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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., University 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 Nutriti o n We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1957 ABSTRACT An attempt has been made to assess the n u t r i t i v e value of the proteins of a number of weed seeds which are considered as noxious under the Canada Feeding Stuffs Act, but which have been fed successfully to ruminants i n the form of heat processed refuse screenings. These weed seeds contain isothiocyanates which may or may not be toxic to animals but which were found to be unpalatable to the r a t . For this reason i t was found necessary to take two approachs to the problem of determining the n u t r i t i v e value of these weed seeds; a d i r e c t approach involving animal assays with rats and an i n d i r e c t approach involving assessment of the esse n t i a l amino acid content of these weed seeds. In carrying out these objectives a study of the properties of the isothiocyantes themselves and of the various methods for evaluation of protein q u a l i t y v,ere also undertaken. For the d i r e c t approach involving animal assays, an attempt was made to develop a procedure which would remove the isothiocyanates from samples of these weed seeds. A procedure involving auto-hydrolysis of the weed seed with water followed by extraction with 70% ethanol was evolved. This treatment reduced the ijsothiocyanate content of the weed seeds and rendered them palatable to the r a t . Using samples prepared i n thi s manner the proteins of a number of the weed seeds were assayed for their net protein u t i l i z a t i o n according 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 isothiocyanates would be successful, an i n d i r e c t approach was also taken for the evaluation of the proteins of weed seeds. A number of weed seeds were analysed, f o r their content of es s e n t i a l amino acids using the microbiological assay methods proposed by Barton - Wright. From this data, a chemical evaluation of the proteins was c a r r i e d out through computation of "essential amino acid.indices" and "chemical scores" according 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 evaluations indicated that although s l i g h t l y lower i n value the proteins of weed seeds compare favourably with those of soybean and linseed as plant proteins of moderately high b i o l o g i c a l value. In connection with the evaluation of proteins by the M i l l e r and Bender method a study was made of the body water -body nitrogen r e l a t i o n s h i p i n the U.B.C. colony of r a t s . The body water and body nitrogen content of 58 Wistar and 36 Sprague - Dawley rats were determined and regression equations c o r r e l a t i n g body nitrogen to body water were calculated. These investigations confirmed the findings of other workers that the nitrogen to water r a t i o varies with such constancy that the nitrogen content of an animal can be calculated from a knowledge of i t s water content and age or body weight. i i In presenting t h i s thesis i n p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by his representative. It i s understood •that copying or publication of t h i s thesis f o r f i n a n c i a l gain s h a l l not be. allowed without my written permission. Department of {^CVUSTUL/ ^/ej^y^aO The University of B r i t i s h Columbia, Vancouver Canada. Date ^6Uf /, t> 7 ACKHOW LEDGEMEN T The w r i t e r wishes to take t h i s opportunity to thank D r . B . A . Eagles , Dean of the Facul ty of 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 Science, fop his permission to undertake t h i s project and.' for the use of departmental f a c i l i t i e s . For his guidance, Interest and enthusiasm throughtout the course of this, s tudy, the author expresses her- s incere apprec ia t ion to D r . A . J . Wood!, Professor of Animal Science. An expression of grat i tude i s a lso extended1 to* D r . Kifcts, f or his c r i t i c i s m and many h e l p f u l suggestions. The f i n a n c i a l assistance provided by funds from; the Ganada Department of A g r i c u l t u r e and a grant from the Pres ident 's Committee on Research; are g r a t e f u l l y acknowledged. The w r i t e r a lso wishes to express her thanks to the Plant Products D i v i s i o n , Canada Department of A g r i c u l t u r e fo r the ir assistance i n supplying the samples of weed seeds used i n t h i s s tudy. v i TABLE OF COMTEMTS PAGE I. Introduction ....................... 1 A. Seed Samples ...•..».•.•..•••••••»..•... 3 II. Attempt to Remove IsoThloey/amtes; from Represent a tive Samples of Weed; Seedis ..••....» 5 A. Introduction ••••••••••••• 5 B. Literature Review • 5 (a) isothiocyanates »•••••••••••••••• 5 (b) t o x i c i t y 11 C. Experimental ••••»••»»•» ........... 17 (a) Determination of isothiocyanates . 17 (b) Extraction procedure 20 I I I . Evaluation of the Protein Quality of Representative Samples of Weed Seeds . 28 A. Introduction 28 B. Literature Review •••«•• ...... 28 (a) Proteins ....... .... 28 (b) Protein Evaluation Methods 31 1. Protein E f f i c i e n c y Ratio ... 31 2. Rat Repletion Method 32 3. l i t r o g e n Balance ••••••••••• 33 i . Protein Minima ......... 3h i i . B i o l o g i c a l Value 35 h» Tissue Proteins 37 5. Microbiological •••.••••••»• 3® (c) Plant Proteins versus Animal Proteins hO G. Experimental ••••••••• hO (a) The l e t Protein U t i l i z a t i o n of a Number of Weed Seed Samples , *0 1. Assay Method • MD i . Animals • hi i i . Diets ... hi i i i . Procedure «•••«• h2 2. Calculations *+3 3. Results hh i i i PAGE k. 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 IV. The Essential Amino Acid Content of Certain Noxious Weed Seeds • • • • • • • • • • • 50 A. Introduction 50 B. Experimental 52 (a) The Analysis of the Essential Acid-Content 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 . . . . . ® . . . . . . e . 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 let ProteinnUtilization Values . . . . . . 62 V. Summary • » • • . • • • • • • » . . ® « . . . • • » < » . • • • 66 ¥1. Appendix 69 I. The Nitrogen : Water Relationship in Albino Rats 70 (a) Introduction . . . . . . . . . . . . . . . . . . . . 70 (b) Experimental Animals . . . . . . . . . . . . 71 (c) Carcass Analysis 72 (d) Results . . . . • . . • • • . . • • • • » . . 7k (e) Calculations » • • • • . » • • . • • • • • • • • • • 79 VII. Bibliography 87 i v LIST OF TABLES TABLE PAGE I Isothiocyanate Determinations •••»••••••• » 19 II Change i n Protein Content of Samples During Extraction Process • .. 23 I I I Weight Losses Associated with the Extraction Procedures .. ••••••«•• 2k IT Protein Content of Extracted Samples .... k2 V The Net Protein U t i l i z a t i o n of a Number of Proteins k5 VI The Amino Acid Content of the Proteins of Certain Noxious Weed Seeds; and Certain Standard Protein Sources 55 VII Reported Values f o r the Amino Acid Composition of a Number of Proteins ••••••»••••••••••••»»•• 56 VIII Computation of the Modified E s s e n t i a l Amino Acid Index ... 61 IX E s s e n t i a l Amino Acid Indices and Chemical Scores f o r the Proteins of Weed Seeds 63 X E s s e n t i a l Amino Acid Indices andi Chemical Scores for a Number of Proteins ••••••••»..••• 6k XI Protein and Fat Composition of a Number of Weed Seed Samples ..... ••«•••••••. • 69 XII : Body Weight - Body Water - Body Nitrogen Relationship i n Female Wistar Rats •••••••••••• 75 XIII Body Weight - Body Water r Body Nitrogen Relationship i n Male Wistar Rats 76 XIV Body Weight - Body Water - Body Nitrogen Relationship i n Female Sprague - Dawley Rats 77 XV Body Weight - Body Water - Body Nitrogen Relationship i n Male Sprague - Dawley Rats .... 78 XVI Regression Equations Expressing the Relationship of Body Nitrogen to Body Water i n Albino Rats XVII A Comparison of the Nitrogen Content of Wistar Rats Determined Chemically with Nitrogen Values Calculated from Regression Equations ....••.»•• 86 v INTRODUCTION THE NDTRITIVE VALUE OF CERTAIN NOXIOUS WEED SEEDS INTRODUCTION For the past f i v e years heat processed refuse screenings have been used successfully as a ruminant animal feed i n B r i t i s h Columbia and several of the western states. To date, some 60,000 tons of the processed material has been fed successfully. Under the present Canada Feeding St u f f s Act c e r t a i n of the weed seeds i n refuse screenings are considered as noxious and a number of regulations control t h e i r sale and use as a commercial l i v e s t o c k feed. There i s adequate evidence (5,53-76) to show that many of these weed seeds contain isothiocyanates which may or may not be toxic when consumed by the domestic animals. Addition-a l information on the t o x i c i t y and n u t r i t i v e value of these weed seeds i s needed before suitable amendments can be made to the Canada Feeding Stuffs Act. The work to be reported herein was undertaken to provide a quantitative assessment of the n u t r i t i v e value of c e r t a i n of these weed seeds. Work on the t o x i c i t y of the material i s proceeding elsewhere, under the supervision of the Canada Department of Agriculture. The weed seeds under study from the family §rae±fer±a@ are characterized by the presence of the sharp pungent flavour-ed isothiocyanates. Reports have also appeared i n the l i t e r a t u r e on the presence of possible toxic factors i n a number of the weed seeds and i n other members of thi s family, (rapeseed, t u r n i p s ) . I t was expected that an attempt might - 2 -have to be made to remove these palatability and/or toxic factors before animal feeding experiments could be carried 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 rat there was complete refusal by the animals to accept the diets. The weight losses and resulting death of the animals could be accounted for on the basis of the inanition associated with the feed refusal rather than on the basis of toxicity, A similar pseudo-toxicity has been noted i n this laboratory in diets containing more than 7 parts per million 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. It was necessary to remove these taste factors before any attempt could be made to determine the nutritive value of the weed seeds by biological methods. Since there was no assurance that the removal of the isothiocyanates would be successful, methods for evaluation of the nutritive value of a protein not involving animals were sought. To avoid the problem of palatability i n animals, therefore, an attempt was made to assess the nutritive value of the various weed seeds by a micro-biological method. The procedure proposed by Anderson and Williams (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 - 3 -response of 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 for the assessment of growth response. This method has since been c r i t i z e d by Sheffner (118) as i t was found that the value obtained with these methods d i d not corre la te we l l with the b i o l o g i c a l value of the pro te in as determined by animal t e s t s . In recent years a number of methods have been proposed which attempt to corre la te the n u t r i t i v e value of pro te in with t h e i r content of e s s e n t i a l amino a c i d s . In order to assess the n u t r i t i v e value of the weed seeds by th i s method, i t was necessary to analyse the weeds seeds for t h e i r content of e s s e n t i a l amino a c i d s . The problem of determining the n u t r i t i v e value of the weed seed, therefore , resolved i t s e l f i n t o three p a r t s : I An attempt to extract the offending isothiocyanate or taste factors from samples of the weed seeds. I I An evaluat ion of the pro te in q u a l i t y of representat ive samples of weed seeds by an es tabl i shed b i o l o g i c a l assay procedure. For these assays samples^which are palatable for the laboratory r a t , were prepared by the e x t r a c t i o n procedure evolved i n Sect ion I . I l l An analys i s of the e s s e n t i a l amino ac id content of a number of weed seeds. A chemical evaluat ion of the proteins can then be ca lcu la ted from t h e i r amino a c i d composit ion. WEED SEED SAMPLES: The samples of weed seeds used i n the present studies were generously suppl ied by the Plant Products D i v i s i o n , _1+ -Production Service of the Dominion Department of Ag r i c u l t u r e . The common and s c i e n t i f i c names (99) of these weed seeds are presented i n the following l i s t . Pertinent data on the protein and f a t content of these seeds are presented i n the Appendix I. Common Name White mustard Wild mustard Brown mustard False Flax Tumble mustard S c i e n t i f i c Name Brassica h i r t a Moench or Brassica alba Boiss or Sinapls alba L Brassica kaber (DC) L.C. Wheeler or Brassica arvensis Ktze or Sinapls arvensiT" L Brassica .juncea (L) Coss Camellna microcarpa Andrz Sisymbrium altlssimum L Norta al t i s s l m a B r i t t o n Stinkweed Thlaspl arvense L ATTEMPT TO REMOVE ISOTHIOCYANATES FROM REPRESENTATIVE SAMPLES OF WEED SEEDS - 5 -II ATTEMPT TO REMOVE ISOTHIOCYANATES FROM REPRESENTATIVE SAMPLES OF WEED SEEDS A. INTRODUCTION: In preliminary experiments, when the ground or f a t extracted samples of weed seeds were incorporated into com-plete r a t i o n s , they were not accepted by the r a t . This r e f u s a l was attributed to a p a l a t a b i l i t y f a c t o r which i s known to be associated with the presence of mustard o i l s or isothiocyanates i n these seeds. I f these weed seeds are to be assessed f o r th 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 to remove these taste f a c t o r s . However, before t h i s was possible, a study of the occurence and properties of the isothiocyanates themselves seemed advisable. A method f o r the determination of the isothiocyanate content of the various weed seeds was also sought. Determinations of the isothiocyanate content of a number of the weed seeds i s desirable i n order to obtain basic values against which attempts to remove the isothiocyanate can be assessed. With these f a c t s , at hand, a procedure was evolved to remove the isothiocyanates from the weed seeds i n order that these samples could be fed to r a t s fo r b i o l o g i c a l evaluation t e s t s . B. LITERATURE REVIEW (a) isothiocyanates The presence of a number of v o l a t i l e isothiocyanates i n the tissues, and e s p e c i a l l y the seeds of the Cr u c i f e r a and other plants, has been established. S i n i g r i n , the precursor - 6 -of the isothiocyanate of black mustard (Brassica nigra (L) Koch) was is o l a t e d i n 1839,(33), and S i n a l b i n from white mustard (Brassica alba Boiss) i n 1831, (33). U n t i l recently no proof of the structure of these various Isothiocyanates had been offered. In 1953, Kjaer and co-workers (5,53-76) began an extensive series of studies on the occurence, i s o l a t i o n , synthesis and i d e n t i f i c a t i o n of the isothiocyanates. Using a paper chromatographic procedure (73) f o r the separation and i d e n t i f i c a t i o n of the thiourea derivatives of the .isothiocyanates, they investigated the seeds and the fresh parts of various plants f o r t h e i r content of isothiocyanates. Included i n the study were numerous seed samples belonging to the Cruciferae, Resedaceae,Tropaelaceae,Capparldaceae. Phytolaccuceae and Euphorbiaceae. The isothiocyanates occur i n nature as thioglucosides, that i s i n connection with a molecule of glucose and the bisulphate ion. These n a t u r a l l y occurlng isothiocyanate containing glucosides can be c l a s s i f i e d into two groups, according to whether or not t h e i r isothiocyanates are v o l a t i l e with steam. Within the former group, the occurence of a l l y l , (+) - sec-butyl, benzyl and B-phenylethyl isothiocyanates. has been established f o r some time. Recently Kjaer et a l (5,53-76) have demonstrated the widespread occurence of addi t i o n a l isothiocyanates. including>butenyl; iso-propyl; methyl; the *f-methythio - and Y-methylsul phony 1 form s ; of butyl and propyl isothiocyanate. and many others. - 7 -In many Cruciferae there i s an enzyme known as myrosinase (myrosin or sinigrinase) which hydrolyzes only the natural mustard o i l thioglucosides. Myrosinase has been shown to consist of two enzymes, a myrosulfatase which s p l i t s o f f bisulphate (usually as the potassium s a l t ) and a thioglucosidasse which s p l i t s the thioglucoside linkage. The following scheme shows how the hydrolysis of s i n i g r i n (the glucoside of brown mustard) may proceed - C6HUO5 C 3 % -S i n i g r i n N = C 0 - SC3K myrosulfatase H 0 2 C H N = a ,s -'OH + KH SO. merosinigrin thioglucosidase + H 2 0 CH = CH - CH_ - N = C =? S + C,H 0 2 2 6 12 a l l y l i s o t h l o c y a n a t e glucose U n t i l recently, the accepted structure f o r the myronate ion was that proposed by Gadamer i n 1897 (33,^9) i n which the isothiocyanate i s linked to D-glucose and the sulfate anion. - 8 -(structure I) R-N = c ' 6 1 1 5 ^oso3~ However, i n 1956 E t t l i n g e r and Lundeen (33) demonstrated a revised s t r u c t u r a l expression f o r the mustard o i l glucosides (structure II) and propose that the mustard o i l s (III) are formed i n nature by an enzyme-actuated Lessen re-arrangement (23). The complete formulation of s i n i g r i n as a B-glucopyranoside i n presented structure IV. R - C - SC.HO_. || 6 1 1 * R - N = C = S N - OSO-j~ II I I I myronate ion isothiocyanate CH = CH-CH -C 2 2 s o 2 o H S-C — I HCOH I HOCH I HCOH I HC I CH OH 2 0 IV s i n i g r i n Kjaer (53) noted the chemical s i m i l a r i t i e s between na t u r a l l y occuring isothiocyanates and the common < amino - 9 -acids and suggested a possible biochemical r e l a t i o n s h i p . E t t l i n g e r and Lundeen ( 5 3 ) have noted the resemblance i n configurations of amino acids and mustard o i l glucosides (compare tyrosine and glucosinalbate). Since isoleucine and natural (+) 2 - b u t y l isothiocyanate, belong to the same stereochemical series they have suggested that the correspond-ing glucoside could, presumably, be related through (+) 2 methyl butyric a c i d . During the course of t h e i r investigations of n a t u r a l l y occuring isothiocyanates, Kjaer and workers (5>55>57»58,59> 68,75) established the following, which i s of i n t e r e s t to the present study of the weed seeds. The seeds of brown mustard (Brassica .juncea (L) Coss) and stinkweed (Thlaspi  arvense L) were found to contain a l l v l i s o t h i o c v a n a t e (5)« Kjaer et 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 isothiocyanates i n enzyme treated rape seeds. (Brassica napus L) proving the main constituent to be 3-butenyl-lsothiocyanate (VI). However, as these series of investigations continued a d d i t i o n a l n a t u r a l l y occuring isothiocyanates made possible the establishment of a complete picture of the mustard o i l of rapeseed (55). Six i n d i v i d u a l glucosides are present, three of which occur i n very small amounts. They are: glucoiberin (or a c l o s e l y a l l i e d 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 (traces), glucorapin, glucobrassicanapin and glucorasturtin (traces) y i e l d i n g (-) 3 methyl-sulphinylpropyl (XI), mustard o i l (or a related species), (-) - 5 v i n y l - 2 - o x a z o l i d i n e t h i o n e (X), p-hydroxybenzyl, (VIII), 3-butenyl, (VI), *f-pentenyl (VII), - 10 -and 2-phenyelethyl (IX) Isothiocyanate, respectively, on enzymatic hydrolysis. CH.=CHGH0NCS 2 * — CH 2 — NH a l l y l isothiocyanate GH_ = CH - GH C = S CH 2 = CHCH2CH2NCS X ( * ) -5-Vinyl-2-ozazolidinethione VI 3-butenyl isothiocyanate C H 1.3 0 4—s CH =^HCH0CHJ3H0NCS CH 0 2 2 2 2 -2 VII ' 2 H--pentenyl isothiocyanate ^ HO \_J> CH2N =C=S XI (•) 3 methylsulphinylpropyl isothiocyanate VIII P-hydroxybenzyl isothiocyanate CH^-CH0-N=C=S <^_^> CH2-CH2N=C=S CH^ IX XII 2-phenylethyl isothiocyanate iso-propyl isothiocyanate CH I 3 0 « - S — NCS XIII - 11 -( S t r u c t u r e XIII on p r e c e e d i n g page ) XIII ( - ) - 10 - methylsulphinyldeeyl Isothiocyanate Besides traces of the v o l a t i l e i s j2-propyl-isothiocyanate, (XII) (59) seeds of white mustard (Brassica alba Boiss) were found to contain a non-volatile isothiocyanate i d e n t i f i e d as p-hydroxybenzvl-isothiocvanate (VIII) (75) present as the aglucone of the glucoside s i n a l b i n . (53-8). Seeds of the cruciferous species Camelina microcarpa Andiz, commonly known as False f l a x contain two isothiocyanate glucosides. (68). The chief constituent, glucocamelinin has been i d e n t i f i e d as a glucoside containing ( - ) - 10 - methylsulphinyldeeyl isothiocyanate (XIII) as the aglucone. At t h i s time, no report has been presented of the isothiocyanate content of Tumble mustard (Sisymbrium altlssimum L ) . However, two species of the Sisymbrium genus, S. sophia L and S. strictissimum L have been reported to contain glucosides of a l l y l , and sec-butyl and iso-propvl (traces only) i s o t h i o -cyanates, respectively (59)• (b) To x i c i t y A number of the weed seeds under study belong to the genus Brassica of the family Gruciferae . The seeds of rape (Brassica  nanus L) have been u t i l i z e d for animal feeds for a number of years. A comparison of the seeds of rape and mustard i s , therefore, warranted. - 12 -In recent years the n u t r i t i v e value of rapeseed has received considerable study mainly i n connection with evidence presented that rapeseed oilmeal contains one or more toxic f a c t o r s . In 1901 Sjollema (119) i s o l a t e d a C isothiocyanate, 5 which he claimed to have i d e n t i f i e d as crotonylisothiocyanate, as a constituent of the esse n t i a l o i l f r a c t i o n of 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 . In 1920, Viehover (123) et a l found isothiocyanates 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 rabb i t s . In 19*+1, Kennedy and Purges, (hk, h5, 52) reported hyperplastic changes i n the thyroid, and changes i n the p i t u i t a r y s imilar to those following thyroidectomy, as a r e s u l t of feeding Brassica seeds to r a t s . Delayed maturation of ovaries i n immature rats was also reported. Evidence based on rats (^, *f5, 52), rabbits (123), chicks (30, 77, 122), turkey poults (17), swine (100) and possibly c a t t l e (11) have suggested that some toxic factor (or factors) exist i n rapeseed oilmeal. These toxic factors usually mani-f e s t themselves i n the form of a thyroid hyperplasia (goitre) and growth depression. In addition there i s suspicion on the part of a number of workers (12, 27) that more than one toxic factor may be involved since extensive kidney, l i v e r , adrenal and growth Involvements have arisen 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 nor, i n f a c t , to the Brassica family, nor does the evidence indicate that the same factor i s involved i n a l l members of the Brassica - 13 -family. Soybean and linseed have also received i n v e s t i g a t i o n i n regard to possible goitrogenic f a c t o r s . (26, 82). A number of reports have been presented which d i r e c t attention to the possible e f f e c t s of these 'goitrogens' d i r e c t l y on tissues and Independently of thyroid function. Bach (11) found a d e f i n i t e retardation of tissue oxidation of ascorbic acid when either 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 present i n concentrations of 5.7 x 10.hM. Benda (11) reported that r e s p i r a t i o n i n guinea pig l i v e r s l i c e s was depressed by 5 0 percent by small concentrations (10~2M) of a l l y l i s o t h i o c y a n a t e , and Berg and F l i c h s t e i n (11) reported i n h i b i t i o n of dehydrogenation reactions by a l l y l i s o t h i o c y a n a t e . B a c t e r i o l o g i c a l investigations ( 5 6 ) have indicated a very pronounced e f f e c t of both cherroline Of- methylsulphonyl-propyl-isothiocyanate) and erysoline (y-methylsulphonylbutyl isothiocyanate) on a rather large s e l e c t i o n of pathogenic bacteria (plant pathogenic bacteria) 0+3). In one of the reports • x'; Kjaer et a l , (76) mentioned that a number of these isothiocyanates w i l l be tested for t h e i r a n t i -thyroid a c t i v i t y . However, no report on this i n v e s t i g a t i o n has been found except for the statement i n paper No. II ( 5 ) of this series that animal tests have indicated p r a c t i c a l l y no antithyroid a c t i v i t y of the n a t u r a l l y occuring lsothlocavanates. Marine et a l (81) found no goitrogenic a c t i v i t y when a l l y l , e t h y l or phenyl-isothlocyanates were fed to r a b b i t s . To date, the evidence regarding the t o x i c i t y of i s o -thiocyanates i s c o n f l i c t i n g . No conclusions concerning the - ih -t o x i c i t y of the isothiocyanates themselves can be made. Recently, however, i t has been suggested that another compound may be responsible f o r the antithyroid a c t i v i t y found i n rapeseed and other meals. Astwood (6) states that up to 19^9 only two antithyroid compounds have been found i n plants. These were thiourea and 5>5 - dimethyl - 2 - thioxazolidone found i n Laburnum angyroides L (Golden chain) and Conringia  o r i e n t a l i s (Hare's ear mustard). ( ^9) . Recently, Astwood (6) found another goitrogenic substance i n rutabaga, which was l a t e r shown to be L -5 v l n y l - 2 - t h i o x a z o l i d o n e . Astwood (6) presented some evidence that might indicate that the thioxazolidone i s formed from some precursor by enzymatic action. Astwood (6) found that when the root OP.' seed of the rutabaga was f i r s t b o i led i n water the antithyroid a c t i v i t y was destroyed and no thio-oxazolidone could be i s o l a t e d . This suggests that the formation of this compound may involve an enzymatic reaction. P i t t Rivers (107) proposed that the i s o -thiocyanates and thio-oxazolidones may be Interrelated, the thio-oxazolidone a r i s i n g upon oxidation and r i n g closure. Kjaer, (63) i n testing the hypothesis, found that glucoconringiin (XIV) the glucoside of the c r u c i f e r Conringia  o r i e n t a l i s (L) Andrez, was ( i n f a c t , ) the natural precursor of 5 ,5-dimethyl -2-oxazoldinethione (XVI). The glycoside, glucoconringiin, affords glucose, sulphuric aeid and 2-hydroxy-2-methyl-propyl Isothiocyanates (XV) on enzymatic hydrolysis. The mustard o i l c y c l i s e s spontaneously to 5»5-dimethyl -2-oxazolidinethione as presented by the following scheme. - 15 -CH„ CH -C-CH -N=C 3 2 | — 0 — OH H OH I I I I S -CH-C-C-C-C-CH -OH I I I ) 2 H OH HH o - s o 2 - o - K myrosinase XIV glucoconringiin CH e y e l i s a t i o n C - NH 1 3 l l CH -C- CH„-N=C=S > CH -C C = S 3 1 2 / V OH CH 3 XV XVI 2-hydroxy-2-methyl-propyl 5 , 5-dimethyl -2-oxazolidine-isothiocvanate thione Because the reaction i s spontaneous the 2-hydroxy-2-methyl-propyl isothiocyanate has not been i s o l a t e d . Evidence i s a vailable f o r the existence of additional 2-oxazolidine-thione producing glucos:ides i n plants. (6). Enzymatic hydrolysis of the 2-hydroxy-3-butenyl isothiocyanate glucoside, p r o g o i t r i n (glukorapiferin) i s responsible for the production of the goitrogenic ( - ) - 5-vinyl - 2-oxazolidinethione, previously i s o l a t e d from seeds and fresh parts of various Brassica species including rape, but excluding the three v a r i e t i e s of mustard - 16 -tested ( 6 ) . Although established as goitrogenic, controversies exi s t as to whether 2-oxazolidinethione are growth depressants ( 11 ) . Wetter (125) suggested another route by which toxic compounds might a r i s e from the isothiocyanates. The g o i t r o -genic? thioureas could be derived from the reaction of i s o t h i o -cyanates with ammonia, the ammonia a r i s i n g from various sources i n the animal body. For example, ammonia i s formed i n the in t e s t i n e from deamination of amino acids by micro-organisms. To date, no experimental evidence has been presented i n support of this postulate. Attempts have been made by a number of workers to counter-act or remove the toxic or goitrogenic f a c t o r . A degree of 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 following treatments with rapeseed: cooking (12 1 *) , steaming ( 11 , 81), iodide (108 ,11 , 30) (79 ) , protamone (17) steaming plus a n t i b i o t i c s ( 11 ) , water extraction (12 , 27 , 30 , *+2) , (8 l ) , and l i m i t i n g the amount of rapeseed oil-meal i n the r a t i o n (11 , 3 0 ) . The majority of the research suggests that l i m i t a t i o n of use i s the only s a t i s f a c t o r y and practicable method yet available to counteract the t o t a l e f f e c t of the rapeseed oil-meal f a c t o r s . ( 1 1 ) . - 17 -c » EXPERIMENTAL: (a) Determination of isothiocyanates. A number of methods have been described f o r the quanti-ta t i v e estimation of mustard o i l s i n seeds. These methods have been reviewed recently by Andre (k). The commonly employed methods involve removal of the mustard o i l by steam d i s t i l l a t i o n , with the formation of a thiourea when the d i s t i l l a t e i s c o l l e c t e d i n ammonia. The thiourea may be ex-tracted and/or estimated by various means. The 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 sulphur. (27 , 102, 121) . Recently, Wetter (12M-) has modified the argen-timetric method fo r estimation of mustard o i l s ; the p r i n c i p a l modification being made i n the treatment p r i o r to steam d i s t i l l a t i o n . Indirect determination of isothiocyanates based on the sulphates produced from the glucosides by r e s o l u t i o n reaction (115) or based on the absorption of iodine by the seed f i l t r a t e s (83) have also been used. Kjaer and co-workers (73) have developed a paper chromatographic technique f o r demonstrating the presence of isothiocyanate i n various plant tissues and have developed an* u l t r a v i o l e t spectrophotometrie method (59) f o r a quantitative evaluation of the Isothiocyanates as thioureas. This procedure takes into account the s p e c i f i c isothiocyanate i n contrast to the other methods i n which the v o l a t i l e mustard o i l i s usually calculated as a l l y l i s o t h i o c y a n a t e . Using the method proposed by Kjaer (59)» a number of the weed seeds were assayed f o r their - 18 -content of v o l a t i l e isothiocyanates. These determinations were c a r r i e d out to obtain basic values against which attempts to remove the isothiocyanates could be assessed. The r e s u l t s are presented i n Table I. The values obtained f o r white mustard, stinkweed and brown mustard are comparable to those reported by Kjaer (when the variations i n procedure are taken into accountt he accuracy of methodis reported to be about $P%). The values f o r rapeseed are low compared to reported values but Wetter (12*f) has indicated that the isothiocyanate:: content of rapeseed varies considerably depending on the source and v a r i e t y . Kjaer, ejt aj, (58) reports that i n d u s t r i a l l y produced rapeseed cakes showed great variations i n their content, apparently dependent on the conditions employed during their manufacture. TAB IS I ISOTHIOCYANATE DETERMINATIONS Seed Isothiocyanate mg/100 gm. Published Reference After f a t - f r e e values number extraction sample mg/100 gm. with 70% f a t - f r e e ethanol sample ~ Argentine Rapeseed 3-butenyl. 29,0 lOOmg/lOOgm (58) 0.3 to 1.3% (121+) 21.2 White Mustard iso-propyl 8.3 3 ( 5 9 ) 2.0 Stinkweed a l l y l 286 327 (5) Brown Mustard a l l y l 6lh 518 (5) Wild Mustard a l l y l * 5 A Refuse screenings U.B.C. Ration No-72 a l l y l * 27.0° * calculated as a l l y l isothiocyanate since type of isjojihioeyanate not yet 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 to remove the 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 are o n l y s l i g h t l y s o l u b l e i n water but are s o l u b l e i n e t h e r , a l c o h o l and other o r g a n i c s o l v e n t s . (^7>). I n order to determine 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 r e -moving these i s o t h i o c y a n a t e s from the seed, a number of 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 separate samples of the raw ground seeds i n c l u d e d the f o l l o w i n g : B o i l i n g Po^nt d i e h l o r e t h y l e n e - 37.0°C. e t h y l e t h e r 3^«6°C. petroleum ether 20o-l*O°C. acetone 56.1°G. a l c o h o l - e t h a n o l 78«5°0. carbon t e t r a c h l o r i d e 76«8°C. water 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 , a l l these treatments met w i t h complete f a i l u r e . The f a i l u r e to remove the o f f e n d i n g i s o t h i o c y a n a t e s can probably be a t t r i b u t e d to the f a c t that they are present i n the weed seeds i n conjugated f o r m . When the samples were h y d r o l y s e d w i t h water a t 37°C. then steam d i s t i l l e d there was some l o w e r i n g of the i s o -t h i o c y a n a t e content 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 of procedures i n v o l v i n g s teaming, c o o k i n g , or water e x t r a c t i o n have been proposed i n an attempt t o remove the g o i t r o g e n i c f a c t o r from these seeds. These have met w i t h v a r y i n g degrees of s u c c e s s . ' However, i t was f e l t t h a t t h i s - 21 -rigourous treatment might lead to protein damage and hence preclude a v a l i d assessment of the b i o l o g i c a l value of the protein. After many exploratory experiments, the following extraction procedure was envolved. 1. the ground seed samples were allowed to steep at 37° -hO°G. for 3 to h hours (Holly and Sandberg). (dependent upon the hydrolytic a c t i v i t i e s of the myrosinase present i n the seeds). 2. the sample was then dried i n a tunnel dryer at 80 -8|?0C. to constant weight (2*+ - *+8 hours). 3. by mixing i n a Waring blender the dried sample was extracted three times with 70% ethanol (by volume, s p e c i f i c gravity 0.8900). The excess alcohol was removed between each extraction by spinning i n a basket centrifuge. h, the alcohol extracted sample was dried i n a tunnel dryer at 80 - 85° to a constant weight. 5. the dried sample was then placed i n a macro-soxhlet and extracted 8 - 1 0 times with ethylene d i c h l o r i d e to remove f a t . 6 . a f t e r defatting, the samples were allowed to a i r dry at room temperature to remove the solvent. 7. the solvent-free meal was then ground i n a hammer m i l l to pass a No 20 mesh seive. Samples of white mustard, iBra'ssj-'Ca alba B o i s s ) , stinkweed, (Thlaspiarviense L), rapeseed (Brassica, napus L ) . - 22 -soybean (Glycine soya)» and lineseed (Linomusitalissium), were extracted by the preceeding method. White mustard and stinkweed were selected as representative samples of the weed seeds. As a member of the Cruciferae family, rapeseed was chosen f o r comparative purposes. Lineseed and soybean are established as plant proteins of good q u a l i t y and were chosen for comparative purposes. Table II presents a picture of the change of nitrogen content during the process. As indicated by the nitrogen determinations c a r r i e d out on the extract, l i t t l e nitrogen ( 0 . 5 - 0 .9 gms per 100 gms of raw ground seed) was l o s t during the extraction procedure with alcohol. Table III indicates the losses that occurred during the extraction procedure. No s p e c i a l attempts were made to make the procedure quantitative and so the major losses (except during f a t extraction) may be accounted f o r through mechanical losses. Organoleptic tests of the weed seed meals prepared by this method revealed a great reduction i n the b i t t e r flavoured constituents. Isothiocyanate determinations on two of the prepared meals indicate some reduction i n the isothiocyanate l e v e l s but can not be considered to be too conclusive i n t h i s respect since 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 at low concentration 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 analysis of this aspect of the problem were planned but this was not the major problem under study i n the present i n v e s t i g a t i o n . Of major concern, the prepared meals now proved to be acceptable - 23 -TABLE II CHANGE IN PROTEIN CONTENT OF SAMPLES DURING EXTRACTION PROCESS % Protein (NX 6.2?) Seed Ground Seed Extracted with 70% Ethanol 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 III WEIGHT LOSSES ASSOCIATED WITH THE EXTRACTION PROCEDURES Weight Weight afte r Weight afte r Weight a f t e r F i n a l of auto- Ethanol Fat Y i e l d Seed Sample Ground hydrolysis Extraction E x t r a c t i o n Sample with water with Ethylene - d i c h l o r i d e  gm gm per cent gm per cent gm per cent per cent Loss Loss Loss White Mustard 1800 I693 5,9 1211 2^.3 800 33 .9 U-7.0 Stinkweed 2000 I89I 5 A 1517 15.7 969 36.2 51.0 Rapeseed 1800 1600 11.1 1V00 12.5 815 ^1.7 *+5*2 Linseed 2000 1826 8.7 M 2 22.7 89I 36.9 M+.5 Soybean 2000 1800 10.0 1527 15.2 1275 16.5 63.8 _ 25 - . to the r a t s . I t i s known that the 70% ethanol removes a c e r t a i n portion of the weedseed protein (probably from the prolamine f r a c t i o n ) . In f a c t , i t may be that the isothiocyanates are conjugated i n some manner with the prolamins and hence an explanation may be provided f o r the successful use of 70% ethanol as an extractant, F r o l i c h (*+2) has reported that a considerable amount of the toxic p r i n c i p l e i n rapeseed can be extracted with 70% ethanol. I t i s also known that denaturation of the protein w i l l occur during t h i s procedure, Beckel, B u l l and Hopper, ( 1 0 ) , working with soybean, set up the following quantitative expression of the degree of denaturation by heat and moisture using water d i s p e r s i b i l i t y of the nitrogen of the meal as a c r i t e r i o n . % % water d i s p e r s i b l e N - non-% denaturation =* 100 1 - i n treated 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 - non-protein N The extent of denaturation by heat and moisture i s dependent on the r e l a t i v e combination of these two f a c t o r s . Data of these workers indicate that a c r i t i c a l temperature probably exists at each r e l a t i v e humidity below which very l i t t l e denaturation takes place, but above which denaturation i s r a p i d . With soybean, 26% denaturation occured a f t e r heating f o r 25 hours at 80°C. and 100% r e l a t i v e humidity. However, Evans and St. John, ( 3 ^ ) , i n studying the e f f e c t of denaturation on the s o l u b i l i t y of soybean i n various - 26 -solvents showed that the percentage of t o t a l nitrogen soluble i n 70% ethanol decreased with the denaturatlon of the protein (the percentage of non-soluble nitrogen increased). On the bases of the above report i t seems safe to conclude that the percentage of the protein extracted by 70% ethanol should be small (3-5% of the i n i t i a l ground seed). Next to heat i n importance as a denaturating agent i n the processing of soybeans or other plant proteins i s the choice of solvent. Markley (82) has reviewed a number of studies carried out on solvent extraction of soybeans. These studies permit the conclusion that low moisture content and a solvent with a low b o i l i n g point are of primary importance i n a l l f a t extraction procedures tested. Ethanol extraction has been shown to improve the flavour and colour of soybean f l o u r . The use of ethylene d i c h l o r i d e as a f a t solvent has been c r i t i z e d by a number of workers. However, the decrease i n n u t r i t i v e value which occurs i s mainly with animal proteins (28). T o x i c i t y i n trichloroethylene extracted soybean meal has been reported ( 1 0 6 ) . Depending on the severity and time of heating the heat treatment of proteins r e s u l t s i n changes to t h e i r chemical properties, p a r t i c u l a r l y i n t h e i r s o l u b i l i t i e s . These chemical changes are associated with changes i n n u t r i t i o n a l a v a i l a b i l i t y . The n u t r i t i o n a l e f f e c t of heating proteins i s generally a depressing one, and the s e n s i t i v i t y to heat damage varies from the extreme s e n s i t i v i t y of the proteins - 27 -of milk to the appreciable resistance of the proteins of 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 value by destroying a tr y p s i n i n h i b i t o r and thus increasing 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 for the b i o l o g i c a l quality of a protein are meaningless unless they are considered against a background of protein metabolism i t s e l f and the methods which are available for protein evaluation. For this reason, a number of assay methods f o r the b i o l o g i c a l evaluation of proteins w i l l be considered before dealing with the evaluation of the weed seed protein themselves. B f LITERATURE REVIEW (a) Protein The n u t r i t i v e value of a feedingstuff can be determined only by an i d e n t i f i c a t i o n of the contained nutrients, their quantitative proportions i n the feed and their a v a i l a b i l i t y to man or animal, f o r whom the feed i s intended. In determining the n u t r i t i v e value of a feedstuff, a l l these factors should be considered. However, since Boussingault (l 1*) f i r s t analysed food and animal feeds for their nitrogen content, i n f e r r i n g from the r e l a t i v e nitrogen content the r e l a t i v e n u t r i t i v e value of the food, the protein content of such materials has received f i r s t consideration i n the determination of the n u t r i t i v e value of a feedstuff. Proteins are synthesized from their constituent amino acids. In the plants, n i t r a t e s and ammonium s a l t s are used as the i n i t i a l nitrogenous compounds. In the case of - 29 -animals, however, the constituent amino acids must be a v a i l -able i n the d i e t , with the exception of some that can be ;•- 4/ synthesized i n the body from simpler compounds. In animal feeding, therefore, plant products are valued f o r t h e i r content of available protein as well as f o r t h e i r energy content. The presently held concept of protein metabolism i s one of a dynamic equilibrium or of a continuous anabolism and catabolism of proteins within the l i v i n g animal. The proteins of the l i v i n g c e l l are being constantly broken down and new protein molecules are being r e b u i l t i n part from amino acids recovered from the hydrolysis of previous tissue proteins, e s p e c i a l l y those of 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 part from the amino acids of the d i e t . This concept has been well reviewed by A l l i s o n (2) , Block (18) and others. The r e a l i t y of this dynamic state 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 ( 38 ) , i n studying protein metabolism promulgated the idea that there are two d i f f e r e n t types of protein metabolism. The protein metabolism associated with the excretion of creatinine and to a les s e r extent with u r i c acid he defined as endogenous. The excre-t i o n of creatinine tends to be constant and independent of the d i e t . The other type of protein metabolism, characterized by the excretion of urea nitrogen and a function of the ingested amino acids, was termed "exogenous" protein metabolism. With the concept of a dynamic equilibrium i n protein - 30 -metabolism such separation of food from body nitrogen i s d i f f i c u l t and often unnecessary. However, c e r t a i n experi-mental designs, which have been reviewed by M i t c h e l l , (9^,95) make use of these concepts to separate endogenous and exogenous metabolism. The concept of proteins 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 contrasts with the u n i t a r i a n theory of a hundred years ago, which maintained that regardless of source there was only one kind of protein (11*0 . At that time there was l i t t l e basis f o r b e l i e v i n g that proteins d i f f e r e d n u t r i t i o n a l l y . The observations that proteins from vegetable sources were less r e a d i l y available than those from animal sources could be explained by the differences i n the d i g e s t i b i l i t y of food of varying f i b r e content. I t was not u n t i l the end of the nineteenth century when Osborne ( l l 1 * ) observed that the amino acid composition of some proteins d i f f e r e d markedly from other p u r i f i e d proteins that attention was directed to possible differences i n the n u t r i t i v e value of proteins from various sources. Studies by Osborne and Mendel (103) , and by Rose (111,112) of the differences i n dietary needs f o r the various amino acids have emphasized the need for assessing the b i o l o g i c a l value of a protein. As a r e s u l t of numerous investigations, the amino acids may be c l a s s i f i e d into three groups! e s s e n t i a l (indispensable), semi-indispensable, and non-essential, (dispensable). The absolute quantities of these e s s e n t i a l amino acids which - 31 -must be supplied i n the d i e t w i l l change with the species of animal and w i l l vary widely with the p h y s i o l o g i c a l state of the animal and among groups i n the same species. The primary function of dietary protein i s to f u r n i s h a mixture of amino acids of the proper pattern f o r the synthesis of tissue proteins (2). A l l methods for estimating the n u t r i t i v e value of a protein evaluate t h i s function, e i t h e r d i r e c t l y or i n d i r e c t l y . The measurement of the d i g e s t i b i l i t y of dietary proteins and the subsequent absorption of the amino acids are usually an i n t e g r a l part of most determinations of n u t r i t i v e value. This i s p a r t i c u l a r l y important since the methods fo r determination of n u t r i t i v e value of proteins are designed to measure the degree of retention of that portion of the d i e t a r y nitrogen which i s absorbed i n t o the body of the animal. The nitrogen retained may be determined as a function of growth, nitrogen balance or r e p l e t i o n measurements of the whole or parts of the animal. A number of reviews by A l l i s o n (2) and others ( 8 ^ , 9 2 , 9 ^ * 9 5 ), have reported on the various methods f o r the b i o l o g i c a l evaluation of proteins. PROTEIN EVALUATION METHODS I PROTEIN EFFICIENCY RATIO The way i n which d i f f e r e n t proteins support growth i n young rats has been the most general and widely used c r i t e r i o n of protein value. The method o r i g i n a l l y developed by Osborne, Mendel and Ferry (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 gain i n body weight and nitrogen retention. The method involves addition of a protein to a basaal d i e t at a concentration - 32 -that i s known to he i n s u f f i c i e n t f o r optimal growth, measure-ment of food consumption and growth rate and c a l c u l a t i o n of the protein e f f i c i e n c y r a t i o . This r a t i o i s defined as the gain i n body weight per gram of protein or of nitrogen consumed. In an attempt to standardize the procedure many modifica-tions have been developed. These have included regulating the protein and c a l o r i c content of the d i e t and c o n t r o l l i n g the methods of feeding, whether ad li b i t u m paired feeding, forced feeding or adjustment of protein to body weight (2) . The p o s s i b i l i t i e s of a low c o r r e l a t i o n between weight gain and nitrogen retention 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 discussed the shortcomings of 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 of d i e t and food intake on the protein, fats and water content of the t i s s u e s . In spite of t h i s major d i f f i c u l t y i n determining composition of the gain, the rate of growth of young animals has been one of the most s a t i s f a c t o r y methods to evaluate the contribution of a given pattern of amino acids f o r protein synthesis. II THE RAT REPLETION METHOD The technique of f i r s t depleting and then r e p l e t i n g the protein stores of an adult r a t have been developed by Cannon and others (25), into a rapid, accurate and usefu l determination of the n u t r i t i v e value of protein. This method uses a basic p r i n c i p l e common to many bio-assays, that i s , the production of a d e f i c i t followed by a measurement of the extent of - 33 -replacement of that d e f i c i t . Depletion i s accomplished by-feeding a protein-free d i e t u n t i l the experimental animals have l o s t 2% of t h e i r i n i t i a l weight. The animals are then fed nitrogen i n the te s t d i e t and the rate of r e p l e t i o n measured. Excellent c o r r e l a t i o n has been obtained between gain i n weight during r e p l e t i o n and the regeneration of blood, l i v e r or carcass proteins, making weight recovery alone a good measure of n u t r i t i v e value. The r a t r e p l e t i o n method has not only been of value i n the determination of the n u t r i t i v e value of natural foodstuffs but i t has also been useful i n the study of basic problems i n protein n u t r i t i o n . I l l NITROGEN BALANCE Although the growth of young animals and the gain i n weight of protein-depleted animals are correlated with retention of dietary nitrogen i n the body, nitrogen balance i s a more d i r e c t measurement of nitrogen retention. By d e f i n i t i o n , nitrogen balance i s the difference between die t a r y nitrogen intake and nitrogen excreted or N _ N N N balance = intake - ( urinary + f e c a l ) I f the nitrogen intake i s greater than the nitrogen excreted, then the organism i s gaining protein and i s i n positive balance. I f the nitrogen intake i s less than the nitrogen excreted, then the animal i s l o s i n g protein and i s i n negative balance. Only i f the nitrogen intake equals that excreted, i s the organism i n protein equilibrium. - 3h -However, the quantity of dietary protein needed to maintain nitrogen equilibrium w i l l vary with the magnitude of the protein stores as well as with the amino acid composition of the d i e t . Thus, the protein requirements f o r nitrogen e q u i l i b -rium are influenced by the previous dietary h i s t o r y of the animal. Nitrogen balance i s the sum of gains and losses from a l l the tissues of the body. The dynamic nature of protein metabolism makes possible the maintenance of some tissues at the expense of others. I t i s possible f o r an animal to be i n p o s i t i v e balance and yet to be l o s i n g nitrogen from one or more tissues. The p o s s i b i l i t y of such adaptive changes taking place must always be considered during experiments involving balance. Various modifications of the nitrogen balance method have been developed. PROTEIN MINIMA Melnick and Cowgill (85,86) determinede the minimum amount of nitrogen necessary to maintain nitrogen equilibrium and developed t h i s into a quantitative procedure. This protein minima procedure has been used with variations by many workers to determine the n u t r i t i v e value of proteins and protein hydrolysates and also to study the quantitative relationships between amino acids i n a dietary mixture. The v a r i a b i l i t y of protein minima fo r nitrogen equilibrium which may r e s u l t from differences i n d i e t and p h y s i o l o g i c a l state of the animals has been emphasized i n a discussion by A l l i s o n . ( 2 ) . -35 -BIOLOGICAL VALUE Thomas (93) has developed a more fundamental and less variable procedure f o r measurement of the n u t r i t i v e value of a dietary protein. He defined the percentage of absorbed nitrogen retained i n the body of the animal as the " b i o l o g i c a l value". This value (B.V.) can be expressed mathematically by the following equation. B.V. = lOOx I - (F-Fm) - (U - Ue) I - (F - Fm) Where I i s nitrogen intake, F i s f e c a l nitrogen, Fm i s metabolic f e c a l nitrogen, U i s urinary nitrogen and Ue i s endogenous urinary nitrogen. A l l these variables can t h e o r e t i c a l l y be determined at the same time from a nitrogen -balance experiment. M i t c h e l l and his associates (93) have done a great deal i n developing and applying this concept to the measurement of the n u t r i t i v e value of dietary proteins and amino acid mixtures. I t i s customary to measure the excretion of urinary and f e c a l nitrogen while feeding a protein free d i e t , to provide values f o r the Ue and Fm i n the above equation. The v a r i a b i l i t i e s encountered i n th 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 of endogenous and metabolic nitrogen. In an attempt to obviate these d i f f i c u l t i e s a small amount of protein, usually whole egg i s included i n the r a t i o n when Ue and Fm are being determined. I t i s assumed that whole egg protein i s complete-l y digested and u t i l i z e d . Another attempt to eliminate much of the v a r i a t i o n associated with the catabolism of tissue - 36 -protein, has been to correlate endogenous nitrogen with excretion of cre a t i n i n e . Other modifications of the nitrogen balance method have been developed. These have been reviewed elsewhere by A l l i s o n . ( 2 ) . The c a l c u l a t i o n of nitrogen balance indices has the same signi f i c a n c e as 'net protein value' which was defined by M i t c h e l l (9*+) as the product of b i o l o g i c a l value and digest-i b i l i t y . Another procedure combines nitrogen balance studies with those of r e p l e t i o n of depleted l a b i l e protein stores. The c l a s s i c a l method (Thomas M i t c h e l l ( 9 3 ) ) of determining the b i o l o g i c a l value of proteins i s both long and tedious, involving N-balances on the experimental animal with test and control d i e t s , a t y p i c a l procedure giving three r e s u l t s a f t e r seven weeks. Using the basic p r i n c i p l e of t h i s c l a s s i c a l method, M i l l e r and Bender ( 1 6 , 8 9 ) , have revised the procedure eliminating many of the time consuming nitrogen determinations. The method i s based on carcass analysis and gives the net protein u t i l i z a t i o n (N.P.U.) which i s the product of b i o l o g i c a l value and d i g e s t i b i l i t y . The N.P.U. value, or that proportion of the nitrogen intake which i s retained by the animal, can be derived from body nitrogen determinations by the following equation. NPU = B - (Bk - Ik) I Where B and Bk are the t o t a l body Nitrogen, of the animals on the test and non-protein diets respectively, and I and Ik are the intake of Nitrogen i n the two groups. Ideally the - 37 -measurement of body Nitrogen should be made on a single r a t fed both the test protein and the non-protein d i e t . This i s impossible but the equivalent can be achieved by use of suitable control groups. At the end of the experimental period (usually 7 to 10 days) the animals are k i l l e d and body - N determined. The body - N may be determined by Kjeldahl digestion of the whole carcass or, a l t e r n a t i v e l y , from the water content of the animal since the r a t i o of water to nitrogen i s constant at any given age when the body water and body nitrogen 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 indicated that there i s no s i g n i f i c a n t difference between the values f o r body N determined d i r e c t l y or those calculated from a regression formula r e l a t i n g body nitrogen to body water over a defined age range. A high c o r r e l a t i o n was found between protein e f f i c i e n c y r a t i o s and net protein u t i l i z a t i o n of proteins, (13), the r e l a t i o n being very s i m i l a r to that found by Block and M i t c h e l l , (21), between protein e f f i c i e n c y r a t i o (PER) and b i o l o g i c a l value (BV). , TISSUE PROTEINS Nitrogen balances can be misleading, ;since they may be the sums of algebraic gains and losses. Two proteins producing the same nitrogen balance c a n . f i l l the protein stores of an animal d i f f e r e n t l y . A somewhat simpler and more d i r e c t approach to the study of the e f f e c t of dietary protein on loss and gain of body protein has been to measure changes occuring i n a s p e c i f i c t i s s u e . Maintenance of so c a l l e d normal plasma protein concentration, a decrease or increase - 38: -i n the albumen-globulin r a t i o and the protein content of the l i v e r are some of a number of s p e c i f i c tissues methods which have been studied. (2) The values obtained by these methods may not p a r a l l e l those obtained by others since the amino acid requirements f o r r e p l e t i o n of the protein of a s p e c i f i c tissue w i l l vary according to the structure of the protein and the p o s i t i o n i t holds i n the dynamic state of the body. Proteins l o s t from tissues through depletion represent loss i n enzyme systems. The p o s s i b i l i t y of using a c t i v i t i e s of enz yme systems to determine the n u t r i t i v e value of proteins was stimulated by the work of M i l l e r . (90) Approximately 30 enzyme systems are reduced by protein depletion. However, one of the most l a b i l e of a l l l i v e r enzyme systems was found to be xanthine oxidase. A method to correlate xanthine oxidase a c t i v i t y with n u t r i t i v e value of the protein has been developed by Litwack, Williams, Chen and Elvehjem. (78,79) This method i s s t i l l i n i t s formative stage. Its p o s s i b i l i t i e s have been discussed by Williams (126)* MICROBIOLOGICAL In recent years a number of workers have attempted to develop simpler and more rapid tests f o r n u t r i t i v e value based upon the growth of protozoa or micro-organisms. (3,32,35,*+6, 50,87,120) The growth response of the p r o t e o l y t i c protozoan Tetrahymena g e l i i to both hydrolysed and unhydrolysed proteins has been correlated to n u t r i t i v e value of protein by Anderson and Williams ( 3 ) , and others ( 3 2 , 3 5 ) . This growth response - 39 -has been measured i n a number of ways including acid production, (35), reduction of the dye triphen$tetrazolium chloride and actual count of the protozoa. I t i s known that the order of release of amino acids and the extent of hydrolysis play an important part i n the u t i l i z a t i o n of the dietary proteins. To simulate more c l o s e l y the fate of proteins i n vivo, Halevy and Grossowiez, 0+6), used i n v i t r o digestion with pancreatin and assayed the growth promoting a c t i v i t y of the hydrolysate f o r a s t r a i n Streptococcus  f a e c a l l s which required the ten amino acids considered as e s s e n t i a l f o r the r a t , Metz, Rennert and Cole, (87), combined the p r o t e o l y l i c action of pepsin with that of the bacterium, Pseudomonas areruginosa while T e e r i , Virchou and Loughlin, (120) used the enzymes normally employed by the animal i n digestion (pepsin, typsin, pancreatin and erepsin) with S f a e c a l i s . The preceding discussion indicates that the methods fo r evaluation of protein 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 protein molecules themselves. The precise structure and mode of synthesis of the proteins i s not yet f u l l y resolved. The i n t e r p l a y of amino acid metabolism with carbohydrate and f a t metabolism, as w e l l as the r e l a t i o n of one amino acid to another further complicates the procedures for protein evaluation. For these reasons none of the methods described i s free from l i m i t a t i o n s . There i s as yet no single procedure f o r determining the absolute n u t r i t i v e value of a protein. - IfO -PLANT PROTEIN VERSUS ANIMAL PROTEIN. A l l assay procedures indicate that, i n general, proteins from animal sources are superior to those from plant sources. However, exceptions, such as gelatin, do occur. The b i o l o g i c a l value of animal proteins vary according to t h e i r content of connective t i s s u e . In the plants the proteins from the seeds tend to be superior to those from leaves or roots. Most of the whole seeds do not show wide differences from one another, but some, such as soybeans and peanuts are d i s t i n c t l y superior. Most cereal proteins are d e f i c i e n t i n l y s i n e . Corn i s also d e f i c i e n t i n tryptophan while soybeans are lacking i n methionine. The difference i n the r e l a t i v e quantities of the amino acids present i n plant proteins as compared to that of the animal proteins which w i l l be synthesized from the plant materials account f o r the main discrepancy between the b i o l o g i c a l value of plant proteins compared to that of animal proteins. C. EXPERIMENTAL (a) THE NET PROTEIN UTILIZATION OF A NUMBER OF WEED SEED .SAMPLES . 1. ASSAY METHOD On the basis of 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 to o f f e r the greatest promise fo r the present studies. This method has the advantage of requiring a r e l a t i v e l y small amount of material and requiring a short experimental period. A number of modifications were - te -made to the procedure. Animals; Rats of the Wistar 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 at 21 days at a weight of about U-0 gms., and fed f o r one week on stock d i e t so that they weighed 55-65 grams at the beginning of the experiment. The rats were randomized into groups, each experimental group consisting of 3 females and 3 males. The t o t a l i n i t i a l weights of a l l groups were care-f u l l y adjusted so that each group t o t a l l e d the same weight within 2-3 grams. The number of experimental groups varied according to the number of proteins under test (usually 8 -10). The animals were housed i n i n d i v i d u a l pans and were provided with fresh water d a i l y . D i e t s : A number of substitutions were made to the basal non-protein d i e t of M i l l e r and Bender. (89). Composition of Basal Non-Protein Diet  M i l l e r and Bender Substituted (Robertson & Wood) Margarine 15 Lard 22.5 Potato Starch 10 Corn Starch 61 .0 Glucose 15 Sucrose 7 .5 Vitamin Mixture 5 Vitamin Mix (NBC) 1.5 S a l t Mixture (Hawk Salts U.S.P. No.2 7 .5 Oserc&Summerson 5 100.0 gms 1 9 W Rice Starch 50 100 gm. The meals with reduced isothiocyanate content as prepared by the preceeding extraction method (Section I - C (b)) were used to ascertain the b i o l o g i c a l value of weed seed proteins. - 1+2-The protein content (N x 6.25) of these extracted meals, as determined by the Kjeldahl method (102) are presented i n Table IV. TABLE IV. Protein Source Per Gent Protein (M x 6.25) White Mustard 1+2.3 Stinkweed 23.7 Rapeseed M+.6 Linseed 3I+.3 Soybean 52.9 The protein under test was added to the basal d i e t i n the required amount to give 10 per cent l e v e l of protein (N x 6.25) Diets containing 10 per cent casein were included i n each group of assays as a protein of known b i o l o g i c a l value. M i t c h e l l and Carmen (97) introduced the procedure of feeding a low l e v e l of completely assimilable protein i n place of the non-protein diet of the c l a s s i c a l procedure, thereby avoiding any p h y s i o l o g i c a l changes due to deprivation. As an al t e r n a t i v e c ontrol, therefore, a d i e t containing h- per cent egg albumen was included i n the assays along with the non-protein d i e t . The nitrogen content of a l l the diets were determined by a standard method.(102), Procedure: One of the r a t groups was fed on the non-protein d i e t and each of the remaining groups pn separate test or control proteins for 5 to 7 days. At the end of the experi-mental period, the t o t a l weight change and 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 with chloroform, and body water was determined by making in c i s i o n s into the abdominal, thoraic and c r a n i c a l c a v i t i e s and drying the carcasses to a constant weight at 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 feed consumption for each r a t were recorded and the corresponding body water, body nitrogen and nitrogen intake values were calculated. 2 , CALCULATIONS The nitrogen content of the bodies of the rats was calculated from the water content. Over the age range 33-57 days M i l l e r and Bender (15,89) found a high 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 this r e l a t i o n s h i p by the equation y = 2,92 * 0 .02x where y = N ( i n grams) x 100 H 2 0 ( i n grams) and x = age i n days. Examination of the nitrogen to water r a t i o i n the Wistar s t r a i n of rats from the U.B.C. colony v e r i f i e d t h i s r e l a t i o n -ship. Body water and body nitrogen determination were c a r r i e d out on 33 rats over the age range ^-lOO gms. This r e l a t i o n -ship may be expressed by the exponential equation. 1.08 y = 27 ,9x where y = mgs body nitrogen + s p = 3«7^ x = gms body water - s r = 3 .61 This r e l a t i o n s h i p has been discussed more f u l l y i n the Appendix I. This equation was used throughout to calculate the body nitrogen of each r a t from i t s water content. The net protein u t i l i z a t i o n (N.P.U.) of the protein samples was calculated by applying the equation of M i l l e r and Bender (16,89), N.P.U. B - l k ) where B and B^ are the t o t a l body nitrogen of the animals on the test and non-protein diets respectively, and I and I k are the intake of nitrogen i n the groups. 3, RESULTS The N.P,U. expressed as percentages of the f i v e extracted protein sources are presented i n Table V. Casein and egg albumen were included as contro l s . Commercial samples of linseed, soybean meal and a number of f i s h meals were included i n these assays f o r comparative purposes. The values obtained f o r the net protein u t i l i z a t i o n i ndicate that white mustard and rapeseed compare favourably with linseed and soybean as plant proteins of moderately high b i o l o g i c a l value. The N.P.U. value f o r stinkweed i s somewhat lower than that of the other two Cruciferae. This may be partly due to a lower d i g e s t i b i l i t y . However, the lower N.P.U. value also r e f l e c t s a l i m i t i n g l e v e l of an e s s e n t i a l amino a c i d . Amino acid analysis (Part III) has indicated that stinkweed contains sub-optimal amounts of h i s t i d i n e (Table V I ) . White mustard and rapeseed contain s l i g h t l y higher l e v e l s of methionine than does soybean, soybean being d e f i c i e n t i n both methionine and cystine. This probably accounts f o r the s l i g h t l y higher N.P.U. values obtained f o r these proteins than f o r soybean. The lower N.P.U. value f o r the ethanol extracted soybean meal as compared to that for the commercial sample - k* -TABLE V THE NET PROTEIN UTILIZATION OF A NUMBER OF PROTEINS _ N # P # t J # V a l u e s Protein Source N e t Protein U t i l i z a t i o n M i l l e r & Bender % of Diet - — — — (lif ,88 ,89) Assay Number 1 2 3 *+ 5 Mean SD Value SD Casein (Vitamin free) 8^.2 81.0 73.1*- 7^.2 78.2 5.2 (Crude) 60.0 5.5 Egg Albumen 90.9 78.*+ 72.1 80.5 6.7 82.5 *+.8 Soybean ^3.8 58.5 51.2 10.*+ Soybean heated 63.8 55.7 59.8 5.7 at 100$ f o r 30 min. Linseed 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 White Mustard 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 Herring Meal 100.1 6H-. Whale Meal L 0 .5 Dog F i s h Meal 80.2 52.5 Shrimp 55A - 1+6 -indicates that a c e r t a i n amount of the amino acids may have been removed during the extraction process. However, the f a c t that the N.P.U. value was improved after autoclaving the raw extracted meal at 100°C for 30 minutes may show that the trypsin i n h i b i t i n g factor present i n raw soybean was responsible f o r part at least of this low u t i l i z a t i o n . It has been suggested that application of heat to raw soybean produced a phenomenal increase i n the b i o l o g i c a l value of i t s protein, largely because the heat caused the methionine-cystine f r a c t i o n to become available f o r u t i l i z a t i o n by the animal. However, the concept of a heat l a b i l e trypsin i n h i b i t o r i n raw soybeans now explains the apparent greater methionine deficiency of raw as compared to a properly heat-treated soybeans or soybean meal. Sevtral investigators (80 , 82) have reported the r e s u l t s of feeding experiments designed to determine how to properly cook or heat raw soybeans and raw defatted soybean o i l meal to obtain the optimum b i o l o g i c a l or n u t r i t i v e value of the protein. Moist heating such as autoclaving has been the most e f f e c t i v e laboratory method fo r improving the n u t r i t i v e value of the protein. Overheating has 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 acids and vitamins. This subject i s well reviewed by Block et a l . ( 20 ) . Fritz,Kramke and Reed (kl) reported that best r e s u l t s were obtained when ground raw soybeans were autoclaved at 15 pounds pressure f o r 20-30 minutes. Evans and McGinnis (80) found an increase i n the n u t r i t i v e value of the protein a f t e r autoclaving ..a raw,, defatted soybean meal at 100, 110, - k7 -or 120°G. fo r 30 minutes. However, the protein n u t r i t i v e values were lower when soybean o i l meal was autoclaved at 130°C. fo r 30 - 60 minutes than when i t was autoelaved at 100= 120°C. For these reasons the raw ethanol extracted, defatted soybeans were autoclaved at 100°C. for 30 minutes to determine i f 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. value obtained f o r the extracted soybeans i n the i n i t i a l assays. As may be noted from Table V t h i s treatment yielded a 15 per cent improvement i n the N. P. U. value. k. DISCUSSION E f f e c t of Protein Treatment on N.P.U. Value. 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) for the evaluation of the proteins. Forbes and Yohe (kO) studied the e f f e c t of previous treatment of the animals on the net protein value. Their data showed that neither excess protein of good q u a l i t y nor a moderate deficiency of excellent q u a l i t y protein e f f e c t s the r e s u l t s obtained when the carcass nitrogen i s determined by d i r e c t analysis. However, when the body nitrogen was c a l c u l a t e d from the body water content, Forbes and Yohe (kO) obtained more variable r e s u l t s f o r < the net protein values. Higher r e s u l t s were obtained when the animals were moderately d e f i c i e n t i n protein at the s t a r t of the t e s t . Their c r i t i c i s m cannot be given too great weight since the M i l l e r and Bender procedure uses normal not protein depleted r a t s . . However, they used Albino rats of the Sprague Dawley s t r a i n while Bender and M i l l e r - if 8 -used a hooded variety f o r the derivation of the equation r e l a t i n g body N to body H 2 0 with age. Forbes and Yohe did not take into consideration the s t r a i n difference which Dreyer (31) has recently shown may a f f e c t the nitrogen water r a t i o . E f f e c t of Energy Intake on the B i o l o g i c a l Value of Proteins Forbes and Yohe (39)' also studied the e f f e c t of energy intake on the b i o l o g i c a l value of proteins fed to r a t s . When diet s containing 10 per cent protein were fed at *+£, 60 , or 8.0 gm. d a i l y , i t was found that the l e v e l of food intake had a highly 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 value. The b i o l o g i c a l values obtained at N-.O gm. intake were lower than those obtained at 6 .0 or 8 .0 gm. intake. However, the b i o l o g i c a l value of the proteins studied did not vary s i g n i f i c a n t l y between intake l e v e l s of 6 .0 and 8.0 d a i l y . In the present work the feed intake of the experimental rat s always exceeded 6.0 gms per day. Forbes and Yohe (39) ascribed the low bio -l o g i c a l values obtained at k-.O gm. intake to u t i l i z a t i o n of a portion of the dietary protein for energy to support weight maintenance of the animals. In connection with a study of the r e l a t i o n between protein e f f i c i e n c y r a t i o (P.E.R.) and net protein u t i l i z a t i o n (N.P.U.), Bender (13) also studied the ef f e c t of food intake on the values 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 with food intake.; f a l l i n g when the food consumption was reduced. However, from studies with amino acid mixtures, the N.P.U. was found to be independent of food intake. The food consumption was, i n f a c t , dependent upon the n u t r i t i v e value of the protein. Foods of high protein value were consumed i n greater quantities than those of low value. The difference between food consumption on the poorest and on the best protein was about 100 per cent. Bender (13) also showed that N.P.U's. can be correlated w i t h negative P.E.R.'s (loss of weight i n g/g protein eaten). When the N.P.U. i s less than kO, rats are unable to grow on the experimental d i e t . These low N.P.U. values are probably a better i n d i c a t i o n of the value of proteins of low q u a l i t y than the P.E.R. Because of a reduced consumption of proteins of low. b i o l o g i c a l value, the P. E. R.'s may be consequently underestimated. Length of Experimental Period The r e l a t i v e l y short length of the experimental period i s one of the advantages of the M i l l e r and Bender method fo r protein evaluation. For the measurement of P.E.R., Fixsen (37) stated that a long experimental period i s e s s e n t i a l , short Experiments giving higher values than the long ones, and 60 days being the minimum duration required f o r accuracy. However, i n nitrogen balance studies shorter periods appear, to be preferable. M i t c h e l l (9*+) has pointed out that as "the composition of the gains put on by growing animals progressively changes with age ..... the best way of assuring the desiredrequality i n the composition of gains i s to use animals of the same age, weight and previous treatment and to conduct the experiments f o r as short periods of time - it9a -as may be required for an accurate measure of the actual gain i n organized t i s s u e " . In their studies, M i l l e r and Bender (89) used 10-day and 7-day experimental periods. In these studies 7-day and 5-day periods were used. This period appears to be adequate. Does the nitrogen water r a t i o remain constant i n rats deprived of protein? Does loss of water p a r a l l e l loss of nitrogen i n a protein deprived r a t or does edema ensue? These are questions of dispute i n the M i l l e r and Bender procedures^ 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 investigations on the chemistry of n u t r i t i o n are emphasizing the significance of the amino acids as the fundamental factors i n a l l problems i n which hitherto the r o l e of i n t a c t proteins has been involved (103). This trend i s most evident i n the development of chemical methods f o r the evaluation of a protein based on i t s content of e s s e n t i a l amino acids. Since there are many advantages that would r e s u l t from a successful chemical evaluation of food proteins, a number of methods have been proposed to correlate the amino acid content of a protein with i t s n u t r i t i v e value. The c l a s s i f i c a t i o n of the amino acids occuring i n proteins on the basis of their 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 , into e s s e n t i a l and non-essential dietary components preceded the s e l e c t i o n of a protein or protein mixture whose amino acid constituents would be completely u t i l i z e d In digestion or metabolism. Kuhnau (77) chose human milk as the standard fo r the evaluation of proteins. Block and M i t c h e l l (96) selected the pattern of amino acids i n whole egg protein as the standard for growth since this protein has been shown - 51 -to be nearly p e r f e c t l y u t i l i z e d i n digestion and metabolism. In an attempt to judge trie n u t r i t i v e adequacy of a protein by the severity of i t s l i m i t i n g amino acid, Block and M i t c h e l l (96) devised a chemical score taken as the smallest percentage of an e s s e n t i a l amino acid based on i t s content i n egg protein. e x i s t between e s s e n t i a l and non-essential amino acids, such as are known to gocur between methionine and cystine, phenyl-alanine and tyrosine, 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 modified e s s e n t i a l amino acid index. The c o r r e l a t i o n between the b i o l o g i c a l value of protein and their chemical appraisal by these methods has been reviewed by M i t c h e l l and others ( 2 , 9 D . the n u t r i t i v e value of c e r t a i n noxious weed seeds the e s s e n t i a l amino acid content of a number of weed seeds were determined. The following weed seeds were chosent In order to consider the in t e r r e l a t i o n s h i p s which may As part of the investigations undertaken to determine Black mustard Brassica nigra (L) Koch White mustard B r a s s i c a l alba Boiss Brassica kaber (DC) L.C. Wheeler Wild mustard Tumble mustard Sisymbrium altissimum L Stinkweed Thlasni arvense L Western False Flax Camellna microcarpa - 52 -Samples of casein (N.B.C.) and raw soybean (Glycine soya), linseed (Linum usitatisslmum) and rapeseed (Brassica nanus) were included f o r comparative purposes. B EXPERIMENTAL (a) The Analysis of the E s s e n t i a l Amino Acid Content of  a number of Weed Seeds. (1) Preparation of Samples. Ground samples of the seeds were defatted by extraction i n a Soxhlet with petroleum ether. The nitrogen content of these samples as determined by Maero Kjeldahl technique are presented i n Table VI. (2) Assay method. The microbiological assay procedures outlined by Barton -Wright ( 9 ) were followed f o r the determination of the amino acid content of these samples. i Preparation of Hydrolysates For the assay of the e s s e n t i a l amino acids; h i s t i d i n e , l y s i n e , leucine, isoleucine, valine, phenylalanine methionine, tryplophane and threonine and the semi-dispensable amino acids, arginine and cystine; acid hydrolysates (2.5N HC1) were prepared. For the assay of tryptophane, a procedure involving enzymatic hydrolysis with pepsin (GBI 1:10,000 polency) trypin (GBI, h U.S.P.) and erepsin (G.B.I) was followed. An a l k a l i n e hydrolysis (5N NaOH) of the material was used f o r the assay of tyrosine. - 53 -i i Assay Procedure, Basal media for the assay of each amino acid were pre-pared according to the compositions outlined by Barton -Wright ( 9 ) , A standard solution of the amino acid to be assessed was made up to f a l l within the assay range. A l l concentration l e v e l s of the standard solu t i o n and extracts were set up i n t r i p l i c a t e , Hydrolysates were assayed at f i v e concentration l e v e l s (usually 500 mg to 2,500 mg of sample). The t o t a l volume of each tube was adjusted to 10 ml. with water. The tubes were capped with aluminium thimbles and s t e r i l i z e d i n an autoclave at 10 pounds pressure f o r 10 minutes. After addition of the inoculum, the tubes were incubated i n a water bath at 30°C. f o r 72 hours. At the end of the incubation period, the l a c t i c acid produced i n the tube was t i t r a t e d against 0.1N sodium hydroxide solution using an electrometric procedure (pH meter). i i i Organisms Stock cultures of the organisms required for the assays were obtained from the Department of Dairying, U.B.C. and included: Lactobacillus arabinosus 17 /5 (SOI1*-) Leuconostoc mesenteroides P 60 (80^2) Streptococcus f a e c a l i s ( l a c t i s Rogers) (80^-3) The organisms were c a r r i e d as stab cultures on l i v e r -Tryptone Agar (Barton - Wright) and sub-cultured at weekly i n t e r v a l s . For the inoculum, a transfer was made from a stab culture to a tube of r i b o f l a v i n standard medium (Barton -- 5h -Wright), ( 9 ) . This was incubated f o r 18 to 20 hours at 30°C. A saline suspension of the washed organisms was used to inoculate the tubes, (3) Calculations. For the standard curve, the logarithm of the dose (micro-grams of amino acid) was plotted against the logarithm of the response ( m i l l i l i t e r s of O.lNaOH), The corresponding values ( i n micrograms) f o r the response of the hydrolysates were read from t h i s curve. The grams of amino acid per 16 g. of nitrogen were calculated. A mean value and standard devi-ation were computed fo r each sample. (1+) Results. The amino acid content of the proteins of the various weed seeds are presented i n Table VI. The values f o r cystine are not included because d i f f i c u l t i e s were encountered i n the determination of this amino acid . The values obtained for the controls did not agree well with those reported by other workers. Cystine i s sen s i t i v e to acid hydrolysis when the material contains considerable quantities of carbohydrates. The destruction of cystine by prolonged acid hydrolysis has been recognized by a number of workers. In an attempt to avoid this destruction Riesen e_t aj. (109) autoclaved the samples f o r only three hours i n contrast to the usual 6 - 1 0 hours prescribed by Barton-Wright ( 9 ) . Another procedure to minimize the destruction of this amino acid recommends separate autoclaving of the medium and samples. (109) . TABLE VI THE AMINO ACID CONTENT OF THE PROTEINS OF CERTAIN NOXIOUS WEED SEEDS AND CERTAIN STANDARD PROTEIN SOURCES Calculated i n g. per 16 g. Nitrogen Amino Casein Soybean Linseed Rapeseed Brown Stink- Tumble Western White Wild Acid Mustard weed Mustard False Mustard Mustard , Flax Nitrogen* 13.62 7.71 5.85 7.1+ 5.9+ 3*79 6.59 5.89 6.^3 6.00 Arginine 3.8+0.2 7.7+0.3 7.7±0.7 6.110.9 6.1++0.5 6.3+0.1 7.1+0.1+ 7.3+0.+ 5.2±0.3 6.6+O.+ H i s t i d i n e 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 2.6+0.3 2.1+0.05 Leucine 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±0.5 5.3+0.2 Iso-Leucine 6.+0.2 5.0+0.1 +.5+0.1 +.+0.1 +.2+0.2 k.2+0.Okk.3+0.3 +.4+0.+ k.0+0.033.8+0.1 Lysine 9.3+0.+ 6.1+0.3 3.6±0.3 5.2+0.2 5.6+0.5 5.6+0.5 +.2+0.2 +.5+0.6 6.0+0.+ 5.1+0.2 Methionine 3.1+0.3 1.3+0.1 1.5+0.1 1.0+0.2 1.6+0.1 1.3+0.3 1.5+0.02 1.2+0,05 1.5±0£>3 I.++O.O5 Phenyl- : ' ' ', ! • alanine k.k±0.5 3.9+0.3 1.2±0.3 3.1+0.2 2.8+0.3 2.9±0.k 3.3+0.2 3.2+O.+ 3.0+0.2 3.0+0.3 Tyrosine 3.6 2.6+0.+ 2.0+0.1 1.9+0.1 2.1+0.3 2.+0.3 2.2+0.2 2.2+0.2 2.k±0.2 2.0+5T2 Threonine +.2+0.2 3.1+0.2 2.8+0.2 3.1+0.2 3.0+0.2 3.5+0.3 3.3+0.2 3.0+0.2 3.3+0.3 2.8+0.2 Tryptophane 2.2+0.3 1.5+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 0.7+0.07 0.8+0.1 Valine 6.7+0.+ +.7+0.2 +.+0.3 +.6+0.0+ +.2+0.3 +.+0.3 +.2+0.3 +.5+0.2 +.5+0.3 +.2+0.2 + Nitrogen gm per 100 g. f a t - f r e e sample - 56 -TABLE VII REPORTED VALUES FOR THE AMINO ACID COMPOSITION OF A NUMBER OF PROTEINS Calculated i n g. per 16 g. Nitrogen Amino Acid Casein Soybean Linseed/ Rapeseed Reference No, (1+8) 0 8 ) (101) (1+8) ( W (1+9) (i+8) (1+8) ( W (1) Arginine k.2 3.7 ^.1 3.^ 5.8 9.9 9.5 6.9 5.6 H i s t i d i n e 3.2 3.0 2.5 1.1 2.3 2.7 1.8 1.9 2.6 ~7 Leucine 9.k 10.5 9.9 5.1 3.8. 6.6 6.3 6.1+ 7.5 Iso-Leucine 6,2 7.1 6.5 2.U, k.7 5.6 i+.l 3.k 3.7 Lysine 7.7 7.9 6.6 1.8 5 .8 3.5 3.3 2.0 3.5 Methionine 2.9 3.1 3.k l.»+ 0.5 2.0 1.5 1.2 2.3 1.1 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 6 A 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 analysis of the amino acid content of casein, soybean, linseed and rapeseed which have been reported i n the l i t e r a t u r e (1, 19, *+8) are presented i n Table VII. These values are included as an i n d i c a t i o n of how w e l l the values obtained i n these studies agree with those published by other workers. When one considers the variations which may a r i s e between samples and between technical procedures of various laboratories, good agreement between our data and data reported elsewhere i s evident. However, i n a l l cases, the values obtained for phenylalanine and tyrosine tend to.be s l i g h t l y lower than the reported values. It i s r e a l i z e d that greater accuracy would have been obtained i f these assays had been repeated several times. However, only 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 values of the proteins by this method was sought. As indicated i n Table VI the amino acid content of the weed seeds tends to follow the general pattern f o r rapeseed (Brassica napus L); Although s l i g h t l y lower i n a majority of the samples, the e s s e n t i a l amino acid content of the weed seeds compares favourably with the general pattern found i n soybean. White mustard and wild mustard tend to be lower i n tryptophane content than the other seeds assayed. In Stinkweed 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 than that obtained for the other weed seeds. Although the protein of stinkweed compares favourably with those of the other weed seeds studied, stinkweed does not appear to be a good protein source because of i t s low nitrogen content. - 58 -As expected, the contents of a l l the es s e n t i a l amino acids i n the plant proteins were lower than the corresponding values for casein. The sulphur containing amino acids, methionine and cystine are usually the l i m i t i n g amino acids i n plant proteins. The values found for this acid 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 values found i n soybean. However, methionine i s the l i m i t i n g amino acid i n soybean. In a l l cases, the l e v e l of methionine i s less than the i d e a l require-ment considered to be present i n whole egg protein of kO g.methionine per 16 grams of nitrogen. (b) Chemical Scores and E s s e n t i a l Amino Acid Indices f o r  the Various Weed Seed Proteins. The amino acid analyses were carried out with the inte n t i o n of correlating amino acid content of the proteins of weed seeds with their n u t r i t i v e value. This c o r r e l a t i o n between amino acid analyses and b i o l o g i c a l value of a protein has been considered thoroughly by M i t c h e l l and Block (21 , 9 1 , 9 6 ) , by Kuhnau ( 77 ) , and by Oser (105) . M i t c h e l l and Block (96) selected the pattern of amino acids i n egg proteins as the standard for growth since d i g e s t i b i l i t y and u t i l i z a t i o n of this protein has-been found to be complete, or nearly complete i n most experiments. Data have indicated that, up to a c e r t a i n l e v e l , a l l of the amino acids absorbed during digestion of egg protein are retained 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 in the literature Indicates that the amino acid in 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 in the order of decreasing nutritive efficiency on the basis of increasing percentage deficits in their respective limiting essential amino acids1'. The nutritive value of a protein may be expressed as a "chemical score" equal to the smallest percentage of an essential amino acid in the protein based on its content in 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 in the test protein to those in egg protein were calculated, a l l percentages over 100 being reduced to 100, preventing excess amino acid in 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 is - 60 -omitted from consideration, because i t i s neither e s s e n t i a l i n the s t r i c t sense of the term, nor does i t have an obligate precursor among the es s e n t i a l amino acids. The rate of i t s synthesis from precursors, with reference to the rate of growth of the animal may be adequate f o r normal growth, depending upon the species and stage of growth. On the other hand, cystine and tyrosine are included because t h e i r presence i n the protein w i l l diminish the requirements for phenylal-ine and methionine, respectively. If the protein under consideration contains higher l e v e l s of cystine or tyrosine than the standard protein then the le v e l s of these amino acids are decreased to the corresponding amount present i n the standard protein. The'"corrected egg rat i o s " a r e thencalculated. An example of the c a l c u l a t i o n of the esse n t i a l amino acid index for casein i s considered i n Table VIII. Fisher (36) maintained that some of the amino acids of whole egg protein might be present i n excess of the minimal requirements of the animal, thus giving a lower value for the protein by chemical score method than by b i o l o g i c a l value method. Using the data presented by Block and M i t c h e l l (21) f o r amino acid content and b i o l o g i c a l value of proteins, Fisher estimated a series of minimal amino acid requirements which he coll e c t e d under the heading of 'ideal protein'. When the esse n t i a l (and semi-dispensable) amino acid requirements for maintenance, and for maintenance and growth have been determined with s a t i s f a c t o r y accuracy, they can be used i n place of some reference protein of superior q u a l i t y , - 61 -TABLE VIII COMPUTATION OF MODIFIED ESSENTIAL AMINO ACID INDEX (based on Amino Acid analysis - Table VI) Amino Acid Content per 16 grams Nitrogen Egg Corrected Logarithms Amino Acids Whole Egg Protein Casein Ratio Ratio of Corrected Ratios Gram Gram Percent Percent H i s t i d i n e 2.h 2.7 112.0 100 2.0000 Lysine 7.0 9.3 133.0 100 2.0000 Phenylalanine 6.3 *+.*+ 70.0 Tyrosine +.5 3.6 80.0 Phenylalanine plus tyrosine 10.8 8.0 7+.0 7+ 1.8692 Tryptophane 1.5 2.2 11+6.0 100 2.0000 Methionine 3.1 77.0 Cystine 0.3 12.5 Methionine plus cystine 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 95 1.9777 Is©leucine 7.7 6.1+ 83.O 83 1.9191 Valine 7.2 6.7 93.0 93 1.9685 Average logarithm 1.9389 Modified E s s e n t i a l Amino Acid index 86.9 Chemical Score 53 Limiting Amino Acid - cystine, methionine - 62 -i n computing e s s e n t i a l amino acid indices. When thi s i s accomplished i t w i l l be possible to base e s s e n t i a l amino a c i d indices on a firmer basis than can be done at present. Using the amino acid pattern of the whole egg protein of M i t c h e l l ( 9 1 ) e s s e n t i a l amino acid indices and chemical scores were computed for each of the weed seed and control p r o t e i n ^ studied. These calculations were based on amino acid data obtained during these studies. (Table VI). The scores and indices obtained are presented i n Table IX. E s s e n t i a l amino acid indices and chemical scores were also calculated f o r the control proteins; casein, soybean, linseed and rapeseed, using amino acid analysis data reported i n the l i t e r a t u r e (Table V I I ) . These values are presented i n Table XI. With the control proteins, values f o r cystine obtained from the l i t e r a t u r e were included i n the c a l c u l a t i o n of the essen t i a l amino acid indices and chemical scores. Unfortunately, values for cystine could not be included when ca l c u l a t i n g the indices and scores for the weed seed proteins. Correlation of Modified E s s e n t i a l Amino Acid Indices with  Net 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 acid indices (Table X) with the net protein u t i l i z a t i o n values (Table V) indicates a high degree of c o r r e l a t i o n between these two methods f o r evaluation of proteins. The chemical scores indicate that the proteins 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 value 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 Acid Analyses - Table VI) Based on Whole Egg Protein M i t c h e l l Protein Source E s s e n t i a l Amino Acid Index Chemical Score Limiting Amino Acid Casein *» 86.9 53.0 Methionine Soybean 9 72.7 51.5 Methionine Linseed ° 56.9 29.6 37.5 Phenylalanine Methionine Rapeseed ° 66.9 +6 +7.5 Phenylalanine Methionine Brown Mustard 61.9 +0.0 Methionine Stinkweed 60.7 32.5 Methionine Tumble Mustard 61.8 37.5 Methionine Western False Flax 59.8 30.0 Methionine White Mustard 60.8 37.5 Methionine Wild Mustard 57.+ 35.0 Methionine Cystine included from published data. - -TABLE X ESSENTIAL AMINO AG ID INDICES AND CHEMICAL SCORES FOR A NUMBER OF PROTEINS (based on reported Amino Acid analyses - Table VII) Based on Whole Egg Mi t c h e l l Protein Protein Reference Number E s s e n t i a l Amino Acid Index Chemical Score Limiting Amino Acid Casein 1+8 86.9 50 Methionine and Cystine Soybean Meal 19 7 .^3 1+0.6 Methionine Linseed Meal 1+8 69.2 37.5 Methionine Rapeseed 1 66.8 ^3.7 Methionine Herring Meal ho 8U..3 Phenylalanine and Tyrosine - 65 -amino acid i n a l l the weed seeds studied. The r e l a t i v e agreement between the ess e n t i a l amino acid indices obtained for a l l the weed seed proteins does not account f o r the high net protein u t i l i z a t i o n value obtained f o r soybean i n contrast to the lower N.P.U. value for stinkweed. Errors i n analysis would account for the s l i g h t discrepancies between indices and scores based on the data from these studies and those based on the data reported elsewhere. However, from the expected c o r r e l a t i o n between the ess e n t i a l amino acid indices and the net protein u t i l i z a t i o n values, one could predict that the proteins of brown mustard, tumble mustard, wild mustard and western f a l s e f l a x would compare with those of while mustard and stinkweed as plant proteins of moderately high b i o l o g i c a l value, but s l i g h t l y lower i n value 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 fully. 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 in Table I and compare with those reported in 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 -with the reduced isothiocyanate content were used i n these assays. The N.P.U. values obtained for these assays are presented i n Table V and indicate that the proteins of white mustard (Brassica alba Boiss) and rapeseed (Brassica napus L) compare favourably with linseed and soybean as plant proteins of moderately high b i o l o g i c a l value. The N.P.U. value for stinkweed (Thlaspi arvense L) was s l i g h t l y lower than that of the other two Cruciferae. Representative samples of the weed seeds were assayed for the i r content of esse n t i a l amino acids. Microbiological assay procedures ( 9 ) were used. The values foor the weed seeds were similar to the general pattern of amino acids found i n rapeseed (Brassica napus L). Although s l i g h t l y lower i n a majority of the samples, the essential amino acid content of the weed seeds compared favourably with that found i n soybeans. Using the data for the es s e n t i a l amino acid content of the weed seeds, a "chemical evaluation" of the proteins of weed seeds was ca r r i e d out. An "es s e n t i a l amino acid index" and "chemical score" was calculated for each weed seed according to the method proposed by M i t c h e l l . The "scores" and "indices" obtained are presented i n Table IX and indicate that proteins from the weed seeds under study were similar i n value but lower i n quality than casein and soybean protein. C a l c u l a t i o n of the ''chemical scores" indicated that methionine was the l i m i t i n g amino acid i n soybean and a l l the weed seeds studied. The "essential amino acid indices" (Table X) obtained from the es s e n t i a l amino acid analysis were compared with the "net protein u t i l i z a t i o n " values obtained from the rat assays. A - 68 -high degree of c o r r e l a t i o n was found between these two methods fo r evaluation of proteins. From this c o r r e l a t i o n one could predict that the proteins of brown mustard (Brassica .iuncea (L) Coss), wild mustard (Brassica kaber (DC) L. C. Wheeler), tumble mustard (Sisymbrium altissimum L) and western f a l s e f l a x (GameUna microcarpa Andrz) compare with those of white mustard (Brassica alba Boiss) and stinkweed (Thlaspi arvense L) as plant proteins of moderately high b i o l o g i c a l value, but s l i g h t l y lower i n value than soybean. APREHD.EC - 69 -TABLE XI PROTEIN AND FAT COMPOSITION OF A NUMBER OF WEED SEED SAMPLES Per Cent Protein (N x 6.2?) P e r C e n t F a t Fat Free Basis extracted with Protein Source Ground Raw Seed (extracted with petroleum petroleum ether ether. Brown Mustard 22.8. 37.1 38.2 (Brassica .luncea L Coss) Stinkweed 16.5 19.7 33.+ (Thlasni arvense L) Tumble Mustard 32.6 +1.2 20.+ (Sisymbrium altissimum L) Western False Flax 22.6 36.8 38.7 (Cameling micro-car oa, AndraJ , White Mustard 28.8 39.5 30.9 (Brassica albq Boiss) Wild Mustard 27.+ 37.5 27.1 (Brassica kaber [DC) L.C.Wheeler Rapeseed 2 8 A M+.6 36.2 (Brassica nanus L) Linseed 21.3 35.3 39.7 (Linum usitalissium) Soybean 38.5 +8.2 21.6 (Glycine soya) Casein 86.3 - 7 0 -Appendix I THE NITROGEN : WATER RELATIONSHIP I I ALBINO RATS INTRODUCTION In connection with the determination of the net protein u t i l i z a t i o n of a number of proteins by the method of M i l l e r and Bender (89) i t was; found necessary to study; the r e l a t i o n s h i p of body nitrogen to body water i n the U.B.C. colony of Albino rats;. Moult on (98) has shown that body/ constituents including; the nitrogen and water content, are a constant proportion of 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 regular manner with age. The r e l a t i v e water content of the f a t - f r e e mass decreases; very r a p i d l y from conception 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 constant concentration i s reached. On the other, the content of protein (nitrogen) and ash are correspondingly increased u n t i l a point where there i s nearly a constant composition of water, protein and ash i n the f a t - f r e e mass. In studies with male albino r a t s , Babineau and Page (7) found water to represent 72 percent of the f a t - f r e e mass. This constant was found to be completely independent of the magnitude of the f a t depots. From the data presented by Moulton ( 9 8 ), M i l l e r and Bender (89) concluded that the p a t i o of nitrogen to water i s a constant and therefore the nitrogen content of the body could be derived from the water content. In testing this they analysed 197 rats of a Mack and white hooded s t r a i n f o r t o t a l body nitrogen and body water. They found that - 7© -the nitrogen to water ratio (NtH^O) varied with sueh constancy that i t could be used for calculating carcass nitrogen from a knowledge of the water content and age, Babineau and Page (7) have since shown that i n physiologically normal animals, the non-fat dry matter can be predicted from total body water with a high degree of accuracy. Miller and Bener, (89) however, doubted whether their calculated regression equation for N:H20 values, correlated with the age i n days, would also be applicable to other rat colonies. Analysis of a small number of albino rats by Forbes and Yohe did indeed indicate the existence of strain differences. Recently, Dreyer (3D has reported further large scale findings on the s t a b i l i t y of the relationship between the N:H20 ratio and age of albino rats. He has reported observed differences between the N:H 0 ratios of 2 male and female animals. Miller and Bender (89) on the other hand reported no sex differences. The present study was undertaken to investigate the relationship between total body water and body nitrogen in the U.B.C. rat colony and was an essential prerequisite to the use of the Miller and Bender procedure. EXPERIMENTAL ANIMALS The animals studied included both the Wistar and Sprague-Dawley strains of albino rats bred i n the U.B.C. rat colony from rats originally obtained from the-Wistar Institute, - 72 -Philadelphia and the Sprague-Dawley Company res p e c t i v e l y . The rats analysed ranged i n weight from 5 to 300 grams and i n age from b i r t h to 87 days. In the weight range 5 to 30 grams ( b i r t h to 18 days) l i t t e r mates (two females and two males) were analysed at each age group. Over the weight range ho to 300 grams, s i x l i t t e r s of Wistar and four l i t t e r s of Sprague-Dawley were used. Animals were drawn at random from these l i t t e r s vwhen they reached the desired weight. A ca r e f u l record was kept of the age of each r at i n these weight ranges. A number of rats (fourteen) i n the weight range (*+0 to 100 grams) which were used i n preliminary studies were also obtained by random s e l e c t i o n from the main colony. For the 250 and 300 gm. weight ranges, some of the rats were obtained from the stock colony. Only approximate ages-could be obtained f o r some of the rats i n th i s group. A f t e r weaning, a l l the rats were kept on the stock d i e t (U.B.C. Ration No. 18) u n t i l they were s a c r i f i c e d f o r an a l y s i s . A t o t a l of 8h r a t s , 36 Sprague-Dawley and 58 Wistar were analysed. The average number of rats per weight group was four but varied from two to ten. CARCASS ANALYSIS Body water was determined a f t e r k i l l i n g the rats with chloroform. Incisions were made int o the c r a n i a l , thoracic and abdominal c a v i t i e s , and the carcasses were dried to a constant weight at 105°C;. fo r ^8 hours. I n t e s t i n a l contents were included. Nitrogen was determined by the Kjeldahl method (102).. The small r a t s , up to 15* grams dry weight, were completely digested with approximately 10 ml concentrated H 2S0^ per gram dry matter and 10 gram cat a l y s t (10 g. K 2S0^, 0.1-0.3g. CUSO^.5"H20) according to the Gunning Method (102). With the carcasses of the larger r a t s , over 15 grams dry weight, d i f f i c u l t i e s were.encountered i n obtaining homogenous samples f o r the determination of t o t a l nitrogen. To avoid t h i s , K jeldahl digestion of the whole carcass was followed. In preliminary studies, the dried carcasses were ground i n a mortar and the entire ground rat was transfered i n 10-15 gm. portions to a Kjeldahl f l a s k f o r dig e s t i o n . The digested material was then recombined. A f t e r d i l u t i o n to 500 ml, duplicate 25 or 50 ml aliquots (depending on size of the rat) were taken f o r d i s t i l l a t i o n . This procedure was carri e d out on lk rats as indicated i n Tables. Following th i s procedure v i o l e n t frothing occured during digestion and another method f o r sampling was sought. A procedure suggested by Sarett and Snipper (116) was followed f o r preparing the carcasses of the remainder of the large anima^for nitrogen determinations. The dried carcass of each animal was weighed i n a tared pint Mason j a r . Approximately 100 ml of d i s t i l l e d water, was added and the mixture was autoclaved at 15 l b s . pressure f o r h hours. Each carcass was homogenized using a V l r T i s homogenizer ("Aero-Seal Chemixer"). Aliquots of the weighed homogenates were transferred to a 500 ml or 800 ml Kjeldahl f l a s k , the weight of the aliquot being determined by weight d i f f e r e n c e . By d i v i d i n g the homogenate into three to s i x portions, depending on the dry weight of the r a t , the t o t a l carcass homogenate was digested. As i n the preceding method, approximately 10 ml. concentrated RvjSO^ . per gram rat dry carcass was used. The vigorous frothing which occured during digestion of the dry carcass was mainly avoided. Care was also taken to maintain the digestion 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 hours of heating. The mixture was digested u n t i l i t cleared (pale green colour) and thereafter f o r about 30 minutes. The aliquots were not recombined, but were d i l u t e d to 500 ml and aliquots containing approximately 1 gm. of dry matter were taken f o r the estimation of ammonia. The mean of the calculated mgs. N per rat for each aliquot was taken as the nitrogen content of the r a t . RESULTS: The r e s u l t s obtained f o r the moisture and nitrogen content of 36 Sprague Dawley and 58 Wistar rats are presented i n Tables XII,XIII, XIV and T able XV, respectively. These results were used f o r c a l c u l a t i o n of nitrogen; water r a t i o s , regression equations and further s t a t i s t i c a l treatment. - 75 -T A B L E x i r : B O D Y W E I G H T - B O D Y W A T E R - B O D Y N I T R O G E N R E L A T I O N S H I P I N F E M A L E W I S T A R R A T S Weight Age Water Nitrogen gmN/gm H o 0 xlOO gas. days gms. mg. _2 T i l TT^O " 77 . 1.52 82 . 1 .5 * 218 2.14-223 r 2 .11 "+35 2 .70 "+97 2.80 716 3 .08 778 3 .31 1,000 3.66 1,113 3.69 1,056 3.58 1,120 3.6*f 1,219 3.68 ' ^28 3.62 5 .51* 0 *+.72 6.00 0 5.07 6.32 0 5.32 12.8 8 10.k 12.9 8 10.3 21 .1 lk 16 .1 23.6 lk 17.7 32 .8 18 23.2 33.6 18 23 i 5 *k>.7** mm mm 27.3 kl.l 22 30 .1 lfLj-.O** mm mm 29 .5 1+5.6** am mm 30.7 1+6.0 29 33 .1 50.9 29 36.6 52.9 29 37 .3 58.3 32 k2& 5 8 . 5 * * 39 .2 61.M-** — 39.»+ 71.0 >+3 50.6 77.9 31 57.1 81 .7 k$ 59 .5 88.8 36 63.4 98.O** --. 70.3 99.8 38 7 2 . k 1 0 2 . 3 * * 75 .0 139.9 51 96.2 207.1 207 - 237 129.1+ 236.2 207 - 237 11+8.8 . H7 3.87 1,6/21 3 .82 1 , ^ 0 3.67 1,561+ 3 .96 1,783 3 .52 1,95*+ 3 A 2 2,327 3 .91 2,428 3 .82 2,828 1+.02 3,000 1+.11+ 2,99k 3.9? ^,375 k.5k 6,202 1+.79 7,01+7 1+.73 N = 29 * i n t e s t i n a l contents removed ** nitrogen determination on ground dry carcass - 76 -TABLE XIH BODY WEIGHT - BODY WATER - BODY NITROGEN RELATIONSHIP IN MALE WISTAR RATS. Weight Age Water gms days gms 5 . 8 V * 0 + . 9 3 6 . 0 0 0 5 . 1 2 6 . 2 3 0 5 . 2 6 1 3 . 6 8 1 1 . 0 1 3 . 6 8 1 0 . 9 21.1+ lk 16 A 21+.3 lk 18 . 5 3 3 A 18 2 3 . 9 3 6 . 0 18 2 5 . 6 + 2 . 5 2 2 3 1 A 5 6 . 8 * * 2k + 0 . 1 ~~ + 0 . 1 5 7 . 3 3 6 + 1 . 5 5 8 . 9 2 9 + 2 . 5 6 1 . 9 * * Mia + 1 . 3 6 5 . 9 * * -- + 7 . 7 7 0 . 1 3 2 5 1 . 5 7 2 . 2 * * 5 3 . 3 3 1 5 7 . 1 8 0 . 2 * * mm mm 5 9 . 0 9 0 . 8 * * mm mm 6 5 . 7 k3 6 7 . 9 9 5 . 2 * * 7 0 . 2 9 9 . 9 +3 73 A 1 0 3 . 3 3 6 7 5 . 7 1 + 6 . 3 5 1 1 0 3 A 1 9 8 . 6 6 8 1 3 9 . 5 2 6 3 . 7 81 1 7 7 . 6 2 8 6 . 7 6 1 - 9 5 1 9 2 A Nitrogen mgms 6 5 7 3 8 2 23*+ 2+2 1+6I 51k 800 8 5 6 1 , 1 2 9 1 + 8 5 llkkQ 1 , 5 6 0 2. 2 , ' 2 , ' ? , , 7 8 0 , 9 6 5 , 9 5 2 . 1 2 1 , 1 2 8 ^ , 8 2 6 , 5 8 + ,92^-, 9 9 + - , 5 + 5 8 , 8 7 8 7 , + 0 6 N/HgO Ratio gm N/gm H 20 X100 1 . 3 2 1 A 1 1 . 5 6 2.12 2.21 2.80 2 . 7 8 3.3J+ 3 . 3 + 3 . 5 9 3.70 3.61 3 . 7 5 3 . 8 3 3 . 7 2 ' 3 . 7 3 3.81 3 . 6 6 3 . 7 1 3.60 3 . 7 6 +.16 3 . 6 8 3 . 9 8 3 . 9 5 + . 5 5 + . 6 9 + . 9 9 3 . 8 5 N = 29 * i n t e s t i n a l contents removed ** nitrogen determination on ground dry carcass - 77 -TABLE XIV BODY WEIGHT - BODY WATER - BODY NITROGEN RELATIONSHIP IN FEMALE SPRAGUE - DAWLEY RATS Weight Age Water Nitrogen N/H20 r a t i o gms days gms mgns gmN/gmHpO xlOO 5.78 0 4 .95 97 1.95 - # 9 t ; 13.35 6 10.65 282 2.64 13.47 6 10.85 291 2.68 20.17 10 15.54 473 3.04 21.30 10 16.24 499 3.06 28.81 18 21.02 767 3 .65 30.97 18 22.69 839 ' 3 .69 38.8 23 2 8 . 5 1,050 3.68 52.1 30 37 .9 1,432 3 .77 64.1 30 45 .1 1,797 3.98 71.3 33 50.8 1,927 3.79 81.2 37 58.3 2,294 3 .93 91.4 40 65 .9 2,600 3 ; 94 99.0 42 72.2 2,963 4 .10 140.0 52 98.6 4,266 4.32 190.9 75 128.0 6,158 4.81 237.1 125 - 156 150.7 7,115 ^ . 7 2 N = 18 - 78 -TABLE XV BODY WEIGHT - BODY WATER - BODY NITROGEN RELATIONSHIP IN MALE SPRAGUE - DAWLEY RATS Weight Age Water Nitrogen N/HpO r a t i o gms. days gms. mgms. gmN/gm H9Q XlOO 6.33 0 5.38 106 1.96 6 .35 0 5.+2 107 1.96 11.26 6 9.22 229 2.+8 1+.67 6 11 .71 299 2 .55 22.55 10 17.25 53+ 3.09 2^.13 10 -18.1*3 5+9 2.98 31.76 18 23.+9 870 3.70 3+.77 18 25.+2 9W 3 .71 iri I 2 3 32 . 7 M 7 5 3 .59 iH 12 ? 7 ' 5 1 ^ 3 76 8 ? 1 ?? S i ' 1 7 9 ? 3 .62 ° 3 . l 33 60 . 5 2 ,290 3.78 90.1 35 66.0 2 503 \ n% 106.0 IfO 77.0 3 127 t{l 2+1.3 87 165.3 7,1+1 J 298.3 87 198.9 & 5 9 Coo N = 18 CALCULATION In formulating an equation fo r the c a l c u l a t i o n of body nitrogen from water content, M i l l e r and Bender (89) regressed, the r a t i o of body nitrogen to body water with age. They used a two variable l i n e a r c o r r e l a t i o n but i n actual f a c t they had three v a r i a b l e s ; water, nitrogen and age. Over the r e l a t i v e l y short age range 33-57 days, M i l l e r and Bender (89) obtained a l i n e a r r e l a t i o n s h i p given by the equation: H2omgms x 1 0 0 a 2 , 9 2 + 0 , 0 2 x> w h e r e x = a S e i n days. However, examination of a d d i t i o n a l r e s u l t s covering the age range 0 - 503 days indicated the r e l a t i o n between age and (N:H2(P)100 to be c u r v i l i n e a r , suggesting an e x p o n e n t i a l form ( 89 ) . Chronological age i s not always a good index of the actual p h y s i o l o g i c a l age of an animal. To quote Brody (24) "A given chronological or physical time unit has a d i f f e r e n t p h y s i o l o g i c a l or functional s i g n i f i c a n c e f o r d i f f e r e n t organisms, fo r d i f f e r e n t organs i n the same organism, at d i f f e r e n t ages, and under d i f f e r e n t conditions." The 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 growth rate of an animal and, therefore, one animal may be p h y s i o l o g i c a l l y older than another animal at the same chronological age. B a i l e y (8) showed that the f a t - f r e e body mass of mice maintained on a low plane of n u t r i t i o n f o r a considerable length of time (25 - 36.days) contained more body water and less body nitrogen than the f a t - f r e e mass of mice maintained > N, - 80 -on a higher plane of n u t r i t i o n . From t h i s he concluded, that at: the same body weight, animals on a low plane of 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 younger but c h r o n o l o g i c a l l y o l d e r than animals on a higher plane of n u t r i t i o n . For t h i s reason, 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 t o body water were c a l c u l a t e d i n t h i s study. The d a t a f o r body water and body n i t r o g e n c o n t e n t were p l o t t e d on a r i t h m e t i c , a r i t h - l o g and l o g - l o g graph paper. An apparent s t r a i g h t l i n e was obtained w i t h the l o g - l o g r e l a t i o n s h i p ( F i g u r e I ) . This; I n d i c a t e d t h a t the r e l a t i o n -s h i p between n i t r o g e n and water c o u l d fee expressed fey the following; e q u i v a l e n t e q u a t i o n s : E x p o n e n t i a l Y. = aX f e Logarithmic l o g ¥ = l o g a * fe l o g X This was not s u r p r i s i n g s i n c e equations of t h i s type have been used e x t e n s i v e l y f o r e x p r e s s i o n of b i o l o g i c a l d a t a by Brody ( 2 * + ) and others ( 5 1 ) . The term "allometry" 1 has been used by a number of workers to d e s i g n a t e t h i s form of e q u a t i o n used f o r e x p r e s s i n g r e l a t i v e growth. Allometry i s a mathematical technique f o r c h a r a c t e r -i z i n g the r e l a t i o n s h i p s of s i z e or c o n c e n t r a t i o n of two p a r t s or c o n s t i t u e n t s of the animal body ( 5 1 ) . The l o g a r i t h m i c form of the equation d e s c r i b e s a s t r a i g h t l i n e w i t h slope b. The exponent b i s the r a t i o of the percentage change i n Y to the c o r r e s p o n d i n g percentage change i n X. When b = 1, the changes of the two measurements - 81 -10000 5ooo 2000 CO 0 CO txO 1000 Sprague-Dawley Y=26T6Xl.09 _ + sr=3.oo 2.91 -s r =. . Wistar Y=27.9X 1 , 0 8 +Sr=3.7+ -S r=3.6l a •H c Q) bO O U 4-5 o 4-> XI •H © 500 200 100 Wistar Y=6.+3X1,^L Spra gue-Dawley +Sr=+.08 -S r=3.92 1 LO Y=10.2XJ-# +Sr=+.10 50 1 1 • ' • ' » ' 10 20 50 100 200 Weight of Water i n Grams Figure I - Body Water - Body Nitrogen Relationships i n the WistaSr and Sprague-Dawley Strains of Albino Rats maintain a proportional.constancy, such that the constant a = Y . When b*> 1 , the percentage increase of Y i s more rapid than that of X, while when b<^l, the reverse i s true. Where b ^ l , the "a" c o e f f i c i e n t i s simply a suitable m u l t i p l i e r without any obvious b i o l o g i c a l s i g n i f i c a n c e . 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 nitrogen appeared to be best described by an equation of t h i s form. Brody (2h) considers the representation of data by a log - log 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 fo r expressing b i o l o g i c a l data. Equations of t h i s type are considered to have greater 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 Y = a + b X used by M i l l e r and Bender (89) and Dreyer ( 3 D . As indicated i n Figure I when the data f o r body nitrogen and body water were plotted on a log - log g r i d , an apparent change or "break" occurred i n both the Wistar and Sprague-Dawley stra i n s i n the region of approximately 30 grams body weight. Brody (2*+) has discussed these changes i n the value of the exponent "b", which take place i n growth curves, expecially during early growth. For some instances abrupt changes of t h i s sort are known to be coincident with a w e l l -defined p h y s i o l o g i c a l event, such as the onset of puberty. In the present study the change i n the slope of the l i n e coincided with the approximate date of weaning. Because these "breaks" occurred, equations regressing body nitrogen to body water were calculated by the method of le 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 calculated f o r the hO - 100 gm. range since t h i s i s the weight range under consideration i n the M i l l e r and Bender (89) procedure. The -equations which were calculated to express this r e l a t i o n s h i p are presented i n Table 16. In a l l cases, Y represents the milligrams of body nitrogen and X represents the grams of body water. The f a c t that the exponent "b" i s greater than one i n a l l cases, indicates that the increase of nitrogen i s proceeding at a greater rate than the increase of water i n the animal body. In other words, the animal body i s becoming progressively dehydrated but at a dec 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 to 30 gms. and hO - 100 gms. indicates that the percentage increase i n nitrogen r e l a t i v e to the increase i n water i s greater i n the younger rats than i n the older ones. The s l i g h t discrepancies which occur i n the values of b over the weight ranges hO - 100 gms. and hO - 300 gms. may be accounted f o r by the increase i n the standard errors of estimate when the larger group Is considered. This error i s probably associated with the d i f f i c u l t i e s i n the determination of t o t a l body water i n these larger animals. A comparison was made of the nitrogen (N) values calculated from t h i s exponential regression equation and the nitrogen (N) values calculated from the equations presented by M i l l e r and Bender (89) and by Dreyer ( 3 D . Standard deviations were calculated f o r the differences between the N calculated by - r84-TABLE XYI Regression Equations Expressing the Relationship of Body Nitrogen to Body Water i n Albino Rats. Body Standard Error of C o e f f i c i e n t Rat Weight Equation Estimate of C o r r e l a t i o n S t r a i n N Range >, r grams Y = aX D +S R -S R LogY.logX Wister 18 b i r t h - 3 0 Y = 6.43X 1 ,^ L 4.08 3.92 20 40-300 Y = 24.4X 1 , 1 2 5.48 5.19 0.961 33 40-100 Y = 2 7 . 9 X 1 * 0 8 3.74 3.61 0.99^ 1.14 40 40-300 Y = 22.IX 8.48 7.82 0.994 Sprague ), Q Dawley 16 b i r t h - 3 0 Y = 10.2X * 4.10 3.94 O.998 13 40-100 Y = 26.6X 1' 0 9 3.00 2.91 O.996 0.996 - 85 -these equations and the actual N determined. These findings are reported i n Table XVII and indicate that the N contents calculated from the exponential equation r e l a t i n g body nitrogen to body water are more accurate than the values obtained from the equations of M i l l e r and Bender (89) and Breyer (31) which r e l a t e nitrogen to water r a t i o with age. T "ABLE Wfl A Comparison of the Nitrogen Content of Wistar Rats Determined Chemically with Nitrogen Values Calculated from Regression Equations Age Sex Body Nitrogen Calculated Days Composition Y = 2 7 . 9 X 1 ' 0 8 • Y=2.92+0.02x Y=3. +331+0.011+9X (1) M i l l e r B e n d e r (2) (Dreyer) (2) Nitro Water Nitro gen Devia Nitro Devia Nitro Devia grams gen mg tio n gen t i o n gen t i o n mg mg mg mg mg mg . 31 M 32 M 36 M 36 M +3 M +3 M 31 F'> 32 F 36 F ?8 F +3 F +3 F 57.1 51 .5 +1.5 75.7 67 .9 73.+ 57.1 +2.if 63.+ 72.+ 50.6 59 .5 2121 1965 1560 299+ 2826 292+ 195+ 1621 2*+28 3000 1783 2327 2202 1969 1560 2985 2655 2888 2202 1597 2+65 28*f9 1932 2302 81 'A 0 9 171 36 2+8 2lf 37 1+9 25 2021 1833 1510 2755 2566 277+ 2021 1509 2307 266+ 1791 224-9 100 132 50 239 260 150 67 112 121 336 8 78 222h 201+ 16+7 3005 2766 2990 2221+ 1658 2517 2895 2061 21+2+ 103 +9 87 11 60 66 270 37 89 105 278 97 Standard deviation 110.1+ mg N 1) where X = mg N, Y = gm H 2 0 2) where X = gmN/gm H 2 0 X 100, X = age i n days 165.2mg N 131.8 mg N BIBLIOGRAPHY - 87 -BIBLIOGRAPHY 1. 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