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Sweat nitrogen losses of young adult females in nigeria with different levels of dietary protein intake
NUTRITION RESEARCH, Vol. 12, pp. 199-208,1992 0271-5317/92 $5.00 + .00 Printed in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
SWEAT NITROGEN LOSSES OF YOUNG ADULT FEMALES LEVELS OF DIETARY PROTEIN INTAKE.
IN NIGERIA WITH DIFFERENT
Gloria N. Egun, PhD. 1 and Tola Atinmo, PhD. Department of Human Nutrition, college of Medicine, University of Ibadan, Nigeria.
ABSTRACT Nitrogen balance studies were carried out in two separate experiments involving a total of fifteen acclimatized young women. The aim was to determine the effects of different levels of protein intake on sweat nitrogen losses. The losses were determined through the collection of 24 hour total body sweat samples under controlled environmental conditions. In nine of the subjects fed on graded level of protein for a ten-day period at a time, sweat nitrogen losses varied with nitrogen intake, being 5.45~0.79, 6.36Z0.70, 6.65~0.73 and 7.10Z0.87mg N/kg/day at protein intakes of 0.3, 0.4, 0.5 and 0.6 protein/kg/day, respectively. Obligatory nitrogen loss in sweat extrapolated from linear regression analysis of nitrogen intake versus sweat nitrogen loss was 4.20 mg N/kg/day. When the subjects were fed a single level of protein (0.6 g/kg/day) for forty days, the mean sweat nitrogen loss of the women was 9.45• mg/kg/day. Thus the recommended allowance for sweat nitrogen losses (3-4 mg N/kg/day) in estimating protein requirement by the 1985 FAO/WHO/UNU expert report is an underestimation under conditions prevailing in tropical countries. Key words:
Sweat Nitrogen,
Protein Intake,
Nitrogen
Balance.
INTRODUCTION
Most studies undertaken to assess protein requirements have either excluded protein needed for the integumental nitrogen losses as negligible and unlikely to be affected by diet (i) or have used 3-4 mg N/kg/day for sweat nitrogen losses as suggested by the 1985 FAO/WHO/UNU report (2). The estimate used in the FAO/WHO/UNU report was derived mainly from studies conducted under moderate ambient temperatures and correspond to obligatory losses (3). The applicability of this figure for N-balance studies conducted under conditions of high environmental temperatures such as exists in the tropics and at protein intakes in the submaintenance to maintenance range has been questioned, since increased sweating and nitrogen intake affect skin losses (4-6). Furthermore, loss of nitrogen via one route may be compensated for by changes in the loss of nitrogen via another. A compensatory reduction of urinary nitrogen when sweat nitrogen loss increased has been observed by many
Address correspondence to : Dr. Gloria N. Egun, Department of Food Science and Technology, University of Reading, Whiteknights, P.O.Box 226, Reading RG6 2AP, United Kingdom.
199
G.N. EGUN and T. ATINMO
200
i n v e s t i g a t o r s (7-97. Therefore, unless all routes of n i t r o g e n losses measured, erronous conclusions may be drawn from a p p a r e n t n i t r o g e n b a l a n c e and in consequence r e q u i r e m e n t s underestimated.
are
The purpose of this study was to estimate the sweat n i t r o g e n losses of subjects living under tropical climatic conditions w h e r e i n c r e a s e d s w e a t i n g may alter total nitrogen needs. U n i v e r s i t y s t u d e n t s were studied, as a basis of c o m p a r i s o n with similar studies e l s e w h e r e (3,4 and 9). Also t h e y p r o v i d e an average estimate for sweat loss under c o n d i t i o n s p r e v a i l i n g in d e v e l o p i n g countries, as their a c t i v i t y pattern is moderate.
METHODS
AND M A T E R I A L S
Subjects Fifteen young w o m e n all p h y s i c a l l y healthy p a r t i c i p a t e d in the study. The subjects were all fully a c c l i m a t i z e d to a high e n v i r o n m e n t a l temperature. They lived under close supervision in t h e i r hall of r e s i d e n c e and e n g a g e d in normal routine daily a c t i v i t i e s w h i c h included their full academic schedule, while m a i n t a i n i n g a r e a s o n a b l e constant level of physical activity during the study periods. The subjects w e r e aged b e t w e e n 21 and 32 yea~s. T h e i r body weights and heights ranged between 45 to 70 kg and 154 to 170 cm, respectively. Body surface e s t i m a t e d from a nomograph was b e t w e e n 1.40 and 1.80 m . Body w e i g h t was m e a s u r e d daily before breakfast i~ order to assess the a d e q u a c y of caloric intake. The e n v i r o n m e n t a l conditions were u n c o n t r o l l e d w h i l e t e m p e r a t u r e and relative humidity were recorded at a nearby e x p e r i m e n t a l station. F r o m the readings provided, average values were r e c o r d e d for periods of sweat collection. Diet The diets were s t a n d a r d i z e d based on foods h a b i t u a l l y consumed and g i v e n in meal p a t t e r n s which the subjects were a c c u s t o m e d to, that is, 7-8 am, 1-2 pm and 7-8 pm. Throughout the study, all subjects were r e q u i r e d to adhere to the experimental diet and eat only the food p r e p a r e d by the investigators. Table 1 shows the c o m p o s i t i o n of the diet. A d j u s t m e n t s in the protein intake were made by i n c r e a s i n g the d i e t a r y beef intake. Complete vitamins or m i n e r a l s w e r e lacking. E n e r g y intake for individual subject was kept constant t h r o u g h o u t the entire study periods. Experimental
Desiqn
Two nitrogen b a l a n c e studies were carried out. the first e x p e r i m e n t (Expt. i) was based on short-term feeding of g r a d e d levels of p r o t e i n at 0.3, 0.4, 0.5 and 0.6 g p r o t e i n / k g / d a y for a p e r i o d of i0 days per p r o t e i n level. The second experiment (Expt. 2) was a 4 0 - d a y c o n t i n u o u s feeding of a single level of protein.
201
SWEAT NITROGEN AND PROTEIN INTAKE
TABLE
Composition
of Diet,
1
Ingredients
and Nutrients
Ingredients
(g)
Bread Refined Cane Sugar Margarine Pepper (dried) Tomatoes (fresh) Onions Palmoil Cassava (grated) Beef Vegetables Yam (cooked) Orange flavoured drinks* (bottles) + Nutrients: Protein (g) Energy (kcal) * +
1 botle contains 120 kcal. Lowest protein intake in the study
90 30 30 4 80 40 60 i00 i0 14 200 (2) 15.68 2168.35
(providing
0.3 g protein/kg/day)
In experiment i, nine women participated and the study lasted 56 days consisting of four experimental diet periods each of lO days duration in which the four different dietary levels of protein were fed. The experiment followed the standard protocol for short-term nitrogen balance studies with graded levels of protein as previously described (i0). In experiment 2, the six subjects were fed 0.6 g protein/kg/day for 40 days continuously. This level of protein is an estimated dietary allowance for young university women in Nigeria. The 40 days were arranged into two consecutive 20 day diet periods. The metabolic periods were taken as days 16-20 and 36-40. Sample and Analysis Skin nitrogen loss through sweat was collected for two days at the end of the metabolic period of each experimental dietary level. Sweat was collected by the total body method similar to techniques described by Spence, Abernathy and Ritchey (ii) and Sirbu, Margen and Calloway (12) with slight modifications. Prior to each period of sweat colection, all towels, washed clothes, beddings and clothings to be used by the subjects including their underwears on individual basis were soaked overnight in 0.5% acetic acid and rinsed six times in distilled deionized water to remove any trapped nitrogen to ensure proper estimation of the loss accountable to sweat during the collection periods. Colour fast clothings were chosen to minimize nitrogen contribution from dyes and to maximize absorption of sweat. At the onset of collection, subjects washed their bodies thoroughly, rinsed with distilled water and towel dried
202
G.N. EGUN and T. ATINMO
v i g o r o u s l y w i t h a nitrogen free towel. The bath w a t e r as well as the towel w e r e discarded. The subjects were d r e s s e d in their n i t r o g e n - f r e e c l o t h i n g s c o n s i s t i n g of underwear and t h r e e - q u a r t e r length cotton dresses. E a c h subject was given a m a r k e d towel (wash cloth) w h i c h was used to d r y any v i s i b l e sweat from her face, neck, arm and legs d u r i n g the c o l l e c t i o n period. Subjects were c a u t i o n e d against c o n t a m i n a t i n g their u n d e r w e a r w i t h urine and faeces. The subjects w o r e the same c l o t h i n g for two days. At the end of this period, the subjects r e m o v e d all the c l o t h i n g and bathed thoroughly by s c r u b b i n g w i t h o u t soap (so as not to c o n t r i b u t e any unknown source of nitrogen) in s p e c i f i e d amounts of d e i o n i z e d d i s t i l l e d water, about 20-30 litres, and then towel dried vigorously. A sample of bath water was saved in t h o r o u g h l y rinsed plastic sample bottle with d i s t i l l e d - d e i o n i z e d w a t e r and stored at -20~ until a n a l y s e d for nitrogen. The sweat and skin losses were e x t r a c t e d from the clothings, w a s h cloths, b e d d i n g s and towels by soaking them in s p e c i f i e d volumes of 0.05% acetic acid solution for 48 hours, and a sample of the soaking solution t a k e n in individual labelled plastic c o n t a i n e r s and stored at -20~ until analysed. This method assumes the garments and soaking s o l u t i o n to be at equilibrium. A similar study by C a l l o w a y et al. (3) showed that conventional washing and r i n s i n g p r o c e d u r e s did not c o m p l e t e l y remove the nitrogen from the cloth. However, the d i f f e r e n c e in amount of nitrogen recovered after repeated r i n s i n g was w i t h i n the limits of a c c e p t e d error (+ 10%). U r i n e was collected for each 24 hour p e r i o d and faeces, which were m a r k e d w i t h carmine were pooled for 4 to 5 day periods. Urine was a n a l y s e d for nitrogen and creatinine (which was used to judge the c o m p l e t e n e s s of urine collection) and the n i t r o g e n content of the faeces were determined. The n i t r o g e n content of sweat collection, urine, faeces and food samples w e r e analysed using the m o d i f i e d K j e l d a h l - C u n n i n g - A r n o l d method with c o p p e r and selenium catalysts (13). Blood samples were taken before b r e a k f a s t at the b e g i n n i n g and end of each m e t a b o l i c period. Plasma was analysed for urea nitrogen by the m a n u a l c a r b a m i n o d i a c e t y l reaction of Marsh, F i n g e r h u s t and M i l l e r (14).
RESULTS
In e x p e r i m e n t i, total nitrogen loss through the skin averaged 5.45~0.79, 6.36~0.70, 6.65~0.73 and 7.10~0.87 mg N / k g / d a y on p r o t e i n intakes of 0.3, 0.4, 0.5 and 0.6 g protein/kg/day, respectively. Table 2 shows the mean total nitrogen loss in skin as well as the n i t r o g e n c o n t e n t of each fraction of sweat collection. It was observed that the n i t r o g e n adhering to clothes was much larger than the n i t r o g e n retained on the skin. Clothes nitrogen was 71% of total sweat collection.
SWEAT NITROGEN AND PROTEIN INTAKE
203
TABLE 2 Nitrogen
Content
of Fractions
Analysed
and Total
Skin Loss
in Young
Women.
Component
0.3
Clothes Body Total skin loss
Level of Protein 0.4
3.89~0.63 1.59Z0.19 5.45•
Intake
4.50Z0.64 1.86~0.23 6.36•
(g/kg/day) 0.5
4.76~0.60 1.89• 6.65~0.73
0.6 5.10Z0.69 2.00• + 7.10~0.87
E x p r e s s e d as m e a n • SD of nine subjects (in mg N/kg/day). S i g n i f i c a n t d i f f e r e n c e s in changes w i t h levels of p r o t e i n intake at p < 0.05 (2-way ANOVA). The m e a n sweat nitrogen losses (skin) e x p r e s s e d per 24 hours and per square m e t r e of body surface per 24 hours i n c r e a s e d as the level of d i e t a r y p r o t e i n increased (Table 3). Individual subjects v a r i e d somewhat in m e a n sweat n i t r o g e n values, but differences in m e a n sweat nitrogen losses, as d e t e r m i n e d by analysis of variance (2-way ANOVA) were significant at p < 0.05. Individual v a r i a t i o n in sweat n i t r o g e n values can be a t t r i b u t e d to variations in activity and i n d i v i d u a l characteristics. I n c r e a s e in n i t r o g e n intake resulted in a c o r r e s p o n d i n g increase in skin e x c r e t i o n of nitrogen. The correlation was p o s i t i v e and significant (r = +0.97, p < 0.05). The m e a n regression e q u a t i o n of individual subjects is r e p r e s e n t e d as Y = 4.2 + 0.03X, w h e r e Y is skin n i t r o g e n loss in mg N / k g / d a y and X is nitrogen intake in mg/kg/day. The intercept of the r e g r e s s i o n w h e n intake was zero was 4.2 mg N/kg/day. This was taken as the o b l i g a t o r y nitrogen loss through the skin. TABLE Dietary Nitrogen Young W o m e n . Nitrogen g/day 2.51 3.42 4.38 5.23
Intake
Intake,
Skin Nitrogen
Skin Nitrogen mg/day 286.0Z74.1 327.8Z70.0 349.5Z71.5 370.7~84.1
3 Loss
and P l a s m a
Loss mg/m2/day 182.3Z39.1 209.5~37.2 222.9~35.4 236.4~42.0
Urea Nitrogen
Plasma
in
Urea Nitrogen mg/100ml I
6.09• 6.72Z0.70 + 7.54~i.06 + + 8.29~0.95
Values are e x p r e s s e d as mean ~ SD of nine subjects. M e a n v a l u e s were significantly different from the other the c o l u m n (2-way ANOVA) : p < 0.05 .
values
in
Skin n i t r o g e n loss increased with increase in p l a s m a urea nitrogen (Table 3). A c o r r e l a t i o n of the two showed a p o s i t i v e s i g n i f i c a n t r e l a t i o n s h i p (r = +0.96, p < 0.05). Plasma urea nitrogen i n c r e a s e d
G.N. EGUN and T. ATINMO
204 significantly
w i t h increase
in p l a s m a
intake
(p < 0.05 w i t h 2 - w a y
ANOVA). E n v i r o n m e n t a l c o n d i t i o n s w e r e fairly constant. The t e m p e r a t u r e and o r e l a t i v e h u m i d i t y w e r e b e t w e e n 25.5 and 26.6 C and 79.9 and 83%, r e s p e c t i v e l y . The slight d i f f e r e n c e s o b s e r v e d in t e m p e r a t u r e and r e l a t i v e h u m i d i t y w e r e not s t a t i s t i c a l l y different, thus we can s a f e l y c o n c l u d e that the o b s e r v e d d i f f e r e n c e s in sweat loss w e r e due to the c h a n g e in the level of p r o t e i n intake. A summary of n i t r o g e n b a l a n c e at the r e s p e c t i v e levels of p r o t e i n intake (Table 4) i l l u s t r a t e s the e f f e c t s of ignoring sweat n i t r o g e n losses. N i t r o g e n r e t e n t i o n was o v e r e s t i m a t e d w i t h o u t the i n c l u s i o n of sweat nitrogen. TABLE
4
Mean Urinary, Faecal and Sweat N i t r o g e n Losses Four L e v e l s of D i e t a r y P r o t e i n Intake.
and N i t r o g e n
Balance
at
NB
Level I II III IV
NI 48.5~5.7 66.0~7.5 84.5~9.8 + I01.7Z12.5
UN 41.4~4.7 47.0~3.8 53.6+5.7 64.9~4.5 +
FN 15.3~2.77 16.6• 16.5+3.83 16.1~3.82 ~
SN 5.45~0.79 6.36• 6.65~0.73 7.10+0.87 +
I-U-F - 9.7 + 1.0 +12.8 +19.2 +
I-U-F-S -15.24 - 5.45 + 6.25 +12.15
NI = N i t r o g e n intake; UN = U r i n a r y nitrogen; FN = Faecal nitrogen; SN = Sweat nitrogen; NB = N i t r o g e n balance; I-U-F = I n t a k e - U r i n a r y F a e c a l nitrogen; I-U-F-S = Intake - U r i n a r y - Faecal - Sweat nitrogen. * M e a n v a l u e s e x p r e s s e d as m e a n ~ SD of nine subjects. + Mean values significantly d i f f e r e n t from the o t h e r v a l u e s in the c o l u m n (2-way ANOVA) at p < 0.05 . # M e a n v a l u e s not s i g n i f i c a n t l y d i f f e r e n t from the o t h e r v a l u e s in the c o l u m n (2-way ANOVA) at p < 0.05 . $ V a l u e s of n i t r o g e n b a l a n c e s are s i g n i f i c a n t l y d i f f e r e n t (paired t - t e s t at p < 0.05). In e x p e r i m e n t 2, sweat n i t r o g e n was found to be 9 . 7 4 ~ 0 . 4 4 mg N/kg/day. Table 5 shows the mean sweat loss c o l l e c t e d t w i c e d u r i n g the e x p e r i m e n t b e t w e e n days 18-20 (first d i e t a r y interval) and 38-40 (second d i e t a r y interval). Their sweat loss wa~ e x p r e s s e d as mg N / k g / d a y as well as per total body surface area (mg N/m /day). The s u b j e c t s had a fairly c o n s t a n t sweat loss which did not change w i t h time. A t m o s p h e r i c t e m p e r a t u r e was also found to be fairly constant t h r o u g h o u t the p e r i o d of the experiment. M e a n t e m p e r a t u r e of the two p e r i o d s of sweat c o l l e c t i o n was r e c o r d e d at 24.5~
205
SWEAT NITROGEN AND PROTEIN INTAKE TABLE
5
Sweat N i t r o g e n L o s s e s of Six Young W o m e n an 4 0 - D a y C o n t i n u o u s 0.6 g P r o t e i n / k g / d a y Nitrogen in Sweat
Loss
mg N / d ~ y mg N m/ /day mg N / k g / d a y
Days Mean 546.90 333.10 9.74
16-20 SD 83.87 25.13 0.42
Days Mean 547.60 343.40 9.75
Feeding
on
38-40 SD 88.04+ 27.04+ 0.35
All v a l u e s are e x p r e s s e d as m e a n ~ SD of six subjects. No s i g n i f i c a n t d e f f e r e n c e b e t w e e n days of c o l l e c t i o n (paired t - t e s t at p < 0.05).
DISCUSSION
M i t c h e l l a n d H a m i l t o n (15) w e r e the first to p r o p o s e that sweat n i t r o g e n loss be included in c a l c u l a t i o n s of human p r o t e i n r e q u i r e m e n t b e c a u s e t h e y could not o b s e r v e any c o m p e n s a t o r y d e c r e a s e in u r i n a r y n i t r o g e n w h e n sweat nitrogen i n c r e a s e d d u r i n g a hot and h u m i d period. T h u s a 13-14% increase in p r o t e i n r e q u i r e m e n t was p r o p o s e d for hot humid t r o p i c a l areas b a s e d on s u b s e q u e n t studies (4). This b e c a m e a c o n t r o v e r s i a l issue as many other w o r k e r s w e r e able to o b s e r v e a c o m p e n s a t o r y r e d u c t i o n of u r i n a r y n i t r o g e n w i t h fully a c c l i m a t i z e d men d u r i n g s w e a t i n g p e r i o d s (5,7,8,9). It was also o b s e r v e d that the i n c r e a s e d sweat n i t r o g e n loss did not result in i n c r e a s e d total n i t r o g e n losses after a p e r i o d of a d a p t a t i o n (9), thus the p r o t e i n r e q u i r e m e n t s of p e o p l e living in the tropical areas are p o s s i b l y not h i g h e r t h a n t h o s e living in the t e m p e r a t e zones. Therefore, it is very e s s e n t i a l t h a t the n i t r o g e n loss t h r o u g h sweat should be d e t e r m i n e d in n i t r o g e n b a l a n c e studies. In our study, the amount of daily n i t r o g e n loss t h r o u g h the skin by the s u b j e c t s a v e r a g e d 286, 328, 350 and 371 mg N / k g / d a y for the p r o t e i n i n t a k e s of 0.3, 0.4, 0.5 and 0.6 g/kg/day, respectively. On c o n t i n u o u s f e e d i n g of the e s t i m a t e d p r o t e i n a l l o w a n c e of 0.6 g/kg/day, sweat n i t r o g e n loss ranged b e t w e e n 483 to 700 mg N/kg/day. These v a l u e s w e r e m u c h lower t h a n e x p e c t e d from results of e a r l i e r w o r k e r s w h o s t u d i e d sweat loss in hot and humid conditions. C o n s o l a z i o et al. (4) r e p o r t e d a v a l u e of 2~63 g / d a y while M i t c h e l l and H a m i l t o n (15) r e p o r t e d 764 mg N / k g / d a y / m . The e x p l a n a t i o n for our lower v a l u e s is that our s u b j e c t s w e r e c o m f o r t a b l e and fully acclimatized. T h e y did not do an~ h a r d p h y s i c a l w o r k and the t e m p e r a t u r e was not e x t r e m e (about 25 C), thus t h e y w e r e not under any stress. Our studies w e r e c o n d u c t e d b e t w e e n the m o n t h s of April and July, w h e n e n v i r o n m e n t a l t e m p e r a t u r e s are u s u a l l y not s e v e r e in the southern part of N i g e r i a (where the studies w e r e conducted). L a c k of a c c l i m a t i z a t i o n may have b e e n r e s p o n s i b l e for the h i g h e r sweat n i t r o g e n values found in C o n s o l a z i o and M i t c h e l l and H a m i l t o n ' s studies, since they were c a r r i e d out in temporary, a r t i f i c i a l l l y hot
206
G.N. EGUN and T. ATINMO
e n v i r o n m e n t s in t e m p e r a t e climates. Thus it is not a p p l i c a b l e to persons living in t r o p i c a l countries. Nevertheless, our v a l u e s are m u c h higher than v a l u e s o b t a i n e d by Calloway et al. (3) for subjects in the t e m p e r a t e climates. They observed a mean value of 112 m g / d a y as o b l i g a t o r y sweat n i t r o g e n loss and 149 mg N / k g / d a y for a p r o t e i n intake of 75g. Thus the 1985 FAO/WHO/UNU expert C o m m i t t e e (2) set up the a l l o w a n c e for sweat n i t r o g e n loss in the calculation of p r o t e i n r e q u i r e m e n t e s t i m a t i o n b a s e d on the Calloway estimates. However, our range of values compare a d e q u a t e l y with results of investigators who w o r k e d on highly a c c l i m a t i z e d subjects in tropical areas. Darke (16) r e p o r t e d a mean 24 hour sweat loss to be 254 mg of nitrogen in eight A f r i c a n males e n g a g e d in s e d e n t a r y activities. Freyberg and Grant (17) found a v e r a g e daily n i t r o g e n loss of 372 mg. Huang et al. (9) o b s e r v e d a range of 650-750 m g / d a y w h i l e W e i n e r et al. (18) obtained similar figures for a c c l i m a t i z e d T a n z a n i a n men under hot conditions. Huang et al. (9) further o b s e r v e d a seasonal change in the sweat n i t r o g e n loss. Higher skin n i t r o g e n losses were observed d u r i n g high e n v i r o n m e n t a l t e m p e r a t u r e s but then there was a c o m p e n s a t o r y significant r e d u c t i o n in u r i n a r y n i t r o g e n loss. Thus increased skin n i t r o g e n loss in sweating does not n e c e s s a r i l y increase total nitrogen losses. D a t a from this study reflected similar o b s e r v a t i o n by C u t h b e r t s o n and G u t h r i e (19) that increasing the nitrogen content of the diet caused an increase in the skin excretion of nitrogen. Dermal n i t r o g e n loss in our study i n c r e a s e d with dietary protein s i g n i f i c a n t l y (p < 0.05) and dermal n i t r o g e n was well related to the blood u r e a nitrogen. Similar results w e r e o b s e r v e d by Calloway et al. (3). N i t r o g e n b a l a n c e s calculated without regard to losses in sweat were o v e r e s t i m a t e d by about 8.0%. This resulted in an u n d e r e s t i m a t i o n of p r o t e i n requirements. It is imperative that the sweat loss should be e s t i m a t e d at d i f f e r e n t levels of dietary p r o t e i n for e f f e c t i v e c a l c u l a t i o n of p r o t e i n requirements. Also the 1985 F A O / W H O / U N U allowance for sweat is an u n d e r e s t i m a t i o n under conditions p r e v a i l i n g in t r o p i c a l areas.
ACKNOWLEDGEMENTS
The authors are grateful to the United Nations University, U n i v e r s i t y of Ibadan and Nigerian Institute of Social and Economic R e s e a r c h (NISER) for making grants available for this work. We are grateful to P r o f e s s o r D.H. Calloway and P r o f e s s o r N. S c r i m s h a w for their v a l u a b l e suggestions, advice and critical e v a l u a t i o n of the manuscript. We w o u l d like to acknowledge the c o - o p e r a t i o n of the subjects.
SWEAT NITROGEN AND PROTEIN INTAKE REFERENCES
1.
Irwin MI, Heysted DM. A conspectus of research of protein requirement of man. J. Nutr. 1971; 101:385-430.
2.
Energy and protein requirements. Report of joint FAO/WHO/UNU Adhoc Expert Committee. WHO Tech. Ser. 1985:No. 724.
3.
Calloway DH, Odell ACF, Margen S. Sweat and miscellaneous nitrogen losses in human balance J. Nutr. 1971; 101:775-86.
studies.
4.
Consolazio DF, Nelson RA, Matoosh CO, Harding RA, Canham JE. Nitrogen excretion in sweat and its relation to nitrogen balance requirements. J. Nutr. 1963; 79:399-404.
5.
Ashworth A, Harrower AD~. Protein requirements in tropical countries: Nitrogen balance. Br. J. Nutr. 1967; 21:833-43
6.
Howat PM, Korslund MK, Abernathy RP, Ritchey SJ. Sweat nitrogen losses by and nitrogen balance of preadolescent girls consuming three levels of dietary protein. Am. J. Clin. Nutr. 1978; 28:879-82.
7.
Yamada T. Could an increased output of nitrogen in sweat result in an increase in protein requirement in muscular exercise? IX International Congress of Nutrition, Mexico 1972.
8.
Huang PC, Chong HC, Rand WM. Obligatory urinary and faecal nitrogen losses in young Chinese men. J. Nutr. 1972; 10:1605-14.
9.
Huang PC, Loc HoW. Protein requirements of men in a hot climate: decreased urinary nitrogen losses concomittant with increased sweat nitrogen losses during exposure to high environmental temperature. Am. J. Clin. Nitr. 1975; 28:494-501.
i0.
Atinmo T, Mbofung CMF, Egun G, Osotimehin B. Nitrogen balance study in young Nigerian adult males using four levels of protein intake. Br. J. Nutr. ; 60:451-58.
Ii.
Spence NP, Abernathy RP, Ritchey SJ. Excretion of nitrogen in sweat by preadolescent girls consuming protein diets. Am. J. Clin. Nutr. 1972; 25:275-78.
12.
Sirbu ER, Margen S, Calloway DH. Effect of reduced
low
207
208
G.N. EGUN and T. ATINMO p r o t e i n intakes on n i t r o g e n loss from the human integument. Am. J. Clin. Nutr. 1967; 20:1158-65.
13.
A O A C (Association of O f f i c i a l A g r i c u l t u r a l Chemists) " O f f i c i a l M e t h o d s of A n a l y s i s " 1970; 10th ed. AOAC, W a s h i n g t o n DC.
14.
M a r c h WH, F i n g e r h o t B, M i l l e r H. A u t o m a t e d and m a n u a l d i r e c t m e t h o d s for d e t e r m i n a t i o n of b l o o d urea. J. Clin. Chem. 1965; ii (6):624-27.
15.
M i t c h e l l HH, H a m i l t o n TS. The e x c r e t i o n u n d e r c o n t r o l l e d e n v i r o n m e n t a l c o n d i t i o n s of n i t r o g e n and m i n e r a l s in human subjects, w i t h p a r t i c u l a r r e f e r e n c e to c a l c i u m and iron. J. Biol. Chem. 1949; 178:345-61.
16.
D a r k e SJ. The c u t a n e o u s loss of n i t r o g e n c o m p o n e n t s A f r i c a n adults. Br. J. Nutr. 1960; 14:115-19.
17.
F r e y b e r g RH, Grant RL. Loss of m i n e r a l s t h r o u g h the skin of normal humans w h e n sweat is avoided. J. Clin. Invest. 1937; 16:729-31.
18.
W e i n e r JS, W i l s o n JOC, E i - N e i l H, W h e e l e r EF. The e f f e c t of w o r k on d i e t a r y intake on sweat n i t r o g e n l o s s e s in a hot climate. Br. J. Nutr. 1972; 27:543-52.
19.
in
C u t h b e r t s o n DP, G u t h r i e WSW. The effect of v a r i a t i o n s in p r o t e i n and salt intake on the n i t r o g e n and c h l o r i d e c o n t e n t s of sweat. Biochem. J. 1934; 28:1444-53.
Accepted for publication January 22,1992.
SWEAT NITROGEN LOSSES OF YOUNG ADULT FEMALES LEVELS OF DIETARY PROTEIN INTAKE.
IN NIGERIA WITH DIFFERENT
Gloria N. Egun, PhD. 1 and Tola Atinmo, PhD. Department of Human Nutrition, college of Medicine, University of Ibadan, Nigeria.
ABSTRACT Nitrogen balance studies were carried out in two separate experiments involving a total of fifteen acclimatized young women. The aim was to determine the effects of different levels of protein intake on sweat nitrogen losses. The losses were determined through the collection of 24 hour total body sweat samples under controlled environmental conditions. In nine of the subjects fed on graded level of protein for a ten-day period at a time, sweat nitrogen losses varied with nitrogen intake, being 5.45~0.79, 6.36Z0.70, 6.65~0.73 and 7.10Z0.87mg N/kg/day at protein intakes of 0.3, 0.4, 0.5 and 0.6 protein/kg/day, respectively. Obligatory nitrogen loss in sweat extrapolated from linear regression analysis of nitrogen intake versus sweat nitrogen loss was 4.20 mg N/kg/day. When the subjects were fed a single level of protein (0.6 g/kg/day) for forty days, the mean sweat nitrogen loss of the women was 9.45• mg/kg/day. Thus the recommended allowance for sweat nitrogen losses (3-4 mg N/kg/day) in estimating protein requirement by the 1985 FAO/WHO/UNU expert report is an underestimation under conditions prevailing in tropical countries. Key words:
Sweat Nitrogen,
Protein Intake,
Nitrogen
Balance.
INTRODUCTION
Most studies undertaken to assess protein requirements have either excluded protein needed for the integumental nitrogen losses as negligible and unlikely to be affected by diet (i) or have used 3-4 mg N/kg/day for sweat nitrogen losses as suggested by the 1985 FAO/WHO/UNU report (2). The estimate used in the FAO/WHO/UNU report was derived mainly from studies conducted under moderate ambient temperatures and correspond to obligatory losses (3). The applicability of this figure for N-balance studies conducted under conditions of high environmental temperatures such as exists in the tropics and at protein intakes in the submaintenance to maintenance range has been questioned, since increased sweating and nitrogen intake affect skin losses (4-6). Furthermore, loss of nitrogen via one route may be compensated for by changes in the loss of nitrogen via another. A compensatory reduction of urinary nitrogen when sweat nitrogen loss increased has been observed by many
Address correspondence to : Dr. Gloria N. Egun, Department of Food Science and Technology, University of Reading, Whiteknights, P.O.Box 226, Reading RG6 2AP, United Kingdom.
199
G.N. EGUN and T. ATINMO
200
i n v e s t i g a t o r s (7-97. Therefore, unless all routes of n i t r o g e n losses measured, erronous conclusions may be drawn from a p p a r e n t n i t r o g e n b a l a n c e and in consequence r e q u i r e m e n t s underestimated.
are
The purpose of this study was to estimate the sweat n i t r o g e n losses of subjects living under tropical climatic conditions w h e r e i n c r e a s e d s w e a t i n g may alter total nitrogen needs. U n i v e r s i t y s t u d e n t s were studied, as a basis of c o m p a r i s o n with similar studies e l s e w h e r e (3,4 and 9). Also t h e y p r o v i d e an average estimate for sweat loss under c o n d i t i o n s p r e v a i l i n g in d e v e l o p i n g countries, as their a c t i v i t y pattern is moderate.
METHODS
AND M A T E R I A L S
Subjects Fifteen young w o m e n all p h y s i c a l l y healthy p a r t i c i p a t e d in the study. The subjects were all fully a c c l i m a t i z e d to a high e n v i r o n m e n t a l temperature. They lived under close supervision in t h e i r hall of r e s i d e n c e and e n g a g e d in normal routine daily a c t i v i t i e s w h i c h included their full academic schedule, while m a i n t a i n i n g a r e a s o n a b l e constant level of physical activity during the study periods. The subjects w e r e aged b e t w e e n 21 and 32 yea~s. T h e i r body weights and heights ranged between 45 to 70 kg and 154 to 170 cm, respectively. Body surface e s t i m a t e d from a nomograph was b e t w e e n 1.40 and 1.80 m . Body w e i g h t was m e a s u r e d daily before breakfast i~ order to assess the a d e q u a c y of caloric intake. The e n v i r o n m e n t a l conditions were u n c o n t r o l l e d w h i l e t e m p e r a t u r e and relative humidity were recorded at a nearby e x p e r i m e n t a l station. F r o m the readings provided, average values were r e c o r d e d for periods of sweat collection. Diet The diets were s t a n d a r d i z e d based on foods h a b i t u a l l y consumed and g i v e n in meal p a t t e r n s which the subjects were a c c u s t o m e d to, that is, 7-8 am, 1-2 pm and 7-8 pm. Throughout the study, all subjects were r e q u i r e d to adhere to the experimental diet and eat only the food p r e p a r e d by the investigators. Table 1 shows the c o m p o s i t i o n of the diet. A d j u s t m e n t s in the protein intake were made by i n c r e a s i n g the d i e t a r y beef intake. Complete vitamins or m i n e r a l s w e r e lacking. E n e r g y intake for individual subject was kept constant t h r o u g h o u t the entire study periods. Experimental
Desiqn
Two nitrogen b a l a n c e studies were carried out. the first e x p e r i m e n t (Expt. i) was based on short-term feeding of g r a d e d levels of p r o t e i n at 0.3, 0.4, 0.5 and 0.6 g p r o t e i n / k g / d a y for a p e r i o d of i0 days per p r o t e i n level. The second experiment (Expt. 2) was a 4 0 - d a y c o n t i n u o u s feeding of a single level of protein.
201
SWEAT NITROGEN AND PROTEIN INTAKE
TABLE
Composition
of Diet,
1
Ingredients
and Nutrients
Ingredients
(g)
Bread Refined Cane Sugar Margarine Pepper (dried) Tomatoes (fresh) Onions Palmoil Cassava (grated) Beef Vegetables Yam (cooked) Orange flavoured drinks* (bottles) + Nutrients: Protein (g) Energy (kcal) * +
1 botle contains 120 kcal. Lowest protein intake in the study
90 30 30 4 80 40 60 i00 i0 14 200 (2) 15.68 2168.35
(providing
0.3 g protein/kg/day)
In experiment i, nine women participated and the study lasted 56 days consisting of four experimental diet periods each of lO days duration in which the four different dietary levels of protein were fed. The experiment followed the standard protocol for short-term nitrogen balance studies with graded levels of protein as previously described (i0). In experiment 2, the six subjects were fed 0.6 g protein/kg/day for 40 days continuously. This level of protein is an estimated dietary allowance for young university women in Nigeria. The 40 days were arranged into two consecutive 20 day diet periods. The metabolic periods were taken as days 16-20 and 36-40. Sample and Analysis Skin nitrogen loss through sweat was collected for two days at the end of the metabolic period of each experimental dietary level. Sweat was collected by the total body method similar to techniques described by Spence, Abernathy and Ritchey (ii) and Sirbu, Margen and Calloway (12) with slight modifications. Prior to each period of sweat colection, all towels, washed clothes, beddings and clothings to be used by the subjects including their underwears on individual basis were soaked overnight in 0.5% acetic acid and rinsed six times in distilled deionized water to remove any trapped nitrogen to ensure proper estimation of the loss accountable to sweat during the collection periods. Colour fast clothings were chosen to minimize nitrogen contribution from dyes and to maximize absorption of sweat. At the onset of collection, subjects washed their bodies thoroughly, rinsed with distilled water and towel dried
202
G.N. EGUN and T. ATINMO
v i g o r o u s l y w i t h a nitrogen free towel. The bath w a t e r as well as the towel w e r e discarded. The subjects were d r e s s e d in their n i t r o g e n - f r e e c l o t h i n g s c o n s i s t i n g of underwear and t h r e e - q u a r t e r length cotton dresses. E a c h subject was given a m a r k e d towel (wash cloth) w h i c h was used to d r y any v i s i b l e sweat from her face, neck, arm and legs d u r i n g the c o l l e c t i o n period. Subjects were c a u t i o n e d against c o n t a m i n a t i n g their u n d e r w e a r w i t h urine and faeces. The subjects w o r e the same c l o t h i n g for two days. At the end of this period, the subjects r e m o v e d all the c l o t h i n g and bathed thoroughly by s c r u b b i n g w i t h o u t soap (so as not to c o n t r i b u t e any unknown source of nitrogen) in s p e c i f i e d amounts of d e i o n i z e d d i s t i l l e d water, about 20-30 litres, and then towel dried vigorously. A sample of bath water was saved in t h o r o u g h l y rinsed plastic sample bottle with d i s t i l l e d - d e i o n i z e d w a t e r and stored at -20~ until a n a l y s e d for nitrogen. The sweat and skin losses were e x t r a c t e d from the clothings, w a s h cloths, b e d d i n g s and towels by soaking them in s p e c i f i e d volumes of 0.05% acetic acid solution for 48 hours, and a sample of the soaking solution t a k e n in individual labelled plastic c o n t a i n e r s and stored at -20~ until analysed. This method assumes the garments and soaking s o l u t i o n to be at equilibrium. A similar study by C a l l o w a y et al. (3) showed that conventional washing and r i n s i n g p r o c e d u r e s did not c o m p l e t e l y remove the nitrogen from the cloth. However, the d i f f e r e n c e in amount of nitrogen recovered after repeated r i n s i n g was w i t h i n the limits of a c c e p t e d error (+ 10%). U r i n e was collected for each 24 hour p e r i o d and faeces, which were m a r k e d w i t h carmine were pooled for 4 to 5 day periods. Urine was a n a l y s e d for nitrogen and creatinine (which was used to judge the c o m p l e t e n e s s of urine collection) and the n i t r o g e n content of the faeces were determined. The n i t r o g e n content of sweat collection, urine, faeces and food samples w e r e analysed using the m o d i f i e d K j e l d a h l - C u n n i n g - A r n o l d method with c o p p e r and selenium catalysts (13). Blood samples were taken before b r e a k f a s t at the b e g i n n i n g and end of each m e t a b o l i c period. Plasma was analysed for urea nitrogen by the m a n u a l c a r b a m i n o d i a c e t y l reaction of Marsh, F i n g e r h u s t and M i l l e r (14).
RESULTS
In e x p e r i m e n t i, total nitrogen loss through the skin averaged 5.45~0.79, 6.36~0.70, 6.65~0.73 and 7.10~0.87 mg N / k g / d a y on p r o t e i n intakes of 0.3, 0.4, 0.5 and 0.6 g protein/kg/day, respectively. Table 2 shows the mean total nitrogen loss in skin as well as the n i t r o g e n c o n t e n t of each fraction of sweat collection. It was observed that the n i t r o g e n adhering to clothes was much larger than the n i t r o g e n retained on the skin. Clothes nitrogen was 71% of total sweat collection.
SWEAT NITROGEN AND PROTEIN INTAKE
203
TABLE 2 Nitrogen
Content
of Fractions
Analysed
and Total
Skin Loss
in Young
Women.
Component
0.3
Clothes Body Total skin loss
Level of Protein 0.4
3.89~0.63 1.59Z0.19 5.45•
Intake
4.50Z0.64 1.86~0.23 6.36•
(g/kg/day) 0.5
4.76~0.60 1.89• 6.65~0.73
0.6 5.10Z0.69 2.00• + 7.10~0.87
E x p r e s s e d as m e a n • SD of nine subjects (in mg N/kg/day). S i g n i f i c a n t d i f f e r e n c e s in changes w i t h levels of p r o t e i n intake at p < 0.05 (2-way ANOVA). The m e a n sweat nitrogen losses (skin) e x p r e s s e d per 24 hours and per square m e t r e of body surface per 24 hours i n c r e a s e d as the level of d i e t a r y p r o t e i n increased (Table 3). Individual subjects v a r i e d somewhat in m e a n sweat n i t r o g e n values, but differences in m e a n sweat nitrogen losses, as d e t e r m i n e d by analysis of variance (2-way ANOVA) were significant at p < 0.05. Individual v a r i a t i o n in sweat n i t r o g e n values can be a t t r i b u t e d to variations in activity and i n d i v i d u a l characteristics. I n c r e a s e in n i t r o g e n intake resulted in a c o r r e s p o n d i n g increase in skin e x c r e t i o n of nitrogen. The correlation was p o s i t i v e and significant (r = +0.97, p < 0.05). The m e a n regression e q u a t i o n of individual subjects is r e p r e s e n t e d as Y = 4.2 + 0.03X, w h e r e Y is skin n i t r o g e n loss in mg N / k g / d a y and X is nitrogen intake in mg/kg/day. The intercept of the r e g r e s s i o n w h e n intake was zero was 4.2 mg N/kg/day. This was taken as the o b l i g a t o r y nitrogen loss through the skin. TABLE Dietary Nitrogen Young W o m e n . Nitrogen g/day 2.51 3.42 4.38 5.23
Intake
Intake,
Skin Nitrogen
Skin Nitrogen mg/day 286.0Z74.1 327.8Z70.0 349.5Z71.5 370.7~84.1
3 Loss
and P l a s m a
Loss mg/m2/day 182.3Z39.1 209.5~37.2 222.9~35.4 236.4~42.0
Urea Nitrogen
Plasma
in
Urea Nitrogen mg/100ml I
6.09• 6.72Z0.70 + 7.54~i.06 + + 8.29~0.95
Values are e x p r e s s e d as mean ~ SD of nine subjects. M e a n v a l u e s were significantly different from the other the c o l u m n (2-way ANOVA) : p < 0.05 .
values
in
Skin n i t r o g e n loss increased with increase in p l a s m a urea nitrogen (Table 3). A c o r r e l a t i o n of the two showed a p o s i t i v e s i g n i f i c a n t r e l a t i o n s h i p (r = +0.96, p < 0.05). Plasma urea nitrogen i n c r e a s e d
G.N. EGUN and T. ATINMO
204 significantly
w i t h increase
in p l a s m a
intake
(p < 0.05 w i t h 2 - w a y
ANOVA). E n v i r o n m e n t a l c o n d i t i o n s w e r e fairly constant. The t e m p e r a t u r e and o r e l a t i v e h u m i d i t y w e r e b e t w e e n 25.5 and 26.6 C and 79.9 and 83%, r e s p e c t i v e l y . The slight d i f f e r e n c e s o b s e r v e d in t e m p e r a t u r e and r e l a t i v e h u m i d i t y w e r e not s t a t i s t i c a l l y different, thus we can s a f e l y c o n c l u d e that the o b s e r v e d d i f f e r e n c e s in sweat loss w e r e due to the c h a n g e in the level of p r o t e i n intake. A summary of n i t r o g e n b a l a n c e at the r e s p e c t i v e levels of p r o t e i n intake (Table 4) i l l u s t r a t e s the e f f e c t s of ignoring sweat n i t r o g e n losses. N i t r o g e n r e t e n t i o n was o v e r e s t i m a t e d w i t h o u t the i n c l u s i o n of sweat nitrogen. TABLE
4
Mean Urinary, Faecal and Sweat N i t r o g e n Losses Four L e v e l s of D i e t a r y P r o t e i n Intake.
and N i t r o g e n
Balance
at
NB
Level I II III IV
NI 48.5~5.7 66.0~7.5 84.5~9.8 + I01.7Z12.5
UN 41.4~4.7 47.0~3.8 53.6+5.7 64.9~4.5 +
FN 15.3~2.77 16.6• 16.5+3.83 16.1~3.82 ~
SN 5.45~0.79 6.36• 6.65~0.73 7.10+0.87 +
I-U-F - 9.7 + 1.0 +12.8 +19.2 +
I-U-F-S -15.24 - 5.45 + 6.25 +12.15
NI = N i t r o g e n intake; UN = U r i n a r y nitrogen; FN = Faecal nitrogen; SN = Sweat nitrogen; NB = N i t r o g e n balance; I-U-F = I n t a k e - U r i n a r y F a e c a l nitrogen; I-U-F-S = Intake - U r i n a r y - Faecal - Sweat nitrogen. * M e a n v a l u e s e x p r e s s e d as m e a n ~ SD of nine subjects. + Mean values significantly d i f f e r e n t from the o t h e r v a l u e s in the c o l u m n (2-way ANOVA) at p < 0.05 . # M e a n v a l u e s not s i g n i f i c a n t l y d i f f e r e n t from the o t h e r v a l u e s in the c o l u m n (2-way ANOVA) at p < 0.05 . $ V a l u e s of n i t r o g e n b a l a n c e s are s i g n i f i c a n t l y d i f f e r e n t (paired t - t e s t at p < 0.05). In e x p e r i m e n t 2, sweat n i t r o g e n was found to be 9 . 7 4 ~ 0 . 4 4 mg N/kg/day. Table 5 shows the mean sweat loss c o l l e c t e d t w i c e d u r i n g the e x p e r i m e n t b e t w e e n days 18-20 (first d i e t a r y interval) and 38-40 (second d i e t a r y interval). Their sweat loss wa~ e x p r e s s e d as mg N / k g / d a y as well as per total body surface area (mg N/m /day). The s u b j e c t s had a fairly c o n s t a n t sweat loss which did not change w i t h time. A t m o s p h e r i c t e m p e r a t u r e was also found to be fairly constant t h r o u g h o u t the p e r i o d of the experiment. M e a n t e m p e r a t u r e of the two p e r i o d s of sweat c o l l e c t i o n was r e c o r d e d at 24.5~
205
SWEAT NITROGEN AND PROTEIN INTAKE TABLE
5
Sweat N i t r o g e n L o s s e s of Six Young W o m e n an 4 0 - D a y C o n t i n u o u s 0.6 g P r o t e i n / k g / d a y Nitrogen in Sweat
Loss
mg N / d ~ y mg N m/ /day mg N / k g / d a y
Days Mean 546.90 333.10 9.74
16-20 SD 83.87 25.13 0.42
Days Mean 547.60 343.40 9.75
Feeding
on
38-40 SD 88.04+ 27.04+ 0.35
All v a l u e s are e x p r e s s e d as m e a n ~ SD of six subjects. No s i g n i f i c a n t d e f f e r e n c e b e t w e e n days of c o l l e c t i o n (paired t - t e s t at p < 0.05).
DISCUSSION
M i t c h e l l a n d H a m i l t o n (15) w e r e the first to p r o p o s e that sweat n i t r o g e n loss be included in c a l c u l a t i o n s of human p r o t e i n r e q u i r e m e n t b e c a u s e t h e y could not o b s e r v e any c o m p e n s a t o r y d e c r e a s e in u r i n a r y n i t r o g e n w h e n sweat nitrogen i n c r e a s e d d u r i n g a hot and h u m i d period. T h u s a 13-14% increase in p r o t e i n r e q u i r e m e n t was p r o p o s e d for hot humid t r o p i c a l areas b a s e d on s u b s e q u e n t studies (4). This b e c a m e a c o n t r o v e r s i a l issue as many other w o r k e r s w e r e able to o b s e r v e a c o m p e n s a t o r y r e d u c t i o n of u r i n a r y n i t r o g e n w i t h fully a c c l i m a t i z e d men d u r i n g s w e a t i n g p e r i o d s (5,7,8,9). It was also o b s e r v e d that the i n c r e a s e d sweat n i t r o g e n loss did not result in i n c r e a s e d total n i t r o g e n losses after a p e r i o d of a d a p t a t i o n (9), thus the p r o t e i n r e q u i r e m e n t s of p e o p l e living in the tropical areas are p o s s i b l y not h i g h e r t h a n t h o s e living in the t e m p e r a t e zones. Therefore, it is very e s s e n t i a l t h a t the n i t r o g e n loss t h r o u g h sweat should be d e t e r m i n e d in n i t r o g e n b a l a n c e studies. In our study, the amount of daily n i t r o g e n loss t h r o u g h the skin by the s u b j e c t s a v e r a g e d 286, 328, 350 and 371 mg N / k g / d a y for the p r o t e i n i n t a k e s of 0.3, 0.4, 0.5 and 0.6 g/kg/day, respectively. On c o n t i n u o u s f e e d i n g of the e s t i m a t e d p r o t e i n a l l o w a n c e of 0.6 g/kg/day, sweat n i t r o g e n loss ranged b e t w e e n 483 to 700 mg N/kg/day. These v a l u e s w e r e m u c h lower t h a n e x p e c t e d from results of e a r l i e r w o r k e r s w h o s t u d i e d sweat loss in hot and humid conditions. C o n s o l a z i o et al. (4) r e p o r t e d a v a l u e of 2~63 g / d a y while M i t c h e l l and H a m i l t o n (15) r e p o r t e d 764 mg N / k g / d a y / m . The e x p l a n a t i o n for our lower v a l u e s is that our s u b j e c t s w e r e c o m f o r t a b l e and fully acclimatized. T h e y did not do an~ h a r d p h y s i c a l w o r k and the t e m p e r a t u r e was not e x t r e m e (about 25 C), thus t h e y w e r e not under any stress. Our studies w e r e c o n d u c t e d b e t w e e n the m o n t h s of April and July, w h e n e n v i r o n m e n t a l t e m p e r a t u r e s are u s u a l l y not s e v e r e in the southern part of N i g e r i a (where the studies w e r e conducted). L a c k of a c c l i m a t i z a t i o n may have b e e n r e s p o n s i b l e for the h i g h e r sweat n i t r o g e n values found in C o n s o l a z i o and M i t c h e l l and H a m i l t o n ' s studies, since they were c a r r i e d out in temporary, a r t i f i c i a l l l y hot
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e n v i r o n m e n t s in t e m p e r a t e climates. Thus it is not a p p l i c a b l e to persons living in t r o p i c a l countries. Nevertheless, our v a l u e s are m u c h higher than v a l u e s o b t a i n e d by Calloway et al. (3) for subjects in the t e m p e r a t e climates. They observed a mean value of 112 m g / d a y as o b l i g a t o r y sweat n i t r o g e n loss and 149 mg N / k g / d a y for a p r o t e i n intake of 75g. Thus the 1985 FAO/WHO/UNU expert C o m m i t t e e (2) set up the a l l o w a n c e for sweat n i t r o g e n loss in the calculation of p r o t e i n r e q u i r e m e n t e s t i m a t i o n b a s e d on the Calloway estimates. However, our range of values compare a d e q u a t e l y with results of investigators who w o r k e d on highly a c c l i m a t i z e d subjects in tropical areas. Darke (16) r e p o r t e d a mean 24 hour sweat loss to be 254 mg of nitrogen in eight A f r i c a n males e n g a g e d in s e d e n t a r y activities. Freyberg and Grant (17) found a v e r a g e daily n i t r o g e n loss of 372 mg. Huang et al. (9) o b s e r v e d a range of 650-750 m g / d a y w h i l e W e i n e r et al. (18) obtained similar figures for a c c l i m a t i z e d T a n z a n i a n men under hot conditions. Huang et al. (9) further o b s e r v e d a seasonal change in the sweat n i t r o g e n loss. Higher skin n i t r o g e n losses were observed d u r i n g high e n v i r o n m e n t a l t e m p e r a t u r e s but then there was a c o m p e n s a t o r y significant r e d u c t i o n in u r i n a r y n i t r o g e n loss. Thus increased skin n i t r o g e n loss in sweating does not n e c e s s a r i l y increase total nitrogen losses. D a t a from this study reflected similar o b s e r v a t i o n by C u t h b e r t s o n and G u t h r i e (19) that increasing the nitrogen content of the diet caused an increase in the skin excretion of nitrogen. Dermal n i t r o g e n loss in our study i n c r e a s e d with dietary protein s i g n i f i c a n t l y (p < 0.05) and dermal n i t r o g e n was well related to the blood u r e a nitrogen. Similar results w e r e o b s e r v e d by Calloway et al. (3). N i t r o g e n b a l a n c e s calculated without regard to losses in sweat were o v e r e s t i m a t e d by about 8.0%. This resulted in an u n d e r e s t i m a t i o n of p r o t e i n requirements. It is imperative that the sweat loss should be e s t i m a t e d at d i f f e r e n t levels of dietary p r o t e i n for e f f e c t i v e c a l c u l a t i o n of p r o t e i n requirements. Also the 1985 F A O / W H O / U N U allowance for sweat is an u n d e r e s t i m a t i o n under conditions p r e v a i l i n g in t r o p i c a l areas.
ACKNOWLEDGEMENTS
The authors are grateful to the United Nations University, U n i v e r s i t y of Ibadan and Nigerian Institute of Social and Economic R e s e a r c h (NISER) for making grants available for this work. We are grateful to P r o f e s s o r D.H. Calloway and P r o f e s s o r N. S c r i m s h a w for their v a l u a b l e suggestions, advice and critical e v a l u a t i o n of the manuscript. We w o u l d like to acknowledge the c o - o p e r a t i o n of the subjects.
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Accepted for publication January 22,1992.