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Protein intake and urinary excretion of proteinderived metabolites in aging female vegetarians and nonvegetarians. a
a
M E Kunkel & R E Beauchene a
Department of Nutrition and Food Sciences, College of Home Economics, University of Tennesse, Knoxville 37996-1900. Published online: 02 Sep 2013.
To cite this article: M E Kunkel & R E Beauchene (1991) Protein intake and urinary excretion of protein-derived metabolites in aging female vegetarians and nonvegetarians., Journal of the American College of Nutrition, 10:4, 308-314, DOI: 10.1080/07315724.1991.10718157 To link to this article: http://dx.doi.org/10.1080/07315724.1991.10718157
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Protein Intake and Urinary Excretion of Protein-Derived Metabolites in Aging Female Vegetarians and Nonvegetarians M. Elizabeth Kunkel,« PhD, FACN and Roy E. Beauchene, PhD, FACN Department of Nutrition and Food Sciences, College of Home Economics and Agricultural Experiment Station, The University of Tennessee, Knoxville
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Key words: aging, energy intake, protein intake, protein metabolism, nitrogen excretion, vegetarian Relationships among age, protein intake, and urinary excretion of protein-derived metabolites were studied in 125 vegetarian and nonvegetarian women ages 40-92. There were 63 women in the vegetarian (AV) group and 62 women in the nonvegetarian (NV) group. Average daily intakes of energy and total animal and vegetable protein were calculated from 7-day dietary records. Twenty-four-hour urine samples were analyzed for total nitrogen, urea, creatinine, hydroxyproline, and inorganic sulfate. Energy intakes for the two groups were similar. AVs consumed less total and animal protein and more vegetable protein than NVs, even though both groups consumed more than the RDA for protein. No significant differences existed between the groups in the urinary excretion of total nitrogen, urea nitrogen, hydroxyproline, or inorganic sulfate. Energy and protein intakes and total nitrogen excretion were lower in older AVs than in younger AVs, while those of NVs increased between 40 and 55 years of age, and decreased among the older NV women. The relationship between these variables and age in NVs was more accurately described by polynomial rather than linear regression models. Abbreviations: AV = vegetarian, HOP = hydroxyproline, N = nitrogen, NV = nonvegetarian, RDA = recommended dietary allowance
INTRODUCTION
METHODS
Lactoovo- and pure (vegan) vegetarian diets are generally considered to be nutritionally adequate and have been reported to contain ample amounts of total protein [1-7] and indispensable amino acids [8]. In addi tion, vegetarian subjects have been reported to utilize their dietary protein at least as well as nonvegetarians [2,9], although differences in urea excretion have been found [10]. In addition to the effect of diet, protein meta bolism may also be affected by age [11,12] and body protein mass [13]. This study was concerned with the relationships among age, protein intake, and the urinary excretion of protein-derived metabolites in older adult vegetarian and nonvegetarian females.
The sample consisted of 125 vegetarian and non vegetarian females who were recruited primarily through religious and civic organizations. Of the 63 women who were vegetarians (AVs), 57 were lactoovovegetarians and six were vegans. Given the small size of the vegan group, lactoovovegetarians and vegans were pooled into the vegetarian group. The remaining 62 subjects were nonvegetarians (NVs). All women were ambulatory. On a general health questionnaire, none of the subjects reported any physical or metabolic impairments that would have affected any of the parameters measured in this study. AVs were defined as those subjects who typically consumed no
Presented, in part, at the 22nd Annual Meeting of the American College of Nutrition. ♦Present address: Food Science Department. College of Agricultural Sciences. Clemson University, Clemson, South Carolina 29634-0371. Addressreprintrequeststo Roy E. Beauchene. Department of Nutrition and Food Sciences. College of Home Economics. The University of Tennessee, Knoxville, Tennessee 37996-1900.
Journal of the American College of Nutrition, Vol. 10, No. 4, 308-314 (1991) © 1991 John Wiley & Sons, Inc.
CCC 0731 -5724/91/040308-07$04.00
Protein Status in Older Women Table 1. Descriptive Data of Subjects
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Parameter Age (years) Mean 1 SEM Range Duration of vegetarianism (years) Mean ± SEM Range Height (cm) Mean ± SEM Range Weight (kg) Mean 1 SEM Range
meat products but did use egg and dairy products. After the details of the study were described to them, each subject signed an informed consent form. Each subject was provided measuring cups, a ruler, and forms in addi tion to oral and written instructions for recording food intake for 7 days. Food intakes were recorded as close approximations of the amounts consumed, with the inclusion of brand names and methods of preparation where applicable. Subjects were asked to supply recipes when possible. A dietary history was also recorded for each subject. Any questionable entries on the food records were checked against the dietary histories; if further clarifications were needed, the entries were discussed directly with the sub ject. The food intakes were summarized and nutrient in takes determined by mainframe computer using a com puter program developed at The University of Tennessee which is based on the composition data found in USDA Handbook No 8 [14], with additional information sup plied by various food manufacturers. Each item in Hand book No 8 was coded as to animal, vegetable, or mixed protein source. Mixed protein sources included cakes, cookies, breads, and pasta entries. Intakes were ex pressed as a mean for the 7-day recording period. Each subject collected a 24-hr urine sample on the third or fourth day of the week in which the 7-day dietary record was kept. The sample was collected in an acid-rinsed polyethylene bottle containing 5 ml of a 10% (w/v) thymol-2-propanol solution. Completeness of the urine samples was determined based on creatinine excretion. The urine was analyzed for urea nitrogen by the Berthelot reaction [15-17], for creatinine by Folin's method [18], for hydroxyproline (HOP) by a modification of the
Vegetarians (n = 63)
Nonvegetarians (n = 62)
57.4 ± 1.4 36.0-92.0
61.511.3 41.0-82.0
36.4 ± 4.8 2.0-70.0
—
161.810.8 146.7-174.6
160.710.9 137.1-172.7
63.2+1.6 39.1-98.0
66.1 11.5 44.5-99.1
method of Bergman and Loxley [19], for total nitrogen by the macro-Kjeldahl method [20], and for inorganic sulfate by the method of Swaroop [21]. Height, weight, and skinfold thickness were measured on each subject. Height was measured to the nearest quarter inch and weight to the nearest pound with the subject dressed in light indoor clothing and wearing no shoes. Skinfold thickness was measured to the nearest mm using Lange (Cambridge Scientific Industries Inc., Cambridge, MD) calipers and following the method described by Tanner and Whitehouse [22]. Triplicate readings were taken on the left triceps of each subject midway between the tip of the acromion process and the top of the radius. Data were analyzed using the Statistical Analysis Sys tem (SAS) [23]. The relationship between a response variable and age was characterized with a model con taining linear, quadratic, and cubic terms for age as well as a dietary classification variable, i.e., AV or NV. Also included in the model were the linear, quadratic, and cubic interactions with the classification variable. If in teractions between age and the classification variable were found to be significant (p < 0.05), then separate relationships for each group were estimated and are presented graphically. For those response variables without significant interaction, the significance of dif ferences among least square mean values, adjusted for the mean age of the sample, were tested using Duncan's New Multiple Range Test [24]. Least square mean values for response variables with significant interaction (ener gy and protein intakes, total urinary nitrogen) were cal culated for comparison with literature values.
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309
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Fig. 1. Regressions of body weight and triceps skinfold thickness on age, NVs represented by a solid line and AVs by a dotted line. Equations for the regression lines and their significances are as follows: Body weight: NV = 177.07 - 3.30(age) + 0.02(age 2 ), AV = -18.78 + 2.97(age) - 0.02(age 2 ); triceps skinfold thickness: NV = 78.65 - 1.23(age) + 0.01(age 2 ), AV = -24.86 + 1.85(age) - 0.02(age 2 ).
RESULTS Table 1 gives descriptive data for the subjects. Regression equations describing the relationships be tween age and body weight and for age and skinfold thickness for the AV and NV groups are shown in Figure 1. The NVs tended to have a lower weight with increas ing age until about age 70, when a tendency toward in creasing weight was observed. The reverse was true in the AVs, who tended to have the greatest body weight at age 60 years and then decline. There was a significant difference between AV and NV groups in skinfold thick ness which was related to age, the quadratic function of age, and the interaction term between age and type of diet. The NVs showed a gradual decrease in skinfold thickness throughout the age span studied. The AVs had
310
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50
60 AGE
70
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Fig. 2. Regressions of energy and protein intakes and total uri nary nitrogen excretion on age, NVs represented by a solid line and AVs by a dotted line. Equations for the regression lines and their significances are as follows: Energy intake: NV = -1724.91 + 978.05(age) - 16.55(age2) + 0.091 (age3), AV = 1818.30 + 10.38(age) -0.427(age2) + 0.003(age3). The overall shapes of the energy intake curves are different (p < 0.05); decrements with age were significant in both groups (p < 0.01); differences in mean energy intakes between NVs and AVs were not significant (p > 0.05). Protein intake: NV = -576.76 + 32.85(age) 0.54(age2) + 0.003(age3), AV = 153.44 - 4.60(age) + 0.07(age2) - 0.0004(age3). The overall shapes of the protein intake curves are different (p < 0.05); differences with age are significant in both groups (p < 0.01); NVs consumed more protein than AVs (p < 0.001). Urinary nitrogen: NV = -155.44 + 8.10(age) O.tfCage2) + 0.0O07(age3), AV = -8.48 + 0.84(age) - 0.01(age2) + 0.00007(age3). The overall shapes of the curves are different (p < 0.05); neither differences with age nor mean differences in N excretion between NVs and AVs were significantly different (p > 0.05).
a peak skinfold thickness about age 60 followed by a gradual loss. Regression equations describing the relationships be tween age and energy and total protein intakes and be tween age and urinary total nitrogen for AV and NV groups are shown in Figure 2. These variables exhibited
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Protein Status in Older Women Table 2. Energy and Protein Intakes of Aging Female Vegetarians and Non vegetarians1 Dietary component
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Energy (kcal/day) Total protein (g/day) Animal protein (g/day) Vegetable protein (g/day) Mixed protein (g/day)3
Vegetarians (n = 63) 1500 ±42 a2 53.6 ± 1.6a 14.8 ± 1.6a 29.1 ±1.1» 10.5 ±0.6"
Nonvegetarians (n = 62) 1524 ±42 a 66.5 ± 1.6b 45.8±1.7b 10.8±l.l b 10.3±0.6a
Values ate means ± SEM adjusted for mean age of samples (59.4 years). Means in a row not sharing a common superscript are significantly different (p < 0.05). Includes foods containing both animal and vegetable proteins.
Table 3. Excretion of Urinary Components by Aging Female Vegetarians and Nonvegetarians1 Metabolite Total nitrogen (g/day) Urea nitrogen (g/day) Creatinine (g/day) Hydroxyproline (mg/day) Inorganic sulfate (g/day)
Vegetarians (n = 63)
Nonvegetarians (n = 62)
8.20 ± 0.36a2 6.92 ± 0.36a 1.31±0.05a 32.1 ± 1.7a 1.49±0.07a
9.00 ± 0.36a 7.77 ± 0.36a 1.54±0.05b 35.5 + 1.7a 1.57±0.07a
Values are means ± SEM adjusted for mean age of samples (59.4 years). Means in a row not sharing a common superscript are significantly different (p < 0.05).
significant age-associated differences and interactions between the dietary groups. The general shape of all three curves within a dietary group is similar. For ex ample, energy intake, protein intake, and nitrogen excre tion of NVs showed increases until age 50-55 years, followed by decreases until about 70 years, with a ten dency to increase thereafter. The AVs showed small gradual decreases in protein intakes and urinary nitrogen with age. Mean daily energy intakes in NVs varied from 1660 calories at age 55 to a low of 1340 calories at age 70; highest energy intakes for AVs were observed at 45 years (1660 cal) and the lowest at 70 years (1370 cal). Differences in energy intakes between AVs and NVs at corresponding ages were not significant. Mean daily total intakes of protein of NVs varied from 71 g at age 55 to 61 g at age 70; values for AVs ranged from 56 g at age 45 to 52 g at age 70. Total protein intakes were significantly higher for NVs than AVs. Total urinary nitrogen peaked at 9.2 g day"1 in NVs; at 45 years it was
7.6 g day 1 , and at age 70, 7.5 g day 1 . Total urinary nitrogen for AVs ranged from 8.6 g day 1 at age 50 to 7.8 g day"1 at age 70. Comparisons at specific ages revealed no significant differences in total nitrogen excretion be tween AVs and NVs. Age-adjusted mean daily protein intakes from animal, vegetable, and mixed sources are shown in Table 2. The AV women consumed significantly less animal protein and more vegetable protein than the NV women. These differences were significant regardless of whether the protein intake data were expressed as an absolute amount or as a percentage of either energy or total protein intake. Age-adjusted excretions of urinary components are given in Table 3. Urea nitrogen, HOP, and inorganic sul fate excretions did not differ significantly among the groups. Vegetarian subjects excreted significantly less creatinine than the NV subjects.
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DISCUSSION Energy intakes for both groups were less than intakes reported for subjects in the Ten-State Nutrition Survey [25], the USDA Nationwide Food Consumption Survey [26], and the Second Health and Nutrition Examination Survey (HANES Π) [27]. Energy intakes of subjects in the present study were also less than those obtained using duplicate weighing and chemical analysis techni ques [5], dietary records [1,6,7,28,29], or food frequency questionnaires [4]. The lack of significant differences in body weight between AVs and NVs in the present study agrees with results reported by other researchers [10,2931] but disagrees with results reported by Tylavsky and Anderson [4]. The significantly lower triceps skinfold thickness in AV than in NV subjects agrees with findings by Rouse et al [29], while Armstrong et al [10] did not report a significant difference in skinfold thickness be tween vegetarian and nonvegetarian subjects. Activity levels of the subjects in the present study were not measured. The age-adjusted mean total protein intakes of both groups exceeded the 0.8 g/kg of body weight RDA [32] and the 0.6 g/kg body weight standard used by the FAO/WHO/UNU [33]. The mean protein intake of AVs was less than the 1 g/kg of body weight standard used for the Ten-State Nutrition Survey [25] and HANES II [27] and also less than the 0.9 g/kg of body weight standard used for the USDA Nationwide Food Consump tion Survey [26]. The adequacy of the RDA for protein for older men and women has been questioned [34] and various alternative requirements proposed [12,35]. Pellett [36] discussed the changes in data on amino acid requirements and in digestibility and concluded that the "two changes in opposite directions have the effect of leaving the allowance of US dietary protein almost un changed." Uauy et al [12] estimated the mean protein requirement for elderly women to be 0.83 g/kg body weight day 1 . Using that standard, protein requirements were calculated to be for AVs 44 g day 1 and for NVs 55 g day 1 ; both groups exceeded these requirements. Both groups consumed less protein than the vegetarian and nonvegetarian subjects used by Hardinge et al [l], Beilin et al [6], and Rouse et al [29], but had similar intakes to the subjects used by other researchers [4,5,7,28,30]. Even though the total protein intake of the vegetarian subjects was less than that of the nonvegetarian subjects, the excretion of urea nitrogen between the groups did not differ significantly (Table 3). Mean total urinary nitrogen multiplied by 6.25 (equivalent to protein excreted in the form of nitrogen) amounted to 87 and 97% of the mean protein intake of NVs and AVs, respectively.
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Urinary N excretion accounted for only 76% of the daily N intake in the male and female vegetarians used by Abdulla et al [5]. Factors which may contribute to the apparent differences in metabolism of protein by AVs and NVs are the use of available food composition data for many of the nonmeat entrees consumed by AVs and/or the use of a single 24-hr urine sample to estimate nitrogen excretion during the week in which dietary records were kept. Since fecal nitrogen was not deter mined, it is impossible to assess actual nitrogen utiliza tion. Nitrogen utilization has been reported to be more efficient in persons consuming a vegetarian diet than in persons consuming a nonvegetarian diet [9], and to be relatively unaffected by age [37]. Armstrong et al [10] reported that vegetarians excreted significantly less urea than nonvegetarians. However, no protein intake data for their subjects were reported. The lower creatinine excretion in vegetarians may reflect dietary influences upon the excretion of this com pound or differences in lean body mass between AVs and NVs. Crim et al [38,39] reported that the body creatine pool and hence creatinine excretion was decreased by feeding a creatine-free (nonmeat) diet. Intake of animal protein was significantly correlated with creatinine ex cretion in both AVs (r = 0.44; p < 0.001) and NVs (r = 0.33; p < 0.01). Lin et al [9], working with female sub jects, noted lower creatinine excretions in vegetarians than in nonvegetarians. They attributed this difference to a low protein reserve and turnover in the vegetarians as a result of adaptation to a low protein intake. Delanghe et al [40] stated that "prolonged depletion of dietary creatine results in decreased creatinine production, owing to insufficient endogenous compensatory creatine synthesis," and proposed reference values for excretion of creatinine in vegetarians. The creatinine excretion values of AV and NV subjects were higher than these proposed reference values [40]; however, the female subjects used in establishing these reference values were younger (mean age 35.3 ± 9.8 years) than the subjects used in the present study. The mean HOP excretions by all groups were in agreement with those reported by Allison et al [41] and Saleh and Coenegracht [42] for older adults. Since dietary HOP is largely of animal origin and is excreted quantitatively in the urine [43], HOP excretion by AVs would essentially represent endogenous collagen meta bolism. The significant correlation (r = 0.31; p < 0.01) between HOP excretion and animal protein intake in AVs may indicate consumption of at least some HOP by AV subjects. Urinary inorganic sulfate values for both groups are similar to those reported by most other researchers for adult subjects [44-46]. Bodwell et al [44] found that
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inorganic sulfate excretion in adult males was increased by feeding diets limited in one or more of the indispen sable amino acids or containing high levels of sulfurcontaining amino acids. However, Sabry et al [47] noted that urinary sulfate excretion by young adult males was lower when they were fed a rice and beans diet than a bread and eggs diet. The sulfate values reported by Sabry et al [47] were much less than those observed in the present study. The inorganic sulfate excretion by AVs did not differ from that of NVs, even though the vegetarian diet has the potential for being limiting in the sulfur-con taining amino acids. It should be pointed out that the textured vegetable protein products which were con sumed extensively by the vegetarian subjects in this study were frequently fortified with the limiting amino acid or contained egg albumin as a binding agent. It has been generally accepted that age-associated changes in physiological functions are best represented as a linear function of age [48]. In the present study, polynomial regression models were found to more ac curately describe the relationship between age and some variables. The regression lines presented were not drawn for the full range of subjects because of the inherent tendency of a polynomial to magnify variations at the extremes of the data. The increase in energy and protein intakes until the sixth decade followed by decreases in the seventh decade for NVs as compared to the gradual decreases observed for these variables with age in AVs may reflect, in part, sampling bias. Of the NV subjects, some of the youngest consumed very low levels of calories while some of the 50-60 year olds consumed very high levels. In addition, the data on the 70 year or over NV women may not be representative because of the small number (n = 8). The methodology employed to collect dietary data has been reviewed by Pao and Cypel [49]. Although no one method appears ideal for all types of studies, the 7-day dietary record would appear well-suited for studies of the present type. In this study the intake data are sup ported by the excretion data, i.e., the general shapes of the curves obtained when energy or protein intake and urinary nitrogen were plotted vs age are very similar to one another in either the AV or NV group. However, the existence of an interaction between the groups is ap parent from the groups, i.e., the AV intakes and excretion tend to drift down gradually with age whereas those of the NVs exhibit a definite initial rise and then a descent beginning about age 55 years. On the basis of these data, it would appear that both vegetarian and nonvegetarian groups had adequate levels of protein intake. The implications of the findings of such adequacy with the judicious use of vegetable pro tein in a time of increasing cost of protein are obvious.
Women
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Protein Status in Older Women niques." New York: Harper and Row, pp 292-296, 1967. 19. Bergman I, Loxley R: The determination of hydroxyproline in urine hydrolysates. Clin Chim Acta 27:347-349, 1970. 20. Hawk PB: "Physiological Chemistry." New York: McGraw-Hill, 1965. 21. Swaroop A: A micromethod for the determination of uri nary inorganic sulfates. Clin Chim Acta 46:333-336, 1973. 22. Tanner JM, Whitehouse RH: Standards for subcutaneous fat in British children. Br Med J 1:446-450, 1962. 23. Barr AJ: "Statistical Analysis System." Raleigh: SAS In stitute Inc., 1976. 24. Steele RGD, Torrie JH: "Principles and Procedures of Statistics." New York: McGraw-Hill, 1960. 25. Centers for Disease Control, US Department of Health, Education, and Welfare: 'Ten-State Nutrition Survey, 1968-1970: I. Dietary," DHEW Publication No (HSM) 72-8133. Atlanta: Centers, for Disease Control, 1972. 26. Consumer and Food Economics Research Division, Agricultural Research Service: "Food and Nutrient Intake of Individuals in the United States, Spring 1965," Household Food Consumption Survey, Report No 11. Washington, DC: Agricultural Research Service, 1972. 27. National Center for Health Statistics, Carroll MD, Abraham S, Dresser CM: "Dietary Intake Source Data: US 1976-1980," Vital and Health Statistics, Series 11, No 231, DHHS Publication No (PHS) 83-1681, Public Health Service. Washington, DC: US Government Printing Of fice, March 1983. 28. Gibson RS, Anderson BM, Sabry JH: The trace metal status of a group of postmenopausal vegetarians. J Am Diet Assoc 82:246-250, 1983. 29. Rouse IL, Armstrong BK, Beilin LJ: The relationship of blood pressure to diet and lifestyle in two religious populations. J Hypertension 1:65-71, 1983. 30. Hunt IF, Murphy NJ, Henderson C, Clark VA, Jacobs RM, Johnston PK, Coulson AH: Bone mineral content in postmenopausal women: comparison of omnivores and vegetarians. Am J Clin Nutr 50:517-523, 1989. 31. Nieman DC, Underwood BC, Sherman KM, Arabatzis K, Baroosa JC, Johnson M, Shultz TD: Dietary status of Seventh Day Adventist vegetarian and nonvegetarian elderly women. J Am Diet Assoc 89:1763-1769, 1989. 32. Food and Nutrition Board, National Research Council: "Recommended Dietary Allowances," 9th ed. Washington, DC: National Academy of Sciences, 1989. 33. Food and Agriculture Organization/World Health Or ganization/United Nations University: "Energy and Protein Requirements," Report of a joint FAO/WHO/UNU expert consultation. Geneva: World Health Organization, 1985. 34. Gersovitz M, Motil K, Munro HN, Scrimshaw MS, Young VR: Human protein requirements: assessment of the ade quacy of the current recommended dietary allowance for
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dietary protein in elderly men and women. Am J Clin Nutr 35:6-14, 1982. 35. Young VR, Bier DM, Pelle« PL: A theoretical basis for increasing current estimates of the amino acid require ments in adult man, with experimental support. Am J Clin Nutr 50:80-92, 1989. 36. Pelle« PL: Protein requirements in humans. Am J Clin Nutr 51:723-737, 1990. 37. Cheng AHR, Gomez A, Bergan JG, Lee TC, Onckeberg F, Chichester CO: Comparative nitrogen balance study be tween young and aged adults using three levels of protein intake from a combination wheat-soy-milk mixture. Am J Clin Nutr 31:12-22, 1978. 38. Crim MC, Calloway DH, Margen S: Creatine metabolism in men: Urinary creatine and creatinine excretion with creatine feeding. J Nutr 105:428-438, 1975. 39. Crim MC, Calloway DH, Margen S: Creatine metabolism in men: creatine pool size and turnover in relation to creatine intake. J Nutr 106:371-381, 1976. 40. Delanghe J, De Slypere JP, De Buyzere M, Robbrecht J, Wierne R, Vermeulen A: Normal reference values for creatine, creatinine, and carnitine are lower in vegetarians. Clin Chem 35:1802-1803, 1989. 41. Allison DJ, Walker A, Smith QT: Urinary hydroxyproline: creatinine ratio of normal humans at various ages. Clin Chim Acta 14:729-734, 1966. 42. Saleh AEC, Coenegracht JM: The influence of age and weight on the urinary excretion of hydroxyproline and cal cium. Clin Chim Acta 13:445^452, 1968. 43. Meilman E, Urivetzky MM, Rapoport CM: Urinary hydroxyproline peptides. J Clin Invest 42:40-49, 1963. 44. Lakshmanan F, Perera WDA, Scrimshaw NS, Young VR: Plasma and urinary amino acids and selected sulfur meta bolites in young men fed a diet devoid of methionine and cystine. Am J Clin Nutr 29:1367-1371, 1976. 45. Bodwell CE, Schuster EM, Brooks B, Womack M: Biochemical indices in humans of protein nutritive values. I. Urinary inorganic sulfate excretions at a high protein intake level. Nutr Rep Int 18:125-133, 1978. 46. Freyberg RH, Block WD, Fromer MF: A study of sulfur metabolism and the effect of sulfur administration in chronic arthritis. J Clin Invest 19:423^t35, 1940. 47. Sabry ZI, Shadarevian SB, Cowan JW, Campbell JA: Relationship of dietary intake of sulphur amino acids to urinary excretion of inorganic sulphate in man. Nature 206:931-933, 1965. 48. Barrows CH, Beauchene RE: Aging in nutrition. In Al banese AA (ed): "Newer Methods of Nutritional Bio chemistry." New York: Academic, pp 163-194,1970. 49. Pao EM, Cypel YS: Estimation of dietary intake. In Brown ML (ed): "Present Knowledge in Nutrition," 6th ed. Washington, DC: International Life Sciences Institute — Nutrition Foundation, pp 399-406, 1990. ReceivedJuly 1989; revision accepted July 1990.
VOL. 10, NO. 4
Journal of the American College of Nutrition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uacn20
Protein intake and urinary excretion of proteinderived metabolites in aging female vegetarians and nonvegetarians. a
a
M E Kunkel & R E Beauchene a
Department of Nutrition and Food Sciences, College of Home Economics, University of Tennesse, Knoxville 37996-1900. Published online: 02 Sep 2013.
To cite this article: M E Kunkel & R E Beauchene (1991) Protein intake and urinary excretion of protein-derived metabolites in aging female vegetarians and nonvegetarians., Journal of the American College of Nutrition, 10:4, 308-314, DOI: 10.1080/07315724.1991.10718157 To link to this article: http://dx.doi.org/10.1080/07315724.1991.10718157
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Protein Intake and Urinary Excretion of Protein-Derived Metabolites in Aging Female Vegetarians and Nonvegetarians M. Elizabeth Kunkel,« PhD, FACN and Roy E. Beauchene, PhD, FACN Department of Nutrition and Food Sciences, College of Home Economics and Agricultural Experiment Station, The University of Tennessee, Knoxville
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Key words: aging, energy intake, protein intake, protein metabolism, nitrogen excretion, vegetarian Relationships among age, protein intake, and urinary excretion of protein-derived metabolites were studied in 125 vegetarian and nonvegetarian women ages 40-92. There were 63 women in the vegetarian (AV) group and 62 women in the nonvegetarian (NV) group. Average daily intakes of energy and total animal and vegetable protein were calculated from 7-day dietary records. Twenty-four-hour urine samples were analyzed for total nitrogen, urea, creatinine, hydroxyproline, and inorganic sulfate. Energy intakes for the two groups were similar. AVs consumed less total and animal protein and more vegetable protein than NVs, even though both groups consumed more than the RDA for protein. No significant differences existed between the groups in the urinary excretion of total nitrogen, urea nitrogen, hydroxyproline, or inorganic sulfate. Energy and protein intakes and total nitrogen excretion were lower in older AVs than in younger AVs, while those of NVs increased between 40 and 55 years of age, and decreased among the older NV women. The relationship between these variables and age in NVs was more accurately described by polynomial rather than linear regression models. Abbreviations: AV = vegetarian, HOP = hydroxyproline, N = nitrogen, NV = nonvegetarian, RDA = recommended dietary allowance
INTRODUCTION
METHODS
Lactoovo- and pure (vegan) vegetarian diets are generally considered to be nutritionally adequate and have been reported to contain ample amounts of total protein [1-7] and indispensable amino acids [8]. In addi tion, vegetarian subjects have been reported to utilize their dietary protein at least as well as nonvegetarians [2,9], although differences in urea excretion have been found [10]. In addition to the effect of diet, protein meta bolism may also be affected by age [11,12] and body protein mass [13]. This study was concerned with the relationships among age, protein intake, and the urinary excretion of protein-derived metabolites in older adult vegetarian and nonvegetarian females.
The sample consisted of 125 vegetarian and non vegetarian females who were recruited primarily through religious and civic organizations. Of the 63 women who were vegetarians (AVs), 57 were lactoovovegetarians and six were vegans. Given the small size of the vegan group, lactoovovegetarians and vegans were pooled into the vegetarian group. The remaining 62 subjects were nonvegetarians (NVs). All women were ambulatory. On a general health questionnaire, none of the subjects reported any physical or metabolic impairments that would have affected any of the parameters measured in this study. AVs were defined as those subjects who typically consumed no
Presented, in part, at the 22nd Annual Meeting of the American College of Nutrition. ♦Present address: Food Science Department. College of Agricultural Sciences. Clemson University, Clemson, South Carolina 29634-0371. Addressreprintrequeststo Roy E. Beauchene. Department of Nutrition and Food Sciences. College of Home Economics. The University of Tennessee, Knoxville, Tennessee 37996-1900.
Journal of the American College of Nutrition, Vol. 10, No. 4, 308-314 (1991) © 1991 John Wiley & Sons, Inc.
CCC 0731 -5724/91/040308-07$04.00
Protein Status in Older Women Table 1. Descriptive Data of Subjects
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Parameter Age (years) Mean 1 SEM Range Duration of vegetarianism (years) Mean ± SEM Range Height (cm) Mean ± SEM Range Weight (kg) Mean 1 SEM Range
meat products but did use egg and dairy products. After the details of the study were described to them, each subject signed an informed consent form. Each subject was provided measuring cups, a ruler, and forms in addi tion to oral and written instructions for recording food intake for 7 days. Food intakes were recorded as close approximations of the amounts consumed, with the inclusion of brand names and methods of preparation where applicable. Subjects were asked to supply recipes when possible. A dietary history was also recorded for each subject. Any questionable entries on the food records were checked against the dietary histories; if further clarifications were needed, the entries were discussed directly with the sub ject. The food intakes were summarized and nutrient in takes determined by mainframe computer using a com puter program developed at The University of Tennessee which is based on the composition data found in USDA Handbook No 8 [14], with additional information sup plied by various food manufacturers. Each item in Hand book No 8 was coded as to animal, vegetable, or mixed protein source. Mixed protein sources included cakes, cookies, breads, and pasta entries. Intakes were ex pressed as a mean for the 7-day recording period. Each subject collected a 24-hr urine sample on the third or fourth day of the week in which the 7-day dietary record was kept. The sample was collected in an acid-rinsed polyethylene bottle containing 5 ml of a 10% (w/v) thymol-2-propanol solution. Completeness of the urine samples was determined based on creatinine excretion. The urine was analyzed for urea nitrogen by the Berthelot reaction [15-17], for creatinine by Folin's method [18], for hydroxyproline (HOP) by a modification of the
Vegetarians (n = 63)
Nonvegetarians (n = 62)
57.4 ± 1.4 36.0-92.0
61.511.3 41.0-82.0
36.4 ± 4.8 2.0-70.0
—
161.810.8 146.7-174.6
160.710.9 137.1-172.7
63.2+1.6 39.1-98.0
66.1 11.5 44.5-99.1
method of Bergman and Loxley [19], for total nitrogen by the macro-Kjeldahl method [20], and for inorganic sulfate by the method of Swaroop [21]. Height, weight, and skinfold thickness were measured on each subject. Height was measured to the nearest quarter inch and weight to the nearest pound with the subject dressed in light indoor clothing and wearing no shoes. Skinfold thickness was measured to the nearest mm using Lange (Cambridge Scientific Industries Inc., Cambridge, MD) calipers and following the method described by Tanner and Whitehouse [22]. Triplicate readings were taken on the left triceps of each subject midway between the tip of the acromion process and the top of the radius. Data were analyzed using the Statistical Analysis Sys tem (SAS) [23]. The relationship between a response variable and age was characterized with a model con taining linear, quadratic, and cubic terms for age as well as a dietary classification variable, i.e., AV or NV. Also included in the model were the linear, quadratic, and cubic interactions with the classification variable. If in teractions between age and the classification variable were found to be significant (p < 0.05), then separate relationships for each group were estimated and are presented graphically. For those response variables without significant interaction, the significance of dif ferences among least square mean values, adjusted for the mean age of the sample, were tested using Duncan's New Multiple Range Test [24]. Least square mean values for response variables with significant interaction (ener gy and protein intakes, total urinary nitrogen) were cal culated for comparison with literature values.
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Fig. 1. Regressions of body weight and triceps skinfold thickness on age, NVs represented by a solid line and AVs by a dotted line. Equations for the regression lines and their significances are as follows: Body weight: NV = 177.07 - 3.30(age) + 0.02(age 2 ), AV = -18.78 + 2.97(age) - 0.02(age 2 ); triceps skinfold thickness: NV = 78.65 - 1.23(age) + 0.01(age 2 ), AV = -24.86 + 1.85(age) - 0.02(age 2 ).
RESULTS Table 1 gives descriptive data for the subjects. Regression equations describing the relationships be tween age and body weight and for age and skinfold thickness for the AV and NV groups are shown in Figure 1. The NVs tended to have a lower weight with increas ing age until about age 70, when a tendency toward in creasing weight was observed. The reverse was true in the AVs, who tended to have the greatest body weight at age 60 years and then decline. There was a significant difference between AV and NV groups in skinfold thick ness which was related to age, the quadratic function of age, and the interaction term between age and type of diet. The NVs showed a gradual decrease in skinfold thickness throughout the age span studied. The AVs had
310
10
4 0
■ / / -
50
60 AGE
70
(years)
Fig. 2. Regressions of energy and protein intakes and total uri nary nitrogen excretion on age, NVs represented by a solid line and AVs by a dotted line. Equations for the regression lines and their significances are as follows: Energy intake: NV = -1724.91 + 978.05(age) - 16.55(age2) + 0.091 (age3), AV = 1818.30 + 10.38(age) -0.427(age2) + 0.003(age3). The overall shapes of the energy intake curves are different (p < 0.05); decrements with age were significant in both groups (p < 0.01); differences in mean energy intakes between NVs and AVs were not significant (p > 0.05). Protein intake: NV = -576.76 + 32.85(age) 0.54(age2) + 0.003(age3), AV = 153.44 - 4.60(age) + 0.07(age2) - 0.0004(age3). The overall shapes of the protein intake curves are different (p < 0.05); differences with age are significant in both groups (p < 0.01); NVs consumed more protein than AVs (p < 0.001). Urinary nitrogen: NV = -155.44 + 8.10(age) O.tfCage2) + 0.0O07(age3), AV = -8.48 + 0.84(age) - 0.01(age2) + 0.00007(age3). The overall shapes of the curves are different (p < 0.05); neither differences with age nor mean differences in N excretion between NVs and AVs were significantly different (p > 0.05).
a peak skinfold thickness about age 60 followed by a gradual loss. Regression equations describing the relationships be tween age and energy and total protein intakes and be tween age and urinary total nitrogen for AV and NV groups are shown in Figure 2. These variables exhibited
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Protein Status in Older Women Table 2. Energy and Protein Intakes of Aging Female Vegetarians and Non vegetarians1 Dietary component
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Energy (kcal/day) Total protein (g/day) Animal protein (g/day) Vegetable protein (g/day) Mixed protein (g/day)3
Vegetarians (n = 63) 1500 ±42 a2 53.6 ± 1.6a 14.8 ± 1.6a 29.1 ±1.1» 10.5 ±0.6"
Nonvegetarians (n = 62) 1524 ±42 a 66.5 ± 1.6b 45.8±1.7b 10.8±l.l b 10.3±0.6a
Values ate means ± SEM adjusted for mean age of samples (59.4 years). Means in a row not sharing a common superscript are significantly different (p < 0.05). Includes foods containing both animal and vegetable proteins.
Table 3. Excretion of Urinary Components by Aging Female Vegetarians and Nonvegetarians1 Metabolite Total nitrogen (g/day) Urea nitrogen (g/day) Creatinine (g/day) Hydroxyproline (mg/day) Inorganic sulfate (g/day)
Vegetarians (n = 63)
Nonvegetarians (n = 62)
8.20 ± 0.36a2 6.92 ± 0.36a 1.31±0.05a 32.1 ± 1.7a 1.49±0.07a
9.00 ± 0.36a 7.77 ± 0.36a 1.54±0.05b 35.5 + 1.7a 1.57±0.07a
Values are means ± SEM adjusted for mean age of samples (59.4 years). Means in a row not sharing a common superscript are significantly different (p < 0.05).
significant age-associated differences and interactions between the dietary groups. The general shape of all three curves within a dietary group is similar. For ex ample, energy intake, protein intake, and nitrogen excre tion of NVs showed increases until age 50-55 years, followed by decreases until about 70 years, with a ten dency to increase thereafter. The AVs showed small gradual decreases in protein intakes and urinary nitrogen with age. Mean daily energy intakes in NVs varied from 1660 calories at age 55 to a low of 1340 calories at age 70; highest energy intakes for AVs were observed at 45 years (1660 cal) and the lowest at 70 years (1370 cal). Differences in energy intakes between AVs and NVs at corresponding ages were not significant. Mean daily total intakes of protein of NVs varied from 71 g at age 55 to 61 g at age 70; values for AVs ranged from 56 g at age 45 to 52 g at age 70. Total protein intakes were significantly higher for NVs than AVs. Total urinary nitrogen peaked at 9.2 g day"1 in NVs; at 45 years it was
7.6 g day 1 , and at age 70, 7.5 g day 1 . Total urinary nitrogen for AVs ranged from 8.6 g day 1 at age 50 to 7.8 g day"1 at age 70. Comparisons at specific ages revealed no significant differences in total nitrogen excretion be tween AVs and NVs. Age-adjusted mean daily protein intakes from animal, vegetable, and mixed sources are shown in Table 2. The AV women consumed significantly less animal protein and more vegetable protein than the NV women. These differences were significant regardless of whether the protein intake data were expressed as an absolute amount or as a percentage of either energy or total protein intake. Age-adjusted excretions of urinary components are given in Table 3. Urea nitrogen, HOP, and inorganic sul fate excretions did not differ significantly among the groups. Vegetarian subjects excreted significantly less creatinine than the NV subjects.
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Protein Status in Older Women
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DISCUSSION Energy intakes for both groups were less than intakes reported for subjects in the Ten-State Nutrition Survey [25], the USDA Nationwide Food Consumption Survey [26], and the Second Health and Nutrition Examination Survey (HANES Π) [27]. Energy intakes of subjects in the present study were also less than those obtained using duplicate weighing and chemical analysis techni ques [5], dietary records [1,6,7,28,29], or food frequency questionnaires [4]. The lack of significant differences in body weight between AVs and NVs in the present study agrees with results reported by other researchers [10,2931] but disagrees with results reported by Tylavsky and Anderson [4]. The significantly lower triceps skinfold thickness in AV than in NV subjects agrees with findings by Rouse et al [29], while Armstrong et al [10] did not report a significant difference in skinfold thickness be tween vegetarian and nonvegetarian subjects. Activity levels of the subjects in the present study were not measured. The age-adjusted mean total protein intakes of both groups exceeded the 0.8 g/kg of body weight RDA [32] and the 0.6 g/kg body weight standard used by the FAO/WHO/UNU [33]. The mean protein intake of AVs was less than the 1 g/kg of body weight standard used for the Ten-State Nutrition Survey [25] and HANES II [27] and also less than the 0.9 g/kg of body weight standard used for the USDA Nationwide Food Consump tion Survey [26]. The adequacy of the RDA for protein for older men and women has been questioned [34] and various alternative requirements proposed [12,35]. Pellett [36] discussed the changes in data on amino acid requirements and in digestibility and concluded that the "two changes in opposite directions have the effect of leaving the allowance of US dietary protein almost un changed." Uauy et al [12] estimated the mean protein requirement for elderly women to be 0.83 g/kg body weight day 1 . Using that standard, protein requirements were calculated to be for AVs 44 g day 1 and for NVs 55 g day 1 ; both groups exceeded these requirements. Both groups consumed less protein than the vegetarian and nonvegetarian subjects used by Hardinge et al [l], Beilin et al [6], and Rouse et al [29], but had similar intakes to the subjects used by other researchers [4,5,7,28,30]. Even though the total protein intake of the vegetarian subjects was less than that of the nonvegetarian subjects, the excretion of urea nitrogen between the groups did not differ significantly (Table 3). Mean total urinary nitrogen multiplied by 6.25 (equivalent to protein excreted in the form of nitrogen) amounted to 87 and 97% of the mean protein intake of NVs and AVs, respectively.
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Urinary N excretion accounted for only 76% of the daily N intake in the male and female vegetarians used by Abdulla et al [5]. Factors which may contribute to the apparent differences in metabolism of protein by AVs and NVs are the use of available food composition data for many of the nonmeat entrees consumed by AVs and/or the use of a single 24-hr urine sample to estimate nitrogen excretion during the week in which dietary records were kept. Since fecal nitrogen was not deter mined, it is impossible to assess actual nitrogen utiliza tion. Nitrogen utilization has been reported to be more efficient in persons consuming a vegetarian diet than in persons consuming a nonvegetarian diet [9], and to be relatively unaffected by age [37]. Armstrong et al [10] reported that vegetarians excreted significantly less urea than nonvegetarians. However, no protein intake data for their subjects were reported. The lower creatinine excretion in vegetarians may reflect dietary influences upon the excretion of this com pound or differences in lean body mass between AVs and NVs. Crim et al [38,39] reported that the body creatine pool and hence creatinine excretion was decreased by feeding a creatine-free (nonmeat) diet. Intake of animal protein was significantly correlated with creatinine ex cretion in both AVs (r = 0.44; p < 0.001) and NVs (r = 0.33; p < 0.01). Lin et al [9], working with female sub jects, noted lower creatinine excretions in vegetarians than in nonvegetarians. They attributed this difference to a low protein reserve and turnover in the vegetarians as a result of adaptation to a low protein intake. Delanghe et al [40] stated that "prolonged depletion of dietary creatine results in decreased creatinine production, owing to insufficient endogenous compensatory creatine synthesis," and proposed reference values for excretion of creatinine in vegetarians. The creatinine excretion values of AV and NV subjects were higher than these proposed reference values [40]; however, the female subjects used in establishing these reference values were younger (mean age 35.3 ± 9.8 years) than the subjects used in the present study. The mean HOP excretions by all groups were in agreement with those reported by Allison et al [41] and Saleh and Coenegracht [42] for older adults. Since dietary HOP is largely of animal origin and is excreted quantitatively in the urine [43], HOP excretion by AVs would essentially represent endogenous collagen meta bolism. The significant correlation (r = 0.31; p < 0.01) between HOP excretion and animal protein intake in AVs may indicate consumption of at least some HOP by AV subjects. Urinary inorganic sulfate values for both groups are similar to those reported by most other researchers for adult subjects [44-46]. Bodwell et al [44] found that
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inorganic sulfate excretion in adult males was increased by feeding diets limited in one or more of the indispen sable amino acids or containing high levels of sulfurcontaining amino acids. However, Sabry et al [47] noted that urinary sulfate excretion by young adult males was lower when they were fed a rice and beans diet than a bread and eggs diet. The sulfate values reported by Sabry et al [47] were much less than those observed in the present study. The inorganic sulfate excretion by AVs did not differ from that of NVs, even though the vegetarian diet has the potential for being limiting in the sulfur-con taining amino acids. It should be pointed out that the textured vegetable protein products which were con sumed extensively by the vegetarian subjects in this study were frequently fortified with the limiting amino acid or contained egg albumin as a binding agent. It has been generally accepted that age-associated changes in physiological functions are best represented as a linear function of age [48]. In the present study, polynomial regression models were found to more ac curately describe the relationship between age and some variables. The regression lines presented were not drawn for the full range of subjects because of the inherent tendency of a polynomial to magnify variations at the extremes of the data. The increase in energy and protein intakes until the sixth decade followed by decreases in the seventh decade for NVs as compared to the gradual decreases observed for these variables with age in AVs may reflect, in part, sampling bias. Of the NV subjects, some of the youngest consumed very low levels of calories while some of the 50-60 year olds consumed very high levels. In addition, the data on the 70 year or over NV women may not be representative because of the small number (n = 8). The methodology employed to collect dietary data has been reviewed by Pao and Cypel [49]. Although no one method appears ideal for all types of studies, the 7-day dietary record would appear well-suited for studies of the present type. In this study the intake data are sup ported by the excretion data, i.e., the general shapes of the curves obtained when energy or protein intake and urinary nitrogen were plotted vs age are very similar to one another in either the AV or NV group. However, the existence of an interaction between the groups is ap parent from the groups, i.e., the AV intakes and excretion tend to drift down gradually with age whereas those of the NVs exhibit a definite initial rise and then a descent beginning about age 55 years. On the basis of these data, it would appear that both vegetarian and nonvegetarian groups had adequate levels of protein intake. The implications of the findings of such adequacy with the judicious use of vegetable pro tein in a time of increasing cost of protein are obvious.
Women
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