Nutritional significance of the rats

3 NUTRITIONAL SIGNIFICANCE OF THE FATS*

Harry J. Deuel, Jr. I . INTRODUCTION

UNTIL recently, many nutrition workers have considered fats as optional components of the diet. I t was believed that an equally good nutritional condition obtained in the absence of fat as when this foodstuff was present, provided t hat the diet contained sufficient calories and was other~'ise complete. However, since the classical discoverT of BVRR and B u ~ ul that certain unsaturated fatty acids are essential for growth and, in fact, for survival, it has been more generally recognized that fats may serve as a required nutrient, not only as a source of the essential fatty acido but probably also in a var'.'ety of other ways. A number of important functions of fat are generally recognized. In the first place, fat is the foodstuff which possesses the greatest caloric density of any of the foodstuffs. In the dry state, it yields 9,3 large caloriest per gin, as contrasted with values of 4-1 calories for protein and carbohydrate. Moreover, fat is hydrophobic and usually occurs without water, while both protein and carbohydrate are hydrophilic. Therefore, in nature, the ratio of caloric value in foods between fat on the one hand and protein and carbohydrate on the other hand, is usually greater than the 2 . 3 : 1 ratio which exists between these foodstuffs in the dry state. I t is obvious that fat is the foodstuff to be employed in increasing amounts when the bulk of a diet must be controlled. A second function of fat i.s as a carrier of the fat-soluble vitamins. Not only does fat act as an excellent solvent for the carotenoids, vitamins A, vitamins D, and the tocopherols, but it also protects them from oxidation to a much greater extent than if they. were present in an aqueous suspension. Moreover, the presence of fats in the ga4trointcstinal tract concomitantly with the fat-soluble vitamins may aid in the absorption of the latter. Phospholipids, which occur to a greater or lesser extent in most natural fats, furnish cholhm, necessary for the synthesis of xnethionine, and as a general transmethylating agent. The importance of dietary fat is not confined to the fat-soluble vitamins, but its presence is also vital in some of the water-soluble vitamins. Thus, EvA.~s and LEPKOVSKY~2~ demonstrated in 1929 that fats exhibit a thiamin-sparing action, although it was later reportcd (3) that this foodstuffdid not possess any riboflavinsparing activity. On the otherhand, the pyridoxine requirement has been shown to be dependent Upon the amount of fat in the diet. c4h c5~ * Contribution No; 324, D e p a r t m e n t of Biochemistry and Nutrition. University of Southern California. t The t~,rm, calories, as used in this chapter refers to kl]ocalorles (1000 small calorics).

99

Nutritional Significance of the Fats Fats are necessary in the diet to improve the palatability of the food. Low-fat diets prove very unappetizing ; under conditions in which such a diet is required, it may bring about a vo!untary reduction in food consumption. STARLING {e} stated that the reduced food intake in Great Britain (luring World War I was definitely associated with the low-fat content of din dict. A considerable number of the population lost weight because "of their failure to consume an adequate number of calories to maintain an energy equilibrium on the high-carbohydra~, low-fat diet then available. Baked foods are likewise virtually impossible without fats. II. P H Y S I O L O G I C A L

I~UI~ICTIONS IN ~,VHICII ]PATS P L A Y

A ROLE

1. Fats as the Source o! Essential Fatty Acids

1. Discover v of the necessity of essential fatty acids The first proof t h a t the exclusion of fats from the diet resulted in a fat-deficiency syndrome was obtained as recently as 1926 b y EvA.nS and Burnt. tT~ Shortly thereafter, MeAtus et al. 18~ likewise noted t h a t rats receiving fat in their diets grew b e t t e r then1 did animals on fat-free regimens; however, t h e y failed to report on any deficiency symptoms in the rats receiving the fat-free diet. I t remained for B ~ a and BURR m to demonatrate clearly for the first time t h a t this p a t h o l o ~ e a l deficiency was a new dietary disease which was not go be ascribed to lack of vitamins A or D. When young rats are subjected to a fat-free diet, t h e y grow at a normal rate for several weeks. However, after 3 or 4 weeks, the growth curve begins to flatten as compared with t h a t of rats receiving cottonseed oil, until a plateau is reached after 9 to 10 weeks, followed b y a moderate decline in weight. (9~ SVhen the rats are eon+Anued on the fat-free regimen for a prolonged period, their weight m a y decline furfl~er, and premature death occurs. BURR ~°) has listed the typical symptoms in the rat as follows: (1) marked r e t a r d a t i o n in growth; (2) development of scaly skin and caudal necrosis; (3) kidney lesions with coneomitant haematuria ; and (4) death. K i d n e y lesions were found in 100°~ of the rats examined at autopsy, while the incidence of caudal necrosis was variable. Other less specific lesions which were noted include histological changes in the ovaries, uterus, and other tissues, resulting in poor ovulation, impaired reproduction and lactation in the female, and complete sterility in the male. Deficient rats likewise exhibit ifistological abnormalities of the epidermis. WmLIA.~lSO.nm~ has shown t h a t the epidermis of rats on fat-free diets becomes thicker and more differentiated t h a n that of normal rats. The ~tratum granultJsum becomes especially distinct, and the horny layer thick. This syndrome can be entirely prevented when essential f a t t y acids are given. Another s y m p t o m voted by BURR was an excessive water intake. A high metabolic rate also obtains, associated with a high respi.ratory quotient. BURR and Be'm0 TM reported somewhat later t h a t tim fat-deficict,ey s y m p t o m s disappeared immediately, and that a dramatic recovery from the deficiency 100

Fats as the Source of Essential Fatty Acids condition occurred when lard, cod-liver oil, or liver was fed. It was shoals that the curative agents were such unsaturated acids as linoleic, hnolenic and arachidonic acids. It was suggested that the varying potencies of different fats in curing the tat-deficiency symptoms might be correlated ~ith the proportion of the component "essential" fatty acids in the particular fat under consideration. 2. Compounds active as essential faIty acids

The curative effects on the so-caUed fat deficiency disease could be traced to specific f a t t y acids present in the neutral fats. This was evident immediately after the first recognition of the deficiency. The glycerol m o i e t y was shown to be completely without prophylactic action. ¢1~ Saturated f a t t y acids such as stearic, palmitic, myristic, laurie, and lower acids were likewise found to be inactive, as was evident from the nega¢ive results obtained with coconut oil, with hydrogenated coconut oil, and with m e t h y l stearate. (12~ I n fact, EvANs and LEPKOVSKY(13) and SINCLAIR(14) have reported t h a t the feeding of hydrogenated coconut oil to rats on an otherwise fat-dcficicnt diet does not alleviate the fatdeficiency symptoms, but in fact actually accentuates them. As a result, the essential f a t t y acid requirement fi)r gro~%h is increased b y the concomitant ingeslion of such saturated fats. DEUEL and co-worl:ers (~5~ have recently confirmed the earlier findings as regards the deleterious effect of hydrogenate(l coconut oil when administered to rats suffering from fat-deficiency symptoms. SI_~CT~¢II~(16) has reported t h a t diets containing a considerable proportion of elaidin accentuate the essential f a t t y acid ".leficicncy in the same way as does h y d r o g e n a t e d coconut oil. Although oleic acid was at first t h o u g h t to have some shght activity as a therapeutic agent, ~°') Bt'RR, B~RR, and M~LLE~(17~ later showed it to be completely inactive in this respect. The fact t h a t the saturated f a t t y acids cannot cure the fat-deficiency sbmdrome is entirely reasonable, as deduced from our knowledge of intermediary metabolism, since the diet employed on which the deficiency develops contains both c a r b o h y d r a t e and protein. B a t h of these foodstuffs are readily conrertible to fats having such saturated f a t t y acids. This fact has been repeatedly demonstrated over recent years. (is) Since unsaturated acids such as oleic aAd m a y normally be derived from the saturated acids, [1.~ it is likewise obvious t h a t oleic acid could not be expected to exert a curative effect. The f a t t y acids which 1)revent or cure the fat-deficiency s y m p t o m s are therefore compounds which cannot be synthesized de novo in the body, and which must be obtained from the dict. The most effective unsaturated f a t t y acids which are classed with these so-called "essential" f a t t y acids are: the dicnoic acid, linoleic acid, c1°), (in 9,12-octadecadicnoic acid, the tricnoic acid, linolenic acid, (17~ 9,12,15octadccatrienoic acid, and the naturally-occurring tetracnoic acid, arachidonic acid,~:0L (2a} 5,S,11,14-eicosatctracnoic acid. A list of the unsaturated acids which have been tested for curative action in fat-(icficiency disease is given in Table 1. I01

Nutritional Significance of the Fata

3. Comparative biol~otency of eJsenlial fatty acids a. Requirement for linoleate Most of the studies on the curative effects of the essential acids have been made with linoleic acid, but there is no general agreement as to the optimum dosage required by rats. According to BURR,(1°~ no absolute values can be given for the amount of linoleate which should be'considered as optimum. I t has been repeatedly shm~n that amounts of linoleate as low as 5 mg will produce definite growth when tested x~ith rats on the fat-deficient diets, while BURR et al. ~°~ and HUME and associates (m reported marked response in body weight gains' when doses of 10 to 20 nag of the essential acid were fed. BURR and collaboratol's (e°l cited a figure of 40 mg as the optimum dosage, while Hu)tE et al. have recorded 42 mg (21) as the linoleate requirement. On the other hand, I~LtRTn~(2s) states that 30 mg of linoleate, or slightly less, is the most satisfactory level, and this conclusion has been supported by ~IACKENZIE and co-workers. ~ ) However, other workers reported augmented growth when the linoleate intake was considerably higher, as compared with that at lower dosages. TURPEINEN (24) found that 100 mg was the optimum value for linoleate in female rats. More recent experiments of the DEVEL group have placed the figure for linoleate requirement even higher for male rats. Although GREENBERG and associates (2s) originally postulated, on the basis of a straight line curve when log dose and body weight gain were plotted, that the optimum requirement fi~r male rats was at least 100 mg daily, later results indicated that the figure exceeded 100 mg. (°-9) Ultimately it was shown t ha t the maximum growth level was not attained even when 200 mg dosages were administered, nS) It was also found that the daily doses of 400 mg of methyl linoleate were toxic, although the possibility exists that the toxicity might be related to the high intake of methyl groups. Further experiments are necessary to establish the opti~num figure for linoleate in male rats. (a) Sex difference in requirement--One interesting development which was reported by GI~EE_~BERG el eL1.~29~was the demonstraticn of a sex difference in the requirement for essential acids. Although the optimum level for mate rats exceeds 200 mg per day, ns} that for females is only a fraction of this value. According to earlier work, (2s~the requirement based on growth response appeared to be in the neighbourhood of 10 to 20 mg for females ; in later experiments, the highest growth was obtained on a 50 nag dosage, while growth was actually depressed when the linoleate intake was increased to 100 mg per day. The differences in essential filtty acid requirement between the male and female rats were later shown to be independent of the ~-t.oeopherol reqmremen~." - cas~

b. Bequiremem for linoIenate Although the term, "essential fi~tty acids" is generally used, BURR''°~ considers that any one of the acids alone may serve to cure the deficiency symptoms. Bv1~R and associates (m reported t hat tile growth-pronmting effects of linoleie and linolenic aeidswere additive. 3IARTI~-,°'5~ howexer, disagrees with this view. 104

Fats aq the Source of Essential Fatty Acids I t was later found that linolenic acid appeared t o b e m o r e active than linoleic acid in its anti-dermatitis action, and equal to it in its grown,h-promoting activity. ~°~ In recent experiments of GREENBERG e2 al., ~2sl linolenic acid, fed alone, w a s found to b e m a r k e d l y inferior to linoleie acid, both a s a growth-promoting agent and in its curative effect on the dcrmatitis of fat-deficiency disease. Thus, no appreciable gro~th response was noted when the daily dosage of linolenate w a s 10 mg, although the same dosage of linoleate gave a marked response. However, when as small an amount as 10 mg of linoleate was administered to the rat on the fat-deficient diet, together with 10 mg of linolenate, the g r o ~ h response w a s as great as t h a t to 20 mg of linoleate alone. GREE~BERGet al. ~2s~ postulate that a "sparking action on the part of linoleate is required before linolenate can play its role as an essential acid." c. Requirement f o r arachidonate

Although arachidonic acid is not a normal component in vegetable fats, it is considered by TURPEI-~EN!23) and SMEDLEY-MACLEA.~and N~:~.~c~'z~that it is the principal unsatllrated f a t t y acid required by the animal organism. As the result of a number of independent investigations by B.taKI et a l., ~361 by Nu.wN and SMEDLEY-~[ACLEAN,(37) by REISER,(38), (39l and by I~IECKEttO~'F and coworkersJ 4°1 it has been established that both linoleic and linolenic acids undergo an interconversion in the animal organism to give rise to arachidonic acid. WIDMER and HOL.~L~.~¢4~ reported that, when fat-deficient rats were given linoleate supplements for two months, increased levels of arachidonate were found in tim heart and liver. In negative control rats which received stearate or oleate, no increase in polyunsaturated ~cids in these organs could be noted.* ]f arachidonic acid is the ultimate unsaturated acid required by the animal organism, one would expect it to exhibit the highest biopotency of any of the essential f a t t y acids. Although the experimental results have been somewhat divergent in demonstrating such a superiority, the bulk of evidence would seem to suppor~ the vie~]~oint that arachidonic acid is superior to other unsaturated acids in supplying the need for essential fatty acids. TURPEINEN,¢-~3lin 1938, reported that arachidonic acid possesses three times the biopotcncy of linoleic acid when tested by the gain-in-weight teclmique. Using the same index of activity, Hu.~tE and co-workers ~°-~l found the ratio of biopotencies of methyl arachidonate and methyl linoleate to be 2 ; 1. On the other hand, in the earlier experiments of BURR, BURR, and .~[ILLER(17l an inferior growth-promoting activi+,y was actually noted for methyl arachidona~ when fed with a mixture of methyi linoleatc and methyl linolcnate. These workers later indicated that the methyl arachidonate sample was one of poor quality. * E d i t o r ' s N o t e : T h e m e t a b o l i c i n t e r c o n v c r s i o n s o f t h e esgcntiM f a t t y a c i d s a n d t h e fi~ctors aff,'cting th,.m arc subj,'ors o u t s i d e t h e scope of the p r e s e n t d i s c u s s i o n . T h e r e a d e r will l l n d t h e ~ discussc:l in l h e f o l l o w i n g papt, rs: (1) l'roc. T h i r d Cottf. Research A m e r . 3[eat I n s t . (1951) 1. (2) Fette ,ttz¢l ~eifen 53 (1951) 332. (3} T h e Vit,imin.~, A c a d e m i c Press, N e w "~'ork, (in pr,,ss). (4) A r c h i v e s l::iochem. ]liopt~ys. :17 (1952) 90; ibi,l. 41 (1952) 266. See also chapt~.r o n A u t o x i d a t i o n of F a t s a n d R~'lated S u b s t a n c e s , .,~.ction o a BiologiCal Significance of Lipids, p. 92.

105

:Nutritional Significance of the Fate However, BtrRR and collaborators, (2°~ using some of the same arachidonate a s had been used by TURPEI~EN,12a~were unable to confirm the fact t h a t arachidonic acid exhibits a superior biopotency as compared with linoleic acid. Using an especially pure sample of methyl arachidonate and a new procedure for the bioassay of essential fatt,: acids, GREEYB~RG and co-workers ~29~have reccntly reported that, whcn administered alone, methyl arachidonate possesses a biopotency 3.5 times that of linoleic acid. On the other hand, when the biologicM activity of arachidonate was calculated from the gro~%h response observed when arachidonate and linoleate were fed together, the ratio of biopotencies of arachidonate to linoleate was 6-2 : 1. The weight of evidence would seem to indicate that arachidonic acid has the highest biopotency of the several essential acids.

4. Procedures for determini'ng the. esseT~tialfatty acids a. Chemical nzetlugd.sfor the determination of esse~vtial fatty acids When a large proportion of the fatty acids present in a fat consist of one or more essent,_'al f a t t y acids, as 0ceurs in the case of the common vegetable fats, quantitative analyses can be obtained by the use of fractional distillation under a high vacuum. Subsequent identification of the fractions can be made from saponification equivalents, iodine and thiocyanogen numbers, refractive indices, ultraviolet, infrared, or Raman spectral patterns or, in special cases, by the use of other physical or chemical constants. ~4z-44) Fractional crystallization of acetone solutions of the fatty acids at low temperatures, as developed in the laboratories of J. B. BROW~, have been useful in the separation of such naturally-occurring unsaturated acids as oleic, linoleic, hnolcnic, erucic, and ricinoleic acidsJ 45~, (4~ The above procedures may be applied jointly to identify and to furnish quantitative data on the amount of unsaturatcd acids present, when they occur in comparatively large amounts. However, thcse mcthods are of little value for the estimation of the esse;atial acids when the latter are present in the low concentration at which they occur in some vegetable fats, as well as in most animal fats. In the latter case, the procedure of BRICE etal. (47) has been most useful. This involves the isomerization of the unsaturated fatty acids which are nonconjugated to the corresponding conjugated acids, by heating with alkali. The concentration of each of the conjugated acids can be asccrtained by spectrol)hotometric analysis. However, the information obtained by chemical analysis may not always be valid. Dienoic, trienoic, and tetracnoic acids exist which do not possess biopotency, although they may contribute to the values obtained by spectrophotometric analysis. I t would thus appear that the exact evaluation of the potency of an unknown oil, especially when the pol3nansaturated acids are present in minimum amount, can be determined only by bioassay methods. b. Bioaasay procedure for the ddermi~ation of e~ssenlial fatty acids A bioassay similar to that cmployed for vitamin A can be used to determine essential acids. That this procedure is wdid became evident when GRi~e~BF.RG etal. ("-s) demonstrated t h a t the curve for the log dose plotted against gain-in106

Fats as the Source of E~ential Fatty Acids weight, when doses of 5, 10, 20, and 50 mg of linoleate were administered to male rats having a fat deficiency, was a straight line after 3 weeks, continuing up to 12 weeks. The results of the control tests as recorded by GREENBERG and associate~ (28) are plotted in Fig. 1. In tests on a group of hydrogenated fats (margarines and shortenings), butters, and cottonseed oil, in which the essential acids were determined both spectrophotometrically, by the procedure of BRICE et al., ~4~ and by the bioassay procedure, there was a fairly good agreement in general. However, although the essential acid content of one shortening prepared b y selee~ive hydrogenation was very close when determined by the chemical and bioassay methods (2.1% QO

o

D

iOO

C

~

, 20

O1 -2C o

I

l

I

O'2

0-4

O'b

I 0-8

I

I

l

I-O

1,2

1"4

LOG DOSE

I

f

I-b i'J~ M~LLICRM4$

I ~O

Fig. 1. The gain-in-weight of previously depleted male rata plotted against the log dose of methyl linol-ate fed for 3, 6, 9, 12, and 15 weeks. Doses of methyl linoleate were 5, 10, 20, and 50 mg daily2 ~8~

vs. 2-46% respectively), a wide difference in values was obtained in another sample prepared by non-selective hydrogenation. In the latter case, only 6.8% of essential acids were found by the spectrophotometric procedure, while a figure of 13% was obtained by the bioassay procedure, t15) 5. E,~se~vtiaZfatty acid co~vtcnt of various fats and oils

Linoleic acid is the main essential fatty acid found in vegetable oils, altheugh in several instances (linseed oil and hempseed oil) large proportions of li.nolenie acid likewise occur. The essential fatty acids are usually present in highest concentrations i.u animal fats if large quantities of fats containing them have previously been fed to the animals. Small amounts of arachidonic acid have alto been reported in many animal fats. In general, the amount of essential t~cids is lowe~- ill animal than in vegetable fats. Table 2 summarizes the maximum amounts Of essential fatty acids which have been repor',ed in the more common vegetable and animal fats. 107

5~

.

Perilla scc(l 15slachio n u t Poppysced Rapeseed Safflower seed Sesame seed Soybean. Sm~flower seed Tobacco seed T o m a t o seed . Thorn applo Walnut . W a t e r m e l o n seed

l)e/tllllt

Almond Avocado Brazil a u t Cocoa b u t t e r Coconut Corli Cottonseed (~rapesced (-;ral)cfruit seed H a e k b c r r y tree seed 1 [empsecd | Ansced . Olive Palm

I"effetablefat

i

i

I

i

"i .l

'i •I

*1

•i •!

i

10"3 22.8 21 '1 2.6 39.1 50.,t 73'0 51.4 7~;'7 (;S.8 46-7 | 5.0 10.9 27-4 33"6 20'0 62"2 29"0 78.0 40-4 58.8 68"0 74'5 38"2 74"5 75'5 65"8

19.9

(%)

Linolelg

10"0

8.1

3"5

24.3 60'9

0'1

(%)

Linolenie

E,~sentlal acids

t

B u t t e r tilt I (~s* B u t t e r fat I I "~) Egg yolk (hen) l{empseed oil diet Linseed oil diet Various diets P h o s p h a t ides Goose fat (body) 1ten tilt (body) ttuman fat O× d e p o t fat Pig d e p o t fat Soybean oil diet P e a n u t diet Cottonseed oil diet (12°+o) Cottonseed oil diet (8°~) Cotton,~ccd oil diet (40/o) Sheep d e p o t tilt Milk fat (cow) Milk fat (goat) Milk tilt ( h u m a n )

A ~imal fnl,+ :

~.8 1.5 q.O

"i i

i 'i

41"9 24'9 • 21'7 8"2 19'3 21'3 ll'O 5"3 15'6 38.(,) 19.7 26.8 18.2 13-3 5"0

3.2 4.0

5.0 5.8 4.6 5.1 2-2 7.1

0

Linolcic (%)

•1

i

Coconut oil .I Margarine oil I Margarino oil I [ i Margarine oil I1[ Margarine plus 1.5°]o c o t t o n s e e d oil , Shortcnix~g (selective) . "1 S h o r t e n i n g (non-selective) .I

Hydrogctmtcd fitta :(16~

H ydrogcm, ted and animal ]at~

°°

5.4

0.5

10,0 17-4 2.9

0"9 1-2

Linolenie (%)

Essential acids

Table 2. Relative m a x i m u m a m m t n t s o f esse~tial f a t t y acids in various vegetable and a n i m a l f a t s ~4~)

0'4

0-9 1"0 0"5 2.1

2.3

0"2 0'2

(%)

Arachidonie

Fats as the Source of Essential F a t t y _~cid~

6. Essential fatty acid requireme~vl in animals other than the rat Although most of the experimental work on fat-deficiency has been carried out on the rat, there is every evidence t h a t this dietary deficiency is a widespread one which m a y occur in a variety of different species. Thus, WHITE and eoworkerg ~9~ reported a syndrome in mice deprived of essential f a t t y acids which is similar to t h a t produced in the rat. DECKER and his co-workers ~Sm have recently published extensive reports of essential f a t t y acid deficiency in mice. RvssE~J~ and his eoUaborators were unable to produce an essential f a t t y acid deficiency in chickens. ~a~ However, it is possible tha~ the linoleic acid present in the cornstarch m, ~a~ m a y have been sufficient to prevent severe deficiency symptoms. REXSER~ has recently shown the essential nature of polyunsatu. rated f a t t y acids b y experiments in which he used more highly purified diets in which sucrose was substituted for starch as a c a r b o h y d r a t e energy source. I t has not. been possible to produce the fat-deficiency symptoms in the hog, although H m D r r c n , LEA, and PEI~ELTY~'~°-~ are of the opinion t h a t the pig requires linoleic acid. However, the ELLIS gwoup ~3-56~ reported t h a t the iinoleate content of lard dropped from a normal value of 7 to 15% to 1.2% when the diet of the hogs was practically fat-free. Moreover, when the diet contained vegetable oils high in essential f a t t y acids (such as soybean), the depot fat of the pig was found to contain as much as 39% of linoleie acid and 0-5°/O of linolenic acid. ~5~ A similar condition obtains in the egg yolk fat of the hen fed on hempseed oil. ¢57~ GULLlCKSCgNand collab,>rators wedng of the iodine number of the blood lipi
~utritional Significance of the Fats administration of lard, the improved clinical condition was associated with an increase in the hnoleate content of the blood, but not necessarily with an augmentation of the arachidonate. (es~ The data on the essential f a t t y acid content of blood serum of normal and of eczematous patients are summarized in Table 3.

Table 3. The li~oleate and arachidonate in the blood serum of normal ,ubjec~ and of eczen,~tous patients (~2~ Cond~

Number o]

of patients

% of total acids

pooled samples

Linoleate

A rachidon~

18 8 35 12

3.20 4"20 4.80 5-20

1-34 1'60 2.83 2-90

I

Young patients with eczema ill Adult patients with eczema Control subjects I . Control subjects II "1 i

Althocgh it would appear probable t hat a correlation exists between the essential fatty acid level in the serum and the occurrence of certain types of eczema, in the baby and even in adult man, there is no other evidence which indicates she necessity of these substances in man. For example, ]3ROW~" and co-workers (71J carried out an experiment on a single normal male subject who maintained himself for six months on a low-fat diet; no harmful effects were noted, but a 50% reduction in blood linolcate and arachidonate was observed. This decrease was entirely out of proportion to the drop in total blood lipids. I t is apparent that the common animals can be divided into two categories. In one group, which includes mice, rats, and dogs, the deficiency of essential fatty acids in the diet is followed by cessation of ~o~-th, by kidney lesions, by effects on the epidermis and, in some cases, by death. In the second class, which includes pigs, eslves and cows, and man, the only symptom which can be observed is the effect on the unsaturated acids in the blood which follows a prolonged fat-free dietary regimen. As would be expected from the results on rats, this effect is much more readily produced in the young animals than it is in the adults. The reason for the differences between the two categories of animals is probably to be ascribed to variations in the level of requirement. In the rat the optimum level is extremely high, being in excess of 200 mg for a young adult male rat. I f a similar requirement obtains for man, this would correspond to a value of 13.5 gm daily if comparative dosages are based upon surface area. One must conclude th at the animals in the second group in all probability require essential fatty acids, but that the required level is considerably lower.

7. ~lIelaboli~ effects of essential fatty acMz The main function of the essential f a t t y acids would appear to be related to the metabolism of the skin. Just how these acids act to carry on this fimetion has not been determined. 110

The Relationship of Fat Intake to Protein Metabolism One of the results of f a t t y acid deficiency which has been repeatedly recognized is the high level cfmetabohc activity which obtains. KU~'KV.Land WrLL~aS(m have recently reported t h a t essential fatty acid deficiency in the rat~ causes a. m~rked increase in the cytochrome oxidase activity in the liver, as well as a slight augmentation in choline oxidase activity; no alteration occurs in the extent of succinic oxidase oxidation, but a marked decrease in endogenous activity results. These authors are of the opinion that the increased, cytochrome oxidase activity in the hver, and possibly also in the tissues, may largely account for the increased meOtabolic rate. I t is beheved that this may likewise represent a specific metabolic function of the essential f a t t y acids. It i s likewise possible t h a t linoleic acid plays a role in the metabolism of choline. Thus, ENGEL(74} has suggested that this dienoic acid is essential to enable choline to exert its lipotropic effect. SMEDLEY-]~IAcLEANand Nu~.~ (2~} reported that fat was not laid d o ~ in body fat depots in +~he absence of arachidonic acid. Another possible role of the essential fatty acids has been suggested by other work of S.~TEDLEY-~IAcLEA.Nand NuN-_~,~vs}in which Walker tumour tissue was implanted in rats fed on a normal diet. Coincident with the development of the tumours, there was a marked fall in the ratio of highly unsaturated f a t t y acids to the fat-free dry weight in the subcutaneous tissues. This is interpreted to mean tt.at, when new tissue is formed in the rat, arachidonic or some similar acid is used up in the promotion of this growth. The acid necessary for this process is dra~wa from the subcutaneous reservoir. The phenomenon of gro~rth may well be associated, to a gTeater or lesser extent, with the metabolism of the essential f a t t y acids. The problem of the mechanism of the action of essential fatty acids in the animal body is thus as yet largely unsolved. 2. The Relationship of Fat Intake to Protein Yletabohsm For a number of years, there has been a general agreement that carbohydrate rather than fat possesses a protein-sparing action. ]n the classical experiments of GRXHAM LUSK,~7~.' it was shown that, on a constant protein intake, the urinary nitrogen level was depressed when carbohydrate was ingested concomitantly, but not when fat was the additional foodstuff eaten. However, in more recent experiments, SwA~so~ and her co-workers ~ have been able to demonstrate that fat plays an important role in one phase of protein metabolism in which carbohydrate appears to be without effect. V,rhen rats were given protein-free diets over a sufficient period, until the urinary nitrogen had reached the so-called "wear and tear quota" or nitrogen minimum, the sparing action on protein continued when the caloric intake was redllced to 50~o of that required, provided fat was included in the ration. Moreover, on the fat-containing diet, the nitrogen excretion was only slightly elevated when the caloric inf~al
Nutritional Significance of the Fata excessive loss of protein was d e m o n s t r a t e d , as determined b y u r i n a r y nitrogen values, when the diet furnished only. 25% of the required calories. Similar rest~ts were also obtained when metliionine was incorporated into t h e proteinfree dict. These d a t a are s u m m a r i z e d in Table 4. Table 4. Nitrogen excretion of rats r ~ e i r i n g protein-free diet alone or with methionine and which was fat-free or contained 2 0 % fat at 100, 75, 50, 250o of required caloric levels (77) Per cent .Basal N.excretion Experirr~entalN.excretion Differenc~e caloric requirement fed Fag-freediet 20°of at diet Fat-freediet 20%fat diet Fat-free diet 20%fag diet Protein.free diet alone

100 75 50 25

mg 280 272 283 249

mg 295 310 265 290

mg 185 211 594 818

mg 210 263 289 544

315 310 298 329

177 204 266 577

188 215 256 523

mg -- 95 -- 61 ~- 311 ~ 5~9

mg -- 85 -47 -! 24 -'- 254

--

--

Pro~inffree diet + methionine

100 75 50 25

295 270 257 28l

118 --

66 +

9

127 --

,

~- 296

95

42 -}- 194 --

i

SAMUELS, GILMORE, and 1-{EINECKE(Ts) have d e m o n s t r a t e d the sparing action of flit on protein metabolism, b y t h e use of an e n t i r e l y n e w t e c h n i q u e . R a t s were fed on a diet containing 8 0 % of the t o t a l calories as c a r b o h y d r a t e , protein, or fat for 28 days preceding a period of fasting. I t was reported t h a t t h e lowest nitrogen excretion obtained in the group which had previously received the f a t diet. Moreover, the rat.s which had originally received the fat diet prior to the fast survived the longest. This p h e n o m e n o n is, in all probability, to be ascribed to the greater sparing action of the fat on protein metabolism ; the p r e - m o r t a l rise in protein metabc, lism is thus retarded, x~fith a resultant prolongation of the survival period. The results of these tests are s u m m a r i z e d in T a b l e 5. One possible explanation for the striking results given in T a b l e 5 is t h a t the previous dietary history conditions the e n z y m e systems which are active during a subsequent fast period. (79) I n the case of the rats which h a v e received exogenous fat as the sole source of calories during the 28-day period prior to the fast, the e n z y m e systems are completely adjusted to the processes involved in the efficient utilization of fat. When the supply of fitt is endogenous r a t h e r t h a n exogenous, no alterations in oxidation p a t t e r n are invoh'ed, a n d t h e same metabolic processes proceed without interruption. I n the case of the r a t s which have previously received either the protein or the carl)ohydrate regimen, a new 112

The Relationship of Fat Intake to Protein Metabolism

Table 5. The at,erafte nitrocjen excretion of rats on special diets and durina a subsequent period of fasting with voluntary activity, and of rats undergoing/vio!en~ exercise ~Ts~ .

i

Period during fast

Category

1Vitrogen excretion in gram/day/rat* 1

Protein diet

!

] Carbohydra2e diet l

I

.Fat diet

I

(days)

f

Total nitrogen excretion /

1-369 :l: 0.38 0.3375 -4- 0-00530-3165 ± 0-0080 (on diet) 0-4 10.2547 ~: 0-0314,0.1770 4- 0.00680.1104 :t: 0.0057 6-11 0.1427 i 0"039210'1318 i 0-0111 0-1004 4- 0-0094 13~Jeath -0-2286 ± 0-0131'0-1407 :t: 0.0272 Before

Nitrogen excretion during 1

work (rag/100 revolutions)~ ,

0-0521 ~ 0-00950.0465 i 0-0058 0.0342 ± 0.0044

m

i

* Including Standard Error of the Mean

pattern of oxidation must be set up as soon as the glycogen ~tores and stores of deposit protein are exhausted. Finally, t h e shorter survival of the rats which had previously received the protein diet might be explained b y the fact t h a t the animals were unable to adjust themselves to an economical use of protein during fasting because they had become accustomed to a high protein intake with a large wastage of this foodstuff in the pre-inanition period. Another approach to the question as to the protein-sparing action of fat is the s t u d y of efficiency of growth in y o u n g animals on low-fat and on high-fat diets. :FRENCH, BLACK, and SwiFt ~s°) have reported more efficient and more rapid gains-in-weight in rats receiving a low-protein diet when 30~o of fat was present than when only 2 % of this foodstuff was involved. GEIOER (81~ has receni.ly shown t h a t the sparing effect of eystine on protein metabolism occurs only when fat is present in the diet. This confirms an earlier report of SALMON,(82~ ROGERS, :F~RC,USO_~, FRIEDGOO]), and VA~s, (83~ experimenting with proteindepleted rats which were then subjected to 70~o partial hcpatectomy, found that the nitrogen balance was more favourable a n d t h a t greater increments of liver protein accrued in rats kept on a diet composed of 30% of fat than on one containing 3')/0 of this foodstuff. Such results were likewise obtained when the caloric intake of the animals on the 30o/0 fat diet was restricted to the ad libitum consumption of the 3 % fat regimen. A possible exI)lanation for the behaviour o f fat on protein metabolism m a y be deduced from the e x p e r i m e n t s of PEARSON and PA-~'ZER.(u~ These workers reported t h a t a significantly greater loss o f phenylalanine, valine, lysine, and methionine obtained in the urine and faeces o f growing rats on a fat-free dict than on one which contained 8o/o of corn o i l Moreover, similar but less striking differences in favour of the fat diet were registered in experiments with adult rats. The report of SCHWL~.~IER and McGAvACK(sS~ indicat,cs t h a t the proteinsparing action of fats m a y likewise have application to man. I t was found t h a t 113

Nutritional Significance of the Fats

the only condition under which a low nitrogen excretion could be maintained in human subjects on protein-free diets, fed at considerably below the levels required for calorie equilibrium, was when the rations contained considerable propor ;ions of fat. Although one must continue to accept the fact that carbohydrate serves to spare protein on mixed diet.s, it seems necessary to include an additional corollary that fat and fat alone may like~ise exert, a protein-sparing action when animals are subjected to low-protein or protein-free diets. 3. Growth as Related to Fat Intake

1. Experiments in which ad libitum diets were employed There is considerable evidence in the literature to prove that the growthpromoting properties of diets are pro~essively improved as the proportion of

F!

C.~.c

2~ 24C

2cc

0 J60 120

i i,

i

~

,,

-2,

COTTON

SEED OIL

MARGARINE FAT

Fig. 2. The average body weight of weanling male rats at the start (to top of lower bla;dll space), after 3 weeks (to top of solid black), after 6 weeks (to top of stippled area), after 12 weeks (to top of cross-line area), and af,~er 18 weeks (to top of upper blank area). The figures in tho lower blank area are the diet numbers (described in text), tss~

fat is increased. HOAGLANDand S N I D E R {$el w e r e the first to prove that, on diets containing 5, 30, or 55% of four types of lard, rats grew best on the regimens containing 30% and 55% of fat. In later tests carried out with incrcased proportions of vitamins, these workers csT) confirmed their earlier results; ~hen steam-rendered lard was fed at 5, 15, 30, or 54% levels in the diet, optimum gro~th obtained in the animals receiving the 30% fat diet, and minimum growth was noted in the group receiving the 5% fat diet. DEUEL and co-workers :8a) have also noted that weanling rats fed on diets containing severM levels of cottonseed oil or margarine fat showed tile best growth when the fa+ocomprised 20 to 40% of the total weight of the diet. The authors suggest that tl:e optimum 114

Growth as R e l a t e d to F a t I n t a k e

level of fat as estimated from these tests would be approximately 30% by weight, or 50% of the calories. (~) Fig. 2 illustrates the mean body weights, at several periods, of weanling rats fed over an 18-week interval on 0% fat without (diet 60a) or with (diet 60b) essential fatty acids, or with diets containing fat at

2OC

)6o

$o

4o

MALES

FEMALES

Fig. 3. The averag e b o d y weight of weanling male and female ra t s a t the s t a r t (to top of lower b l a n k space), after 12 weeks on restricted calories (to top of solid bl a c k area), after 3 weeks on diets ad libilum (to top of stippled area), after 6 weeks on diets ad libitum (to top of cross-lined area), aft<~r 9 weeks on diet~ ad libiturn (to top of u p p e r stippled area), and after 12 weeks on diets ad libltura (to top of upper b l a n k area).'*°) The fa t was cot tonseed oil.

the following levels: 5% (diet 61); 10% (diet 62); 20% (diet 63); 30% (diet 65); 40% (diet 64); 50°/o (diet 66); and with a mixture of margarine fat (7-2%) and cottonseed oil (2-8%) (diet 67) and with the stock diet (S). 2. Expcrimenls in which paired-feeding tcst~ were employed

In the experiments of FORBES and co-workers (gz) carried out with pair-fed rats on isocaloric diets containing 2, 5, 10, or 30% of fat (mostly lard), the gains-inweight, the digcstibility of the fat, and the retention of nitrogen displayed proportionally greater efficiency as the amount of fat in the diet was increased. In another series of tests on growing rats, carried out with 2, 10, and 30% fat but with increasing proportions of vitamins as compared with that in the earlier series, F0aBES and collaborators (9~') proved that the greater efficiency of the diets with increasing fat eontent Was associ~ted with significant gains of fat and energy and with a decrease in the total heat production. In expcriments with mature rat~, again employing diets containing 2, 10, and 30% of fat, FOanES ct al. (~a) notcd that the fat content of the diet had litC,le effect on the utilization of protein, but that the energy loss aauociated with the utilization of isocaloric 115

"Nutritional Significance of the Fats quantities of the diets decreased with the increasing fat content of the ration. In an extension of this work, FRENCH and co-workers (s°~ demonstrated t h a t the high-fat diet (30~/o) produced better .results, from the standpoint of energy utilization, than did the low-fat diet (2~/O), when the protein level was reduced from 2 2 o to 7 o . There was an increased weight gain; filrthermore, greater increases in fat content and energy, and a decrease in heat production, were associated with the high-lab regimen. In contrast to the uniformly superior results obtained with bigh-fat as compared with low-fat diets, which have been recorded by a number of investigators, HOAGLA:~D, SNIDER, and SWIFT(s4~ have recently been unable to find statistically significant differences in the rate and efficiency of gro~%h of young male rats fed diets containing 4, 9, or 14% of the diet (5.0, 10-98, or 18.27% of the calories respectively).

3. Experiments on fat fcedi~g following periods of restricl(~d food intake The improvement in nutrition and growth which results from a generous inclusion of fat in the diet has likexvise been demonstrated by SCHEER et al., (9°~ who varied the experimental conditions. When weanling rats were stunted by drastic restriction of the food intake to such an extent that practically no increase in body weight occurred over a period of 12 weeks, and the rats were then allowed to eat the various diets ad libitum, markedly superior growth was exhibited by the rats receiving 10, 20, and 40% of fat in the diet, as compared with that recorded for the animals receiving 0°/o or even 5% of cottonseed oil. This was particularly evident during the last period investigated (9 to 12 ~vecks after the start of ad libitum feeding). Essentially similar results were reported by SCHEER et al. (9~) in later studies on young adult rats. When these rats were subjected to severe caloric restrictions, the loss-in-weight of the rats receiving the high-flit diets was less than in the case of those receiving isocaloric low-fat diets. During the period of caloric restriction, a lower nmrtality rate was likewise observed in the group of rats receiving the high-fat diet. than in those on the low-fat regimen..Moreover, the rats on high-fat rations made a better recovery during a subsequent period when ad libitum feeding was reinstituted than did those on the low-fat diets. Finally, PEARSON and PANZER~84)have shown that rats fed a diet containing 8% of corn oil or lard ad libitum gained 29}/O more in body weight than did rats maintained on a similar diet minus the fat but with the addition of ethyl linoleate.

4. "Associative dy~andc action" as an explanation of the greater e~qcienzy in utilization of fat di,~Is The best explanation for the greater efficiency in growth which has been demonstrated for high-fat diets, as compared with isocaloric low-fat regimens, is the peculiar effect exerted by fats on the specific dynamic action of other foodstuffs. This behaviour of fats, which was first discovered by Follt~ES and Swlr*r, (9~ has been termed by them the "associative d.ynamic effect." It has long been known that, following the ingestio,l of fi)o(lstuffs, an increased 116

G r o w t h as R e l a t e d t o F a t I n t a k e

respiratory metabolism obtains over and above the basal metabolic level, du~hlg the period of their absorption, transport, and interconversion in the body. This is known as the "specific dynamic action." Proteins exert the greates¢ effect in this respect, followed, in turn, by carbohydrate and fat. Under certain conditions, such as external cold, the heat arising from the specific dynamic action of the foodstuffs m a y be utilized for warming the body, in lieu of a stimulation of the metabolism by shivering or by chemical regulation, and may thus produce the extra heat required to accomplish this purpose. (97) Under these circumstances, the specific dynamic effect would not represent waste energy, but would rather tend to result in greater efficiency in the utilization of foodstuffs. However, under most conditions, the specific dynamic effect represents a direct loss of energy to the body. When the specific dynamic effect is not needed for warming the body, due to an elevated environmental temperature, this excess heat is lost. Moreover, the energy produced by the specific dynamic effect of protein cannot be used to derive energy for mechanical work. (gs, (99) ttowever, ANDERSON and LUSK(99) did demonstrate that, when carbohydrate was consumed during work, no specific dynamic effect was exerted. There is no conclusive evidence as to the effects which would be noted, under these con(litions, with ingested fat. Thus, it should become apparent that any situation which ~ n d s to reduce the specific dynamic effect of foodstuffs will bring about an increased efficiency in utilization of the foodstuffs. It has been generally accepted that, under conditions favourable for demonstrating the specific dynamic effect, the activity due to each of the foodstuffs is additive. Some years ago, 5It']~LtN and LuSK(1°°) demonstrated, in tests on dogs, that the extra heat produced wken glycine (20gin), glucose (70 gin), and fat (75gm) were fed together was i)raetieally identical with the sum of the specific dynamic effects obtained when each of the foodsLuffs was fed separately. Ahhough the data of MrRLIN and LUSK appear quite conclusive, it is possible that the deductions may have 1seen erroneous, since the calculations were based only upon a 2-hour period (at which time the specific dynamic action was believed to be at a maximum) rather than upon the entire period during which the effect was exerted..Moreover, one might likewise question whether or not the period of maximum extra heat production might have been modified when the three foodstuffs were given together, sinr'e the presence of fat might be expected to alter the rate at which p,otein and carbohydrate could be absorbed. In their studies on rats, FORUES and SWIFT~96)have overcome these criticisms by determining the heat production over the entire period during which the dynamic effects were being exerted. The new procedure, according to ¢he~ workers, is more representative of comnmn nutritive practice, as compared with the measurement of the (lvnamic effects of single-tout- meals.. Moreover, the results are more significant, and are prol)al)ly more accurate, inasmuch as they are dcternlined as differences between the amounts of heat produce(1 at established levels of metab~)lism, without concern as to the beginning, maximum or termination of the specific dynamic effects of a single meal. Finally, no confusion arises in regard to the dynamic effects of the catabolized body nutrients. 117

Nutritioual Significance of the Fats T h e specific di}mamie effects per 1000 calorics were found to be as follows when the foodstuffs were fed separately to rats: beef protein, 323 calories; glucose, 202 calories; and lard, 160 calories. When glucose and protein ~'ere fed together, the response obtained agu~ed quite closely with the additive effects o f t h e two foodstuffs calculated from the pre~ious figures for the quantities of each foodstuff fed. On the other hand, whenever lard was fed, the calculated values were always considerably greater t h a n those actually found. Thus, when beef protein and lard were fed simultaneously, only l l 3 e x t r a calories were produced per 1000 calories fed, compared with a computed value o f 244 calories. This sparing effect was likewise n o t e d to a somewhat lesser degree when fat was fed with glucose alone or with glucose 0nd protein. I t would appear t h a t fat was t h e only one of the three foodstuffs to exhibit this so-called "associative d3mamic effect." A graphic presentation of the data of FO~tBES and S w a r t cge) is given in Fig. 4, while Table 6 supplies a breakdo~-n in the partition of the average daily intake of eneIgy by the rat. Table 6. Partition of average daily intake of energy per rat during 70 days (x°l~ Diet.s containing fat at let,de of Ca~gory

2%

o, 5/0

10%

30%

~854 799 [912 143

~854 799 713 342

854 799 456 599

2854 799 657 1398

Metabolizable calories

!601

t605

6i0

2615

Energy output, calories: In faeces In urine A~ heat .

128 125 H95

126 123 q65

121 123 154

123 116 2155

Energy retained, calories

406

440

456

460

Energy intake, calories: Gross

Protein . Carbohydrate Fat

T h e decreased expenditure of total calories which occurs when fat r e p l a c e s c a r b o h y d r a t e in isocaloric diets is sufficient to account for the greater efficiency with which high-fat diets are utilized for growth, as compared ~ t h low-fat diets. Although the authors do not pose an explanation as to the cause of the effect of fat on specific d3mamic actions it is possible t h a t the prolongation in the absorption period of protein and carbohydrate would lessen the m a x i m u m concentration o f these components, and thus reduce their specific dynamic action. FORBES el al. (96~ suggest t h a t it is thus not necessary to diminish the protein c o n t e n t of the diet (luring hot~ weather in order to ensure a low heat increment; r a t h e r one need o ~ y substitute fat for some of the carbohydrate. However, 118

Growth as Related to F a t Intake there is no p r o o f t h a t fat exerts a n "associative d y n a m i c action" in m a n as it does in the rat. 9EEF PR3TEIN

~090

i

1

CERE LOSE !000

202 I ['6019oo 1

CERELOSE 432 BEEF PROIEIN 566

BEEF PROTEIN LARD

LA~D

CERELOSE LARD

5/)3 437

4q5 $05

CE RELOSE BEEF PROTEIN LARD

300 394

306

Fig. 4. Dynamic effeet~ per 1000 eMories of gross energy of nutrients as affected b y nutrient combination2 °6j

5. Sexual maturity as related to dietary fat Although sexual m a t u r i t y is not as obvious an indication o? growth as gain-inweight, nevertheless it is a factor which m u s t necessarily be dependent upon this physiological change. T h e completion of sexual m a t u r i t y can be determined with reasonable precision in the female r a t from the opening of the vagina. The t i m e at which sexual m a t u r i t y occurs has not ordinarily been considered as one of the indices of nutrition, b u t t h e r e is no reason w h y it should not afford a satisfactory indication of this state. I n the e x p e r i m e n t s of DEUEL a n d co-workers, (ss) it was sho~la that, whereas nol'mal female rats did not become sexually mature, on a fat-free diet, until t h e y were a p p r o x i m a t e l y 81 d a y s of age, m a t u r a t i o n t o o k place as early as 70 d a y s

Table 7. The effect of fat level in the diet on the time whelz sexual maturity deretops in the female rat .Average time in days to maturity after start of ad libitum feeding in diets c~ntaining: Previous diet

o%/~t

5%f,a

~0%.f~

20%J~

49-0

45.5

44-8

44.1

11-9

10-5

7-7

9-8

4 0 °. O/ J

fat

I N o r m a l r a t s , 3 w e e k s old a t s~art, o n / diets ad libit,zm f r o m w e a n i n g '88) /

I

Restricted rats, 15 weeks old at start, after 12 weeks on restricted calories, anti then giyen diets ad libitum tg°~

60.2

14-7

119

Nutritional Significance of the Fats after birth in the group receiving 5 % of fat in the diet, and at 65 days in the animals receiving the 40% fat regimen. In rats which had received calories restricted sufficiently to p r e v e n t appreciable g r o ~ t h over a 12-week period following weaning, sexual m a t u r i t y did not obtain in a n y case, irrespective o f the a m o u n t of fat in the diet. However, when the rats had been allowed to consume the diet ad libitum, sexual m a t u r i t y occurred most p r o m p t l y in the rats on the 20% fat ration, folh,wed by those on the 40 ,°/o fat regimen. The d a t a are summarized in Table 7. 4. Pregnancy and Lactation as Indices oi the Nutritional Value of Fat The opinion is practically unanimous among nutrition workers t h a t pregnancy offers a better index of the nutritional value of a diet t h a n does g r o ~ h alone. I t is also generally agreed t h a t a still more precise evaluation of the nutritive value of a foodstuff m a y be obtained from an examination of the response of lactation. A n u m b e r of different workers have reported t h a t reproductive failure invariably ens'aes in rats raised to m a t u r i t y on fat-free diets. (x°2-1°4~ After prolonged gestation period and excessive vaginal bleeding, the young are generally born dead, or t h e y die shortly after birth. However, QUACKENBUStt and his co-workers (1°o found that, if the female rats on the fat-deficient diet were given as small an a m o u n t as 100 mg of ethyl linoleate 3 weeks before t h e y were bred, a normal gestation period was noted, and up to 83% of the young were weaned. ]~_UM3IEROWand associates ~a°5) reported that, when the fat-free diet was replaced by 5 % h y d r o g e n a t e d fat, the animals gave birth to living young which did not live more t h a n 72 hr; however, when the diet contained 5% of corn oil, 85% of the young were weaned. The mothers and newly-born young were deficient in araehidonic acid, but not in fat p e r se. The main differences occurred in the phospholipid fraction of the young. After corn oil, the phosl)holipids in the young contained 5 to 10 times more arachidonic acid t h a n did those from rats on a fat-free dict. The phospholipids from rats which had received hydrogenated fat contained twice as much of the acid as 0id those from the rats on a fat-fcee regimen. Although it is obvious t h a t m a r k e d differences obtain in pregnancy and lactation on fat-free diets, as contrasted ~ ith the results on fat diets, it is somewhat morc difficult to demonstrate variations in these physiological functions when different levels of dietary fat are fed. Thus, no differences in reproductive and lactation performance eouht be noted i,1 normal rats receiving 5 to 40°,/0 of fat. The results in all cases were fimn(t to bc superior to those obtained on a fat-free diet. ~ss) However, when rats were first subjected to ~ 12-week period of undernutrition followed b y an ad libit~on feeding period, SClfEER ct a U 9°~ reported t h a t the best performance (both in the n u m b e r in the litter and in the ~reaning weights) was obtained in the group receiving 400,'o of cottonseed oil in the diet. The data based upon these experiments are recorded in Table 8. Although the weaning weights of the individual rats do not exhibit very great differences, the total li~t.er weight of the rats receiving the 40% fat diet is 120

Work

Capacity

as an Index of the Nutritional

Value of Fat

Table 8. Reproductive performance of female rats recot~ring from a period of undernulrition i~°~ ,,,,,

,

Di~ IVumber

A t~rage Fat conterg

i

i

I

t at~ per litter

Average litter we~h$

3days

21 days

47.7 64.3 60-8 72-3

152 219 167 230 283

Average rat weight

3 days

21 days

7-1 8"4 7-2 8"1

gm 26-1 32-4 27-8 31-8 30.6

% 60b* 61 62 63 64

0 5 10 2O 40

5.3 6.8 7-7 8.5 8-9

* Containing linoleie acid.

approximately twice t h a t of the rats receiving the fat-free diet supplemented with linoleate. Confirmation of the improved effect of high-fat diets on pregnancy was obtained b y SCHEER et al. (gs) in another series of tests in which adult rats were first subjected to caloric restriction to such a degree t h a t the animals lost approximately one,half of their body weight over a 12-week period. After this interval, the animals were continued on the diets containing the several fat levels, but the food was given ad libitum. Breeding tests were conducted immediately following recovery. The following percentage of fertility was noted in the groups on the several.diets : 0 % fat, _90°//o," 5 % fat, 0 °/," 10~o fat, -,ga°//o," 20~o fat, 80% : and 40% fat, 80%. However, in no case were the mothers able to raise their progeny to the age of 21 days. The evidence afforded b y a number of types of experiments would seem to indicate the great ilnportance of fat in rendering normal pregnancy and lactation PoSsible. To what e x t e n t this favourable effect is to be traced to the essential f a t t y acid content of fat is not known. Although a 5% cottonseed oil diet would provide more than the required linolcate for the female rats, the best performances ir, pregnancy and lactation have been recorded wlmn 20% or 400/0 cottonseed oil diets were given. Therefore, it would appear t h a t some fact~)r in flits, in addition to tlm essential f a t t y acid content, is responsible for their beneficial effect under these conditions. 5. Work Capacity as an Index of the Nutritional Value of Fat Although c a r b o h y d r . t e h'-s long been regarded as the main source of energy for muscular exertion, a n u m b e r of observations indicate t h a t fat m a y play an even more important rote in this physiological function. KRoe.~ and LINDI{ARD(1°61 reported, niany years ago, t h a t work could be accomplished b y man approximately 10°/0 more efficiently when carbohydrate served as a source of calorics t h a n when fats were exclusively metabolized. On the other hand, these results entirely contradicted the exhaustive studies of ASDERSOX and LUSK, (99~ which were carried out with dogs. The latter investigators reported that, from a caloric standpoint, dogs ran with equal efficiency in the respiration cMorimetcr, 121

,X'utritional Significance of the Fats irrespective of whether t h e y had a R e s p i r a t o r y Quotient of 1-0 and so were using c a r b o h y d r a t e almost exclusively, or whether t h e y had a non-protein R.Q. o f a p p r o x i m a t e l y 0.71, which would indicate t h a t fa$ was being used almost exclusively. The latter condition was a t t a i n e d after a 13-day fast. In view of the conflicting d a t a of these two groups of investigators, the question of the source of fuel for muscle work can still not be considered as a settled problem.. E i t h e r species difference accounts for the variation (and this would seem improbable), or the conditions in one o f the experiments were not adequate for testing the hypothesis. More recently, the problem has been reinvestigated, using total working capacity as the index for comparison. W h e n rats receiving diets containing several levels of cottonseed oil or margarine fat were subjected to exhaustive swimming tests, it was found t h a t those on the higher fat levels had a greater capacity for work. The procedure for testing physical capacity, as developed b y SCHEER, D O R S T , C O D I E , and S O U ' L E , (107) w a s employed. This involved the determination of the duration of the rats' capacity for swimming when increasing loads were given to t h e m at successive 3-minute intervals. All animals had been adjusted to the same specific gravity before the s t a r t of the test. The results of these tests are summarized in Table 9. Table 9. The effect of fat level of the diet on the u,orklmj capacity c~s delermi~ed by a su,immbu} test (ss) i

of rats,

i

Period

Duration~( ett~mperincent secondsoffaton(bydietSweight):*c°ntaining the foUowing 61

62

Stock

60a 0%

60b 0 o~ ;o

aOo

lo%

i u,ee"]~" 512 6 (5)

580 (5)

802 (6)

777 (5)

1025 1:65)2 (4)

921 (5)

955 (4)

(5)

850 (17)

1084 (19)

846 (19)

1068 (15)

806 (10)

--

1 1 7 4 1065 (8) (25)

876 (4)

14%

63 ~0%

65 30 o/ ,'o

64

40%

6{}

50%

Cottonseed oi2 d / a

Female

6

Male

Male

8O5 (5)

•

12

645 (_°0)

.

12

485 (23)

692 (5)

922 • (3)

1223

Morgarinefatdi~ Male

--

--

1122 (lO)

• The fi~ures in parentheses represent the ntlmber of rxl-'rimcnt~ hlchlded in the average. Values in Italics were shown to be ~tatistically greater than those on diets O)a and {50b.

The work capacity was shown to be statistically greater in most cases in which there were sufficient tests to support this mathematical evaluation on the fatcontaining diets, as contrasted with t h e performance on the fat-free regimen. 122

Survival Time as an Index of the .N'utritional VaJue of Fat

.Furthermore, longer periods of work were noted in some cases in which higher fat diets were used than when the fat was present in lower amounts. Thus, it was found that the rats receiving the 20% level of margarine fat (diet 63) did better than those on the 10~o level (diet 62) which, in turn, had a statistically greater physical capacity than did the animals on the fat-free diet (diet 60a).( ss~ In later experiments by SCHEERand collaborators) ~°7~it was shown that physical c~pacity decreased markedly when the caloric intake was severely restricted. Under such conditions, the level attained was independent of the fat content of the diet. SA.~tVELS, GII~ORE, and REINECKE(78) have compared work capacity and survival of rats during fasting, following a 28-day period throughout which the rats had received diets containing 80~/o of the calories from fat, carbohydrate, or protein. Not only was spontaneous activity in general greater in the case of the rats which had previously received the fat diet, but also the total forced activity in this group greatly exceeded t h a t in the other groups. The latter phenomenon is partly to be ascribed to the longer period of survival by the fat-fed group. These data are summarized in Table 10.

Table 10. Effect of pr~'ious diet on spontaneous and forced activity and on the period of survival of ralz durimj fa.stin9 ~Ts~ Previou~ diet* Category

S p o n t a n e o u s a c t i v i t y , cage revolutions: 2-4 days 9-11 d a y s . 16--18 d a y s . Forced activity, cag6 revdutions Survival time, d a y s

Fa$

Carbohydrate

6730 4 - 915 (12) 8800 4- 1020 (13) 7960 4- 738 (7)

40404- 411(16)

19700 :t: 1780 (12)

1 3 8 0 0 ± 1630 (14)

6465 ± 1650 (9)

18-3 4- 0.44 (12)

15"5 4- 0-46 (13)

10'2 4- 1-68 (9)

8660 4- 1017 (14) 8 6 5 0 ± 0000 (3)

Protein

3600 47220 :i:

262 (7) 500 (4)

(0)

* Including Standard Error of the Mean. The figures in mrentheses designate the number of the rats in each group.

6. Survival Time as an Index o! the Nutritional Value o! Fat

The ler,gth of life and the duration of survival during fasting are two nutritional indices which are of importance in the evaluation of the nutritive value of a foodstuff. Although there are no data available to answer the question as to whether or not the lfigh-fat diets have a superior effect on longevity as compared with other foodstuffs, the recent experiments of SAMUELS, GILMORE, and ]~EI~ECKEits} have indicated t h a t fat is the best protective foodstuff in preparation for a subsequent fast period. V,rhen rats were fed on diets containing 80% of fat, carbohydrate, or protein for 28 days prior to fasting, the survival (luring the period of inanition was statistically greater when the pre-fimt diet was fat 123

.N'utritiona! Significance of the Fats than when it was carbohydrate, while the latter foodstuff was again distinctly superior to protein as judged from the survival time. These data are summarized in Table 10. The longer survi\'al time following pre-feeding with fat, as compared with that after tim administration of protein or carbohydrate, is probably to be traced to the fact t hat a metabolic pattern is developed during the pre-inanition period, when fat is given, which is especially favourable for a prolonged survival. SA~tt'r.LS et al. (~8~ showed that a marked sparing action affecting the protein metabolism obtained during the fasting period when fat had been the previous food, as compared with that when the pre-inanition regimen bad consisted of carbohydrate. (~°s) ROBERTS and SA.~ItZELS~1°9~, (u0~ reported t h a t rats which had become accustomed to a high-fat diet had a markedly lower susceptibility to insulin injection than did animals which were receiving a high-carbohydrate ration. This resistance to insulin on the part of the fat-fed rats was ascribed to a prolonged retention of liver glycogen in these animals; it was later suggeste(l cm~ that rats on a diet in which 85% of the calories are derived from fat develop a sparing action for liver glycogen. In support, of this theory, it was found that hepatectomized rats which had prcviousl S received a fat regimen were free from h3q3oglycemic symptoms for a longer period after the opcrati(m than were rats which had received a highcarb(~hydrate diet prior to the removal of the liver. In experiments on isolated tissues, GILMORE and SAMt'ELS~;9~ noted 5hat e. difference in glucose utilization 9btained in tissues removed from animals previously on the fat diet and from rats in which carbohydrate had been the pre-fasting regimen. These experiments support the earlier data of LUNDBAEK and STEVE-XSO~',cu2~ who reported that the diaphragm of a carbohydrate-fed rat utilized glucose in vitro at approximately twice the rate which occurred in the muscle of the fat-fed rat, when both tissues were immersed in ,~ glucose medium. TE)IPLETOS and ERSHOFr (113~investigated the relative influence of the several foodstuffs on survival, making use of another procedure. When the stress employed was thyroxine, it was fi)und that rats fed fat (margarine fat) or carbohydrate (sucrose) lived appreciably longer than those which received protein (casein). No significant differences were noted in control tests when saline was injected instead of the thyroxine solution. Moreover, when another stress was used, namely a low temperature, and the rats on the several diets were exposed to an eavironmep.t of 2°C, tlic fat-fed and carbohydrate.fed rats had greater resistance than did the protein-fed znimals. On the other hand, no difference related to diet was observed when the temperature to which the rats were exposed was 23°C, 7. The Effect of Dietary Fat on Vitamin Requirements 1. T h i a m i n and fat

I t has long been recognized t h a t tile metal),~lis:nl of thiamin and t hat of tilt are int.erreh~ted. As early as 1928, Ew~xs and LEPKOVSKY~O-L~u4~discovered that, on diets adequate except fi)r their content of vitamin B1, the onset of polyneuritie 124

The Effect of Dietary Fat on Vitamin Requ;remcnt~

syrnptoms was retarded on high-fat diets; on the other hand, when highcarbohydrate diets were employed, polyneuritis developed a t an accelerated rate. P r o o f t h a t the behaviQur of fat is a generalized effect was afforded b y the fact t h a t m a n y vegetable and animal fats were found to be equally efficacious in retarding the onset of polyneuritis. Thus lard, coconut oil, synthetic coconut oil, hydrogenated coconut oil, partially hydrogenated cottonseed oil (Crisco), cottonseed oil, and perilla oil were all effective. (115~, (116~ The only fat which did not possess a thiamin-sparing action was hydrogenated pcrilla oil; this product, which melted at 67.5°C, was believed to be inactive, because of its low digestibility. (ns~ ]n addition to digestibility, several other factors are of importance as related to the sparing action on thiamin. In the first place, unsaturation p e r se does not seem to play a role, except when complete saturation of a fat is associated with its defective absorption, as has already been mentioned. Thus, Evn.~s and LE]'KOVSK¥ (116~ found t h a t fats having an iodine number of only 8 spared t h i a m i n as effectively as did a highly unsaturated oil with an iodine value of 187. • Such saturated triglyceridcs as tricaprylin and trimyristin were found to be very effective in sparing vitamin Bx, although ~.ristearin was inactive. ~z~r) The latter fat is now known to be practically unabsort)able b y the rat. (11sl, (ng~ According to EvA.~s and LEPKOVSKY, (116) neither the precise melting point nor the degree of saturation of the several other natural f a t t y acids appears to influence this activity. On the other }rand, SALMON and GOOD.~tA~(lz°: demonstrated t h a t the variations in the thiamin-si~,aring action could be demonstrated in synthetic esters of the f a t t y acids; the m a x i m u m vitamin-sparing action was found with the Cs-fatty acid ester. In addition to the fact t h a t the s y m p t o m s of polyneuritis are delayed in rats b y the presencc of fat in vitamin B~-free diets, it has also been shown t h a t other symptoms associated with thiamin deficiency arc retarded b y this dietary procedure. Thus, MAcDo.XALD and McHE_~RX"(~-~) were able to demonstrate, in 1940, t h a t the onset of bradycardia, which is characteristic of thiamin deficiency in the rat, was ~tlso postponed when fat was included in the diet. I t was likewise shown t h a t a n o t h e r symptom of thiamin deficiency which is typical of the polyneuritic syndrome, namely the augmented excretion of bisulphite-hinding substances, is also modified by the presence of fat in the diet. (L~°-r'a) This latter effect docs not appear to be caused by an interaction hetwccn the fat and thinrain in the intestine, since it is also observed when the thiamin is administered parcnterally. (~'4) L a t c r experiments of BA.~En,II and YUDKIS (123~demonstrated t h a t k i d n e y slices from rats on difterent diets invariably showed a defective oxidation of p y r u v a t c , which could be corrected by the addition of thiamin to the respiring tissue. This condition ol)tairicd irrespective of the diet of the rat from which the tissue had been lemoved. I t is therefore evident t h a t the behaviour of fat in inhibiting the production of the p y r u v a t e (bisu!phitebinding) compounds is to be a t t r i b u t e d to a (lecrcase in the mc'.abolism of the substances from x~hich the compounds are ,h, rived rather than to an alteration i n the 1)athway fifllowcd b y their oxidation. According to MEL.~ICK 125

Nutritional Significance o f the Fats

and :F1EI,D,(12a~ fats which show the sparing action do not contain any thiamin. Apparently the presence of fat in tl~e gastrointestinal tract can influence the bacterial synthesis of thiamin. Thusl WU[PPLE and CHURCH(126~ were able to show that, on a thiamin-deficient diet, the faeces of rats contained some thiamin if lard was incorporated into the diet. However, when the lard was omitted. from the ration, the thiamin acth~ity of the stools was completely lost. In human subjects, carbohydrate ingestion has been s h o ~ to cause a decrease in the output of thiamin in the urine, which would seem to indicate that a greater utilization of thiamin occurs under these conditions. On the other hand, in spite of the fact that a high-fat diet reduces the thiamin requirement in experimental animals, (2~, (x27~,(12s~ it has been impossible to demonstrate that such increases in fat consumption augment the urinary excretion values of this .¢itamin.(129~, (~a0~

In later work, ~VmPPLE and CHURCHclal) have adduced evidence to indicate that thiamin has a function in the synthesis of fatty acids from carbohydrate. The demonstration that this transformation is continually taking place may be attributed to SCHOE.N'HELMERand RITTENBEttG, (la2) who carried out experiments in mice, using tracer substances. These authors have shown that there is a rapid turnover not only of the phosphorus-containing lipids of the kidney and liver but also of the fat deposits ; this necessitates a continuous conversion of carbohydrates into fatty acids under normal dietary conditions. If, when L,t is fed, the extent of the carbohydrate --> fat conversion is reduced, one might naturally expec t thiamin to be spared. SCIIOENItEI3IER and P~ITTENBERG(132) look on the carbohydrate -+ fat change as a normal physiological method whereby the nutrients are storcd until needed for oxidation. Inasmuch as food is taken only at intervals in the case of most animals, it must be deposited for a short period before it i3 further metabolized. Since thcrc are insufficient facilities for storing the excess carbohydrate as glycogen, most of the excess.carbohydrate is immediately changed to fatty acids. These are temporarily laid down in the fat depots, to be utilized for absorption during the post-absorptive periods.

2. Riboflavin and fat The interrelation between fat and riboflavin (vitamin B2) is not as clear-cut as in the ease of thiamin. Evx.xs et al. TM suggested in 1934 that fat has no sparing action on the riboflavin requirements in the rat comparable to that observed with thiamin. On the other hand, .~,~ANNERINGand his co-workers ~1a3~,~134~have postulated that actually more riboflavin is required by rats when they are receiving a high-flit diet than when they are on a low-fat ration. A possible explanation ;or the augmentation of the riboflavin requirement in the presence of high-fi~t diets, which has been suggested by ELg'EHJE-',I,~13"~ is that the intestinal s)mthesis of riboflavin is inhibited when fat is substituted for dextrin in the diet. On the other hand, CZACZKESand GU('GE.~-tIEIM(la~} reported that rats maint,ained on a low-protein diet excreted so much riboflavin that they fidled to maintain a functional level in their tissues, and ultimately died of ariboflavinosis. 126

The ]Effect of Dietary Fat on Vitamin Requirements lVhen the protein or fat content of such diets was increased, the excretion of riboflavin was reduced, while a decreased fat content increased the excretion. These authors attribute such variations to differences in the extent of synthesis of this vitamin in the intestinal flora. I t was shown t h a t rats on high-protein and high-fat diets need at least t~-ice as much riboflavin for the maintenance of the riboflavin level in the organs and in the urine as do rats kept on a "normal" diet. POTTERand associates (x37) ha~ e been unable to show a similar increase in riboflavin requirements on high-fat diets when puppies were the experimental animals. Fa~ has been sho~a to have a stimulatory effect on the g r o ~ h of bacteria, as demonstrated by microbiological methods for the determination of riboflavin.~xas), ( ~

3. Pantothenic acid and fat Deficiencies in pantothenic acid, pyridoxine, and essential fat t y acids result in the production of forms of dermatitis which are similar in appearance. According to S.~L-~Io-~,(14°) the actions of all of these es~ntial components are related, and all three are required to cure the dermatitis caused by pantothenic acid deficiency. STOTZ (141} has suggested another role for pantothenic acid. According to this investigator, the vitamin in the form of a co-enzyme A, called co-acetylase, may be involved in fatty acid synthesis. The presence of this enz3~ne, now kno~,~n as "acetylase," was first proved by Ln~th.~" and collaborators. (14z) These workers are of the opinion th at acetylase is important both in pyruvate and in acetate metabolism.(143), (144) Moreover, SOODAK and LIP.~t~-~-~(14~) have deduced proof t h a t the co-enzyme A is required for the formation of acetoacetate. All these Considerations lead one to the conclusion that pantothenic acid is of importance in the biosynthesis of fat in the animal body. The amount of this vitamin required is obviously a function of the amount a n d type of fat in the diet. In the microbiological assay for pantothenic acid, fat exbibits the same stimulating effect that it does in the case of the assay for riboflavin. (4~. c5)

4. Pyridoxine (vitamin Be) and fat A close relationship exists between the requirement for fat and for pyridoxinc.(4), (s) As has already been mentioned, the skin symptoms which originate when essential fatty acids are absent from the diet are similar to those which have been noted in pyridoxine deficiency. Bn~crI and GY6RGY(4) have shown ?,hat the dermatitis which occurs on a diet low in pyricloxine but containing 10°/o of fat can be cured when small amotmts of lard are added. In f~ct, somewhat later B ~ c ~ ~14e) was able to show that the addition of fat to the diet delayed t:hc onset of dermatitis produced by a pyrido.~:ine deficiency. In some cases, it complet,ely prevented the skin condition, evcn up to the time of death. SAL~o.~, ~147) likewise, demonstra'~ed that vitamin B~ and fats supplement each other in curing dermatitis. Most of the cvidence indicates that the effectiveness of the fat is directly proportional to its degree of unsaturation and thus 127

Nutritional Sigrdficance of the Fat8 presumably to the essential fatty acid content. Tile latter explanation might readily account for the fact that lard, which ordinarily has a relatively high essential fatty acid content, will be effective in alleviating pyridoxine deficiency, whereas butter, which has a relatively low essential fatty acid content, is somewhat less efficacious. SCHNEIDER{14s} attributed the low anti-dermatitis potency of rancid butter to the destruction of its linoleic acid. A number of workers have demonstrated that all commonly occurring essential acids are effective ill curing pyridoxine (leficieney. Of these several acids, linoleic acid appears to be the most effective, ~4~ whereas both linolcnie acid and araehidonic acid have been shown to be less satisfactory in clearing up the dermatitis. S.~L.~Io:~',(~47~and SCHNEIDER and co-workers, n ~ agree that linoleic acid possesses a greater potency in this respect than does linolcnic acid. They point out that the effectiveness of several, natural fats in clearing up the acrodynia is in proportion to their linoleate content. Corn oil was shown to have a ~reatcr ~ "tlvaty than linseed oil ; other fats exerted a curative effect proportional to their linolcate content, while co(t-liver oil was completely ineffective. According to RICHaRDSO.~ Ct at., ~a~ methyl ara(dfidonate and linoleie acid were usefid in clearing up the dermatitis caused by pyridoxine deficiency, t)ut did not afford permanent proteclion. The latter wcwkcrs suggest that the Lmsaturat.ed acids do not replace pyridoxine but simply delay the onset of the skin symptoms. SAR3IA and associates (~s~-) have shown that the growth inhibition in rats caused by feeding diets containing suboptinml amounts of psa'i(loxal or pyridoxine is accentuated when oleic acid is added to the ration. Hoxvever, this inhibitory effect of oleate can be counteracted by the administrat.ion of additional quantities of vitamin Ba. Non-essential fatty aei(ls have been shown to increase the deficiency symptoms in the rat. Thus, the administration of hydrogenated coconut (:)il(l~-ls) to rats suffering from fat-deficiency symptoms resulted not only in greater loss in weight but also in a more rapi(t mortality. Elaidin has likewise been shown to accentuate this essential f a t t y acid deficiency.~16) The question which naturally arises is whether or not the essential fatty acid and pyridoxine deficiencies are entirely similar. Although MrDt:S and coworkers n'~a) emphasized the interrelation between the two types of deficiency, their results might be interpreted to mean that two ditterent factors are involved. Thus, when the diet was lacking in both of these components, a relief from the deficiency was obtained by the admini.stration of eithcr ethyl linolcate or pyridoxine. However, the resulting growth response was loss with maximum amounts of ethyl linoleate than it was with optimum (loses of pyridoxine. Best results were ()btained when both of these components were inclu(le(l in the diet simultaneously. One possible explanation of the importance of p3widoxine in fat metah~dism is t h a t this faet(w is required by the mammal in the synthesis of fat from protein, n~) UMBRE1T and Gt:NSALUSns:) have denumstratcd that vitamin B, is one of the "essential steps in the prodnction of fat from carl~ohytlrate; O¢

(-

"

.

*

128

The Effect of Dietary F a t on Vitamin Requirement8

the importance of this ~-itamin in fat synthesis is therefore immediately evident,.

5. Niacin, folic acid, biot;*t, and fat I t has long beeen known t h a t both niacin and folie acid are synthesized in the intestine of the rat. ELVr.HaV-.~tclas~ has shoula t h a t the rate of this synthesis is accelerated on a diet containing fat. H e reported t h a t b u t t e r f a t had a greater value t h a n did corn oil. In addition to its sparing action on thiamin and pyridoxine, fat exerts a similar action on niacin. According to SAL.~IO.~(s2~, (147) the requirement for nicotinic acid is lower when fat is the chief source of calories ~han when the diet is p r e d o m i n a n t l y carbohydrate. It. now appears evident t h a t biotin hkewise exerts a pronounced effect on fat metabolism. As early as 1927, BoAs (lss* reported t h a t practically no body fat was present in the tissues of rats which had suffered severe biotin deficiency. On the other hand, the administration of this vitamin to rats increases fat synthesis; i t will likewise provoke a f a t t y infiltration of the liver. (xS:~ These f a t t y livers have a high cholesWrol content and resemble those occurring after the feeding of a fraction from beef liver. ~54) PAVCEK and SneLL ~Ss) have demonstrated t h a t biotin is readily destroyed b y rancid fats, b u t t h a t tocopherol is an excellent protective agent to prevent this destruction. I t has been suggested t h a t biotin is changed to biotin sulphoxide when this inactivation takes place, c159~ The resulting biotin compound no longer supports the growth of Laclobacillu.s easel, although it can still stimulate the growth of yeast. (aSs~ Biotin plays a major role in the growth of micro-organisms. In most cases, the requirement varies with the a m o u n t of oleic acid or other u n s a t u r a t e d acids present in the medium. This subject is discussed in a later section (see p. 137).

6. Fat.soluble vitami~ amt fat Since fats are excellent media for the solution and stabilization of the fatsoluble vitamins, it is obvious t h a t the quality and q u a n t i t y of fat in the d,.'et are of prime importance in the absorption of these vitamins from the gastrointestinal tract. In the case of vitamin A, MUELDER and KELLY ¢~6°~ have shown t h a t the absorption o[ the vitamin b y depleted rats was aided by dietary fat, atthougb its subsequent utilization was independent of the level of this foodstuff. On the t~ther hand, it was shown b y RI'SSELr~ and collaborators ~st~ t h a t the utilization of vitamin A in the chicken was not related to the fat level of the diet, although the absorption of carotene was dependent upon the presence of this substance. Although, under most conditions, preformed vitamin A has been considered to have the same biological ~'alue ~vhcn administered as the ester or as the free alcohol/~s'* W r r K and SEYI(~,NE {163-165) have rct)orted a n u m b e r of instances in which this was not the case. Thus, when difficultly absorbed oils or waxes were employed, as the dib~ents, vitamin A was invariably best iJtilized when given in the form of the free alcohol. When vitamin A deposition in the livers of chicks 19_9 IO

.~utxStional Significance of the Fat~

was used as the criterion o f v i t a m i n A utilization, (~e3) the results were practically identical w h e n cottonseed, corn, sardine, or cas*or oil was the diluent, irrespective of whether v i t a m i n A was fed as the alcohol or as the acetate. On the other

hand, when the solvent was basking shark oil, sardine oil, jojoba seed oil, ethyl l a u r a t e , or m i n e r a l oil, t h e a c e t a t e was c o n s i d e r a b l y less r e a d i l y u t i l i z e d tffan was t h e free alcohol. I n all cases, t h e n a t u r a l v i t a m i n A es~,er was d e f i n i t e l y inferior t o t h e o t h e r t w o f o r m s of v i t a m i n A e m p l o y e d i n t h e s e t e s t s i n c a u s i n g v i t a m i n A d e p o s i t i o n i n t h e l i v e r of chicks. T a b l e 11 gives a s u m m a r y o f t h e s e i n t e r e s t i n g results.

Table 11. The average storage of vitamin A in the livers of vitamin A-depleted chicks after the feeding of 30,000 units of vitamin .4 in the form of alcohol, acetate, o r ~ m t u r a l ester, i n three d i v i d e d d o s e s w i t h s e v e r a l d i l u e n t o i l s (a63) Vitamin A per liver after Seriez No.t

Diluent oil*

I

I

Vitamin A stored after

f

Alcohol Alcohol jI Acetate !t Natural:' ester

Acetate

Natural ester

Vitamin A fed in 3 doses of 2 nd each with concentration of 50(;0 un/ts,'ml units

Basking shark Castor Corn Corn Cottonseed Ethyl ]aurate Jojoba seed Mineral oil Sardine oil

I I

II I

lI lI I I

4510 8210 7150 1O670 7490 8830 9850 4860 3930

uni~ 2390 8020 5970 10080 6830 4240 4770 2160 391;0

u~its

o,o

O! /O

%

1880 5090 4940 7130 5320 3660 2440 1465 1390

15.0 27-4 23.8 35.6 25.0 29.4 32'8 16.2 13-1

8.0 26-7 19-9 33-6 22.8 14-1 15'9 7-2 13.2

6-3 17-0 16-5 22.8 17"7 12-2 8-1

4.9 4.6

Vitamin A fed in 3 doses of 0'1 nd each with concentration of 100,000 unilzl, rrd

l I

Corn Jojoba seed

i

1I ]I

10380 9170

11690 10490

$320 84O0

34.6 30.5

39-0 35.O

l

27-7 28-C

" * Control tests on chicks giveneach diluent oil without vitanfin A yieldednegativeresults for vitamin A in the liver in all eases. ~f Series I, Sew Hampshire chicks, 52 days old, 10 (.hicks per group; Series II, white Leghorn cockerels, 56 days old, 12 chit'ks per group. .4 s o m e w h a t s i m i l a r s i t u a t i o n o b t a i n s i n t h e r a t , as was s h o w n b y l a t e r t e s t s of

WEEK and SEViC;_~w.(16~) Only 0"4 ml (loses of the solutions were employed in

this case, as compared with 2-0 ml in the chick tests. Although no differences in t h e degree of u t i l i z a t i o n of v i t a m i n A were n o t e d w h e n it was g i v e n as t h e fl'ee alcohol or as t h e a c e t a t e , r e s p e c t i v e l y , a m a r k e d l y inferior r e s p o n s e o c c u r r e d w h e n t h e ' v i t a m i n A was a d m i n i s t e r e d as t h e n a t u r a l ester in j o j o b a oil. F i n a l l y , in u n p u b l i s h e d e x p e r i m e n t s o f t h e a u t h o r , (1~6) it was f o u n d t h a t v i t a m i n d e p l e t e d r a t s e x h i b i t e d less g r o w t h , d i n i n g a 2 S - d a y b i o a s s a y p e r i o d , w h e n v i t a m i n A was a d m - n i s t e r e d as t h e n a t u r a l e s t e r i n j o j o b a oil t h a n w h e a v i t a m i n A alcohol was used w i t h t h i s s o l v e n t . I n cases in which c o t t o n s e e d oil was 130

The Effect of Dietary Fat on Vitamin Requirements employed as the diluent, no differences in growth response were observed when different forms of the vitamin were employed. I t has recently been demonstrated that the action of vitamin A may be conditioned by the form in which it is administered, in the case of human subjects. WEEK and SEVIG.~'EU65~ concluded, on the basis of vitamin A tolerance curves in the blood, t h a t the rate of absorption of vitamin A by male subjects, after 134,000/~g of vitamin A had been given in 50 gm of margarine, was best with the alcohol, somewhat less satisfactory with the acetate, and poorest ~ith a mixture of natural esters. The experiments with female subjects showed that the vitamin A alcohol was significantly better than the acetate but not superior to the natural ester. The utilization of fl-carotene by the rat depends upon the solvent in which it is fed. (1¢n, (los) FnAPS and 5[EINKE, (le9) using a liver storage test, found that carotene was less readily utilized when present in vegetables than when fed as a component of cottonseed off. KnEc~x and VmT~_'cE.~~:°) found that, in the case of human subjects, the absorption of carotene from carrots was less efficient than that from butterfat. RUSSELL et al. (~s~) reported that fl-carotene is efficiently utilized by the chick only if fat is also present. According to Btn~'s et al., ~17r) both carotene and vitamin A were more effective in the rat when fed with a diet containing 5% of lard than when given with a fat-free diet which was supplemented with 0.1 gm of corn oil daily. The utilization of carotene by rats has been shown to be improved by incorporating it in margarine. (lr') It is suggested that this latter effect is potentiated by the emulsifying agents present in this product. I t has long been known that the presence of mineral oil in the diet has a deleterious effect on the utiliza'~ion of some of the fat-soluble vitamins. Some interference may obtain in the utilization of vitamin A, ~3) while the depressing effect on the absorption of carotene is greaterY TM BURNS and associates (~75) have noted a decreased utilization of carotene when as small an amount as 0.08% of mineral oil was present; some effect on vitamin A utilization obtained when the level of mineral oil in the diet was increased to 0.16%. When 0-32% of mineral oil was incorporated in the diet, the utihzation of vitamin A and car6tene conld not be improved by increasing the level of dietary fat from 5 to I0%. I t has also been reported that the utilization of vitamins D and K is impaired by the presence of petrolatum in the g,~trointestinal tract, c~76) BACO~ and collaborat.ors~:n have noted that a retardation in ~o~-t.h obtains ~dthin 2 to 3 weeks when 10% mineral oil is included in a fat-free ration. It is suggested that this effect is related to the development of an essential fatty acid deficiency. The hydroxy fatty acids may also have a depressing effect on rats. Thas, NI:i;]tTINGALE C[ al. ¢l;;a)" reported avitaminosis K in rats when dihydroxystearic acid triglyceride was incorporated in the diet to the extent of 25O,/o. It is possible that the hydroxy acid may interfere with the synthesis of vitamin K which normally occurs in the intestine. No adequate answer is as yet available to explain the antirachitic effect of 131

Nutritional Significance of the Fats fats on rats receiving high-calcium, low-phosphorus, rachitogenie diets, as reported by BOOT~, HE.','RY, and Ko~'. (l:s~ These workers found that vegetable fats devoid of vitamin D and triglycerides resynthesized from the fatty acids of butter and peanut oil exerted an antirachitic effect differing from that of vitam;_n D. It is possible that a differential effect of the several fats on calcium excretion via the intestine, as demonstrated by C~E-~'o and her co-workcrs, mg~ might offer an explanation for this phenomenon. 8. The Effect of Dietary Fat on Galactose Absorption

SCHANTZ, ELVEHJEM, and HART (1:9~ first called attention to the fact t h a t a galactosuria occurred in rats and other animals when t h e y were fed on a liquid skimmed milk diet. On the other ha n d , i t was shown that the excretion ofgalactose did not occur when a whole milk diet was fed. Moreover, when 3 or 4% of fat was added to the skimmed milk, the galaetosuria was completely prevented. I t was suggested t h a t fat may play a role in the metabolism of lactose and galactose other than by altering its rate of absorption. In a later report, ,~CHANTZand KREWSO.~ (~8°~ reported that several synthetic even-chain f a t t y acids containing 12 or more carbon atoms were equally effective in preventing galactose excretion in the urine. On the other hand, ZIALCITA and 5IITCHELL(lsl} failed to confirm the reported effect of fat, as such, on the metabolism of galactosc. T h e y likewise suggest t h a t there is no reasonable deduction, based upon the available facts of animal nutrition, t h a t such an effect would be expectcd. Corn oil, but not butterfat, was found to be able to decrease galactosuria by about 2 5 0 , on a diet, containing 48% of lactose. These workers suggest the possibility t h a t this effect may be dug to some non-glyceride component in the corn oil. In a later communication of the Wisconsin group, GEYER Ct al. (1~'~ extended the earlier work, employing skim milk and synthetic diets containing lactose or galactose. I t was concluded t h a t fat decreased galactosuria, and hence increased the utilization of galactose. These conflicting viewpoints of the several observers leave the question open as to whether the fat effect on galacto~uria originates primarily in the absorptive or in the metabolic phase. The results e f NIEFT and DEUEL (lsa~ throw some further light on the problem. Their data strongly support the concept t h a t the absorption mechanism is primarily involved. It is suggested t h a t the delayed evacuation of the stomach caused by the presence of fat might slow down the rate at which lactose (or galactose) reaches the intestine, and hence might result in a lower galactosemia, with a consequent increase in retention. The effect was shown to be unrelated to an upset in the rate of hydrolysis of lactose. Moreover, at a 20% fat level, the relative effectiveness of butterfat, cottonseed oil, and corn oil in reducing the loss of galactose in the urine was found to be identical. Although favouring tlie the~)ry t h a t fat acts in the absorptive phase of galactose metabolism, NIEFT and DEUEL "8~) |Gave the question open as to whether or not the metabolic phase is involved, especially since the latter hypothesis might be an explanation for the greater ketolytic effect of galactose as compared with glucose, reported for rats (~s4~ and for man. (lss~ 132

Fatty Acids as Required Growth Factors for Bacteria 9. Fatty Acids as Required Growth Factors for Bacteria

1. Olclc acid as a substitute for biotin There is considerable evidence, based upon microbiological studies, t h a t the metabolism of biotin and t h a t of fat are related. On a f a t t y acid-free medium, the groxvth of most bacteria requires the presence of biotin. However, ff oleie acid is supplied, normal gTowth m a y obtain, even in the complete absence of biotin. COHE.n and co-workers (ls~ were the first to report t h a t oleic acid is required b y bacteria. These workers sllowed t h a t this unsaturated acid is essential for Co,'ynebacterium dipl~theriae. I t was subsequently demonstrated to be a prerequisite for the gro~vth ofClostridium tetaM and Clostridium wdcMi. WmL~AMS, BaOQt-IST, and SNELLtls:~ reported, in 1947, that cultures of several types of lactic acid bacteria required oleic or linolcic acids, or a combination of both, while the even-numbered saturated acids from Ca to Cls were completely inactive. In the case of Lactobacillus b~dgaricus, although oleic acid was found to be essential for gro~vth, its growth-promoting action could be observed only within a narrow range of concentration and within certain T H limits. A particular strain of Lactobacillus isolated from the caecal contents of rats was also sho~vn to require olcic -~cid for growth/~ss) This organism could be repeatedly transferred on synthetic media only when oleic acid was present. WILLL~)tS and FIEG~:R{189~were the first to call attention to the similarity in the stimulating action of oleic acid an(1 of biotin on Lactobacillws ca,~ei. Although a maximum greyish (as determined from acid production) could be demonstrated in the presence of lipids, no s5-rJthesis of biotin occurred. (~9°~ I t was suggested that the bacteria adapt themselves to either the biotin-free or the biotin-low environment. These results render it doubtful that the f a t t y acids are concerned with the biosynthesis of biotin in the intestine. This agrees with the data of el'tEL, {~9~) who reported a relatively constant excretion of biotin in the urine on biotin-free diets, while the sudden va,'iations were related to biotin changes in the dict. Although Laclobacillus arabinosJs, L. easel, and Strej)lococcus faecalis can grow in the complete a bsence of biotin, aspartic acid as well as unsaturated acids must be present in the me(lia. (lg:) On the other hand, I~ROQUISTand S.NELL{192) noted t h a t unsaturated acids alone were necessary to insure grox~-th in Lac/obacillus ferme~ti and Clo~Tridium btdyricum. The latter organism was shown to synthesize asparti c acid when grown in the presence of oleic acid and in the absence of biotin. I t is now believed t h a t the biotin-like component in acid-ilydrolyzed plasma is an unsaturated acid. Although TRACER (19s) believed that this ether-soluble substance was a component of the nomsaponifiable residue, HC~'MANN and AXELRODc194~ were unable to confirm this hypothesis. The latter workers reported t h a t the biotin-like substance in beef and h u m a n plasma occurred exclusively in the saponifiable portion of the lipid, lVhcn the fraction was esterificd with diazomethane, the resulting methyl esters obtained by distillation 133

Nutritional Significance of the Fats

possessed high biological activity. On a weight basis, oleic acid was shown to have about ~he same potency as the plasma distillate. It was also found that the liquid acid fraction of the plasma, which contains oleic, linoleic, and arachidonie acids, had a greater grouch activity for the l.~ctobacilli than did the original fraction, although the saturated acids were also found to have some synergistic action.(~s~ There are a number of h)Totheses as to the reason why biotin and unsaturated f a t t y acids can replace each other as requirements for bacterial growth. TRA~ER~198~concluded that biotin functions in the synthesis of f a t t y compoands or of substances containing f a t t y acids, such as lecithin. RvBI~ and SCHE~Ea (~97~ demonstrated that biotin is primarily concerned with the s)~nthesis of one or more fatty acids. Thus, the latter investigators were able to show t h a t such biotin antagonists as homobiotin, trishomobiotin, homobiotin sulphone, and trishomobiotin sulphone have no depressing effect on the growth of Lactobacillus casci on a biotin-free medium, provided oleic acid is used as a biotin substitute. In addition, WILL~IS and FIEGER(198} noted that avidin, sufficient to combine with 1000fig of biotin, did not prevent lipid stimulation of L. ca.~ei in the absence of biotin. This would indicate that oleic acid is not active by virtue of effecting a synthesis of biotin. It is therefore apparcnt that biotin normally functions in bacterial metabolism to bring about the synthesis of oleic acid; biotin antagonists can have no inhibiting action on growth whe:~ the products, ordinarily synthesized by the aid of biotin, are furnished in adequate amounts. Although oleic acid functions as a substitute for biotin with many bacteria in in vitro tests, it does not behave in the same manner in vivo. For example, TRAGER(19a~found that oleic acid injected intramuscularly in chicks did not have the same effect in reducing biotin deficiency produced by the ingestion of avidin (raw egg white) as has biotin or the fat-soluble fraction from hydrolyzcd plasma. Moreover, WrLLL~IS and ]~'IEGEg(~'°°~ demonstrated that the lipids from rice polishings, which have a biotin-like activity in the case of L. ,'asei a~:d L. arabi~,osus, will not reduce the biotin-deficiency symptoms in white Leghorn chicks. 2. Comparative pote~cies of octadecenoic acids A large number of isomers of oleie acid, both geometric and positional, can serve as adjuncts in the growth Of bacteria in lieu 0f oleic acid. In the first place, the trans-isomer of oleic acid, elaidic acid, has been found by a number of workers (2°1-2°5) to possess approximately the same biopotency as oleie acid, although AXELROD et al. ~lgs) had assigned to it a somewhat lower potency, and ~VILLIASISand FIEGER~19°) have reported that it has a somewhat greater effect than has olcic acid. The location r~f t h e double bond does :lot markedly influence the activity of the cis.octadecenoic acids. Tiros, petrosclinic acid (6-octadecenoic acid),(2~, (20s~, c205~ cis-S-octadccenoic act(l, ~e°a~ vaccenic acid (ll-octadecenoie acid),t201~, t205~and cis.12-¢)ctadecenoic acid (2°~ all had about the same growthstimulating actmn as did oleic acid, although the c/s-8 acid appeared to exert a 134

F a t t y Acids as Reqtfired Growth Factors for Bacteria

somewhat greater effect. However, the cis-4-octadecenoic acid has been reported to be inactive, as has also the trans-isomer. [~) In addition to the activity exhibited by the cis.octadecenoic acids, the trans forms are, in many cases, equally potent, although marked variations have been noted. As reported earlier, elaidic acid is equally as potent as oleic acid. However, CHESG and associates ¢2°5) found t h a t elaidic acid possessed the maximum potency of the trans-isomers tested; as the double bond shifted in either direction from the 9 position, a reduction in potency obtained, until at the 6 position the acids were completely iqactive. This was likewise true of 17octadecenoic acid, which forms no geometric isomers. These results are summarized in Table 12.

Table 12. Biotin-like effect of octadecenoie acids and llnolelc acid, alone and with biotin, usi,~g Lactoba~;illws arabinosws (2°s) trans.Acid

c~-Ac~

Compound Me~ing point °C

Biotin equiva. lent ( rn/~ gin) per rag fatty acid Alone

With biotin

11"6

12'4

14-7 10'3

13"4 10"2

ll-Ochadecenoic acid

12"0

11'4

12-Octadecenoic 17-Octa(lecencic c/s-9, 10-, cis-12, acid (Linoleie)

10"8 0"0

13'4 1"8

5"3

6"9

I

6-Octadccenoic acid 7-Octadecenoic acid $-Octadecer,~;c acid

.! 29-8 . -., 22-7-'23-8

9-Octadccenoic acid

/ ( .!

10-Octadcccnoic acid

•

,

13-0 --

/ 13-0-14-0 / acid .' 2 6 - 8 - 2 7 - 6 acid* l 55.5-56-1 13-Octadeeadienoic'[ . . .

J

Melting point °C

53-0 43-5--14.5 51-5-52-3 44-,5--45-5 41-5-42-5 52-O-52-6 43-5-44.5 39.0 52-O-53-0

Biotin equivalent(mu gin) per mg fatty acid Alone

With biotin

0'4 2"7 2"8 11"5 9-6 6"I

3.7 15"2 9"1 10"4 9.8 11-0

2"6

7.3

1"5 2.7

7-2 15.2

"

* .N'o geometric isomers of this compound. Included in c/s column for convenience.

3. Comparative potency of other unsaturated acids and related compounds AXL~OD etal. (19s)have found that a number of compounds related to oleic acid are likc~ ise effective stimulants for the growth of LactobacilIus arabinosus. The folJowing activities expressed ill micrograms of biotin per milligram of fatty acid were obtained: olcic acid, 6-0; methyl oleate, 3-2; oleic acid amide, 6"3; oleyl alcohol, < 0-05; elaidic acid, 1-0; vaccenic acid, 1'2; dihydroxystearic acid (94°C), < 0.05; and dihydroxystearic acid (130-7°C), -< 0.05. Linolcic acid (9,12-octadccadicnoie acid) possesses a high biotin-like activity, (192)but it is somewhat lower than t h a t of olcic.¢lo~L (~0.0),(~3), ¢~) Linolenic acid (9,12,15-octadccatrienoic acid) is like,vise effective in stimulating bacterial growthjiO~,, (~95), ¢~.-), ¢~3), (~o6)but its potency is only a fraction of that of oleic 135

Nutritional Significance of the Fats

acid. cl~s~ Azelaie acid (HOOC(CH2)TCOOH) and pelargonie acid (CHs(CHz) , COOH), which are oxidation produc'~s of oleic acid, are completely inactive. Activity in substituting for biotin obtains ~ith unsaturated fatty acids other than those having 18 carbon atoms. Thus, palmitoleic acid (9-hexadecenoi~ acid) was sho~-a by HAssI_','Ey et al. ~°~ to stimulate acid production in the case of L. arabinosws in a manner similar to that of oleic acid. When Lactobacillus bifidus was used, this acid was likewise able to stimulate gro~th when present in a low concentration. However, at a higher concentration, Palmitoleic acid brought about a growth inhibition of these bacteria, in contrast to oleic acid, which accelerates grou~th at both low and high concentrations. BOUC,ItTON and POLLOCK alone ~m~) and with How)d~D (~°~-~,~o.0~ likcwise reported a positive stimulation of the diphtheria bacilli on the part of cis- and trans-pahnitoleic acids as wcll as by the odd-carbon acids, cis- and trans-heptadec.9-enoic acids On the other hand, a number of unsaturated acids are inactive. Thus, BGUGYITON"and POLLOCK(~m~reported negative results with the following: cisundec-9-enoic acid; cis-undcc-lO-enoic acid; cis- and trans-myristoleic acid (9.tetradeeenoic acid); and cis- and traws-gadoleic acid (9-eicosenoic acid). Erucic -acid (cis-13-doeosenoic acid) and brassidic acid (trans-13.docosenoic acid) were reported to be inactive, as were also :~-, fl-hydroxyoleic acid and ricinolcic acid (12-hydroxy-9-octadecenoic acid). (2°3~

4. The effect of saturated acids on bacterial growth In contradistinction to the activity of a number of unsaturated fatty acids tested as substitutes for biotin as a growth factor in bacterial cultures, the saturated f a t t y acids have been fimn~ to be completely inactive when supplied alone. Thiz is true not only of the various even-carbon acids from Cs to C~6,~sT~ but also of stearic acid (octadecenoic acid). ~195~ Laurie and myristic acids have been reported by WrLLIA.~ISand FtEaFR, ¢a9°'~also, to be inhibitory. HASSIN'E~ and colleagues ¢2°s~ noted that growth of both Gram-positive and Gram-negative organisms was inifilfited by Cs and C10 saturated fatty acids, but that no significant depressing action was found on the part ~f C~, CG, Ca6, and Cls saturated acids. On the other hand, the Ca2 saturated acid inhibited only the Gram-positive organisms, while some inhibiting action likewise obtained in the ease of the C~4 acid. On the other hand, the results of CHEX'a and co-workers ~5) indicate that the saturated aci;ls (principally palmitic and stearie acicls) have a synergistic action on bacterial gro~-t.h when present along with oleic acid. This effect is discussed below.

5. Growth stimulation of bacteria, produo~d by mixed laity acids from various natural and artificial fate In the study of tile biotin-like activities of the mixed f a t t y acids prepared from a number of fats, it was found that their activities could not be correlated with the content of oleie and linoleie acids unless account was taken of tile synergistic action of the saturated acids. The following formula, which takes into account 136

F_~tty Acids as Required Growth Factors for Lower Organisms the variation in activity of oleic and linoleic acids and corrects for the synergistic action of the saturated acids, was found to give excellent results in predicting the activity of the fatty acid mixtures. Thus, the biotin equivalent, as micrograms of bi6tin per milligram of fatty acids, equals: (c}/° °leie × 10"3) (-+- (%1 hn°leie 0 ×0 35)"

X

1.00 4- 2.3 °/° saturated a c ifdast )t yi O ( )

By the use of this formula, the calculated biotin-like effect of mixed f a t t y acids can be estimated, A comparison of the estimated and found values of fatty acids from several fats is given in Table 13.

6. Effects of u nsatu rated fatty acids on growth of bacteria other than the biotin-like effect In the extensive studies of KoDmnn, (2°9~it has been shown that most fatty acids possess an inhibitory effect on the growth of various Gram-positive organisms. This behaviour applies to ehe saturated and unsaturated acids alike. Laurie and myristic acids inhibit bacterial growth in concentrations of 1 : 100,000. In the case of the unsaturated acids, the cls-forms have a marked retarding effect on growth, and an increased effect is noted in the case of polyunsaturated acids. Thus, toxicity occurs in the following order: oleic acid < linoleic acid < linolenic acid. The bacteriostatic action of the unsaturated acids was shown to be reversed by surface-active agents such as lecithin, sterols, tocopherols, 1)rotcins, and similar compounds. On the other hand, esters of the fatty acids were not bacteriostatic. In contradistinction to the reversal of the inhibitory effect caused by the surface-active agents on the unsaturated acids, these agents were unable to reactivate the saturated acids. 10. Fatty Acids as Required Growth Factors for Lower Organi.qms Tr~AGEe,~196J found that oleic acid can replace biotin in the gro'~vth of the larvae of the :yellow fever mosquito, A~des ae.qypti. In the absence of biotin, larval growth was very slow, and metamorI)hosis did not occur. V~qlen biotin was present to the extent of 50 m/z mg per ml of medium, excellent g r o ~ h was obtained. Relatively low concentrations of oleie acid, an oil from hydrolyzed plasma, lecithin, and related compounds, when used in place of biotin, supported larval growth comparable with that obtained with the lower effective doses of biotin. On the other hand, oleic acid and Tween 80, either al(me or with. aspartie acid, produced some growth response on the part of cholinel,,ss Neurospora crassa (red bread mouM), although they were unable to replace biotin completely. ~210) FmtENKEL and BLEWm~r~zm reported that linoleic acid and :~-tocophcrol are required by the 3T.editctrancan flour moth, Ephestia kuehniella. Linoleie acid is needed for the normal development of the wings and f~r suceessfifl emergence, while tocopherol is necessary as an antioxidant fiJr linoleic acid, and to promote

137

i

.I

:!

92 26 12 13 59 21 38 32

%

(1)

,Saturated

3 5

7 2

55 66

2 52 7 62§

O/ /0

.(3)

Linoleic

6 22 8l 25 37 74

0 / /0

(2)

Oleic

Composition of total mindedfatty acids

2.0 9.2 12.0 7.7 9.1 11.8 8.2 10.6

mlt gin~rag fatty acid

Found(4)

0.7 5'0 8"7 5'5 4.0 7"8 6.1 6"9

"'P gm/mg fatty acid

A nticipalcd from (2))+(5 (3)

Biotin equivalents of ,nixed fatty acids

• ((4.) - (5)/(~,)) × loo. t Based ilpon thc composition of the f a t t y acid m i x t u r e , using tile f o r m u l a given a b o v e . ((4) - (7)/(7)) × 100. Including 7°,~ Iinolenlc acid,

C o c o n u t oil Cot tons(,cd oil Olive oil S o y b e a n oil Butterfat . Margariao fat Shortenilag C S l m r t e n i n g 1)

Fatty acids from:

|l.,

54

186 8t 38 40 127 51 34

%

(6)

Increase*

0.9 1.7

2.2 2.4

2.0 3.2 3.2 3"1

Ratio

(6): (1)

Synergistio effeCtacidof s saturated

,,, .1.

11.2 7.2 0.4 11.7 11.3 12.0

8.0

2.2

fatty acid

roll ,qm ling

Calculatcd¢ (7)

".

+ I

-- 3

+ 7

-- 9 15 + 7

- - 12

-- 27

+

?/o

Deviation ~

Biotin equivalents of mixed fatty acids

Table 13. Biotin.like activity of fl~tty ac;ds from several fats and oils as found by ~nicrobioloffical assay and by calculation from fatty acid composition 12°s~

Thyrotoxicosis and Dietary Fe,t

rapid gro~th. On the other hand, arachidonic acid has no effect on wings or on emergence, but it does have a striking effect on grouch. ~2x2~ Its effect closely resembles t h a t of doeosahexenoic acid from cod-liver oil. On the basis of the linoleate composition of body oil, it is ccncluded that Ephestia, which requires linoleate, does not synthesize this acid. On the other hand, another insect which does not require linoleate, namely the meal beetle, Ten,rio, is apFarently able to synthesize this dienoic acid. ~21a~ III. THE EFFECT OF DIETARY FAT ON THE RESPONSE TO STRESS FACTORS There is considerable support for the thesis that the presence of fat in the diet may exert a beneficial effect in times of physiological stress. Thus, during periods of gro~th, pregnancy, and lactation, animals in general responded better when fat was incorporated in the diet. Moreover, when rats were subjected to complete inanition until they died, it was found that the most prolonged survival followed prefeeding the animals with fat. ~,~rhen the diet during the pre-fast period was carbohydrate or protein, the survival tinm was curtailed. Moreover, when rats were subjected to physical exertion to the point of complete exhaustion, a greater physical capacity was noted in the rats receiving a high-fat diet than in those on a fat-low regimen. These phases of fat nutrition have been developed in Section II. It is likewise of considerable interest to observe how diet will modify the response of animals to external forms of stress. Thus, the present discussion will centre around the question as to whether or not dietary fat can modify the normal response to thyrotoxicosis, x-radiation, cold environmental temperatures, and similar forms of stress. 1. Thyrotoxicosis and Dietary Fat

When an excess of the purified thyroid hormone Or desiccated thyroid powder is given to rats and to otheI animals, as well as when the thyroid gland becomes sufficiently hyperactive, certain readily reproducible symptoms develop. Suppression of growth normally occurs concomitantly with an increased basal metabolism. The heart rate is increased proportionally with the rise in the basal metabolic rate. Inhibition in ovarian development occurs in young rats. On the other hand, the kidneys, adrenal glands, and heart become considerably h3I)ertrophied. The beneficial effect of the inclusion of fat in the diet of hyperthyroid animals was first noted by ABELIN and his co-workers, c2t4-216) These workers reported that a high-fat diet was able to counteract the increased metabolic rate, as well as to prevent the reduction in liver glycogen which foll~,ws the administration of the thyroid hormone.: A number of workers {il'-21'a) have confirmed x~BELIN'S work on rats, While BErG tz2°~ has corroborated the protective action of fat in dogs, manifested by a reduction in the basal metabolic rate following h3~perthyroidism. One explanation for the protective action of fats in thyrotoxieosis is that they furnish the necessary essential acids. However, not only linoleie acid, but also 139

Nutritional Significance of the F a t s t h e non-essential oleic acid, w a s sho~-n t o p r e v e n t t h e rise m m e t a b o l i s m . ¢22x) On t h e o t h e r h a n d , GUE~RA ~°'°'2~correlated t h e p r o t e c t i v e a c t i o n o f f a t w i t h its u n s a t u r a t i o n . A c c o r d i n g t o ZAIN, ¢½23), ~224) l:'noleic acid b u t n o t stearic acid prevent~ t h e loss o f liver g l y c o g e n after massive closes o f t h y r o i d e x t r a c t s . Oleic acid w a s s h o w n t o o c c u p y an i n t e r m e d i a t e position. :It is o f course possible t h a t t h e n e g a t i v e effect o f stearic acid in affording p r o t e c t i o n a g a i n s t t h y r o t o x i e o s i s in t h e a b o v e studies m a y h a v e b e e n due t o t h e failure o f t h e s a t u r a t e d acid t o be absorbed. T h e r e c e n t r e p o r t s o f EI~SHOrF ~22s~ as well as o f GREENBERG a n d DEUEL ¢22s} h a v e s h o w n t h a t t h e beneficial effect of fat in p r e v e n t i n g gro~vth inhibition o f rats is r a t h e r generalized. Tiros olive oil, c o t t o n s e e d oil, p e a n u t oil, corn oil, s o y b e a n oil, h y d r o g e n a t e d c o t t o n s e e d oil, w h e a t g e r m oil a n d lard, all c a u s e d a significantly g r e a t e r g r o w t h o f r a t s t h a n o b t a i n e d on t h e fat-low ration, irrespective o f t h e essential f a t t y acid c o n t e n t o f t h e variQus substances. I-~-%) H o w e v e r , in these tests fat fidled t o p r e v e n t inhibition o f o v a r i a n d e v e l o p m e n t , or t h e e n l a r g e m e n t o f t h e kidneys, a d r e n a l glands, a n d h e a r t . :In t h e e x p e r i m e n t s o f GREENBERG a n d DEUEL, (226) n o t o n l y did t h e presence o f fat p e r m i t n o r m a l g r o w t h in t h e presence of desiccated t h y r o i d p o w d e r , but. also it r e d u c e d m o r t a l i t y t o 0. T a b l e 14 gives a s u m m a r y o f t h e l a t t e r results. Table 14.

T h e m e a n w e i g h t s a ~ d m o r t a l i t y o f r a t s o n l o w - f a t a~ut h i g h - f a t d i e t s w i t h o u t o r w i t h t h y r o i d p o w d e r (22e~ Desiccated [ thyroid ..... present ,

Ba*al diet

Body weight Gain in weigl~

.lIortality

41-8 43"8

gin 203-3 141-7 134.0 271-7 280.2

gm 162-2 98.0 90-0 229-9 236.4

%

173.3 138.7 11.t-0 207-0 216.3

50 83 0 0

46-3 45.3 43.8 44-7 43-8

134.7 ± 7.41 161-0 135-2 ± 4-1 I 121-0 ] 17-5 =L 10-5 ! 135-0 166.5 -- 3.91, 200-7 175.8 ± 7-4i 208-3

114-7 75-7 91.2 156-0 164.5

0 50 83 0 0

Start

I ~ After ' 4fter 6 u,eel~'s* ! . . i" i '' wee~

3Isle rate

•I

Low-fat Low-fatt . High-fat . High-fat .

i

gin

gin

I

Lo ~'-fat

0 + -~0 +

41-1 43"7 44'0

?: ~± ~ ±

4.0 2.4 3-1 14-9 7-5

0

Female rata

Low-fat Low-fat Low-fat¢ High-fat High-fat

. . . . .

]

.] "1 ] .

i

0 + -÷0 +

•

!

i

* ]ncluding Standard Error of the Mean.

? Group in which the lw'el of B vi::~mins was twi('e that in other groups. T h e r e a s o n for t h e p r o t e c t i v e a c t i o n o f fat on t h y r o t o x i e o s i s has n o t b e e n established. ERSHOrF ~225~ suggested the possibility t h a t t h e fat m a y f u r n i s h essential f a t t y acids a n d t h u s alleviate a deficiency which m a y arise as a r e s u l t 140

X - R a d i a t i o n I n jinx- a n d D i e t a r y F a t o f a n i n c r e a s e d i ' a t t y a c i d r e q u i r e m e n t . H o w e v e r , since a p r a c t i c a l l y f a t - f r e e l i v e r p r e p a r a t i o n w a s a l s o s h o w n t o h a v e a beneficial effect, ERSHOFF does n o t b e l i e v e t h a t t h e f a t t y a c i d d e f i c i e n c y t h e o r y is a p l a u s i b l e one. O n t h e o t h e r h a n d , GREE~'BERO (2z7~ w a s a b l e t o d e m o n s t r a t e u p r o t e c t i v e a c t i o n a g a i n s t t h y r o t o x i c o s i s in t h e r a t o n t h e p a r t o f b o t h m e t h y l l i n o l e a t e a n d c o t t o n s e e d oil. I t h a s also b e e n s u g g e s t e d t h a t t h e b e n e f i c i a l effect o f f a t in t h i s c o n d i t i o n m a y r e s u l t f r o m s y n t h e s i s o f a n a n t i t h y r o t o x i c f a c t o r b y t h e i n t e s t i n a l flora, c2z7~ A c c o r d i n g t o TEMPLETON a n d ERSHOFF, m3~ b o t h f a t a n d c a r b o h y d r a t e a r e s u p e r i o r t o p r o t e i n (casein) in p e r m i t t i n g s u r v i v a l o f r a t s r e c e i v i n g t h y r o x i n e .

2. X - R a d i a t i o n I n j u r y a n d D i e t a r y Fat DECKER a n d a s s o c i a t e s ~2~s~ were t h e first t o s h o w t h a t a f a t - d e f i c i e n c y s y n d r o m e c a n b e p r e c i p i t a t e d in m i c e b y t h e a p p l i c a t i o n o f x - r a d i a t i o n . Mice r e c e i v i n g fat were found to be more resistant to injury from x-radiation than were animals o n a f a t - f r e e r e g i m e n . I n a n e x t e n s i o n o f t h i s w o r k , CHENG e~ al. ~2~ h a v e c o n f i r m e d t h e r e s u l t s on r a t s ; m o r e o v e r , i t w a s f o u n d t h a t , in y o u n g r a t s , o n l y t h e m a l e a n i m a l s were p r o t e c t e d b y f a t wtfi]e, in t h e case o f t h e o l d e r a n i m a l s , f a t f e e d i n g p r o d u c e d beneficial effects e q u a l l y in b o t h sexes. T h e p r o t e c t i o n a f f o r d e d b y f a t w a s e x h i b i t e d e q u a l l y well in g r o u p s o f r a t s r e c e i v i n g d i e t s c o n t a i n i n g 2 % o f c o t t o n s e e d oil a n d in t h o s e in w h i c h 15% o r 3 0 % o f f a t w a s p r e s e n t . I n l a t e r w o r k , (23°) m e t h y l l i n o l c a t e in doses as low a s l 0 m g was s h o ~ t o b e efficacious as a p r o p h y l a c t i c a g e n t , a l t h o u g h t h i s d o s a g e o f l i n o l e a t e is far b e l o w t h e o p t i m u m level for gro~%h. T h e r e s u l t s o f t h e s e i n v e s t i g a t i o n s a r e s u m m a r i z e d in T a b l e 15. Table 15. The average length of survival and average exposure to x-radialion of old and of young rats on different diets a~ad subjecZed to repeated sublethal doses o f x.radiation m29~ i

Average day of death*

A veroge exposure per rat in r*

Series, sex, and diet

~,ries I (18-month rats) : Diet 1 (fat.fl-ee) . Diet 2 (15% cottonseed oil) Diet 3 (30% cottonseed oil) ,Series I I (3.month rats): Diet 1 (fat-free) Diet "2 (2°o eottmLseed oil) Diet 3 (30% eotto1~seed oil)

.~lale rats

Fe~t~alerats

.llale rats

Female rat~

34-3 ~ 3"3

39 ± 4"3 76 ± 7.0

1235 ± 54 1860 ± 112

1270 ! 77 1950 ± 128

70"6 ± 6-1

5"22

4-52

5"06

4"55

63"7 ~ 7-1 3"61

69 ± 5-3

1710 ± 119

1800 ~ 111

4-42

363

51 i 3-1 65 ~ 3-0 3"25 67 p: 3-2

61 ± 3-6 67 ~ 2-6 62 ± 3-4

1770 ± 99 2205 ~ 92 3.22 2290 ~ 82

020

4-07

3"60

1-35

t

3"92 1980 ~ 84 2170 ± 62

1.82 2080 ! ~7 082

* -Inch.ling the ~tandard Error of tile Mean. Tile bold face flgur(~ are tile ratios of Mean Differenceand X';(.%.E.M.)'c0n;roi+-(S.E.M.)'exp. When this figure exceeds 3.0, statistical significanceis indicated.

141

:Nutritional Significance of the Fats

3. Cold Stress and Dietary Fat It has been know,an for many years that metabolism is greatly increased by cold. As early as 1881, Ki)Lz(23n demonstrated that the liver glycogen of a dog disappeared almost completely if the animal was exposed to prolonged and severe chilling. In studies on human subjects exposed to intense cold, MITCHEI~L and co-workers (232) found that a high-fat diet is cuperior to a high-carbohydrate regimen in maintaining general psychomotor performance and visual efficiency, as wcU as in allowing a maximum speed of tapping. Superiority of the high-fat meals over high-carbohydrate rations in maintaining tissue temperatures in a cold environment appears to be related to a decrease in heat emission rather than to any resulting increase in heat production. A temporary deposition of dietary fat in the subdermal tissues follo~ing a fat meal may be related to this phenomenon. However, the fat diet was not appreciably more effective in preventing the cooling of the skin. Likewise, 2 0 0 0 of the basal calorics were required for the maintenance of body weight on the high-fat diet, as contrasted with a figure of 188~/o for the high-carbohydrate diet.

4. Hepatectomy and Dietary Fat One of the most critical forms of stress which can be applied is that resultin~ from partial removal of the liver. This is because the liver is important in so many physiological functions which it is inapossible or difficult to delegate to other organs. Thus, it serves as a s~orehouse for glycogen, for the fat-soluble vitamins, as well,as for the water-soluble vitamins, and to a certain extent for fats. Moreover, it is the chief site of the formation of ketone bodies, of deamination, of oxidation of ulic acid (in dogs and lower animals), ~f urea synthesis, and of many detoxication reactions. It aids in the utilization of fructose. Moreover, this organ seives as the chief organ for the synthesis of serum albumin and serum globulin. As a result of stinmlation by vitamin K, prothrombin is produced in the liver cells. One very important function of this organ is the excretion of such products as bile pigments, cholesterol, and rela~cd compounds. Hepatic tissue is responsible for the mediation of so many vital functions that animals cannot long survive the removal of this organ. However, 3IANX,~23z~,~za4~ many years ago, surmounted the immediate difficulties inherent in the removal of the liver. He was able to maintain life fi~r as long as l l hr in dogs in which a collateral circulation had been established as the result of a reverse Eck fistula, pcrforraed several weeks before the hcpatcctomy was carried out. ~L~RKOWITZand SosKI.~~°-zh~were able to develop a similar degree of collateral circulation by a preliminary part;al ligation of both the vena ca ca and the portal vein. Although these procedures extend the life Gf the a~,imals following the removal of the liver, it has been impossible to alter the fatal outcome by dietary means, except that survival is extended for many hours when the normal blood sugar level is maintained by means of periodic injections of glucose. However, this stinmlatory effect of glucose soon loses its value, and the animals no longer recover following its administration. In spite of the inability of animals to survive the complete removal of the

142

Miscellaneous Stress Factors and Dietary Fat liver, even u n d er the most satisfactory experimental conditions, rabbits are able to recover if as small an amount as 30~o of the .functioning tissue of the liver remains, c236) ,4£ter partial hepatectomy, the rate of regeneration of the liver is extremely rapid. Such experimental conditions as partial hepatectomy therefore provide an ideal stress for the study of the role of the diet. In the case of rats, BRUES et al. (23~) reported that regenerotion of liver tissue proceeded at a slower rate when fat was included in the diet than when protein or carbohydrate was the chief dietary component. Because of the importance of bile in the utilization of fat, the use of fat diets has generally been avoided in studies of liver regeneration. However, ROGERS and associates ~s3) have recently reported th at moderately high-fat diets are as efficacious in permitting recovery of rats following a 70~o partial bepatectomy as are low-fat rations. When rats on the 30% fat diet were limited in food intake to t hat of the animals on the 3% fat regimen, a significantly greater n~:ogcn balance and a higher increment in liver protein and in body weight obtained, When the consumption of food was ad libit~m in both series of tests, the animals on the high-fat diet did considerably better than those on the low-fat intake. 5. Miscellaneous Stress Factors and Dietary Fat

One stress factor which can be counteracted much more effectively by fat than by other foodstuffs is that caused by the administration of 2,4.dinitrotoluene (DNT). C~AYTO~"and BAt'~A~'~ ('038) demonstrated t hat mice and rats grew less and died more rapidly when fed a low-fat diet along with DNT than when fed the same amount of the nitro compound per calorie in a diet containing 5 or 30% of added fats. I t was beliered t h a t the harmful effects o~:the low-fat diets were to be ascribed to the low calorie intake of the animals. The beneficial effect of fat in minimizip.g the toxic action of DNT in mice was demonstrated with cottonseed oil, corn oil, Crisco, hydrogenated coconut oil, lard, and butter-fat. Rancid cottonseed oil was found to augment the toxicity of DNT. LUNDBAEK and STEVESSO~(0-39.reported that, after hypotha]amie injury, the ~:cight gain of the rats suffering from hypothalamic h3~perphagia was greater on a high-fat, non-carbohydrate diet than on a high-carbohydrate diet. The weight gain per calorie was likewise shown to be greater when fat was the main foodstuff than when carbohydrate was the principal nutrient. It was suggested that this may be attributed to the fact that the animals on the high-fat diet do not experience a loss of energy entailed by the transformation of carbohydrate into fat. IV. :FACTORSALTERI_~'O.THE NUTRITIONALVALUE OF FAT 1. Sex as a Factor in Fat Utilization and Requirement

Much evidence has developed in recent years indicating that a sex difference obtains not only in the distribution of lipids and glycogen in animal tissues but also in the metabolism of fats. Most recently it has been shown that variations in the essential fatty acid requirement occur between male and female rats. 143

Nutritional Significance of the Fats

1. Sex differenca, in "l¢~tonuria K e t o n u r i a is obtained more quickly, and occurs with a higher degree of intensity, in the female t h a n in the male. DEUEL and GVLICK(~°~ were the first to report t h a t the ketone b o d y o u t p u t based on grams per square metre of body surface per d a y a v e r a g e d 5 times as much in women during fasting as in men.. A similar sex difference was later demonstrated in rats and guinea pigs when acctoacetic acid, butyric acid, or other ketogenic acids, in the form of sodium salts, were administered to the fasting animals. (2~1) B y means of this procedure, a so-called "exogenous" ketonuria was produced which has proved of value in the investigation of fat metabolism. BEACH and associates (2.2) have recently recorded a similar sex difference in the ketonuria of rats suffering from alloxan diabetes. Moreover, when rats with f a t t y livers are subjected to a fast, an " e n(" lo~, ~e nous " ketonuria occurs during the inanition period, even when no supplementary kctogenic acids are fed. Under these conditions, ,lso, the level of ketone bodies excreted is considerably higher in the casc of the female rats t h a n in the male animals. ('4a) The relation of the variation in ketonuria to sex is further suggested b y the demonstration t h a t the high ketonuria ordinarily noted in the normal female rat is reduced, following ovariectomy, to less t h a n the value for male animals. (244) 2. Sex variation in tissue lipids DEUEL and associates (24~) reported t h a t tlle total tissue lipids of female rats on several fat diets exceeded those of males on similar regimens, while the tissue content of protein and water was somewhat higher in the males. The most pronounced variations in lipid composition have been noted, b y various investigators, in the liver. Higher levels of liver lipid in the female are invariably associated with a lower glycogen concentration in this organ. A number of investigators have recorded higher liver fat levels in females t h a n in males under a variety of conditions. (~'4aL (246-251) Many workers ~eaa), {246-248), (252), (25~) have also reported t h a t liver glycogen is consistently lower in the m a t u r e female t h a n in the male. However, a sex difference was not observed in immature or old rats (2~ or in female rats following ovaricctomy; (2:'4~ in fact, in the latter case, the level was slightly higher for the fcmales. A possible clue ~0 the correlation between the variation in composition of lipids in the tissues and the ketonuria has been observed in rats having f a t t y livers. Although the liver lipids of unfasted rats on choline-frce diets were markedly higher in females than in the m~des, a greater drop in liver lipids occurred in the female than in the male during a subsequent 5-day fasting period, concomitantly with a higher kctonuria. (2~'~) This was interpreted as indicating t h a t thc greater production of ketone bodies in the females x~as associated with a more rat)id removal of the lipid from the liver. ~2~5) Another expression of sex difference in lipid storagc is to 1)c found in the studies of LORENZ,CHAIKGFF,and ENTENMAN.(2~6) These workers reported t h a t tile level of neutral fat was much higher in the livers of laying hens than in the non-laying fowl or in male birds. Although no similar variation in phosl)holipid 144

Digestibility and Absorption of Fats as Related to their Nutritional Value and cholesterol in the liver obtained under these conditions, a significant increase in these components, as well as in neutral fat, was noted in the blood.

3. Sex differences in re~luirernents for essential fatty acids Sex differences in composition and metabolism m a y be reflected in the require: m e n t for essential lipids. This was recently demonstrated b y G~EENBER~ et el. (29~ Whereas the optimum level for linoleate intake exceeds 200 mg per day in the male rat, (15~ t h a t for females based upon gro~%h response appeared at first to be a p p r o x i m a t e l y l0 to 20 mg per day. (28~ However, in later experiments, a value of 50 mg was noted for female rats. Closely associated with the difference in linoleate requirement is the sex difference in susceptibility to x-radiation injury. Whereas, in the case of old rats, equal protection was afforded b y fat in the case of both sexes, young adult female rats were not protected b y fat in the diet, whereas young adult males were protected. (2a°~ Since it has also been demonstrated t h a t protection from x-radiation injury is afforded by the administration of linoleate, ('~°) the question arises as to whether or not a sex variation in the a m o u n t of this essential acid in the tissues m a y account for the discrepancy between tlm sexes.

2. Digestibility and Absorption of Fats as Related to their Nutritional Value The importance of fat in nutrition is obviously related to its ability to be digested and absorbed. Thus, in order to serve as a source of calories, fat must gain entrance into the tissues before it can be oxidized and yield its potential energy to the ti~ues. I n a like manner, the utilization of fat is necessary before it can serve in its other capacities. I t has already been noted t h a t hydrogenated perilla oil and tristearin do not possess the thiamin-sparing action of the other fats, because of their low digestibility. The e x t e n t to which a fat is utilized (i.e. its digestibility) is expressed as the coeffcient of dige~tibility. This term refers to the percentage of the foodstuff which is absorbed. This value is calculated b y dividing the fat absorbed (fat ingested minus fat cxcrct:cd) by the fat ingested. A correction figure is usually applied to to~al: faecal fat to correct for the so-called "metabolic fat." This latter fraction is composed of lipid-like material which is excreted irrespective of the a m o u n t of fat in t h e diet; it can readily be determined from the lipid content of the faeces obtained when the subject is on a lipid-free diet.

1. Tl~e dige~tibility, in man, of vegetable arxl animal fats ~elting below 50°C Practically all vegetable and animal fats melting at less than 50°C are almost completely digested b y the normal individual.. The most comprehensive s t u d y of this p r o p e r t y was made in an extended series of tests carried on by the Office of Home Economics of the U.S. D e p a r t m e n t of Agriculture under the direction of the late Dr. C. F. L.A_NGWORTIIYand of Dr. A. D. HOL.~tES. In these tests, the fat under consideration was mixed with ~ fat-free corn starch blancmange and was fe,.t with other foods of low fat content (oranges, whole wheat biscuit, sugar, black coffee) over a 3-day period, during which the faeces were collected for analysis. Although no allowance was made for the faecal flit'excreted as soaps

145 XZ

Nutritional Significance of the Fats in these tests, more recent data would seem to indicute that this additional correction would not appreciably change the findings in the fats and oils covered in the present category. Thus, DEVF~Let al. (257) obtained results for the coefficients.of digestibility of cottonseed oil and rapeseed off in human subjects, when account was taken of the soap excretion, which are identical with figures found in earlier tests in which this value was not determined. Another feature of thdse more recent tests was the use of a new technique involving a much longer experimental period, and the use of an appetizing mixed diet in which the experimental fat comprised 90% of the total fat. Table 16 gives a summary of the results which have been reported for the digestibility of several vegetable and animal fats in man. According to the results given in Table 16, it would appear that most animal and vegetable fats are well digested by man when taken in amounts of 50 to 100 gm daily. With the exception of avocado fat and toasted oil, which have coefficients of digestibility of 88 and 91 respectively, the average figures for the 34 vegetable fats varied between 94 and 99, and those for the 18 animal fats ranged between 93 and 99.

2. The digestibility, in man, of vegelable a~d animal fats melting above 50°C When the melting points of fats exceed a critical value of approximately 50°C, the digestibility is much less than that observed for lowcr-melting fats. LA_~'GWORTHY and HoLnIES(~-6~ called attention to the fact that the high-melting fats could not be assimilated as completely as the low-mel~ing fats, while DEVEL and ]IO~IES ~26~)suggested that an inverse relationship exists betweer, the extent of digestibility and the melting point of fat. Data on utilization of some highmelting fats are included in Table 17. ,Mthough the coefficients of digestibility of the several natural high-melting fats (deer, mutton, oleostearin) and of several of the higher-melting hydrogenated fats are definitely lower than those for the vegetable and animal fats reported in Table 16, they are to be regarded as m~ximum rather than minimum values for human subjects. This is because allowance was not made for the portion excreted in the faeces as soaps which, in tiffs case, would probably represent a fairly large fi~lre. The discrepancies between the reported values for digestibilities in man and in other animals can probably be attributed to this fact. Further experiments arc necded to clarify the absolute extent of digestibility of the several higher-melting fats in man.

3. The digestibility of oleomargarines in man Hydrogenation per se does not lower the digestibility of the fat unless the resulting mix%ure has a sufficiently high melting point. Moreover, when completely hydrogenated corn, cottonseed, or peanut oil, melting at above 60°C, is mixed with untreated oil so that the melting point of the resulting blend is under 50°C, the coefficient of digestibility is not appreciably lowered below that of the liquid fats. In a study of the digestibilities of the older types of oleomargarine obtainable 146

g~

Almond . A p r i c o t kernel Avoca(lo lJtack wa|~lut Brazil n u t lluttcrnut Charlock C h e r r y kernel Cocoa b u t t e r Coconut Coconut Cohune Corn . Cottonseed Cottonseed Cupuassu English walnut 1Iempseed /lickory nut Japanese mustardaeed Java ahnond 51elan seed . Olive . l ' a l m kernel P e a c h kernel ]'canut l)ccan l'oppysec(l . Pumpkinsccd Rapeseed

Vegetable .fats :

Fat tested

258 . 259 260 258 260 258 201 259 262 262 263 260 261 262 257 260 258 260 258 261 264 259 262 26O 259 262 258 260 259 261 257

Reference

60

82

78

50

73 100 62 98 104

41

95 79 60

84

86 60 41 78

52 80

58 59 51 64 28

84 40

gill 70 70 100 56

Average daily fat intake

J

97 98 88 98 96 95 99 98 95 98 89 99 97 98 97 94 98 98 99 96 97 98 98 98 97 98 97 96 98 99 99

Cat.cleat of diffestibility

Becf. Brisket Dutter Butter Butter Chicken Cod-liver Egg-yolk Fish . Goat's butter Goose Hard palate Horse Kid . • Lard Milk f a t Oleo . Ox marrow O x tail Turtle

A)) imal felts:

Sesame Soybean Soybean Sunflowerseed Teaseed Tomato seed Wa~rmolon seed

Vegetable fat.s: (contd.)

Fat tested

"1

.!

265 266 265 2G3 267 266 264 260 206 268 266 268 268 268 265 266 268 268 268 268

262 261 263 261 264 259 264

Reference

lO0 80 lOO 20 40 95 47 83 O0 45 95 90 65 62 90 78 rio 87 77 40

gm 90 82 21 90 49 57 30

fal intake

Avcraffe daily

Table 16. Average coej~cienls, in man, of digesflbilily of vegetable and animal fit~s nielting below 5&C

97 94 97 99

97 07

9495 9g 95 94 94 95

9~

9"/

0'1

93 07 97 S9

98 98 94 96 91 96 95

Coe~cient of diffestibility

N u t r i t i o n a l S i g n i f i c a n c e of t h e F a t s

Table 17. Average coe l~eients of dige~'tibility, in man, of some Mgh-mdtin 9 animal fats and some hydrogenated veitelable fats i

Fat te~txd

l?eferenee

daily Coefficientof Melting point Average fat intake digestibility

265

°C 50

gm

Mutton

53

88

Oleostearin

268

50

68

80

Deer

264

51"4

46

82

Hydrogenated fats : Cottonseed

270

Peanut.

270

Cam

270

35

84

97

46

89

95

37 39 43 50 52

76 78 91 59 62

98 96 96 92 79

33

78 74 44

95 95 88

45.8 47-8 48"1 50-0

53 76 49

96 94 94 87

43-0 43-2 51"1

74 80 90

39 49 54

103

43 50 l¢]end(,d hydrogenated fats:* Cottonseed (12-5 : 87.5)t (18-8 : 81.2) (23.5 : 76.5) (22.1 : 77-9) Peanut. (6-2 : 93-8) (9.1 : 90.9) (33.3 : 6~.7) CA)In

(9.1 : 90.9) (25-0 : 75.0) (30.8 : 69-2)

264

Ol

264 .!

I

i

97 97

93

2(;4 105 92

(.;5 93 ,(}2

• A portion of the fats wa~ almost co ,petely saturated with hydrogen and was blended ~ i t h sufficient untreated oil to yield mixtures with the melting points indicated. ? TIlE Vfllllt'Sin parentheses indicate tile respective prollorti(ms of eomldetely saturated fat and untreated oil in tile fat blend. i n 1915, HOLMES (2;1) r e p o r t e d t h a t t h e y w e r e w e l l u t i l i z e d . T h e v a l u e s f o r t h e 0 9 /o/o o l e o oil, 7 % l a r d , ooo~ v e g e t a b l e s e v e r a l t y p e s s t u d i e d w e r e as f o l h n v s : (i) ( -" oils, 1 -o,°o/ m i l k f a t ) , 9 7 ; (it) (41°,o o l e o oil, 3 2 % l a r d , _940//o p e a n u t oil, 3 % m i l k f a t ) , i~3", a n d (iii) ( 6 7 % o l e o oil, 3' 3 /Oo/ c o t t o n s e e d oil, 0-1 °~,,o m i l k flit),* 97. A h i g h d i g e s t i b i l i t y f o r m a r g a r i n e f a t w a s a l s o s h o w n t o h o l d t r u e in t h e c a s e o f a m o d e r n t y p e o f p r o d u c t in w h i c h t h e fat. is e x c l u s i v e l y a p a r t i a l l y h y d r o g e n a t e d v e g e t a b l e oil. <+-6;~ T t m s , t h i s p r o d u c t , w h i c h h a d a W i l e y m e l t i n g p o i n t o f 94 t o 95°F, was found to be digested to the extent of 96.7% when taken by normal * These are HOL.~tES'figares, which add up to more than 100%. 148

Digestibility and Absorption of Fats as Related to their Nutritional Value men and w o m e n in average a m o u n t s of 86 gm daily. All of the tests on oleomargarine recorded here are open to the same criticism as are the other experiments on h u m a n subjects, namely, t h a t faecal soaps were not determined. However, it seems probable t h a t this would not have influenced the result, inasmuch as a high digestibility was noted for this fat, in rats, in tests which took into consideration the loss of fat in the faeces as soaps (see Table 20). LANGWORTltY12~9} has s.ummarized most of these d a t a elsewhere.

4. The digestibility of fats in animals other than man I t is generally agreed t h a t the p a t t e r n of digestibility of fats is similar in man, dog, and rat. On the other hand, McCAY and PArL (27e~ have reported t h a t guinea pigs digest a n u m b e r of the eommon fats quite poorly. In later studies, these workers (e73~ found t h a t rabbits and sheep utilized fat to a somewhat greater e x t e n t t h a n did guinea pigs. Table 18 gives the average coefficients of digestibility of various fats as reeorded for several species of animals. Although the coefficients of digestibility in man and in lower animals are in general a ~ e e m e n t , a n u m b e r of striking differences exist. In the first place, eastor oil is readily digested in rats, rabbits, sheep, and guinea pigs, and exerts no catharsis in these animals; on the other hand, practically none is utilized in m a n when sufficient is given to produce a eathartic action. However, it is possible that, when administered in amounts too small to produce catharsis, it might be well utilized in the h u m a n subject. In spite of the practically complete digestibility of castor oil in rats, STEWART and SIYCLAm c27s~ were unable to demonstrate a n y trace of its prineipal f a t t y acid, rieinoleie acid, in the phospholipids of the small intestine, liver, or musc]e or in the triglyeerides of the liver. This result can 0nly be interpreted as evidence of the rapid metabolism of this h y d r o x y acid. A second difference between man and the rat concerns the coefficient of digestibility of ra-t)eseed oil. DEUEL, CItENG, and ~[OREtlOUSEO811 have reported that, in the rat, crude rapeseed oil was utilized to the e x t e n t of only 770/0, while t h e figure obtained for the refined oil was ..~oo/ - / o - This is the lowest digestibility which has been recorded for a triglyceride fat, liquid at ordinary temperatures, which is not an intestinal irritant. This finding is in line with an earlier observation (283) t h a t the rate of absorption of rapeseed oil from the gastrointestinal t r a c t of the rat is the lowest for any of the common liquid fats. The low digestibility of rapesced oil in the rat wouhl likewise explain its poor showing in the growth tests reported b y BOER. ¢2s4) The faulty utilization of rapeseed oil in the' rat m a y well be related to its composition. Ig contains 40 to 50% trieruein. (-~8~ Since no failure in lipolysis was nvtcd by DEUEL el aI., (0"8'~ the low digestibility m a y be ascribed to the difficulty in absorption of erucic acid. Th e poor digestibility of rapeseed oil in the rat is in striking contrast to its behaviour in man.. HOLMrS c2n1~ originally reported a figure of 99 for the coefficient of digestibility i n man. Although this investigator (lid not take into eonsi(leration the possible excretion of split products as soaps, the failure to do so cannot be the cause of the d i v e r g e n c y between the results for the rat and for 149

Nutritional

Significance of the Fat~

Tabl¢ 18. Average coefficients of digestibility of some fala in several species of aninuds Caefftcients of digestibility Mdting point

Fat fed

Beef tallow. Butter Butter C a s t o r oil Cacao b u t t e r Cacao b u t t e r C o c o n u t oil . Coconut oil. C o c o n u t oil . C o c o n u t oil, h y d r o g e n a t e d Cod-liver oil C ~ m oil C o r n oil C o t t o n s e e d oil C c t t o r ~ e e d off, h y d r o g e n a t e d Cotton.¢eed oil, h y d r o g e n a t e d . C o t t o r ~ e d oil, h y d r o g e n a t e d . C o t t o n s e e d oil, h y d r o g e n a t e d . C o t t o m ~ e d oil, h y d r o g e n a t e d . Crisco Lard . Lard . Lard, bland i Lard, hydrogenated Lard, hydrogenated Margarine fat Margarine fat M u t t o n tallow Mutton tallow .N'eai~s f o o t oil Oleo s t o c k . • Oleo s t o c k . Olive oil P e a n u t oil, c r u d e P e a n u t oil, rei'med P e a n u t oil, h y d r o g e n a t e d (blended) . . P e a n u t oil, h y d r o g e n a t e d (blended) P e r i l l a oil P e r i l l a oil, h y d r o g e n a t e d R a p e s e e d oil S a l m o n oil . S a r d i n e oil . S o y b e a n oil S o y b e a n oil •

.

°C -34.5 34.5

Guinea

72-0 91.0

Rat

I

Rabb/t

8 -4~.8-3 ~

F

Sheep

Dog

I __

____

a0"7'""t ] 9C.2

28 28 26 26 25 35

94-0

93-8 86-5

38 46 54 62 65 43 37 34 48 55 61 34 47 47

63"3~2v4~* 81"6~2r4~t 95"9 ctr°* 96.5c=~4) t 97 "Sm*~ 94.5tim

l t [ [

87.4

97"0 '~4~* [ ' 98"3'2'*it ] 94.8~'7:~ ~ 91.o,~:a, 94,t;aJ I {

73.8

83"8"°7s~ 68 .7(2;s~ 30.o"'6~§ 24 "0g=rs; 97"31'79~ 94"5 ~zm

[

[ ] 91"0 t2ra~

99~zrlJ 99ttrt~

94(~a~

75.2

94"3~t7'~ [ 63"')~=~ 21.0~'7"

I [

97.0,,-~ I 79-8

99.7~s0~

97.0'~s~ 74 " 6 ~ : ° * I

84.8~:,~ 93.5

48 48

74.0~:~* 94.5

86"7~'~)t [ 98"4(11S~ [ 97"6(~:~ I

91-8 92.6~r~

39

91 "4't~7) [ 97"0~*~ 6.0(llt~

•

67.5

I [

89.Ot*-a~ 94.0 94-5

9 *;'~"*It 98 8""3 5t'~ 98.3,,ro'["

* Fat fed at 5°0 level. ? Fat fed at 15% Ievel. :$ Average of 9 experiments with fat fed at 10%, -a°t°'/o, and 55% of diet. § Average of 6 experiments with fat fed at 10% and 25% of diet. 150

1

Digestibility a n d A b s o r p t i o n of F a t s as Related to their N u t r i t i o n a l Value m a n . DEUEL a n d c o l l a b o r a t o r s / 2 ~ " w h o i n c l u d e d t h e d e t e r m i n a t i o n of faecal soaps i n t h e i r s t u d i e s , o b t a i n e d a coefficient o f d i g e s t i b i l i t y i n m a n i d e n t i c a l w i t h t h a t r e p o r t e d b y HOLliES. O n e c a n o n l y c o n c l u d e t h a t , i n t h e case of r a p e s e e d oil, a v a r i a t i o n obtai:~s b e t w e e n t h e r a t a n d m a n . L t p r e s e n t , t h e r e is n o a d e q u a t e e x p l a n a t i o n f n r t h i s difference. T h e r e s u l t s o n t h e g u i n e a pig c o n s i s t e n t l y p r e s e n t a t h i r d v a r i a t i o n in t h e u t i l i z a t i o n o f f a t b y lower a n i m a l s as c o m p a r e d w i t h m a n . A l t h o u g h cod-liver, olive, a n d s o y b e a n oils are d i g e s t e d t o a b o u t t h e t a m e e x t e n t i n t h e case of t h e g u i n e a pig as i n o t h e r a n i m a l s ( i n c l u d i n g m a n ) , t h e coefficient of d i g e s t i b i l i t y o f corn, c o t t o n s e e d , a n d p e a n u t oils a p p e a r s to be s o m e w h a t d e p r e s s e d in t h i s r u m i n a n t . (eT"~) L a r d has b e e n f o u n d t o be digested t o t h e e x t e n t of o n l y 7 5 . 2 % in t h e g u i n e a pig, as c o n t r a s t e d w i t h 9 6 - 6 % i n t h e r a t 127~) a n d 9 7 . 0 % i n m a n . ~26m

5. The digestibility of simple triglycerides a~ut fatty acids A n u m b e r o f p e r t i n e n t s t u d i e s h a v e b e e n m a d e on t h e d i g e s t i b i l i t y of s i m p l e t r i g l y c e r i d e s a n d f a t t y acids i n s e v e r a l species. T h e r e s u l t s of these s t u d i e s a r e t ~ b u l a t e d i n T a b l e 19.

Table 19. Average coefficients of digestibility of simple triglycerides and fatty acids i7+ rat,, dogs, a~d guinea pigs Meltihg poiut TriglyceriJ.e or fatty ac4dfed

Trilaurin Trimyristin Tripahnitin Tripahnitin Tripahnitin Trist.caria Tristearin Tristearin Laurie acid Myristic acid Palmitic acid Pahnitic acid Palmitic acid Pahnitic acid Palmitic acid Stearic acid . Stearic acid . Stcaric acid . Stearic acid . Stcaric acid . Oleic acid • Elaidic

acid

Pure substance Mixturefed ©C

°C

49 56 66'5

-69

---45 51 -55 59 32 44 -37 44 48 53 --

--

43

--

-70 •

--

-44 53 63 --

--

Rat

97"3m** 76"6m*~ 27-9(n*) 84"0(nS~* 82-0ms)'f 18"9m°) 6"0mS~* 8"0ms~t 81"5(nsJ~ 81"9ms~+ 35-6rag) 39-6IllsJ* 37"lm~J t 31"2msJ§ 23-8mm** 15"8m'j 9"4

95,2si)

10t287)

82<28o)

tnS~*

--

13"31n8~'1 " 21'0ms)§ 19"6m*~

.

--

__

95.41~73~

__

.

--

--

95.6

__

--

Gup~g l rv,]a

Dog

51 55 59

--

.

Coefficient of digestibility

c-+73:

95.0<2rs~ 55.6<=7J) !

* The "t The ** The Tim

fat fat fat fat

]tiikturv mixture mlxtiLre mixture

comqsted conM~ted consisted con.-:isted

of of of of

5% simple trh:Ivceride or f a t t y acid an(I 95% olive oil. 100,, simple triglycerkle or fatty acid and 90% olive oil. 25?0 of f a t t y acid and 75% of olive oil. 1570 of f a t t y acid and ~5% of olive oil.

151

Nutritional Significance of the Fats

I n the absence of other fats in the diet, trilaurin was found to be completely utilized, while trimyristin, tripalmitin, and tristearin were progressively less effectively utilized in the rat. (ng~ In the experiments of ttOA(~LA~'D and S.~IDER, (ns~ in which the triglycerides were fed as 5 or 10% solutions in olive oil, t r i l a u r i n and 'trimyristin were practically completely utilized, while the coefficient of digestibility of tripalmitin was calculated as 84. On the other hand, Che digestibility of tristearin was estimated to be only 6 and 8%, respectively, when fed as a 5% or 10% solution in olive oil. Although the results of L Y y ~ s (2se~ indicate a high digestibility for tripalmitin in the dog, AR.~SCmSK (2s~) reported a m i n i m u m utilization of tristearin in this species. In most cases, f a t t y acids are poorly utilized when administered as such or in solution in olive oil. Palmitic acid was shown to have a relatively high digestibility in the dog; this is in line witll the high utilization of tripa!mitin in t h a t species. However, this result is in sharp contrast to the results on rats. PAt=L and McCA:~"(:r:~ have demonstrat.ed an interesting species difference ;n the utilization of oleic acid and its trans-isomer, claidic acid. Whereas the rat can utilize both of these acids equally well, the guinea pig was able to absorb claidic acid ,,uly to the extent of 55-6°o, in contrast to the complete digestibility of oleie acid in this species (95%).

6. Factors iT~fluencing the digestibility of fats a. The effect of age and vex .............. No systematic studies have been made on the effect of age or sex on the digeStibility of fats. However, it is evident from the studies of HOLT and his coworkerst2~3), (2ss), t2s9) t h a t the digestibility of this foodstuff is less satisfactory in infants and young children t h a n it is in older chihtrcn and adults. GORDO_~ and 5IcNA)L*RA (°-9°~ noted a high excretion of fat in the faeces of premature infants, while WILLIAS,IS(291) and HARRISO.~ and SIIELDO~ (-°92~have reported t h a t cven somewhat older children have a slightly lower digcstit~ility coefficient for fats t h a n do adults. b. The effect of species Considerable differences in the digestibility of fats with high melting points, of castor oil, of rapeseed oil, and of lard have been noted between the levels for man, rats, guinea pigs, and for other animals, respectively. These are listed in Tables 16~-to 19. c. The effect of melting point I t has generally been reported that, for fats melting above some critical temperature, which is approximately 50°C, an inverse relationship obtains between tile melting point of the fat and tile coefficient of digestibility. CHEX[; and her associates t''9~ have reported t h a t this relationship exists in the case of the simple trifflycerides (Table 19). Fig. 5 illustrates the relationship between the percentage of unabsorbed fat and the melting point, of tile fat. In analyzing the relationship of the melting point of a fat to its digestibility ip 152

Digestibility a n d A b s o r p t i o n of F a t s as R e l a t e d to theh- N u t r i t i o n a l Value r a t s , D E U E L (~3) h a s s h o w n t h a t ,

in the

ease of the higher melting fats, the

greatest increase in the excretion of fat occurs in the soap fraction. results are summarized

These

i n T a b l e 20.

P a U L a n d M c C A Y (::a) s t a t e t h a $ t h e m e l t i n g p o i n t o f f a t is a d e t e r m i n i n g f a c t o r , i n s o f a r a s u t i l i z a t i o n is c o n c e r n e d , i n £he g u i n e a p i g b u t n o t in t h e c a s e

,oor Zl

18

BO

14

6C £3

,9, ° Z

40

/

ZC

0 46

I~ I

.50

l

54

l

I

I. I

I

58 62 M.P.C.

I

56

!

I

I

70

74

Fig.. 5. The relationship of mehing point to digestibility of simple triglycerides (solid square), and of samples of hydrogenat~-d lard (solid triangle), when calcium and magnesium were present in the diet. The result of lhe lcsts with calcium.magnesium-low diets is represented in each ease by similar characters which arc not filled in. The numbers beside each character indicate the number of earbo.'l at.ores in the fatly acid component of the simple t riglyeerides2 nl~

Table 20. Comparison o f the effect of melting paine on th~ dige~stibility of some rmtural and hydrogemtt(d fals in rats, and the distribution of excreted fa.t~ between neutral fats and soaps (293~ Faecal/at Fat fed

Melting t point

Fat intnke

~rt~ 0

°C F a t - f r e e diet Margarine rat 1)rime st(~am lard . Criseo . . Bland lard H y d r o g e n a t e d cottonseed oil

H y d r o g e n a t e d la'rd

34

37 43 48 46 65 55 61

.

10-7 12-2 12-3 12-8 14-9 12-3 15-7 13-5 13-2

* Including the Standard Error of the Mean.

153

6oe/~cietU of

Ne~aralfat + fatty acids

Soap8

gm

ym

0.18 0-24 0.21 0.23 0.34 0.73 0-62 6-31 0.58 2.21

0-088 0.30 0-48 0.35 0-68 2.09 3.80 6-93 4.90 8-89

d igcstibility*

97-0 96-6 97.3 94.3 $3.4 68.7 24-0 63.2 21-0

+ ± ~ ± = ± -~ --

0-4 1-4 0-3 1-8 1.4 2.7 2-6 1-2 2.6

Nutritional Significance of the Fats of rabbits and sheep. On the other hand, HOAGL/LNDand S.~IDER(274} have pointed out t h a t their experiments, do not support the thesis that a definite relationship exists between melting point and digestibility. Thus, it was noted that mutton tallow (m.p., 47:C) had a higher digestibility than cacao butter (m.p., 28°C). Moreover, cacao butter, butterfat, and coconut oil, each melting below body temperature, had widely different coefficients of digestibility. (2u~ HOAGLAND and S N I D E R (118) a r e of the opinion that the stcaric acid content rather than the melting point is the factor which controls digestibility. However, the experiments of )~ATTILand HmGL~S, (204) summarized in Table 21, speak against this h)q3othesis. One must conclude that, whereas melting point is an important consideration in determining the digestibility of a fat, it is not the sole factor, and other conditions such as composition must influence it.

d. The effect of the structure of the fat Natural fats are known to exist largely in the form of mixed triglycerides rather than as simple triglycerides. The question is pertinent as to whether the digestibility of a fat depends upon its constituent acids or whether it is a function of the arrangement of such acids in the triglyceride molecules. The experiments of ~IATTIL and HmGL~'S(0"94~have definitely proved that the second possibility is the one which is of importanc~ in establishing the utilization of the fat. Thus, as shown in Table 21, the extent of digestibility varies markedly, depending

Table 21. Digestibility of oleic and stearic acids whe~, fed as simple or as mixed triglycerid~ .cz94~ Fat

Cocffcient of

Fat fed I~ food

lrl

fOe~8

digestibility

gltl

gm 51-0 79.2

29.5 48.4

42.2 } 37-6 Av. 39.9

Distearomonoolein, 15°,~,

94-2 24.5

39.7 9.0

57.9}

Tr~stcarin, ,)~°t'/o, triolein, 10%

87.5 35-9

27-4 11"3

~J8.7 }

Monostearodiolein, 15~o

89-4 14.6

24-3 3'9

72.8 } 73"3 Av. 73.0

Tristearin, 10°~; triolein, 5Oo

63.3 Av. 60-6

68-5 Av. 68-6

upon whether the fatty acids are given to the animal in the form of mixtures of the simple triglyceridcs or as mixed triglyccrides containing a similar proportion of fatty acids.

e. The effect of polymeri~tion of the fat When fats are heated at 275 ° to 300°C over a period of time, polymerization occurs. As a result of this trealment, the 0dour and taste characteristic of 154

Digestibility and Absorption of Fats as Related to their l~'uh4tional Value unsaturated glyeerides are removed; at the same time, the oils develop a considerable degree of viscosity. This alteration in properties is associated with a change in structure, as is indicated by the fact that the degree o f unsaturation, as determined from the iodine number, decreases to a considerable degree as polymerization progresses. Several studies have recently been published which indicate that polymerization may influence the physiological as well as the physical properties of a fat. LASSEN, BACON,and D u ~ ~2s'~) reported that the coefficient of digestibility of sardine oil, heated at 250°C for various periods of time, was depressed from 98-3 for the untreated oil (iodine value 177.7), the values for the several fractions being as follows: iodine value, 155.5, 96.0: iodine value, 138-1, 89-5; and iodine value, 124-1, 84-8. :Roy (295) reported similar but less pronounced effects on san-~ples of cow glee, buffalo ghee, lard, and hydrogenated peanut oil when the fats were subjected to 250 °, 275 °, or 300°C. No effect was noted when a temperature of 200°C was employed, and the values were only slightly altered at 250°C.

fi Th~ effect of food,stuffs fed concomitantly with fat There are a number of ways in which the digestibility of fat may be influenced by ether foodstuffs present at the time of digestion and absorption. The most important substances in this category are inorganic salts, which form insoluble soaps and thus prevent absorption of a considerable proportion of the fatty acids. Mineral oil has already been mentioned as an indigestible substance which may reduce absorption by removing some absorbable lipids in solution via the intestine. Moreover, when gastric or intestinal irritants are present in the foods, • the length of stay of the food in the intestine may be so curtailed as to preclude a normal utilization. (a) The effect of cc,lcium salts in 1he diet--Although C-~E~-c and her associates cng~ reported that no appreciable effect was exerted on the digestibility of bland lard when calcium salts were present in the diet, the extent of digestibility of most fats was decreased when these salts were included in the ration. These data are summarized in Table 22. The presence, of calcium has a more pronounced effect on the digestibility of trimyristin and trilaurin than on that of the other fats studied. I t was also reported by CHE.XOand associates (Hg~that the extent of digestibility of trilaurin was proportional to the quantity of calcium in the diet. Thus, the tblh)wing coefficients of digestibility were reported in diets containing 6-1, 2-5, 1-17, and 0 mg of calcium per gram of food, respectively: 70-5, 87-2, 89-5, and 97-3. GIvE~s '~9~ was among the first to report that calcium absorption was decreased when the fat utilization was poor. The extent of the soap formation was believed by BOSWORTIt and co-workers (~97~ to be dependent upon the proportion of ionized calcium, although the amount of soap lost in the faeces was obviously a function of the solubility of the calcium soap. Since calcium oleate is more readily soluble in bile than is calcium palmitate or calcium stearate, it will disappear in a larger proportion than the last two soaps, 155

Nutritional Significance of the Fats

Table 22. The effeel of indu.sion of calcium salts in the diet on digestibility of fats m~*

DieU c,mtaining Ca

Ca-free diet~ Fat f ~

Bland lard . Blended lard* Blended lardt Hydrogenated lard~ Itydrogenated lardl Trilaurin Trimyristin Tripahnit in Tristearin . Pahnitie acid Stearie acid Monostearin

+.=~

Mdtinffi point i

°C 47.8 47.9 55.2 55-4 61-0 49 56 66.5 70 63 69 59-9

gm 12-2 12.5 9-1 9.2 10-6 8-8 9.7 9.8 9.0 9.1 9.6 9.5

¢~

gm 0.39 0-37 1-33 1.17 5-98 0-16 1-49 7-51 7-97 4.98 6.92 4.00

gm 0-54 0-57 1-16 1'40 1-99 0"29 1"33 1"27 0.88 2.29 2-78 1-84

+:-

~'~T.

95-8 95.6 80-0 77-9 38-0 97-3 76"6 27.9 18.9 35.6 15-8 47.4

14-1 10-7 11.8 ll.0 8.9 12-8 9.1 9.5 9-7 9.8 10-1

0.25 0.24 1.06 0'51 3.78 0.25 1"12 6.40 7-50 3-89 3-99 3.22

1.g~l6 1.43 2-97 4.67 6.03 2.72 7-46 2.27 1-90 4-60 5"53 5"55

92'4 91"7 66"2 58-0 17-3 70'5 37"7 12.8 10"6 19"8 14-4 20.7

• One part hydrogenated lard to 9 parts prime stcmn lard. Reported in unpulflL~hedcommunication. * One part hydrogenated lard to 2 parts prime ~teal|l l~rck Whole sample partially hydrogenated. e v e n in t h e p r e s e n c e o f a n e x c e s s o f c a l e i m n . S i m i l a r d a t a a r e r e c o r d e d b y BOYD et al. ~s) T i l e d e p r e s s i n g effect o f a h i g h - c a l e i m n i n t a k e on t h e u t i l i z a t i o n o f f a t s o c c u r s p r i m a r i l y in t h e e a s e o f f a t s h a v i n g a r e l a t i v e l y h i g h s t e a r i c a c i d e~*ntent. A c c o r d i n g t o t h e e x t e n s i v e e x p e r i m e n t s c a r r i e d o u t b y t h e U.S. D e p a r t m e n t o f A g A c u l t u r e ,m t h e d i g e s t i b i l i t y o f f a t b y h u m a n s u b j e c t s , p r a c t i c a l l y all f a t s a n d oils o f a n i m a l a n d v e g e t a b l e o r i g i n w i t h m e l t i n g p o i n t s u n d e r 50~C w e r e u t i l i z e d t o t h e e x t e n t o f 9 3 % or b e t t e r , in s p i t e o f t h e f a c t t h a t t h e b l a n c m a n g e u s e d in t h e d i e t w a s m a d e f r o m s k i m m i l k , ~vhich h a s a r e l a t i v e l y h i g h c a l c i u m c o n t e n t I n m o s t o f t h e t e s t s r e c o r d e d in T a b l e 18 on a n i m a l s o t h e r t h a n m a n , c a l c i u m w a s l i k e w i s e p r e s e n t in t h e d i e t . I t is o n l y in t h o s e n a t u r a l f a t s a n d h y d r o g e n a t e d f a t s in w h i c h a sufficient, s t e a r i c a c i d c o n t e n t is p r e s e n t t o i n c r e a s e t h e m e l t i n g p o i n t o f t h e f a t a b o v e 50°C t h a t t h e d e l e t e r i o u s effect o f h i g h c a l c i u m diet.s becomes evident. (b) The ~ffect of protein--Protein is a s e c o n d d i e t a r y c o m p o n e n t w h i c h m a y m o d i f y t h e d i g e s t i b i l i t y o f fat. BARNES, PRIMItOSE, a n d I:~URI~.(29~) were t h e first t o call a t t e n t i o n t o t h e f a c t t h a t f a t s h a v e a l o u e r coefficient o f d i g e s t i b i l i t y in r a t s on a l o w - p r o t e i n d i e t ( 1 4 % ) l h a n on a d i e t a r y r e g i m e n w i t h a h i g i m r p r o t e i n c o n t e n t (30°/0). T h e s e r e s u l t s l i a v e b e e n c o n f i r m e d b y S.tVAGE (3°°} a n d b y SEVERA.NCE. c2;n O n t h e o t h e r h a n d , s e v e r a l o t h e r i n v e s t i g a t o r s c3m-3°a~ h a v e riffled t o d e m o n s t r a t e a n y a l t e r a t i o n in t h e d i g e s t i b i l i t y o f f a t s in d o g s or in h u m a n s u b j e c t s . T a b l e 23 r e c o r d s s o m e o f t h e p o s i t i v e d a t a . 156

Absorption R a t e o f F a t s as Related to their Nutrit;onal Value

Table 23. The cgmparative digestibility of several fats in the case of rats receiving low- or high-protein diets re, "

i

I Fat fed

Six

High.protein diet

I, ow.protein diet

l Fat [ " i "ma~:e -

h

I Coefficient Faecal fall oJdigesti]bility

Fat intake

Coe~cient Faecalfat of digesti] bility

gm 2.09

of/o 6-52 16-74 5-28 19-11

91-9 98-0

F

1-59 1.77 1-82

94-0

1-36

10-20 1.51 7-88

Standard butter c2~9) .

M F

1-80 1-63

20-26 18-86

89.3 92.1

1-89 1-61

9-41 6-09

95"9 97.7

Special butter spread I29Dj

M

2-38 2-00

17-61 19-47

92.4 92-4

2.33 !-86

10.41 10-67

96-2 96-0

2.63 2-64 2.80 2-70

25-4~ 25-98 47-10 38.40

95.5 95.5 86-4 90.8

1.96 2.38 2.19 2-34

11'89 14.35 36-70 29.10

97-6 97-3 89-4 91-7

Steam lard c2t°)

M M

F

F

Peanut oils :,tin Crude . .i M Refined . M Hydrogt,nated . . M Blended hydrogenated :1 M ~,L.

97.6

gm 2.39 2.07 1-86

7•6

98.4 95"4

99'5 96.7

I .

..

,

,

•

g. The effect of emulsifying agents A l t h o u g h it is impossible t o i m p r o v e t h e digestibility o f h i g h l y utilized fats b y th(; use of e m u l s i ~ ' i n g agents, AVGVR a n d co-workers 1278) were able to d e m o n strate a m a r k e d elevation in t h e eoeffieien+,s of digestibility of h i g h - m e l t i n g h y d r o g e n a t e d c o t t o n s e e d oil s a m p l e s in t h e presence o f lecithin. T h e coefficients o f digestibility o f these s a m p l e s in r a t s w i t h o u t and with lecithin were as follows : sample m e l t i n g a t 46°C, 83-8 a n d 87-9; s a m p l e melting a t 54°C, 68.7 a n d 82-8; a n d t h e s a m p l e m e l t i n g a t 65°C, 24-0 a n d 44-2. " T w e e n 8 0 " (PSM or p o l y o x y e C h y l e n e sm'bitan mnnooleate), which is an. espee.~ally effective emulsi~-iltg a g e n t , was shou:n to increase fat utilization in p a t i e n t s whose c a p a c i t y for f a t utilization was low. (a°*) 3. A b s o r p t i o n R a t e o f F a t s as R e l a t e d to their Nutritional V a l u e

A l t h o u g h a b s o r p t i o n a n d digestibility are conshlered b y m a n y people t o be identical, t h e y a c t u a l l y r e p r e s e n t different physiologi(-al functions. A b s o r p t i o n is m e a s u r e d in t e r m s o f rate, while digestibility records the prol)ortion o f ingested m a t e r i a l w h i c h is utilized. Ordinarily, fats ~vhich are q u i c k l y absorbed h a v e a high eoefficient o f digestibility. On t h e o t h e r h a n d , t h e converse m a y n o t necessarily be true. A l t h o u g h no differences m a y be <,bserved betw,:en t h e quantities of t w o fats which can be utilized, as d e t e r m i n e d f r o m digestibility studies when the fats are t a k e n in m o d e r a t e a m o u n t s , a n e n t i r e l y different p i c t u r e m a y be o b t a i n e d if t h e y are fe~l in large doses. T h u s , t h e m a x i m u m a m o u n t of a fat which m a y be 157

Nutritional Significance of the Fats t a k e n without causing a digestive disturbance varies widely. I f the fat is one which is absorbed slowly, diarrhoea m a y occur on the administration of relatively bmall quantities; in the case of a fat having a more r a p i d rate of absorption, tolerance is greater before the onset of the diarrhoea. I t is therefore evident t h a t absorption rates and digestibility coefficients, a l t h o u g h relatO1, afford information on independent physiological responses to fat.

I. Methods for expressing the rate of absorption There are several possible methods b y which the rate of absorption m a y be expressed. One of these procedures, employed by STEE.~BOCK, IRV,'~Y, and WEBER, (z°5~ simply involves recording the percentage of the original dose absorbed during a 4-hr period. As long as the size of the animals, t h e time of absorption, and the dosage employed are uniform, one would expect to obtain fairly consistent results b y this procedure. However, this index loses nmch of its value if a n y one of the above conditions is varied. A seccnd procedure for comparing the rates of absorption is to base t h e m upon the q u a n t i t y per unit surface area of b o d y surface per hour. (2s'~) I n testing this m e t h o d for evaluating absorption, it was demonstrated t h a t fairly c o n s ' a n t rates were obtained for absorption of fats in rats varying widely in sizt., and when different dosages were employed over several time intervals. On the other hand, no consistent results could be obtained b y the use of the index proposed b y STEENBOCK et al. m°5) under the above conditions. Surface area is thus a biometric measurement i m p o r t a n t in assessing not only basal metabolism and the absorption of glucose, b u t also fat absorption•

2. Comparative absorption rates of different fats The results of absorption of 1.5 ml doses of some c o m m o n fats b y adult rats a p p r o x i m a t e l y uniform in size, as calculated b y the index of STEE.~OCK et at., (3°5: are indicated in Table 24, while some comparable studies in which

Table 24. The mean percentages of fats absorbed by previously fasted male rat~s at several periods after feeding 1.5 ~nl of the various fats by stomach tube (~°5~ Absorption* in % of original dose a t : F~ f~d 2 hour#

Butterfat Butter oil Cod liver oil Corn oil Halibut liver oil Lard . Shortening A Shortening B

36'2 ± 37.4 ± 40'8 ~ 28.9 ± 39.4 -24-I ± 26-6 ~ 27-1 ~

1-6 2-3 1-4 0"8 1.6 0-8 1-5 1-8

4 hours 60.3 ± 71.0 ~67.7 ! 58.3 ± 70-2 ± 57.0 :j: 53"8 -52-8 ±

i

• IncludingProbable :Errorof the Meam 158

1-2 1-2 1-9 0-9 2-0 1-5 1"6 2"4

6 hour#

77.2 :t: 2.0 86-4 :j:: 1-7 79-7 :k: 1-5 71-4 J: 2.1 78-1 :k: 1.3 67.5 ± 1.5 68"5 ± 1"7 71-1 -~ 1"5

hOUr~

91"2 ! 95"6 ~ 89"2 ~ 94.4 i 85.4 i 92-3 ! 86"0 i 85-6 ±

12 h.oure

l'l 97-4 :t: 0.4 l'0 0"7 98-2 + 0-4 0-7 97.9 i 0.3 0-9 O-9 97-8 j: 0.4 1-3 98-6 ~ 0"3 1-2 99-6 -t- O'l

A b s o r p t i o n R a t e of F a t s as R e l a t e d to t h e i r N u t r i t i o n a I Valuo

Table 25. The absorption of natural fats and hydrogenated fat~ when fed to rats al a level of 300 mg per 100 em ~ of body surface I

Absorption in m~'/lO0 crat/hr *

S ~ M~imj! point

F~t#a

3 hour8

6 houre

49'6 -4- 2"7 52-6 ± 5"1 42-0 4" 4"5

42.6 4- 2•1 41'6 =k 2-1 43"2 + 2-0

8 hour#

°C Butterfat 'tSt3 Coconut fat ~tSa~ Corn oil ~z°e~ Cottonseed oiP t78j, t..8a~

M F M F .] M I F I

39-8 4" 2-9 --

38.5 4- 0.0 35-0 ± 1-9

-I F

--

•[ M F . F

--

38.5 ± 3.0 37.0 -4- 1.5

43

37.1 ± 2-0

46 54

26-5 ± 1-8 18-0 -4- 1-8

30.0 ± 1-8 26.2 4- 1-9 34.3 ± 0.6 24.7 -i- 2.0 8-5 4" 0.9

48~

34-5 4" 1-7

31-4 -4- 1-6

55 3~

20-7 4- 3-5 44-5 ± 3.3 41.7 _~ 0.0 36-3 4- 1"0 43-2 :~ 9"0

21.6 46.1 36-5 39-7 36.0

.

.

!

m

39.7 4" 1-5 43.3 + 1-3 38.3 ± 2-0

Rapeseed off '2ss~ • •

m

44-9 4" 1-6

P r i m e s t e a m l a r d ~2r°*

Crisco~=~* •Hydrogenated cottonseed oil ~:78~ • F F Hydrogenated lard (~°p F F Margarine fat ~ a ' . M Margarine fat ~'-a° . M Margarine fl.t t ~ , t~o~ . F Margarine faV 2~°', ,~o~ . I F

m

34

34 34

4" 4. ± 4. 4.

3.5 1"9 0.0 1-8 0.0 I 42.8 4, 1.4 i

• hwluding the Standard Eri'or of the Mean. ? Blandlard. T h i s l s a m i x t u r e o f u n h y d r o g e n a t e d l a r d a n d ] a r d h y d r o g e n a t e d t o a m e l t i n g p o i n t a b o v e 4 8 * L .

a b s o r p t i o n is b a s e d u p o n m i l l i g r a m s p e r 1 0 0 c m ~ p e r h o u r a r e g i v e n in T a b l e 25. T h e r a t e s o f a b s o r p t i o n o f b u t t e r f a t , c o c o n u t , corn, a n d c o t t o n s e e d otis a n d p r i m e s t e a m l a r d , a s well as t h a t o f m a r g a r i n e f a t ( T a b l e 25), a r e in t h e s a m e r a n g e in t h e 6- a n d 8 - h r t e s t s , a l t h o u g h , in t h e s t u d i e s a.t t h e 3 - h r i n t e r v a l , b u t t e r a p p e a r s t o b e a b s o r b e d s l i g h t l y m o r e r e a d i l y . On t h e o t h e r h a n d , r a p e s e e d oil is a b s o r b e d m o r e s l o w l y t h a n a r e o t h e r oils; t h i s d i s c r e p a n c y b e c o m e s p a r t i c u l a r l y e v i d e n t in t h e t e s t s c a r r i e d o u t o v e r l o n g e r periods• T h i s is in line w i t h t h e o b s e r v a t i o n t h a t r a p e s e e d oil is p o o r l y u t i h z e d in t h e r a t . n m I n t h e case o f t h e h y d r o g e n a t e d f a t s o t h e r t h a n a m a r g a r i n e f a t , Crisco, a h y d r o g e n a t e d c o t t o n s e e d oil m e l t i n g a t 46°C, a n d a h y d r o g e n a t e d l a r d m e l t i n g a t 48cC a r e a b s o r b e d a t a s l i g h t l y l o w e r r a t e . O n t h e o t h e r h a n d , t h e a b s o r p t i o n r a t e s o f a h y d r o g e n a t e d l a r d m e l t i n g a t 55°C a n d o f a h y d r o g e n a t e d c o t t o n s e e d oil m e l t i n g a t 54°C a r e d e f i n i t e l y l o w e r t h a n t h o s e o f o t h e r fats. I n s p i t e o f t h e w i d e d i s c r e p a n c i e s in absc, r p t i o n r a t e s , m o s t o f t h e f a t s a r e u l t i m a t e l y e q u a l l y well u t i l i z e d , a n d o n l y s m a l l a m o u n t s a r e lost in t h e faeces. E x c e p t i o n s to t h i s c o m p l e t e u t i l i z a t i o n i n c l u d e r a p e s e e d oil ( m e n t i o n e d a b o v e ) , t h e h i g h - m e l t i n g h y d r o g e n a t e d c o t t o n s e e d oil, ~8~ a n d h y d r o g e n a t e d l a r d . ( ' ' ~ I n m o s t cases, t h e r a t e o f a b s o r p t i o n is h i g h e s t for t h e s h o r t e r p e r i o d s , a n d decrcascs toward the end of the test period. There are several possible explanat i o n s for t h i s p h e n o m e n o n . I n t h e first p l a c e , t h e h i g h e s t c o n c e n t r a t i o n o f f a t 159

.N'utritional Significance of the Fats obtains during the early period, and this presumably increases the rate of absorption to some extent. (°-s3~ Moreover, the rate of absorption of the several glycerides, in any single natural fat, differs according to their respective fatty acid content. Presumably this would mean that the most rapidly absorbed glycerides would be utilized first. This explanation might account for the faster absorption of butter during the earlier periods when the short-chain glycerides were first being utilized. Also, this same reasoning would apply to the results with rapeseed oil. During the early period, when the Clvtriglycerides are being absorbed, the rate of absorption of rapeseed oil compares favourabty with that of other natural fats. However, when the residue becomes largely tricrucin, the speed of absorption is markedly retarded. It is a moot question as to what relationship exists between the rate of absorption and the nutritional value of a fat. In the first place, when fats are absorbed at a rapid rate, large amounts can be tolerated without producing a diarrhoea or, presumably, other unfavourable symptoms. On the other hand, such rapid absorption might tend to bring about a greater alimentary lipemia than would be observed in the case of more slowly absorbed fats. Moreover, the advantagcs of the greater satiety which appears to be associated with a longer retention of a foodstuff like fat in the gastrointestinal tract, as compared with carbohydratc or protein, are counteracted by the shortening of the absorption pcriod. In the case of fats which are more slowly absorbed, one can argue that the presence of fat in the gastrointcttinal tract over a longer period has a beneficial effect, in that it cxtends the period of absorption, and thus may entail less of a burden on the organism during any one period. It is only when the rate of absorption of a fat is so slow that the process cannot be completed while the fat is in the part of the gut where the absorption is actively taking place (and hence some is lost in the faeces) that a slow rate of absorption becomes a factor in lowering the nutritional value of a fat. ASNEGE~S and IvY (3°s~ rcported that an incrcase in the proportion of fat prolongs the gastric evacuation time. No significant differences were found between the effect of lard and of vegetable oil, respectively, on gastric emptying time.

3. Factors affecting the tale of absorption of fa.ts a. The effect of age According to IRWIN ctal., ~ neither age nor sex is a factor in regulating the rate of absorption of fats, at least within the range of thci: experiments, namely from 4 to 7 months. However, with the technique employed by these investigators, which involves feeding 1-5 ml of the fat, irrespective of the size of the rat, the index of .~.bsorption would most certainly have been much lower had very young rats been tested. Thus, DEUEL and collaborators ('~s3~reported that, whvn 1100 mg of fat were fed to male rats weighing 270 gin, 43.2°,,0 was absorbed in 3 hr, compared with a value of 23-1°,~ when the same dosage was administered to young rats weighing an average of 63 gin. However, when the absorption index was based upon surface area, the values for thcse groups become 43"3 and 160

Absorption Rate of Faus as Relatc~ to their :Nutritional Value 56.1 mg per 100 cm ~ of surface area per hour, respectively. Moreover, when a standard dosage of 300 mg per 100 cmo- of b o d y surface was used, the comparative values for males were 43.3 4- 1-8 mg (270 gm rats) and 53-8 4- 2.6 mg (74 gm rats); the results obtained with females were 38-1 4- 3.2 mg (150 gm rats) and 43.7 4- 2-1 mg (64 gin rats). One can only conclude t h a t the rate of absorp¢ion of fats is not influenced b y the size of the rat (and hence the age) over a fairly wide range if the results are expressed in terms of surface area. The size of the dosage would appear to be of less consequence, although the most consistent results were obtained when this factor, also, was adjusted to surface area. In the case of man, however, extreme variations in age have been shox~-n to influence the rate of absorption of fat. SOBEL and co-workers, (31°~ as well as TIDWELL et al. C3m have demonstrated t h a t newborn babies and infants under one year of age absorb fats quite inefficiently, as compared with older children. On the other hand. BECKER and associates (3~) reported t h a t fat is absorbed or metab~dized more slowly in the aged t h a n in younger subjects. Thns, the ehylomicron count was found to remain elevated for a prolonged interval after the feeding of fats to aged subjects.

b. The effect of sex I n spite of the fact t h a t sex plays a role in the metabolism of fats, as well as in the requirement for essential f a t t y acids (p. 104), it does not, apparently, exert a n y effect cn the rate of absorption of fats. cO-s3~,(3o9) IRwI.~ et al. ~3°9~have likewise reported t h a t pregnancy does not influence the rate of absorption of fats.

c. The nature of tI~e fat There is no d o u b t t h a t the nature of the fat is of prime importance in establishing the rate of absorption. I f one subscribes to the Frazer hypothesis o f fat absorption, then variations in physical properties m a y well be the factors responsible for differences in absorption rate. On the other hand, if fats must be split prior t o ~bsorption, according to the Vcrz,4r theory, then differences in absorption rate between different fats m a y reflect vari'~tions in the rate of hydrolysis, as well as i n t h c rate of absorption, of the resultant f a t t y acids. (a) The rate of absorption of simple lriglycerides--Although most of the triglyeerides found in natural!y-occurring fats are mixed, information on the effect of the type of f a t t y acid chain on absorption can best be obtained with simple triglycerides. These d a t a can be obtained only for triglycerides with f a t t y acids of C12 or lower, since the high melting point of the longer-chained compounds practically precludes their administration.. I t is well k n o ~ t h a t such highmelting fitts as tripahnitin and tristearin are Utilized only to a minor extent. Thus the rate of absorption would be minimal. Table 26 gives a summ,qry of absorption tests on simple triglyceridcs composed of Co to C1: acids. There are two interesting features to be noted in Table 26, The first one is the extremely rapid ~bsorption rate of triacetin and tributyrin. I t is also evident that, as the n u m b e r of carbons i n the evcmcliain acids increases, there is a progressive decrease in the rate of absorption. The second phenomenon of note 161 12

~ut.r~tlona] gignifieaflee of the FafA

~'able 26. The absorption rates of simple triglycerides ~n ]a.st~ng rais over th ree-hour period# sm~ Absorption in ~ : ' 1 0 0 crnt/hr *

F~ f~

Triaeetin Tripropionin Tributyrin Triisovalerin Trivalerin Trieaproin Triheptylin Tricap~lin Tricaprin Trilaurin

i¢

Even,chain fats

68q

Odd-chain fatJ

+ 1-4 02) 31-4 ± 2-1 (12)

4 5 5 6

65.0 + 2-5 O0) 45"7 :k 2"5 (fi)~

32-9 ± 2-3 (9) 54"5 ± 1-5 (9) I

I 28-0 ~ 1"6 (10)

7

8 10 12

45"9 ~ 4-1 (8) t

21"9 (5)

• Including Standard Error of the Mean. The figures in parentheses represent the number of tests. ff Forked-chain acid classed as even-chain. $ Diarrhoea in all 20 tests.

is the marked retardation in absorption rates of the triglyeerides containing an odd number of et:rbons as compared with the triglyeerides having an even number of carbons. However, tripropionin (Ca), trivalerin (C5), and triheptylin (C7) all have practically the same absorption rates; t h e y do not exhibit the progressive decrease found for t r i b u t y r i n (C4), tricaproin (C~), and trieaprylin (C8). Triisovalerin, although containing an odd number of carbons, appears to fall in line with the even-chain acids rather t h a n with the odd-chain acids. (b) The rate of absorption of fatty acids--It is not known whether the behaviour of the simple triglycerides, as regards absorption, is clue to some property inherent in the triglyeeride molecule, or whether it is related to the f a t t y acids themselves. ~he results in Table 27 indicate t h a t the soaps exhibit, to some extent, the same pattern of absorption as do the corresponding triglycerides. Although this would at first seem to support the VerzAr Lipolytic Theory concerning the mechanism of the absorption of fats, it sheuld be recalled t h a t the Partition Theory of Frazer likewise predicates the hydrolysis of the t riglycerides which eontain water-soluble f a t t y acids. Moreover, it is n o t certain t h a t the administration of the lower f a t t y acids in the form of soaps involves a strictly physiological situation. The result~ in general support the thesis t h a t variations in absorption rate of the simple triglycer!des are related to the speed of absorption of the f a t t y acids. However, acetate exhibits a slow rate of absorption, in contradistinction to t h e rapid rate of absorption of triaeetin. This alteration may, in part., be due to the use of the sodium salts of the shorter-chain fittty acids, since the free acids are tc~) corrosive to be employed. The lower rate of removal of the odd-chain acids, propionic, valeric, and heptylie, as compared with the eorrespondiug even-chain values, can be observed in the ease of the soaps. The short duration of the tests when the soap solutions were employed was due to the fact t h a t iL was impossible 162

Absorption Rate of Fats as Related to their Nutritional Value

Table 27. The rate of absorption of the lower fatty acids by female rats (sl') Ab6orption in u~g]100 eml/hr * Fatty acid fed l hour

3 houra

Acids fed as sodium 8alto in 20;0 o, aqueou~ aolutiot~ in dose~ of 100 rag/100 em z Acetic . Propionic Butyric Butyric[~ Valeric Caproie Heptoic

26-6 21-4 39-7 45-0 23.3 38.0 25.8

-I± 44-t± 4-

1.0 i.2 1-5 2.6 1-9 1.7 1-5

(10 I (16) (18) (16) (11) (11) (20)

Acids fed in free state in dGe~s of approxirnatdy 100 rag/100 em z (in l-h~ur teste) and 200 mgt'100 eras (in 5.hour tea~t,~) Caprylie Nonylie Capric . Undecylic Laurie . Tridecylie

37.3 4- 1-5 ('-)3)

34.4 ± 2.3 (24) 19.2 21.3 2.7 20.8

~_ -± :k

1.5 3.4 1.2 3,2

(22) (15) (I0) (15)

46-0 32-8 22"6 21.4

444±

1.7 1"3 2.1 0"5

(10) (23) (ll) (9)

3.8 4- 0.7 (1o) t

* Including Standard Error of the Mean. Figures in parentheses represent the number of tests hicluded in the average. 5" Diarrhoea developed In 16 of,18 rata before S ham's.

to administer doses of sufficient magnitude to insure a residue in the gut after a period longer t h a n one hour. (c) The effect of therneltinq point of the fat on the rate of absorption--Probably the most i m p o r t a n t factor which influences the rate of absorption is the physical state of the fat which, in turn, is related to its melting point. Few observations are available o n . t h e rate of absorption of fat melting at over 50°C, because of the difficulty of administering the fat in liquid form without killing the test animal. Howev.er, in the tests on digestibility in which the fat was eaten as part of the diet mixture~ the utilization m a y in some cases be so low as to indicate a rate of absorption approaching 0. Intermediate values have been noted for hydrogenated lard (melting a t 55~C) and for hydrogenated cottonseed oil (melting at 54°C) (see Table 25).

d. The role of the adreTwcortical bonnet.s I t is now recognized t h a t tile hormones of the suprarenal cortex are important in fat absorption.. VERZAR and LaSZT (315~, ~31~)were lhe first t o demonstrate tile influence of tile adrenal glands on fat absorption. In an extensive series of investigations, these workers showed t h a t the absorption of fat in. rats was iI~hibited b y adrenalectomy, and t h a t the normal function o f tbe gland, in 163

Nutritional Significance oi" the ~'ats respect to fat absorption, could be restored by the administration of cortical extract, but not of ascorbic acid, which is one of the chief active agents of the adrenal medulla. I t was postulated that thc adrenocortical hormones control the phosphorylation of fat in the intestinal mucosa; this reaction was considered to be essential for the absorption of fat. The mechanism was like~ise related to the presence of riboflavin (vitamin B2), since the activity of the adrenal cortex was found to be diminished in riboflavin deficiency.~31~ On the other hand, BA_R.~ES and co-workers(31s), c319~ failed to note any decrease in the absorption of mcthyl esters.of the fatty acids of corn oil or of corn oil itself, after the removal of the adrenal glands of rats, if tile animals were maintained on salt solution. This would assign the adrenal glands to a secondary role in the absorption of fats, as had previously been demonstrated in the case of glucose. In the latter instance, if dehydration and the consequent circulatory disturbances following adrenalcctomy were avoided by the administration of Rubin-Krick solution, or by that of sodium chloride solution, no disturbance in carbohydrate absorption couhl be noted after extirpation of the adrenal glands. 132°~ However, more recent results have supported the concept of VERZ.~R and LASZT that the adrenal cortex plays a primary role in fat absorption. Thus, BAYETTAand associates ~7~ reported that the reduction in the absorption rate of fat amounted to 38% of the normal in untreated adrenalectomizcd rats, while it was still 2-i~o of the normal in salt-treated animals from which the adrenals had been removed. The lowering in absorption was associated with an accumulation of fatty acids in the intestine. ~3°7~ Both of these deficiencies were correctcd by the oral administration of cortin. In later work of BAR_XES el a l . ~321~ a decreased rate of absorption was reported for emulsified hydrogc,mtcd cottonseed oil after adrenalectomy, although no deviation from the normal was observed for certain other oils in operated rats. BAVETTAand DEUEL(3°6} were unable to confirm this latter work, and suggested that the failure of BARNES and co-workers (321)to note decreased absorption, in the case of ~ majority of the fats tested following adrcnalectomy, may have been due to the fact that large (and probably old) rats were used; it has long been recognizecl t hat corvical deficiency is much more critical in younger animals than in old ones. In spite of the fact t hat adrenalectomy retards the absorption of fats composed principally of C1G and C18 f a t t y acids, ]~AVETTAand I~EUFL~306~reported that no difference obtained in the rate of absorption of tributyrin in normal and in salt-treated adrenalectomized rats. In later work, BAVETTA(a~-~-Jf,)und t h a t no sig,~ificant depression in absorption resulted from extirpation of the adrenal glands in the case of tricaproin (C6) or tricaprylin (Ca). Sodium butyrate [m~) and sodium caproate ~a22) were absorbed equally well in nt~rmal and in operated rats. However, the absorption of eaprylic (Cs) and capric (Clo) acids, when they were fed as the frcc acids, was found to be decreased following adrenalectomy "It was concluded that the adrenal cortex was concerned only with the absorption of the long-chain fatty acids which are insoluble in an aqueous medium. 164_

Absorption Rate of Fats as Related to their Nutritional Value

e. The effect of emulsifying agents I t has recently been demonstrated t h a t the presence of emulsifying agents will increase the rate of absorption as well as the coefficient of digestibility of difficultly absorbed fats. Although some emulsifying agents will also accelerate t h e rate of absorption of readily absorbed fats such as limpid cottonseed oil, t h e y cannot increase the coefficient of digestibility, in view of the fact t h a t these fats, even without the emulsifer, are practically completely utilized. The commoner emulsifying agents in foods include lecithin, mono- and diglycerides, and polyoxyethylene sorbitan monooleote (PSM, marketed under the trade name of " T w e e n 80"}. (a) The effect of lecithln--Commercial lecithin has been wide]T employed as an emulsifying agent in a v a r i e t y of foods, over the past several decades. B y the use of this phospholipid, it has been possible to prepare extremely fine emulsions which possess a remarkable degree of stabihty. At~t-R and her co-workers ~27s~ were able to demonstrate t h a t fats were removed from the intestines more rapidly if t h e y contained lecithin, t h a n they were if this phospbolipid was absent. ~,~qmn cottonseed oil was fed to fasting female rats, the absorption rate (expressed in milligrams per 100 cm 2 body surface per hour) was increased by lecithin from 47-8 ± 2.3 to 61.8 =i= 3.7 in the 2-hr tests, and from 38-5 to 59.9 in the 3-hr experiments. The results on highmelting hydrogenated cottonseed oil were equally decisive in showing the accelerating effect of lecithin. The values for absorption of the sample melting at 46°C were as follows for the fat without and with lecithin, respectively: 3-hr tests, 26-5 + 1"8 and 48.9 -q- 3.0; 6-hr tests, 24-7 ~ 2.0 and 36-7 =t= 2.2. In the case of the sample of hydrogenated cottonseed oil melting at 54°C, the r e s u l t s were as follows: 3-hr tvsts, 1S.0 ~ 1-S and 30.1 =~ 2-8; and 6-hr tests, 8"5 -4- 0.9 and 21-8 ~ 1"5. I t could be shown statistically t h a t these differences were highly significant. ' A n o t h e r experimental procedure which has been used to demonstrate the favourable behaviour of lecithin in aiding the absorption of fat is its effect on the susceptibility to diarrhoea. When doses of fat amounting to from 300 to 600rag per 100 cm ~- body surface were given to rats, a fairly high incidence of diarrhoea was noted. Thus, in a series of 50 tests in which cottonseed or hydrogenated cottonseed oil was used, diarrhoea occurred in 20 cases, or 40°//0. On the other band, only 4 of 40 rats subjected to tcsts with lecithin-containing fat, fed at the same level as those described abo\'e, developed diarrhoea. This represents an incidence of 10~//o, or one-fourth of the control value. (b) The effect of potyo.cyethyIene sorbitan monooleate ( P S M or "Twecn 8 0 " ) - - N o data are available in regard to PS3I comparable to t h a t recorded above for lecithin. However, do.','r:s ct al. c3°~ reported on the fat absorption i n a group of patients with nutritional difficulties secondary to subtotal gastrectomy, carried out t o correct duodenal ulcer, as well as in a case of sprue. I t is believed that the improvement in absorption is accompEshed through the etIect of PS3I on surface tension, which fitcilit.ates the production of a fat emulsion composed of finer fat droplets t h a n would otherwise be the case. 165

Nutritional Significance of the Fats

f. The effect of inhibitors Substances such as monoiodoacetie acid and phlorhizin, which are believed to inhibit phosphorylation, have like~Sse been found to decrezse the rate of fat or f a t t y acid absorption in rats. (a) Experiments with ,nonoiodoacetic acid--Although there is little d o u b t t h a t monoiodoacetate interferes with the absorption of glucose, galactose, and fructose, in contradistinction to its inactivity in delaying the absorption of t.he pentoses, xylose, and arabinose, (3-~3~there is some question as to whether or not it acts to inhibit the absorption of fat. VERZLa and 3IcDot'GALL'317~ reported the results on rats given 3-5 ml of olive oil without monoiodoacetic acid (A), or with doses of this compound in the a m o u n t of 0-07 to 0.1 mg per gm body weight (B). After 6 hr, the following comparative results were noted : fat absorbed, A, 1.5 gm, B, 0 gm ; fat in stomach, A, i.5 gm, B, 3 gm ; fat in intestine, A, 0.5 gm, B, 0-5 gm. The failure in absorption was not due to gastric retention of fat in toddacetate-treated rats, since these rats had an inability to absorb the fat when it was introduced directly into the inteztinc. Monoiodoacetic acid was found to have no inhibitory action on lipase ;(324~ in fact, half of the fat in the intestine of the monoiodoacetate-treated rats was found to be hydrolyzed. On the other hand, the monoiodoacetate tests have been severely criticized b y KL~'GHOFFEI¢ 325) as well as b y [3HYELL and HOBER, (326) on thc grounds t h a t this substance is very toxic and produces bleeding and irreversible damage to the intestinal mucosa. I t was pointed out t h a t not only was the absorption of fat and of glucose inhibited after the administration of monoiodoacetate, b u t also t h a t of the pentoses anct even of sodium chloride was reduced. The absorption is not believed to be influenced by monoiodoacetate in either of the latter groups. (b) Experiments with phlorl;izin--VE~z_~a and LASZT(a'~7~ were the first to demonstrate t h a t an inhibition of fat absorption occurs in the rat following the irjection of the glucosidc, phlorhizin. When phlorhizin was given to rats, i*owas found t h a t the absorption of fat was reduced practically to 0 over a 6-hr period, irrespective of whether the fat was given per os or was injected into the intestine. (3zs~ Not only was the absorption of fat prevented but a similar inhibition in the abs(~rption of f a t t y acids was noted. In the case of phlorhizin, one cannot level the same criticism as regards toxicity as applies to monoiodoacetate, inasmuch as phlorhizin is less toxic, c3~ As in the case of monoiodoacetate, the inhibitory action of phlorhizin has bcen ascribed to its interfercnce with phosphorylation. (al:~ The difficulty resulting from both drugs is not ascribed to the inability to transfer the f a t t y acid to the intestinal mucosa, but rather to the absence of synthesis of the trigly(~ride within the cell. According to VERZ.;m and 3IcDouaALL, the formation of triglycerides must inrolve an intermediate phosphorylation which cannot occur in the presence of inhibitors. FRAZER,(3"s°~however, discounts the validity of ~he experiments with phlorhizin, as well as with monoiodoacetate, as the doses used were excessively large and caused extensive damage to the intestinal mucosa. The subject of the mechanism of fat absorption will be treated in a chapter b y BER(:STRb.Si in Volume 3 of this series. 166

The Comparative Nutritive Value of Vegetable anc~ of Animal Fats

4. The Comparative Nutritive Value of Vegetable and o! Anlms| Fats 1. General remar'l~s Since fats would appear to h~ve a primary significance, not only as sources of calories, but also for m a n y varied physiological functions, it is of considerable importance to ascertain whether or not the need for fats can be met equally well by vegetable or b y animal fats. From a chemical standpoint, vegetable and animal fats are composed of the same type of f a t t y acids and glycerides. However, the animal fats, in general, contain more stcaric acid t h a n do the vegetable tilts, while the reverse is true insofar as the unsaturated acids, linoleic and linolcnic, are concerned. There is some indication t h a t the m a n n e r of distribution of f a t t y acids in the triglyceride molecules varies somewhat in the animal and vegetable fat molecules, respectively. Thus, HmDITCn (28~) has pointvd out t h a t the principle of "even distribution" obtains in the case of vegetable fats, while a random arrangement is presented b y tlle saturated acids (bu~ not b y the u n ~ t u r a t e d acids) in tLe case of animal fats. However, since the animal is able to hydro.lyze and tG resynthesize the triglycerides, there is no reason to suppose tlmt any variation in metabolism would obtain because of the slightly different pattern of assembling the f a t t y acids. Some of the differences in nutritional value, formerly ascribed to fats, are now recognized to be related to variations in their content of fat-soluble vitamins. Whereas vitamin A as such is never found in the vegetable fats, it may, in some cases, be present in the animal fats. In the latter instance, the highest concentrations occur in the liver fats, espco_'ally in fish liver oils, while the storage fats of a majority of animals m a y be completely devoid of this vitamin. In contradistincticn to the distribution of vitamin A, that of the carotenoids (some of Wlfich are provitamins A), is almost entirely reversed. Although both ~- and ~-carotenc are sometimes found in animal fats, (s31), (33~.)including butterfat, (3~) and another provitamin A, cr)~ptoxanthin, has been demonstrated in egg yolk ~3~4~ and in butter, ~33"~)the provitamins are more frequently found in vegetable oils than in animal fats. For example, palm oil is an excellent commercial source of fl-carotene. Vitamins D are found exclusively in animal fats although, when present, they occur in minimal quantities except in some fish liver oils. On the other hand, the tocopherols (vitamin E) have a much wider distribution, and occur in greater amounts, in the vegetable oils than t h e y do in the animal fats. The same s t a t e m e n t can be made concerning the occurrence and distribution of the so-called essential unsaturated f a t t y acids.

2. The com2arative anw,a~Yz of vitamins and of ~ssenZia~fatty acids in anima~ and veget~zbrefats a. Vitamin A and provitamin A B u t t e r is the commonest source of vitarain A among the animal foods. According to a recent co-operative study, (336) tile average vitamin A co:Jtent of buttvr produced in the United States somewhat exceeds 15,000 I.U. per pound, altlmugh it is recognized t h a t the q u a n t i t y of vitamin A varies with season, 167

:h'utritional Significance of the Fate breed of the animals, and their diet. The highest concentration of vitamin A in any animal tissues occurs in the livers of certain fishes. Cod-liver oil (Gadus rnorrhua), which has always been regarded as a standard source of this component, has only a relatively low concentration, namely 600 I.U. per gm. The liver oils from a number of fishes such as the Atlantic mackerel (Scomber scombrus), barracuda (Sphyraena argenlea), yellow,-tail (Seriola dorsalis), and the yellowfm tuna (Neothunnus n!uvropterus) contain between 30,000and S0,000 I.U. per gm. (337~ Even higher quantities of vitamin A are found in other fish liver oils. These include bonito (Sarda chilensi's), 120,000 I.U.; cabrilla pinta (Epinephelus analogus), 170,000 I.U.; swordfish (Xiphias gladius), 250,000 I.U.; ishinagi (Stereolepis ishinagi), 300,000 ].U. ; black sea bass (Stereolepis gigas), 600,000 I.U. ; and several varieties of shark, of which the soupfin (GaleorMuus zyoptcrus) has been reported as containing 6~0,000 I.U., (33s~and in one case 800,000 I.U. (339~ In addition to its occurrence in the liver of fishes and other animals, this vitamin is present in egg-yolk, in lung tissue, and in kidney tissue. Some may also be present in corpora lutea, although it is probably chiefly in the form of carotene. The retina, which has long been recognized as the site in which an exceedingly high concentration of a vitamin A-like compound occurs, is now known to contain vitamin A aldehyde. Although the plants are the principal source of the carotenoids, these provitan~ius frequently occur there with a minimum amount of fat. Such is the case with turnip greens, spinach, and broccoli, as well as with such yellow, orange, or rcd-coloured foods as squash, carrots, apricots, persimmons, and tomatoes. On the other hand, of the commercial vegetable oils, red palm oil has the highest fl-carotene content. ]~UCKLEY(340) has recorded values as high as 1900 I.U. per gm in the oil of the fresh fruit, while KAUFSIA3"N(zal} has noted a value of 3000 I.U. Vitamin A occurs in margarine fat both as preformed vitamin A and as the provitamin. In the case of the United States, the average vitamin A content in margarine was formerly set at 9000 i.U. per lb (20 I.U. per gin). This figure was established as the standard for fortification of margarine by the Food and Drug Administration, in 1941, when it was believed that this value represented the average vitamin A content of butter, ca*2~ ttowcvcr, since the higher figure for thc vitanain A level in butter has been published, (aa6~ practically all margarine produced in the United States contains this higher level of vitamin A (15,000 I.U. per lb or 33 I.U. per gin). Since legislation has been passcd repealing the restrictions on the sale of co]ourcd margarine, some varieties of margarine now sold contain carotene. Such sources of provitamins A may now be included as portion of the vitamin A declared on the label of the product. (3~3) It has recently been demonstrated that margarine is an especially effective vehicle for facilitating the utilization of fl-carotche by the rat. (17.,~

b. Vitamin D Vitamin D is only rarely found in common animal fats, and never in vegetable fats. Small amounts may be present in butter. On the other hand, the most 168

The Comparative Nutritive Value of Vegetable and of A~imal Fats

concentrated sources of this vitamin are certain fish liver oils which 'have been widely employed as practical sources for therapeutic use. The values for the vitamin D in fish liver oils vary from 0 LU. in sturgeon liver oil to approximately 40,000 I.U. per gm in the liver oil of the bluefin tuna (Thunnus thynnus). Liver otis such as those from the swordfish (XipMas gladius) and from the yellowfin tuna (Neothunnus macropterns), contain approximately 10,000 I.U. per gin, while cod-liver oil (Gadus ~norrhua), long regarded as the standard medicine for rickets, contains only 100 I.U. per gm. Among the vitamin D-containing oils listed by BILLS(s44~ in decreasing order of their vitamin potency, cod-liver oil holds twenty-third place. Since it has recently been demonstrated that vitamins D can be formed by irradiation of the provitamins D which occur in foodstuffs, this procedure has become widespread; consequently the availability of the vitamin in the foods has been increased. Thus, vitamin D is formed not only by irradiation of 7-dehydrocholesterol in animal fats but also by similar treatment of the ergosterol and diliydroergosterol in yeast, as well as of the diliydrosi'~osterol in numerous vegetable fats.

c. Tocopherols (vitamins E) Both animal and vegetable fats arc sources of tocopherols. The most ~ffective source of these compounds is wheat germ oil, which contains as much as 550 mg per 100 gm. (s4s) This quantity is more than 20 times that of the most conccntrated animal source, namely cod-liver oil, which contains a maximum of 06 mg per 100 gin. (s46) Thc comi)arative amounts of tocopherols present in some common vegetable and animal fats are listed in Table 28. The comparative activity of oils from the standpoint of their physiological action may vary according to the distribution of the tocoI)herols. From the

Table 28. The toa)pherol (vitamin E) contemptof some rey'dable and animal fa/s ,

,

i

- -

Od or fal

,,

Tocopherol

,

0 il or .fat

Tocopherol

rag/100gin

rag/100 gm 90 80 56, 5 212, 152 175-110 200 4 0 0 - 3 1 0 , 520 550

R i c e b r a n , r e f i n e d ~34~) Safflower, c r u d e '**Sj S e s a m e , r e f i n e d '3's), [a*~) S o y b e a n , c r u d e (a**) S o y b e a n , r e f i n e d : ~ ) , la=*) Soybean phosphatide 'a'~

Vegetable f a t s :

3 B a b a s s u , c r u d e (34s~ . 50 Castorta~) 3 I C o c o n u t , re f'med (~4s~ 3 1 C o c o n u t , h y d r o g e n a t e d 43.6) 119 C ~ r n , c r u d e (~46) 95 W h e a t gcmn, cru(ic's'~). (am C o r n , r e f i n e d 4s45~ 110 1Vheat g e r m , s o l v e n t e x t r a c t e d (a's~ C o t t o n s e e d , ~,Vesson ~ 6 ) C o t tozL~v.~d, r e f i n e d (s*sj 90 23 A n i n ~ l fl~t.s: Linseed,~¢~ Olive(3*~-3*?) i 20, 25, 8 C o d - l i v e r oil (~*) . 50 L a r d , p r i m e s t e a m ~a*s~, ~a~ P a l m , c r u d e (3111 P e a n o t , crude (34~ P e a n u t , refinedt3*5~

48

:Pecan, r e f i n e d Ca4s) R i c o b r a n , eru,le 'x's)

:

45

100

5 I a n g o n a s h a r k l i v e r oil ~*~) Oleo oil ¢'~*~) • S o u p f i n s h a r k l i v e r oil ~

t[

J

169

26 2..~-0-5, 10 2

i

4

Nutritional Significance of the Fat8

standpoint of antisterility effect (which is generally considered to be tlle true vitamin E effect), :t-tocopherol (5,7,8-trimethyltocol), fl-tocopherol (5,8-dimethyltocol), y-tocopherol (7,S-dimetlayltocol), and 5-tocopherol (8 methyltocol) are effective in decreasing order. On the other hand, in terms of their antioxidant action (which is referred to as the tocopherol effect), t h e activity is in reverse order, being greatesL for the b-compound and least for the ~-isomer. The proportion of the several component tocopherols present in any oil will obviously determine its antisterility potency or its antioxidant activity. Although it is not ce=tain that vitamin E plays a role as an antisterility agent in the human subject, it most certainly has other important functions in man. It is of great practical importance in the protection of fats from oxidation; the relatively greater resistance of the vegetable oils to rancidity as compared with animal fats is a reflection of the variation in tocoplierol content. Moreover, the relatively low tocopherol content of olive oil renders it one of the vegetable fats most susceptible to oxidation. Vitamin E is apparently stable to the hydrogenation process;(3sl) in fact, a number of workers (3~2-~541have considered that bydrogenated fats are more reliable sources of tocopherols than is the unhydrogenated product.

d. U~asaturatedfatty acids The vegetable fats in general have much higher proportions of the so-called "essential" acids than do the animal fats (see Table 2, p. 108). Linoleic and linolenic acids are the only biologically active unsaturated acids in vegetable fats. Animal fats m a y include not only linoleic and linolenic acids, but also arachidonic acid, which exhibits the greatest biopotency of the 3 acids. 3. The comparative dig~'tibility and absolT~tion of vegetable and animal fats Digestibility and absorption are important physiological indices of nutrition. No differences in the behaviour of vegetable vs. animal fat can be demonstrated in either of these flmetions. Thus, the coefficients of digestibility of 34 vegetable fats melting below 50°C varied between 94 and 99, with the exception of avocado fat (SS) and teaseed oil (91). On the other hand, the variations in digestibility of 18 animal fats were between 93 and 99 (see Table 16, p. 147). Moreover, there was no apparent difference in digestibility of the higher-melting fats, which were more difficultly absorbed irrespective of whether they were of vegetable or of animal origin (Table 17, p. 148 and Table 18, p. 150). Finally, no appreciable differences in the ra~s of absorption were observed between vegetable and animal fats. From the standpoint of utilization, it would appear that animal and vegetable fats are of equal nutritional value.

4. Growth tests u'ith vegetable and animal fu:s Gro~,h is the physiological response which has most often been accepted as an index of nutritional value. In a large number of studies for the evaluation of the nutritional value of vegetable and animal fats this index has been employed. 170

The Comparative Nutritive Value of Vegetable and of Animal Fat~ a. E x p e r i m e n t s with normal rat$

I n our earlier discussion in this review, it was pointed out t h a t greater weight gains obtain in animals which receive a fat-containing diet t h a n in animals in whose case a fat-free regimen is employed. The question is a pertinent one as to whether or not vegetable and animal fats can function equally well in supplying this need. The chief controversy has been as to whether or not b u t t e r f a t possesses a specific gro~-th-promoting effect. On the positive side of the argument, SCItANTZ, ELVEHJEM,and HART (z55) repol~ed t h a t weanling rats grew b e t t e r over a 6-weeks period when t h e y were fed on liquid skimmed milk homogenized ~ i t h b u t t e r f a t t h a n when corn oil, cot.tonseed oil, coconut oil, or soybean oil was homogenized into the fat. The differences in gro~th-promoting power of the fats largely disappeared after tile 6-weeks period following weaning. I t was later report~dlZS~), (z~7) t h a t the s a t u r a t e d f a t t y acid fraction of butter was more effective in producing growth t h a n was a comparable a m o u n t of the whole butter, while the volatile and u n s a t u r a t e d f a t t y acid fractions were ineffective. The unsaturated acid fraction served effectively for growth after hydrogenation. (357) I t was concluded t h a t the superior gro~'th on b u t t e r f a t observed in the earlier experiments ~a55) was t o be ascribed to the presence of certain longchain saturated acids which were present in b u t t e r f a t b u t not in other fats. However, two independent groups of investigators (3ss), (3~9) have failed to confirm the finding t h a t the s a t u r a t e d f a t t y acid fraction of butter possesses a n y specific effect on gro~%h. GEYER c t a l . (3~°) also reported a superior grox~%h-promoting action on the part of the liquid portion of b u t t e r which remained after the separation of the solid glyceridcs in acetone solution a t -- 4°C, in one experiment. In a repetition of the ~ s t , t h e y failed to confirm the original findings. ,lACK and HL~Sm~W (ael; likewise noted better growth on the liquid fraction of b u t t e r than on other fractions, provided care was t a k e n to prevent oxidative destruction. I t is obvious t h a t the earlier reports of ScI~.,.~TZ et aI., (3Ss), (a~6) in which the superior effect of b u t t e r f a t on gro~vth is ascribed to saturated rather than to unsaturated acids, and tlm lat.er studies in which it was traced to the unsaturated acids,¢3~0~, (s61) are contradictory. Moreover, the results obtained by DEUEL and collaborators (s62~ on whole b u t t e r do not bear out this finding. BOER (a~s) also reported superior g r o ~ t h in rats when b u t t e r f a t was fed, as contrasted with results obtained with olive oil. However, the choice of the vegetable oil was an unfortunate one, since this fat contains very little tocophcrol and consequently readily becomes rancid. DEUEL and co-workers (a~*-~ likewise found tha~ the weight gains of rats fed an olive oil sample which ha~l become rancid were inferior to those observed when other vegetable oils or butterfats were employed, l~oEtt and collaborators ~csa~ reported t h a t rapeseed oil wa,~ inferior to b u t t e r f a t in promoting growth in rats. This finding has been confirmed b y DEUEL and associates ~3s~ as well as b y EL'LER, ECLER, and L15DE.~IA-'~.~as~ The low gro~vth-promoting ability of rapeseed oil has been correlated with its low digestibility irt the rat. c's~ 171

Nutritional Significance of the Fats

In contradistinction to the positive results obtained with butterfat, a number of negative reports fail to confirm the hypothesis that the growth-promoting action of butterfat is superior to that of vegetable fats. Thus, DEUEL and co-workers (ss2~ reported that no st~'perior gro~%h could be observed at any period over 12 weeks following weaning, in male or female rats fed buttercontaining diets, as compared with rations containing corn, cottonseed, olive, peanut, or soybean oils or a margarine fat. In these tests, dried skimmed milk powder was employed as a vehicle for the fat, instead of liquid skimmed milk. The fact that the gains-in-weight in all cases represented true gro~'th was confirmed by the determination of the rate of bone growth. In addition to using the gain-in-weight as a comparative index, it was also established that a similarly efficient utilization of the several diets obtained. Moreover, analyses of the tissues of the rats raised on the several dietary regimen~ indicated a similar distribution of protein, fat, carbohydrate, and ash. (245) One is therefore forced to the conclusion that the extent of gro~-th obtained by means of the several vegetable fats is identical with that of the rats receiving butterfi~t. Several suggestions have been proposed to account for the better growth observed by SCHA~'TZet al. (a55~ on the butter diets than in eases in which other fats were fed. In the first place, there is a possibility that a greater food consumption may have been responsible, since it was sho~vn that rats prefer a diet of bland fat flavoured with diacetyl (butter flavour) to one which is unflavoure(t. (36s~ In the tests on vegetable fats by DEUEL ~t al., ~36:~ cited abo.'e, butter flavour was used; this probably accounts for the uniformly high food consumption in the several dietary groups. BOUTWELLand collaborators (36~) included diacetyl with the corn oil. Although they failed to improve the food 'consumption sufficiently to Cause growth identical with that in the butterfat tests, there was an appreciable increase in food consumption as a result of edding this flavouring. EULER, ELrLER, and SABERG(368) likewise gave a negative answer to the question as to whether or not butterfat has a specific gro~-th-promoting property. These workers noted better grox~'th when oleomargarine was the dietary fat than when butter served in that capacity. More recently, EULEI¢ et al. (3~9~ have extended their studies on growth with butter and margarine diets over a period of 700 days; growth was equally satisfactory with tim two fats over the entire period. Other workers, who have demonstrated a similar gro~vth-promoting behaviour of vegetable fats and of butter, include DEUEL et al.,(3s2L (aTo~ ZL~LCITA and .~,[ITCItELL,(371) HENRY and co-workers, tsSs~ and LASSEN and

BACON.(372) (a) The ~ffect of vaccenic acid on growth--Several suggestions ]lave been made as to the reason for the greater growth of rats on butterfat diets as compared with those containing vegetable fats. For example, vacccnic acid, l l octadecenoic acid, has recently been cited as the acid in butterfat responsible for the superior nutritional value of this fat. ]_~OER,JANSEN, and KFNTIE(28~1note(l that summer butter contains appreciable amounts of this unsaturated acid, while it is absent from winter butter, as well as from Vegetable fats. The presence of the acid in 172

The Couaparative Nutritive Value of Vegetable and of .~aimal Fats b u t t e r f a t and in animal fats has been confirmed b y a number of workers, cs:3-3"s) while GEYER et al. ~3~5) demonstrated its absence in vegetable fats. However, h y d r o g e n a t e d ;'egetable fats have been sho~na to contain relatively large amounts of vaccenic acid. (3~), c3u) The first r e p o r t of the high nutritional value of vaccenic acid was t h a t of BOER e¢ al.;(~6) these workers found t h a t the g r o ~ h of rats on a rapeseed oil diet was increased if the rations were supplemented with vaccenic acid prepared from China wood oil. I t was further sugge~ed t h a t the failure of the E~rLER group(a~8), (sTy) to confirm the earlier r e p o r t of BOER (3e3) and of BOER and J-~NSEN(3~s) on the superiority of b u t t e r f a t over margarine or olive oil could be a t t r i b u t e d to the probable lack of vaccenic acid in the winter b u t t e r produced in Sweden. However, a n u m b e r of reports have failed t o confirm the fact t h a t vaccenic acid is a dietary adjunct. Thus, DEUEL and associates, ~3~4) using the same procedure as BOER and co-workers, (es4) confirmed the fact t h a t rapeseed oil produced a poorer grm~th t h a n did cottonseed oil. However, the addition of vaccenic acid to either the rapeseed oil or the cottonseed oil diets did not augm e n t growth. Similar negative results have been recorded b y EULER e.f a/., (365~ as well as b y NATlt, BAt~KI, ELYEttJEM, and HART. (a~9) In fact, BOER and co!laborators! as°) have rcccntly retracted their earlier statement. I t is possible t h a t vaccenic acid m a y have been mistaken b y BOER el al. ~3~ for 12-octadecenoic acid, since these two unsaturated acids have quite similar properties. However, investigations on the nutritional value of this acid, b y DEt'EL eta/., ~a:m have failed to demonstrate any specific beneficial effects on the part of the l a t t e r acid. I t is therefore a p p a r e n t t h a t there is no convincing evidence t h a t vaccenic acid is specifically required by the animal organism. (b) Experiments on prematurely-wea~¢cl rats--In their original report on the superior nutritional value of milk fats as contrasted with vegetable fats, BOUTWELL and associates (as:) noted t h a t the need for the particular f a t t y acids present in b u t t e r f a t could no longer be demonstrated if the aninmls werc k e p t on the stock diet until they were 30 days o f age, instead of being weaned and placed on the test diets at the age of 21 days. These investigators further stated t h a t this need for butt.er was accentuated in rats placed on the test diets at 14 days of age rathcr than at the usual 21-day perio d. However, tests in the author's ]ab(,ratory (as~) indicated t h a t such prematurely-weaned rats grew as rapidly on complete dicts containing corn, cottonseed, olive, peanut, or soybean oil, or a vegetable margarine fat, as on one containing a butterfat. Moreover, ZIALCITAand ,~,IITcIIELL(37D were able to raise rats as effectively on corn oil a s on :t b u t t e r f a t when the ar, imals were weaned at the age of 7 days and were continued thereafter on synthetic dicts. The evidence would therefore tend to discount the age factor as regards the ~equirement for butterfat.

b. Comparative effectiveness in an.imals receiving restricled die1~ Another procedure fc~r the evaluation of the nutritional merit of a foodstuff is t o compare the results when a restricted diet is employed with those when ttle 173

Nutritional Significance of the Fats

foodstuff So be tested is fed. Under such conditions, if a specific fat is required for growth, the need for it may not be satisfied during periods of restricted food intake. I-Iowever, it was found t ha t the rate of growth of rats on diets containing several vegetable oils or a vegetable margarine fat, when fed at such a level t h a t growth occurred at a subnormal rate, was similar to t h a t obtained on a diet containing a butterfat. ~3s~ During a subsequent period, when the same die:ts were given ad libitum, the response of the rats to the vegetable fat diets was similar to t h a t of the animals on the butterfat regimen.

c. Experiments on rats receiving growth hormone A procedure for nutritional evaluation, which is the converse of t h a t in which restricted feeding is employed, involves the determination of whether or not the diets under investigation 'will be satisfactory at abnormally high levels of gro~%h. I t is conceivable that a substance might be satisfactory for growth under normal conditions but that it might fail under conditions of stress caused by a high rate of gro~-th. As an example of the latter, ERSHOFF and DEUELcas3} reported t h a t the lack of vitamin A can be accentuated when the gro~-th hormone is injected into rats on a vitamin A-free diet. In their experiments, the symptoms of at-itaminosis A appeared sooner, and an earlier death resulted under such conditions. When an active pituitary gro,~'th hormone was injected into rats, it was found that diets containing corn, cottonseed, olive, peanut, or soybean oils, or a margarine fat, supported the increased growth as effectively as did one containing butterfat. (3s~)

d. Growth and nutrition experimenls on children LEICHENGER, EISENBERG, and CAnLSO_n~384) have recently reported the results of a two-year test carried out on children in two orphanages. In one group, margarine served exclusively as the table spread while, in the second case, a comparable diet was used except t h a t butter ieplaced the margarine. I t was found th at the well-being of the children during the test, and Cheir g r o ~ h during the two-year period, were similar in the two groups. It would therefore appear that, provided the nutritional identity of the butterfat diet and the dic~s containing vegetable fats is c~tablished, the results on rats are also applicable to man.

5. Preg~ancy and lactation performance of rats on vegetable or animal fats Other nutritional indices which have been used to compare vegetable and animal fats include their efficiency in animals during pregnancy and lactation. As indicaWd earlier, growth, pregnancy, and lactation aff-rd progressively more critical methods for evaluation of the nutritional value of foodstuffs. It is a ~ell-known fact that fat-low or fitt-free diets are especially unsatisfactory for lactatio,l.(as~L ~as6) t t o w e v e r , SURE~3s;~ found t h a t satisfi~ctory lactation could not bc produced on otherwise iTmd~uate diets by btTtterfat, lard, hydrogenated oil, olive oil, or wheat germ oil. On the other hand, 3[AYNARD anti RAS.~tUSSE-~~3ss) reported that, on adequate diets, improvement in lactation 174

The Comparative Nutritive Value of Vegetable and of Animal Fats obtained in rats when corn or eoconut oil was added to o f;.t-free diet, although hydrogenated coconut oil was found to be ineffective,cn~* There is some question as to what factor in fat is responsible for the beneficial effect of this foodstuff on milk production. According to QUACKENBUSH et of., ~1°~ linoleic acid is necessary for satisfactory lactation. I f this is true, vegetable fats should be preferable for this function since, in general, they contain more essential f a t t y acids than do the animal fats. This would also explain the findings of LOOSLI and associates 13~* t h a t hydrogenated coconut oil is ineffective. On the other hand, these latter workers (n~) were unable to demonstrate an improvement in lactation of rats on a fat-free diet when 125 mg of ethyl linoleate were administered daily. Irrespective of what causes improved lactation when fat is fed, there is evidence of the equivalence of vegetable and animal fats in effecting this phenomenon. Thus, DEUEL et al. ~39°1reported t hat rats fed on diets containing a series of vegetable oils, or a vegetable margarine fat, presented essentially the same reproductive capacity and the same increase in lactation as did animals on a butterfat regimen. These data are summarized in Table 29.

Table 29. Comparative nutritional value of vegetable oils, a vegetable fat, ~argarine and butterfat a.s indiczlted by pregnancy and lactation tests ~39°~ Skimmed

milk powder

mixed with one of the f o l l o w i n g f a t s

P r e g ~ , a n c y tests ] Animal~ bred

L~tter8 cast

29 31 31 35 21 32 28

29 28 30 33 18 31 26

L a e a t i o n tests . . . IR a t s died beforel . . . . • ", l v e r. a g e o - a n y I ue.anzn a~ z i t ~ v e r a. f f e . l - a n y w e t g h t p e r rat dags wetgtd per rat* - -

gm 33.4 (84)

gill

Butterfat C o r n oil C o t t o n s e e d oil Margarine fat Olive o;1 P e a n u t oil S o y b e a n oil .

. . . . ,[ .] i.t

9.09 9.06 9-15 7.57 7.88 8-03 7-60

23 1 2 8 1 4 1 i

~ ,

32'9 34-4 34-8 35-2 33-6 34-5

(112) (98) (112) (70) (84) (98)

i

* Figures in parentheses indicate the number of rats weaned. Only seven-rat litters included in averages.

EULER, EULER, and RONSESTAM-SXBERG(369} have reported an experiment carried out over 18 months which indicates a marked superiority of margarine fat over butterfat as demonstrated by reproduction tests. The total weight at 28 days of all offspring produced over the year and one-half period was 8508 gm for the group on the butterfat diet as compared with 15,956 gm for those on the margarine fat regimen. Although the supplementation with vitamin E improved the response of the butterfat group in a subsequent series of tests, the weights of the supplemented butterfat group did not equal t hat of the unsupplemented margarine group. The results obtained from pregnancy and lactation tests d o not indicate any nutritional response to animal fats w!fich is not shared by the vegetable fat group. 175

Nutritional Significance of the Fats

6. Growth and reproduction of rats over many generations on a diet containing a vegetable margarine

One procedure for estimating the r~utritional value of foodstuffs which combines grm~th, pregnancy, and lactation tests is the so-called "multigeneration" method. Much more information can be gleaned from a test of this kind carried out over a number of generations than can be obtained from growth, pregnahcy, or lactation studies on a single generation. :It is conceivable that a diet which would be satisfactory for grm~th or even for reproduction and lactation during one generation might be unsatisfactory after several generations, over which deficiencies could develop. SHER.~tA_~"and C_~,IPBELL(391), (392) devised a simple diet which allowed rats to continue ~ o w t h , reproduction, and lactation and which at the last report had progressed successfully to the sixty-seventh generation, and was still continuing. (aga~ The diet "A" which was employed to the fortieth rat generation by these workers consisted of whole milk powder (1/6) and ground whole wheat (5/6), plus NaC1 to the extent of 2% of the weight of the wheat. The " B " diet employed after the fortieth generation contained one-third whole milk powder and two-thirds ground whole wheat with 2~/o of sodium chloride. SIIER3I&Nand CAMPBELL(394) reported that the life span of the animals on diet B was prolonged significantly over that of the rats on diet A. A somewhat comparable test has been under way in the author's laboratory since April, 1940, during which the rats have received the same (list as diet B used by SHER.~A.~ and C~WBELL, except that skimmed milk powder containing margarine tilt in an amount comparable to the butterfat in whole milk powder was employed. At the end of 10 generations, the rate of growth of the male and fem,le rats was considerably higher than at the start of the test. ('~95) Moreover, fertility and lactation have been maintained at a high level over the entire period. In a later report, (396~ after 25 generations, it was shown that the same high level of growth and reproduction had continued. At the present time (July, 1953), the experi,nent has successfully progressed to the thirty-seventh generation. These results offer cogent evidence of the equivalence of butterfat and of vegetable margarine fat when tested by the most rigorous experimental procedure, namely, that of the multigeneration experiment. 5. The Comparative Nutritive Value of Mono-, Di-, and Triglycerides Although it is generally agreed that mono- and diglyceridcs arc intermediates in fat digestion and in fat absorption, as well as in its intermediary metabolism, no evidence as to their nutritional value has been available until recently. BRAUN a n d SItREWSBURY(397} were the first to report on this factor. They noted that monostearin and monolinolein were nutritionally approximately equivalent to lard in producing growth in rats, when fed at a level of 8 oz .:o- A_~IESand coworkers ~39s~ reported no difference in growth, reproduction, or lactation performance between rats fed mono- or triglyeerides prepared fl'om cottonseed oil, and those fed the refined cottonseed oil. The monoglyeerides were isolated by molecular distillation. 5Iore recently, 3IATTSOY e2 al. c'~9~ have investigated the 176

The Influence of the Type of Fat on the Toxicity of Alloxan mmm-, di-, and triglycerides isolated from soybean oil and from coconut oil, as well as the purified mono- and triglycerides of olcic, stearic, and laurie acids. These authors concluded that mono-, di-, and triglyeerides of corresponding fatty acid composition were nutritionally equivalent. The caloric efficiencies of the mono- and triglycerides of both stearie and laurie acids were found to be low. These workers attributed this fact entirely or partially to poor absorption. In later investigations, MATTSO.~ and associates [4°m isolated and identified, by Unequivocal methods, as much as 16% of monoglyceride and 36% of diglyceride in the total lipid,~ separated from the lumen of the intestine of rats, following the feeding of triglyceride. This supports the work of FRAZER and S.~IMo.~s,(~°1. who previously interpreted the high hydroxyl value of the lipids isolated from the lumen of the intestine as indicative of the presence of monoglycerides.

6. 2he Influence of the Type of Fat on the Toxicity of Allo~an HovssAY and .-~IARTfNEZ{402) were the first to point out that the toxic and diabetogenic action of alloxan may be considerably influenced by dietary factors. Thus, in the white rat, low-protein and high-lard diets increased the toxicity of alloxan (recorded as mortality during l0 days following the injection of the drug), while high-protein and high-coconut oil diets had a protective effect. On the other hand, GY6RGY and llo~E (4°a~ reported a high toxicity in rats, as demonstratcd by early mortality (within 2 (lays) and haemoglobinuria, on diets free from yeast and high in lard or coconut oil. The early mortality was significantly reduced by the inclusion of yeast Or tocopherol in the diet. Moreover, tocopherol was found to prevent the development of haemoglobinuria caused by alloxan. In a re-evaluation of this problem, RODRIGUEZand ,I2~REHL(4°~) confirmed the increased toxicity occasioned by low-protein diets in alloxan poisoning. On the other hand, it was found that the inclusion of coconut oil or of such short-chain acids as caprylic acid in the diet resulted in a marked decrease in the diabetoge~fic action of alloxan. In contradistinction to these results, it was shown that diets containing large amounts of lard produced a high incidence of diabetes, as well as a high mortality. The results were not influenced by tocopherol, in contrast to those of GY6Rc,Y and :ROSE.~a) Strangely enough, the results on palmitic acid showed the lowest mortality and the lowest incidence of diabetes of any of the fats examined. The divergent behaviour of coconut oil ard lard is believed to be due to a difference between the method of metabolism of a fat composed primarily of long-chain acids (lard) and one containing a large proportion of short-chain acids (coconut oil). V. POSSIBLE DELETERIOUS EFFECTS OF FAT IN THE DIET

1. Cholesterol Deposition and Fat Intake One of tile most serious criticisms which has been levelled against tile inclusion of fat in the diet is tile charge that its contimled ingestion results in increased levels of body cholesterol. Animal fats have been considered to be the more serious offenders, because they contain preformed cholesterol, while vegetable 177 z3

Nutritional Significance of the Fats fats are less harmful in that the phytosterols in these products cannot be absorbed or changed to cholesterol, c4°5-t°s~ even when fed ~ i t h bile salts. (t°s~ However, it is believed that fat ~oer se also contribu[es to the production of cholesterol, which is formed synthetically in the animal body. Acetic acid, related to the two-carbon fragment, which is bcheved to be formed during the metabolism of fat, has been cited as the precursor of cholesterol. Under stich conditions, animal and vegetable fats would share equally in furnishing the building stones for the synthesis of cholesterol. While there is adequate e~idence to prove that cholesterol can originate from acetic acid, (4°9) Gne should not forget that acetic acid may also arise directly from carbohydrate, as well as from fatty acids. Moreover, it can be formed indirectly from this carbohydrate by breakdo~vn of the body fat originally s3mthesized from this foodstuff. ALFr~-SLATER and her collaborators m°) have recently sho~vn that the rate at which cholesterol is synthesized in rats is not influenced by diet; as rapid an increase in deuterocholesterol obtained in the blood and hver of rats on a fat-free dict as in animals receiving a 30% fat regimen. 2. Toxic Products Yr.om Heated Fats

It has already been pointed out t h a t one of the factors which influence digestibility of fats is pol3mmrization. When fi~ts are heated to 275°C or higher, a polymer formation takes place. According to LASSEN, BACON, and Du~-~ ~2s2~ and Ro:t-, ~35~ the coefficients of digestibility of such fats are decreased in pro. portion to the extent of polymerization which has occurred. CR~trrO.~ and MmL.~, ~4m in 1946, were the first to observe that the feeding of linseed oil previously heated at 275°C, in the presence of inert gas, resulted in a high death rate. Several other workers ¢412~, ~4~3, had previously reported deleterious effects of fats which had been subjected to high temperatures, i n some cases, paralysis resulted similar to that described by ~LkCKENZIEe~ al. ~41'~ as occurring in vitamin E deficiency. However, it must be considered that the oils used by these workers had in all cases been subjccted to heating in the presence of air, resulting in oxidation. C~[Pwo_~ and his associates ~415J later showed that a decrease in wcight gains per 100G calorics ingested occurred in rats when the diets contained corn, herring, linseed, peanut, rapeseed, or soybean oil which had been subjected to heating at 275°C anaerobically. A marked effect was noted for peanut oil, in which the gain-in-weight per 1000 calorics ingested decreased from 87 for the unheated oil to 30 f'or the oil heated at the above temperature for 30hr. In the case of linseed oil, the gain-in-wcight amounted to only 4 gm per 1000 calorics for the oil heated for 12 hr, as contrasted with a value of SO for the control values for this fat. On the basis of later work from this same laboratory, ~4te~it was suggested that the primary cause of the reduced nutritivc value was the presence of one or more dimcric fatty acid radicals; these are be]ievcd to act in some way inimical to the well-being of the animals. Still more recently, CRA.~trTO.Wc! al. c41~ concluded that the toxicity of hcated fats may result from the formation of cyclized or branched-chain acids. Although polymerization results when fats are heated at 275°C, thcre is no 178

Toxicity of Rancid Fats

evidence which can be detected by nutritional tests that the temperatures to which fat is ordin..arily subjected in cooking cause injury to the fat. R o y tns~ reported no decrease in digestibility in several fats subjected to 250°C. Likewise, DEUEL et al. c41~ found tha~ margarine fat, which had been heated at 205°-210~C for a tong period during Which a number of batches of potato chips were prepared in it, gave as effective g r o ~ h in young rats, when incorporated in their diet to the extent of 40%, as did unheated oil. 3. Toxicity of Rancid Fats

The effects of rancid fats and products of fat oxidation has been discu~cd in the chapter on autoxidation of fats and related substances, page 51. 4. YlisceUaneons Conditions in which Fat Ingestion is Contraindicated According t o L I and FREE:~AN, ¢418) a greater degree of leucopaenia was noted in rats ~ubjected to benzene when they were maintained on a high-fat diet than when a low-fat diet was fed. The leucocyte count was reduced, irrespoccive of the protein level in the diet. A marked hacmotytic anemia has been demonstrated by DAws and GROSS~419~ in dogs, as a result of the daily administration of 60 gm of fat and of l0 mg per kg of choline. This was accompanied by significant elevation of the icteric index. A somewhat similar condition was shown to obtain in two human subjects who ingested ¼lb of butter and 400 mg of choline for breakfast; the depression in the red blood cell count continued for 36 hr, after which a reticulocytosis occurred. ~[cLEAN and BEVERIDGE~4~) have noted that increasing levels of fat in the diet augmented the extent of liver necrosis in rats on a basal necrogenic diet. On the other hand, the carbohydrate level of the diet was withot~t effect on this condition. VI. 0PTIML~ LEVELS OF FAT IN THE DIET

1. Conclusions Based upon E=periments on Rats

The beneficial effects of fat in the diet are so Varied in character as t~ cause one to question whether fats are only optional components of the rations or whether they should be regarded as obligatory constituents of the diet. The figure at which the optimum fat level is established depends to some extent upon the mcthod of evaluation. By most of the mcthods employed in determining the nutritional value of foodstuffs, somewhat better results were obtained when the diet contained 20 to 40% of fat by weight. However, in many instances, the results obtained were quite satisfactory when only 10°/o of the diet consisted of fat. One question which arises is whether:or not all types of tat can fulfil the nutritior:al requirements equally well. At the present time, the data available are insu ftieient to answer this question. In the case of margarine fat and cottonseed oil, the best results were obtained on the 200/0 and 400/0 fat diets. The determination of the optimum amount of fat as a source of essential fatty 1."9

.N,'utritional Significance of the Fats acids leads to different results, depending upon the particular situation involved. The requirement for the essential acids for gro~-th varies with sex, being higher with males th an with females. In the former case, it probably exceeds 200 mg per day, while in the latter instance the figure is about 50 mg daily. On the other hand, when the essential acids serve as protective agents against x-radiation injury, amounts as small as 10 mg per day have been shown to be effective in rats; however, the ability to withstand x-radiation injury was found to be progressively increased when 10, 20, 50 and 100 mg of linoleate was given daily, c421~ I t seems probable that the beneficial effects of fats are not to be traced exclusively to their essential f a t t y acid content. One experiment, ~9~ which indicates this fact, was carried out with female rats previously depleted of fat. After the administration of 20 mg of linoleate daily over a number of weeks until a plateau in body weight was reached, the dosage was increased to 60 mg without causing any further increase in weight. However, when 10% of cottonseed oil was added to the diet, a marked growth-stimulating effect obtained, which continued over a number of weeks. These results led to the conclusion that fat possesses some beneficial action not traceable to the essential f a t t y acids or tocopherol which it contains. 2. Conclusions Based upon Experiments on Man It is difficult to determine how far the results obtained with rats can be translated into terms of human nutrition. According to BRANDT, (422) the daily intake of fat by the British increased from 99 gm per day before World War I to a figure of 124 gm by 1934. A similar fat consumption also obtained for the Americans and Germans during the latter period ; fat accounted for 30% of the calories in their dietaries at that time. In sharp contrast to this relatively high intake by the Occidentals, the intake for Orientals has been shown to be quite low. According to ~BRANDT,(422) the Japanese consumed a, diet before the war containing only 6 to 10°~) of fat calorics. Moreover, SHE.~(42~)estimates that the soldiers of South China in World War II ate fat to the amount of only 3% of their caloric intake, wlfich is less than one-tenth of the 40°/0 which H o w e (42.~ calculates as the average consumption of the American soldiers in World War II. The Food and Nutrition Board of the National Research Council (United States) ~4"5) has made the following suggestions as to the human requirement for fat: FAT. There is available little information concerning the human requirement for fat. F a t allowances must be based at present more on food habits than cn physio]c,6ical requirements. While a requirement for certain unsaturated f a t t y acids (the linolcic and arachktonic acids of natural fats) has been amply demonstrated with experimental animals, the human need for these f a t t y acids is not kno~-n. In spite of the paucity of information on this subject, there are several factors which make it desirable (1) t bat fat be included in the diet to the extent of at least 20 to 25 per ~ of the total calories and (2) that the fat intake incluae essential unsaturated f a t t y acids 180

Refereuces

to t h e extent of at least l per cent of the total calories. At higher levels of energy expenditure, e.g. for a very active person consuming 4500 calories and for children and for adolescent persons, it is desirable that 30 to 35 per c e ~ of the total calories be derived from fat. Since foodstuffs such as meat, cheese, nuts, etc., contribute fat to the diet, it is necessary to use separated or "visible" fats such as butter, oleomargarine, lard, or shortenings to supply only one-third to one-half the amounts indicated. Much experimental work is still required to determine what quantities are to be considered as the optimum for man. I t is self-evident that the capacity for fat is limited and that, when this quantity is exceeded, gastric distress follows. It is of considerable importance to determine to what extent tolerance for fat vaiies with the type of fat and the method of preparation of the food into which it is incorporated. VII.

CONCLUDING I-~EM.ARKS

The more moJern concept of the role of fat in nutrition would seem to assign more importance to this foodstuff than was formerly the case. Instead of being regarded merely as a concentrated source of calories, this foodstuff is now considered to be an essential requirement for many physiological processes of the body. Thus, it possesses the capacity to spare protein, which is not shared by carbohydrates. Prefeeding with fat enables rats to adjust their metabolism in such a way as to extend their survival period during a subsequent fast far beyond that possible in rats prefed with other foodstuffs. The commanding role which fat plays in metabolism is demonstrated by such physiological phenomena as the rate of gro~%h, Pregnancy, lactation, and physical capacity. Moreover, the importance of fat in the diet can be demonstrated by its beneficial effects in such conditions of stress as thyrotoxicosis, x-radiation, cold, and partial hepatectomy. Although the importance of fat can, in part, be ascribed to the essential unsaturated acids which it furnishes, it is believed that there are b.enefici~.l effects over and abo~:e t h a t of the essential fiitty acids. The whole field of the importance of fat in nutrition is one concerning which our information is still fragmentary. We can look forward to continued elucid~ttion of this field in the future. ]~.EFERENCES n) :BURR, G. O. an d BURR, M. M.; J . B i o l . C h e m . 82 (1929) 345 c~ EVANS, It. M. an d LEPKOVSKY, S.; g. Biol. Che~n. 83 (1929) 269 (3~ an d ~IURPHY, E . A . ; ,1. B i o l . C h e m . 107 (1934) 443 (4~ BIRClt, W. W. a n d GYORGY, P.; Bioche~n. J . 30 (1936) 304 (~' IIoGA~', A. G. an d RICHARD~O.',', L. R . ; .V~dure, I, ond. 13(} {1935) 186 • ~*~ STARLIS6, E. H . ; B r i l . M u d . J . 2 (1918) 105 •:~ Evn.~'s, II. M. and Bt'ItR, G. O.; Proe. Soc. exp. Biol., N . Y . 24 (1926-27) 740 (8~ l~,lC.~*II.~,A. J.,.ANDEI~ON, ~,V. E. a n d .~IE~N'DIZ.L, L. B.; J . B i o l . Chum. 82 (1929) 247 191 DEUEL, H. J:, Jr., GI,,EENBERG, S. ~1"., CALBERT, C. E., SAVAGE, E. E. and Ft*KUI, T . ; J . N u t r i t i o n 40 (1950) 351 t,o~ BURR, G. O.; F e d . -Proc. 1 (19t2) 2 2 i

181

:Nutritional Significance of t h e F a t s (11) (i,) (13) (14) (Is}

~Vrl~so~, R . ; Biochem. J . 35 (1941) 1003 Bt'RR, G. O. a n d BURR, M. M.; J . Biol. Chem. 86 (1930) 587 EVANS, H. M. a n d LEPKOVSKY, S.; J . Biol. Chem. 96 (1932) 157 S L i c e R , R. G.; J. B i o l Chem. 114 (1936) xciv DEUEL, H. J., J r . , GREENBERG, S..-~I., .~kNISFELD, L. a n d ~IELNICK, D.; J . N u t r i t i o n 45 (1951) 535 (]6} SL',,'CLAIR,R. G.; J . Nutrition 19 (1940) 131 (17) B~*RI:~,G. O., BURR, M. M. a n d MILLER., E. S.; J. Biol. Chem. 97 (1932) 1 (is) DEUEL, H. J., Jr. a n d MOKEHOUSE, M G.; Advances in Carbohydrate Chemistry 2 (1946) 119 (19} SCHOE.~rrELXrER, R. a n d RI~rE~'BERG, D.; J. Biol. Chem. 113 (1936) 505 (20) BURR, G. O., :BitowN, 5. B., KASS, J. P. a n d LUNDBEI~G, x,V. O. ; Proc. Soc. exp. Biol., N . Y . 44 (1940) 242 (2~) HV.~rE, E. M., NUNN, L. C. A., S-MEDLEY-)IACLEAN, ]. a n d S.X[ITH, H. H . ; Biochem. J. 34 (1940) 879 (22) SM:EDLEY-~IACLEAN,I. a n d NUN~*, L. C. A.; Biochvm. J. 34 (19t0) 884 {231 TURPEINEN, O.; J . Nutrition 15 (1938) 351 {0.4) ; Droc. Soc. exp. Biol., N . Y . 37 (1937) 37 (~s) 5IAnTI~', G. J . ; J. Nutrition 17 (1939) 127 (2~} TA.N'GE,U.; Sc$. Papers Inst. pT~ys, chem. t?zsex:rch 20 {1932) 13 (o_:) ~ ; Sci. Papcrs Inst. phys. chem. lIvscarch 36 (1939) 2 (2S) GR.EENBERG, S. ~[., CALBERT, C. E., SAV'txGE, E. 13. a n d DEUEL, H. J., J r . ; J. N u t r i t i o n 41 (1950) 473 (29} , ~DEuEL, H. J., Jr. a n d BROW~', J. B.; J. Nutrition 45 (1951) 521 (30) HU)m, E. 5I., NUb.'~', L. C. A., S-',[EDLEY-)[ACI~EA~N', 1. a n d S_~IITH, H. I f . ; Biockem. J . 32 {1938) 2162 (31) EvA~-S, 1t. M. a n d LEPKOVSKY, S.; J . Biol. Chem. 96 (1932) 143 [32) BURR, G. O.; Cl~emi.stry and Medicine, Univ. Minnesota Press 1940, p. 101; Fed. Proc. 1 (1942)224 (a3) KARRER, IJ. a n d KOENIG, t1.; Helv. chim. Acta 26 (i943) 619 (~) ~IACKENZIE, C. G., ~[ACKENZIE, J. ~B. a n d McCoLLUM, E. V.; Biochem. J . 33 (1939) 935 135) ANISFELD, L., GREEN'BERG, S. ~ [ a n d DEU-EL, r[. J., J r . ; J. N , d r i t i o n 45 (1951) 599 (36) BARKI, ~7o H., COLLINS, ~5~.A., HART, E. B. and ELVEHJE),f, C. A . ; Proc. Soc. exp. Biol., N . Y . 71 (1949) 694 {37} Nu,xN, L. C. A. a n d SSfEDLEY-~IACLEAN, I . ; Biochc,n. J. 32 (!93S) 2178 (as) REISER, R . ; J . N~trition 42 (1950) 319, 325 {a*) _ _ : ; j . Nutrition 44 (1951) 159 (40) RIECKEHOFF, I. G., HOLSI.AN, ]~. T. a n d BURR, G. O.; Arch. Biochem. 20 (1949) 331 (¢1) ~,VIDMER, C. a n d HOLX/AN, IR. T.; Arch. BiocIwm. 25 (1950) 1 (42) 11ARPER, D. A., If~DITC~, T. P. a n d TERELESKI, J. T.; Y. Soc. chem. Ind. 56 (1937) 310 T (43) LONGENECKER, 1-1. E. ; Oil ffrbd Soap 17 {1940) 53 (¢~) HILDITCH, W. P., in H E ~ R , G. a n d SCHS.WFELD, I t . ; Chemie u n d Technologie dcr Fctle und Feltprodukte, 2 n d ed., Vol. I, Springer Berlin, 1937, pp. 60 et seq. (*~) BRow.,% J. 13.; Chem. Re'vs. 29 (1941) 333 (4~) SSIITH, F. A. a n d BROW.W, g. B.; Oil and Soap 22 (1945) 277 (47) BRICE, B r A., S'~VAIN, M . L,, SCr[AEr'FER, B. B. and AULT, ~V. C. ; 0~.~ atld S o a p 22 (1945) 219 {as) DEUEL, 1I. g., Jr. in BAILEY, A. E . ; Cottonseed and Cottonseed Products Interscience, New York 1948, p. 763 182

Referencam ~ag~ Wlirrz;, E. A., FoY, J . . R . a n d CF~¢ECEDO, L. R. ; Proc. 8oc. exp. Biol., N . Y . 54 (1943) 301 ~50~ DECKER, A. B., FIT L~RtrP, D. L. a n d MEAD, J. F . ; J . Nu~rilion 41 (1950) 507 ~sl) RUSSELL, W. C., TAYLOR, M. W. a n d POLSKIN, L. J . ; J . Nutrition 19 (1940) 555 (52) ]~ILDITCH, T. P., LEA, C. H. a n d PEDELTY, ~V. "~[.; Biochem. J. 33 (1939) 493 (~3) ELLIS, N. R . a n d HANKINS, O. G.; J. Biol. Chem. 66 (1925) 101 ~s4) _ _ a n d ISBELL, H. S.: J. Biol. Chem. 69 (1926) 219 qss) ; ,1. Biol. Chem. 69 (1926) 239 (56) ~ a n d ZE=r.T~R,J. H . ; J. Biol. Chem. 89 (1930) 185 ~6v) CRUICKSItANK, E. M.; Biochem. J. 28 (1934) 965 (ss) GULIACKSON,T. ~V., FOUNTAINE, F. C. a n d FITCH, J. B.; J. DairySc$.~24 (1941) 315 [sg) GIBsoN, G. a n d ~IUFF.~IAN,C. F . ; M~chigan Shdc Coll. Agric. app!. Sci., Agric. Exp. Sta. Quart. Bull. 21 (1939) 258 (~0) MAYNARD, L. A., GARDNER, K. E. a n d HODSO.~, A.; CorneU Univ. Agric. Exp. Sta., Bull. No. 7 (Mar. I0, 1939) 22 (61) ItA.~SEN, A. E. a n d I,VIESE, H. F . ; Proc. Soc. exp. Biol., N . Y . 52 {1943) 205 ce2) GRLER, F. vON; Biochem. Z. 97 {1919) 311 ~6a~ HANSEN, A. E.; Amcr. J . Diseases Children 53 (1937) 933 (64) GINSBEKG, J. ]~., BERNSTEL~,", C., Jr. a n d IOB, L. V.; Arch. Der~ra~ol. Syphilol. 36 (1937) 1033 ~s) TA~n, S. 3. a n d Z~KO.~, S. J . ; J . Amer. mvd. Assoc. 105 {1935) 1675 ~ HA-~'SEN, A. E.; Proc. Soc. exp. Biol., N . Y . 30 (1933) 1198 (~) FABER, H. K. a n d ROBERTS, D. B.; J . Pedi~d. 6 (1935) 490 (~s) FL~'ERUD, C. W., KE~LE~t, R. L. a n d WIE~E, H. F. ; Arch. Der~wdol. Syphilol. 44 (1941) 849 ~*~) ]IA,~'SEN, A. E.; Proc. Soc. exp. Biol., N . Y . 31 (1933) 160 (70) CORNBLEET, T. a n d PACE, E. R , ; Arch. Dcr~r~:tol. Sypbilol. 31 (1935) 224 ( ' ~ ) B~ow~', W. R., HANSEN, A. E., BURRO, G. O. a n d ~IcQb'ArCRIE, I.; J. Nutrition 16 (1938) 511 ~:~) BROW~', W. I~. a n d tt~.~SE.~, A. E . ; Proc. Soc. exp. Biol., N . Y . 36 (1937) 113 ira) KU:,'KEL, H. 0. a n d ~V~LLI~£S, J. N., Jr.; J. Biol. Chem. 189 (1951) 755 [v4) ENGEL, n . W . ; J . Nutrition 24 (1942) 175 {~5~ SM:EDLEY-~IACLEAN,I. a n d ~'t'N.~, L. C. A.; B~ocI~em. J. 35 (1941) 983 (n) LusK, G.; Z. Biol. 27 (1890) 459 ~ ) "~VzLL.~AN, W., BRUSH, M., CLA~K, It. a n d SW~.~SON, P . ; Fed. Proc. 6 (1947) 423. Also personal c o m m u n i c a t i o n to the a u t h o r (~8) SAMUELS, a . T., G IL.~:ORE~ R. C. a n d REINECKE, R . M.; J. Nutrition 36 (1948) 639 (la) GIL.MORE, ];[. C. a n d Sa)~UELS, L. T.; J. Biol. Chem. 181 (1949) 813 (80) FRENCH, C; E., BLACK¢ A. a n d SWIFT, R. W . ; J. Nutrition 35 (1948) 83 (s~) GEIGER, E . ; Personal com,-~mnication to the a u t h o r (1952) (s.~) SAL~ION, ~V. D.; J. Nedrition 33 {1947) 155 (S3) I{OGER.%C. S., I~]RGUSON, C. C., FRIEDGOOD, C. E. a n d VAIn% IL M.; A~wr. J . i'l, ysiol. 163 (1950) 347 c~a~ PEAI~qO~', P. B. a n d PA,','ZER, F.; J. Nutrition 38 (!949) 257 [a~) Sclrwl.~tER, D. a n d :AIcGAVACK,T. t I . ; New YorkStateJ..~lled. 48 (1948) 1797 lS~l ~IOAGLAND, R. and ,qNIDER, G . G . ; U.S. Dept. Agric., Tech. Bull. No. 725 (1940) (s~) , J. Nulrltion 22 (1941)65 (Be) DEUEL, H. J., Jr., .~IE.
Nutritional Significance of the F a t s (~1) FORBES, E. B., SWIFT, R. W., ELLIOTT, R. F. and JA.MES, W. H.; J. Nutrition 31 (1946) 203, 2 i 3 (~2) _ _ __, JAstxs, W . H., BRAT;ZLER, J. W. and BL.'.CK, A.; J. Nutrition 32 (1946) 387 (ga) --, THACKER, E. ft., S.~HTtt, V. F. and FRE:NCH, C. E.; J. Nutrition 32 (1946) 397 (9,1 IIOAGLA?ZD, 1:{., SNIDE:R, G. G. aI~d SWIFT, C. E . ; J . N u t r i t i o n 47 (1952) 399 (951 SCHT:ER, B. T., CODIE, J. F. and DEU~L, H. J., J r . ; J . N u t r i t i o n 33 (1947)641 1~61 FORBES, E. B. an d SWIFT, R. W . ; J . N u t r i t i o n 27 (1944) 453 (97} RUBNER, 3I.; D i e Geselze des E n e r g i c v e r b r a u c h s bei der Ern~ihrung, Leipzig and Vienna, 1902, p. 109; cited b y LusK, G. T h e S c i e n c e o f N u t r i t i o n , Saundei~, P h i l a d e l p h i a a n d London, 4th ed. 1928, p. 278 (gs} ; S i l z u n g s b e r . p r e u s s . A k a d . TVisscnsch. 16 (1910) 316 (99} A N D E I ~ O N , R. J. a n d LUSE:, G.; J . B i o l . Chem. 32 (1917) 421 ~1o01 MURLIN, 5. R. an d LUSK, G.; J . B i o l . C h e m . 22 (1915) 15 (lol} SWIFT, R. W. an d BLACK, A . ; J . A m e r . Oil Chem. Soc. 26 (1949) 171 (lo2} EVANS, H. 5I., LEPKOVSKY, S. and MURPHY, E . A . ; J . B i v l . C h e , l . 106 (1934) 431 (1o3) .~IAEDER, E. C.; A m d . . r ~ v c . 70 (1937) 73 (lo4) QUACKENBUSH, F. ~,V., KU.'ffM-EROW, F. A. a n d S'rEENBOCK, 1t.; J . N u t r i t i o n 24 (1942) 213 (~o~ KC.~.~rEROW, F. A., P.'~\', H. P. and HICK.~tAN, II. P . ; J . N u t r i t i o n 46 (1952) 489 (lo~) ]~:ROGH, A. an d LINDHARD, 3.; B i o c h e m . J . 14 (1920) 290 (107) SCHEER, B. T., I ) O ~ T , S., CoDm, J. F., and SOULE, D. F . ; A m e r . J . I ' h y s i o l . 149 (1947) 194 (los) ]:{OBERTS, S. an d S~tUEL% L. T.; A m c r . J . _Physiol. 158 (1949) 57 (lo9) --; B u l l . M i n ~ w s o t ~ ~wd. F o u ~ u t a t i o n 4 (1944) 55 {~o} _ _ ; Proc. Soc. exp. Biol., N . Y . 53 (1943) 207 (~) -an d I~{EINECKE:, 1{. 5I.; A m c r . J . P h y s i o l . 140 (1944) 639 (H~) LUNDBAEK, K. and STEVENSON, J. A. F . ; F e d . I)roc. 7 (1948) 75 (11~) TEMPLETON, H. A. a n d EI~SItOFF, B. 1:[.; A m e r . J . P h y s i o l . 159 (1949) 33 (l~a) EVANS, H. M. an d LEPKOVSKY, S.; S c i e n c e 68 (1928) 298 (~a~) , J . B~ol. C h e m . 92 (1931) 615 ; J . B i o l . C h e m . 96 (1932) 165 ; J . B i o l . C h e m . 96 (1932) 179 (11~) IIoA(;LAND, R. and SNIDER, (]. G.; J . N u i r i t i o n 26 (1943) 219 (~x~} Ch:ENG, A. L. S., )IOREtIOUSE, 3I. G. a n d DE:UEL, H. J., J r . ; d. Nutrition 37 (1949) 237 (x~0} SAL~ION, W. D. and GOODM-A.~, ,T. G.; J . N u t r i t i o n 13 (1937) 477 (~i) MACDONALD, D. G. It. a n d MCItENRY, E. \V.; A ~ w r . J . l~hysiol. 128 (1940) 608 (~2~) BANERJI, G. G.; B~ochem. J . 34 (1940) 1329 (x2a) ~ an d YUDKIN, J . ; B i o c h e m . J . 36 (1942) 530 (x~) EVANS, It. M. and LEPKOVSKY, S.; J . B i o l . Chem. 99 (1932) 235 (12z) 3IE:LNICK, D. an d FIELD, ]~., ~r.; J. N u t r i t i o n 17 (1939) 223 (12~} WItIPPLE, D. V. a n d CHURCH, C. F . ; J. Biol. Chem. 109 (1935) xcviii {127) ARNOLD, A. and ELVEItJE3I, C. A.; A m e r . J . P h y s i o l . 126 (1939) 289 (~.os} STnIN, F. E., ARNOLD, A. ~nd ELVI~HJE.~[, C. A.; J . N u t r i t i o n 17 (1939) 485 (x~} CAmLL, W. 5I.; J . N u t r i t i o n 21 (1941) 411 (~3o) REINHOLD, J. G., NICHOLSON, ~. T. L. a n d 0'SH~:A.-ELsoM, K . ; J . ~Vulrition 28 (1944) 51 (~3~) WmPPLE, D. V. a n d CHL:RCH, C. F . ; J . B i o l . C h e m . 114 (!936) cvii (~a~) SCHOE:.X'H:EI.~ER,R. a n d ]{~TTENBERO, D.; J . B i o l . Chcm. 114 (1936) 381 (~a3) .-~IANNERINO,G. J., L:~PTON, 5I. A. and ELVEItJEM, C. A.; Proc. Soc. e2rp. Biol., N . Y . 46 (1941) 100 (la*) , OR-SINI, D. a n d ELVEHJEM, C. A . ; J . N u t r i t i o n 28 (1944) 141 (~x~}

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184

References (lss) ]~LVEHJE~, C. A . ; J. Amc."r. /")ietef. Assoc..'22 (1946) 959 ~l.~e) CZACZKES, J. ~7. and GUG6E.~HE.I~t, K . ; J. Biol. Chem. 162 (1946) 267 ils~ PO~'ER, R. L., AXmla~OD, A. E. and ELV~HJE.~, C. A. ; J . NutriJion 24 (1942) 449 {lss) ~BAUERNFEIND, ,~. C., SOTIER, A. L. a n d BORUFF, C. S.; I n d . Eng. Chem., Anal. Ed. 14 (1942) 666 ilagJ STRO~'O, F. M. a n d CAI~PENTER,L. E . ; Ind. Eng. Chem., Anal. Ed. 14 (1942) 909 1140~ Snl.~ON, W . D . ; J. Biol. Chem. i40 (1941) cix ~141) STOTZ, E.; J . Anazrr. Oil Chem. Soc. 26 {1P49) 341 cl,~) LIP~.~'~', F., K^PLAN, N. O., ,NOVb:LLI, G. D., TUTTI.E, L. C. a n d GUIRARD, B . M . ; J . Biol. Chem. 167 (1947) 869 [~43, NOVEL~J, G. D. a n d L r e . ~ s , n , F . ; Arch. Biochem. 14 (1947) 23 u44~ --, J. Biol. Chem. 171 (1947) 833 ~145~ SOODAK, M. a n d LIP~J~N, F . ; J. Biol. Chem. 175 {1948) 999 (146) BIRCH, T. W . ; J . Biol. Chem. 124 (193~) 775 ~,.4~) ShiP, ON, W . D . ; J. Biol. Chem. 123 (1938) civ (118) SCHNEIDER, n:. A.; Proc. Soc. exp. Biol., N . Y . 44 (1940) 266 ~14~) QUACEE.~BUSH, F . ~V., STEENBOCK, It., KL~.~IEROW, F. A. a n d PLATZ, t~. R . ; J. Nutrition 24 (19~2) 225 [lS0} SCHNEIDER, H., STEENBOCK, l:[. a n d PLATZ, B. R. ; J. Biol. Chem. 132 (1940) 539 (151) I:~ICHARDSON, L. R., ]~OGAN, A. G. a n d ITSCHNER, K. F . ; Univ. Missouri Agriz. E x p . Sta. Research B~dl. 333 (1941) 3; Cl~em. Abst. 36 {1942) 2591 (152) SAR.~A, P. S., S.','ELL, E. E. a n d E[.VEHJE~i, C. A.; J. Nulrition 33 (1947 121) {153) )IEDES, G., KELLER, D. C. a n d KURKJIAN, A.; Arch. Biochem. 15 (1947) 19 (154) 5IctIE.','~Y, E. ~V. a n d GAvI~', G.; J. Biol. Chem. 134 (1940) 683 ( 1 5 5 | USIBRE~T, "~V. ~V. a n d GUNSALUS, ]. C.; J. Biol. Chem. 159 (1945) 333 (158} BOAS, M. A . ; Biochem. J. 21 (1927) 712 (157} GAv].x-, G. a n d ~ICHENRY, E. "~¥.; J. Biol. Chem. 141 (1941) 619 (158} PAVCEK, P. L. a n d SHELL, G. M.; J. Biol. Chem. 146 (1942) 351 (159) ~(,ELVILLE, D. ~B.; Vi~4lmzn8 and Hornmnvs 2 (1944) 29 (180) hIUELDER, K. D. a n d ]~ELLY, E.; J. Nutrilion 23 (1942) 335 (161) RUSSELL, W . C., TAYLOR, M. ~V., WALKER, H. A. a n d POI~K~N, L. J . ; J . ~Vulrilion 24 (1942) 199 ~*~) KRL~GS';AD, H. a n d IJE, 5.; Tidsskr. K j e m i , Bergvcsen 2 (1942) 57; Chem. Abst. 38 (1944) 2367 (l~a) WEEK, E. F. a n d SEVIGNE, F. ~.; J. NulritAon 39 (1949) 233 ~ ; J . Nutrition 39 (1949) 251 ~ -; J. Nutrilion 40 (1950) 563 ~ ) DEUEL, H. J., Jr. a n d G~EENnEIlC~, S~ M.; U n p u b l i s h e d results (1951) (~:) LE.~SE, E . J., LEA.SE, J. (;., STEENBOCK, H. a n d BAU.~i~'~.'% C. A.; J. N~driJion 17 (1939) 91 ~,s~ SP[ERMA~', H. C.; J. N~drition 22 {1941) 153 (1~) :FRAPS, G. S. a n d .-~EINKE, W. W . ; Food Rvsearch 10 (19.t5) 187 (~70) KREULA, 5[. a n d VIRTANEN, A. I.; Upsala LgikarefJren. FSrh. 45 (1939) 3 5 5 ; cited b y MELNICK, D. a n d O~:ER, B. L.; V i t a m i n s and Hormones 5 (1947) 39 ~1~1) BURNS, 3I. J., HAUGE, S. ~'[. a n d QUACKENBUSFI, F. W . ; Arch. Biochem. 30 (1951) 341 (17~} DEUEL, 1t. J., J r . , G~EE.~nERG, S. M., SAVAGE, E. E. a n d MEz2,'ICg, D.; J . Nutrition 43 (1951) 371 (173) RO~VNTREE, J. I:; J , Nulrilion 3 (1931) 345 ~ a ) CURTIS, H. C. a~:d KLINE, E . M.; .Arch. lnlvrn~d Mcd. 63 {1939) 54 ~ ) BUR~'s, M. 5., IIAuGE, S..51. a n d QUACKENBUSH, F. W . ; Arch. Biochem 30 (1951) 347 ~'~) ANONY.~ICUS; J . . t i m e r . m~d. Assoc. 131 (1946) 1426

185

.N'utritional Significance of the F a t s {x~7} BAcoN, E. K., I~SSEN, S., GREENDERG, S. ,~I., ~IEHL, J. ~V. a n d DEUEL, H. J., J r . ; J . N u t r i t i o n 47 (]952)'383 (177a) NIGHTINGALE, G., IA:)CKHART, E. E. a n d HARRIS, R. S.; A r c h . B i o c h e m . 12 (1947) 381 {l~s} BOOTH, R. G:, HE.~'I~Y, K. M. a n d KON, S. K . ; B i o c h e m . J . 36 (1942) 445 cx~9} SCHaNTZ, E. J., ELX~EHJE-~r, C. A. a n d ItART, E. B.; J . B i o l . C l w m . 122 (1938) 381 {is0} _ _ a n d KREWSO.~, C. F . ; Proe. Soc. exp. Biol., N . Y . 42 (1939) 577 (181) ZIALCITA,L. P., Jr. a n d ~IITCHEI,L, It. H . ; J . N u t r i t i o n 30 (1945) 147 {1s2~ GEY-ER, R. P., BOUT~VELL, R. K., ELVEHJE~, C. A. a n d ItART, E. B.; J . B i o l . Chem. 162 (1946) 251 {xsa~ NIEY'r, M. L. a n d DEUEL, H. ft., J r . ; J . B i o l . Chem. 167 {1947) 521 {lS4} B u r r s , J. S.; J . B i o l . Chem. 105 (1934) 87 {lSS} DEUEL, H. J., Jr., GULICK, :~I. a n d B u ~ ' s , J. S.; J . B i o l . Chem. 98 (1932) 333 {lS6} COHEN, S., SNYDER, 5. ~. a n d MuEr.T.~a, J. H . ; J . Boot. 4l (1941) 581 {187} ~VILLIA.~f~,~,V. L., BROQUIST, H. I ). a n d SNELL, E. E.; J . B i o l . Chem. 170 {1947) 619 {18s} ~VH1TEItILL, ~'k. R., OLESON, 5. J. a n d SUBBARC~V,Y. ; A r c h . B i o c h e m . 15 (1947) 31 {189} WILLIASIS, V. R. a n d FIEGER, E . A . ; 1 ~ . E~g. Ch,'m., A~u~l. E d . 17 (t945) 127 {19o} , J . B i o l . Chem. 166 (1946) 335 {191} 0PPEL, T. W . ; J . clin. I n v e s t . 21 (1942) 630 {lt~! BRoQvis'r, H. P. a n d SNELL, E. E.; J . B i o l . Chem. 188 (1951) 431 {193~ TRAGFR, ~V.; Pro{. Soc. exp. B i o l . , N . Y . 64 (1947) 129 {~94~ ItOF.~tA~'N, K. a n d AXELROD, A. E . ; A r c h . B i o c h e m . . 1 4 (1947) 4~2 (195) fi.XELROD, .'k. E., 3IITZ, ~I. a n d IIoF.~LA,NY, K . ; J . B i o l . Chem. 175 (1948) 265 {19s~ TRAGER, "~V.; J . B i o l . Chem. 176 (19t8) 1211 (1~73 RUBIN, S. H. a n d SCI[EINE~t, ft.; A r c h . B i o c h e m . 23 (1949) 400 {a~s~ WH.LIA.~tS, V. R. a n d FIEGER, E. A.; J . Biol. Chem. 170 (1947) 399 {199~ TRAGER, W . ; J . B i o l . Chem. 176 (1948) 133 (2o0~ WILL~A.~IS,V. R. a n d FIEGER, E . A . ; ft. B i o l . Chem. 177 (1949) 739 {20~} BOUGItTON', B. W. a n d POLLOCK, 3I. 1~.; B i o c h c m . J . 50 {1952) xxxi {202} POLLOCK, ~I. R., ]:[O~VARD, G. H. a n d BOUGHTON, B. x,V.; B i o c h e m . J . 44 (1949) lii {203~ , HOWAP.D, G. ~-. a n d ]]ouGtrroY, B. XV.; B i o c h e m . J . 45 {1949) 417 {204) .~'~ELROD, ~_. E., HOFMANN, K. a n d DAUBERT, 1]. F . ; J . B i o l . Chem. 169 (1947) 761 {205} CHENC~, A. L. S., GREENBEI~G, S. ~I., DEUEL, H. J., $r. a n d 3IELNICK, D.; J . B i o l . Chem. 192 (1951) 611 ,_~on} ~IOREIS, F. W. a n d LYNES, K. 5.; B i o c h c m . J . 43 {1948) xlv {20:} ttASSINEN, ft. B., DURBIN, G. T. a n d ]~ERNHART, F. ~V.; A r c h . B i o c l w m . 25 (1950) 91 ~os, --, A r c h . B i o c h e m . 31 (1951) 183 ~.o9} I{ODICEK, E . ; S y r u p . Soc. exp. B i o l . No. I I I (1949) 217 {~o~ ttOD.qON, A. Z.; J . Biol. Chem. 179 (1949) 49 {.on} FR.~NKEL, G. a n d BLEWEa-T, 3I.; J . exp. Biol. 22 (1946) 172 {~} , l~iochem. J . 41 (1947) xviii {2~} ; B i o c h c m . J . 41 (1947) 475 1214} ?tBELI.N', I.; K i l n . IVochsch. 5 (1926) 367 ~21~} ~ _ _ , KNU('HEL, )I. a n d SPICIiTIN, "~¥.; B i o c h c m . Z . 228 (1930) 189 ~*} a n d I~_t~R.STEINER, P . ; B i o c h c m . Z . 19,~ (192S) 19 (.I17} HOFF.~IAN, ] . , DE I]OFF3IANN, E. 5. a n d TALESNIK, ~.; ReL,. 2Ileal. Chile 123 (1945) 392 186

References itts~ $Iug'oz OR~Z, E . ; A c c i S n de la tiroxlna sobre la c u a n l i a del vwtabolismo en ratas somelidas a dietas fleas en grosa y relativa,wrde pobres en colina. Tesis C i r u j a n o - D e n t i s t a U n i v . de Chile, Inst, Fisiol. (1944} $2121 SPALIA)NI CIALDEA, W. ; Ir~fltt('rt47i42d~ dietas grasas sobre la elevacion del eonsumo de ox~jcno en ratas tratadas con tiroxina. Tesis Quimico F a r m a e e u t i c o Univ. de Chile, I n s t . Fisiol. (1945) 12201 BERG, ft.; Z. ges. exp. M e d . 93 (1934) 143 (221} KEESER, E.; K l i n . lVochsch. 17 (1938} 1100 12221 GU'r_~RA, E. Z.; A c c i d n inhibidora de diversas grasas dietvticas (saturadas y no saluradas) sobre elevacion del consurno de oxbjeno produclda p o t la tiroxina. Tesis Medico-Cirujano U n i v . de Chile, I n s t . Fisiol. (1947) 122a~ ZAIN, ]:I.; K l i n . Wochsch. 15 (1936} 1722 i~u~ ; A r c h . exp. Pedhol. 187 (1937) 302 1~5~ ERSHOFF, B. H . ; J . N u t r i t i o n 39 (1949) 259 ~226) GREENBER£~, ,_q.M. a n d DEUEL, H. J., Jr.; J. Nu~'~:ii~on 42 (1950) 279 ~2271 ; J . N u t r i t i o n 47 (1952) 31 ~22sl DECKER, A. B., FILLERUP, D. L. a n d MEAD, J. F . ; J. N~drffion 41 (1950) 507 1~2~1 CH:ENG, A. L. S., KRYDER, G. D., BEItGQUIST, L. and DEUEL, H. or., J r . ; J . N u t r i t i o n 48 (1952) 161 12301 DEX~EL, H . . I . , Jr,, CItE.~'G, A. L. S., KRYDER, G. D. a n d BINGE3fANN, ~[. E . ; Scicncc 117 (1953} 254 (2sl) KffLZ, E . ; Pfliig. A r c h . gcs. Physiol. 24 (lSS1) 46 12321 MITCIIELL, ]_{. It. t GLICK3fAN, N., LA3IBERT, E. R., KEETON, R. XV. a n d FAHNESTOCK, )I. K . ; A m e r . J. Pl~ysiol. 146 (19t6) 8~ 12s31 MA.~:¢, F. C.; A m e r . J . Physiol. 55 (1921) 285 12a4~ ; A m e r . J . Med. Sci. 161 (1921) 37 12ssl ~IARKOWITZ, J. a n d SO.~K~N, S.; Proc. Soc. exp. Biol., N . Y . 25 (1927) 7 12a61 McMaST};R, T. D. a n d DRURY, D. R.; J . exp. 3!ed. 49 (1929) 745 ~23~ BRUES, A. M., DRb~Rn", D. R. a n d BRUES, M. C.; A r c h . Patlwl. 22 (1936) 6"58 (2a8~ CLAYTON, C. C. a n d BAU,~L~.~N,C. A.; Arch. B i o c h e m . 16 (1948) 415 I ~ LUNDBAEK, K. a n d STEVE.~SO.~', 5. A. F . ; A m e r . J . Physiol. 151 (1947) 530 i~0} DEUEL, It. J., Jr. a n d GULICK, 3I.; J . Biol. Chem. 96 (1932) 25 (~} B u r r s , J. S. a n d DEUEL, H. 5 , J r . ; J . Biol. Chem. 100 (1933) 415 12a~1 ]:{EACH, E. F., ]~RADSHAW, P. J. a n d BLATtLERWICK, N. R.; A ~ c r . J . P h y s i o l . 166 (1951) 364 [2~s~ DEUEL, H. J., Jr., ttALLn~.~', L. F. a n d MURRAY, S.; J. ~,iol. Chem. 119 (1937) 257 I~} GRUNEWALD, C. F , CUTLER, C. ~ . a n d DEUEL, t~. J., J r . ; J. Biol. Chem. 105 (1934) 35 i~a~} DEUEL, It. J., Jr., tItJ~L.~[A.~*, L. F,, Movi~r, E., MAVTSON, F. 1I. a n d WU, E. ; J . N u t r i t i o n 27 (1944} 335 (24~1 GRE~SHEL~mR,E . ; J . N u t r i t i o n 4 (1931) 411 12~71 PONSFORD, A. I~. a n d SMEDLEY-~IACLEAN, I.; Biochem. J. 26 (1932) 1340 12~s} DEUEL, H. J., Jr., GULICK, M., GRUNE~VALD, C. F. a n d CUTLER, C. I:L; J . Bivl. C~wm. 104 (1934) 519 12491 OKEY, R., GILLUS[, H. L. a n d YOKELA, E . ; J . Biol. Chvm. 107 (1934) 207 ~2501 BAR.N'ES, :R. H., ~IILLER; E. S. a n d BURR, G. O.; J . Biol. Chem. 140 (1941) 247 125~1 :BEST, C. 1I., RIDOUT, J. H., I)A~I'rERSON, 5. ?,I. a n d LUCAS, C. C.; Biocltem. J . 48 (1951) 448 12521 DEUEL, ]-[. J., Jr., BUTTS, J. S., IIALL.~IAN,L. F., 3[URRAY, S. a n d BLUNDEN, H. ; J . Biol. Chem. 119 (1937) 617 ~31 BLATttER'WICK, N. R., ]~RADStL*-B', P. J., CULLI3[0RE, O. S., n w I ' ; O , 3I. E., LA~
187

N u t r i t i o n a l Significance of the F a t s c25~ DEUEL, H. J. Jr., 3[URRAY, S.,-I"-[ALL.M~N, L. F. a n d TYLER, D. B . ; J. Biol. Chem. 120 (1937) 277 ~2~6~ LORENZ, F. W., CHAIKOFF, I. L. a n d ENTENMAN, C.; J . Biol. Chem. 123 (1938) 577 {257} DEUEL, H. 3., Jr., JOHNSON, R. M., CALBERT, C. E., GARDNER, J. a n d T ~ o ~ s , B.; J . N u l r i t h m 38 (1949) 369 (2~s~ ttOI-~[ES, A. D.; U.S. Dept. Agric. Bull. N o . 630 (16th Apr. 1916) (~sg~ ; U.S. Depf. Agr~c. Bull. No. 781 (31st May 1919) (260~ . a n d DEUEL, H. J., J r . ; J. Biol. Chem. 41 (1920) 227 (2e1~ ; U.S. Dept. Agriv. Bull. N o . 687 (28th JuDe 1918) (262~ LANGWORTHY, C. f . a n d HOI2~ES, A. D.; U.S. Dept. Agriv. Bull. No. 505 (13th Feb. 1917) {2s3) HOLT, L. E., Jr., TID~ELL, H. C., KIRK, C. h~., CROSS, D. hi. a n d NEALE, S.; J. Pediot. 6 (1935) 427 t2ea~ DEUEL. H. J., ,Tr. a n d H O T . ~ s , A. D.; U.S. Dept. Agrio. Bull. No. 1033 (27th J u l y 1922) ~26~ LANGWORTn*', C. F. a n d HOLLIES, A. D.; U.S. Dept. Agric. Bull. No. 310 (9th Nov 1915) t~sG, --, U.S. Dept. Agric. B~ll. No. 507 (24th March 1917) (28~ DEUEL, ]_t. J., J r . ; J. N u t r i t i o n 32 (1946) 69 (26s~ ]IoI.~[ES, A. D.; U.S. Dept. Agric. Bldl. No. 613 (25th Apr. 1919) ~269~ LANGWORTItY, C. F . ; Ind. E~g. Chem. 15 (1923) 276 ~270, HOL.~tES, A. D. a n d DEUEL, H. 3., J r . ; A ~ v r . J. Physiol. 54 (1921) 479 [271~ ; Boston ~wd. and surg. J. 192 (1925) 1210 ~2~ McCAY, C. M. a n d PAUL, H . ; J. N u tr i ti o n 15 (1938) 377 [2~3~ P.~UL, 1I. a n d 5IcCAY, C. ~I.; Arch. Biochem. 1 (1942) 247 {274~ HOAGLAND, I:[. a n d SNIDER, G. G.; J . N u t r i t i o n 25 (1943) 295 (275) STE'O,'ART,W. C. a n d SINCLAIR, R. G.; A r c h . Biochem. 8 (1945) 7 (~Te~ RGCKWOOD, E. \V. a n d SI~CKES, P. B . ; J. Amvr. reed. Assoc. 71 (1918) 1649 (277) SEVERANCE, E. ; U n p u b l i s h e d results, 1952 ~27s~ AUGUR, V., ROIJ~.~IAN, II. S. a n d DEUEL, If. J., Jr.; J. N u t r i t i o n 33 (1947) 177 (2:~) CROCKETT, M. E. a n d DEUEL, LI. J., J r , ; J. Nutrition 33 (1947) 187 12s0) CALBERT, C. U., GREENBERG, S. ~ . , KRYDER, G. a n d DEUEL, ]-l. J., J r . ; Food R~search 16 (1951) 294 ~2s~ DEUEL, H. J., Jr., CHI~NG, A. L. S. a n d 5IoREHOU.~E, M. G.; J . Nutrition 35 (1948) 295 {282} LASSEN, S., ]=~ACON,E. K. a n d DUNN, H. J . ; Arch. Biocbcm. 23 (1949) 1 ~2sa) DEUEL, H. J., Jr., tIALLMAN, L. a n d LEONARD, A.; J. Nutrition 20 (1940) 215 (28~) I$OER, J., JANSEN', S . C. P. a n d KEN'TIE, A.; J. Nutrition 33 (1947) 339 (.~s~) HILDrrcH, T . P . ; The Chemived Constitution of Not~ral Fo~s Wiley, New York, 2nd ed. 1947 (.*sG~ L'~.IAN, J. F . ; J. Biol. Chem. 32 (1917) 7 t2sT~ ARNSCHINK, L.; Z. Biol. 26 (n.s. 8) (1890) 434 . [2ss~ ltOLT, L. E., COURT.N'EY, A. ?,I. a n d FALES, H. L.; A m c r . J. Diseases Children 17 (1919) 241 (2s~ , A m e r . J. Diseases Children 18 (1919) 107 [2~0~ GORDON, I[. 1t. a n d McNA)~AR.a, H . ; A m e r . J. Discetses Children 62 (1941) 328 (2~1} 1V1LLIAMS, H. 11., ENDICOTT, ]~..N., SHEPHERD, hE L., GALBRAITI:I, ~ . a n d ),I~ACY, I. C.; J..V~drition 25 (1943) 379 (2s~ IiARRI.~ON, G. A. a n d SHFLDON', \V. 1'. II.; Arch. Discetse~ Childhood 2 (1927) 338; cited b y ~VII.LiA-~IS,H. H. et at. ; J. Ntdrilion 25 (1943) 379 (a931 DEI'EL, 1:[. J., Jr. in ~.[ARKLEY, K. S.; Soybe~t~s and Soybean Products I n t e r science, New York, 1951 vol. 1I 727 (=~t~ MATrIL, K. F. a n d I.[IGGIN.% J. "~V.; J. N~drilion 29 (1945) 255 I88

Referencea

~.'95} ROY, A.; .Ann. Biochcm. exp. Med. (India) 4 (1944) 71 (296~ GIVE.~S, M. I t . ; J . Biol. Chem. 31 (1917) 441 t297~ BOSWORTH, A. W., BOWDITCH, H . I. a n d G1BLIN, L. A .; A~ner. J . D/~eases Children 15 (1918) 397 ~29a~ BOYD, O. F., CRU~t, C. L. a n d L ~ I A N , J. F . ; J. Biol. Chem. 95 (1932) 29 ~29a~ BARNES, R. H., PRL~ROSE, M. F. a n d BURR, G. O.; J. Nutrition 27 (1944) 179 (300) SAVAGE, E. E. ; A Comparative Sludy of the Utilization of Jojoba and Cottonseed Oil in the Rat. Tbesis, U n i v . S o u t h e r n Calif., 1951 [a01~ JT~GERROOS, B. I:I.; Skand. Arch. Physiol. 13 (1902) 375 caoz~ CInITENDEN, R. H . ; Physiological Economy in %'utrition St~kes, .N'ew Y o r k , 1905 caoa) COFFEY, R. ft., MANN, F. C. a n d BOLL.AlAN, ft. L.; Amer. J. dlgeshve Diseases 7 (1943) 141 (3(}4) ~ONES, C. 5~., CULXrER, P. J., DRU:,ISIEY, G. D. and RYAN, A. E . ; A n n . irdcrnal Med. 29 (1948) 1 {305) STEENBOCK, H., IB,~VIN, M. H. a n d vtVEBER, J.; J. Nutrition 12 (1936) 103 130a) BAVETrA, L. an d DEUEL, It. J., J r . ; Anwr. J. Physiol. 136 (1942) 712 {307} , ]~ALI~MAN,L,, DEb~EL, ]=[. J-, J r . a n d GREELEY, P. O.; Amer. J. Physiol. 134 (1941) 619 (aoa) ANNEOEFa~, ft. It. and IvY, A. C.; A~u"r. J. Physiol. 150 (1947) 461 t309) I][~XVIN,M. 11., STEENbtOCK, H. a n d TEMPLIN, V. M.; J . Nutrition 12 (1936) 85 (310) SOBEL, A. E., BES.M[AN, L. a n d KRAMER, B . ; Anle-'r. ,1. Discascs Children 77 (1949) 576 "~?~ TIDX~ELL, It. C., ltOLT, L. E., J r . , FARROW, H. L. an d NEALE, S.; J. Pedial. 6 (1935) 481 {a12~ BECKER, G. H., .'~IEYER, J. a n d NECtmLES, H . ; Gastroenterology 14 (1950) 80 ~a~a~ DEUEL, H. ft., fir. a n d HALL.~L~N, L . ; J . Nutrition 20 (1940) 227 1314~ . , HALLMAN,: L. and I{EIFMAN, A.; J. Nutrition 21 (1941) 373 ta15~ VERZ~-R, F. an d LASZT, L.; Biochem. Z. 276 (1935) 11 ~a1¢.~ ....... Biochcm. Z. 278 (1935) 396; 288 (1936) 3 5 1 , 3 5 6 ~s:7~ ~ an d McDout:ALL, E. ft.; Absorption from the Intestine Iz)ngmans Green, L o n d o n .New Y o r k 1936 isis) BAR~'ES, R. H., WicK, A. N., MILLER, E. S. a n d MACKAY, E. M. ; Proc. Soc. exp. Biol. N . Y . 40 (1939) 651 (s~ , M~LL~R, E. S. and BURR, G. O.; A~wr. J. Physiol. 126 (1939) 127 P {32o) DEUEL, I~: J., Jr., I~ALL3IAN, L. F., ~IURRAY, S. an d SA-M-LIEL~_,L. T.; J. Biol. Chem. 119 (1937) 607 ~a~ BA~I,~'ES, R. H. , ]RUsO~*F, I. I. a n d BURR, G. O.; Proc. Soc. exp. Biol., N . Y . 49 (i942) 84 ~-~ BAVETTA, L. ; Am~r. J. Physiol. 140 (1943) 44 (s~s~ WVBRANDT, W. a n d LASZT, L . ; Biochem. Z. 259 11933) 398 ta24} BARTH, F . ; Biochem. Z. 270 (1934) 63 ta25) KLINGHOFFER, K. A.; J. Biol. Chem. 126 (1938) 201 (a2~ 0H.~ELL, R. an d H(iBER, R . ; J . cell. comp. Physiol. 13 (1939) 161 ~a.,~ VERZ.~, F. and LASZT, L.; Biochem. Z. 276 (1935) 1 ~a~ ; Biochem. Z. 270 (1934) 35 ( s ~ BECK, L. ~V.; J. Biol. Chem. 143 (1942) 403 ~aa0~ FRAZEIt, A. C.; Physiol. Rev. 26 (1946) 103 ira1} ZECHSIEISTER, L. a n d Tuz~oN, P . ; Ber. dlseb, chem. Ges. 67 (1934) 154 ~as~ _ ; Erqebn. Physiol. 39 (1937) 117 ~aaa) GILLA.~L A. E. an d EL I{iDI, M. S.; Biochem. J. 31 (1937) 251 (aa,~ _ _ _ and 11EILBRON, I. 3I.; Biocht'm. J. 29 (1935) 1064 ~s:~ _ _ and IIEILB~O.~', I. M.; Biocbem. J. 29 (1935) 834 (~a~ A.~o.~Y.~ous; U.S. Dept. Agric. misceU. Pub. No. 571 (1945) 189

N u t r i t i o n a l Significance of the F a t s {aJTJ ROSENBERG, It. R . ; Chemistry and Physiology of lhe Vitamins Interscience, ,New York (1945) (sasj RIPLEY, W. E. a n d BOLO.~rEY, R. A. ; Calif. Dept. Nat. Resources, Div. Fish and Game, Bu. Mari~w Fish cries, Fish Bull. No. 64 (1946) 39 t3ag~ SWKIN, L. A. a n d McKERCHER, B. It. ; Fisheries Research Bd. Canada, Prog. Repts. Pacific Coast Sta. No. 65 (19455 67; Chem. Abst. 40 (1946) 1681 ~a40~ BUCI-:LEY, T. A.; Malayan agric. J. 24 (19365 485 ~aaD KAUFMANN, H. P . ; Forte u. Scifcn 48 (1941) 53; cited b y DEUEL, 1t. J., Jr. a n d GREENBERG, S. M.; Fortschr. Chem. organ. Naturstoffe 6 (19505 1 (a42~ M~IJ~R, W. B.; Federal Register 6 No. 111 (Tth J u n e . 1941) 2761 ( F o r d a n d Drug A d m i n . P a r t 45) ~a~a~ TItURSTON, T . L . ; Federal 1h~glstcr 17 No. 100 (21st May 1952) 4613 (Food a n d Drug Admin. P a r t 45) (a44~ BILL% C. E.; Physiol. R w . 15 (1935) 1 (a4s~ BMLEY, A. E.; Industrial Oil and Fat Prodvzts Interscience, New York (1945) (3a6~ QUACEENBUSH, F. W., GOTTLEIB, 11. L. a n d STEENBOCK, I:[. ; I n d . E,ug. Chem. 33 (19415 1276 la47~ KARRER, P. a n d KELLER, I t . ; Helv. chim. Acta 21 (19385 1161 ,a48} JENSEN, J. L., I:[ICKMAN, K. C. D. a n d ItARmS, P. L.; Proc. Soc. exp. Biol., W.Y. 54 (1943) 294 ta4,} CItIPAULT, J. I{., LUNDBERG, W. O. and :BURR, G. O.; Arch. Biochem. 8 (1945) 321 t~50~ I~OBESON, C. D. a n d BAXTER, J. G.; J. Amvr. chem. Soc. 65 (19435 940 ~a515 MILLE~t, It. G.; J. Nutrition 26 (1943) 43 [3525 Cu)r)~Ixc.s, M. J. a n d hIA2"rlLL, It. A.; J. N~drition 3 (1931) 421 (asa) EVANS, 1t. M. a n d BURR, G. O.; J. Amvr. ,:wd. Assoc. 89 (1927) 1587 (a545 WEBER, J., IRWIN, M. t t . a n d STEENBOCK, I L ; Anwr. J. l)bysiol. 125 (19395 593 {a55) SCHANTZ, E. J., ELVEHJEM, C. A. a n d HART, E. B , ; J . Dairy Sci. 23 (1940) 181 (3~6} - - , J~OL'TV.'ELL, Pt. K., ELVEIIJESI, C. jk. a n d ItART, E. B.; J . Dairy Sci. 23 (1940) 1205 {357} BOUTXVELL,R. K., GEYER, R. P., ELVEILIES[, C. A. a n d HART, E. B. ; J. Dairy Sci. 24 (1941) 1027; 26 (1943) 429 (35s) HENRY, K..~1., KON, S. K., ItlLDITCIt, T. P. a n d h[EARA, ~[. L.; J. Dairy Research 14 (1945) 45 ; cited b y DEUEL, H. g., Jr. a n d GREEN'BERG,S. ~[. ; Forlschr. Chem. organ. Naturstoffe 6 (1950) 60 (asg} JACK, E. L., IIENDER~SON, J. L., REID, D. F. a n d LEPKOVSKY, S.; J. Nutrition 30 (1945) 175 (3~0} GE~ER, R. P., GEYER, B. R., DEll
190

References (a:0) I)EUEL, H. 3., fir., GREENBEItO, S. M., STRAUB, E., FUKL'I, T., GOODI.~O, C. M. a n d BROWN, C. F . ; d. NulrUion 38 (1949) 361 taxi) ZIAI,CITA, L. P., Jr. a n d )1ITCH:ELL, 1:~. I t . ; Science 100 (1944) 60 cJv.*~ LassE.N, S. and BACON, E. K . ; J. N~driNon 39 (1949) 83 ( 3 : 3 ) BEBTaA.'~, S. H . ; Biochem. Z., 197 (1928) 433 ~3:4) GROS.~FELD, J. a n d SI.~n~tER,A.; Z. Unters. Lebens~dttel 59 (1930) 237; Chem. Abst. 24 {1930) 5173 tats) GEYER, R. 1:>., HATH, H., BARKI, V. H., ELVEHJEbl, C. A. a n d MART, E. B . ; J. Biol. Chem. 169 (1947) 227 (376) BOER, ft., JA.X'SE~-, B. C. P., KENTIE, A. a n d KNOL, H. W . ; J . Nutrilion 33 (1947) 3 5 9 c3:~ Et:I.ER, B. v., EULER, H. v. a n d SXB~G, I . ; Ern2ihrung 8 (1943) 257 (aTs~ BOER, J. a n d JANSEN, B. C. P.; Vocding 2 (1941) 204; cited b y BOEB, ,1., JANSEN, B. C. P. a n d KENTIE, A.; J. Nutrition 33 (1947) 339 {379) NATH, ]=]'., BARKI, V. H., ELVEHJE3I, C. A. a n d HABT, E. B . ; J. N~drilion 36 (1948) 761 43s0~ BOEB, ft., G11oow, E. H, and JANSEN, B. C. P . ; Voeding 9 (1948} 60; Chem. Abst. 42 (1958) 7847 ~aSl) DEUEL, H. J., Jr. a n d Mox~la'r, E . ; J. Nulrilion 29 (1945) 237 ~jso.) , tIE.','I)tHCK, C. a n d CBOCKE~'r, M. E.; J. Nutrilion 31 (1916) 737 ~3sa) ERSttOFF, B. H. a n d DEUEI.., H. J., Jr.; Endocrinology 36 (1945) 280 t3s4) LEICIIENGER, It., EI.~ENBEBG, G. a n d CARL'~ON, A. J.; J . A~ver. med. Assoc. 136 (1948) 388 c3,5) ANOEI~ON, XV. 1[. a n d XVIIJ.JAMS, H. H . ; Physiol, R w . 17 (1937) 335 tJs6~ MEres, E. B.; Physiol. Rev. 2 (1922) 204 (.~s~ SURE, B.; J. Ntdrflion 22 ~1941) 499 {*ss) :~IAYSARD,L. A. a n d :RA~MUSSE.~',E.; J. Nutrition 23 (1942) 385 ~3s9) LOOSLI, J. K., LINGENFELTER, J. I{., THOMAS, J. W. a n d .~IAYNARD, L. A.; J. Nutrition 28 (1-944) 81 ~.~o~ DEUEL, H. J., Jr., MOVlTT, E. a n d ]~ALI2IAN, L. F . ; J . N~drLt~on 27 (1944) 5O9 {391) SHER.XEAN,H. C. a n d CAM-PBELL, H. L.; J. Biol. Chem. 60 (1924) 5 ~ --, J. Nutrition 2 (1929, 1930) 415 ~*~) - a n d TRUPI,, H. Y.; Proc. nat. Acad. Sei., ]Vash. 35 (1949)90 (394) _ _ ~illd 'CAMPBELL, H. L.; .jr. ~V~dr~tion 14 (1937) 609 (3~5) I)EUEL, I~. J., Jr. tIALLMAN, L. F. a n d Movi~r, E.; J. Nutrition 29 (1945) 309 (~9B) ~ , GREENBERG, S. ~I., SAVAGE, E. E. a n d BAV]ETTA, L. A. ; J..\'tt~r~lion 42 (1950).239 ~7~ BRAUN, W. Q. a n d SHI~EW~I:IURY,C. L.; Otl o n d S o a p 18 (1941) 249 t39s) .kJvEs, S. R., O'GRADY, 31. P., EMBItEE, N. D. a n d ]tARBI~, P. L.; J. Anl~'r. O~/ Chem. Soc. 28 (1951) 31 (a~9~ MATrSON, F. H., BALm, F. J. a n d BECK, L. W . ; J. Am,,rr. 05l Chore. Soc. 28 (1951) 386 (4oo) _ _ , I~ENEDIC~, J. H., 3IAI:tTIN, J. B. a n d BE~K, L. ~V.; J . Ntdrition 48 (1952) 335 N0~ FRAZER, A. C. a n d SA.~OI~S, H. G.; Biochem. J. 39 (1945) 122 ~400.~ IIou.~.~AY, B. A. a n d 3IARTINEZ, C.; Science i05 (1947) 548 No~) GviL".GY, P. a n d ROSE, C. S.; Science 108 (1948) 716 (.to~t) IIODlilGUEZ, R. 1{. and KRI-?HL, W. A : ; Am,yr. J. Physiol. 169 (1952) 295 (4o5) SCIIJNFIEIME:II, ]{.; Hoppe-Seyl. Z. 180 (1929) 16, 24, 32; 1S5 (1929) 119 N0a~ _ _ and YUASA, D.~ Hoj;pe-Seyl. Z. IS0 {1929).19 No:~ BEHtiI.X'(;, lI. V. a n d SCH0.'~:I.~tE~, 1{.; Hoppe-Seyl. Z. 192 (1930) 97 Nos~ SCHiiNHEDtEFt, ]{., BEIiRINO, ]I. V. a n d Ilu)DrEL, K . ; Hoppe-Scyl. Z. 192 (1930) 117

191

N u t r i t i o n a l Significance o f t h e F a t ~ ~40ml BLocrL K . a n d 1RITTE.~q~ER6, D.;" J . Biol. Chem. 143 (1942) 297; 145 (1942) 625; 159 (1945) 45; 160 (1945) 417 ~4101 ALFIN-SLATER, If. B., SCrtOTZ, 5I. C., SI~'~fODA, F., a n d DEUEL, H. J., J r . ; J . Biol. Clwm. 195 (1952) 311 (~111 CRX~tPTON, E. W . a n d MILLArt, J . ; U n p u b l i s h e d work, 1946; cited b y CRA~PTO~ el al. ; J. Nutrition 43 (1951) 431 ~1~1 1ROFFO, A. I t . ; Bol. Inst. Med. exp. (Baenos Aires) 21 (64) (1944) 1; cited in Biol. Absf. 19 No. 8570 (1945) 922 ~41a) MoRRxs, Lt. P., L.~a-~SE.~', C. D. a n d LJPPINCOTT, S. ~V.; J . nat. Cancer Inst. 4 (1943) 285 (414) ~IACK~NZIE, C. G., .-~[ACKENZIE, J. B. a n d McCoLLU.~I, E. V.; Proc. Soc. exp. Biol., N . Y . 44 (1940) 95 ~4~61 CRA~rPTOX, E. W., F~r~XmR, F. A. a n d BERRYmLL, F . M.; J. Nutrition 43 (1951) 431 1a,61 , CO.~L~fON, R. H., FAI~M-ER,F. A., BERI~'ItILL, F. 3[. a n d WISEBLATT, L.; J . Nutrition 44 (1951) 177 1416a)CRA.~IIYI,ON, U. W., COMMON, 1~. I~., FARMiER, F. A., ~VELLS, A. F., a n d CRAW. FORD, D.; J. Nulrition 49 (1953) 333 'a17~ DEUEL, It. J., Jr., GREENBERG, S. 5I., CALBERT, C. W., BAKER, R. a n d FIS1[EP-, H . R . ; Food Research 16 (1951) 258 ~4~sl LL T. ~,V. a n d FrtEE.~tAN, S.; Amer. J. Physiol. 145 (1945) 158 14191 DAVIS, J. E. a n d GRoss, J. B.; A n w . r . J . Physiol. 144 (19i5) 444 (~201 McLEAN, J. R. a n d ]3EVERIDOE, J. 5I. R . ; J. Nu t ri t i o n 47 (1952) 41 [421~ CI~ENG, A. L. S., RYA..~, 51., ALFIN-SLATER, 1~. B., a n d DEUEL, H. J. J r . ; u n p u b l i s h e d results, 1953 1422) BP.ANDT, K . ; A n n . Amvr. Aead. Polil. Soc. Sci. 225 (1943) 210 (4*.s~ SHEN, T.; Science 98 (1943) 302 (lea~ HowE, P. E . ; A n n . Amer. Acad. Polil. Soc. Sci. 225 (1943) 72 ta.~s~ AsoNYsIOUS; Becomnw~ut~d Dietary Allowa~wcs, F o o d a n d N u t r i t i o n ]3d. N a t . R e s e a r c h Council Circ. No. 129 (Oct. 1948) p. 17

192