The intestinal absorption of glycerol trioctadecenyl ether

368

RIOCHIMICA ET BIOPHYSICA ACTA

BB.4 55433

THE

INTESTINAL

ABSORPTION

OF

GLYCEROL

TRIOCTADECENYL

ETHER

F. SPENER,

F. PALTAUI;

AKD A. HOLASEK

Institute of Physiological Chenzistvql,University (Received November gth, 1907)

of Graz, Gvaz (Austria)

SUMMARY I. Glycerol

in studying

tri-cis-9-octadecenyl

the intestinal

ether is used as a model substance

absorption

of non-hydrolysed

2. m14C]Glycerol trioctadecenyl duct f&a

and to non-operated

ether was fed to rats provided

rats. The radioactivity

organs and the whole carcass was determined. 3. The very poor absorption of [Xlglycerol suggestion

of others that the absorption

with a thoracic

of the lipids of lymph,

trioctadecenyl

of intact

for triolein

triglycerides.

triglycerides

ether confirms

some the

must be very poor.

INTRODUCTION

Micellar

solutions

formed

during

fat digestion

were found

to contain

only

traces of di- and triglycerides1-3. If lipids are absorbed via a micellar phase, as suggested by HOFMANN AND BORGSTR~~M~ and JOHNSTON AND BORGSTR~M~, and confirmed by electron microscopic studies 5- 7, the absorption of intact triglycerides must be poor*. Experiments done by FELDMAN ASD BORGSTR~M~’ in vitro showed that up to 6% of triglycerides were taken up by hamster intestinal rings after incubation with a triglyceride-containing micellar lipid solution. SAVARY AND COKSTANTIN~~ concluded from the absorption triolein,

that

However,

an exact

glyceride

of non-polar

the non-polar

absorbed

Long-chain

n-hexadecane,

triglyceride

quantitative

might

determination

also

fed to rats in a mixture be absorbed

of the amount

to some

with

extent.

of unhydrolysed

tri-

in viva has not yet been performed. trialkyl

should be adequate

glycerol

ethers,

model substances

which

are isosteric

for absorption

to glycerol

experiments.

triesters,

Since they are not

or otherwise changed in the intestinal lumen, their absorption may allow an estimation of the amount of triglyceride absorbed without previous hydrolysis. l”C-Labelled trioctadecenyl glycerol mixed with olive oil was fed to rats and the radioactivity of the lipids of the lymph as well as of some organs and the carcass was determined.

hydrolysed

* The theory of fat absorption has been described in some recent reviewss-‘O. Biochim.

Biophys.

Acta,

152 (1968) 368-371

ABSORPTIONOF GLYCEROLTRIOCTADECENYLETHER

369

METHODS [x-14C]cis-g-0 c t a d ecenyloxy-2,3-di-cis-g-octadecenyloxypropane ([Xlglycerol trioctadecenyl ether) was synthesized from 2,3-di-cis-g-octadecenyloxy-r-propanol and [r-WI&s-g-octadecenyl mesylate as described elsewhere13. The purity of the substance was checked by thin-layer chromatography as follows. Glyerol trioctadecenyl ether with an activity of IOOOOOcounts/min was applied to the plate. After developing with light petroleum-diethyl ether (go : IO, by vol.) and a short exposure to iodine vapour, the spot was scraped off and eluted with diethyl ether. At least 99.5 y0 of the activity was recovered; no activity could be detected on the rest of the plate. Radioactivity was measured with a Tricarb Liquid Scintillation Spectrometer Mod. 3315 (Packard Instrument Company), counting efficiency go.5 %. Quenching was corrected by external standardization. The 14C-labelled glycerol trioctadecenyl ether mixed with olive oil was fed to starved male albino rats (Sprague-Dawley) by gastric intubation under slight ether anesthesia. The animals had free access to food (Altromin R) and water until they were killed 4 days later. Other animals were provided with a thoracic duct cannula according to the of the radioactive method of BOLLMAN, CAIN AND GRINDLEY 14. After administration triether these animals were fed only calcium-free Krebs-Ringer solution. Lymph was collected for about 40 h. The lipids of the lymph and organs were extracted with a zo-fold volume of chloroform-methanol mixture (2: I, v/v) (see ref. 15). Radioactivity of aliquots of the extract was determined after evaporation to dryness in counting vials and dissolving of the residue in 15 ml of scintillation cocktail (5 g PPO and 0.3 g POPOP in IOOO ml toluene). Lymph lipids containing IOO mg of inactive glycerol trioctadecenyl ether as carrier were hydrolysed with 5 o/o KOH in methanol. After boiling for z h, water was added and the non-saponifiable material was extracted with several portions of diethyl ether. From the acidified water phase the fatty acids were extracted. Both fractions had to be further purified by preparative thin-layer chromatography on Kieselgel H (Merck, Darmstadt) using light petroleum-ether-acetic acid (yo: IO: I, by vol.) as developing solvent. The glycerol trioctadecenyl ether and the fatty acid fractions were scraped off and eluted with ether. The solutions were evaporated and the residue dissolved in scintillation fluid. RESULTS Two rats each were fed solutions of 5 %, IO y. and 20 %, respectively, of 14Clabelled glycerol trioctadecenyl ether in olive oil. After 4 days the animals were killed and the radioactivity of the lipids of heart, liver, lung, spleen, kidney and small intestine, as well as of the rest of the carcass, was measured. After administration of o.8.10~ to 1.23.10~ counts/min, up to IOOO countsjmin could be found in the liver. All other organs contained only trace amounts of radioactivity (see Table I). 4 rats with a thoracic duct fistula were fed a 5 y. mixture, and z rats a 30% mixture, of the labelled glycerol trioctadecenyl ether and olive oil. The total lymph Biochim.

Biophys.

Acta,

152 (1968) 368-371

1:.SPENEK, F. PALTAUF, A. HOLASEK

370

flow amounted to about Szo ml per animal; the fat content ranged from 6j to 105 “:, of the administered olive oil. l;or the radioactivity of lymph lipids see Table II. The radioactivity

of lung and liver lipids of some of these animals

was tested

and was found to be less than IOO counts/min. Lymph lipids of rats Nos. II and 12 were subjected to alkaline hydrolysis and separated into non-saponifiable and fatty acid fractions

which were further

purified

by thin-layer

chromatograplly

because

each fraction was contaminated by the other. The pure triether fraction contained 27 0; of the total radioactivity while 55 o/Owas in the fatty acid fraction. The rest of the radioactivity dividual

was spread over the plate and could not be attributed

compound

because

of the low counting

to one in-

rates.

RJJCOVERYOF R.,DIOACTI”1T\~FROM R.1TS AFTISRhUMINISTRATI”N OF I*J(‘ GLY(‘ISKOI.TKIO<‘T.\T~HcT;sI’I. lST”iiK :14C1Glycerol trioctadeccnvl ether together with olive oil in the indicated percwtagc (\v/\v) \vas iiven to rats by gastric tu&. After 4 days the rats were killed, the whole carcass or the organs and the rest of the carcass were homogenized and extracted. The radioactivity found in the organs was extremely low and is not shown in the table, with the exception of the liver. Hat ,Yo.

1

LO

I.Ig’lo”

1850

2

LO

r.2j.106

1870

3

IO

4 5

10

5

9.3.105

G

5

8.j.

*

IITO *

1230

0.1

*

970

105

0.10;

IO+0

*

Id

0.11) I

i-?

0.104

Not determined.

TABLE

II

RADIOACTIVITY OF LYMPH TO

*

8.105 8.2

* 610

RATS

WITH

A

THORACIC

LIPIDS DUCT

AFTER

4DMINISTRATION

OF

!l”c]CLYCEROL

TRIOCThDECEKYL

ETHER

FISTULA

The rats were fed a mixture of radioactive glycerol trioctadecenyl ether and olive oil by gastric intubation. Lymph was collected for 40 h, extracted and the radioactivity of total lipids was measured. The amount of triether absorbed is calculated from the radioactivity found in the lymph. It is not identical to the amount of triether isolated from the lymph, since some of the radioactivity is contained in a fatty acid fraction, as judged by thin-layer chromatography (see text). _ -Rat

Administered

No.

[WT]Glycerol tvioctadecenyl ether in olive oil (“(,)

Found ‘K ~~&m?vol trioctadecenyl ether fpmol~s)

26.8 26.8 26.8 26.8

5 5 5 5 30 30

i

9 IO

II I2

147.8 147.8

__.

Counts/mm

Comtslmin

9.5 105 9.5.105 9.5.105 g.5.105 5.3.106 5.3’ 106

990 1190 I290

01) of a.dministewlt vndloactli,it\j

O.TO.$

% lGlycero1 tr*imtadecen~,l ether absovhPd (~twd?s x I@) 2;

0.125

3s

.3fJ 2s

1530

0.136 0.106 0.02c)

4-k

1200

0.02.3

3-l

1010

~_~.

____~

DISCUSSION

Experiments Biochim.

Bioph.vs.

Acta,

with both operated 152 (1968)

368-371

and non-operated

rats indicate

that less than

ABSORPTION OF GLYCEROL TRIOCTADECENYL 0.14%

of orally

administered

testine.

Provided

glycerol

in their micellar involved range,

solubility

in lipid namely

about

tion,

is in accordance

Different

be ruled

Because

of the poor

The only feeding olive

ties is proved by analysis The

discrepancy

experimental

conditions

The totally to rats

together

alonelz.

This

prerequisite

which

its absorption

phase

substances

way

fed to the dependent.

can not be made. the results

specific

absorbable

after

activity

radioactive

not be excluded

in

impuri-

with

of FELDMAN

by hamster

in part

and

certainty

intestinal

AND BORGrings

be due to the very in the physical

the

from

different properties

is compared

with

trioctadecenyl

when

of the paraffin might

its physical ether is poorly

fed is a

also

be

properties absorbed.

role in lipid absorption

of a molecule. or not

may

be determined

such as, e.g., cell membrane.

shall

of glycerol their

extent

triglycerides

Although

must play an important

character

well when given

lesser

solubility

non-polar

of magnitude.

is absorbed

absorption

to be absorbed

to a much

that

the glycerol

absorption

but

the micellar

by some receptor

in lipid

was found

that

and order

a substance

the intestinal

same

after all, to differences

size or shape therefore,

or perhaps

may

or triolein

conclusion

to the amphiphilic

Whether micellar

acid

to those of a paraffin,

in addition solubility

triglycerides

n-hexadecane

oleic

led to the

The molecular

oil were

between

and the finding

are absorbed

and perhaps,

non-polar

in a similar

are similar

of the

of

absorp-

and triether.

with

for

our results

triglycerides

containing

the triester

pinocytosis

experimentsh-7.

on this question

ether

process

the same

of lipid

is concentration

small difference

in-

lipids.

between

up to 6%

solution

absorbed

a final statement trioctadecenyl

a simple

ether in olive

of this substance

within

process

microscopic

by these data. This source of error could

STR~M’~ that a micellar

electron

rat

markedly

or any other

be absorbed

important

trioctadecenyl

by

do not differ

membrane

must

; at least the absence of readily

of the lymph

is absorbed

fat. By these data

out is the very

glycerol II)

recent

the uptake

absorption

30%

oil (see Table

between

with

fact to be pointed 5 and

triglycerides

of glycerol

whether

ether triethers

by the intestinal

intact

out as a quantitatively

quantities

rats to determine

trioctadecenyl

0.1 o/o of the ingested

may

371

and the isosteric

or uptake

absorption,

fat droplets which

glycerol

triesters

ETHER

micellar

be elucidated

by

di- and tri-alkyl solubility

current ethers

by The

its micellar role of the

experiments and similar

by

model

in vitro.

REFERENCES I 2 3 4 5 6 7

8 9 IO II 12

13 14 15

A. R. A. J.

F. HOF&IANN AND B. BORGSTRGM, Federation Proc., 21 (1962) 43. REISER AND H. C. Fu, Biochim. Biophys. Acta, 116 (1966) 563. F. HOFMANN AND B. BORGSTR~M, J. Clin. Invest., 43 (1964) 247. N. JOHNSTON AND B. BORGSTRGM, Biochim. Biophys. Acta, 84 (1964) 412. C. T. ASHWORTH AND J. F. LAWRENCE, J. LipidRes., 7 (1966) 465. E.-W. STRAUSS, J. Lipid Res., 7 (1966) 307. D. G. MCKAY, H. KAUNITZ, I.CSAVOSSY AND R. E. JOHNSON, Metabolism, 16(1967) III. J. M. JOHNSTON, in R. PAOLETTI AND D. KRITCHEVSKY, Advances in Lipid Research, Vol. I, Academic Press, New York, 1963. p. 105. J. R. SENIOR, J. Lipid Res., 5 (1964) 495. A. M. DAWSON, Brit. Med. Bull., 23 (1967) 495. E. B. FELDMAN AND B. BORGSTRBM, Lipids, I (1966) 128. P. SAVARY AND M. J. CONSTANTIN, Biochim.Biophys. Acta, 137 (1967) 264. F. PALTAUF AND F. SPENER, Chem. Phys. Lipids, (Ig67), submitted. J. L. BOLLMANN, J. C. CAIN AND J. H. GRINDLAY, J. Lab. Clin. Med., 33 (1948) 1349. J. FOLCH,M. LEES AND G. H.SLOANE-STANLEY, J.Biol. Chem., 226(1957)497. Biochim.

Biophys.

Acta,

152 (1968)

368-371