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In vivo body composition studies in rats: Assessment of total body protein
Appl. Radiat. Isot. Vol. 49. No. 5/6, pp. 731-732, 1998
Pergamon P l h S0969-8043(97)00209-1
!i~ 1998 ElsevierScience Ltd. All rights reserved Printed in Great Britain 0969-8043/98 $19.00+ 0.00
/n Vivo Body Composition Studies in Rats: Assessment of Total Body Protein S. Y A S U M U R A
.1, I. E. S T A M A T E L A T O S 2, C. N. B O O Z E R 3, R. M O O R E I a n d R. M A ~
~Medical Department, Brookhaven National Laboratory, Upton, NY 11973, U.S.A., -'Institute of Nuclear Technology and Radiation Protection, National Centre for Scientific Research 'Demokritos', Agia Paraskevi, Athens, 153 10, Greece and 3St Lukes Roosevelt Hospital, Columbia University, New York, NY 10025, U.S.A. The precision and accuracy of a prompt-gamma neutron activation facility developed to assess total body protein in rats is estimated. The coefficient of variation of nitrogen measurement, as estimated by repeated measurements on 15 rats, was 5.5% for an equivalent dose of 60 mSv (Q = 20). Good agreement was observed in comparing the results of in vivo neutron activation analysis and chemical carcass analysis performed by the Kjeldahl method. The application of the technique in comparing the effect of a low-fat and a high-fat diet on body protein in rats is demonstrated. © 1998 Elsevier Science Ltd. All rights reserved.
Introduction Total body protein (TBP) is a central parameter in in vivo body composition studies (Wang et al., 1991). An established technique to determine TBP in vivo is by assessing total body nitrogen (TBN). Although significant attention has been given to this in humans (Sutcliffe, 1996), less attention has been directed to such studies in small experimental animals (Preston et al., 1985; McNeill et al., 1988). However, small animal studies (e.g. in the rat) may provide important data on hi vivo body composition, complementary to those of human studies (Yasumura et al., 1993). We have developed a Prompt G a m m a Neutron Activation Analysis ( P G N A A ) facility optimized for TBN measurements in small animals having a body weight in the range 0.5 to 1 kg (Stamatelatos and Yasumura, 1995). The scope of the present work was to estimate the precision and accuracy of in vivo TBP assessment in rats at the BNL P G N A A facility. Moreover, we wished to demonstrate the application of the technique in investigating the effect of two different nutrition regimes in rats.
Method The small animal P G N A A nitrogen facility incorporates a 252Cf neutron source (2 x l0 s n s ~) within a heavy water neutron moderator assembly. This combination of the neutron source and heavy water provides the optimum thermal neutron fluence
within the small animal body size, while reducing gamma background from the facility. The 10.83 MeV nitrogen prompt-gamma rays are measured using two NaI(T1), 5.12 cm thick x 15.4 cm diameter, scintillation detectors. Measurements were made for 7200 s for an equivalent dose of 60 mSv (Q = 20). Cylindrical bottle phantoms of various sizes matching the dimensions of the animals and containing known quantities of nitrogen solution (1-5% by weight) in the form of urea were used for calibration. The M C N P - 4 A Monte Carlo transport code system was used to model the nitrogen facility and to calculate thermal neutron fluences as a function of the phantoms' geometrical shape and size. TBN was determined in two groups of Wistar albino rats. The first group, consisting of 15 animals (av. weight 608 g), was used to determine the in vivo precision of the two systems by taking three repeated measurements of each of the animals. The second group, of nine animals (av. weight 678 g), was used to investigate the effect of a high-fat diet on TBP. All these animals were fed a powder diet containing 12% fat by weight. After 30 days, five of the animals were switched to a high-fat diet containing 78% fat by weight. After completing the in vivo measurements, four animals from the first group and all nine animals from the second group were killed and chemically analysed for nitrogen by Kjeldahl digestion.
Results and Discussion The coefficient of variation of in vivo nitrogen measurement was 5.5%, as derived by three repeated
*To whom all correspondence should be addressed. 731
732
S. Yasumura et al.
Table I. Comparison of nitrogen assessment by PGNAA and chemical analysis Rat No. BW......... (g) N~/BW x 10 : Nrona^/BW x 10-2 1 2 3 4
570.4 608.6 626.0 476.6
3.13 2.88 3.17 3.22
3.12 2.76 2.95 3.44
Table 2. Comparison of rats on high-fat and low-fat diet High-fat diet Low-fat diet No. of animals 5 4 BW (g) 704 _+36 679 + 22 TBN (g) 20.0 _+0,44 20.3 _+ 1.50 [TBN/BW] × 100 2.91 _+0.10 3.08 + 0.23
'7~ 7.5 ~,-~ 7.1 ,~
Cylindrical/t/
/
~
6.7 6.3 lzl
5.9 5.5
. . . .
250
I
. . . .
350
I
. . . .
450
I
. . . .
550
I
....
650
I
. . . .
750
8.50
3
Phantom volume (cm) measurements on 15 animals. This variation is attributed to counting statistics (3.5%) but also to small differences in each animal's positioning a n d shape between measurements. Table 1 compares the results o f in vivo nitrogen assessment and chemical carcass analysis. G o o d agreement is evident between the two m e t h o d s when the appropriate M C N P calculated correction factors are applied on the P G N A A nitrogen measurements (Fig. 1). Nitrogen m e a s u r e m e n t sensitivity depends on the activating thermal n e u t r o n fluence, the scatter and a t t e n u a t i o n o f g a m m a rays within tissue and the detector efficiency. The correction factors derived account for the size and shape differences between the animal and the cylindrical calibration p h a n t o m s . Table 2 compares T B N values between the high-fat and the low-fat diet in rats. N o differences in T B N / B W were observed between the two groups, despite some difference in body weight, with the high-fat diet animals gaining more weight t h a n the low-fat diet group.
Conclusions The results of the present study indicated that & and chemical carcass analysis give c o m p a r a b l e results in assessing T B P in rats. However, P G N A A has the a d v a n t a g e of overall simplicity and, most particularly, of enabling serial vivo P G N A A
Fig. I. Calculated thermal neutron fluence.
measurements to be taken on the same animal; indeed, the low radiation dose permits repeated measurements as often as necessary. Preliminary results are presented suggesting that there are no significant differences in T B P when the effect o f a high-fat diet a n d a low-fat diet are compared.
References McNeill, K. G., Wang, H. Y. and Waana, C. (1988) In vivo measurement of nitrogen in small animals. J. Radioanal. Nucl. Chem., Articles 124, 251. Preston, T., Reeds, P. J., East, B. W. and Holmes, P. H. (1985) A comparison of body protein determination in rats by in vivo neutron activation and carcass analysis. Clin. Sci. 68, 349. Stamatelatos, I. E. and Yasumura, S. (1995) Prompt-gamma neutron activation facility for measuring body nitrogen in small animals. Appl. Radiat. Isot. 46, 269. Sutcliffe, J. F. (1996) A review of in vivo experimental methods to determine the composition of the human body. Phys. Med. Biol. 41, 791. Wang, Z., Pierson, R. N. and Heymsfield, S. B. (1991) The five level model: a new approach to organizing body-composition research. Am. J. Clin. Nutr. 56, 19. Yasumura, S., Jones, K., Spanne, P., Schidlovsky, G., Wielopolski, L., Ren, X., Glaros, D. and Xatzikonstantinou, Y. (1993) In vivo animal models of body composition in aging. J. Nutr. 123, 459.
Pergamon P l h S0969-8043(97)00209-1
!i~ 1998 ElsevierScience Ltd. All rights reserved Printed in Great Britain 0969-8043/98 $19.00+ 0.00
/n Vivo Body Composition Studies in Rats: Assessment of Total Body Protein S. Y A S U M U R A
.1, I. E. S T A M A T E L A T O S 2, C. N. B O O Z E R 3, R. M O O R E I a n d R. M A ~
~Medical Department, Brookhaven National Laboratory, Upton, NY 11973, U.S.A., -'Institute of Nuclear Technology and Radiation Protection, National Centre for Scientific Research 'Demokritos', Agia Paraskevi, Athens, 153 10, Greece and 3St Lukes Roosevelt Hospital, Columbia University, New York, NY 10025, U.S.A. The precision and accuracy of a prompt-gamma neutron activation facility developed to assess total body protein in rats is estimated. The coefficient of variation of nitrogen measurement, as estimated by repeated measurements on 15 rats, was 5.5% for an equivalent dose of 60 mSv (Q = 20). Good agreement was observed in comparing the results of in vivo neutron activation analysis and chemical carcass analysis performed by the Kjeldahl method. The application of the technique in comparing the effect of a low-fat and a high-fat diet on body protein in rats is demonstrated. © 1998 Elsevier Science Ltd. All rights reserved.
Introduction Total body protein (TBP) is a central parameter in in vivo body composition studies (Wang et al., 1991). An established technique to determine TBP in vivo is by assessing total body nitrogen (TBN). Although significant attention has been given to this in humans (Sutcliffe, 1996), less attention has been directed to such studies in small experimental animals (Preston et al., 1985; McNeill et al., 1988). However, small animal studies (e.g. in the rat) may provide important data on hi vivo body composition, complementary to those of human studies (Yasumura et al., 1993). We have developed a Prompt G a m m a Neutron Activation Analysis ( P G N A A ) facility optimized for TBN measurements in small animals having a body weight in the range 0.5 to 1 kg (Stamatelatos and Yasumura, 1995). The scope of the present work was to estimate the precision and accuracy of in vivo TBP assessment in rats at the BNL P G N A A facility. Moreover, we wished to demonstrate the application of the technique in investigating the effect of two different nutrition regimes in rats.
Method The small animal P G N A A nitrogen facility incorporates a 252Cf neutron source (2 x l0 s n s ~) within a heavy water neutron moderator assembly. This combination of the neutron source and heavy water provides the optimum thermal neutron fluence
within the small animal body size, while reducing gamma background from the facility. The 10.83 MeV nitrogen prompt-gamma rays are measured using two NaI(T1), 5.12 cm thick x 15.4 cm diameter, scintillation detectors. Measurements were made for 7200 s for an equivalent dose of 60 mSv (Q = 20). Cylindrical bottle phantoms of various sizes matching the dimensions of the animals and containing known quantities of nitrogen solution (1-5% by weight) in the form of urea were used for calibration. The M C N P - 4 A Monte Carlo transport code system was used to model the nitrogen facility and to calculate thermal neutron fluences as a function of the phantoms' geometrical shape and size. TBN was determined in two groups of Wistar albino rats. The first group, consisting of 15 animals (av. weight 608 g), was used to determine the in vivo precision of the two systems by taking three repeated measurements of each of the animals. The second group, of nine animals (av. weight 678 g), was used to investigate the effect of a high-fat diet on TBP. All these animals were fed a powder diet containing 12% fat by weight. After 30 days, five of the animals were switched to a high-fat diet containing 78% fat by weight. After completing the in vivo measurements, four animals from the first group and all nine animals from the second group were killed and chemically analysed for nitrogen by Kjeldahl digestion.
Results and Discussion The coefficient of variation of in vivo nitrogen measurement was 5.5%, as derived by three repeated
*To whom all correspondence should be addressed. 731
732
S. Yasumura et al.
Table I. Comparison of nitrogen assessment by PGNAA and chemical analysis Rat No. BW......... (g) N~/BW x 10 : Nrona^/BW x 10-2 1 2 3 4
570.4 608.6 626.0 476.6
3.13 2.88 3.17 3.22
3.12 2.76 2.95 3.44
Table 2. Comparison of rats on high-fat and low-fat diet High-fat diet Low-fat diet No. of animals 5 4 BW (g) 704 _+36 679 + 22 TBN (g) 20.0 _+0,44 20.3 _+ 1.50 [TBN/BW] × 100 2.91 _+0.10 3.08 + 0.23
'7~ 7.5 ~,-~ 7.1 ,~
Cylindrical/t/
/
~
6.7 6.3 lzl
5.9 5.5
. . . .
250
I
. . . .
350
I
. . . .
450
I
. . . .
550
I
....
650
I
. . . .
750
8.50
3
Phantom volume (cm) measurements on 15 animals. This variation is attributed to counting statistics (3.5%) but also to small differences in each animal's positioning a n d shape between measurements. Table 1 compares the results o f in vivo nitrogen assessment and chemical carcass analysis. G o o d agreement is evident between the two m e t h o d s when the appropriate M C N P calculated correction factors are applied on the P G N A A nitrogen measurements (Fig. 1). Nitrogen m e a s u r e m e n t sensitivity depends on the activating thermal n e u t r o n fluence, the scatter and a t t e n u a t i o n o f g a m m a rays within tissue and the detector efficiency. The correction factors derived account for the size and shape differences between the animal and the cylindrical calibration p h a n t o m s . Table 2 compares T B N values between the high-fat and the low-fat diet in rats. N o differences in T B N / B W were observed between the two groups, despite some difference in body weight, with the high-fat diet animals gaining more weight t h a n the low-fat diet group.
Conclusions The results of the present study indicated that & and chemical carcass analysis give c o m p a r a b l e results in assessing T B P in rats. However, P G N A A has the a d v a n t a g e of overall simplicity and, most particularly, of enabling serial vivo P G N A A
Fig. I. Calculated thermal neutron fluence.
measurements to be taken on the same animal; indeed, the low radiation dose permits repeated measurements as often as necessary. Preliminary results are presented suggesting that there are no significant differences in T B P when the effect o f a high-fat diet a n d a low-fat diet are compared.
References McNeill, K. G., Wang, H. Y. and Waana, C. (1988) In vivo measurement of nitrogen in small animals. J. Radioanal. Nucl. Chem., Articles 124, 251. Preston, T., Reeds, P. J., East, B. W. and Holmes, P. H. (1985) A comparison of body protein determination in rats by in vivo neutron activation and carcass analysis. Clin. Sci. 68, 349. Stamatelatos, I. E. and Yasumura, S. (1995) Prompt-gamma neutron activation facility for measuring body nitrogen in small animals. Appl. Radiat. Isot. 46, 269. Sutcliffe, J. F. (1996) A review of in vivo experimental methods to determine the composition of the human body. Phys. Med. Biol. 41, 791. Wang, Z., Pierson, R. N. and Heymsfield, S. B. (1991) The five level model: a new approach to organizing body-composition research. Am. J. Clin. Nutr. 56, 19. Yasumura, S., Jones, K., Spanne, P., Schidlovsky, G., Wielopolski, L., Ren, X., Glaros, D. and Xatzikonstantinou, Y. (1993) In vivo animal models of body composition in aging. J. Nutr. 123, 459.