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Maternal protein malnutrition in the rat: Effect on protein and two enzymes in the milk
NUTRITION RESEARCH, Vol. 6, pp. 437-442, 1986 0271-5317/86 $3.00 + .00 Printed in the USA. Copyright(c) 1986 Pergamon Press Ltd. All rights reserved.
MATERNAL PROTEIN M~LNUTRITION IN THE RAT: EFFECT ON PROTEIN AND TWO ENZYMES IN THE MILK John A. Sturm~nl,ph.D., Evelyn Devine2~ph.D., Oscar Resnick ,Ph.D., Peter J. Morgane3,Ph.D.
Department of Developmental Biochemistry 1 Department of Human Genetics New York State Office of Mental Retardation and Developmental Disabilities Institute for Basic Research in Developmental Disabilities 1050 Forest Hill Road Staten Island, New York 10314 Worcester Foundation for Experimental Biology 3 222 Maple Avenue Shrewsbury, Massachusetts 01545 ABSTRACT We have examined milk samples from lactating rats fed a low protein (6Z casein) diet and a normal protein (25~ casein) diet. Total protein content of the milk was reduced. More detailed examination of the milks by two dimensional SDS polyacrylamide electrophoresis indicated that selected proteins are present in different amounts, rather than an overall, across-the-board reduction. Activity of sulfhydryl oxidase was substantially reduced, whereas activity of Y-glutamyltranspeptidase, another membrane-bound skim milk enzyme, was not. These results suggest that the quality of milk is influenced by the maternal diet. KEY WORDS: 7-glutamyltranspeptidase, malnutrition, sulfhydryl oxidase.
milk protein,
protein
INTRODUCTION
Malnutrition is a major health problem in many regions of the world and results in compromised mental functions, reduced resistance to infection, and greatly increased infant mortality (1,2,3). Many investigators have attempted to study this problem experimentally using the rat as a model, and a variety of sophisticated paradigms have reproduced various facets of the human problem (4,5, 6,7). Maternal malnutrition affects the infant both prenatally in ute~o and postnatally via the milk. A number of studies have investigated the effects of maternal malnutrition on the milk of both humans and rats by measuring quantity and quality, and, generally, have produced results suggesting an adverse, undesirable effect (8,9,10,11,12). As part of a larger study examining the effects of severe maternal rat protein malnutrition on the offspring, as an animal model for small-for-Eestational-age neonates, in which the deficits are irreversible (13), we now report the effects on various parameters in the milk. 437
J.A. STURMAN et a l .
438
MATERIALS AND METHODS Dietary and rearin~ conditions The diets and rearing conditions were the same as those described in detail previously (13). Briefly, virgin female albino Sprague-Dawley rats, age 60-70 days (Charles River Laboratories, Inc.) were fed isocaloric (4.3 kcal/g) diets containing either very low (6% casein) or normal amounts (25% casein) of protein. Briefly, the 25% and 6% casein diets are isocaloric and complete with respect to vitamin and mineral content which have been previously listed in detail (14,15). Casein is the complete source of protein and consists of 87% protein that is fully utilizable. It also contains 1.5% fat and 11.5% water. The casein diets contain a supplement of L-methionine (0.4% by weight) since casein has a low proportion of this amino acid. The caloric deficit of the low protein diet is made up by additional carbohydrate (69% in the 6% diet vs 51% in the 25% diet). Both the 25% and 6% diets are higher in fat content than is rat chow (15% vs 5%, approximately). Both diets have relatively less water than rat chow. This dietary paradigm was commenced 5 weeks prior to mating with a normally fed male and continued through gestation and lactation. At parturition, each litter was culled to 8 pups and randomized with other litters from dams fed the same diet and bern on the same day. Throughout the study all rats were given food and water ad libitum and maintained on a 12h light:dark cycle (lights on 07.00 - 19.00h). Five litters on each diet were studied. Mil ki n~ nroce dur e Milk samples were taken at 10 days after birth frum dams on both dietary regimens. The dams were separated frcm the pups at 09.00h by putting them in a separate cage with food and water. At 13.00h each dam was injected intraperitoneally with 10 mg sodium pentobarbital (Nembutal, Abbott Laboratories, North Chicago, Ill.) and allowed to rest for 10 rain. Oxytocin (0.5 IU in 0.5 ml 0.9% saline) was injected intraperitoneally immediately before milking. Drops of milk were gently squeezed from the nipples and aspirated into a sterile plastic container until approximately I ml was obtained. The samples were stored at -70~ until analyzed. Five milk samples from dams in each group were analyzed as described below. Protein determination Total protein concentration in milk was determined Bradford (16) using a 1:200 dilution of milk with water. serum albumin was used as standard.
by the method of Crystalline bovine
Sul fhvdrv i oxida se Activity of sulfhydryl oxidase was measured by following the disappearance of free substrate sulfhydryl groups (0.4 mM Ellman's reagent as substrate) using 5,5'-dithiobis(2-nitrobenzoic acid) at 412 nm in a recording spectrophotometer at 37~ (17). - Glutamvltr ans De Dtidase Activity of Y-glutamyltranspeptidase was measured by following the release of p-nitroaniline from I mM y-glutamyl-p-nitroanilide at 405 nm in a recording spectrophotometer at 30~ (18). Two-dimensional
~el electronhoresis
Protein composition of the milk was examined by two-dimensional gel electrophoresis by the method of O'Farrell (19) as modified by Anderson and
MATERNAL MALNUTRITION AND MILK
439
Anderson (20). Samples were subjected to isoelectric focusing for 14,000 volt hours during which a pH gradient of 4 to 8 was obtained. The first dimension gels were incubated in SDS equilibration buffer (10% glycerol-2% mercaptoethanol-2% SDS-0.125 M Tris HC1, pH 6.8) for 15 rain and placed directly on SDS polyacrylamide gels (10-20% gradient) for second dimension electrophoresis at 36 volts per gel for 5 hours. Proteins were visualized by the sensitive color silver stain of Sammons et al. (21). RESULTS AND DISGJSSION The total protein concentration in the milk of the control rats (25% casein) was slightly ~naller than values previously reported for rats c o n s ~ i n g a synthetic diet containing 30% protein (soybean) (22). The total protein concentration in the milk of the protein malnourished dams (6% casein diet) was reduced by 30% compared with that of the controls (25% casein diet) (Table I). The offspring suckling such mothers are thus receiving a lower concentration of protein as well as a reduced volt, he since severely protein malnourished dams are significantly ~ a l l e r than their well-nourished counterparts (6,13). A recent study in healthy lactating h ~ a n s reported a decrease in the milk protein content when the protein content of the maternal diet was reduced from 20% to 8% (11). TABLE I Effect of Maternal Protein Malnutrition on Protein Content of Milk and the Activities of Two Milk Enzymes
Diet
Protein (mg/ml )
Sulfhydryl
Oxida se Y- Glutamyltr anspe pt ida se (~mol es/min/ml )
6% 25%
49.7 + 8.2 72.9 + 6.9
2.44 • 0.38 4.34 + 0.39
Significance
P < 0.0125
P < 0.0005
Each value represents the mean + S.D. was determined by Student's t test.
of 5 separate
8.96 + 1.31 7.50 • 2.04 P < 0.15
samples.
Significance
Milk contains a number of proteins with enzymatic activity, and we selected two enzymes in milk for comparison of specific proteins that might be altered in protein malnutrition. There are many enzymes present in milk although little is known about their function. Such enzymes may play a role in changing the composition of the milk itself, they may function in the intestine, or may be absorbed by intestinal epithelial cells and transported within the body to target tissues. Sulfhydryl oxidase is a membrane-bound enzyme in skim milk which catalyzes the net synthesis of disulfide bonds and thus may alter the structure and/or function of milk proteins or intestinal proteins that have functional sulfhydryl groups (6,23). y-Glutamyltranspeptidase was selected for measurement for comparison as another membrane-bound skim milk enzyme. We found a reduction of more than 40% in the activity of sulfhydryl oxidase in the milk of protein malnourished dams (6% casein diet) compared with that in the controls (25% casein diet), whereas there was no change in activity of y-glutamyltranspeptidase (Table I). This data, obtained from the same samples, provides clear evidence that maternal protein malnutrition affects milk proteins differentially. The protein composition of the milks was examined in more detail by two-dimensional SDS polyacrylamide electrephoresis. Equal amounts of protein
440
J.A. STURMAN et a l .
Figure I. IWo-dimensional SDS polyacrylamide gels of samples of milk from lactating rats fed the low protein (6% casein) diet (a) and the high protein (25% casein) diet (b). 100 Jug of protein was used on each gel. The proteins in the upper circle represent the casein complex and those in the l ~ e r circle the lactalbumlns. The arrows point to other proteins that are clearly altered quantitatively between the two samples.
MATERNAL MALNUTRITION AND MILK
441
(100 ~g) were examined in each case. Samples from each group gave consistent patterns, illustrated by the examples in Fig. I. By comparison with previous studies on h ~ a n milk proteins using Coomassie blue staining (24) we can prestmptively identify the casein complex and lactalbumins. All of the components of the casein complex are relatively reduced in the milk from the protein malnourished dams where the lactalbumins are differentially altered, some increased and some decreased. Careful comparison of the stained gels reveals changes in the relative concentrations of individual proteins, some increased and some decreased, between the samples (Fig. l). Although there appear to be several quantitative differences between the milks from p r o t e i n malnourished dams and control dams, qualitatively most proteins are present in samples of both milks. The silver stain is so sensitive that it shows the presence of hundreds of proteins in milk, some of which may be minor constituents such as enzymes or growth factors, most of which are unidentified. Despite the lack of identity of individual proteins, our observations emphasize the complexity of milk and provide a qualitative confirmation of differential effects of maternal diet upon milk composition. ACKN GWLEDG E ~ N T S This research was supported by the Office of Mental Retardation and Developmental Disabilities of the State of New York, and by NIH grant HD 06364 (to O.R.). We thank Jonathan Kastin for expert technical assistance during this work. REFERENCES I.
Galler, J.R., Ramsey, F., Solimano, G., Lowell, W.E., and Mason, E. The influence of early malnutrition on subsequent behavioral development. I) Degree of impairment in intellectual performance. J. Am. Acad. Child Psych. 22:8-15, 1983.
2. Galler, J.R., Ramsey, F., Solimano, G., and Lowell, W.E. The influence of early malnutrition on subsequent behavioral development. II) Classroom behavior. J., A~. Acad. Child Psych. 22:16-22, 1983. 3. Galler, J.R., Ramsey, F. , and Solimano, G. The influence of early malnutrition on subsequent behavioral development. III) Learning disabilities as a sequel to malnutrition. Ped. Res. 18:309-313, 1984. 4. Morgane, P.J. , Miller, M. , Kemper, T. , Stern, W. , Forbes, W. , Hall, R. , Bronzino, J., Kissane, J., Hawrylewicz, E., and Resnick, O. The effects of protein malnutrition on the developing central nervous system in the rat. ~eurosci. Behav. Rev. 2:137-230, 1978. 5. Crnic, L.S. Effects of nutrition and enviror~ent on brain biochemistry and behavior. Nutr. Environ. 2:129-145, 1983. 6. Resnick, 0., and Morgane, P.J. Animal models for small-for-gestational-age (SGA) neonates and infants-at-risk (IAR). Devel. Br. Res. 10:221-225, 1 983. 7. Nwankwo, M.U., and Schuit, K.E. Effects of maternal protein deprivation on the nutritional status and neutrophil function of suckling neonatal rats. J. Infect. Dis. 151:23-32, 1985. 8. Mueller, A.R., and Cox, W.M., Jr. The effect of changes in diet on the volt,he and composition of rat milk. J. Nutr. 3_1:249-259, 1946.
442
J.A. STURMANet al.
9. Jelliffee, D.B., and Jelliffee, E.F.P. The volume and composition of human milk in poorly nourished communities. Am. J. Clin. NuSr. 3_1:492-515, 1 978. I0. Crnic, L.S., and Chase, H.P. Models of infantile undernutrition in rats: Effects on milk. J. Nutr. 108:1755-1760, 1978. 11. Forsum, E., and Lonnerdal, B. Effect of protein intake on protein and nitrogen composition of breast milk. Am. J. Clin. Nutr. 3_~:1809-1813, 1 980. 12. Miranda, R. , Saravia, N.G. , Ackerman, R. , Murphy, N. , Berman, S., and McMurray, D.N. Effect of maternal nutritional status on immunological substances in heman colostrtl~ and milk. Am. J. Clin. Nutr, 3.7_:632-640, 1 983. 13. Resnick, O, , Morgane, P.J. , Hasson R, Miller, M. Overt and hidden forms of chronic malnutrition in the rat and their relevance to man. NeuroscL Biobehav. Rev. 6:55-75, 1982. 14. Jalowiec, J.E., Chisholm, J.A., Forbes, W.B., Morgane, P.J., and Resnick, O. Feeding and drinking in rats maintained on a low protein diet. Brain 2:223.229, 1977. 15. Rogers, O.R., and Harper, A.E. Amino acid diets and maximal growth in the rat. J, Nutr. 87:267-273, 1965. 16. Bradford, M.M. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254, 1976. 17. Isaacs, C.E., Pascal, T., Wright, C.E., and Gaull, G.E. Sulfhydryl oxidase in human milk: Stability of milk enzymes in the gastrointestinal tract. Ped. Res. 18:532-535, 1984. 18. Griffith, O.W., and Tate, S.S. The apparent glutathione oxidase activity of y-glutamyltranspeptidase. J. Biol. Chem. 255:5011-5014, 1980. 19. O'Farrell, P.H. High resolution two-dimensional proteins. J. Biol. Chem. 250:4007-4021, 1975. 20. Anderson, N.G., and Anderson, N.L. Analytical fractions. Anal. Biochem. 85:331-340, 1978.
electrophoresis
techniques
for
of
cell
21. Sammons, D.W., Adams, LoD., and Nishizawa, E.E. A unique silver staining process for color characterization of polypeptides. ElectroDhoresis 2 : 135-141, 1981. 22. Keen, C.L. , Lonnerdal, B., Clegg, M., and Hurley, L.S. Developmental changes in composition of rat milk: Trace elements, minerals, protein, carbohydrate and fat. J. Nutr, 2:226-236, 1981. 23. Janolino, V.G., and Swaisgood, H.E. Isolation and characterization of s u l f ~ d r y l oxidase from bovine milk. J. Bio~. Chem. 250:2532-2538, 1975. 24. Anderson, N.G., Powers, M.T., and Tollaksen, S.L. Proteins of hi, an milk. I. Identification of major components. Clin. Chem. 28:1045-I055, 1982. Acceptef for publication February 21, 1986.
MATERNAL PROTEIN M~LNUTRITION IN THE RAT: EFFECT ON PROTEIN AND TWO ENZYMES IN THE MILK John A. Sturm~nl,ph.D., Evelyn Devine2~ph.D., Oscar Resnick ,Ph.D., Peter J. Morgane3,Ph.D.
Department of Developmental Biochemistry 1 Department of Human Genetics New York State Office of Mental Retardation and Developmental Disabilities Institute for Basic Research in Developmental Disabilities 1050 Forest Hill Road Staten Island, New York 10314 Worcester Foundation for Experimental Biology 3 222 Maple Avenue Shrewsbury, Massachusetts 01545 ABSTRACT We have examined milk samples from lactating rats fed a low protein (6Z casein) diet and a normal protein (25~ casein) diet. Total protein content of the milk was reduced. More detailed examination of the milks by two dimensional SDS polyacrylamide electrophoresis indicated that selected proteins are present in different amounts, rather than an overall, across-the-board reduction. Activity of sulfhydryl oxidase was substantially reduced, whereas activity of Y-glutamyltranspeptidase, another membrane-bound skim milk enzyme, was not. These results suggest that the quality of milk is influenced by the maternal diet. KEY WORDS: 7-glutamyltranspeptidase, malnutrition, sulfhydryl oxidase.
milk protein,
protein
INTRODUCTION
Malnutrition is a major health problem in many regions of the world and results in compromised mental functions, reduced resistance to infection, and greatly increased infant mortality (1,2,3). Many investigators have attempted to study this problem experimentally using the rat as a model, and a variety of sophisticated paradigms have reproduced various facets of the human problem (4,5, 6,7). Maternal malnutrition affects the infant both prenatally in ute~o and postnatally via the milk. A number of studies have investigated the effects of maternal malnutrition on the milk of both humans and rats by measuring quantity and quality, and, generally, have produced results suggesting an adverse, undesirable effect (8,9,10,11,12). As part of a larger study examining the effects of severe maternal rat protein malnutrition on the offspring, as an animal model for small-for-Eestational-age neonates, in which the deficits are irreversible (13), we now report the effects on various parameters in the milk. 437
J.A. STURMAN et a l .
438
MATERIALS AND METHODS Dietary and rearin~ conditions The diets and rearing conditions were the same as those described in detail previously (13). Briefly, virgin female albino Sprague-Dawley rats, age 60-70 days (Charles River Laboratories, Inc.) were fed isocaloric (4.3 kcal/g) diets containing either very low (6% casein) or normal amounts (25% casein) of protein. Briefly, the 25% and 6% casein diets are isocaloric and complete with respect to vitamin and mineral content which have been previously listed in detail (14,15). Casein is the complete source of protein and consists of 87% protein that is fully utilizable. It also contains 1.5% fat and 11.5% water. The casein diets contain a supplement of L-methionine (0.4% by weight) since casein has a low proportion of this amino acid. The caloric deficit of the low protein diet is made up by additional carbohydrate (69% in the 6% diet vs 51% in the 25% diet). Both the 25% and 6% diets are higher in fat content than is rat chow (15% vs 5%, approximately). Both diets have relatively less water than rat chow. This dietary paradigm was commenced 5 weeks prior to mating with a normally fed male and continued through gestation and lactation. At parturition, each litter was culled to 8 pups and randomized with other litters from dams fed the same diet and bern on the same day. Throughout the study all rats were given food and water ad libitum and maintained on a 12h light:dark cycle (lights on 07.00 - 19.00h). Five litters on each diet were studied. Mil ki n~ nroce dur e Milk samples were taken at 10 days after birth frum dams on both dietary regimens. The dams were separated frcm the pups at 09.00h by putting them in a separate cage with food and water. At 13.00h each dam was injected intraperitoneally with 10 mg sodium pentobarbital (Nembutal, Abbott Laboratories, North Chicago, Ill.) and allowed to rest for 10 rain. Oxytocin (0.5 IU in 0.5 ml 0.9% saline) was injected intraperitoneally immediately before milking. Drops of milk were gently squeezed from the nipples and aspirated into a sterile plastic container until approximately I ml was obtained. The samples were stored at -70~ until analyzed. Five milk samples from dams in each group were analyzed as described below. Protein determination Total protein concentration in milk was determined Bradford (16) using a 1:200 dilution of milk with water. serum albumin was used as standard.
by the method of Crystalline bovine
Sul fhvdrv i oxida se Activity of sulfhydryl oxidase was measured by following the disappearance of free substrate sulfhydryl groups (0.4 mM Ellman's reagent as substrate) using 5,5'-dithiobis(2-nitrobenzoic acid) at 412 nm in a recording spectrophotometer at 37~ (17). - Glutamvltr ans De Dtidase Activity of Y-glutamyltranspeptidase was measured by following the release of p-nitroaniline from I mM y-glutamyl-p-nitroanilide at 405 nm in a recording spectrophotometer at 30~ (18). Two-dimensional
~el electronhoresis
Protein composition of the milk was examined by two-dimensional gel electrophoresis by the method of O'Farrell (19) as modified by Anderson and
MATERNAL MALNUTRITION AND MILK
439
Anderson (20). Samples were subjected to isoelectric focusing for 14,000 volt hours during which a pH gradient of 4 to 8 was obtained. The first dimension gels were incubated in SDS equilibration buffer (10% glycerol-2% mercaptoethanol-2% SDS-0.125 M Tris HC1, pH 6.8) for 15 rain and placed directly on SDS polyacrylamide gels (10-20% gradient) for second dimension electrophoresis at 36 volts per gel for 5 hours. Proteins were visualized by the sensitive color silver stain of Sammons et al. (21). RESULTS AND DISGJSSION The total protein concentration in the milk of the control rats (25% casein) was slightly ~naller than values previously reported for rats c o n s ~ i n g a synthetic diet containing 30% protein (soybean) (22). The total protein concentration in the milk of the protein malnourished dams (6% casein diet) was reduced by 30% compared with that of the controls (25% casein diet) (Table I). The offspring suckling such mothers are thus receiving a lower concentration of protein as well as a reduced volt, he since severely protein malnourished dams are significantly ~ a l l e r than their well-nourished counterparts (6,13). A recent study in healthy lactating h ~ a n s reported a decrease in the milk protein content when the protein content of the maternal diet was reduced from 20% to 8% (11). TABLE I Effect of Maternal Protein Malnutrition on Protein Content of Milk and the Activities of Two Milk Enzymes
Diet
Protein (mg/ml )
Sulfhydryl
Oxida se Y- Glutamyltr anspe pt ida se (~mol es/min/ml )
6% 25%
49.7 + 8.2 72.9 + 6.9
2.44 • 0.38 4.34 + 0.39
Significance
P < 0.0125
P < 0.0005
Each value represents the mean + S.D. was determined by Student's t test.
of 5 separate
8.96 + 1.31 7.50 • 2.04 P < 0.15
samples.
Significance
Milk contains a number of proteins with enzymatic activity, and we selected two enzymes in milk for comparison of specific proteins that might be altered in protein malnutrition. There are many enzymes present in milk although little is known about their function. Such enzymes may play a role in changing the composition of the milk itself, they may function in the intestine, or may be absorbed by intestinal epithelial cells and transported within the body to target tissues. Sulfhydryl oxidase is a membrane-bound enzyme in skim milk which catalyzes the net synthesis of disulfide bonds and thus may alter the structure and/or function of milk proteins or intestinal proteins that have functional sulfhydryl groups (6,23). y-Glutamyltranspeptidase was selected for measurement for comparison as another membrane-bound skim milk enzyme. We found a reduction of more than 40% in the activity of sulfhydryl oxidase in the milk of protein malnourished dams (6% casein diet) compared with that in the controls (25% casein diet), whereas there was no change in activity of y-glutamyltranspeptidase (Table I). This data, obtained from the same samples, provides clear evidence that maternal protein malnutrition affects milk proteins differentially. The protein composition of the milks was examined in more detail by two-dimensional SDS polyacrylamide electrephoresis. Equal amounts of protein
440
J.A. STURMAN et a l .
Figure I. IWo-dimensional SDS polyacrylamide gels of samples of milk from lactating rats fed the low protein (6% casein) diet (a) and the high protein (25% casein) diet (b). 100 Jug of protein was used on each gel. The proteins in the upper circle represent the casein complex and those in the l ~ e r circle the lactalbumlns. The arrows point to other proteins that are clearly altered quantitatively between the two samples.
MATERNAL MALNUTRITION AND MILK
441
(100 ~g) were examined in each case. Samples from each group gave consistent patterns, illustrated by the examples in Fig. I. By comparison with previous studies on h ~ a n milk proteins using Coomassie blue staining (24) we can prestmptively identify the casein complex and lactalbumins. All of the components of the casein complex are relatively reduced in the milk from the protein malnourished dams where the lactalbumins are differentially altered, some increased and some decreased. Careful comparison of the stained gels reveals changes in the relative concentrations of individual proteins, some increased and some decreased, between the samples (Fig. l). Although there appear to be several quantitative differences between the milks from p r o t e i n malnourished dams and control dams, qualitatively most proteins are present in samples of both milks. The silver stain is so sensitive that it shows the presence of hundreds of proteins in milk, some of which may be minor constituents such as enzymes or growth factors, most of which are unidentified. Despite the lack of identity of individual proteins, our observations emphasize the complexity of milk and provide a qualitative confirmation of differential effects of maternal diet upon milk composition. ACKN GWLEDG E ~ N T S This research was supported by the Office of Mental Retardation and Developmental Disabilities of the State of New York, and by NIH grant HD 06364 (to O.R.). We thank Jonathan Kastin for expert technical assistance during this work. REFERENCES I.
Galler, J.R., Ramsey, F., Solimano, G., Lowell, W.E., and Mason, E. The influence of early malnutrition on subsequent behavioral development. I) Degree of impairment in intellectual performance. J. Am. Acad. Child Psych. 22:8-15, 1983.
2. Galler, J.R., Ramsey, F., Solimano, G., and Lowell, W.E. The influence of early malnutrition on subsequent behavioral development. II) Classroom behavior. J., A~. Acad. Child Psych. 22:16-22, 1983. 3. Galler, J.R., Ramsey, F. , and Solimano, G. The influence of early malnutrition on subsequent behavioral development. III) Learning disabilities as a sequel to malnutrition. Ped. Res. 18:309-313, 1984. 4. Morgane, P.J. , Miller, M. , Kemper, T. , Stern, W. , Forbes, W. , Hall, R. , Bronzino, J., Kissane, J., Hawrylewicz, E., and Resnick, O. The effects of protein malnutrition on the developing central nervous system in the rat. ~eurosci. Behav. Rev. 2:137-230, 1978. 5. Crnic, L.S. Effects of nutrition and enviror~ent on brain biochemistry and behavior. Nutr. Environ. 2:129-145, 1983. 6. Resnick, 0., and Morgane, P.J. Animal models for small-for-gestational-age (SGA) neonates and infants-at-risk (IAR). Devel. Br. Res. 10:221-225, 1 983. 7. Nwankwo, M.U., and Schuit, K.E. Effects of maternal protein deprivation on the nutritional status and neutrophil function of suckling neonatal rats. J. Infect. Dis. 151:23-32, 1985. 8. Mueller, A.R., and Cox, W.M., Jr. The effect of changes in diet on the volt,he and composition of rat milk. J. Nutr. 3_1:249-259, 1946.
442
J.A. STURMANet al.
9. Jelliffee, D.B., and Jelliffee, E.F.P. The volume and composition of human milk in poorly nourished communities. Am. J. Clin. NuSr. 3_1:492-515, 1 978. I0. Crnic, L.S., and Chase, H.P. Models of infantile undernutrition in rats: Effects on milk. J. Nutr. 108:1755-1760, 1978. 11. Forsum, E., and Lonnerdal, B. Effect of protein intake on protein and nitrogen composition of breast milk. Am. J. Clin. Nutr. 3_~:1809-1813, 1 980. 12. Miranda, R. , Saravia, N.G. , Ackerman, R. , Murphy, N. , Berman, S., and McMurray, D.N. Effect of maternal nutritional status on immunological substances in heman colostrtl~ and milk. Am. J. Clin. Nutr, 3.7_:632-640, 1 983. 13. Resnick, O, , Morgane, P.J. , Hasson R, Miller, M. Overt and hidden forms of chronic malnutrition in the rat and their relevance to man. NeuroscL Biobehav. Rev. 6:55-75, 1982. 14. Jalowiec, J.E., Chisholm, J.A., Forbes, W.B., Morgane, P.J., and Resnick, O. Feeding and drinking in rats maintained on a low protein diet. Brain 2:223.229, 1977. 15. Rogers, O.R., and Harper, A.E. Amino acid diets and maximal growth in the rat. J, Nutr. 87:267-273, 1965. 16. Bradford, M.M. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254, 1976. 17. Isaacs, C.E., Pascal, T., Wright, C.E., and Gaull, G.E. Sulfhydryl oxidase in human milk: Stability of milk enzymes in the gastrointestinal tract. Ped. Res. 18:532-535, 1984. 18. Griffith, O.W., and Tate, S.S. The apparent glutathione oxidase activity of y-glutamyltranspeptidase. J. Biol. Chem. 255:5011-5014, 1980. 19. O'Farrell, P.H. High resolution two-dimensional proteins. J. Biol. Chem. 250:4007-4021, 1975. 20. Anderson, N.G., and Anderson, N.L. Analytical fractions. Anal. Biochem. 85:331-340, 1978.
electrophoresis
techniques
for
of
cell
21. Sammons, D.W., Adams, LoD., and Nishizawa, E.E. A unique silver staining process for color characterization of polypeptides. ElectroDhoresis 2 : 135-141, 1981. 22. Keen, C.L. , Lonnerdal, B., Clegg, M., and Hurley, L.S. Developmental changes in composition of rat milk: Trace elements, minerals, protein, carbohydrate and fat. J. Nutr, 2:226-236, 1981. 23. Janolino, V.G., and Swaisgood, H.E. Isolation and characterization of s u l f ~ d r y l oxidase from bovine milk. J. Bio~. Chem. 250:2532-2538, 1975. 24. Anderson, N.G., Powers, M.T., and Tollaksen, S.L. Proteins of hi, an milk. I. Identification of major components. Clin. Chem. 28:1045-I055, 1982. Acceptef for publication February 21, 1986.