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The nutritive value of certain noxious weed seeds Robertson, Mary Chalmers 1957

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THE NUTRITIVE VALUE OF CERTAIN NOXIOUS WEED SEEDS by MARY CHALMERS ROBERTSON B.A., U n i v e r s i t y  of B r i t i s h Columbia,  1953  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In the D i v i s i o n of Animal  Nutrition  We a c c e p t t h i s t h e s i s as conforming to the r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA April  1957  ABSTRACT An attempt  has been made t o assess the n u t r i t i v e v a l u e  of the p r o t e i n s o f a number of weed seeds which a r e c o n s i d e r e d as noxious under the Canada Feeding S t u f f s A c t , but which have been f e d s u c c e s s f u l l y to ruminants processed r e f u s e s c r e e n i n g s .  i n the form o f heat  These weed seeds c o n t a i n  i s o t h i o c y a n a t e s which may or may n o t be t o x i c t o animals but which were found to be u n p a l a t a b l e to the r a t .  For this  r e a s o n i t was found necessary t o take two approachs  to the  problem o f determining the n u t r i t i v e v a l u e of these weed seeds; a d i r e c t approach i n v o l v i n g animal assays w i t h r a t s and an i n d i r e c t approach i n v o l v i n g assessment a c i d content of these weed seeds.  o f the e s s e n t i a l amino  I n c a r r y i n g out these  o b j e c t i v e s a study o f the p r o p e r t i e s of the i s o t h i o c y a n t e s themselves and of the v a r i o u s methods f o r e v a l u a t i o n o f p r o t e i n q u a l i t y v,ere a l s o  undertaken.  For the d i r e c t approach i n v o l v i n g animal assays, an attempt was made to develop a procedure which would remove the i s o t h i o c y a n a t e s from samples of these weed seeds.  A  procedure i n v o l v i n g a u t o - h y d r o l y s i s o f the weed seed w i t h water f o l l o w e d by e x t r a c t i o n w i t h 70% e t h a n o l was e v o l v e d . T h i s treatment reduced the ijsothiocyanate content o f the weed seeds and rendered them p a l a t a b l e to the r a t .  Using samples  prepared i n t h i s manner the p r o t e i n s of a number of the weed seeds were assayed f o r t h e i r n e t p r o t e i n u t i l i z a t i o n a c c o r d i n g to the method proposed by M i l l e r and Bender (89 ) .  i  Since there was no assurance  that attempts  to remove  the i s o t h i o c y a n a t e s would be s u c c e s s f u l , an i n d i r e c t  approach  was a l s o taken f o r the e v a l u a t i o n of the p r o t e i n s o f weed seeds.  A number o f weed seeds were analysed, f o r t h e i r  o f e s s e n t i a l amino a c i d s u s i n g the m i c r o b i o l o g i c a l methods proposed  by B a r t o n - Wright.  content  assay  From t h i s d a t a , a  c h e m i c a l e v a l u a t i o n o f the p r o t e i n s was c a r r i e d out through computation  o f " e s s e n t i a l amino a c i d . i n d i c e s " and "chemical  s c o r e s " a c c o r d i n g to the method proposed  by M i t c h e l l .  A comparison and c o r r e l a t i o n of the animal assays and chemical e v a l u a t i o n s i n d i c a t e d t h a t although s l i g h t l y  lower  i n value the p r o t e i n s o f weed seeds compare f a v o u r a b l y w i t h those of soybean and l i n s e e d as p l a n t p r o t e i n s of moderately high b i o l o g i c a l value. In c o n n e c t i o n w i t h the e v a l u a t i o n of p r o t e i n s by the M i l l e r and Bender method a study was made of the body water body n i t r o g e n r e l a t i o n s h i p i n the U.B.C. colony o f r a t s . The body water and body n i t r o g e n content o f 58 W i s t a r and 36 Sprague - Dawley r a t s were determined  and r e g r e s s i o n equations  c o r r e l a t i n g body n i t r o g e n to body water were c a l c u l a t e d . These i n v e s t i g a t i o n s confirmed  the f i n d i n g s o f other workers  t h a t the n i t r o g e n to water r a t i o v a r i e s w i t h such  constancy  that the n i t r o g e n content o f an animal can be c a l c u l a t e d a knowledge of i t s water content and age or body  ii  weight.  from  In p r e s e n t i n g the  this thesis in partial fulfilment  requirements f o r an advanced degree at the  of  University  of B r i t i s h Columbia, I agree t h a t  the  L i b r a r y s h a l l make  it  and  study.  freely available  for reference  I  further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may  be granted by the  Department or by h i s r e p r e s e n t a t i v e .  Head o f  my  It i s understood  •that copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not  Department o f  be. allowed without my  {^CVUSTUL/  The U n i v e r s i t y of B r i t i s h Vancouver Canada. Date  ^6Uf  /,  t> 7  ^/ej^y^aO  Columbia,  written  permission.  ACKHOW LEDGEMEN T  The w r i t e r wishes to take t h i s o p p o r t u n i t y t o thank Dr.  B . A . E a g l e s , Dean of the F a c u l t y o f A g r i c u l t u r e and  chairman of the D i v i s i o n of Animal S c i e n c e ,  fop his  permission  to undertake t h i s p r o j e c t and.' f o r the use of departmental facilities. For  h i s g u i d a n c e , I n t e r e s t and enthusiasm  the course of this, s t u d y ,  throughtout  the author expresses her- s i n c e r e  a p p r e c i a t i o n to D r . A . J . Wood!, P r o f e s s o r of Animal An e x p r e s s i o n o f g r a t i t u d e i s a l s o extended  1  Science.  to* D r . Kifcts, f o r  h i s c r i t i c i s m and many h e l p f u l s u g g e s t i o n s . The  f i n a n c i a l a s s i s t a n c e p r o v i d e d by funds from; the  Ganada Department of A g r i c u l t u r e and a grant from the P r e s i d e n t ' s Committee on Research; are g r a t e f u l l y  acknowledged.  The w r i t e r a l s o wishes to express her thanks t o  the  P l a n t Products D i v i s i o n , Canada Department of A g r i c u l t u r e f o r t h e i r a s s i s t a n c e i n s u p p l y i n g the samples used i n t h i s  study.  vi  of weed seeds  TABLE OF COMTEMTS PAGE I.  Introduction A.  II.  .......................  Seed Samples  ...•..».•.•..•••••••»..•...  3  Attempt t o Remove IsoThloey/amtes; from Represent a t i v e Samples o f Weed; Seedis A.  Introduction  B.  L i t e r a t u r e Review (a)  ..••....»  5  •••••••••••••  5  •  isothiocyanates  »••••••••••••••••  (b) toxicity Experimental ••••»••»»•» ........... (a) D e t e r m i n a t i o n o f i s o t h i o c y a n a t e s . (b) E x t r a c t i o n procedure E v a l u a t i o n o f the P r o t e i n Q u a l i t y o f R e p r e s e n t a t i v e Samples o f Weed Seeds . C.  III.  1  A.  Introduction  B.  L i t e r a t u r e Review •••«•• ...... (a) Proteins ....... .... (b) P r o t e i n E v a l u a t i o n Methods 1. Protein Efficiency Ratio ... 2. Rat R e p l e t i o n Method 3. l i t r o g e n Balance ••••••••••• i . P r o t e i n Minima ......... i i . B i o l o g i c a l Value h» Tissue P r o t e i n s 5. M i c r o b i o l o g i c a l •••.••••••»• (c) P l a n t P r o t e i n s versus Animal Proteins Experimental ••••••••• (a) The l e t P r o t e i n U t i l i z a t i o n of a Number of Weed Seed Samples , 1. Assay Method • i . Animals • i i . Diets ... i i i . Procedure «•••«• 2. Calculations 3. Results  G.  5 5 11 17 17 20 28 28  iii  28 28 31 31 32 33 3h 35 37 3® hO hO **0 MD hi hi h2 * + 3 hh  PAGE k.  IV.  Discussion .. h7 Effect of Protein Treatment on N.P.1'. Value . . . . . . . . . . . . *+7 Effect, of Energy Intake on the Biological Value of Proteins. *f8 Length of Experimental Period *+9  The Essential Amino Acid Content of Certain Noxious Weed Seeds • ••••••••••  50  A.  Introduction  50  B.  Experimental 52 (a) The Analysis of the Essential AcidContent of a Number of Weed Seeds. 52 1. Preparation of Samples . . . . . 52 2. Assay Method . . . . . . . . . . . . . . . 52 i . Preparation of hydrolysates • • • • • » • • . • • 52 i i . Assay procedure . . . . . . . . 53 i i i . Organisms . . . . . ® . . . . . . . 53 3. Calculations . . . . . . . . . . . . . . o 5>+ k. Results . . . . . . . . . . . . . . . . . . . . 5*+ (b) Chemical Scores and! Essential Amino Acid Indices for the Various Weed Seed' Proteins ......... 58 (c) Correlation of Modified Essential Aad.no Acid Indices with l e t ProteinnUtilization Values . . . . . . 62 e  V.  Summary  ¥1.  Appendix I.  VII.  •» • • . • • • • • • » . . ® « . . . • • » < » . • • •  66 69  The Nitrogen : Water Relationship i n Albino Rats (a) Introduction . . . . . . . . . . . . . . . . . . . . (b) Experimental Animals . . . . . . . . . . . . (c) Carcass Analysis (d) Results . . . . • . . • • • . . • • • • » . . (e) Calculations » • • • • . » • • . • • • • • • • • • •  70 70 71 72 7k 79 87  Bibliography  iv  LIST OF TABLES TABLE I II III  IT V VI  VII VIII  PAGE I s o t h i o c y a n a t e Determinations  XI  23  Procedures  2k  ..  ••••••«••  P r o t e i n Content of E x t r a c t e d Samples  55  Reported Values f o r the Amino A c i d Composition of a Number o f P r o t e i n s ••••••»••••••••••••»»••  56  Computation  k5  o f the M o d i f i e d E s s e n t i a l Amino A c i d 61  ...  E s s e n t i a l Amino A c i d I n d i c e s and Chemical Scores 63  the P r o t e i n s o f Weed Seeds  E s s e n t i a l Amino A c i d I n d i c e s andi Chemical Scores f o r a Number o f P r o t e i n s ••••••••»..•••  .....  •  69  ••••••••••••  75  ••«•••••••.  : Body Weight - Body Water - Body N i t r o g e n Body Weight - Body Water r Body N i t r o g e n 76  R e l a t i o n s h i p i n Male W i s t a r Rats XIV  Body Weight - Body Water - Body N i t r o g e n 77  R e l a t i o n s h i p i n Female Sprague - Dawley Rats XV  6k  P r o t e i n and F a t Composition o f a Number of Weed  R e l a t i o n s h i p i n Female W i s t a r Rats XIII  .... k2  The Net P r o t e i n U t i l i z a t i o n o f a Number of Proteins The Amino A c i d Content o f the P r o t e i n s o f C e r t a i n Noxious Weed Seeds; and C e r t a i n Standard P r o t e i n Sources  Seed Samples XII  19  E x t r a c t i o n Process • .. Weight Losses A s s o c i a t e d w i t h the E x t r a c t i o n  for X  »  Change i n P r o t e i n Content o f Samples During  Index IX  •••»•••••••  Body Weight - Body Water - Body N i t r o g e n R e l a t i o n s h i p i n Male Sprague - Dawley Rats  ....  XVI  R e g r e s s i o n Equations E x p r e s s i n g the R e l a t i o n s h i p of Body N i t r o g e n to Body Water i n A l b i n o Rats  XVII  A Comparison of the N i t r o g e n Content of W i s t a r Rats Determined C h e m i c a l l y w i t h N i t r o g e n Values C a l c u l a t e d from R e g r e s s i o n Equations ....••.»••  v  78  86  INTRODUCTION  THE NDTRITIVE VALUE OF CERTAIN NOXIOUS WEED SEEDS INTRODUCTION For  the past f i v e years heat processed r e f u s e  screenings  have been used s u c c e s s f u l l y as a ruminant animal f e e d i n B r i t i s h Columbia and s e v e r a l o f the western s t a t e s .  To  date, some 60,000 tons o f the processed m a t e r i a l has been fed s u c c e s s f u l l y .  Under the present  Canada Feeding S t u f f s  Act c e r t a i n o f the weed seeds i n r e f u s e screenings a r e considered  as noxious and a number o f r e g u l a t i o n s  control  t h e i r s a l e and use as a commercial l i v e s t o c k f e e d . There i s adequate evidence (5,53-76) to show t h a t many of these weed seeds c o n t a i n i s o t h i o c y a n a t e s  which may o r may  not be t o x i c when consumed by the domestic a n i m a l s . a l information  on the t o x i c i t y and n u t r i t i v e v a l u e  weed seeds i s needed before  Additiono f these  s u i t a b l e amendments can be made  to the Canada Feeding S t u f f s A c t . The work t o be r e p o r t e d h e r e i n was undertaken t o provide of the n u t r i t i v e v a l u e  a q u a n t i t a t i v e assessment  o f c e r t a i n o f these weed seeds.  Work  on the t o x i c i t y o f the m a t e r i a l i s proceeding elsewhere, under the s u p e r v i s i o n o f the Canada Department o f A g r i c u l t u r e . The weed seeds under study from the f a m i l y  §rae±fer±a@  are c h a r a c t e r i z e d by the presence o f the sharp pungent f l a v o u r ed i s o t h i o c y a n a t e s .  Reports have a l s o appeared i n the  l i t e r a t u r e on the presence o f p o s s i b l e t o x i c f a c t o r s i n a number o f the weed seeds and i n other members o f t h i s (rapeseed, t u r n i p s ) .  family,  I t was expected t h a t an attempt might  - 2 -  have to be made to remove these p a l a t a b i l i t y and/or toxic factors before animal feeding experiments could be c a r r i e d out.  Preliminary studies did indeed show that removal of the  isothiocyanate was necessary.  When rations containing the  ground weed seeds were fed to the laboratory r a t there was complete r e f u s a l by the animals to accept the d i e t s .  The  weight losses and r e s u l t i n g death of the animals could be accounted f o r on the basis of the i n a n i t i o n associated with the feed r e f u s a l rather than on the basis of t o x i c i t y ,  A  s i m i l a r pseudo-toxicity has been noted i n this laboratory i n d i e t s containing more than 7 parts per m i l l i o n of selenium (29).  For these reasons, i t was found necessary to  study the isothiocyanates or mustard-oils  present i n these  weed seeds and attempt to remove them from the representative samples of weed seeds under study.  I t was necessary to  remove these taste factors before any attempt could be made to determine the n u t r i t i v e value of the weed seeds by b i o l o g i c a l methods.  Since there was no assurance that the  removal of the isothiocyanates would be successful, methods for evaluation of the n u t r i t i v e value of a protein not involving animals were sought. To avoid the problem of p a l a t a b i l i t y i n animals, therefore, an attempt was made to assess the n u t r i t i v e value of the various weed seeds by a micro-biological method. proposed by Anderson and Williams  The procedure  (3) for the evaluation of  protein quality using the protozoan, Tetrahymena pyriformis was  studied.  D i f f i c u l t i e s were encountered i n the growth  - 3response  o f the organism to the i n t a c t p r o t e i n and i n the  e x t r a c t i o n of the dye used f o r the assessment o f growth response.  T h i s method has s i n c e been c r i t i z e d by S h e f f n e r  (118) as i t was found that the v a l u e obtained w i t h these methods did  not c o r r e l a t e w e l l w i t h the b i o l o g i c a l v a l u e o f  the  p r o t e i n as determined by animal t e s t s . In r e c e n t years a number o f methods have been proposed which attempt to c o r r e l a t e the n u t r i t i v e v a l u e o f p r o t e i n w i t h t h e i r content o f e s s e n t i a l  amino a c i d s .  I n o r d e r to assess  the n u t r i t i v e v a l u e of the weed seeds by t h i s method, i t necessary  to analyse  the weeds seeds f o r t h e i r content  essential  amino a c i d s .  of  The problem o f d e t e r m i n i n g the n u t r i t i v e v a l u e o f weed seed, t h e r e f o r e , I  resolved i t s e l f  i n t o three  was  the  parts:  An attempt to e x t r a c t the o f f e n d i n g i s o t h i o c y a n a t e  or  t a s t e f a c t o r s from samples of the weed s e e d s . II  An e v a l u a t i o n of the p r o t e i n q u a l i t y of  representative  samples o f weed seeds by an e s t a b l i s h e d b i o l o g i c a l assay procedure. for  Ill  F o r these assays samples^which are p a l a t a b l e  the l a b o r a t o r y r a t , were prepared by the  extraction  procedure evolved i n S e c t i o n  I.  An a n a l y s i s o f the e s s e n t i a l  amino a c i d content o f a number  of  weed s e e d s .  A chemical e v a l u a t i o n o f the p r o t e i n s can  then be c a l c u l a t e d from t h e i r amino a c i d c o m p o s i t i o n . WEED SEED SAMPLES: The  samples o f weed seeds used i n the present  studies  were generously s u p p l i e d by the P l a n t Products D i v i s i o n ,  _1+  Production Service The  -  o f the Dominion Department o f A g r i c u l t u r e .  common and s c i e n t i f i c names (99)  presented i n the f o l l o w i n g l i s t .  o f these weed seeds a r e  P e r t i n e n t data on the  p r o t e i n and f a t content o f these seeds a r e presented i n the Appendix I . Common Name  S c i e n t i f i c Name  White mustard  Brassica h i r t a or B r a s s i c a a l b a or S i n a p l s a l b a  Moench Boiss L  W i l d mustard  B r a s s i c a kaber (DC) L.C. or B r a s s i c a a r v e n s i s Ktze or S i n a p l s arvensiT" L  Brown mustard  Brassica  False  Camellna microcarpa  Flax  .juncea  (L) Coss Andrz  Tumble mustard  Sisymbrium a l t l s s i m u m L Norta a l t i s s l m a Britton  Stinkweed  T h l a s p l arvense  L  Wheeler  ATTEMPT TO REMOVE ISOTHIOCYANATES FROM REPRESENTATIVE SAMPLES OF WEED SEEDS  - 5 II  ATTEMPT TO REMOVE ISOTHIOCYANATES FROM REPRESENTATIVE SAMPLES OF WEED SEEDS A.  INTRODUCTION: I n p r e l i m i n a r y experiments, when the ground o r f a t  e x t r a c t e d samples o f weed seeds were i n c o r p o r a t e d p l e t e r a t i o n s , they were n o t accepted  by the r a t .  i n t o comThis r e f u s a l  was a t t r i b u t e d t o a p a l a t a b i l i t y f a c t o r which i s known t o be a s s o c i a t e d w i t h the presence o f mustard o i l s o r i s o t h i o c y a n a t e s i n these seeds.  I f these weed seeds a r e t o be assessed f o r  t h e i r n u t r i t i v e value by animal t e s t s , an attempt must be made t o remove these t a s t e f a c t o r s .  However, b e f o r e  this  was p o s s i b l e , a study o f the occurence and p r o p e r t i e s o f the isothiocyanates  themselves seemed a d v i s a b l e .  the d e t e r m i n a t i o n  o f the i s o t h i o c y a n a t e content  weed seeds was a l s o sought. content  Determinations o f the i s o t h i o c y a n a t e  a g a i n s t which attempts t o remove the  i s o t h i o c y a n a t e can be a s s e s s e d . a procedure was evolved weed seeds i n order  With these f a c t s , a t hand,  t o remove the i s o t h i o c y a n a t e s from the  t h a t these samples c o u l d be f e d t o r a t s  for b i o l o g i c a l evaluation  (a)  o f the v a r i o u s  o f a number o f the weed seeds i s d e s i r a b l e i n o r d e r  to o b t a i n b a s i c v a l u e s  B.  A method f o r  tests.  LITERATURE REVIEW isothiocyanates The  presence o f a number o f v o l a t i l e i s o t h i o c y a n a t e s i n  the t i s s u e s , and e s p e c i a l l y the seeds o f the C r u c i f e r a and other p l a n t s , has been e s t a b l i s h e d .  S i n i g r i n , the p r e c u r s o r  - 6 of the i s o t h i o c y a n a t e o f b l a c k mustard ( B r a s s i c a n i g r a (L) Koch) was i s o l a t e d i n 1839,(33), and S i n a l b i n from white mustard ( B r a s s i c a a l b a B o i s s ) i n 1831, (33). no proof o f the s t r u c t u r e o f these v a r i o u s had  U n t i l recently Isothiocyanates  been o f f e r e d . I n 1953, K j a e r and co-workers (5,53-76) began an  extensive  s e r i e s o f s t u d i e s on the occurence, i s o l a t i o n ,  s y n t h e s i s and i d e n t i f i c a t i o n o f the i s o t h i o c y a n a t e s .  Using  a paper chromatographic procedure (73) f o r the s e p a r a t i o n and  i d e n t i f i c a t i o n o f the t h i o u r e a d e r i v a t i v e s o f the  .isothiocyanates, they i n v e s t i g a t e d the seeds and the f r e s h p a r t s o f v a r i o u s p l a n t s f o r t h e i r content Included  of isothiocyanates.  i n the study were numerous seed samples  belonging  to the C r u c i f e r a e , Resedaceae,Tropaelaceae,Capparldaceae. Phytolaccuceae and Euphorbiaceae. The  i s o t h i o c y a n a t e s occur i n nature as t h i o g l u c o s i d e s ,  that i s i n connection bisulphate ion.  These n a t u r a l l y o c c u r l n g  containing glucosides according  w i t h a molecule o f glucose  and the  isothiocyanate  can be c l a s s i f i e d i n t o two groups,  to whether o r not t h e i r i s o t h i o c y a n a t e s a r e v o l a t i l e  w i t h steam.  W i t h i n the former group, the occurence o f a l l y l ,  (+) - s e c - b u t y l , b e n z y l and B - p h e n y l e t h y l has been e s t a b l i s h e d f o r some time.  isothiocyanates.  Recently Kjaer e t a l  (5,53-76) have demonstrated the widespread occurence o f a d d i t i o n a l isothiocyanates. including>butenyl;  iso-propyl;  methyl; the *f-methythio - and Y-methylsul phony 1 form s ; o f b u t y l and p r o p y l i s o t h i o c y a n a t e . and many o t h e r s .  - 7 I n many C r u c i f e r a e there i s an enzyme known as (myrosin or s i n i g r i n a s e ) which h y d r o l y z e s  o n l y the  mustard o i l t h i o g l u c o s i d e s . Myrosinase has c o n s i s t of two bisulphate  been shown to  ( u s u a l l y as the potassium s a l t ) and  the h y d r o l y s i s of s i n i g r i n  mustard) may  natural  enzymes, a m y r o s u l f a t a s e which s p l i t s o f f  which s p l i t s the t h i o g l u c o s i d e l i n k a g e . shows how  myrosinase  The  a  thioglucosidasse  f o l l o w i n g scheme  (the g l u c o s i d e  of brown  proceed  C  3 %  N = C  -  Sinigrin  -  C6HUO5  0 - SC3K myrosulfatase H  C H N = a  2  0  ,s 'OH  merosinigrin  + KH  SO.  thioglucosidase H0 +  2  CH = CH - CH_ 2  2  - N = C =? S  + C,H 0 6 12  allylisothlocyanate  glucose  U n t i l r e c e n t l y , the accepted s t r u c t u r e f o r the myronate i o n was  t h a t proposed by Gadamer i n 1897  the i s o t h i o c y a n a t e anion.  (33,^9) i n which  i s l i n k e d to D-glucose and  the s u l f a t e  - 8 -  (structure I) R-N  = c'  6  1  5  1  ^oso ~ 3  However, i n 1956 E t t l i n g e r and Lundeen (33)  demonstrated  a r e v i s e d s t r u c t u r a l e x p r e s s i o n f o r the mustard o i l g l u c o s i d e s ( s t r u c t u r e I I ) and propose t h a t the mustard o i l s ( I I I ) a r e formed i n nature by an enzyme-actuated Lessen re-arrangement (23).  The complete f o r m u l a t i o n o f s i n i g r i n as a  B-glucopyranoside  i n presented  R - C - SC.HO_. || * N - OSO-j~ 6  1  s t r u c t u r e IV.  R - N = C = S  1  II  III  myronate i o n  CH = CH-CH -C  2  2  isothiocyanate  H S-C  I  —  HCOH  I  0  HOCH  so o 2  I  HCOH  I  HC I CH OH  2  IV sinigrin Kjaer  (53)  noted the chemical  s i m i l a r i t i e s between  n a t u r a l l y o c c u r i n g i s o t h i o c y a n a t e s and the common < amino  -  a c i d s and  -  suggested a p o s s i b l e b i o c h e m i c a l r e l a t i o n s h i p . Lundeen ( 5 3 ) have noted the resemblance i n  E t t l i n g e r and configurations  of amino a c i d s and  (compare t y r o s i n e and and  9  natural  (+)  mustard o i l g l u c o s i d e s  glucosinalbate).  Since isoleucine  2 - b u t y l isothiocyanate,  belong to the same  s t e r e o c h e m i c a l s e r i e s they have suggested t h a t the ing glucoside  could,  correspond-  presumably, be r e l a t e d through  (+)  2 methyl b u t y r i c a c i d . During the course of t h e i r i n v e s t i g a t i o n s of n a t u r a l l y occuring  isothiocyanates,  68,75) e s t a b l i s h e d  K j a e r and  workers  the f o l l o w i n g , which i s of i n t e r e s t to  the present study of the weed seeds. mustard  (Brassica  (5>55>57»58,59>  .juncea (L) Coss) and  The  seeds o f brown  stinkweed  (Thlaspi  arvense L) were found to c o n t a i n a l l v l i s o t h i o c v a n a t e K j a e r e t a l (58)  o r i g i n a l l y demonstrated the presence of  three v o l a t i l e i s o t h i o c y a n a t e s ( B r a s s i c a napus L) p r o v i n g 3-butenyl-lsothiocyanate  i n enzyme t r e a t e d rape seeds.  the main c o n s t i t u e n t  (VI).  made p o s s i b l e  amounts.  be of  occuring  the e s t a b l i s h m e n t of a complete  p i c t u r e of the mustard o i l of rapeseed (55). glucosides  to  However, as these s e r i e s  i n v e s t i g a t i o n s continued a d d i t i o n a l n a t u r a l l y isothiocyanates  (5)«  Six individual  are p r e s e n t , three of which occur i n v e r y They a r e : g l u c o i b e r i n (or a c l o s e l y a l l i e d  small compound,  traces only) p r o g o i t r i n ( g l u c o r a p i f e r i n ) , s i n a l b i n ( t r a c e s ) , glucorapin, yielding  (-)  glucobrassicanapin  and  glucorasturtin  3 methyl-sulphinylpropyl  (or a r e l a t e d s p e c i e s ) ,  (-)  - 5  (traces)  ( X I ) , mustard o i l  vinyl-2-oxazolidinethione  (X), p-hydroxybenzyl, ( V I I I ) , 3 - b u t e n y l ,  ( V I ) , *f-pentenyl  (VII),  - 10 and 2-phenyelethyl (IX) Isothiocyanate, r e s p e c t i v e l y , on enzymatic h y d r o l y s i s . CH.=CHGH NCS 0  *  2  — CH — NH 2  a l l y l isothiocyanate  CH  2  GH_ = CH - GH  C = S X  = CHCH CH NCS 2  2  (*)-5-Vinyl-2-ozazolidinethione  VI 3-butenyl isothiocyanate  CH  1.3  0 4—s  CH 2=^HCH2CHJ3H 2 2NCS 0  CH -2  0  0  VII  '  H--pentenyl isothiocyanate HO \_J>  2  ^ XI  CH N =C=S 2  (•) 3 methylsulphinylpropyl isothiocyanate  VIII P-hydroxybenzyl i s o t h i o c y a n a t e CH^-CH -N=C=S CH^ 0  <^_^> CH -CH N=C=S 2  2  IX  XII  2-phenylethyl isothiocyanate  i s o - p r o p y l isothiocyanate  0 —  CH I 3  «-S  NCS XIII  - 11 ( S t r u c t u r e XIII o n p r e c e e d i n g page  XIII )  ( -  ) - 10  -  methylsulphinyldeeyl Isothiocyanate  Besides t r a c e s of the v o l a t i l e isj2-propyl-isothiocyanate, (59)  (XII)  seeds of white mustard ( B r a s s i c a a l b a B o i s s ) were  found to c o n t a i n a n o n - v o l a t i l e i s o t h i o c y a n a t e i d e n t i f i e d p-hydroxybenzvl-isothiocvanate  (VIII) (75)  present  (53-8).  aglucone of the g l u c o s i d e s i n a l b i n .  as  the  Seeds of  the  c r u c i f e r o u s s p e c i e s Camelina microcarpa A n d i z , as F a l s e f l a x c o n t a i n two  glucoside containing isothiocyanate  ( - ) - 10  -  methylsulphinyldeeyl  (XIII) as the aglucone.  i s o t h i o c y a n a t e content  and  However, two  the  of Tumble mustard (Sisymbrium  altlssimum  have been r e p o r t e d  to c o n t a i n  L  glucosides  s e c - b u t y l and i s o - p r o p v l ( t r a c e s o n l y ) i s o t h i o -  cyanates, r e s p e c t i v e l y (b)  of  species of the Sisymbrium genus, S. sophia  S. s t r i c t i s s i m u m L  of a l l y l , and  (68).  has been i d e n t i f i e d as a  At t h i s time, no r e p o r t has been presented  L).  commonly known  isothiocyanate glucosides.  The c h i e f c o n s t i t u e n t , g l u c o c a m e l i n i n  as  (59)•  Toxicity A number of the weed seeds under study belong to the genus  B r a s s i c a of the f a m i l y G r u c i f e r a e  .  The  seeds of rape ( B r a s s i c a  nanus L) have been u t i l i z e d f o r animal feeds f o r a number of years.  A comparison of the seeds of rape and mustard i s ,  t h e r e f o r e , warranted.  - 12 In r e c e n t years the n u t r i t i v e v a l u e received considerable  study mainly i n c o n n e c t i o n  presented that rapeseed o i l m e a l c o n t a i n s factors.  In 1901  o f rapeseed has  Sjollema  (119)  w i t h evidence  one or more t o x i c  isolated a C  isothiocyanate,  5 which he claimed  t o have i d e n t i f i e d as c r o t o n y l i s o t h i o c y a n a t e ,  as a c o n s t i t u e n t o f the e s s e n t i a l o i l f r a c t i o n o f rapeseed. The  i d e n t i f i c a t i o n was not p o s i t i v e .  et a l found i s o t h i o c y a n a t e s  In 1920,  Viehover  (123)  i n rape and mustard seed and  established their r e l a t i v e t o x i c i t i e s i n rabbits. Kennedy and Purges, (hk, h5, 52) r e p o r t e d  In  19*+1,  h y p e r p l a s t i c changes  i n the t h y r o i d , and changes i n the p i t u i t a r y s i m i l a r to those f o l l o w i n g thyroidectomy, as a r e s u l t o f f e e d i n g B r a s s i c a seeds to r a t s . also  Delayed m a t u r a t i o n of o v a r i e s i n immature r a t s was  reported. Evidence based on r a t s (^,  (30, 77,  122),  c a t t l e (11)  turkey p o u l t s  *f5, 52),  (17),  rabbits  swine (100)  (123),  and p o s s i b l y  have suggested t h a t some t o x i c f a c t o r (or f a c t o r s )  e x i s t i n rapeseed o i l m e a l .  These t o x i c f a c t o r s u s u a l l y mani-  f e s t themselves i n the form of a t h y r o i d h y p e r p l a s i a and  growth d e p r e s s i o n .  (goitre)  I n a d d i t i o n there i s s u s p i c i o n on the  p a r t o f a number o f workers (12,  27)  that more than one t o x i c  f a c t o r may be i n v o l v e d s i n c e e x t e n s i v e and  chicks  kidney, l i v e r ,  adrenal  growth Involvements have a r i s e n i n c e r t a i n experiments. Goitrogenic  substances are not r e s t r i c t e d  to rapeseed n o r ,  i n f a c t , to the B r a s s i c a f a m i l y , nor does the evidence i n d i c a t e that the same f a c t o r i s i n v o l v e d i n a l l members o f the B r a s s i c a  - 13 family.  Soybean and  i n regard to p o s s i b l e  l i n s e e d have a l s o r e c e i v e d goitrogenic  A number of r e p o r t s attention  to the  d i r e c t l y on Bach (11) ascorbic  -  e f f e c t s of these  found a d e f i n i t e r e t a r d a t i o n  'goitrogens'  of t i s s u e o x i d a t i o n  x 10.hM.  Benda (11)  r e s p i r a t i o n i n guinea p i g l i v e r s l i c e s was small concentrations  Berg and  reactions  by  F l i c h s t e i n (11)  (10~ M) of  depressed by  50  allylisothiocyanate.  p r o p y l - i s o t h i o c y a n a t e ) and  (56)  erysoline  i s o t h i o c y a n a t e ) on a r a t h e r  large  have i n d i c a t e d  of the  reports  (y-methylsulphonylbutyl  0+3).  w i l l be  report  on  tested  statement i n paper No.  goitrogenic  To date, the evidence r e g a r d i n g the No  (5)  of  lsothlocavanates.  a c t i v i t y when a l l y l ,  e t h y l or p h e n y l - i s o t h l o c y a n a t e s were f e d t o  thiocyanates i s c o n f l i c t i n g .  II  has  p r a c t i c a l l y no  a n t i t h y r o i d a c t i v i t y o f the n a t u r a l l y o c c u r i n g found no  that  for their a n t i -  this investigation  t h i s s e r i e s t h a t a n i m a l t e s t s have i n d i c a t e d  Marine et a l (81)  mentioned  ;  However, no  been found except f o r the  methylsulphonyl-  • x' K j a e r e t a l , (76)  a number of these i s o t h i o c y a n a t e s activity.  Of-  a  s e l e c t i o n of pathogenic  ( p l a n t pathogenic b a c t e r i a )  In one  reported  r e p o r t e d i n h i b i t i o n o f dehydrogenation  v e r y pronounced e f f e c t of both c h e r r o l i n e  thyroid  of  allylisothiocyanate,  2  Bacteriological investigations  bacteria  function.  a c i d when e i t h e r a l l y l i s o t h i o c y a n a t e or s i n i g r i n was  percent by and  82).  Independently of t h y r o i d  p r e s e n t i n c o n c e n t r a t i o n s of 5.7 that  (26,  have been presented which d i r e c t  possible  t i s s u e s and  factors.  investigation  rabbits.  t o x i c i t y of i s o -  conclusions concerning  the  - ih t o x i c i t y of the i s o t h i o c y a n a t e s Recently, compound may  however, i t has been suggested t h a t another  be r e s p o n s i b l e f o r the a n t i t h y r o i d a c t i v i t y found  i n rapeseed and 19^9  themselves can be made.  o n l y two  Astwood (6)  other meals.  5>5  - dimethyl  found i n Laburnum angyroides L  - 2 -  thioxazolidone  (Golden chain) and  (Hare's ear mustard). ( ^ 9 ) .  Recently,  Conringia  l a t e r shown to be L-5  vlnyl-2-thioxazolidone.  was  Astwood  some evidence that might i n d i c a t e t h a t  (6)  Astwood  found another g o i t r o g e n i c substance i n rutabaga, which  presented  to  a n t i t h y r o i d compounds have been found i n p l a n t s .  These were t h i o u r e a and  orientalis  s t a t e s that up  (6)  the  t h i o x a z o l i d o n e i s formed from some precursor by enzymatic a c t i o n . Astwood (6) rutabaga was was  found that when the r o o t OP.' seed o f  f i r s t b o i l e d i n water the a n t i t h y r o i d a c t i v i t y  destroyed  and no  thio-oxazolidone  suggests that the f o r m a t i o n enzymatic r e a c t i o n . thiocyanates  and  thio-oxazolidone Kjaer,  (63)  c o u l d be i s o l a t e d .  of t h i s compound may (107)  P i t t Rivers  thio-oxazolidones  involve  proposed that the  may  i n t e s t i n g the hypothesis,  found  ( i n f a c t , ) the n a t u r a l  of 5 , 5 - d i m e t h y l - 2 - o x a z o l d i n e t h i o n e (XVI). g l u c o c o n r i n g i i n , a f f o r d s glucose, 2-methyl-propyl I s o t h i o c y a n a t e s  The  Conringia  precursor  glycoside, 2-hydroxy-  on enzymatic h y d r o l y s i s .  The mustard o i l c y c l i s e s spontaneously to 5 » 5 - d i m e t h y l - 2 o x a z o l i d i n e t h i o n e as presented  iso-  that  s u l p h u r i c a e i d and  (XV)  an  closure.  the g l u c o s i d e of the c r u c i f e r  (L) Andrez, was  This  be I n t e r r e l a t e d , the  a r i s i n g upon o x i d a t i o n and r i n g  g l u c o c o n r i n g i i n (XIV) orientalis  the  by the f o l l o w i n g scheme.  - 15 |—0 — OH H OH  CH„  I I I I  S -CH-C-C-C-C-CH -OH CH -C-CH  3  2  2  I I I )  -N=C  H OH HH o-so -o2  myrosinase  K  XIV glucoconringiin  CH 13 CH -C- CH„-N=C=S 3  1  eyelisation >  C - NH l l CH - C C = S  /  2  OH  CH  V 3  XV  XVI  2-hydroxy-2-methyl-propyl isothiocvanate  5,5-dimethyl-2-oxazolidinethione  Because the r e a c t i o n i s spontaneous the 2-hydroxy-2m e t h y l - p r o p y l i s o t h i o c y a n a t e has not been i s o l a t e d . i s a v a i l a b l e f o r the e x i s t e n c e o f a d d i t i o n a l thione producing glucos:ides i n p l a n t s . (6). of the 2-hydroxy-3-butenyl i s o t h i o c y a n a t e  Evidence  2-oxazolidineEnzymatic h y d r o l y s i s  glucoside,  p r o g o i t r i n ( g l u k o r a p i f e r i n ) i s r e s p o n s i b l e f o r the p r o d u c t i o n o f the g o i t r o g e n i c  (-)-5-vinyl-2-oxazolidinethione, previously  i s o l a t e d from seeds and f r e s h p a r t s of v a r i o u s B r a s s i c a i n c l u d i n g rape, b u t excluding  species  the three v a r i e t i e s of mustard  - 16 tested  (6).  A l t h o u g h e s t a b l i s h e d as g o i t r o g e n i c , c o n t r o v e r s i e s e x i s t as t o whether 2 - o x a z o l i d i n e t h i o n e a r e growth depressants ( 1 1 ) . (125) suggested another r o u t e by which t o x i c  Wetter  compounds might a r i s e from the i s o t h i o c y a n a t e s .  The g o i t r o -  genic? t h i o u r e a s c o u l d be d e r i v e d from the r e a c t i o n o f i s o t h i o cyanates w i t h ammonia, the ammonia a r i s i n g from v a r i o u s sources i n the animal body.  F o r example, ammonia i s formed  i n the  i n t e s t i n e from deamination o f amino a c i d s by micro-organisms. To date, no experimental evidence has been presented i n support of t h i s p o s t u l a t e . Attempts  have been made by a number of workers t o c o u n t e r -  a c t or remove the t o x i c or g o i t r o g e n i c f a c t o r .  A degree o f  improvement i n n u t r i t i o n a l q u a l i t y has been observed from the f o l l o w i n g treatments w i t h rapeseed: (11,  1  81), i o d i d e ( 1 0 8 , 1 1 , 30) ( 7 9 ) , protamone (17)  plus a n t i b i o t i c s and l i m i t i n g (11,  cooking ( 1 2 * ) ,  30).  steaming steaming  ( 1 1 ) , water e x t r a c t i o n ( 1 2 , 2 7 , 3 0 , * + 2 ) , ( 8 l ) ,  the amount of rapeseed o i l - m e a l i n the r a t i o n  The m a j o r i t y o f the r e s e a r c h suggests  that  l i m i t a t i o n o f use i s the only s a t i s f a c t o r y and p r a c t i c a b l e method y e t a v a i l a b l e to c o u n t e r a c t the t o t a l e f f e c t o f the rapeseed o i l - m e a l f a c t o r s . ( 1 1 ) .  - 17 c  » (a)  -  EXPERIMENTAL: Determination  of i s o t h i o c y a n a t e s .  A number of methods have been d e s c r i b e d f o r the q u a n t i t a t i v e e s t i m a t i o n of mustard o i l s i n seeds. have been reviewed r e c e n t l y by Andre (k).  These methods The  commonly  employed methods i n v o l v e removal of the mustard o i l by steam distillation, distillate  w i t h the f o r m a t i o n of a t h i o u r e a when  i s c o l l e c t e d i n ammonia.  t r a c t e d and/or estimated  The  the  t h i o u r e a may  by v a r i o u s means.  The  be  ex-  argentimetric  method measures the s i l v e r consumed i n p r e c i p i t a t i n g  the  (27,  the argen-  102,  121).  R e c e n t l y , Wetter (12M-) has m o d i f i e d  t i m e t r i c method f o r e s t i m a t i o n of mustard o i l s ;  the  sulphur.  principal  m o d i f i c a t i o n being made i n the treatment p r i o r to steam distillation.  I n d i r e c t determination  of i s o t h i o c y a n a t e s based  on the sulphates produced from the g l u c o s i d e s by r e s o l u t i o n r e a c t i o n (115) filtrates  (83)  or based on the a b s o r p t i o n of i o d i n e by the seed have a l s o been used.  K j a e r and co-workers (73) chromatographic technique  have developed a paper  f o r demonstrating the presence of  i s o t h i o c y a n a t e i n v a r i o u s p l a n t t i s s u e s and u l t r a v i o l e t spectrophotometrie  method (59)  e v a l u a t i o n of the I s o t h i o c y a n a t e s  have developed an* for a quantitative  as t h i o u r e a s .  This procedure  takes i n t o account the s p e c i f i c i s o t h i o c y a n a t e i n c o n t r a s t to the other methods i n which the v o l a t i l e mustard o i l i s u s u a l l y c a l c u l a t e d as a l l y l i s o t h i o c y a n a t e . by K j a e r  (59)»  Using  the method proposed  a number of the weed seeds were assayed f o r t h e i r  - 18 content of v o l a t i l e i s o t h i o c y a n a t e s .  These d e t e r m i n a t i o n s were  c a r r i e d out to o b t a i n b a s i c v a l u e s a g a i n s t which attempts t o remove the i s o t h i o c y a n a t e s c o u l d be a s s e s s e d . presented i n Table I . stinkweed  The r e s u l t s a r e  The v a l u e s o b t a i n e d f o r white mustard,  and brown mustard a r e comparable t o those r e p o r t e d  by K j a e r (when the v a r i a t i o n s i n procedure  a r e taken i n t o  accountt he accuracy of methodis r e p o r t e d to be about $P%).  The  v a l u e s f o r rapeseed a r e low compared t o r e p o r t e d v a l u e s but Wetter (12*f) has i n d i c a t e d that the isothiocyanate:: content of rapeseed v a r i e s c o n s i d e r a b l y depending on the source and variety.  K j a e r , ejt aj, (58) r e p o r t s t h a t i n d u s t r i a l l y  rapeseed cakes showed g r e a t v a r i a t i o n s i n t h e i r  content,  a p p a r e n t l y dependent on the c o n d i t i o n s employed d u r i n g manufacture.  produced  their  TAB IS I ISOTHIOCYANATE  DETERMINATIONS  Isothiocyanate  mg/100 gm.  3-butenyl.  29,0  White Mustard  iso-propyl  8.3  Stinkweed  allyl  286  327  (5)  Brown Mustard  allyl  6lh  518  (5)  W i l d Mustard  allyl *  5A  allyl *  27.0°  Seed  Argentine Rapeseed  fat-free sample  Published values mg/100 gm. fat-free sample  lOOmg/lOOgm  0.3 to 1.3%  Reference number  (58)  (121+)  3 ( 5 9 )  After extraction w i t h 70% ethanol ~  21.2 2.0  Refuse screenings U.B.C. R a t i o n No-72 *  c a l c u l a t e d as a l l y l i s o t h i o c y a n a t e s i n c e type of isjojihioeyanate not y e t determined.  0  sample not f a t - f r e e .  - 20 (b)  E x t r a c t i o n Procedure P r e l i m i n a r y attempts were made t o remove t h e o f f e n d i n g  i§o_£hiocyanates by s o l v e n t e x t r a c t i o n . I s o t h i o c y a n a t e s a r e o n l y s l i g h t l y s o l u b l e i n water b u t a r e s o l u b l e i n e t h e r , a l c o h o l and o t h e r o r g a n i c s o l v e n t s .  I n order to  (^7>).  d e t e r m i n e i f any s i n g l e s o l v e n t would be e f f e c t i v e  i n re-  moving these i s o t h i o c y a n a t e s from the s e e d , a number o f s o l v e n t s were t e s t e d .  The s o l v e n t s used on s e p a r a t e samples o f t h e  raw ground seeds i n c l u d e d t h e f o l l o w i n g : diehlorethylene  B o i l i n g Po^nt  -  37.0°C.  ethylether  3^«6°C.  petroleum ether  20 -l*O°C.  acetone  56.1°G.  a l c o h o l - ethanol  78«5°0.  carbon t e t r a c h l o r i d e  76«8°C.  o  w a t e r a f t e r h y d r o l y s i s a t 37°C. As i n d i c a t e d by o r g a n o l e p t i c t e s t s , met w i t h complete f a i l u r e .  a l l these treatments  The f a i l u r e t o remove t h e  o f f e n d i n g i s o t h i o c y a n a t e s c a n p r o b a b l y be a t t r i b u t e d t o t h e fact form.  t h a t they a r e p r e s e n t i n the weed seeds i n c o n j u g a t e d When t h e samples were h y d r o l y s e d w i t h w a t e r a t 37°C.  t h e n steam d i s t i l l e d t h e r e was some l o w e r i n g o f t h e i s o t h i o c y a n a t e c o n t e n t as i n d i c a t e d by t a s t e t e s t s ,  As mentioned  e a r l i e r , a number o f procedures i n v o l v i n g s t e a m i n g , c o o k i n g , o r water e x t r a c t i o n have been proposed i n an attempt t o remove t h e g o i t r o g e n i c f a c t o r from these s e e d s .  These have met w i t h  v a r y i n g degrees o f s u c c e s s . ' However, i t was f e l t  that t h i s  -  r i g o u r o u s treatment might  21  -  l e a d to p r o t e i n damage and hence  p r e c l u d e a v a l i d assessment  of the b i o l o g i c a l v a l u e o f the  protein. A f t e r many e x p l o r a t o r y experiments, the f o l l o w i n g e x t r a c t i o n procedure was envolved. 1.  the ground hO°G.  seed samples were allowed to s t e e p a t 37° -  f o r 3 to h hours  ( H o l l y and Sandberg).  (dependent  upon the h y d r o l y t i c a c t i v i t i e s of the myrosinase  present  i n the s e e d s ) . 2.  the sample was then d r i e d i n a t u n n e l d r y e r a t 80 8|? C. to c o n s t a n t weight 0  3.  (2*+ - *+8 h o u r s ) .  by mixing i n a Waring b l e n d e r the d r i e d sample was e x t r a c t e d t h r e e times w i t h 70% e t h a n o l (by volume, s p e c i f i c gravity  0.8900).  The excess a l c o h o l was removed between  each e x t r a c t i o n by s p i n n i n g i n a basket h,  the a l c o h o l e x t r a c t e d sample was d r i e d i n a t u n n e l d r y e r a t 80 - 85°  5.  centrifuge.  t o a c o n s t a n t weight.  the d r i e d sample was then p l a c e d i n a macro-soxhlet and extracted 8 - 1 0  times w i t h e t h y l e n e d i c h l o r i d e t o remove  fat. 6.  a f t e r d e f a t t i n g , the samples were allowed t o a i r d r y a t room temperature  7.  to remove the s o l v e n t .  the s o l v e n t - f r e e meal was then ground  i n a hammer  mill  to pass a No 20 mesh s e i v e . Samples o f white mustard, stinkweed,  iBra'ssj-'Ca a l b a B o i s s ) ,  (Thlaspiarviense L ) , rapeseed  (Brassica, napus L ) .  - 22  -  soybean ( G l y c i n e soya)» and  l i n e s e e d ( L i n o m u s i t a l i s s i u m ) , were  e x t r a c t e d by  method.  the preceeding  White mustard and  stinkweed  were s e l e c t e d as r e p r e s e n t a t i v e samples of the weed seeds. a member of the C r u c i f e r a e f a m i l y , rapeseed was comparative purposes.  Lineseed  and  As  chosen f o r  soybean are e s t a b l i s h e d  as p l a n t p r o t e i n s of good q u a l i t y and were chosen f o r comparative purposes. Table content  I I presents  during  a p i c t u r e of the change of  the p r o c e s s .  As i n d i c a t e d by  the  determinations  c a r r i e d out on the e x t r a c t , l i t t l e  (0.5  per 100  - 0.9  gms  gms  of raw  nitrogen nitrogen nitrogen  ground seed) was  lost  during  during  the  the e x t r a c t i o n procedure w i t h a l c o h o l . Table  I I I i n d i c a t e s the l o s s e s t h a t o c c u r r e d  e x t r a c t i o n procedure.  No  s p e c i a l attempts were made to make  the procedure q u a n t i t a t i v e and d u r i n g f a t e x t r a c t i o n ) may  so the major l o s s e s  (except  be accounted f o r through mechanical  losses. Organoleptic  t e s t s of the weed seed meals prepared by  t h i s method r e v e a l e d a great r e d u c t i o n i n the b i t t e r constituents.  Isothiocyanate  determinations  on two  flavoured of  the  prepared meals i n d i c a t e some r e d u c t i o n i n the i s o t h i o c y a n a t e l e v e l s but can not be c o n s i d e r e d  to be  too c o n c l u s i v e i n t h i s  r e s p e c t s i n c e the a n a l y t i c a l method i s not  too s a t i s f a c t o r y  a t low c o n c e n t r a t i o n of t h e - i ^ t h i o c y a n a t e s A d d i t i o n a l a n a l y s i s of t h i s aspect  of the problem were planned but  major problem under study i n the present major concern, the prepared meals now  t h i s was  investigation.  proved to be  not  the  Of  acceptable  - 23 -  TABLE I I CHANGE IN PROTEIN CONTENT O F SAMPLES DURING EXTRACTION PROCESS  % P r o t e i n (NX Seed  Ground Seed  Extracted with 70% E t h a n o l  6.2?) Eihanol extracted Dichlor©ethylene extracted  White Mustard  28.8  29.6  *f2.3  Stinkweed  16.5  16.3  23.7  Rapeseed  28A  29.8  ¥+.6  Linseed  21.3  23.9  3^.3  Soybean  38.5  h5.5  52.9  -  2^  -  TABLE I I I WEIGHT LOSSES ASSOCIATED WITH THE EXTRACTION PROCEDURES  Seed Sample  Weight Weight a f t e r Weight a f t e r Weight a f t e r F i n a l of autoEthanol Fat Yield Ground h y d r o l y s i s Extraction Extraction Sample w i t h water with Ethylene dichloride gm gm per cent gm per cent gm per cent per cent Loss Loss Loss  White Mustard 1800 I693  I89I  33.9  U-7.0  1517 15.7  969 36.2  51.0  1V00 12.5  815 ^1.7  *+5*2  M 2  89I  36.9  M+.5  1527 15.2 1275 16.5  63.8  5,9  1211  5A  Stinkweed  2000  Rapeseed  1800 1600  11.1  Linseed  2000 1826  8.7  Soybean  2000 1800  10.0  2^.3  22.7  800  _ 25 - . to the r a t s . I t i s known t h a t the 70% e t h a n o l removes a c e r t a i n p o r t i o n of the weedseed p r o t e i n (probably from the prolamine  fraction).  I n f a c t , i t may be t h a t the i s o t h i o c y a n a t e s a r e conjugated i n some manner w i t h the prolamins  and hence an e x p l a n a t i o n  may be p r o v i d e d f o r the s u c c e s s f u l use o f 70% e t h a n o l as an extractant,  Frolich  (*+2) has r e p o r t e d t h a t a c o n s i d e r a b l e  amount of the t o x i c p r i n c i p l e i n rapeseed  can be e x t r a c t e d  w i t h 70% e t h a n o l . I t i s a l s o known t h a t d e n a t u r a t i o n o f the p r o t e i n w i l l occur d u r i n g t h i s procedure,  B e c k e l , B u l l and Hopper, ( 1 0 ) ,  working w i t h soybean, s e t up the f o l l o w i n g q u a n t i t a t i v e e x p r e s s i o n o f the degree o f d e n a t u r a t i o n by heat and moisture u s i n g water d i s p e r s i b i l i t y o f the n i t r o g e n o f the meal as a criterion. % d e n a t u r a t i o n =* 100  The  1 -  % % water d i s p e r s i b l e N - noni n t r e a t e d sample ' protein N % water d i s p e r s i b l e N ^ i n o r i g i n a l sample - nonprotein N  extent o f d e n a t u r a t i o n by heat and moisture i s  dependent on the r e l a t i v e combination  o f these two f a c t o r s .  Data o f these workers i n d i c a t e t h a t a c r i t i c a l  temperature  probably e x i s t s a t each r e l a t i v e humidity below which v e r y l i t t l e d e n a t u r a t i o n takes p l a c e , but above which d e n a t u r a t i o n is rapid.  With soybean, 26% d e n a t u r a t i o n occured  h e a t i n g f o r 2 5 hours a t 80°C. and 100% r e l a t i v e  after  humidity.  However, Evans and S t . John, ( 3 ^ ) , i n s t u d y i n g the e f f e c t of d e n a t u r a t i o n on the s o l u b i l i t y o f soybean i n v a r i o u s  - 26 s o l v e n t s showed t h a t the percentage o f t o t a l s o l u b l e i n 70% e t h a n o l decreased w i t h  nitrogen  the d e n a t u r a t l o n o f  the p r o t e i n (the percentage o f non-soluble  nitrogen  increased).  On the bases o f the above r e p o r t i t seems safe t o conclude t h a t the percentage o f the p r o t e i n e x t r a c t e d by 70% should be s m a l l (3-5%  o f the i n i t i a l ground  seed).  Next t o heat i n importance as a d e n a t u r a t i n g i n the p r o c e s s i n g  ethanol  agent  o f soybeans o r other p l a n t p r o t e i n s i s  the choice o f s o l v e n t .  Markley (82) has reviewed a number  o f s t u d i e s c a r r i e d out on s o l v e n t e x t r a c t i o n o f soybeans. These s t u d i e s permit the c o n c l u s i o n t h a t low moisture and  content  a s o l v e n t w i t h a low b o i l i n g p o i n t a r e o f primary  importance i n a l l f a t e x t r a c t i o n procedures t e s t e d .  Ethanol  e x t r a c t i o n has been shown t o improve the f l a v o u r and c o l o u r o f soybean f l o u r . The  use o f ethylene  d i c h l o r i d e as a f a t s o l v e n t has been  c r i t i z e d by a number o f workers. n u t r i t i v e value which occurs (28).  However, the decrease i n  i s mainly w i t h animal p r o t e i n s  T o x i c i t y i n t r i c h l o r o e t h y l e n e e x t r a c t e d soybean meal  has been r e p o r t e d  (106).  Depending on the s e v e r i t y and time o f h e a t i n g treatment o f p r o t e i n s r e s u l t s i n changes t o t h e i r properties, particularly i n their s o l u b i l i t i e s . chemical  the heat chemical  These  changes a r e a s s o c i a t e d w i t h changes i n n u t r i t i o n a l  availability.  The n u t r i t i o n a l e f f e c t of h e a t i n g  i s generally a depressing  proteins  one, and the s e n s i t i v i t y t o heat  damage v a r i e s from the extreme s e n s i t i v i t y o f the p r o t e i n s  - 27 of m i l k to the a p p r e c i a b l e r e s i s t a n c e of the p r o t e i n s o f the peanut.  However, i n c e r t a i n of the legume seeds, f o r  example, the soybean heat treatment improves  the n u t r i t i v e  v a l u e by d e s t r o y i n g a t r y p s i n i n h i b i t o r and thus i n c r e a s i n g the d i g e s t i b i l i t y of the meal.  (82)  EVALUATION OF THE PROTEIN-QUALITY OF REPRESENTATIVE SAMPLES OF WEED SEEDS  - 28 III  EVALUATION OF THE PROTEIN QUALITY OF REPRESENTATIVE SAMPLES OF WEED SEEDS  A. INTRODUCTION: The numerical values obtained f o r the b i o l o g i c a l of  quality  a p r o t e i n a r e meaningless u n l e s s they are considered a g a i n s t  a background o f p r o t e i n metabolism i t s e l f and the methods which  are a v a i l a b l e f o r p r o t e i n evaluation. For this  reason,  a number o f assay methods f o r the b i o l o g i c a l e v a l u a t i o n o f p r o t e i n s w i l l be c o n s i d e r e d b e f o r e d e a l i n g w i t h the e v a l u a t i o n of  the weed seed p r o t e i n  B  LITERATURE REVIEW  f  themselves.  (a) P r o t e i n The n u t r i t i v e value o f a f e e d i n g s t u f f can be determined o n l y by an i d e n t i f i c a t i o n of the c o n t a i n e d n u t r i e n t s , t h e i r q u a n t i t a t i v e p r o p o r t i o n s i n the f e e d and t h e i r to man  availability  or animal, f o r whom the f e e d i s intended. I n  determining  the n u t r i t i v e value o f a f e e d s t u f f , a l l these  f a c t o r s should be c o n s i d e r e d . However, s i n c e B o u s s i n g a u l t (l *) f i r s t analysed food and animal feeds f o r t h e i r n i t r o g e n 1  c o n t e n t , i n f e r r i n g from the r e l a t i v e n i t r o g e n content the r e l a t i v e n u t r i t i v e v a l u e o f the food, the p r o t e i n content of such m a t e r i a l s has r e c e i v e d f i r s t c o n s i d e r a t i o n i n the d e t e r m i n a t i o n of the n u t r i t i v e value o f a f e e d s t u f f . P r o t e i n s a r e s y n t h e s i z e d from t h e i r c o n s t i t u e n t amino a c i d s . In the p l a n t s , n i t r a t e s and ammonium s a l t s a r e used as the i n i t i a l nitrogenous compounds. In the case o f  - 29 animals, however, the c o n s t i t u e n t amino a c i d s must be a v a i l a b l e i n the d i e t , w i t h the e x c e p t i o n  o f some t h a t can be ;•- 4/  synthesized  compounds.  feeding,  i n the body from s i m p l e r  t h e r e f o r e , p l a n t products a r e valued  I n animal  f o r their  content o f a v a i l a b l e p r o t e i n as w e l l as f o r t h e i r energy content. The  p r e s e n t l y h e l d concept o f p r o t e i n metabolism i s one  of a dynamic e q u i l i b r i u m o r o f a continuous anabolism and catabolism  o f p r o t e i n s w i t h i n the l i v i n g a n i m a l .  o f the l i v i n g c e l l a r e being p r o t e i n molecules a r e being recovered  The p r o t e i n s  c o n s t a n t l y broken down and new r e b u i l t i n p a r t from amino a c i d s  from the h y d r o l y s i s o f previous  tissue proteins,  e s p e c i a l l y those o f the g a s t r o i n t e s t i n a l t r a c t and i n p a r t from the amino a c i d s o f the d i e t . reviewed by A l l i s o n  (2),  T h i s concept has been w e l l  B l o c k (18) and o t h e r s .  The r e a l i t y  of t h i s dynamic s t a t e has been demonstrated by Schoenheimer  15 (117)  using i s o t o p i c N  .  Approximately 50 years ago, F o l i n ( 3 8 ) , i n s t u d y i n g p r o t e i n metabolism promulgated the i d e a that there a r e two d i f f e r e n t types o f p r o t e i n metabolism.  The p r o t e i n metabolism  a s s o c i a t e d w i t h the e x c r e t i o n o f c r e a t i n i n e and t o a l e s s e r extent w i t h u r i c a c i d he d e f i n e d as endogenous. t i o n o f c r e a t i n i n e tends t o be constant diet.  The other  The e x c r e -  and independent o f the  type o f p r o t e i n metabolism, c h a r a c t e r i z e d  by the e x c r e t i o n o f urea n i t r o g e n and a f u n c t i o n o f the i n g e s t e d amino acids, was termed "exogenous" p r o t e i n metabolism. With the concept o f a dynamic e q u i l i b r i u m i n p r o t e i n  - 30  -  metabolism such s e p a r a t i o n o f food from body n i t r o g e n i s d i f f i c u l t and  o f t e n unnecessary.  which have been reviewed by M i t c h e l l , (9^,95)  mental d e s i g n s , make use  However, c e r t a i n e x p e r i -  of these concepts to separate endogenous  and  exogenous metabolism. The  concept of p r o t e i n s d i f f e r i n g i n q u a l i t y i s  r e l a t i v e l y new.  I t c o n t r a s t s w i t h the u n i t a r i a n theory  a hundred years ago, source there was time there was  which maintained t h a t r e g a r d l e s s  o n l y one k i n d of p r o t e i n (11*0  l i t t l e basis f o r b e l i e v i n g that  differed nutritionally.  The  observations  that  .  At  of  of that  proteins proteins  from v e g e t a b l e sources were l e s s r e a d i l y a v a i l a b l e than those from animal sources c o u l d be e x p l a i n e d  by the d i f f e r e n c e s  i n the d i g e s t i b i l i t y of food of v a r y i n g f i b r e c o n t e n t . was  not u n t i l the end  of the n i n e t e e n t h  It  century when Osborne  ( l l * ) observed that the amino a c i d composition o f some 1  p r o t e i n s d i f f e r e d markedly from other p u r i f i e d p r o t e i n s a t t e n t i o n was  that  d i r e c t e d to p o s s i b l e d i f f e r e n c e s i n the  n u t r i t i v e value  of p r o t e i n s from v a r i o u s  by Osborne and Mendel ( 1 0 3 ) , and  sources.  Studies  by Rose ( 1 1 1 , 1 1 2 ) o f  the  d i f f e r e n c e s i n d i e t a r y needs f o r the v a r i o u s amino a c i d s have emphasized the need f o r a s s e s s i n g  the b i o l o g i c a l v a l u e  of  a protein. As a r e s u l t of numerous i n v e s t i g a t i o n s , the amino a c i d s may  be c l a s s i f i e d i n t o three  semi-indispensable, absolute  groups! e s s e n t i a l ( i n d i s p e n s a b l e ) ,  and n o n - e s s e n t i a l , ( d i s p e n s a b l e ) .  The  q u a n t i t i e s of these e s s e n t i a l amino a c i d s which  - 31 must be s u p p l i e d i n the d i e t w i l l change w i t h the  species  of animal and w i l l v a r y w i d e l y w i t h the p h y s i o l o g i c a l s t a t e of the animal and among groups i n the same s p e c i e s . The  primary f u n c t i o n o f d i e t a r y p r o t e i n i s to f u r n i s h  a mixture of amino a c i d s of the proper p a t t e r n f o r the of t i s s u e p r o t e i n s  (2).  A l l methods f o r e s t i m a t i n g  n u t r i t i v e v a l u e of a p r o t e i n e v a l u a t e d i r e c t l y or i n d i r e c t l y .  The  of d i e t a r y p r o t e i n s and  synthesis  the  this function, either  measurement o f the  digestibility  the subsequent a b s o r p t i o n of the amino  a c i d s are u s u a l l y an i n t e g r a l p a r t of most determinations n u t r i t i v e value.  T h i s i s p a r t i c u l a r l y important s i n c e  methods f o r d e t e r m i n a t i o n  of  the  of n u t r i t i v e value of p r o t e i n s  are  designed to measure the degree of r e t e n t i o n of t h a t p o r t i o n of the d i e t a r y n i t r o g e n which i s absorbed i n t o the body o f the a n i m a l .  The  n i t r o g e n r e t a i n e d may  be determined as a  f u n c t i o n of growth, n i t r o g e n balance or r e p l e t i o n measurements of the whole or p a r t s o f the a n i m a l . Allison  (2) and  (8^,92,9^*95),  others  A number o f reviews have r e p o r t e d  on  by  the  v a r i o u s methods f o r the b i o l o g i c a l e v a l u a t i o n of p r o t e i n s . PROTEIN EVALUATION I  METHODS  PROTEIN EFFICIENCY RATIO The way  i n which d i f f e r e n t p r o t e i n s support  growth i n  young r a t s has been the most g e n e r a l and w i d e l y used of p r o t e i n value.  The  criterion  method o r i g i n a l l y developed by Osborne,  Mendel and F e r r y (10*0, i s based on the c o r r e l a t i o n e x i s t i n g between g a i n i n body weight and  nitrogen retention.  i n v o l v e s a d d i t i o n o f a p r o t e i n to a b a s a a l d i e t a t a  The  method  concentration  -  32  -  t h a t i s known t o he i n s u f f i c i e n t f o r o p t i m a l growth, measurement o f food consumption and growth r a t e and c a l c u l a t i o n o f the p r o t e i n e f f i c i e n c y r a t i o .  T h i s r a t i o i s d e f i n e d as the  g a i n i n body weight p e r gram o f p r o t e i n or o f n i t r o g e n consumed. In an attempt t o s t a n d a r d i z e t i o n s have been developed.  the procedure many m o d i f i c a -  These have i n c l u d e d r e g u l a t i n g  the p r o t e i n and c a l o r i c content  o f the d i e t and c o n t r o l l i n g  the methods o f f e e d i n g , whether ad l i b i t u m p a i r e d  feeding, (2).  f o r c e d f e e d i n g or adjustment of p r o t e i n t o body weight The  p o s s i b i l i t i e s o f a low c o r r e l a t i o n between weight  gain  and n i t r o g e n r e t e n t i o n have been i l l u s t r a t e d by M i t c h e l l (92).  He has d i s c u s s e d  the shortcomings o f t h i s method which  r e s u l t from the d i f f e r e n t i a l e f f e c t o f d i e t and food on the p r o t e i n , f a t s and water content  intake  o f the t i s s u e s .  s p i t e o f t h i s major d i f f i c u l t y i n determining  In  composition o f  the g a i n , the r a t e o f growth o f young animals has been one o f the most s a t i s f a c t o r y methods t o e v a l u a t e  the c o n t r i b u t i o n o f  a g i v e n p a t t e r n o f amino a c i d s f o r p r o t e i n s y n t h e s i s . II  THE RAT REPLETION METHOD The  technique o f f i r s t d e p l e t i n g and then r e p l e t i n g the  p r o t e i n s t o r e s o f an a d u l t r a t have been developed by Cannon and  others  (25),  i n t o a r a p i d , accurate  of the n u t r i t i v e v a l u e  of p r o t e i n .  p r i n c i p l e common t o many b i o - a s s a y s ,  and u s e f u l  determination  T h i s method uses a b a s i c t h a t i s , the p r o d u c t i o n  o f a d e f i c i t f o l l o w e d by a measurement o f the extent o f  - 33 replacement o f t h a t d e f i c i t .  D e p l e t i o n i s accomplished by-  f e e d i n g a p r o t e i n - f r e e d i e t u n t i l the experimental have l o s t 2%  o f t h e i r i n i t i a l weight.  animals  The animals are then  f e d n i t r o g e n i n the t e s t d i e t and the r a t e of r e p l e t i o n measured.  E x c e l l e n t c o r r e l a t i o n has been obtained  between  g a i n i n weight d u r i n g r e p l e t i o n and the r e g e n e r a t i o n  of blood,  l i v e r o r c a r c a s s p r o t e i n s , making weight r e c o v e r y alone measure o f n u t r i t i v e v a l u e .  a good  The r a t r e p l e t i o n method has not  o n l y been of value i n the d e t e r m i n a t i o n  o f the n u t r i t i v e  value  o f n a t u r a l f o o d s t u f f s but i t has a l s o been u s e f u l i n the study o f b a s i c problems i n p r o t e i n n u t r i t i o n . Ill  NITROGEN BALANCE Although the growth of young animals and the g a i n i n  weight o f p r o t e i n - d e p l e t e d  animals are c o r r e l a t e d w i t h  r e t e n t i o n of d i e t a r y n i t r o g e n i n the body, n i t r o g e n balance i s a more d i r e c t measurement  of nitrogen r e t e n t i o n .  By  d e f i n i t i o n , n i t r o g e n balance i s the d i f f e r e n c e between d i e t a r y n i t r o g e n i n t a k e and n i t r o g e n e x c r e t e d N  balance  =  or  _ N N intake - ( urinary  +  N fecal)  I f the n i t r o g e n i n t a k e i s g r e a t e r than the n i t r o g e n excreted,  then the organism i s g a i n i n g p r o t e i n and i s i n  p o s i t i v e balance.  I f the n i t r o g e n i n t a k e i s l e s s than the  nitrogen excreted,  then the animal i s l o s i n g p r o t e i n and i s  i n negative  balance.  that e x c r e t e d ,  Only i f the n i t r o g e n i n t a k e  equals  i s the organism i n p r o t e i n e q u i l i b r i u m .  - 3h However, the q u a n t i t y  of d i e t a r y p r o t e i n needed to m a i n t a i n  n i t r o g e n e q u i l i b r i u m w i l l v a r y w i t h the magnitude of  the  p r o t e i n s t o r e s as w e l l as w i t h the amino a c i d composition of the d i e t .  Thus, the p r o t e i n requirements f o r n i t r o g e n e q u i l i b -  rium are i n f l u e n c e d by the p r e v i o u s d i e t a r y h i s t o r y of  the  animal. N i t r o g e n balance i s the sum all  the  t i s s u e s of the body.  of gains and  The  l o s s e s from  dynamic nature o f  protein  metabolism makes p o s s i b l e the maintenance of some t i s s u e s a t the expense of o t h e r s . i n p o s i t i v e balance and more t i s s u e s .  The  I t i s p o s s i b l e f o r an a n i m a l t o  be  y e t t o be l o s i n g n i t r o g e n from one  or  p o s s i b i l i t y of such a d a p t i v e changes  taking  p l a c e must always be considered  during  balance.  of the n i t r o g e n balance method  Various modifications  experiments i n v o l v i n g  have been developed. PROTEIN MINIMA M e l n i c k and  Cowgill  ( 8 5 , 8 6 ) determinede the minimum  amount of n i t r o g e n n e c e s s a r y to m a i n t a i n n i t r o g e n and  developed t h i s i n t o a q u a n t i t a t i v e procedure.  minima procedure has  and  protein  protein  a l s o to study the q u a n t i t a t i v e r e l a t i o n s h i p s  between amino a c i d s i n a d i e t a r y m i x t u r e .  The  variability  p r o t e i n minima f o r n i t r o g e n e q u i l i b r i u m which may d i f f e r e n c e s i n d i e t and has  This  been used w i t h v a r i a t i o n s by many workers  to determine the n u t r i t i v e v a l u e of p r o t e i n s and hydrolysates  equilibrium  r e s u l t from  p h y s i o l o g i c a l s t a t e o f the animals  been emphasized i n a d i s c u s s i o n by A l l i s o n .  of  (2).  -35 BIOLOGICAL VALUE Thomas (93)  has developed a more fundamental and l e s s  v a r i a b l e procedure f o r measurement o f the n u t r i t i v e of a d i e t a r y p r o t e i n .  value  He d e f i n e d the percentage o f absorbed  n i t r o g e n r e t a i n e d i n the body of the animal as the " b i o l o g i c a l value".  T h i s value  by the f o l l o w i n g  (B.V.) can be expressed m a t h e m a t i c a l l y  equation. B.V. = lOOx  I - (F-Fm) - (U - Ue) I - (F - Fm)  Where I i s n i t r o g e n i n t a k e , F i s f e c a l n i t r o g e n , Fm i s metabolic  f e c a l n i t r o g e n , U i s u r i n a r y n i t r o g e n and Ue i s  endogenous u r i n a r y n i t r o g e n .  A l l these v a r i a b l e s can  t h e o r e t i c a l l y be determined a t the same time from a n i t r o g e n balance experiment.  M i t c h e l l and h i s a s s o c i a t e s  done a great d e a l i n d e v e l o p i n g  and a p p l y i n g  t o the measurement o f the n u t r i t i v e v a l u e and  amino a c i d m i x t u r e s .  (93)  have  t h i s concept  of d i e t a r y proteins  I t i s customary to measure the  e x c r e t i o n of u r i n a r y and f e c a l n i t r o g e n while f e e d i n g a p r o t e i n f r e e d i e t , t o provide v a l u e s equation.  f o r the Ue and Fm i n the above  The v a r i a b i l i t i e s encountered i n t h i s procedure^  a r i s e mainly from the d i f f i c u l t i e s i n the measurement o f endogenous and metabolic  nitrogen.  I n an attempt t o o b v i a t e  these d i f f i c u l t i e s a s m a l l amount o f p r o t e i n , u s u a l l y whole egg  i s i n c l u d e d i n the r a t i o n when Ue and Fm a r e being  determined.  I t i s assumed t h a t whole egg p r o t e i n i s complete-  l y d i g e s t e d and u t i l i z e d .  Another attempt t o e l i m i n a t e much  of the v a r i a t i o n a s s o c i a t e d w i t h the c a t a b o l i s m  of tissue  - 36 p r o t e i n , has been to c o r r e l a t e endogenous n i t r o g e n w i t h e x c r e t i o n of c r e a t i n i n e . Other m o d i f i c a t i o n s of the n i t r o g e n balance method have been developed. (2).  The  These have been reviewed  elsewhere by  c a l c u l a t i o n of n i t r o g e n balance i n d i c e s has  s i g n i f i c a n c e as  'net p r o t e i n v a l u e ' which was  Allison. the same  d e f i n e d by  M i t c h e l l (9*+) as the product of b i o l o g i c a l v a l u e and d i g e s t ibility.  Another procedure  combines n i t r o g e n balance s t u d i e s  w i t h those of r e p l e t i o n of d e p l e t e d l a b i l e p r o t e i n s t o r e s . The  c l a s s i c a l method (Thomas M i t c h e l l ( 9 3 ) ) o f  the b i o l o g i c a l v a l u e of p r o t e i n s i s both long and  determining  tedious,  i n v o l v i n g N-balances on the experimental animal w i t h t e s t c o n t r o l d i e t s , a t y p i c a l procedure seven weeks.  g i v i n g three r e s u l t s  Using the b a s i c p r i n c i p l e o f t h i s  method, M i l l e r and Bender ( 1 6 , 8 9 ) ,  and  after  classical  have r e v i s e d the  procedure  e l i m i n a t i n g many of the time consuming n i t r o g e n d e t e r m i n a t i o n s . The method i s based on c a r c a s s a n a l y s i s and protein u t i l i z a t i o n  g i v e s the net  (N.P.U.) which i s the product o f b i o l o g i c a l  v a l u e and d i g e s t i b i l i t y .  The N.P.U. v a l u e , or t h a t p r o p o r t i o n  of the n i t r o g e n i n t a k e which i s r e t a i n e d by the animal,  can  be d e r i v e d from body n i t r o g e n d e t e r m i n a t i o n s by the f o l l o w i n g equation. NPU  = B - (Bk - Ik) I  Where B and Bk are the t o t a l body Nitrogen, of the  animals  on the t e s t and non-protein d i e t s r e s p e c t i v e l y , and I and are the i n t a k e of N i t r o g e n i n the two  groups.  Ideally the  Ik  - 37 measurement o f body N i t r o g e n should be made on a s i n g l e rat  f e d both the t e s t p r o t e i n and the n o n - p r o t e i n  diet.  T h i s i s i m p o s s i b l e but the e q u i v a l e n t can be a c h i e v e d by use o f s u i t a b l e c o n t r o l groups. period  A t the end o f the experimental  ( u s u a l l y 7 t o 10 days) the animals a r e k i l l e d and  body - N determined.  The body - N may be determined  by K j e l d a h l  d i g e s t i o n o f the whole c a r c a s s o r , a l t e r n a t i v e l y , from the water content o f the animal s i n c e the r a t i o of water t o n i t r o g e n i s constant a t any g i v e n age when the body water and body n i t r o g e n r e l a t i o n s h i p i s known.  M i l l e r and Bender  (89) have i n d i c a t e d t h a t there i s no s i g n i f i c a n t d i f f e r e n c e between the v a l u e s f o r body N determined  d i r e c t l y or those  c a l c u l a t e d from a r e g r e s s i o n formula r e l a t i n g body n i t r o g e n to body water over a d e f i n e d age range.  A high  correlation  was found between p r o t e i n e f f i c i e n c y r a t i o s and n e t p r o t e i n u t i l i z a t i o n o f p r o t e i n s , (13), the r e l a t i o n being v e r y to t h a t found by B l o c k and M i t c h e l l , efficiency ratio  similar  (21), between p r o t e i n  (PER) and b i o l o g i c a l v a l u e  (BV).  ,  TISSUE PROTEINS N i t r o g e n balances  can be m i s l e a d i n g , s i n c e they may be ;  the sums o f a l g e b r a i c gains and l o s s e s . producing  Two p r o t e i n s  the same n i t r o g e n balance c a n . f i l l the p r o t e i n  s t o r e s o f an animal d i f f e r e n t l y .  A somewhat s i m p l e r and more  d i r e c t approach to the study o f the e f f e c t o f d i e t a r y p r o t e i n on l o s s and g a i n o f body p r o t e i n has been t o measure changes occuring i n a s p e c i f i c t i s s u e .  Maintenance o f so c a l l e d  normal plasma p r o t e i n c o n c e n t r a t i o n , a decrease  or increase  - 38: i n the albumen-globulin r a t i o and the p r o t e i n content of the l i v e r are some of a number o f s p e c i f i c t i s s u e s methods which have been s t u d i e d . (2) may  The v a l u e s obtained by these methods  not p a r a l l e l those obtained by o t h e r s s i n c e the amino a c i d  requirements  f o r r e p l e t i o n o f the p r o t e i n o f a s p e c i f i c  w i l l v a r y a c c o r d i n g to the s t r u c t u r e of the p r o t e i n and  tissue the  p o s i t i o n i t h o l d s i n the dynamic s t a t e o f the body. P r o t e i n s l o s t from t i s s u e s through d e p l e t i o n r e p r e s e n t l o s s i n enzyme systems. of enz yme was  The p o s s i b i l i t y o f u s i n g a c t i v i t i e s  systems to determine  the n u t r i t i v e v a l u e o f p r o t e i n s  s t i m u l a t e d by the work o f M i l l e r . (90)  Approximately  30 enzyme systems are reduced by p r o t e i n d e p l e t i o n .  However,  one of the most l a b i l e o f a l l l i v e r enzyme systems was to be xanthine  found  oxidase.  A method to c o r r e l a t e xanthine oxidase a c t i v i t y w i t h n u t r i t i v e v a l u e o f the p r o t e i n has been developed W i l l i a m s , Chen and Elvehjem. i n i t s formative s t a g e . by W i l l i a m s  by  Litwack,  ( 7 8 , 7 9 ) T h i s method i s s t i l l  I t s p o s s i b i l i t i e s have been d i s c u s s e d  (126)*  MICROBIOLOGICAL I n r e c e n t years a number o f workers have attempted  to  develop s i m p l e r and more r a p i d t e s t s f o r n u t r i t i v e v a l u e based upon the growth o f protozoa or micro-organisms. 50,87,120)  The growth response  (3,32,35,*+6,  o f the p r o t e o l y t i c  protozoan  Tetrahymena g e l i i to both h y d r o l y s e d and unhydrolysed p r o t e i n s has been c o r r e l a t e d to n u t r i t i v e v a l u e o f p r o t e i n by Anderson and W i l l i a m s ( 3 ) ,  and others ( 3 2 , 3 5 ) .  T h i s growth  response  - 39 -  has  been measured i n a number of ways i n c l u d i n g a c i d  (35), r e d u c t i o n o f the dye a c t u a l count of the  triphen$tetrazolium  production,  chloride  and  protozoa.  I t i s known that the order o f r e l e a s e o f amino a c i d s the extent  of h y d r o l y s i s p l a y an important p a r t i n the  u t i l i z a t i o n o f the d i e t a r y p r o t e i n s .  To simulate  the f a t e of p r o t e i n s i n v i v o , Halevy and  promoting a c t i v i t y o f the h y d r o l y s a t e  more c l o s e l y 0+6),  Grossowiez,  used i n v i t r o d i g e s t i o n w i t h p a n c r e a t i n and  assayed the growth  f o r a s t r a i n Streptococcus  f a e c a l l s which r e q u i r e d the ten amino a c i d s considered e s s e n t i a l f o r the r a t ,  Metz, Rennert and C o l e ,  Pseudomonas a r e r u g i n o s a  w h i l e T e e r i , V i r c h o u and  bacterium, Loughlin,  used the enzymes n o r m a l l y employed by the animal i n  d i g e s t i o n (pepsin, t y p s i n , pancreatin S  as  (87), combined  the p r o t e o l y l i c a c t i o n of p e p s i n w i t h that of the  (120)  and  and  erepsin)  with  faecalis. The  preceding  d i s c u s s i o n i n d i c a t e s t h a t the methods f o r  e v a l u a t i o n of p r o t e i n are numerous and  diverse.  The d i v e r s i t y  r e f l e c t s the nature of the p r o t e i n molecules themselves.  The  p r e c i s e s t r u c t u r e and mode o f s y n t h e s i s o f the p r o t e i n s i s not y e t f u l l y r e s o l v e d . w i t h carbohydrate and of one  The  i n t e r p l a y of amino a c i d metabolism  f a t metabolism, as w e l l as the  amino a c i d to another f u r t h e r complicates  for protein evaluation. described  the procedures  For these reasons none o f the methods  i s f r e e from l i m i t a t i o n s . There i s as yet no  procedure f o r determining the a b s o l u t e protein.  relation  n u t r i t i v e value  single of a  - IfO -  PLANT PROTEIN VERSUS ANIMAL PROTEIN. A l l assay procedures i n d i c a t e t h a t , i n g e n e r a l ,  proteins  from animal sources a r e s u p e r i o r t o those from p l a n t  sources.  However, e x c e p t i o n s , value  such as g e l a t i n , do o c c u r .  o f animal p r o t e i n s v a r y a c c o r d i n g  connective  tissue.  The b i o l o g i c a l  t o t h e i r content o f  I n the p l a n t s the p r o t e i n s from the seeds  tend t o be s u p e r i o r t o those from leaves  or r o o t s .  Most o f  the whole seeds do n o t show wide d i f f e r e n c e s from one another, but  some, such as soybeans and peanuts a r e d i s t i n c t l y  superior.  Most c e r e a l p r o t e i n s a r e d e f i c i e n t i n l y s i n e .  Corn i s a l s o d e f i c i e n t i n tryptophan w h i l e soybeans a r e l a c k i n g i n methionine. The  d i f f e r e n c e i n the r e l a t i v e q u a n t i t i e s o f the amino  a c i d s present  i n p l a n t p r o t e i n s as compared t o t h a t o f the  animal p r o t e i n s which w i l l be s y n t h e s i z e d m a t e r i a l s account f o r the main d i s c r e p a n c y b i o l o g i c a l value  from the p l a n t between the  o f p l a n t p r o t e i n s compared t o t h a t o f animal  proteins. C.  EXPERIMENTAL  (a) 1.  THE NET PROTEIN UTILIZATION OF A NUMBER OF WEED SEED .SAMPLES . ASSAY METHOD On  the b a s i s o f the l i t e r a t u r e reviewed, the procedure  of M i l l e r and Bender (89) appeared t o o f f e r the g r e a t e s t promise f o r the present  studies.  T h i s method has the advantage  of r e q u i r i n g a r e l a t i v e l y s m a l l amount o f m a t e r i a l and r e q u i r i n g a short experimental p e r i o d .  A number o f m o d i f i c a t i o n s were  - te made t o the procedure. Animals;  Rats of the W i s t a r s t r a i n from the U.B.C. colony-  were used i n a l l the experiments.  The animals were weaned  a t 21 days a t a weight o f about U-0 gms., and f e d f o r one week on s t o c k d i e t so t h a t they weighed b e g i n n i n g o f the experiment.  55-65  grams a t the  The r a t s were randomized  into  groups, each e x p e r i m e n t a l group c o n s i s t i n g o f 3 females and 3 males.  The t o t a l i n i t i a l weights o f a l l groups were c a r e -  f u l l y a d j u s t e d so that each group t o t a l l e d the same weight w i t h i n 2-3 grams.  The number o f e x p e r i m e n t a l groups  a c c o r d i n g to the number o f p r o t e i n s under t e s t 10).  The animals were housed  p r o v i d e d w i t h f r e s h water  varied  (usually 8 -  i n i n d i v i d u a l pans and were  daily.  D i e t s : A number o f s u b s t i t u t i o n s were made t o the b a s a l n o n - p r o t e i n d i e t o f M i l l e r and Bender.  (89).  Composition o f B a s a l Non-Protein D i e t M i l l e r and Bender  Substituted  (Robertson & Wood)  Margarine  15  Lard  Potato S t a r c h  10  Corn S t a r c h  Glucose  15  Sucrose  7.5  V i t a m i n Mix (NBC)  1.5  S a l t s U.S.P. No.2  7.5 100.0 gms  V i t a m i n Mixture S a l t M i x t u r e (Hawk Oserc&Summerson  19W  Rice Starch  5 5  22.5 61.0  50  100  gm.  The meals w i t h reduced i s o t h i o c y a n a t e content as prepared by the preceeding e x t r a c t i o n method ( S e c t i o n I - C ( b ) ) were used to a s c e r t a i n the b i o l o g i c a l v a l u e o f weed seed p r o t e i n s .  1+2-  -  The  (N x 6.25)  p r o t e i n content  determined by Table  of these e x t r a c t e d meals, as  the K j e l d a h l method (102)  are presented  in  IV. TABLE IV. P r o t e i n Source  Per Gent P r o t e i n (M x  White Mustard  1+2.3  Stinkweed  23.7  Rapeseed  M+.6  Linseed  3I+.3  Soybean  52.9  The  p r o t e i n under t e s t was  r e q u i r e d amount to g i v e 10 D i e t s c o n t a i n i n g 10  added to the b a s a l d i e t i n the  per c e n t c a s e i n were i n c l u d e d i n each  M i t c h e l l and Carmen (97)  introduced  f e e d i n g a low l e v e l of completely  value.  the procedure of  assimilable p r o t e i n i n place  d i e t of the c l a s s i c a l procedure, thereby  a v o i d i n g any p h y s i o l o g i c a l changes due  to d e p r i v a t i o n .  a l t e r n a t i v e c o n t r o l , t h e r e f o r e , a d i e t c o n t a i n i n g h- per egg  albumen was  protein diet.  i n c l u d e d i n the assays along w i t h The n i t r o g e n content  determined by a standard Procedure: d i e t and  One  6.25)  per cent l e v e l of p r o t e i n (N x  group of assays as a p r o t e i n of known b i o l o g i c a l  o f the n o n - p r o t e i n  6.25)  As  an  cent  the non-  of a l l the d i e t s were  method.(102),  of the r a t groups was  f e d on the  each of the remaining groups pn separate  c o n t r o l p r o t e i n s f o r 5 to 7 days.  A t the end  mental p e r i o d , the t o t a l weight change and  non-protein t e s t or  of the e x p e r i -  t o t a l feed  -  i*3  consumption f o r each  -  r a t was measured. The animals were k i l l e d  w i t h c h l o r o f o r m , and body water was determined by making i n c i s i o n s i n t o the abdominal, t h o r a i c and c r a n i c a l c a v i t i e s and d r y i n g  the c a r c a s s e s to a c o n s t a n t weight a t 105°C. f o r  k8  hours i n a tunnel  d r i e r . The i n i t i a l weight, f i n a l weight,  dry  weight, and f e e d consumption f o r each r a t were r e c o r d e d  and the c o r r e s p o n d i n g body  water, body  n i t r o g e n and n i t r o g e n  i n t a k e v a l u e s were c a l c u l a t e d . 2 , CALCULATIONS The n i t r o g e n content of the bodies o f the r a t s c a l c u l a t e d from the water c o n t e n t . days M i l l e r and Bender  was  Over the age range 33-57  ( 1 5 , 8 9 ) found a h i g h c o r r e l a t i o n  between the r a t i o of body  N to body water and age.  They  expressed t h i s r e l a t i o n s h i p by the e q u a t i o n y = 2,92  * 0 . 0 2 x where y = N ( i n grams) x H0 2  100  ( i n grams)  and x = age i n days. E x a m i n a t i o n of the n i t r o g e n to water r a t i o i n the W i s t a r s t r a i n of r a t s from the U.B.C. c o l o n y v e r i f i e d  this relation-  s h i p . Body water and body n i t r o g e n d e t e r m i n a t i o n were c a r r i e d out  on 33 r a t s over the age range ^ - l O O  gms.  This  relation-  s h i p may be expressed by the e x p o n e n t i a l e q u a t i o n .  1.08  y = 27,9x + s  p  = 3«7^  - s  r  =  where y = mgs body n i t r o g e n x = gms body water  3.61  This r e l a t i o n s h i p has been d i s c u s s e d more f u l l y i n the Appendix I . This e q u a t i o n was used throughout to c a l c u l a t e the body n i t r o g e n o f each r a t from i t s water c o n t e n t . The net p r o t e i n  u t i l i z a t i o n (N.P.U.) o f the p r o t e i n samples was c a l c u l a t e d by a p p l y i n g the e q u a t i o n o f M i l l e r and Bender (16,89), N.P.U.  B -  l  k  )  where B and B^ a r e the t o t a l body n i t r o g e n o f the animals on the t e s t and n o n - p r o t e i n d i e t s r e s p e c t i v e l y , and I and I  k  are the i n t a k e o f n i t r o g e n i n the groups. 3, RESULTS The N.P,U. expressed as percentages o f the f i v e e x t r a c t e d p r o t e i n sources a r e presented i n Table V. albumen were i n c l u d e d as c o n t r o l s .  C a s e i n and egg  Commercial samples of  l i n s e e d , soybean meal and a number of f i s h meals were i n c l u d e d i n these assays f o r comparative  purposes.  The v a l u e s o b t a i n e d f o r the n e t p r o t e i n u t i l i z a t i o n i n d i c a t e that white mustard  and rapeseed compare f a v o u r a b l y  w i t h l i n s e e d and soybean as p l a n t p r o t e i n s o f moderately b i o l o g i c a l value.  high  The N.P.U. v a l u e f o r stinkweed i s somewhat  lower than that of the other two C r u c i f e r a e . p a r t l y due to a lower d i g e s t i b i l i t y .  This may be  However, the lower N.P.U.  v a l u e a l s o r e f l e c t s a l i m i t i n g l e v e l o f an e s s e n t i a l amino acid.  Amino a c i d a n a l y s i s (Part I I I ) has i n d i c a t e d  stinkweed c o n t a i n s sub-optimal amounts of h i s t i d i n e White mustard methionine  that (Table V I ) .  and rapeseed c o n t a i n s l i g h t l y higher l e v e l s o f  than does soybean,  b o t h methionine and c y s t i n e .  soybean being d e f i c i e n t i n This probably accounts f o r the  s l i g h t l y higher N.P.U. v a l u e s o b t a i n e d f o r these p r o t e i n s f o r soybean.  than  The lower N.P.U. v a l u e f o r the e t h a n o l e x t r a c t e d  soybean meal as compared to that f o r the commercial  sample  -  k*  -  TABLE V THE NET PROTEIN UTILIZATION  _  OF A NUMBER OF PROTEINS  N#P#tJ# V a l u e s  P r o t e i n Source  % of Diet  N  e  t  Protein U t i l i z a t i o n  M i l l e r & Bender  - — — —  (lif,88,89)  Assay Number 1 2 3  Casein (Vitamin free) (Crude)  *+  5  Mean  8^.2 81.0 73. *- 7^.2 78.2 1  SD  Value  5.2 60.0  90.9 78.*+ 72.1 80.5 6.7  Egg Albumen ^3.8  5.5  82.5  *+.8  51.2  10.*+  55.7  59.8  5.7  70.2 73.3 73.3  62.1+  69.8  5.1  Argentine Rapeseed  70.0 77.6 71.5  6h.6 70.9  5.3  Stinkweed  5^.1 5^.5 53.6  57.0 5^.0  1.5  79.0 80.2  72.8 77.3  3.J  Linseed Meal Commercial Sample  70.1  6h.h 67.2  k.O  55.0  5.7  Soybean Meal Commercial Sample  69A  70.2  3.8  56.0  5.7  Soybean Soybean heated at 100$ f o r min. Linseed  63.8  30  White Mustard  H e r r i n g Meal Whale Meal Dog F i s h Meal Shrimp  58.5  SD  100.1 L  6H-.  0.5  80.2 55A  52.5  -  1+6  -  i n d i c a t e s t h a t a c e r t a i n amount of the amino a c i d s may been removed during  the e x t r a c t i o n p r o c e s s .  t h a t the N.P.U. v a l u e was  However, the  improved a f t e r a u t o c l a v i n g  e x t r a c t e d meal a t 100°C f o r 30 minutes may t r y p s i n i n h i b i t i n g f a c t o r present f o r p a r t a t l e a s t of t h i s low  i n raw  have fact  the  show that  raw  the  soybean was  responsible  utilization.  I t has been suggested that a p p l i c a t i o n of heat to  raw  soybean produced a phenomenal i n c r e a s e i n the b i o l o g i c a l  value  o f i t s p r o t e i n , l a r g e l y because the heat caused the methioninec y s t i n e f r a c t i o n to become a v a i l a b l e f o r u t i l i z a t i o n by animal.  However, the concept of a heat l a b i l e t r y p s i n  i n h i b i t o r i n raw  soybeans now  methionine d e f i c i e n c y of raw  e x p l a i n s the apparent  have r e p o r t e d  to determine how  greater  as compared to a p r o p e r l y  t r e a t e d soybeans or soybean meal. 82)  the  heat-  Sevtral investigators  (80,  the r e s u l t s of f e e d i n g experiments designed to p r o p e r l y cook or heat raw  soybeans and  raw  d e f a t t e d soybean o i l meal to o b t a i n the optimum b i o l o g i c a l or n u t r i t i v e v a l u e of the p r o t e i n . autoclaving  Moist  heating  has been the most e f f e c t i v e l a b o r a t o r y method f o r  improving the n u t r i t i v e value of the p r o t e i n . has  Overheating  an adverse e f f e c t upon the n u t r i t i o n a l value of some amino  a c i d s and v i t a m i n s . et a l . (20).  This s u b j e c t i s w e l l reviewed by  Fritz,Kramke and Reed (kl)  r e s u l t s were obtained when ground raw  reported  Block  that b e s t  soybeans were  a t 15 pounds pressure f o r 2 0 - 3 0 minutes. (80)  such as  Evans and  autoclaved McGinnis  found an i n c r e a s e i n the n u t r i t i v e v a l u e of the p r o t e i n  a f t e r a u t o c l a v i n g ..a raw,, d e f a t t e d soybean meal a t 100,  110,  - k7 or 120°G. f o r 30 minutes.  -  However, t h e p r o t e i n n u t r i t i v e  v a l u e s were lower when soybean o i l meal was  autoclaved at  130°C. f o r 30 - 60 minutes than when i t was  autoelaved a t  120°C.  For these reasons  the raw e t h a n o l e x t r a c t e d , d e f a t t e d  soybeans were autoclaved at 100°C. f o r 30 minutes to if  100=  determine  heat treatment would e f f e c t an improvement of the r e l a t i v e l y  low N. P. U. v a l u e obtained f o r the e x t r a c t e d soybeans i n the i n i t i a l assays.  As may  be noted from Table V  this  treatment y i e l d e d a 15 per cent improvement i n the N. P.  U.  value. k. DISCUSSION E f f e c t of P r o t e i n Treatment on N.P.U. V a l u e . A number of c r i t i c i s m s have been made of the method presented by M i l l e r and-Bender (89) proteins. treatment  f o r the e v a l u a t i o n of the  Forbes and Yohe (kO) s t u d i e d the e f f e c t of p r e v i o u s of the animals on the net p r o t e i n v a l u e .  Their data  showed that n e i t h e r excess p r o t e i n of good q u a l i t y nor a moderate d e f i c i e n c y of e x c e l l e n t q u a l i t y p r o t e i n e f f e c t s the r e s u l t s obtained when the c a r c a s s n i t r o g e n i s determined analysis.  However, when the body n i t r o g e n was  by  direct  calculated  from the body water content, Forbes and Yohe (kO) more v a r i a b l e r e s u l t s f o r < the net p r o t e i n v a l u e s .  obtained Higher  r e s u l t s were obtained when the animals were moderately i n p r o t e i n a t the s t a r t of the t e s t .  deficient  T h e i r c r i t i c i s m cannot  g i v e n too great weight s i n c e the M i l l e r and Bender uses normal not p r o t e i n d e p l e t e d r a t s . .  be  procedure  However, they used  A l b i n o r a t s of the Sprague Dawley s t r a i n w h i l e Bender and  Miller  - if 8  -  used a hooded v a r i e t y f o r the d e r i v a t i o n of the e q u a t i o n r e l a t i n g body N to body H 0 w i t h age. 2  Forbes and Yohe d i d  not take i n t o c o n s i d e r a t i o n the s t r a i n d i f f e r e n c e which Dreyer  (31) has r e c e n t l y shown may a f f e c t the n i t r o g e n water  ratio. E f f e c t o f Energy Intake on the B i o l o g i c a l Value o f P r o t e i n s Forbes  and Yohe (39)' a l s o s t u d i e d the e f f e c t o f energy  i n t a k e on the b i o l o g i c a l v a l u e o f p r o t e i n s f e d to r a t s .  When  d i e t s c o n t a i n i n g 10 per cent p r o t e i n were f e d a t *+£, 6 0 , o r 8.0 gm. d a i l y , i t was found  that the l e v e l o f food i n t a k e had a  h i g h l y s i g n i f i c a n t e f f e c t on b i o l o g i c a l v a l u e . v a l u e s obtained a t N-.O gm. i n t a k e were lower o b t a i n e d a t 6 . 0 or 8 . 0 gm.  intake.  The b i o l o g i c a l  than  those  However, the b i o l o g i c a l  v a l u e o f the p r o t e i n s s t u d i e d d i d n o t vary  significantly  between i n t a k e l e v e l s of 6 . 0 and 8 . 0 d a i l y .  I n the present  work the f e e d i n t a k e o f the experimental r a t s always exceeded 6.0  gms per day. Forbes and Yohe (39) a s c r i b e d the low b i o -  l o g i c a l v a l u e s o b t a i n e d a t k-.O gm. i n t a k e to u t i l i z a t i o n o f a p o r t i o n o f the d i e t a r y p r o t e i n f o r energy t o support weight maintenance of the animals.  I n c o n n e c t i o n w i t h a study of the  r e l a t i o n between p r o t e i n e f f i c i e n c y r a t i o  (P.E.R.) and n e t  p r o t e i n u t i l i z a t i o n (N.P.U.), Bender (13) a l s o s t u d i e d the e f f e c t of food i n t a k e on the v a l u e s obtained by these two methods.  The P.E.R. c o r r e l a t e d ! c l o s e l y w i t h food intake.;  f a l l i n g when the food consumption was reduced.  However, from  s t u d i e s w i t h amino a c i d mixtures, the N.P.U. was found independent of food i n t a k e .  to be  The food consumption was,  i n f a c t , dependent upon the  n u t r i t i v e v a l u e of the p r o t e i n .  Foods of h i g h p r o t e i n v a l u e  were consumed i n g r e a t e r q u a n t i t i e s than those of low v a l u e . The d i f f e r e n c e between food consumption on the poorest on the b e s t p r o t e i n was  about 100  per c e n t .  Bender  and  (13)  a l s o showed that N.P.U's. can be c o r r e l a t e d w i t h n e g a t i v e P.E.R.'s ( l o s s of weight i n g/g p r o t e i n e a t e n ) . N.P.U. i s l e s s than kO, experimental d i e t .  r a t s are unable  When the  to grow on the  These low N.P.U. v a l u e s a r e probably a  b e t t e r i n d i c a t i o n of the v a l u e of p r o t e i n s of low than the P.E.R.  quality  Because of a reduced consumption of p r o t e i n s  of low. b i o l o g i c a l v a l u e , the P. E . R.'s may  be  consequently  underestimated. Length of E x p e r i m e n t a l P e r i o d The r e l a t i v e l y s h o r t l e n g t h of the experimental p e r i o d i s one of the advantages of the M i l l e r and Bender method f o r protein evaluation. (37)  For the measurement of P.E.R., F i x s e n  s t a t e d t h a t a long experimental p e r i o d i s e s s e n t i a l ,  s h o r t Experiments  g i v i n g higher values than the long ones, and  60 days being the minimum d u r a t i o n r e q u i r e d f o r a c c u r a c y . However, i n n i t r o g e n balance s t u d i e s s h o r t e r p e r i o d s appear, to be p r e f e r a b l e .  M i t c h e l l (9*+)  has p o i n t e d out  "the composition of the gains put on by growing p r o g r e s s i v e l y changes w i t h age  .....  the b e s t way  animals of a s s u r i n g  the d e s i r e d r e q u a l i t y i n the composition of g a i n s use animals of the same age, weight and p r e v i o u s and  to conduct  the experiments  t h a t as  i s to treatment  f o r as s h o r t p e r i o d s of  time  - it9a as may  be r e q u i r e d f o r an accurate measure o f the a c t u a l  g a i n i n organized Bender  -  t i s s u e " . In t h e i r s t u d i e s , M i l l e r and  (89) used 10-day and 7-day e x p e r i m e n t a l p e r i o d s .  In these s t u d i e s 7-day and 5-day periods were used. T h i s period  appears to be adequate. Does the n i t r o g e n water r a t i o remain c o n s t a n t  deprived  i n rats  of p r o t e i n ? Does l o s s o f water p a r a l l e l l o s s of  nitrogen i n a p r o t e i n deprived These a r e questions procedures^  r a t or does edema ensue?  o f d i s p u t e i n the M i l l e r and Bender  THE ESSENTIAL AMINO ACID CONTENT OF CERTAIN NOXIOUS WEED SEEDS  -50 IV  THE ESSENTIAL AMINO ACID CONTENT OF CERTAIN NOXIOUS WEED SEEDS.  A  INTRODUCTION The  current  trend of i n v e s t i g a t i o n s on the c h e m i s t r y  of n u t r i t i o n are emphasizing the s i g n i f i c a n c e of the amino a c i d s as the fundamental f a c t o r s i n a l l problems i n which h i t h e r t o the r o l e of i n t a c t p r o t e i n s has been i n v o l v e d  (103).  T h i s trend i s most evident i n the development of c h e m i c a l methods f o r the e v a l u a t i o n of a p r o t e i n based on i t s content of e s s e n t i a l Since a  amino a c i d s . there are many advantages that would r e s u l t from  s u c c e s s f u l c h e m i c a l e v a l u a t i o n of food p r o t e i n s , a number  of methods have been proposed to c o r r e l a t e the amino a c i d content of a The  protein with i t s n u t r i t i v e  value.  c l a s s i f i c a t i o n of the amino a c i d s occuring  i n proteins  on the b a s i s of t h e i r b i o l o g i c a l s i g n i f i c a n c e , that i s , i n t o e s s e n t i a l and n o n - e s s e n t i a l d i e t a r y components preceded  the  s e l e c t i o n of a p r o t e i n or p r o t e i n mixture whose amino a c i d c o n s t i t u e n t s would be completely metabolism. Kuhnau (77)  u t i l i z e d I n d i g e s t i o n or  chose human m i l k as the  f o r the e v a l u a t i o n of p r o t e i n s . B l o c k and  Mitchell  s e l e c t e d the p a t t e r n of amino a c i d s i n whole egg the standard  standard (96)  p r o t e i n as  f o r growth s i n c e t h i s p r o t e i n has been shown  -  51  -  to be n e a r l y p e r f e c t l y u t i l i z e d i n d i g e s t i o n  and  In an attempt to judge trie n u t r i t i v e adequacy  metabolism.  of a  protein  by the s e v e r i t y o f i t s l i m i t i n g amino a c i d , B l o c k and (96)  devised a chemical  Mitchell  score taken as the s m a l l e s t percentage  of an e s s e n t i a l amino a c i d based on i t s content i n egg  protein.  In order to c o n s i d e r the i n t e r r e l a t i o n s h i p s which e x i s t between e s s e n t i a l and n o n - e s s e n t i a l amino a c i d s ,  may  such  as are known to gocur between methionine and c y s t i n e , phenyla l a n i n e and t y r o s i n e , M i t c h e l l  (91)  has a procedure f o r  c a l c u l a t i o n of a m o d i f i e d e s s e n t i a l amino a c i d i n d e x . The c o r r e l a t i o n between the b i o l o g i c a l v a l u e of p r o t e i n and  their  c h e m i c a l a p p r a i s a l by these methods has been reviewed by M i t c h e l l and others ( 2 ,  9D.  As p a r t o f the i n v e s t i g a t i o n s  undertaken to determine  the n u t r i t i v e v a l u e of c e r t a i n noxious weed seeds the e s s e n t i a l amino  a c i d content of a number of weed seeds were determined.  The f o l l o w i n g  weed seeds were chosent  B l a c k mustard  Brassica  White  B r a s s i c a l alba B o i s s  Wild  mustard mustard  Brassica  nigra  (L) Koch  kaber (DC) L.C.  Tumble mustard  Sisymbrium a l t i s s i m u m L  Stinkweed  Thlasni  Western F a l s e  Flax  arvense L  Camellna microcarpa  Wheeler  - 52 Samples of c a s e i n (N.B.C.) and raw linseed  (Linum u s i t a t i s s l m u m ) and rapeseed  were i n c l u d e d f o r comparative B  soybean ( G l y c i n e s o y a ) , ( B r a s s i c a nanus)  purposes.  EXPERIMENTAL (a) The A n a l y s i s of the E s s e n t i a l Amino A c i d Content  of  a number of Weed Seeds. (1)  P r e p a r a t i o n of Samples. Ground samples of the seeds were d e f a t t e d by  extraction  i n a Soxhlet w i t h petroleum  ether.  The n i t r o g e n c o n t e n t of  these samples as determined  by Maero K j e l d a h l technique  are  presented i n Table V I . (2)  Assay method. The m i c r o b i o l o g i c a l assay procedures  Wright  (9)  o u t l i n e d by B a r t o n -  were f o l l o w e d f o r the d e t e r m i n a t i o n of the amino  a c i d content of these samples. i  P r e p a r a t i o n of H y d r o l y s a t e s  For the assay of the e s s e n t i a l amino a c i d s ;  histidine,  l y s i n e , leucine, i s o l e u c i n e , v a l i n e , phenylalanine tryplophane and  threonine and  the semi-dispensable amino a c i d s ,  a r g i n i n e and c y s t i n e ; a c i d h y d r o l y s a t e s (2.5N prepared. enzymatic  For the assay of tryptophane,  HC1) were  a procedure  involving  h y d r o l y s i s w i t h p e p s i n (GBI 1:10,000 polency)  t r y p i n (GBI, h U.S.P.) and e r e p s i n (G.B.I) was alkaline hydrolysis assay of  methionine,  tyrosine.  followed.  (5N NaOH) of the m a t e r i a l was  An  used f o r the  - 53 ii  Assay Procedure,  B a s a l media f o r the assay of each amino a c i d were p r e pared a c c o r d i n g to the compositions o u t l i n e d by B a r t o n Wright ( 9 ) ,  A standard s o l u t i o n of the amino a c i d to be  assessed was made up to f a l l w i t h i n the assay range. c o n c e n t r a t i o n l e v e l s of the standard s o l u t i o n and were s e t up i n t r i p l i c a t e ,  All  extracts  H y d r o l y s a t e s were assayed a t f i v e  c o n c e n t r a t i o n l e v e l s ( u s u a l l y 500 mg  to 2,500 mg of sample).  The t o t a l volume of each tube was a d j u s t e d t o 10 ml. w i t h water.  The tubes were capped w i t h aluminium thimbles  and s t e r i l i z e d i n an a u t o c l a v e a t 10 pounds p r e s s u r e f o r 10 minutes. A f t e r a d d i t i o n of the inoculum, the tubes were i n c u b a t e d i n a water b a t h a t 30°C. f o r 72 hours.  At the end of the  i n c u b a t i o n p e r i o d , the l a c t i c a c i d produced i n the tube was t i t r a t e d a g a i n s t 0.1N  sodium hydroxide s o l u t i o n u s i n g an  e l e c t r o m e t r i c procedure (pH meter). iii  Organisms  Stock c u l t u r e s of the organisms r e q u i r e d f o r the assays were o b t a i n e d from the Department of D a i r y i n g , U.B.C. and included: 17/5  (SOI *-)  Leuconostoc mesenteroides P 60  (80^2)  L a c t o b a c i l l u s arabinosus  Streptococcus f a e c a l i s  1  ( l a c t i s Rogers)  (80^-3)  The organisms were c a r r i e d as stab c u l t u r e s on l i v e r Tryptone Agar intervals.  (Barton - Wright) and s u b - c u l t u r e d a t weekly  For the inoculum, a t r a n s f e r was made from a stab  c u l t u r e to a tube of r i b o f l a v i n standard medium (Barton -  - 5h Wright), (9).  incubated f o r 18 to 20 hours at 30°C.  T h i s was  A s a l i n e s u s p e n s i o n of the washed organisms was i n o c u l a t e the (3)  used  to  tubes,  Calculations. For the standard c u r v e , the l o g a r i t h m of the dose (micro-  grams of amino a c i d ) was response  p l o t t e d a g a i n s t the l o g a r i t h m of the  ( m i l l i l i t e r s of O.lNaOH),  ( i n micrograms) f o r the response read from t h i s curve.  The corresponding  values  of the h y d r o l y s a t e s were  The grams of amino a c i d per 16 g. of  n i t r o g e n were c a l c u l a t e d .  A mean value and standard d e v i -  a t i o n were computed f o r each sample. (1+)  Results. The amino a c i d content of the p r o t e i n s of the v a r i o u s  weed seeds are presented i n Table V I .  The v a l u e s f o r c y s t i n e  are not i n c l u d e d because d i f f i c u l t i e s were encountered the d e t e r m i n a t i o n of t h i s amino a c i d . for  in  The v a l u e s o b t a i n e d  the c o n t r o l s d i d not agree w e l l w i t h those r e p o r t e d by  other workers.  C y s t i n e i s s e n s i t i v e to a c i d h y d r o l y s i s when  the m a t e r i a l c o n t a i n s c o n s i d e r a b l e q u a n t i t i e s of  carbohydrates.  The d e s t r u c t i o n of c y s t i n e by prolonged a c i d h y d r o l y s i s has been r e c o g n i z e d by a number of workers. avoid t h i s d e s t r u c t i o n R i e s e n e_t aj. (109)  I n an attempt autoclaved  the  samples f o r only three hours i n c o n t r a s t to the u s u a l hours p r e s c r i b e d by Barton-Wright to minimize  (9).  Another  to  6-10  procedure  the d e s t r u c t i o n of t h i s amino a c i d recommends  separate a u t o c l a v i n g of the medium and samples.  (109).  TABLE VI THE AMINO ACID CONTENT OF THE NOXIOUS WEED SEEDS AND  CERTAIN STANDARD PROTEIN SOURCES  C a l c u l a t e d i n g. per 16 Amino Acid Nitrogen* Arginine Histidine Leucine Iso-Leucine Lysine Methionine Phenylalanine Tyrosine Threonine Tryptophane Valine  +  Casein  PROTEINS OF CERTAIN  g. N i t r o g e n  Soybean Linseed Rapeseed Brown S t i n k - Tumble Western White Wild Mustard weed Mustard F a l s e Mustard Mustard , Flax  7.71 5.85 7.1+ 5.9+ 3*79 6.59 5.89 6.^3 6.00 3.8+0.2 7.7+0.3 7.7±0.7 6.1109 . 6.1++0 5 . 6 .3+0.1 7.1+0.1+ 7.3+0.+ 5.2±0 2.7+0.2 2 . * + + 0 . 2 1.6+0.1 2 . 1 + + 0 . 1 2A+0.2 1.8+0.1 2,1+0.1 2.0+0.1 2. 8.7±0.2 6.++0.1 +.6+0.3 5.7+0.1 5.2+0.+ 6.0+0.k 5.9+Q.+ 5»2±0.2 5.6±0. 6 . + + 0 . 2 5.0+0.1 +5 .+0.1 + . + + 0 . 1 +.2+0.2 k.2+0.Okk.3+0.3 9.3+0.+ 6.1+0.3 3.6±0.3 5.2+0.2 5.6+05 . 5.6+05 . + .2+0.2 +5 .+0.6 6 .0+0 3.1+0.3 1.3+0.1 15 .+0.1 1 .0+0.2 1.6+0.1 1.3+0.3 15 .+0.02 1.2+00 ,5 15 .± 39 .+0.3 1.2±0.3 3 .1+0.2 2.8+0.3 3.3+0.2 3.2+O.+ 3.0+0.2 3.6 2.6+0.+ 2.0+0.1 19 .+0.1 2 .1+0.3 2.++0.3 2.2+0.2 2.2+0.2 +.2+0.2 3.1+0.2 2.8+0.2 3.1+0.2 3.0+0.2 35 .+0.3 3 .3+0.2 3.0+0.2 3. 2.2+0.3 15 .+0.1 2 .0+0.1. 1.-3+0.1 1.1+0.1 1.0+0.1 1.3+0.2 1.3+0.1 0. 6.7+0.+ +.7+0.2 + . + + 0 . 3 +.6+0.0+ +.2+0.3 + . + + 0 . 3 +.2+0.3 +5 .+0.2 +5 . 13.62  '  :  !  •  2.9±0.k  k.k±0.5  N i t r o g e n gm  ' ',  2.k±0  per 100  g. f a t - f r e e sample  - 56 TABLE V I I REPORTED VALUES FOR THE AMINO ACID COMPOSITION OF A NUMBER OF PROTEINS C a l c u l a t e d i n g. per 16 g. N i t r o g e n  Amino A c i d  Casein  Reference No, (1+8)  08)  Soybean  (101)  (1+8)  ( W  Linseed/  (1+9)  (i+8)  (1+8)  Rapeseed ( W  (1)  Arginine  k.2  3.7  ^.1  3.^  5.8  9.9  9.5 6.9 5.6  Histidine  3.2  3.0  2.5  1.1  2.3  2.7  1.8  1.9 2.6  Leucine  9.k 10.5  9.9  3.8.  6.6  6.3  6.1+  7.5 ~7  Iso-Leucine  6,2  7.1  6.5  2.U, k.7  5.6  i+.l 3.k  7.7  7.9  6.6  1.8  5.8  3.5  3.3 2.0 3.5  l.»+  0.5 2.0  1.5  1.2  2.3 1.1  Lysine  5.1  3.7  Methionine  2.9  3.1  3.k  Cystine  0.3  0.5  0,»+  0.6  0.9  1.9  1.9 1.7  Phenylalanine  5.1  5.1  5.2  5.1 2.5 5.7  3.9  k.6  5 . 8 i+.O  Tyrosine  6A  5.if  6.k  i+.l  2.2  2.1+  5.1 2.3  Threonine  5.0  *u6  3.9  3.8  1.9  ^.0  3.9  k.2 i+.5 3 . 8  Tryptophane  1.2  l.i+  1.3  1.3  0,5  1.6  1.1+  1,3  1.6 2.0  Valine  7.k  6.3  7.0 5 A  2.6 »+.2  5.9  k.7  5.8  5.7  - 57 A number of a n a l y s i s of the amino a c i d content of c a s e i n , soybean, l i n s e e d and rapeseed which have been r e p o r t e d i n the l i t e r a t u r e (1,  19,  *+8)  are presented i n Table V I I .  These  v a l u e s are i n c l u d e d as an i n d i c a t i o n of how w e l l the v a l u e s o b t a i n e d i n these s t u d i e s agree w i t h those p u b l i s h e d by other workers.  When one c o n s i d e r s the v a r i a t i o n s which may  between samples and between t e c h n i c a l procedures  of v a r i o u s  l a b o r a t o r i e s , good agreement between our data and r e p o r t e d elsewhere  i s evident.  data  However, i n a l l cases, the  v a l u e s obtained f o r p h e n y l a l a n i n e and s l i g h t l y lower  arise  t y r o s i n e tend  than the r e p o r t e d v a l u e s .  to.be  I t i s r e a l i z e d that  g r e a t e r accuracy would have been obtained i f these assays been repeated s e v e r a l times.  However, o n l y a r e l a t i v e  i n d i c a t i o n of the n u t r i t i v e v a l u e s of the p r o t e i n s by method was  had  this  sought.  As i n d i c a t e d i n Table VI the amino a c i d content of the weed seeds tends to f o l l o w the g e n e r a l p a t t e r n f o r rapeseed ( B r a s s i c a napus L ) ;  Although  s l i g h t l y lower i n a m a j o r i t y of  the samples, the e s s e n t i a l amino a c i d content of the weed seeds compares f a v o u r a b l y w i t h the g e n e r a l p a t t e r n found i n soybean.  White mustard and w i l d mustard tend to be lower i n  tryptophane content than the other seeds assayed. the l e v e l of h i s t i d i n e i s s l i g h t l y lower f o r the other weed seeds.  Although  In Stinkweed  than t h a t o b t a i n e d  the p r o t e i n of  stinkweed  compares f a v o u r a b l y w i t h those of the other weed seeds s t u d i e d , stinkweed  does not appear to be a good p r o t e i n source because  of i t s low n i t r o g e n c o n t e n t .  - 58 As expected,  the contents of a l l the e s s e n t i a l amino  a c i d s i n the p l a n t p r o t e i n s were lower values f o r c a s e i n . methionine  than the corresponding  The sulphur c o n t a i n i n g amino a c i d s ,  and c y s t i n e are u s u a l l y the l i m i t i n g amino a c i d s  i n plant proteins.  The v a l u e s found f o r t h i s a c i d i n the  weed seeds are comparable and, i n some cases such as  rapeseed,  brown mustard, tumble mustard and white mustard, may  be  s l i g h t l y higher than the v a l u e s found i n soybean.  However,  methionine  In a l l  i s the l i m i t i n g amino a c i d i n soybean.  c a s e s , the l e v e l of methionine  i s l e s s than the i d e a l r e q u i r e -  ment c o n s i d e r e d to be present i n whole egg p r o t e i n of kO g.methionine per 16 grams of n i t r o g e n . (b)  Chemical  Scores and E s s e n t i a l Amino A c i d I n d i c e s f o r  the V a r i o u s Weed Seed P r o t e i n s . The amino a c i d analyses were c a r r i e d out with the i n t e n t i o n of c o r r e l a t i n g amino a c i d content of the p r o t e i n s of weed seeds w i t h t h e i r n u t r i t i v e v a l u e .  This c o r r e l a t i o n  between amino a c i d analyses and b i o l o g i c a l v a l u e of a p r o t e i n has been c o n s i d e r e d thoroughly by M i t c h e l l and B l o c k ( 2 1 , and by Oser ( 1 0 5 ) .  91,  96),  by Kuhnau ( 7 7 ) ,  (96)  s e l e c t e d the p a t t e r n of amino a c i d s i n egg p r o t e i n s as  M i t c h e l l and  the standard f o r growth s i n c e d i g e s t i b i l i t y and  Block  utilization  of t h i s p r o t e i n has-been found  to be complete,  or n e a r l y  complete i n most experiments.  Data have i n d i c a t e d t h a t , up  to a c e r t a i n l e v e l , a l l of the amino a c i d s absorbed  during  d i g e s t i o n of egg p r o t e i n are r e t a i n e d i n the body of the animal.  - 59 -  The idea of Mitchell and Block (96) was to judge the nutritive adequacy of the protein by the severity of its limiting essential amino acid deficiency. The data i n the literature Indicates that the amino acid i n the test protein with the greatest deficit, when compared to egg protein does indeed limit the amounts of a l l other essential amino acids that can be used for growth.  Accordingly,  the proteins of foods may be ranked i n the order of decreasing nutritive efficiency on the basis of increasing percentage deficits i n their respective limiting essential amino acids '. 1  The nutritive value of a protein may be expressed as a "chemical score" equal to the smallest percentage of an essential amino acid i n the protein based on i t s content i n egg protein. Similarly, Oser (105) took egg protein as a standard for the calculation of an essential amino acid index which placed emphasis upon the pattern fed as well as the limiting acid. Ratios between the essential amino acids i n the test protein to those i n egg protein were calculated, a l l percentages over 100 being reduced to 100, preventing excess amino acid i n the test protein from overweighting the mean.  A geometric mean  of these ratios was computed equal to the antilogarithm of the mean logarithm of the ratios.  This approach to the calculation  of an essential amino acid index has the advantage of giving consideration itd it he mixture of amino acids being fed to the animal. Recently, Mitchell (91) has developed a modified essential amino acid index.  In computing the revised index, arginine i s  - 60 omitted from c o n s i d e r a t i o n , because i t i s n e i t h e r e s s e n t i a l in  the s t r i c t  sense of the term, nor does i t have an o b l i g a t e  p r e c u r s o r among the e s s e n t i a l amino a c i d s .  The r a t e of i t s  s y n t h e s i s from precursors, w i t h r e f e r e n c e t o the r a t e o f growth of the animal may be adequate f o r normal growth, depending  upon the s p e c i e s and stage of growth.  On the other  hand, c y s t i n e and t y r o s i n e a r e i n c l u d e d because t h e i r i n the p r o t e i n w i l l d i m i n i s h the requirements ine  and methionine, r e s p e c t i v e l y .  presence  f o r phenylal-  I f the p r o t e i n under  c o n s i d e r a t i o n c o n t a i n s higher l e v e l s of c y s t i n e or t y r o s i n e than the standard p r o t e i n then the l e v e l s o f these amino a c i d s are decreased to the corresponding amount present i n the standard p r o t e i n .  The'"corrected egg r a t i o s " a r e t h e n c a l c u l a t e d .  An example of the c a l c u l a t i o n o f the e s s e n t i a l amino a c i d index f o r c a s e i n i s c o n s i d e r e d i n Table V I I I . Fisher  (36)  maintained that some of the amino a c i d s of  whole egg p r o t e i n might be present i n excess o f the minimal requirements o f the animal, thus g i v i n g a lower v a l u e f o r the p r o t e i n by chemical s c o r e method than by b i o l o g i c a l v a l u e method. for  Using the data presented by B l o c k and M i t c h e l l  (21)  amino a c i d content and b i o l o g i c a l v a l u e o f p r o t e i n s ,  F i s h e r estimated a s e r i e s o f minimal amino a c i d  requirements  which he c o l l e c t e d under the heading o f ' i d e a l p r o t e i n ' . When the e s s e n t i a l (and semi-dispensable) amino a c i d requirements f o r maintenance, and f o r maintenance and growth have been determined w i t h s a t i s f a c t o r y accuracy, they c a n be used i n p l a c e o f some r e f e r e n c e p r o t e i n o f s u p e r i o r q u a l i t y ,  - 61 TABLE V I I I COMPUTATION OF MODIFIED ESSENTIAL AMINO ACID INDEX (based  on Amino A c i d a n a l y s i s - Table VI)  Amino A c i d Content Amino A c i d s  per 16 grams N i t r o g e n Whole Egg Casein Protein Gram  Percent  2.7 9.3  112.0 133.0 70.0 80.0  100 100  2.0000 2.0000  7+ 100  1.8692 2.0000  2.h  Lysine  7.0  Phenylalanine  6.3 +.5  *+.*+  10.8 1.5  8.0 2.2  Phenylalanine plus t y r o s i n e  3.6  Methionine  3.1  7+.0 11+6.0 77.0  Cystine  0.3  12.5  Tryptophane  Logarithms of Corrected Ratios  Gram  Histidine  Tyrosine  Egg Corrected Ratio Ratio Percent  Methionine plus c y s t i n e  6.1+  3.+  53.0  53  1.72+3  Threonine  +.3  H-.2  98.0  98  I.9912  Leucine  9.2  8.7  95.0  Is©leucine  7.7  6.1+  83.O  95 83  1.9777 1.9191  Valine  7.2  6.7  93.0  93  1.9685 1.9389  Average l o g a r i t h m M o d i f i e d E s s e n t i a l Amino A c i d index Chemical Score  53  L i m i t i n g Amino A c i d - c y s t i n e , methionine  86.9  - 62 i n computing e s s e n t i a l amino a c i d i n d i c e s .  When t h i s i s  accomplished i t w i l l be p o s s i b l e to base e s s e n t i a l amino a c i d i n d i c e s on a f i r m e r b a s i s  than can be done a t p r e s e n t .  Using the amino a c i d p a t t e r n of the whole egg p r o t e i n of M i t c h e l l ( 9 1 ) e s s e n t i a l amino a c i d i n d i c e s and c h e m i c a l s c o r e s were computed f o r each of the weed seed and c o n t r o l studied.  protein^  These c a l c u l a t i o n s were based on amino a c i d d a t a  o b t a i n e d during  these s t u d i e s .  (Table V I ) .  i n d i c e s obtained are presented i n Table IX.  The s c o r e s and E s s e n t i a l amino  a c i d i n d i c e s and chemical scores were a l s o c a l c u l a t e d f o r the control proteins; casein, using  soybean, l i n s e e d and rapeseed,  amino a c i d a n a l y s i s data r e p o r t e d  (Table V I I ) .  i n the l i t e r a t u r e  These v a l u e s are presented i n Table X I .  W i t h the c o n t r o l p r o t e i n s , v a l u e s f o r c y s t i n e from the l i t e r a t u r e were i n c l u d e d  obtained  i n the c a l c u l a t i o n of the  e s s e n t i a l amino a c i d i n d i c e s and chemical s c o r e s .  Unfortunately,  v a l u e s f o r c y s t i n e c o u l d not be i n c l u d e d when c a l c u l a t i n g the i n d i c e s and scores f o r the weed seed C o r r e l a t i o n of M o d i f i e d Net  proteins.  E s s e n t i a l Amino A c i d I n d i c e s  with  Protein U t i l i z a t i o n Values.  A comparison of the e s s e n t i a l amino a c i d i n d i c e s w i t h the net p r o t e i n u t i l i z a t i o n v a l u e s (Table  (Table  X)  V) i n d i c a t e s a  h i g h degree of c o r r e l a t i o n between these two methods f o r evaluation proteins  of p r o t e i n s .  The chemical scores i n d i c a t e t h a t the  of the weed seeds are s l i g h t l y lower i n b i o l o g i c a l  v a l u e than soybean.  As i n soybean, methionine i s the l i m i t i n g  - 63 TABLE IX ESSENTIAL AMINO AG ID INDICES AND CHEMICAL SCORES FOR THE PROTEINS OF WEED SEEDS (based on Amino A c i d Analyses - Table VI) Based  Protein  Source  on Whole Egg P r o t e i n Mitchell Chemical L i m i t i n g Amino Essential Amino A c i d Score Acid Index  86.9  53.0  Methionine  72.7  51.5  Methionine  56.9  29.6 37.5  Phenylalanine Methionine  Rapeseed °  66.9  Phenylalanine Methionine  Brown Mustard  61.9  +6 +7.5 +0.0  Stinkweed  60.7  32.5  Methionine  Tumble Mustard  61.8  37.5  Methionine  Western F a l s e F l a x  59.8  30.0  Methionine  White  60.8  37.5  Methionine  57.+  35.0  Methionine  C a s e i n *» Soybean L in se ed  Wild  9  °  Mustard Mustard  C y s t i n e i n c l u d e d from p u b l i s h e d  data.  Methionine  -  TABLE X  ESSENTIAL AMINO AG ID INDICES AND CHEMICAL SCORES FOR A NUMBER OF PROTEINS  (based on r e p o r t e d Amino A c i d analyses - Table V I I )  Protein  Reference Number  Based on Whole Egg P r o t e i n Mitchell Limiting Chemical Essential Score Amino A c i d Amino A c i d Index  Casein  1+8  86.9  50  Soybean Meal  19  7^.3  1+0.6  Methionine  Linseed Meal  1+8  69.2  37.5  Methionine  1  66.8  ^3.7  Methionine  Rapeseed H e r r i n g Meal  ho  8U..3  Methionine and Cystine  Phenylalanine and T y r o s i n e  - 65 amino a c i d i n a l l  the weed seeds s t u d i e d .  The  relative  agreement between the e s s e n t i a l amino a c i d i n d i c e s o b t a i n e d f o r all  the weed seed p r o t e i n s does not account f o r the h i g h n e t  p r o t e i n u t i l i z a t i o n v a l u e o b t a i n e d f o r soybean i n c o n t r a s t to the lower N.P.U. v a l u e f o r stinkweed. E r r o r s i n a n a l y s i s would account f o r the s l i g h t d i s c r e p a n c i e s between i n d i c e s and scores based on the d a t a from these s t u d i e s and those based on the data r e p o r t e d elsewhere. However, from the expected c o r r e l a t i o n between the e s s e n t i a l amino a c i d i n d i c e s and the net p r o t e i n u t i l i z a t i o n v a l u e s , one c o u l d p r e d i c t that the p r o t e i n s of brown mustard, mustard, w i l d mustard and western f a l s e f l a x would  tumble  compare  w i t h those of w h i l e mustard and stinkweed as p l a n t p r o t e i n s of moderately h i g h b i o l o g i c a l v a l u e , but s l i g h t l y lower i n v a l u e than  soybean.  - 66 -  SUMMARY  An a attempt has been made to assess the nutritive value of the proteins of a number of weed seeds considered as noxious under the Canada Feeding Stuffs Act.  These weed seeds contain  isothiocyanates which may or may not be toxic to animals but which are not palatable for the rat.  For this reason i t was  necessary to take an indirect approach involving analysis of the essential amino acid content of these weed seeds as well as a direct approach involving animal assays with rats using samples of the weed seeds which had been treated to remove the isothiocyanate content.  In carrying out these investi-  gations, a study of the properties of the isothiocyanates themselves and of the methods for evaluation of proteins was necessary.  These features have been described f u l l y . The  main aspects of the investigations are recorded below. 1.  The isothiocyanate content of a number of weed seeds was determined.  The values obtained are presented i n Table I  and compare with those reported i n the literature. 2.  An attempt was made to develop a procedure for extraction of the isothiocyanates from samples of the weed seeds. A procedure involving autohydrolysis of the weed seeds with water, followed by extraction of the dried sample with 70$ ethanol was evolved.  This procedure reduc ed  the content of isothiocyanates and rendered the weed seeds palatable to the rat. 3.  The protein quality of the weed seeds was evaluated by bio-assay with rats, following the method proposed by Miller and Bender.  The prepared samples of weed seeds  - 67 w i t h the reduced assays.  i s o t h i o c y a n a t e content were used i n these  The N.P.U. v a l u e s obtained f o r these assays a r e  presented i n Table V and i n d i c a t e that the p r o t e i n s of white mustard ( B r a s s i c a a l b a B o i s s ) and rapeseed  ( B r a s s i c a napus L)  compare f a v o u r a b l y w i t h l i n s e e d and soybean as p l a n t p r o t e i n s of moderately stinkweed  high b i o l o g i c a l value.  The N.P.U. v a l u e f o r  ( T h l a s p i arvense L) was s l i g h t l y lower  than that o f  the other two C r u c i f e r a e . R e p r e s e n t a t i v e samples of the weed seeds were assayed f o r t h e i r content of e s s e n t i a l amino a c i d s . procedures ( 9  ) were used.  Microbiological  assay  The v a l u e s foor the weed seeds were  s i m i l a r to the g e n e r a l p a t t e r n o f amino a c i d s found i n rapeseed ( B r a s s i c a napus L ) . Although  s l i g h t l y lower i n a m a j o r i t y of  the samples, the e s s e n t i a l amino a c i d content of the weed seeds compared f a v o u r a b l y w i t h t h a t found i n soybeans. U s i n g the data f o r the e s s e n t i a l amino a c i d content o f the weed seeds, a "chemical e v a l u a t i o n " of the p r o t e i n s of weed seeds was c a r r i e d out.  An " e s s e n t i a l amino a c i d index" and "chemical  s c o r e " was c a l c u l a t e d f o r each weed seed a c c o r d i n g to the method proposed  by M i t c h e l l .  The " s c o r e s " and " i n d i c e s "  obtained a r e presented i n Table IX and i n d i c a t e t h a t p r o t e i n s from the weed seeds under study were s i m i l a r i n value but lower i n q u a l i t y than c a s e i n and soybean p r o t e i n .  C a l c u l a t i o n of  the ''chemical s c o r e s " i n d i c a t e d that methionine was the l i m i t i n g amino a c i d i n soybean and a l l the weed seeds s t u d i e d . The " e s s e n t i a l amino a c i d i n d i c e s "  (Table X) o b t a i n e d from the  e s s e n t i a l amino a c i d a n a l y s i s were compared w i t h the "net p r o t e i n u t i l i z a t i o n " v a l u e s o b t a i n e d from the r a t a s s a y s .  A  - 68 h i g h degree of c o r r e l a t i o n was f o r e v a l u a t i o n of p r o t e i n s .  found between these two methods  From t h i s c o r r e l a t i o n one c o u l d  p r e d i c t that the p r o t e i n s of brown mustard C o s s ) , w i l d mustard mustard  ( B r a s s i c a .iuncea (L)  ( B r a s s i c a kaber (DC) L. C. Wheeler),  (Sisymbrium a l t i s s i m u m L) and western f a l s e  tumble  flax  (GameUna m i c r o c a r p a Andrz) compare w i t h those of white mustard ( B r a s s i c a a l b a B o i s s ) and stinkweed ( T h l a s p i arvense L) as p l a n t p r o t e i n s of moderately h i g h b i o l o g i c a l v a l u e , but lower i n v a l u e than soybean.  slightly  APREHD.EC  - 69 TABLE X I PROTEIN AND FAT COMPOSITION OF A NUMBER OF WEED SEED SAMPLES  Per Cent P r o t e i n (N x 6.2?) F a t F r e e B a s i s e x t r a c t e d with Ground Raw Seed ( e x t r a c t e d w i t h petroleum petroleum ether ether. P  P r o t e i n Source  e  r  C  e  n  t  F  Brown Mustard ( B r a s s i c a .luncea L Coss) Stinkweed ( T h l a s n i arvense L)  22.8.  37.1  38.2  16.5  19.7  33.+  Tumble Mustard (Sisymbrium a l t i s s i m u m L)  32.6  +1.2  20.+  Western F a l s e F l a x (Cameling m i c r o c a r oa, AndraJ  22.6  36.8  38.7  White Mustard (Brassica albq Boiss)  28.8  39.5  30.9  W i l d Mustard ( B r a s s i c a kaber [DC) L.C.Wheeler  27.+  37.5  27.1  28A  M+.6  36.2  Linseed (Linum u s i t a l i s s i u m )  21.3  35.3  39.7  Soybean (Glycine  38.5  +8.2  21.6  Rapeseed ( B r a s s i c a nanus L)  Casein  ,  soya) 86.3  a  t  -70-  Appendix I THE NITROGEN : WATER RELATIONSHIP I I  ALBINO RATS  INTRODUCTION I n c o n n e c t i o n w i t h the d e t e r m i n a t i o n of the net p r o t e i n u t i l i z a t i o n of a number o f p r o t e i n s by the method of M i l l e r and Bender (89) i t was; found n e c e s s a r y to study; the r e l a t i o n s h i p of body n i t r o g e n to body water i n the U.B.C. c o l o n y of A l b i n o rats;. Moult on (98) has shown t h a t body/ c o n s t i t u e n t s i n c l u d i n g ; the n i t r o g e n and water content, are a c o n s t a n t p r o p o r t i o n o f the body/ mass when measured on a f a t - f r e e b a s i s . He showed that the composition of the f a t - f r e e mass changes; i n a r e g u l a r manner w i t h age.  The r e l a t i v e water c o n t e n t of the  f a t - f r e e mass decreases; v e r y r a p i d l y from c o n c e p t i o n to b i r t h and  then l e s s r a p i d l y u n t i l a p r a c t i a l l y c o n s t a n t  c o n c e n t r a t i o n i s reached.  On the o t h e r , the content of  p r o t e i n ( n i t r o g e n ) and ash are c o r r e s p o n d i n g l y i n c r e a s e d u n t i l a p o i n t where there i s n e a r l y a c o n s t a n t c o m p o s i t i o n of water, p r o t e i n and ash i n the f a t - f r e e mass.  In s t u d i e s w i t h male  a l b i n o r a t s , Babineau and Page (7) found water t o r e p r e s e n t 72 percent of the f a t - f r e e mass. completely independent  T h i s c o n s t a n t was  found  t o be  of the magnitude of the f a t depots.  From the d a t a presented by Moulton ( 9 8 ) , M i l l e r and Bender (89) concluded c o n s t a n t and  that the p a t i o of n i t r o g e n t o water i s a  t h e r e f o r e the n i t r o g e n content of the body c o u l d  be d e r i v e d from the water c o n t e n t .  In t e s t i n g t h i s  analysed 197 r a t s of a M a c k and white t o t a l body n i t r o g e n and body water.  they  hooded s t r a i n f o r  They found  that  - 7© -  the nitrogen to water r a t i o (NtH^O) varied with sueh constancy that i t could be used f o r c a l c u l a t i n g carcass nitrogen from a knowledge of the water content and age, Babineau and Page (7) have since shown that i n p h y s i o l o g i c a l l y normal animals, the non-fat dry matter can be predicted from t o t a l body water with a high degree of accuracy. M i l l e r and Bener, (89) however, doubted whether t h e i r calculated regression equation f o r N:H 0 values, correlated 2  with the age i n days, would also be applicable to other r a t colonies.  Analysis of a small number of albino rats by  Forbes and Yohe did indeed indicate the existence of s t r a i n differences.  Recently, Dreyer (3D has reported further  large scale findings on the s t a b i l i t y of the r e l a t i o n s h i p between the N:H 0 r a t i o and age of albino r a t s . 2  He has  reported observed differences between the N:H 0 r a t i o s of 2  male and female animals.  M i l l e r and Bender (89) on the other  hand reported no sex d i f f e r e n c e s . The present study was undertaken  to investigate the  relationship between t o t a l body water and body nitrogen i n the U.B.C. r a t colony and was an e s s e n t i a l prerequisite to the use of the M i l l e r and Bender procedure. EXPERIMENTAL ANIMALS  The animals studied included both the Wistar and SpragueDawley strains of albino rats bred i n the U.B.C. r a t colony from rats o r i g i n a l l y obtained from the-Wistar I n s t i t u t e ,  - 72  Philadelphia  -  and the Sprague-Dawley  Company r e s p e c t i v e l y .  The r a t s a n a l y s e d ranged i n weight from 5 t o 300 grams and i n age from b i r t h to 87 days.  I n the weight range 5  to 30 grams ( b i r t h t o 18 days) l i t t e r mates (two females and two males) were a n a l y s e d a t each age group. range ho t o 300 grams, s i x l i t t e r s of Sprague-Dawley  were used.  Over the weight  of W i s t a r and f o u r  litters  Animals were drawn a t random  from these l i t t e r s when they reached the d e s i r e d weight.  A  v  c a r e f u l r e c o r d was kept o f the age o f each r a t i n these weight ranges.  A number o f r a t s  ( f o u r t e e n ) i n the weight  range (*+0 t o 100 grams) which were used i n p r e l i m i n a r y studies  were a l s o o b t a i n e d by random s e l e c t i o n from the main  colony. For  the 250 and 300 gm. weight ranges, some o f the r a t s  were o b t a i n e d from the stock c o l o n y .  Only approximate ages-  c o u l d be o b t a i n e d f o r some o f the r a t s i n t h i s group. weaning, a l l the r a t s were kept on the s t o c k d i e t  After  (U.B.C.  R a t i o n No. 18) u n t i l they were s a c r i f i c e d f o r a n a l y s i s . t o t a l of 8h r a t s , 36 Sprague-Dawley  A  and 58 W i s t a r were a n a l y s e d .  The average number o f r a t s per weight group was f o u r but varied  from two to t e n .  CARCASS ANALYSIS Body water was determined a f t e r k i l l i n g chloroform.  the r a t s w i t h  I n c i s i o n s were made i n t o the c r a n i a l , t h o r a c i c  and abdominal c a v i t i e s , and the c a r c a s s e s were d r i e d  to a  constant weight a t 105°C;. f o r ^8 hours.  Intestinal  contents  were i n c l u d e d . N i t r o g e n was determined The  s m a l l r a t s , up t o 15* grams d r y weight, were  d i g e s t e d w i t h approximately gram d r y matter and  10  (102)..  by the K j e l d a h l method  completely  10 ml concentrated H S0^ per 2  gram c a t a l y s t  (10  0.1-0.3g.  g. K S0^, 2  CUSO^.5"H 0) a c c o r d i n g t o the Gunning Method (102). 2  With the c a r c a s s e s o f the l a r g e r r a t s , over 15 grams d r y weight, d i f f i c u l t i e s  were.encountered i n o b t a i n i n g  homogenous samples f o r the d e t e r m i n a t i o n o f t o t a l n i t r o g e n . To a v o i d t h i s , K j e l d a h l d i g e s t i o n of the whole c a r c a s s was followed.  I n p r e l i m i n a r y s t u d i e s , the d r i e d c a r c a s s e s were  ground i n a mortar and the e n t i r e ground r a t was t r a n s f e r e d i n 10-15  gm. p o r t i o n s t o a K j e l d a h l f l a s k f o r d i g e s t i o n .  The d i g e s t e d m a t e r i a l was then recombined. to  500  of  the r a t ) were taken f o r d i s t i l l a t i o n .  After  dilution  ml, d u p l i c a t e 25 or 50 ml a l i q u o t s (depending  c a r r i e d out on lk t h i s procedure  on s i z e  T h i s procedure  r a t s as i n d i c a t e d i n T a b l e s .  was  Following  v i o l e n t f r o t h i n g occured d u r i n g d i g e s t i o n and  another method f o r sampling A procedure  suggested  was sought. by S a r e t t and Snipper  (116) was  f o l l o w e d f o r p r e p a r i n g the c a r c a s s e s of the remainder o f the large anima^for of  nitrogen determinations.  The d r i e d  carcass  each animal was weighed i n a t a r e d p i n t Mason j a r .  Approximately  100  ml of d i s t i l l e d water, was added and the  mixture was a u t o c l a v e d a t 15 l b s . pressure f o r h hours.  Each  c a r c a s s was homogenized u s i n g a V l r T i s homogenizer ("AeroS e a l Chemixer"). A l i q u o t s o f the weighed homogenates were t r a n s f e r r e d to a 500  ml or 800  ml K j e l d a h l f l a s k , the weight o f the a l i q u o t  being determined by weight d i f f e r e n c e .  By d i v i d i n g  homogenate i n t o t h r e e to s i x p o r t i o n s , depending  the  on the d r y  weight of the r a t , the t o t a l c a r c a s s homogenate was  digested.  As i n the p r e c e d i n g method, approximately 10 ml. c o n c e n t r a t e d RvjSO^. per gram r a t d r y c a r c a s s was  used.  The v i g o r o u s f r o t h i n g  which occured d u r i n g d i g e s t i o n o f the d r y c a r c a s s was avoided.  Care was  a l s o taken to m a i n t a i n the d i g e s t i o n a t  a r e l a t i v e l y low temperature f o r the f i r s t 1-2 heating.  mainly  The mixture was  hours o f  digested u n t i l i t cleared  green c o l o u r ) and t h e r e a f t e r f o r about 30 minutes. a l i q u o t s were not recombined,  but were d i l u t e d t o 500  and a l i q u o t s c o n t a i n i n g approximately 1 gm.  (pale The ml  o f d r y matter  were taken f o r the e s t i m a t i o n of ammonia. The mean of the c a l c u l a t e d mgs. was  N per r a t f o r each a l i q u o t  taken as the n i t r o g e n content o f the r a t .  RESULTS: The r e s u l t s o b t a i n e d f o r the moisture and n i t r o g e n content of 36 Sprague Dawley and 58 W i s t a r r a t s a r e p r e s e n t e d i n T a b l e s X I I , X I I I , XIV and T a b l e XV,  respectively.  These r e s u l t s were  used f o r c a l c u l a t i o n o f n i t r o g e n ; water r a t i o s , equations and f u r t h e r s t a t i s t i c a l  treatment.  regression  - 75 xir:  T A B L E  BODY  N I T R O G E N  Weight gas. 5.51* 6.00 6.32 12.8 12.9 21.1 23.6 32.8 33.6 *k>.7** kl.l  lfLj-.O**  1+5.6** 1+6.0 50.9 52.9 58.3 58.5**  61.M-**  71.0 77.9 81.7 88.8 98.O** 99.8 102.3** 139.9 207.1 236.2  W E I G H T  -  BODY  R E L A T I O N S H I P  Age days 0 0 0 8 8 lk lk  18 18 mm mm  22 mm mm am mm  29 29 29 32 —  >+3 31 k$  36  --.  38  51 207 - 237 207 - 237  I N  Water gms. *+.72 5.07 5.32 10.k 10.3 16.1 17.7 23.2 23 i 5 27.3 30.1 29.5 30.7 33.1 36.6 37.3 k2& 39.2 39.»+ 50.6 57.1 59.5 63.4 70.3 72. k 75.0 96.2 129.1+ 11+8.8  W A T E R  F E M A L E  -  BODY  W I S T A R  R A T S  Nitrogen mg. T i l 77. 82. 218 223 "+35 "+97 716 778 1,000 1,113 1,056 1,120 1,219 ' ^28 .  H7  1,6/21 1,^0 1,561+ 1,783 1,95*+ 2,327 2,428 2,828 3,000 2,99k ^,375 6,202 7,01+7  N = 29  *  i n t e s t i n a l contents removed  ** n i t r o g e n determination on ground dry carcass  gmN/gm H 0 xlOO _2 TT^O " 1.52 1.5* 2.142.11 2.70 2.80 3.08 3.31 3.66 3.69 3.58 3.6*f 3.68 3.62 3.87 3.82 3.67 3.96 3.52 3A2 3.91 3.82 1+.02 1+.11+ 3.9? k.5k 1+.79 1+.73 o  r  -  76 -  TABLE X I H BODY WEIGHT - BODY WATER - BODY NITROGEN RELATIONSHIP IN MALE WISTAR RATS.  Weight gms 5.8V* 6.00 6.23 13.6 13.6 21.1+ 21+.3 33 A 36.0 +2.5  Age days 0 0 0 8 8  80.2** 90.8** 95.2** 99.9 103.3 1+6.3 198.6 263.7 286.7  +.93 5.12 5.26 11.0 10.9 16 A  lk lk  18 18  22  2k  56.8** 57.3 58.9 61.9** 65.9** 70.1 72.2**  Water gms  ~~  36 29  --  Mia  32 31 mm mm mm mm  k3  +3 36 51 68  81 61 - 95  18.5  23.9 25.6 31A +0.1 +0.1 +1.5 +2.5 +1.3 +7.7 51.5 53.3 57.1 59.0 65.7 67.9 70.2 73 A 75.7 103 A 139.5 177.6 192 A  Nitrogen mgms  N/HgO R a t i o gm N/gm H 0 X100 2  1.32 1A1 1.56  65 73 82 23*+ 2+2  2.12 2.21 2.80  1+6I  2.78  51k  800  3.3J+ 3.3+ 3.59  856 1,129 1+85  3.70 3.61  llkkQ  1,560  ,780 ,965 ,952 .121 2. , 1 2 8 2,' ^ 2 , ,' 8 26 ?, ,58+ ,92^,99+  '  -,5+5 8,878 7,+06  N = 29 *  i n t e s t i n a l contents  removed  ** n i t r o g e n d e t e r m i n a t i o n on ground d r y c a r c a s s  3.75 3.83 3.72 3.73  3.81 3.66  3.71  3.60 3.76  +.16 3.68 3.98 3.95 +.55 +.69 +.99 3.85  - 77 -  TABLE XIV BODY WEIGHT - BODY WATER - BODY NITROGEN RELATIONSHIP IN FEMALE SPRAGUE - DAWLEY RATS Weight gms  Age days  5.78  0  4.95  -  t;  13.35 13.47 20.17 21.30 28.81 30.97 38.8 52.1  64.1  71.3 81.2 91.4 99.0 140.0 190.9 237.1  N = 18  6 6 10 10 18 18 23 30 30 33 37 40 42 52 75 125 - 156  Water gms  #  9  10.65 10.85 15.54 16.24 21.02 22.69 28.5 37.9 45.1 50.8 58.3 65.9 72.2 98.6 128.0 150.7  Nitrogen mgns  N/H 0 r a t i o gmN/gmH O xlOO 2  p  97  1.95  282 291 473 499 767 839 1,050 1,432 1,797 1,927 2,294 2,600 2,963 4,266 6,158 7,115  2.64 2.68 3.04 3.06 3.65 3.69 3.68 3.77 3.98 3.79 3.93 3 ; 94 4.10 4.32 4.81 ^.72  '  - 78 -  TABLE XV BODY WEIGHT - BODY WATER - BODY NITROGEN RELATIONSHIP IN MALE SPRAGUE - DAWLEY RATS  Weight gms.  Age days  Water gms.  6.33 6.35 11.26 1+.67 22.55 2^.13 31.76 3+.77 iri  0 0 6 6 10 10 18 18  5.38 5.+2 9.22 11.71 17.25 -18.1*3 23.+9 25.+2 32.7 ? '  I  iH  2 3  12  8?1 °3.l 90.1 106.0  ?? 33 35 IfO  2+1.3 298.3  87 87  N = 18  7  5  Si'  60.5 66.0 77.0 165.3 198.9  Nitrogen mgms. 106 107 229 299 53+ 5+9 870 9W  M75 1  ^  1 7 9 ?  2,290 2 503 3 127 7,1+1 & 5 9  N/HpO r a t i o gmN/gm H Q XlOO 9  1.96 1.96 2.+8 2.55 3.09 2.98 3.70 3.71 3.59 3 76 3.62 3.78  \ t{l n%  J Coo  CALCULATION In f o r m u l a t i n g an e q u a t i o n f o r the  c a l c u l a t i o n of body Bender (89)  n i t r o g e n from water content, M i l l e r and  regressed,  the r a t i o of body n i t r o g e n to body water w i t h age. a two had  variable  l i n e a r c o r r e l a t i o n but  three v a r i a b l e s ;  water, n i t r o g e n and  obtained a l i n e a r r e l a t i o n s h i p  H o gms x m  a  1 0 0  2 , 9 2  +  0 , 0 2  g i v e n by  >  x  2  w h e r e  However, examination of a d d i t i o n a l range 0 -  age  503  (N:H (P)100 to be 2  Over  days i n d i c a t e d  the  x  the =  a  S  e  they the  Bender equation: i n  days.  r e s u l t s covering  the  r e l a t i o n between age  c u r v i l i n e a r , s u g g e s t i n g an  and  exponential  (89).  form  C h r o n o l o g i c a l age actual physiological "A  age.  fact  range 3 3 - 5 7 days, M i l l e r and  r e l a t i v e l y s h o r t age (89)  i n actual  They used  i s not  age  always a good index of  of an animal.  g i v e n c h r o n o l o g i c a l or p h y s i c a l  physiological  or f u n c t i o n a l  significance  The  under d i f f e r e n t  t h e r e f o r e , one  (8)  showed that the  maintained on a low l e n g t h of time (25  (24)  a different  different  same organism, a t  conditions."  animal may  o l d e r than another animal at the Bailey  for  l e v e l of n u t r i t i o n w i l l a f f e c t the  an animal and,  quote Brody  time u n i t has  organisms, f o r d i f f e r e n t organs i n the d i f f e r e n t ages, and  To  the  be  growth r a t e  of  physiologically  same c h r o n o l o g i c a l  age.  f a t - f r e e body mass of mice  plane of n u t r i t i o n f o r a c o n s i d e r a b l e -  3 6 . d a y s ) contained more body water  l e s s body n i t r o g e n than the  and  f a t - f r e e mass of mice maintained  > N,  - 80 -  on a h i g h e r plane o f n u t r i t i o n . that  at: t h e same body w e i g h t ,  than animals  For  he  younger but  on a h i g h e r p l a n e o f  plotted An  correlating  straight  relationship  l i n e was  (Figure I ) .  i n this  study.  body n i t r o g e n c o n t e n t were  on a r i t h m e t i c , a r i t h - l o g  apparent  of  nutrition.  this reason, r e g r e s s i o n equations  d a t a f o r body w a t e r and  plane  chronologically  b o d y n i t r o g e n t o body w a t e r w e r e c a l c u l a t e d The  concluded,  a n i m a l s on a low  n u t r i t i o n are p h y s i o l o g i c a l l y older  From t h i s  and  l o g - l o g graph  obtained with  the  This; I n d i c a t e d t h a t  paper.  log-log  the  relation-  s h i p b e t w e e n n i t r o g e n and w a t e r c o u l d fee e x p r e s s e d fey t h e following; equivalent equations:  T h i s was  Exponential  Y. =  Logarithmic  log ¥  not s u r p r i s i n g  been used  The  term  to designate growth. izing  fe  = l o g a * fe l o g X  since equations  of t h i s  type  have  e x t e n s i v e l y f o r expression of b i o l o g i c a l data  ( 2 * + ) and  Brody  aX  by  others ( 5 1 ) .  "allometry" this  form  has b e e n u s e d  1  by  of e q u a t i o n used  Allometry i s a mathematical  the r e l a t i o n s h i p s  of s i z e  a number o f w o r k e r s  f o r expressing  relative  technique f o r c h a r a c t e r -  or c o n c e n t r a t i o n of  two  parts  o r c o n s t i t u e n t s o f the a n i m a l b o d y ( 5 1 ) . The  l o g a r i t h m i c form  straight  of the e q u a t i o n d e s c r i b e s a  l i n e w i t h s l o p e b.  the p e r c e n t a g e  change i n  change i n  When b = 1,  X.  Y  The  exponent  b  i s the r a t i o  t o the c o r r e s p o n d i n g the changes o f the  two  of  percentage measurements  - 81 -  10000  5ooo  Sprague-Dawley Y=26T6Xl.09 _  + s =3.oo r  - s . 2.91 r=  . Wistar Y=27.9X +S =3.7+ -S =3.6l  2000  1,08  r  CO  0  r  CO  txO  1000  a •H  c  Q)  500 Wistar  bO O  Y=6.+3X ^ +S =+.08 -S =3.92 1,  U  L  r  4-5  r  o 4->  200  XI •H  ©  Spra gue-Dawley  100  1  Y=10.2X +S =+.10 J  #  LO  r  50  1  1  •  10  20  50  '  • ' »'  100  200  Weight o f Water i n Grams Figure I  -  Body Water - Body N i t r o g e n R e l a t i o n s h i p s i n the WistaSr and Sprague-Dawley S t r a i n s o f A l b i n o Rats  m a i n t a i n a p r o p o r t i o n a l . c o n s t a n c y , such that the c o n s t a n t a = Y .  When b*> 1 ,  the percentage  i n c r e a s e of Y i s more  r a p i d than that of X, w h i l e when b<^l, the r e v e r s e i s t r u e . Where b ^ l , the " a " c o e f f i c i e n t i s simply a s u i t a b l e m u l t i p l i e r without any obvious b i o l o g i c a l  significance.  The r e s u l t s obtained f o r the r e l a t i o n s h i p of body water to body n i t r o g e n appeared of t h i s form.  to be b e s t d e s c r i b e d by an e q u a t i o n  Brody (2h) c o n s i d e r s the r e p r e s e n t a t i o n of d a t a  by a l o g - l o g r e l a t i o n s h i p to be the most s a t i s f a c t o r y method f o r e x p r e s s i n g b i o l o g i c a l d a t a .  Equations of t h i s  type are c o n s i d e r e d to have g r e a t e r p h y s i o l o g i c a l s i g n i f i c a n c e than the l i n e a r form (89)  and Dreyer  Y = a + b X used by M i l l e r and Bender  (3D.  As i n d i c a t e d i n F i g u r e I when the data f o r body n i t r o g e n and body water were p l o t t e d on a l o g - l o g g r i d , an  apparent  change or "break" o c c u r r e d i n both the W i s t a r and SpragueDawley s t r a i n s i n the r e g i o n of approximately 30 grams body weight.  Brody (2*+)  has d i s c u s s e d these changes i n the v a l u e  of the exponent "b", which take p l a c e i n growth c u r v e s , e x p e c i a l l y d u r i n g e a r l y growth.  F o r some i n s t a n c e s abrupt  changes of t h i s s o r t are known to be c o i n c i d e n t w i t h a w e l l d e f i n e d p h y s i o l o g i c a l event, such as the onset of puberty. I n the present study the change i n the s l o p e of the l i n e c o i n c i d e d w i t h the approximate  date o f weaning.  Because these  "breaks" o c c u r r e d , equations r e g r e s s i n g body n i t r o g e n to body water were c a l c u l a t e d by the method of l e a s t squares f o r the weight  ranges; b i r t h to 30 grams,  to. 100  grams, and  - 83 hO to 300 grams.  Equations were c a l c u l a t e d f o r the  hO - 100 gm. range s i n c e t h i s i s the weight range under c o n s i d e r a t i o n i n the M i l l e r and Bender (89) procedure. equations which were c a l c u l a t e d t o express t h i s are presented i n Table 16.  The -  relationship  I n a l l cases, Y r e p r e s e n t s the  m i l l i g r a m s o f body n i t r o g e n and X r e p r e s e n t s the grams o f body water. The f a c t t h a t the exponent "b" i s g r e a t e r than one i n a l l cases, i n d i c a t e s t h a t the i n c r e a s e o f n i t r o g e n i s proceeding a t a g r e a t e r r a t e than the i n c r e a s e o f water i n the animal body.  I n other words, the animal body i s becoming  p r o g r e s s i v e l y dehydrated  but a t a d e c l i n i n g r a t e .  The change  i n slope "b" over the weight ranges, b i r t h t o 30 gms. and hO - 100 gms. i n d i c a t e s t h a t the percentage  increase i n  n i t r o g e n r e l a t i v e t o the i n c r e a s e i n water i s g r e a t e r i n the younger r a t s than i n the o l d e r ones.  The s l i g h t d i s c r e p a n c i e s  which occur i n the v a l u e s o f b over the weight hO - 100 gms. and hO - 300 gms. may be accounted  ranges f o r by the  i n c r e a s e i n the standard e r r o r s o f estimate when the l a r g e r group I s c o n s i d e r e d .  T h i s e r r o r i s probably a s s o c i a t e d w i t h  the d i f f i c u l t i e s i n the d e t e r m i n a t i o n o f t o t a l body water i n these l a r g e r  animals.  A comparison was made o f the n i t r o g e n (N) v a l u e s c a l c u l a t e d from t h i s e x p o n e n t i a l r e g r e s s i o n e q u a t i o n and the n i t r o g e n (N) v a l u e s c a l c u l a t e d from the equations presented by M i l l e r and Bender (89) and by Dreyer  (3D.  Standard d e v i a t i o n s were  c a l c u l a t e d f o r the d i f f e r e n c e s between the N c a l c u l a t e d by  -  r  84-  TABLE XYI  R e g r e s s i o n Equations  Expressing  the R e l a t i o n s h i p o f Body  N i t r o g e n t o Body Water i n A l b i n o R a t s .  Rat Strain  Wister  N  Body Weight Range grams  r  +S  D  18  b i r t h - 3 0 Y = 6.43X ^  33  40-100  Y = 27.9X *  40  40-300  1.14 Y = 22.IX  1,  1  Sprague Dawley  Standard E r r o r o f C o e f f i c i e n t Estimate of C o r r e l a t i o n  Equation >, Y = aX  b i r t h - 3 0 Y = 10.2X *  13  40-100  Y = 26.6X '  20  40-300  Y = 24.4X  1  -S  R  LogY.logX  L  4.08  3.92  0.961  0 8  3.74  3.61  0.99^  8.48  7.82  0.994  4.10  3.94  O.998  3.00  2.91  O.996  5.48  5.19  0.996  ), 16  R  0 9  1 , 1 2  Q  - 85 -  these equations and  the a c t u a l N determined.  These f i n d i n g s  are r e p o r t e d i n Table XVII and i n d i c a t e that the N c o n t e n t s c a l c u l a t e d from the e x p o n e n t i a l e q u a t i o n r e l a t i n g body n i t r o g e n to body water are more a c c u r a t e than the v a l u e s obtained from the equations of M i l l e r and Bender (89) Breyer  (31)  which r e l a t e n i t r o g e n to water r a t i o w i t h  and age.  T "ABLE  Wfl  A Comparison of the N i t r o g e n Content o f W i s t a r Rats Determined C h e m i c a l l y w i t h N i t r o g e n Values C a l c u l a t e d from R e g r e s s i o n Equations Age Days  Sex  Body Composition  Nitrogen  •  Y=27.9X '  (1)  Y=2.92+0.02x MillerBender (2)  Devia tion mg  Nitro gen mg  Devia tion mg  Nitro gen mg  Devia tion mg .  81  2021 1833 1510 2755 2566 277+  100 132 50 239 260 150  222h 201+ 16+7 3005 2766 2990  103 +9 87 11 60 66  2021 1509 2307 266+ 1791 224-9  67 112 121 336 8 78  2221+ 1658 2517 2895 2061 21+2+  270 37 89 105 278 97  1  Water grams  Nitro gen mg  Nitro gen mg  31 32 36 36 +3 +3  M M M M M M  57.1 51.5 +1.5 75.7 67.9 73.+  2121 1965 1560 299+ 2826 292+  2202 1969 1560 2985 2655 2888  31 32 36  F'> F F F F F  57.1 +2.if 63.+ 72.+ 50.6 59.5  195+ 1621 2*+28 3000 1783 2327  2202 1597 2+65 28*f9 1932 2302  ?+3 8  +3  0 8  'A 0 9  171 36 2+8 2lf 37 1+9 25 110.1+ mg N  Standard d e v i a t i o n 1) where X = mg N, Y = gm H 0 2  2) where X = gmN/gm H 0 X 1 0 0 , X = age i n days 2  Calculated  165.2mg N  Y=3. +331+0.011+9X (Dreyer) (2)  1 3 1 . 8 mg N  BIBLIOGRAPHY  - 87 -  BIBLIOGRAPHY 1.  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