J v r m o l o/ Nrurnclwn~i~rr?.1976. VoI. 27. pp. 1361-1 365. Pergamon Press. Printed in Grrat Britain
EFFECTS OF LYSINE AND THREONINE FORTIFICATION OF BREAD DURING GESTATION AND LACTATION ON OFFSPRING BRAIN LIPID DEVELOPMENT H. P. CHASE,N. NOREENWELCH,JOANNA L. GANONG and G. R. JANSEN' University of Colorado Medical Center, Department of Pediatrics, B. F. Stolinsky Laboratories, 4200 East Ninth Avenue, Denver, CO 80220, U.S.A. and ' Colorado State University, Department of Food Science and Nutrition, Fort Collins. CO 80523. U.S.A. (Receioed 4 Febritary 1976. Accepted 4 M a y 1976) Abstract- -Offspring brain lipid development has been studied following the amino acid fortification of wheat diet fed to pregnant and lactating rats. Comparisons are made with a 13% casein diet which contains a similar quantity of protein of known high quality, with a 26% casein diet, and with a standard laboratory diet. Increased maternal energy intake was found, particularly during lactation, with protein quality improvement. Offspring of mothers receiving the unsupplemented bread diet had lower total brain cholesterol, phospholipid, cerebroside-sulfatide, and (for 4 of the 5 other diets) ganglioside-NANA levels in comparison to offspring whose mothers received the other diets. Brain cerebrosidesulfatide and cholesterol concentrations were also lower in the offspring whose mothers received the unsupplemented bread, suggesting impaired myelin formation. Phospholipid and ganglioside-NANA concentrations were not lower. Fortification of the bread diet with lysine resulted in increases in both the total levels and concentrations of cerebroside-sulfatide and cholesterol. When both lysine and threonine were added, the cerebroside-sulfatide and cholesterol levels and concentrations were similar to or greater than values for the two casein diets and the standard laboratory diet. The 2600 casein diet did not result in higher lipid values than obtained with the 13% casein diet. This study demonstrates the adequacy of amino acid supplementation of a maternal cereal protein diet in allowing normal offspring brain lipid development.
CEREALS such as wheat, rice, and corn provide the major sources of protein for people living in preindustrial countries (JANSEN,1974). These foods often provide an adequate quantity of protein, but they are
METHODS
Animals mid diets. The animals used in this investigation are the same as those studied previously (JANSEN & CHASE, 1976; CHASEand JANSEN,1976), with the half of the brain qualitatively deficient in essential amino acids and not used for protein and DNA analyses frozen under may be poorly absorbed. However, they are available nitrogen for the present lipid analyses. The method of in these countries and attempts at improving nutri- breeding is given in detail in the previous reports and will tion should include their utilization. Cereal proteins be only briefly described here. Virgin female SpragueDawley rats (Simonsen Laboratories of Gilroy, CA) weighwill likely provide an increasing share of the world's ing 256 l o g were bred to a male of similar strain, and protein needs as the world population continues to upon finding a breeding plug were started on one of 6 increase. If the quality of cereal proteins can be imdiets: (A) white bread containing 2.1% N (135, protein); proved by amino acid fortification during pregnancy (B) bread plus 0.35Y0 L-lysine monohydrochloride; ( C ) and lactation, improved intrauterine and infant body bread plus 0.69 L-lysine monohydrochloride plus 0.35% and brain growth might be possible. DL-threonine; (D) 137; casein diet; (E) 26'); casein diet and In initial studies (JANSEN& CHASE, 1976; CHASE (F) stock diet (obtained from the Ralston Purina Company, & JANSEN,1976) of the effects of fortification of wheat St. Louis, MO). All diets had similar vitamin, mineral and calorie content. The exact composition of the diets has with lysine or with lysine and threonine during pregnancy and lactation in rats, it was found that mater- been described previously (JANSEN & CHASE,1976). In Table 1, the amino acid composition of white bread nal weight loss was prevented and total offspring birth and weaning weights increased as a result of and casein is compared with the 1973 FAO/WHO reference standard. Lysine is the first limiting amino acid in amino acid fortification. Offspring brain protein and bread, showing a 540,; deficit compared to the reference cellularity, as represented by DNA, increased pro- pattern, and threonine is second limiting. In rat growth gressively as lysine and lysine plus threonine were studies, lysine is clearly first limiting and threonine second added to the maternal bread diet. Brain lipids have limiting in wheat (JANSEN, 1974). As shown in Table 1 the not previously been evaluated following vegetable bread fortified with lysine and threonine is no longer limitprotein diets with or without amino acid fortification. ing in these amino acids. 1361
*
H. P. CHASE,N. NOREENWELCH,JOANNAL. GANONC~ and G. R. JANSEN
1362
TABLEI . AMINOACID COMPOSITION OF WHITE BREAD AND CASEIN TO FAOiWHO REFERENCE STANDARD (mg/g N ) Amino acid
Isoieucine Leucine Lyrine Methionine + Cystme Phenylalanine 1)roslne Thrconine Tr)ptophan Valine
+
COMPARED
Caselnt
F A 0 WHO reference standard:
270
345
250
444
607 six
440
15.51364)
237
201
220
?on
705 297
3no 3 0
103 430
310
While bread.
IW(2641 61
292
340
M)
* Data taken from Amino Acid Content of Foods and Biological Data on Proteins. F A 0 Nutritional Studies No. 24, Rome, Italy, 1970. t Data taken from BRADLEY W. B., Wheat Foods ns Sources of Nutrients. pp. 66-71. The Bakers Digest, October 1967. Values in brackets are estimated levels in the bread as fortified with lysine and threonine in the present investigations. $ Energy and Protein Requirements. Report of a Joint FAOjWHO Ad Hoc Expert Committee. World Health Organization Technical Report Series No. 522. Geneva, Switzerland, 1973. The 13", casein diet was included to compare the bread diet to one of similar protein quantity and known good quality, and the 26"" casein diet to determine if a higher protein diet resulted in enhanced development. The standard white bread was obtained from Continental Baking Company, Denver, CO and was air dried and ground in a Wiley Mill. When fortified with lysine alone. 0.35", L-lysine monohydrochloride was added based on dry weight of the bread. When lysine and threonine were added together the fortification levels were 0.63,; L-lysine monohydrochloride and 0.35"" DL-threonine, again based on dry weight of the bread. The stock diet was included for comparison with results from previous experiments. Three
Ff *
I et
A
RESULTS
Bod!. weights mid food intrike
I I I
B
mothers. with 8 offspring each, were fed one of the 6 diets during their first pregnancy and lactation. All offspring were killed at age 22 days, the brains removed. dissected free of meninges. weighed. and divided longitudinally using the Left half for the DNA and protein determinations and the right half for lipid analyses. Lipid trmrl~srs.Lipids were extracted with initial homogenization in 19 vol of ?: 1 (viv) chloroform-methanol as described by FOLCHet nl (1957) and as utilized previously ef a/.. 1967). Brain lipids were partitioned by Unisil (CHASE (Clarkson Chemical Company, Williarnsport, PA) column chromatography as described by ROUSERet a/. (1967), with cholesterol separated in the chloroform eluate (fraction one). cerebroside-sulfatide in the acetone eluate (fraction two) and phospholipid and lipid NANA in the methanol eluate (fraction three). Column fractions were monitored by TLC to verify that there was no fraction overlap. Quantitative lipid analyses were done using methods described rt a/., 1972). previously (CHASE Statistical analyses. The Student t-test was used for all statistical analyses. Results in the text are referred to as 'higher' or 'lower' only if P was
EFFECTS OF LYSINE AND THREONINE FORTIFICATION OF BREAD DURING GESTATION AND LACTATION ON OFFSPRING BRAIN LIPID DEVELOPMENT H. P. CHASE,N. NOREENWELCH,JOANNA L. GANONG and G. R. JANSEN' University of Colorado Medical Center, Department of Pediatrics, B. F. Stolinsky Laboratories, 4200 East Ninth Avenue, Denver, CO 80220, U.S.A. and ' Colorado State University, Department of Food Science and Nutrition, Fort Collins. CO 80523. U.S.A. (Receioed 4 Febritary 1976. Accepted 4 M a y 1976) Abstract- -Offspring brain lipid development has been studied following the amino acid fortification of wheat diet fed to pregnant and lactating rats. Comparisons are made with a 13% casein diet which contains a similar quantity of protein of known high quality, with a 26% casein diet, and with a standard laboratory diet. Increased maternal energy intake was found, particularly during lactation, with protein quality improvement. Offspring of mothers receiving the unsupplemented bread diet had lower total brain cholesterol, phospholipid, cerebroside-sulfatide, and (for 4 of the 5 other diets) ganglioside-NANA levels in comparison to offspring whose mothers received the other diets. Brain cerebrosidesulfatide and cholesterol concentrations were also lower in the offspring whose mothers received the unsupplemented bread, suggesting impaired myelin formation. Phospholipid and ganglioside-NANA concentrations were not lower. Fortification of the bread diet with lysine resulted in increases in both the total levels and concentrations of cerebroside-sulfatide and cholesterol. When both lysine and threonine were added, the cerebroside-sulfatide and cholesterol levels and concentrations were similar to or greater than values for the two casein diets and the standard laboratory diet. The 2600 casein diet did not result in higher lipid values than obtained with the 13% casein diet. This study demonstrates the adequacy of amino acid supplementation of a maternal cereal protein diet in allowing normal offspring brain lipid development.
CEREALS such as wheat, rice, and corn provide the major sources of protein for people living in preindustrial countries (JANSEN,1974). These foods often provide an adequate quantity of protein, but they are
METHODS
Animals mid diets. The animals used in this investigation are the same as those studied previously (JANSEN & CHASE, 1976; CHASEand JANSEN,1976), with the half of the brain qualitatively deficient in essential amino acids and not used for protein and DNA analyses frozen under may be poorly absorbed. However, they are available nitrogen for the present lipid analyses. The method of in these countries and attempts at improving nutri- breeding is given in detail in the previous reports and will tion should include their utilization. Cereal proteins be only briefly described here. Virgin female SpragueDawley rats (Simonsen Laboratories of Gilroy, CA) weighwill likely provide an increasing share of the world's ing 256 l o g were bred to a male of similar strain, and protein needs as the world population continues to upon finding a breeding plug were started on one of 6 increase. If the quality of cereal proteins can be imdiets: (A) white bread containing 2.1% N (135, protein); proved by amino acid fortification during pregnancy (B) bread plus 0.35Y0 L-lysine monohydrochloride; ( C ) and lactation, improved intrauterine and infant body bread plus 0.69 L-lysine monohydrochloride plus 0.35% and brain growth might be possible. DL-threonine; (D) 137; casein diet; (E) 26'); casein diet and In initial studies (JANSEN& CHASE, 1976; CHASE (F) stock diet (obtained from the Ralston Purina Company, & JANSEN,1976) of the effects of fortification of wheat St. Louis, MO). All diets had similar vitamin, mineral and calorie content. The exact composition of the diets has with lysine or with lysine and threonine during pregnancy and lactation in rats, it was found that mater- been described previously (JANSEN & CHASE,1976). In Table 1, the amino acid composition of white bread nal weight loss was prevented and total offspring birth and weaning weights increased as a result of and casein is compared with the 1973 FAO/WHO reference standard. Lysine is the first limiting amino acid in amino acid fortification. Offspring brain protein and bread, showing a 540,; deficit compared to the reference cellularity, as represented by DNA, increased pro- pattern, and threonine is second limiting. In rat growth gressively as lysine and lysine plus threonine were studies, lysine is clearly first limiting and threonine second added to the maternal bread diet. Brain lipids have limiting in wheat (JANSEN, 1974). As shown in Table 1 the not previously been evaluated following vegetable bread fortified with lysine and threonine is no longer limitprotein diets with or without amino acid fortification. ing in these amino acids. 1361
*
H. P. CHASE,N. NOREENWELCH,JOANNAL. GANONC~ and G. R. JANSEN
1362
TABLEI . AMINOACID COMPOSITION OF WHITE BREAD AND CASEIN TO FAOiWHO REFERENCE STANDARD (mg/g N ) Amino acid
Isoieucine Leucine Lyrine Methionine + Cystme Phenylalanine 1)roslne Thrconine Tr)ptophan Valine
+
COMPARED
Caselnt
F A 0 WHO reference standard:
270
345
250
444
607 six
440
15.51364)
237
201
220
?on
705 297
3no 3 0
103 430
310
While bread.
IW(2641 61
292
340
M)
* Data taken from Amino Acid Content of Foods and Biological Data on Proteins. F A 0 Nutritional Studies No. 24, Rome, Italy, 1970. t Data taken from BRADLEY W. B., Wheat Foods ns Sources of Nutrients. pp. 66-71. The Bakers Digest, October 1967. Values in brackets are estimated levels in the bread as fortified with lysine and threonine in the present investigations. $ Energy and Protein Requirements. Report of a Joint FAOjWHO Ad Hoc Expert Committee. World Health Organization Technical Report Series No. 522. Geneva, Switzerland, 1973. The 13", casein diet was included to compare the bread diet to one of similar protein quantity and known good quality, and the 26"" casein diet to determine if a higher protein diet resulted in enhanced development. The standard white bread was obtained from Continental Baking Company, Denver, CO and was air dried and ground in a Wiley Mill. When fortified with lysine alone. 0.35", L-lysine monohydrochloride was added based on dry weight of the bread. When lysine and threonine were added together the fortification levels were 0.63,; L-lysine monohydrochloride and 0.35"" DL-threonine, again based on dry weight of the bread. The stock diet was included for comparison with results from previous experiments. Three
Ff *
I et
A
RESULTS
Bod!. weights mid food intrike
I I I
B
mothers. with 8 offspring each, were fed one of the 6 diets during their first pregnancy and lactation. All offspring were killed at age 22 days, the brains removed. dissected free of meninges. weighed. and divided longitudinally using the Left half for the DNA and protein determinations and the right half for lipid analyses. Lipid trmrl~srs.Lipids were extracted with initial homogenization in 19 vol of ?: 1 (viv) chloroform-methanol as described by FOLCHet nl (1957) and as utilized previously ef a/.. 1967). Brain lipids were partitioned by Unisil (CHASE (Clarkson Chemical Company, Williarnsport, PA) column chromatography as described by ROUSERet a/. (1967), with cholesterol separated in the chloroform eluate (fraction one). cerebroside-sulfatide in the acetone eluate (fraction two) and phospholipid and lipid NANA in the methanol eluate (fraction three). Column fractions were monitored by TLC to verify that there was no fraction overlap. Quantitative lipid analyses were done using methods described rt a/., 1972). previously (CHASE Statistical analyses. The Student t-test was used for all statistical analyses. Results in the text are referred to as 'higher' or 'lower' only if P was
