Lipid Metabolism in Lubricus Terrestris

Citation preview

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FORDHAM UNIVERSITY G r a d u a t e S c h o o l o f A r t s a n d S cienc es

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19 ..5.Q

This dissertation prepared under my direction by

____ Henryj p . White entitled E ipid Metabolism in Lumbricus t e r r e s t r i s

has been accepted in partial fulfilment of the requirements for the Degree of D°c to r o f P hilosophy

_______ C harles A. B erger, S.J»__ (Faculty Adviser)

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JXPXD METABOLISM .IN JXJIBRICUS TERHESTRIS

BY

HENRY F. WHITE A. B. , Fordham U n iv ersity , *32 M.A., V illanova u o lle g e , *34

PISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE DEPARTMENT OF BIOLOGY AT FORfflAM UNIVERSITY NEW YORK 1950

ProQuest Number: 10992990

All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is d e p e n d e n t upon the quality of the copy subm itted. In the unlikely e v e n t that the a u thor did not send a c o m p le te m anuscript and there are missing pages, these will be noted. Also, if m aterial had to be rem oved, a n o te will ind ica te the deletion.

uest ProQuest 10992990 Published by ProQuest LLC(2018). C opyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C o d e M icroform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346

LIBRARY

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TABLE OF CONTENTS Page LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

iv

LIST OF TABLES

vi

..................

I.

INTRODUCTION..........................

1

II.

MATERIAL AND METHODS.....................

4

III.

OBSERVATIONS AND RESULTS

IV.

DISCUSSION

V.

SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

........................

BIBLIOGRAPHY . . . . . ........

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16 148 160 161

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LIST OF FIGURES %

Figure 1.

2.

5.

4.

5.

6.

7.

8*

Page A graph, showing the photometer read­ in g s ( in microampheres) p lo tted against concentrations o f c h o lestero l (mgms. per 10 c c . o f s o lu t io n ). .............. .

10

A graph, showing th e photometer readings ( in microampheres) p lo tte d again st con­ cen tration s o f l ip id phosphorus (mgms. ...................... per 10 c c . o f s o lu tio n ).

12

A graph, showing the photometer readings (in microampheres) p lo tte d again st con­ cen tration s o f f a t t y acid (mgms. per 5 c c . o f s o lu tio n ). . . . . . . . . . . . . . . . . . . . . . .

14

A graph, showing th e lip id r a tio s be­ tween ch o le ste r o l and lip id phosphorus in skin-m uscle in normal worms, those fed com meal, those fed ch o le ste r o l and corn meal, and starved worms. .................

131

A graph, showing the lip id r a tio s be­ tween c h o le ste r o l and f a tt y acid in skin muscle o f normal worms, those fed corn meal, those fed c h o lestero l and com meal, ................... and starved w orm s..........

133

A graph, showing the lip id r a tio be­ tween f a t t y acid and lip id phosphorus in skin-m uscle o f normal worms, those fed corn meal, those fed ch o le ste r o l and com meal, and starved worms.

135

A graph, shovdng th e li p i d r a tio ch o lestero l and l i p i d phosphorus in te s t in e o f normal worms, those com meal, th o se fed c h o lestero l com meal, and starved worms.

137

between in fed and .........

A graph, showing the li p id r a tio be­ tween c h o le ste r o l and f a t t y acid in in t e s t in e o f normal worms, those fed com meal, th ose fed c h o lestero l and com meal, and starved worms*. . . . . . . . . . . .

139

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Figure 9

Page A graph, showing the lip id r a tio between f a t t y acid and lip id phosphorus in in t e s ­ tin e o f normal worms, those fed corn meal, those fed c h o le ste r o l and com meal, and starved worms* .................

141

«

10.

11.

12.

A graph, showing th e l ip id r a tio between c h o le ste r o l and lip id phosphorus in blood o f normal worms, those fed on com meal, those fed on c h o lestero l and com meal, and starved worms..................................

143

A graph, showing the l ip i d r a tio between c h o le ste r o l and f a t t y acid in blood o f normal worms, those fed com meal, those fed c h o lestero l and com meal, and starved worms............. » • • . • • • « . . . . . ..............

145

A graph, showing the lip id r a tio between f a t t y acid and lip id phosphorus in blood o f normal worms, those fed com meal, those fed ch o le ste r o l and corn meal, and starved worms. .......... .............

147

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LIST OF TABLES Table I. II. III.

IV. V. VI.

V II.

V III. IX*. X*-

XI.

X II.

X III. L

Page Table showing the mean value o f ch o lestero l in skin-m uscle o f normal worm s................................

31

Table showing the mean value o f ch o lestero l in skin-m uscle o f worms fed corn meal...................

33

Table showing the mean value o f ch o lestero l in skin-m uscle o f worms fed ch o lestero l and corn meal* . . ........... *. * *............. ............ . . . . .

33

Table showing the mean value o f ch o lestero l in skin-m uscle o f starved worms. *. . . ...............

37

Table showing the mean value o f ch o lestero l e s te r s in skin-m uscle o f normal w o r m s . . . . . . .

39

Table showing the mean value o f ch o lestero l e s te r s in skin-m uscle o f worms fed corn meal ..........

41

Table showingmean value o f ch o lestero l e s te r s in skin-m uscle o f worms fed choles­ te r o l and corn meal ; ................................

43

Table showing the mean value o f ch o lestero l e s te r s in skin-m uscle o f starved worms . . . . . .

45

Table showing the mean value o f lip id phos­ phorus in skin-m uscle o f normal w o r m s . . . . . . .

47

Table showing the mean value o f lip id phosphorus in skin-muscle o f worms fed ........................... corn meal

49

Table showing the mean value o f lip id phos­ phorus in skin-m uscle o f worms fed chol­ e s te r o l and corn m e a l ................................

51

Table showing the mean value o f lip id phosphorus in skin-m uscle o f starved worms.........................

53

Table showing the mean value o f f a tt y acid in skin-m uscle o f normal worms..........................

55 _J

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Table XXVIII*

XXIX. XXX.

XXXI*

XXXII. XXXIII. XXXIV. XXXV.

XXXVI* XXXVII* XXXVIII.

XXXIX.

XI. XLI. L

Page Table showing the mean value o f lip id phosphorus in in te s t in e o f starved worms. ......................................................

85

Table showing the mean value o f fattyacid in in t e s t in e o f normal worms .........................

87

Table showing the mean value o f fattyacid in in t e s t in e o f worms fed com ................................................. meal

89

Table showing the mean value o f fattyacid in in t e s t in e o f worms fed choles­ te r o l and com meal ......................

91

Table showing the mean value o f f a tt y acid in in t e s t in e o f starved w o r m s . . . . . . . .

95

Table showing the mean value o f choles­ te r o l in blood o f normal wo r ms . . . .

95

Table showing the mean value o f choles­ te r o l in blood o f worms fed corn m e a l . . . . .

97

Table showing the mean value o f choles­ te r o l in blood o f worms fed c h o lestero l and com meal.

99

Table showing the mean value o f ch oles­ te r o l in blood o f starved w o r m

s

.

101

Table showing the mean value o f ch oles­ te r o l e s te r s in blood o f normal worms . . . . .

105

Table showing the mean value o f ch oles­ te r o l e s te r s in blood o f worms fed com meal ...........................

105

Table shoving the mean value o f ch oles­ te r o l e s te r s in blood o f worms fed c h o le ste r o l and com m e a l ......................

107

Table shoving the mean value o f ch oles­ te r o l e s te r s in blood o f starved worms....

109

Table showing the mean value

o f l ip i d _j

v ii r

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Table XIV. XV.

XVI. XVII. XVIII. XIX.

XX. XXI., XXII. XXIII.

XXIV. XXV. XXVI. XXVII*

L

Page Table showing the mean value o f f a t t y acid in skin-m uscle o f worms fed corn m e a l

57

Table showing the mean value o f f a tt y acid in skin-m uscle o f worms fed choles­ te r o l and corn meal .

59

Table showing the mean value o f f a t t y a cid in skin-m uscle o f starved worm s

61

Table showing the mean value o f ch oles­ te r o l in in t e s t in e o f normal worms

65

Table showing the mean value o f c h o lestero l i n in te s t in e o f worms fed corn meal . . . .................

65

Table shovdng the mean value o f ch o lestero l in in t e s t in e o f worms fed c h o le ste r o l and corn meal

67

Table showing the mean value o f c h o lestero l in in te s t in e o f starved worms ...................

69

Table showing the mean value o f ch o lestero l e s te r s in in te s t in e o f normal worms.. . . . . . .

71

Table shofdng the mean value o f ch o lestero l e s te r s in in t e s t in e o f worms fed com meal.

75

Table showing the mean value o f ch o lestero l e s te r s in in t e s t in e o f worms fed choles­ te r o l and com meal

75

Table showing the mean value o f ch o lestero l e s te r s in in t e s t in e o f starved worms .......

77

Table shoidng the mean value o f l ip id phos­ phorus i n in t e s t in e o f normal worms . . . . . . .

79

Table showing the mean value o f l ip id phos- phorus in in te s t in e o f worms fed corn meal.

81

Table showing the mean value.. o f l ip id phos­ phorus in in t e s t in e o f.worms...fed choles­ te r o l and corn meal • • • • • . ................

83

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Table phosphorus in blood o f normal worms..............

111

Table showing the mean value o f lip id phosphorus in blood o f worms fed com . ... m e a l . . . ..........................

113

Table showing the mean value o f lip id phosphorus i n blood o f worms fed choles­ te r o l and corn meal ............

115

Table showing the mean value o f l ip id phos­ phorus in blood o f starved worms.• • . • • • • • • •

117

Table showing the mean value o f f a tt y acid in blood o f normal worms. ............

119

Table showing the mean value o f fa tty acid in blood o f worms fed com meal ..............

121

Table showing the mean value o f fa tt y acid in blood o f worms fed c h o lestero l and com meal ...................

123

Table showing the mean value o f f a tt y acid in blood o f starved worms » • • • • • . . . . • .

125

XLIX*

Table showing a summary o f r e s u lt s ...................

127

L.

Table o f l i p i d r a t io s .............................

129

XLII*

XLIII.

XLIV* XLV* XLVI. XLVII*

XLVIII*

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LIPID METABOLISM IN LUMBRICUS TERRESTRIS

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INTRODUCTION

The number o f in v e stig a to r s o f lip i d metabolism in the in v erteb ra tes i s very few*

Among th ose who have contributed

most to our knowledge are the follow ing: Doree(l909) showed that ch o le ste r o l i s present in the common whelk, Buccinum undatum* De Waele (1930) reported th at the only s te r o l present in earthworms i s c h o le ste r o l. S l i f e r (1952) reported on th e iod in e number o f fa ts in grasshopper eggs? she concluded th at the season o f the year plays a leading r o le in determining the types o f fa ts in eggs o f A crid iae. Bergman (1934) showed th a t c h o lestero l i s absent in o y sters and in i t s p lace an e n tir e ly d iffe r e n t s te r o l, o s tr e a s te r o l, i s p resen t. Bergman and Johnson (1933) studied the red sponge, Microciona p r o life r a . and proved the presence o f microc io n a ste r o l, a s t e r o l, not id e n tic a l with those o f other sponges. Becker (1954) concluded from h is stu d ies o f the b e e tle , Tenebrio m olitor. that f a t in la r v a l and pupal forms dimin­ is h e s during metamorphosis and th a t f a t serves as a supply o f energy during t h is period o f development.

2 r Howes and Ih e llo ck (1937) hold th at the common s n a il, H elix pomatia, needs c h o le ste r o l for the normal metabolism o f calcium and phosphorous* Tatum (1939) discovered th at Drosophila requires c h o le ster o l as w ell as other d ietary fa cto rs fo r normal growth and development* S alisbury and Anderson (1959) in th e ir a n a ly sis o f the l ip id s in C ysticercus fa s c io la r is * concluded that the l ip id content i s a mixture o f phospholipids, c h o le ste r o l, cerebrosides and a small amount o f glycerides*

Of the lip id s

30$ are phospholipids, a mixture o f cephalin and le c it h in , and the proportions o f saturated f a t t y acid to unsaturated are roughly lr l* Evans and G ood liffe (1959) estim ated th at the larvae* o f the b e e tle , Tenebrio m olitor, use 83$ o f th e f a tt y acids in a bran d ie t fed to them. Bahl (1947), studying excretion in the O ligochetes, shed a l i t t l e lig h t on the l i p i d

content o f blood and

eoelomic f lu id . Wilber (1947) stu d ied the e f f e c t o f prolonged starva­ tio n on the l ip id s in Phascolosoma*, He concluded that starva­ tio n decreases a l l lip id s in the worm; r e s u lts in l i t t l e change in the muscle lip id s ; decreases the f a t t y acid and phospholipid content o f the p e r iv isc e r a l f lu id , but does not change i t s c h o le ste r o l content* L

Muscle, th erefore, i s not

a storehouse o f f a t and fa tt y acid , and phospholipids are used fo r energy expenditure* I t i s d i f f i c u l t to draw any conclusions from th ese in ­ v e s tig a tio n s because the r e s u lts have not been obtained "from a s u f f ic ie n t ly la r g e enough number o f stu d ies o f any one p a rtic u la r in v erteb rate phylum, nor from a complete enough study o f the various in verteb rate groups.

From th ese stu d ie s,

i t i s im possible to say th a t the metabolism o f lip id s follow s the same pattern in in v erteb ra tes as in vertebrates* Such stu d ie s serve to emphasize the lack o f d e fin ite information*

Because t h is f i e l d has been so l i t t l e explored,

i t prompted the author to study li p id metabolism in a te r r e s t i a l form, Lumbricus te r r e s tr is *

4

II*

MATERIAL AND METHODS

Specimens o f Lumbricus t e r r e s t r is were obtained in the Spring, Summer and F a ll o f 1948 from the campus o f Fordham U niversity*

During the Winter o f 1948 and Spring o f 1949

they were obtained from the Garolina B io lo g ica l Supply Com­ pany, Elon C ollege, North C arolina.

When th ese worms were

receiv ed , they were placed i n a s p e c ia l g la ss terrarium for th ree weeks before being used, to become acclim ated to the s o il. The in v e s tig a tio n o f t h is problem was divided in to 4 s e r ie s o f experim ents.

The f i r s t s e r ie s was concerned with

the estim ation o f c h o le ste r o l, ch o le ste r o l e s te r s , lip i d phosphorus and f a t t y acid in ®control” anim als.

By 11con trol”

i s meant th a t the animals were allowed to feed fr e e ly on s o il obtained from the campus. way o f food was added.

Nothing foreign to the s o il in the

Ten worms ( A1-A10) were kept in another

g la ss terrarium, which was sh ield ed from the lig h t by card­ board and which contained s ix inches o f s o i l .

Estim ations

o f c h o le ste r o l, c h o le ste r o l e s te r s , lip id phosphorous and f a t t y acid in skin-m uscle preparations, in t e s t in a l t is s u e and blood were made on th ese worms.

The r e s u lts o f the e s t i ­

mations o f c h o le ste r o l, c h o le ste r o l e s te r s , lip id phosphorus and f a t t y acid in th is group o f specimens are designated as normal. L

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The second s e r ie s was the random s e le c tio n o f 10 more worms (B1-B10) which were to be fed pulverized corn meal* The inform ation regarding the amount o f f a t (1*9%) in the com meal was furnished by the manufacturer*

These animals

were placed in oblong, amber colored dish es (2 animals to a dish) and kept in the dark*

The tops o f the d ish es were

covered by perforated f i l t e r paper through which water could be added*

For three days the worms were fed

paper to clean out th e d ig e stiv e tract* Then removed and the d ish es cleaned*

macerated f i l t e r the worms were

A fter returning the worms to

the d ish es, 1 gram o f com meal was p laced in each d ish . in 2 days th is food was consumed.

With­

At in te r v a ls fe c a l matter

and urinary products were removed by means o f a cotton swab, and fresh water supplied*

Estim ations o f c h o le ste r o l,

c h o le ste r o l e s te r s , l ip i d phosphorus and f a t t y acids were then made* The th ird s e r ie s was the s e le c tio n o f 10 more worms (C1-C1Q) which were to be fed a mixture o f ch o le ste r o l and cornmeal*

Again p a irs o f worms were placed in dishes*

The

procedure follow ed fo r th is group was id e n tic a l with th at o f the group in the second s e r ie s , except the d ie t of the worms con­ s is t e d o f 50 mgms. c h o le ste r o l and 1 gram com meal*

Estima­

tio n s o f c h o le ste r o l, c h o lestero l e s te r s , lip id phosphorus and f a tt y acid s were then made* The f in a l s e r ie s con sisted in s e le c tin g 10 more worms L

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D1-D10) again placing p a irs in g la ss d ish es.

These were fed

macerated':- f i l t e r paper fo r three days, during which time the d ig e stiv e tr a c t was cleaned.

These were allowed to starve

fo r two more days but were not permitted to starve to death. The sta rv a tio n p oin t was determined from previous experiments and the norm esta b lish ed by a marked decrease in a c t iv it y . Fecal matter and urinary products were removed at in te r v a ls to prevent any damage to the animals and fresh water sup­ p lie d .

Estim ations o f c h o le ste r o l, c h o lestero l e s te r s , lip id

phosphorus and f a tt y acid were then made. Skin-muscle and in t e s t in a l preparations were obtained by d is s e c tio n , from animals anesth etized by spraying with absolute a lco h o l. in a l l ca ses. region .

The samples o f blood were obtained f i r s t

The specimens were d issected in the esophageal

A fter cu ttin g through the body w a ll, the coelomic

f lu id was allowed to escape and the blood was soaked up by dipping in to i t f i l t e r paper o f a known weight.

The f i l t e r

paper had been kept covered and, when used as a b lo tte r , handled with fo rcep s.

A fter the blood dried, the f i l t e r paper was

reweighed and the dry weight o f the blood ca lcu la te d . The method used for the ex tra ctio n and estim ation o f ch o le ste r o l was th a t o f Bloor (1916).

The tis s u e was ?/eighed,

ground with sand in a mortar u n til a fin e paste r esu lted . The paste was placed in a 50 cc. alcohol ether mixture ( 3 r l ) , heated and allowed to stand fo r 3 days. L

Then measured volumes -J

7 “i r

were drawn o f f and filte r e d *

In estim ating c h o le ste r o l, v o l­

umes o f 20 cc* or 10 cc* o f ex tra ct were placed in a 250 cc* fla s k and heated u n til almost dry*

Next 5 cc* o f chloroform

were added 3 tim es and the so lu tio n again heated u n til near­ l y dry*

To the remaining so lu tio n (made up to 10 cc. with

chloroform) were added 2 c c . o f a c e tic anhydride and concen­ trated sulphuric acid (1 0 :1 ).

This was placed in the dark

fo r 20 minutes a t room temperature*

Five cc. o f th is solu ­

tio n were transferred to a 510 absorption tube and placed in a H e llig e -D ille r Bio-Photo-Colorim eter, model 500, and 5 read­ in g s taken*

The average o f th ese was used as the ty p ic a l

reading o f the extract* For estim ation o f c h o le ste r o l e ste r s the method o f Bloor & Knudson (1916) was used.

Ten c c . or 5 c c . o f ex tra ct

was drawn o f f and 1 cc* o f 1% d ig ito n in so lu tio n was added* This e x tr a ct was evaporated to dryness. o f ether were added* minutes* flask *

To t h is residue 15 cc.

The so lu tio n was slow ly b oiled fo r 5

The clea r ether p ortion was poured in to another This procedure was repeated tw ice, using 5 c c . o f

ether each tim e.

The eth er was b oiled o f f .

form were added to the dry resid u e.

Two cc* o f chloro­

The so lu tio n was b o iled ,

poured in to a 10 c c . graduate and made up to 5 cc. by adding chloroform.

From here on the procedure was the same as for

the estim ation o f ch o lesterol* The method fo r estim ation o f f a t t y acid was th at o f Bloor L

8

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r

(1922)*

E xtracts o f 20 cc* and 10 cc, were transferred to a

100 cc* f la s k .

Two-tenths o f a cc. o f concentrated sodium

hydroxide was added and the so lu tio n evaporated almost to dry­ ness*

To the cool so lu tio n was added ,2 cc. o f d ilu ted su l­

phuric acid (1:3)*

A few drops o f water were added*

taken th at the so lu tio n remain a lk a lin e . on a hot p la te follow ed.

Care was

Complete evaporation

Ten cc, o f alcoh ol were poured on the

residue in the fla sk and b oiled g en tly for 10 m inutes. was repeated using 5 cc. o f a lco h o l.

This

The f il t r a t e s were'com­

bined, evaporated to 3 c c . , transferred to a 10 c c . graduate and brought to a f in a l volume o f 5 cc. with a lco h o l.

The fa tt y

acid ex tra ct was poured through a bare g la s s funnel in to a 200 cc. beaker, containing 100 cc. o f d i s t i l l e d water*. The funnel was rinsed with f lu id from the beaker and the washings added to the beaker.

Ten cc. o f d ilu te hydrochloric acid ( l s3)

were added to the beaker without s t ir r in g .

Readings were taken

a fte r 5 m inutes. For l i p i d phosphorus estim ation , the method o f Youngburg & Youngburg (1930) was employed. were heated to dryness.

Five or s ix cc. o f extract

Then 2.5 c c . o f 10N sulphuric acid

was added to the beaker and d igested u n t il black and fuming* The so lu tio n was cooled and 1 drop o f 50% hydrogen peroxide was added and then i t was heated. the contents were c o lo r le s s .

This was repeated u n t il

To th is cooled so lu tio n 5 cc.

o f water were added and the ex tract b oiled for a minute. L

9

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E x p la n a tio n o f F ig u re F ig . 1 - A g ra p h , showing th e ph o to m eter re a d in g s ( in m icroam pheres) p l o t t e d a g a in s t c o n c e n tr a tio n s o f c h o l e s t e r o l (mgms. p e r 10 c c . o f s o l u t i o n ) .

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