–99 yrs; 62 kg|
7 men; 70–82 yrs; 71 kg
Energy intake; 30 Kcal/kg
First, Chang et al. (1978) describe a study on young and old jail prisoners in Chile who received three levels of protein intake (0.4, 0.8 and 1.6 g/kg) from a wheat-soy-milk source, along with 40 kcal/kg throughout 11 days on each diet. Balance was achieved in both young and old groups on 0.8 gm protein/kg.They “agree with the present recommendation by the National Research Council (RDA) that the intake of 0.8 gm/kg per day from protein of high biological value for adults of all ages allows for individual needs and adaptation.” However, it is accepted that older people need smaller energy intake (see background document on the energy needs of the elderly) and thus the provision of the same caloric intake for older people could lead to a difference in energy balance between the two age-groups. The more favorable energy balance of the older men would allow them to retain dietary protein better and thus could mask a greater need.
Second, Zanni, Calloway and Zezulka (1979) gave two levels of egg protein as well as a protein-free diet to elderly men, and interpolated the resulting N balance data to obtain the point of equilibrium.They concluded that the RDA allowance of 0.59 gm. high quality protein/kg body weight was adequate to sustain N equilibrium.While the energy intake of their subjects (30 kcal/kg) was appropriate for people of their age, the administration of the two protein levels in sequence after the protein-free period is likely to have resulted in more favorable utilization of the dietary protein because of the better response to dietar protein after the subject has been on a low intake.Finally, two studies carried out in the same laboratory on young adults at different times (Maryen and Calloway 1971; Calloway, 1975) gave two levels of requirement, one less than and one similar t that of the older subjects, a point acknowledged by Zanni et al. (1979) in their paper
In contrast to this study, Uauy, Scrimshaw and Young (1978c) carried out studies in a group of elderly men and women to obtain the nitrogen balance response to graded intakes of good quality protein (whole egg), with the objective of identifying minimum levels of high quality protein sufficient to maintain nitrogen equilibrium.The results of two such series of studies, involving men and women, are shown in Fig. 1.It is apparent from these that the nitrogen requirement of nearly all elderly subjects receiving a high quality food protein cannot be met by 0.59 gm/kg body weight, and marginally at 0.8 gm./kg, especially for women (see Fig. 1).However, Zanni et al. (1979) have criticized these studies on the grounds that most of the men had chronic diseases and many were on therapeutic drugs.
Fig.1 Nitrogen balance in 8 elderly males and 7 elderly females on graded levels of egg protein intake.(From date of Uauy et al., 1978c)
Table 4.Nitrogen Balance (NB) for Elderly Men and Women Receiving 0.8 gm Egg Protein/kg Body Wt. for 30 Days (Gersowitz, Motil, Munro, Scrimshaw and Young, 1981)
|Period of 10 days||Intake||Urine||Feces||NB||Number in Negative Balance|
Data are expressed as mg N/kg/day for last 5 days of each 10-day period. N balance includes 5 mg N for skin and other minor losses.
Gersovitz, Motil, Munro, Scrimshaw and Young (1981) report a continuous 30-day study on elderly men and women (over 70 years of age) who received 0.8 gm. whole egg protein/kg. body weight.Energy intake was provided to meet needs of 32 and 29 kcal/kg. respectively for these men and women.Nitrogen output and N balance were measured over the last 5 days of each 10-day segment of the 30-day study (Table 4).During days 6–10, the men were in mean negative N balance, thereafter coming into equilibrium.However, by days 26–30, 3 out of the 7 were still in negative balance.The women had an average negative N balance throughout, 4 out of 8 being negative at the end of the study.This implies that 0.8 gm egg protein/kg is at best marginal and probably inadequate, especially for women (cf. Fig. 1) (who happen also to live longer than men!) It must, however, be admitted that this type of study is subject to severe limitations because of the small differences involved in N balance.Thus the mean N balance for men over the whole 30-day period was -1.8 mg. N/kg/day, or -0.13 gm. N/70 kg. man, which would amount to a loss of 3.9 gm N in 30 days, equivalent to 25 gm body protein per month or 300 gm per year.This last figure would represent 3% of the total protein content of the body.Similar calculations for the elderly women show that the mean daily N balance over the 30-day period (-3.6 mg/kg) represents -0.22 gm N/60 kg woman, which is 6.6 gm.N over a 30-day period.This would represent a monthly loss of 40 gm body protein, or 500 gm per year, which is about 7% of the protein content of the body.Using 40K measurements, Steen et al. (1979) found that their subjects lost 1 kgm. lean body mass between age 70 and 75 years.This can be translated into a yearly loss of 40 gm of protein, equivalent to a daily negative N balance of 002 gm N/70 kg man, which is one-sixth the mean daily negative N balance for our old men (-0.13 gm N/70 kg) and one-tenth that for our elderly women (-0.22 gm/60 kg). This is reminiscent of the frustrations of trying to use energy balance in studying obesity.Garrow (1978) points out that a consistent excess of 3% of energy sources over needs will add many pounds in body weight in one year.
Requirements for Individual Essential Amino Acids
Compared with studies of amino acid requirements in young adults, there have been few definitive studies of the essential amino acid requirements in the elderly.Tuttle, Swendseid and colleagues conducted a series of studies of the essential amino acid requirements of older men.In the first study (1957), with five men aged 52 to 68 years, it was concluded that elderly subjects may have a higher requirement for one or more essential amino acids.A second study by these investigators (1959) in subjects over 50 years of age suggested that the requirements of one or more essential amino acids may also increase as the total dietary nitrogen intake rises.Two sharply differing conclusions have been drawn for the minimal methionine and lysine requirements of the elderly. Tuttle et al. (1965) gave a synthetic L-amino acid mixture patterned as for egg protein to six males, 58 to 73 years of age.Total nitrogen intake was 7g per day.Four subjects receiving no cystine required 2.1–3.0 g methionine to achieve nitrogen equilibrium, thus suggesting S-amino acid needs considerably higher than published estimates for the young adult.Tuttle et al. (1965) also suggest that the requirement for lysine is higher for the elderly.In contrast with these findings, six black men, 65 to 84 years, studied by Watts et al. (1969) required lower intakes of the sulfur-containing amino acids to achieve N balance, compared with the needs of younger subjects.
An alternative approach to requirements for individual essential amino acids is based on measurement of the response of free amino acids in plasma to changing levels of intake.Beyond the level of requirement, higher intakes of the essential amino acids cause plasma levels to rise.This approach has been used to assess the minimum requirements for tryptophan (Young et al., 1979), valine (Young et al., 1972), and threonine (Tontisirin et al, 1974) in young adults.These studies have been extended to older subjects (Young et al., 1972) for varying levels of tryptophan intake.The point of inflection when excess tryptophan was taken was at 2 mg/kg/day, whereas young subjects need more, namely 3 mg per kg per day.This approach was also used to determine the threonine requirement in elderly (Tontisirin et al, 1974).Although these studies suggested that the daily threonine requirement in elderly women, expressed per unit of body weight, is the same as for young men, this probably means that the threonine requirement per unit of total body protein increases with age, since lean body mass is proportionately less in the older subject. This conclusion also applies to the valine requirement, based again on an interpretation of the plasma valine response curve (Young et al., 1972).
From the foregoing it is evident that information about requirements for individual essential amino acids in the aging human is fragmentary and contradictory. This is an unsatisfactory state of affairs; particularly because estimations of the requirements for essential amino acids form the basis on which to design the “protein” component of both normal and therapeutic diets for use in the elderly and to assess the significance of dietary protein quality in this age group.
Recommendations for Protein Allowances for the Elderly
It remains difficult to decide whether the protein requirements of the elderly are the same per kg body weight as for younger subjects, or less or more. The following is an attempt to summarize the evidence on which a decision may be made.
(1) Despite refinements of the parameters and care in execution, nitrogen balance remains a fragile tool for estimating needs, as for example the 30% difference in estimates for young adult males reported at different times by the Berkeley group (Calloway and Margen, 1971; Calloway, 1975).
(2) It is improbable that high intakes of protein can prevent the aging process in adults, since measurements showing loss of lean body mass and of tissue function with age have been made in Western countries in which the consumption of protein by adults is customarily about twice that of the estimated allowance for protein of 0.8 gm/kg body weight.What we need is evidence that populations living at this or even lower levels of dietary protein show accelerated losses of lean body mass and of tissue function, and information on this appears to be lacking.
(3) Energy intake is of considerable importance in determining an adequate allowance of protein for the elderly.First, nitrogen balance is affected by energy balance as well as by protein intake, thus complicating determinations of protein needs from N balance data.Second, the current recommendations (RDA) for protein and for energy intakes of people over 76 years of age provide sharp reductions in energy needs without a change in protein needs.For instance, the recommended energy intake of men falls from 2700 kcal between 23 and 50 years to 2050 kcal after 75 years, but recommended protein intake remains at 56 gm daily, so that protein calories rise from 8% to 11% of the diet.Women show a similar phenomenon.For target groups of elderly with specially low caloric intakes, such as disabled persons living alone, caloric density for protein allowances could be even higher.
(4) Chronic disease is more frequent in the elderly, and can lead to periodic temporary losses of body protein through fever or simply loss of appetite. Such losses have to be replaced from the diet and thus make an added need for dietary protein.In order to obtain some idea of the magnitude of this factor, the frequency of periods of low food intake should be compared in young and elderly people, and whether they relate to minor illnesses and other environmental factors.If it is thought to be significant, then the elderly may require a small repletion allowance built into their protein allowances.
5. In view of these considerations, it is recommended that the protein allowance for people over 60 years should not be less than 12–14% of caloric intake.This intake of protein should more than adequetely meet requirements for individual essential amino acids.
Table 5 illustrates some data for intakes of energy and protein by adults of various ages in Scotland, obtained during the course of extensive studies by Durnin.
Table 5.Protein intakes of groups of adults in Scotland, obtained by weighing all foodstuffs consumed during a 7-day period (data of Durnin and colleagues cited by Munro, 1964 a)
|Group||Mean age (yr.)||Mean energy intake per day (kcal)||Mean protein intake per day|
|Total (g)||As % of total energy intake||Intake per kg body wt (with range) (g/kg)|
|Older workers (heavy work)||60||3430||113||13.2||1.48||(1.24–1.86)|
|Older workers (moderate work)||61||2910||90||12.4||1.31||(0.85–1.80)|
|Retired men (living alone)||73||2050||72||14.0||1.03||(0.78–1.24)|
|Young shop assistants||21||2220||70||12.3||1.21||(0.99–1.63)|
|Pregnant women (first child)||-||2450||75||12.4||1.20||-|
|Elderly women (living alone)||66||1890||62||13.2||1.03||(0.69–1.59)|
Albanese, A. A., Higgens, R. A., Orto, L. A., Zwattoro, D. N., 1957, Geriatrics, 12, 443
Albanese, A. A., Higgens, R. A., Vestal, B., Stephanson, L., and Malsch, M., 1952 Geriatrics, 7, 109
Allen, T. H., Anderson E. C., and Langham W. H., 1960, J. Gerontol, 15, 348
Bricker, M. L. and Smith, J. M., 1951, J. Nutr., 44, 5530
Calloway, D. H., 1975, J. Nutr., 105, 914
Calloway D. H. and Margen, S., 1971, J. Nutr. 101, 205
Cheng A. H. R., Gomez A., Bergan J. G., Lee T. C., Monckeberg F., and Chichester C. O., 1978, Am. J. Clin. Nutr., 31, 12
Cohn, S. H., Vartsky, D., Yasumura, S., Sawitsky, A., Zanzi, I, Vaswani, A. and Ellis, K. J. 1980, Am. J. Physiol., 239, E524.
Forbes G. B., 1976, Human Biol., 48, 161
Forbes G. B., and Reina J. C., 1970, Metabolism, 19, 653
Garrow, J. S. 1978, Energy balance and obesity in man, 2nd edn. Elsevier, New York
Gersovitz M., Munro H. N., Udall J., and Young V. R., 1980, cited by Munro (1981)
Gersovitz M., Motil K., Munro H. N., Scrimshaw N. S., and Young V. R., (1981) Amer. J. Clin. Nutr. (in press)
Horwitt, M. K., 1953, J. Am. Diet. Assoc., 29, 443
Korenchevsky V., 1961, Physiological and Pathological Ageing, (Bourne G. H.:ed) p.541
Kountz W. B., Hofstatter L., and Ackerman P. G., 1951 J. Gerontol 6, 20
Makinoden, T. 1978, Fed. Proc., 37, 1239.
Munro, H. N., 1964, Mammalian Protein Metabolism, Vol II, P. 3 (eds. H. N. Munro and J. B. Allison).Academic Press, New York.
Munro, H. N., 1981, Brit. Med. Bull., 37, 83.
Munro, H. N., and Young, V. R., 1980, Protein Metabolism and Requirements in Metabolic and Nutritional Disorders in the Elderly, ed. A. N. Exton-Smith and F. L. Caird, pp. 13–25.Wright, Bristol.
Munro, H. N., and Young, V. R., 1981, Recent Advances in Clinical Nutrition, edited by A. Howard and I. Baird, pp. 33, Libby, London
Parizkova J., Eiselt E., Sprynorova S., et al., 1971, J. Appl. Physiol, 31, 323.
Recommended Dietary Allowances, 1980, Ninth Edition, NAS-NRC-Washington D. C.
Robert, J. J., Bier D. M., Schoeller, A., Matthews, D. E., Munro H. N., Young, V. R., 1981, (in preparation)
Steen, G. B., Isaksson, B., and Svanberg, A., 1979 J. Clin. Exp. Gerontol,1, 185
Scrimshaw, N. N., Hussein, M. A., Murray, E., Rand, W. M., and Young, V. R., 1972, J. Nutr. 102, 1595.
Scrimshaw, N. S., Perera, W. D. A., and Young, V. R., 1976, J. Nutr., 106, 665
Tontisirin, K., Young, V. R., Rand, W. M., and Scrimshaw, N. S., 1974, J. Nutr., 104, 495
Tuttle, S. G., Swendseid, M. E., Mulcare, D., Griffith, W. H., and Bassett, S. H., 1957, Metabolism, 6, 563
Tuttle, S. G., Swendseid, M. E., Mulcare, D., Griffith, W. H., and Bassett, S. H., 1959, Metabolism, 8, 61
Tuttle, S. G., Basset, S. H., Griffith, W. H., Mulcare, D. B., and Swendseid, M. E., 1965, Am. J. Clin. Nutr., 16, 229
Uauy, R., Scrimshaw, N. S., Rand, W. M., and Young, V. R., 1978a, J. Nutr., 108, 97
Uauy R., Scrimshaw, N. S., and Young, V. R., 1978b, Symp. Proc. Nutr. Soc. Canada, p. 53
Uauy R., Scrimshaw, N. S., and Young, V. R., 1978c, Am. J. Clin. Nutr. 31, 779
Watkin, D. M., 1957 New York Acad. Sci, 69, 902
Watts, J. H., Mann, A. N., Bradley, L., and Thompson, D. J., 1964, J. Gerontol, 19, 370
Young, V. R., Tontisirin, K., Ozalp, I., Lakshamana, F., and Scrimshaw, N.S., 1972, 102, 1159
Young, V. R., Hussein, M. A., Murray, E., and Scrimshaw, N. S., 1971, J. Nutr., 101, 45
Young, V. R., Gersovitz, M., and Munro, H. N., 1981, in Nutritional Approaches to Aging Research (G. B. Moment, ed.) CRC Press.
Zanni E, Calloway, D. H., and Zezulka, A. Y., 1979, J. Nutr., 109, 513