-
Journal of Clinical InvestigationVol. 42, No. 2, 1963
DEMONSTRATIONOF AN INTESTINAL MONOGLYCERIDELIPASE: ANENZYMEWITH
A POSSIBLE ROLE IN THE INTRACELLULAR
COMPLETIONOF FAT DIGESTION *
By JOHNR. SENIORt AND KURTJ. ISSELBACHERWITH THE TECHNICAL
ASSISTANCEOF DOROTHYM. BUDZ
(Froml tie DepartmtcW of Medicine, Harvard Medical School, and
the Medical Services[Gastrointestinal Unit], Massachusetts General
Hospital, Boston, Mass.)
(Submitted for publication August 1, 1962; accepted October 18,
1962)
The concept is well established that digestionof foodstuffs that
occurs within the lumen of theintestine serves to convert
macromolecules tosmaller ones that may be more assimilable
andavailable for transport across the mucosal cell.Not until
recently has it become widely appreci-ated that this intraluminal
digestion is often in-complete and that additional digestion of the
par-tially split fragments may actually occur withinthe mucosal
cell during the process of absorption.Thus, it has been shown
recently (2) that dipep-tides liberated from ingested proteins by
the ac-tion of pancreatic proteolytic enzymes may be ab-sorbed by
the cell and split by mucosal dipeptidases(3). Similarly, it has
been demonstrated that af-ter the intraluminal digestion of starch
to disac-charides, these sugars are hydrolyzed within theintestinal
epithelial cell by specific disaccharidases(4-6). In fact, these
functions appear to be car-ried out at the surface of the
epithelial cells byenzymes localized at the microvilli or brush
bor-ders of the cells (3, 6).
In the case of fat digestion, the ingested lipid,most of it
triglyceride, is hydrolyzed in the lumenof the gut by action of
pancreatic lipase. This en-zyme attacks specifically the ester
bonds joiningthe fatty acyl chain to the two primary (a and
a')hydroxyl groups of the glycerol molecule (7).The result is a
liberation of fatty acids, but thereis also a significant
accumulation of unhydrolyzedmonoglyceride, especially
/3-monoglyceride. Al-
* Investigation supported in part by research grantA-3014 from
the U. S. Public Health Service, and pre-sented in part before the
Eastern Section meeting of theAmerican Federation for Clinical
Research, January 12,1962, Philadelphia, Pa. (1).
t Work done during tenure of a Special Research Fel-lowship of
the National Institutes of Arthritis and Meta-bolic Diseases.
Present address: Philadelphia GeneralHospital, Philadelphia,
Pa.
though some of this /8-monoglyceride may be iso-merized to an
a-monoglyceride and then be sub-ject to further attack by
pancreatic lipase, con-siderable amounts of monoglycerides appear
toescape hydrolysis and remain available for in-testinal
absorption.
The present study demonstrates that mono-glycerides absorbed by
the intestinal mucosal cellmay undergo further cleavage within it
by theaction of an intestinal lipase. Although intestinallipases
were postulated as early as 1892 by Schiff(8) and have recently
been demonstrated moreconvincingly (9, 10), the present results
emphasizethat under reasonably physiologic conditions theintestinal
lipase is most active towards mono-glycerides. Data are also
presented concerningthe properties of the enzyme system and its
lo-calization within the cell compartments. The in-testinal
monoglyceride lipase may have a signifi-cant role in fat
absorption, especially in facilitatingthe completion of fat
digestion within the mucosalcell.
MATERIALS AND METHODS
Mono-, di-, and tripalmitin were obtained from Dr.F. H.
Mattson,' as were a-monolinolein and the mono-glyceride isomers a-
and p-mono-olein, a- and p-mono-palmitin, and a- and 3-monostearin.
A series of DL-a-monoglycerides of varying chain length in the
fatty acidportion was synthesized from the acyl chlorides and
iso-propylidine glycerol under conditions established by Baerand
Fischer (11). Acyl chlorides were prepared by re-fluxing the fatty
acids 2 butyric, caproic, caprylic, capric,lauric, myristic,
palmitic, and stearic with excess thionylchloride, and purified by
distillation under reduced pres-sure of 10 to 15 mmof mercury
obtained by a wateraspiration system. The DL-isopropylidine
glycerol wasmade by exhaustive reflux of dry acetone and
glycerol
1 Biochemical Research Division, Proctor & GambleCompany,
Cincinnati, Ohio.
2 Nutritional Biochemicals Corporation, Cleveland, Ohio.
187
-
JOHN R. SENIOR AND KURT J. ISSELBACHER
with petroleum ether (boiling range, 38' to 58' C)through a
column of glass helices over into a total-refluxcondenser fitted
with a Barrett water trap (12), fol-lowed by distillation of the
product. The monoglycerideswere purified by recrystallization and
silicic acid chro-matography. A similar process was used to
synthesizeDL-a-monopalmitin labeled in the glycerol portion
bystarting with glycerol-1(3)_-C"3 diluted to a specific ac-tivity
of 0.06 juc per umole. Tri- and diglycerides la-beled in the
glycerol portion were prepared by directacylation of glycerol-C" in
dry pyridine with acyl chlo-rides, or with the free acids in
trifluoroacetic acid an-hydride (13), and were purified by elution
from columnsof silicic acid. All solvents were of certified
reagentquality4 and were freshly redistilled before use;
glycer-ides were checked finally for purity on thin layer platesof
silicic acid.
The glyceride substrates were suspended variously inaqueous
solutions of bovine serum albumin,2 Tween 80,5or sodium
taurocholate.6 Crude pancreatic lipase wasobtained
commercially.7
Female albino rats 8 weighing 200 to 300 g and fedon Purina chow
were ordinarily fasted overnight beforeuse. After cervical
dislocation, the abdomens wereopened, and the small intestine was
rinsed in situ withRinger's solution at room temperature and then
chilledto 0' in 0.278 M mannitol solution buffered to pH 7.0with
0.01 M Tris-maleate. After the epithelial cellswere gently scraped
free, 14 ml of mannitol solution perg of scraped cells was added
and an homogenate preparedwith a Potter-Elvehj em tissue grinder,
with a Teflonpestle. After filtration through a double layer of
ab-sorbent gauze, nuclei and cell debris were spun down assediment
at 1,400 X g for 10 minutes, mitochondria, at5,900 X g for 15
minutes, and the microsomal fraction,at 105,000 X g for 60 minutes.
The tissue fractions wereresuspended in appropriate volumes of
isotonic KCl buf-fered to pH 7.0 with 0.01 Mphosphate, and proteins
weremeasured by the biuret reaction or the Lowry method(14).
Isolated brush border preparations were made asdescribed by Miller
and Crane (6). Tissue suspensionsboiled for 15 minutes before
mixture with substrate andmedium were used for control experiments
in which in-activated enzyme preparations were desired.
Assay of the lipolytic activities of the various suspen-sions
was carried out by two methods: 1) liquid scintil-lation counting
of glycerol-C" and lower glycerides pro-duced by hydrolysis of the
glycerides labeled in theglycerol portion, and 2) direct titration
of fatty acids re-leased at a constant pH in an automatic glass
electrodetitrating device.9 In the first method, free glycerol
wasseparated from the glyceride substrates by the chloro-
3 Nuclear-Chicago Corporation, Chicago, Ill.4 Fisher Scientific
Corporation, Boston, Mass.5 Polyoxyethylene sorbitan mono-oleate.6
Organon, Incorporated, West Orange, N. J.7 Sigma Chemical
Corporation, St. Louis, Mo.8 Charles River Laboratories, Boston,
Mass.9 Model TTT-1, Radiometer Corporation, Copenhagen.
form-methanol extraction technique of Folch, Lees, andSloane
Stanley (15), the glycerides remaining in thelower, chloroform
phase, and the glycerol entering theupper, aqueous phase. Samples
of the upper phase wereused for chemical and radioactive assay of
the freeglycerol.
1. Radioactive assay of free glycerol-C" and glycerol-labeled
glycerides. To avoid certain difficulties, bothpractical and
theoretical, in the chemical assay of liber-ated glycerol from
glycerides, glyceride substrates weresynthesized with the glycerol
portion labeled with C'4 inthe 1- or 3-position, as described
above. Glycerides weresuspended in bovine serum albumin solution,
100 mg perml of 0.01 M potassium phosphate buffer at pH 7.4,with
the aid of small amounts of diethyl ether for initialdissolving of
the glycerides and a glass homogenizingvessel with a rotating
Teflon pestle. During homogeniza-tion, the temperature was
gradually raised to 40' C byimmersion of the vessel in a beaker of
warm water, un-til all of the ether was driven off and a uniform
suspen-sion of 1 mg glyceride per ml of albumin solution re-mained.
The incubation media consisted of 1 ml of theglyceride-albumin
suspension; 0.4 ml of fivefold concen-trated, low calcium,
Krebs-Ringer phosphate buffer atpH 7.4 (16) ; and 0.1 to 0.5 ml of
the intestinal tissuesuspensions in isotonic buffer of the same
type, whichwas also used to make the final volumes to 2.0 ml.
In-cubations of the intestinal cell homogenates and resus-pended
subcellular fractions were carried out in conicalPyrex centrifuge
tubes for 30 to 60 minutes at 38' C inair, with occasional shaking.
In some experiments, so-dium taurocholate was added to the media to
a finalconcentration of 20 mMper L.
Incubations were terminated by addition of 15 ml ofice-cold
methanol, followed by 30 ml of chloroform.After 30 minutes or more
at room temperature, 10 mlof 0.05 MKC1 was added and the phases
were allowed toseparate overnight at 4' C. The upper phase was
sepa-rated and samples were taken for counting of the glycerol-C".
The glycerides in the lower, chloroform phase wererecovered after
removal of the solvent under a streamof nitrogen, then redissolved
in chloroform and applied inspots on silicic acid thin-layer
plates. After ascendingchromatography in a solvent system 30 per
cent (by vol-ume) diethyl ether, 69 per cent hexane, and 1 per
centglacial acetic acid, spots were located by brief exposureof the
plates to iodine vapor and identified by compari-son with standard
glycerides. The light brown glyceridespots were marked by needle
scratches on the surround-ing silicic acid, and the iodine was
allowed to disap-pear completely by sublimation. Careful scraping
of thesilicic acid areas bearing the lipids was done by razorblade,
and the scrapings from the glass plates were trans-ferred
quantitatively into counting bottles. Scintillationcounting fluid
was added, made up in 5 ml of 95 per centethanol and 10 ml of
toluene containing 0.3 per cent2,5-diphenyl-oxazole and 0.01 per
cent p-bis-2-(5-phenyl-oxazolyl) benzene,'0 and counting was
carried out in a
10 Pilot Chemicals, Inc., Waltham, Mass.
188
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INTESTINAL MONOGLYCERIDELIPASE
TABLE I
Formation of lower glycerides and free glycerol fromglycerides
by rat-gut microsomes*
Labeled product isolated
Mono-Triglyc- Diglyc- glyc- Glyc-
Substrate eride eride eride erol
%original substrate radioactivityTripalmitin-C14 97.4 1.9 0.4
0.3Dipalmitin-C14 0.3 96.6 1.5 1.6Monopalmitin-C14 0.5 0.3 7.3
91.9
* Incubation conditions as described in Figure 1.
Packard liquid scintillation spectrometer. Recovery ofcounts
from lipids applied to silicic acid plates or fromchromatographed
lipid spots was within 5 per cent of theexpected radioactivity.
In subsequent experiments, the preparation of the tis-sue was
varied to permit assay of activity in whole in-testinal epithelial
cell homogenates, nuclei, mitochondria,microsomes, and cell sap, as
well as in isolated brushborders and the supernatant fluid derived
from them bythe technique of Miller and Crane (6). Other series
ofexperiments were performed at pH levels from 5.0 to 9.5,with
0.125 M Tris-maleate or potassium phosphatebuffer.
2. Titration of fatty acids released. Suspensions of
sub-cellular particles or pancreatic enzyme preparations
wereincubated at a constant temperature of 38° C, and at apH
maintained at 7.0 by the automatic addition of 0.2 to1 mMNaOH by a
radiometer glass-electrode titratingmechanism. The volume of base
required to maintain thepH per unit of time was observed, and this
reflectedthe rate of acid release. Although several systems
weretried as suspending media, including solutions of albu-min, gum
arabic, and Tween 80, the most reproduciblemedium was found to be a
solution of 0.005 M sodiumtaurocholate in 0.002 Mpotassium
phosphate buffer at pH7.0. This medium permitted reasonably good
dispersionof the substrates and good sensitivity to pH
changeswithout undue instability of pH in control
experiments.Series of experiments were carried out wvith
variousglyceride substrates, including monoglycerides with vary-ing
fatty acyl chain lengths, synthesized as describedabove.
Comparisons were made of the rates of fattyacid release from the a-
and ,-isomers of several mono-glycerides, of intestinal tissue
preparations and crude pan-creatic lipase preparations, and of
other pH levels. Re-sults were expressed as mAs equivalents of acid
releasedper minute per milligram protein. Similar techniques
ofcontinuous titration at constant pH have been previouslydescribed
(17), and have the advantage of more closelyestimating the true
initial reaction velocity of the ester-bound hydrolytic
reaction.
RESULTS
ide products of the reaction as well as free gly-cerol, labeled
glycerides were used in which theglycerol portion contained C14 in
the 1- or 3-carbon. When suspensions of these radioactiveglycerides
were incubated with subcellular frac-tions from intestinal
epithelial cells, it was seenthat the monopalmitin-C14 was almost
entirelyhydrolyzed, whereas very little hydrolysis of
thetripalmitin-C14 or the dipalmitin-C14 occurred.When the
glycerides obtained after incubation inthese experiments were
separated on thin-layerplates of silicic acid, it was observed
(Table I)that very little degradation had occurred of
thetripalmitin to di- or monopalmitin, or of dipalmi-tin to
monopalmitin. As might be expected inview of these results, it
appeared that much of themonopalmitin produced from dipalmitin
lipolysiswas further split to free glycerol and palmiticacid.
Repeated localization studies revealed consist-ently that of the
cell fractions, the highest specificactivity in monoglyceride
lipolysis was found inthe microsomes and the mitochondria, which
wereseveral times more active than the whole homoge-nate or the
cell sap. Because of the large amountof the cell protein in the
cell sap, however, thetotal activity in this fraction was
appreciable.When isolated brush borders were prepared,
thesupernatant mixture resulting from sedimentationof the brush
borders resembled the whole homoge-nate in its activity, whereas
the activity in thebrush borders was slight. The specific and
totalactivities are compared in Table II.
TABLE II
Intracellular localization of monoglyceride lipasein rat
intestinal mucosa*
TotalSpecific Protein in activity
Fraction activity fraction in fraction
mimoles %of wholehydrolyzed homogenate
min/mgprotein
Whole homogenate 32.5 100. 100.Nuclei, unwashed 26.0 28.2
22.6Mitochondria 77.9 5.9 14.6Microsomes 67.5 10.3 21.4Cell sap
10.3 55.6 17.7
Sumof fractions 76.5
Isolated brush borders(prepared separately) 21.0 2.0 1.3
* Each incubation was carried out for 10 minutes at 38° C with
2Assay of glyceride hydrolysis with radioactive moles of
monopalmitin-c'4 (112,000 cpm) and 0.4 to 1.0 mg tissuesubstrates.
In order to measure the lower Oycer- protein in 5 per cent albumin
at pH 7.0 in 0.15 Mpotassium phosphate,substrates. In order to
measure the lower glycer- in a total volume of 2.0 ml.
189
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JOHN R. SENIOR AND KURT J. ISSELBACHER
0.5
0.4
p MOLESMONOPALMITIN
SPLIT 0.3
0.2
0.1
05 6 7 8 9 10
pHFIG. 1. CURVEOF MONOGLYCERIDELIPASE ACTIVITY OF INTESTINAL
MUCOSALMICRO-
SOMESAS A FUNCTION OF PH. Each point represents an incubation
carried out for 30minutes at 380 C at the indicated pH, with 0.7
/Amole of mono-palmitin-C1' (40,000cpm) and 0.56 mg microsomal
protein in 0.125 M Tris-maleate and 0.125 M potas-sium phosphate
buffers. The total volume was 2.0 ml.
14J
IN
'4.
0.6
0.5
0.4
0.3
0.2
0.1
00 20 30
MINUTES
FIG. 2. RATE OF HYDROLYSISOF MONOPALMITINBY RATINTESTINAL
EPITHELIAL CELL MICROSOMES. Each incu-bation was carried out for
the indicated time at 38° C,with 1 ,umole of monopalmitin-C1'
(56,000 cpm) and 0.4mg microsomal protein in 5 per cent albumin and
0.15 Mpotassium phosphate buffer, pH 7.0. The total volumewas 2.0
ml.
Although purification and isolation of the mono-glyceride lipase
from the cell fractions was notextensively carried out, rough
characterization ofsome of the properties of the enzyme in
thesecrude fractions was attempted. The pH optimumfor the splitting
of monopalmitin by intestinal epi-thelial cell microsomes was found
to be about 7.8(Figure 1), although appreciable hydrolysis oc-cured
between pH 7.0 and 8.5. Parallel experi-ments using no tissue
preparation were carried outin order to estimate the relative
amount of non-enzymatic hydrolysis; at the more alkaline pHvalues,
increasing nonenzymatic hydrolysis bysaponification was noted. The
addition of 20 mMsodium taurocholate did not produce a markedshift
in the pH optimum of the microsomal mono-glyceride lipase, although
it is known (18) tolower the optimal pH of pancreatic lipase by
about2 pH units.
The rates of lipolysis were fairly linear over avariable range
of enzyme concentrations whenthe amount of monopalmitin-C14
substrate wasconstant. A time study (Figure 2) revealed that
190
-
INTESTINAL MONOGLYCERIDELIPASE
the rate of monopalmitin-C14 hydrolysis by intes-tinal
microsomes was linear for about 20 minutes.The temperature
stability of the microsomal mono-glyceride lipolytic activity was
noteworthy. Itwas repeatedly observed that tissue suspensionscould
be kept for several days at 00 to 40 C with-out appreciable loss of
activity, that only slightloss of activity occurred after 24 hours
at roomtemperature, and that temperatures above 600 Cwere required
for inactivation.
Addition of 20 mMsodium taurocholate pro-duced no enhancement of
monoglyceride lipase ac-tivity in microsomes, mitochondria, or
brush bor-ders from rat intestinal epithelial cells. In fact,some
30 to 40 per cent inhibition of the micro-somal activity was
observed at this concentrationof the conjugated bile salt. Studies
using tri- anddilinolein-C14 (labeled in glycerol portion)
re-vealed that they were as resistant to lipolysis bymicrosomes as
were the saturated higher glycer-ides, despite the much clearer
dispersions yieldedby the unsaturated glycerides in albumin
solution.
Study of glyceride hydrolysis by titration offatty acids
released. In order to follow the lipoly-sis of glycerides over a
period of time withoutterminating the reaction, it was feasible to
meas-ure the fatty acids released by using an automatictitrating
device at constant pH, measured by aglass electrode. This second,
independent assaypermitted quantitation of the fatty acids
releasedfrom ester linkage under initial reaction condi-tions,
since the pH of the medium was maintainedconstant by addition of
enough NaOH to neu-tralize the fatty acids liberated. Under these
con-ditions, there was no accumulation of free, un-ionized fatty
acids and H+ that would tend toreverse the reaction. This technique
permitted as-say of a much wider range of substrates, since
la-beled glyceride substrates were not necessary.Thus it was also
possible to compare rates of hy-drolysis of the series of synthetic
monoglycerideswith varying fatty acyl chain lengths. Titrationcould
not be carried out in albumin solution, pre-sumably because of the
strong buffering capacityof the protein. In the search for suitable
media,10 per cent gum arabic was rejected because ofpH instability
in the absence of any added enzyme;cell sap was found to have a low
but definite lipo-lytic activity of its own; and Tween 80, an
oleicacyl monoester of polyoxyethylene sorbitans, it-
self acted as a substrate. A dilute sodium tauro-cholate
solution weakly buffered by phosphate wasfound to provide
reasonable dispersions of sub-strates, nonenzymatic pH stability,
and sufficientsensitivity to permit titration of the fatty
acidsreleased.
At pH 7.0 and a constant temperature of 380C, the
microsome-catalyzed hydrolysis of mono-glycerides was found to be
maximal (Figure 3)when the fatty acyl chain length was 10
carbonatoms (monocaprin). Monoglycerides of shorteror longer
side-chain lengths showed decreasedrates of splitting. Unsaturation
of the side-chainseemed to have the same effect as shortening
asaturated chain, a double bond having roughly theeffect of two
fewer methylene groups. Thus,rates were comparable for monolinolein
and mono-myristin, and for mono-olein and monopalmitin.
The effect of position of the fatty acyl groupwas estimated by
comparison of the rates of hy-drolysis of the a- and ,-isomers of
monopalmitin,mono-olein, and monostearin. Although theserates
appeared to be about equal in suspendingmedia consisting of dilute
cell sap, in experimentswith taurocholate in the medium, the
,B-isomersappeared to be hydrolyzed more slowly.
Possibleisomerization of the p- to a-isomers during prepa-ration
and assay of the substrates was not ex-cluded. Addition of crude
pancreatic lipase, how-
N.
-4
14J
"It
CARBON ArOMS IN FArTY S/OF-CHAIN OFMONHLYCE//D
FIG. 3. RELATIVE RATES OF HYDROLYSIS OF MONO-GLYCERIDES OF
VARYING FATTY ACYL CHAIN LENGTH BY
INTESTINAL MUCOSALCELL MICROSOMES. In each titrationat 380 C and
constant pH 7.0, there were present 6 umolesof monoglyceride, 1.7
mg microsomal protein, 0.005 Msodium taurocholate, and 0.002 M
potassium phosphate, inan initial volume of 6 ml.
60
50 -
40~~~~~~
50~~~~~~~~~~~
20
10
4 140 10 12 14 16 18
191
-
JOHN R. SENIOR AND KURT J. ISSELBACHER
TRIGL YCER/DES
DIGL YCERIDES
o ( R')CR") CH20-C vVWAAM
NV\AWA C-OCH110 CH20-C WAAAW
I 1i1o CR I
CH20H
RC-OC H11 IH o ol '0 CH20-CR
0
MONOGLYCER/DESCH20H CH20H
IsomerizotionHOCH - R c-C H
I~~~~~~~~11 1.CH20-CR" 0 CH20H
0
GLYCEROLCH20H
CHOH + HOOCR"
CH20H
FIG. 4. SCHEMEOF THE PROGRESSIVEHYDROLYSIS OF TRIGLYCERIDESBY
PANCREATIC LIPASE. The fatty acyl side-chains are symbolized bythe
zigzag lines and by the alkyl abbreviation R. The asterisks
in-dicate reactions thought to be catalyzed by pancreatic
lipase.
ever, accelerated the splitting of a-monoglyceridesbut not
8-isomers under similar conditions.
Results of inhibition studies revealed that themicrosomal
monoglyceride lipase was essentiallyuninhibited by 0.01 Mdisodium
EDTA, but onlyslightly inhibited by potassium fluoride at thesame
concentration. Because of the very crudeenzyme preparations, no
substrate-inhibitor curveswere attempted. No acceleration of the
rate ofmonoglyceride hydrolysis was observed when cal-cium ions
were added in a concentration of 0.005Mper L.
DISCUSSION
The question of whether an intestinal lipase ac-tually exists
has been raised for many years, andindeed, the studies Schiff (8)
reported 70 yearsago on pancreatectomized dogs suggested thatthere
probably was such an enzyme. The greatabundance of highly active
pancreatic lipase bath-ing the intestinal mucosa has previously
obscuredthe demonstration of a separate intestinal lipase.Evidence
for such an enzyme has continued to ac-
cumulate, however, and recently some of the mostconvincing work
has shown pronounced lipolyticactivity in subcellular particles
obtained from hogduodenum (9). In those studies, the
intestinallipase was distinctly different from pancreatic li-pase
in its substrate specificity and response toinhibitors, and
considerable care was taken toavoid contamination of the intestinal
tissue prepa-rations with pancreatic fluid material. Otherstudies
by Tidwell and Johnston (10), usingeverted sacs of hamster small
gut in buffered al-bumin media containing various suspended
glycer-ides, have indicated that preferential splitting ofthe
monoglycerides occurred, with much less hy-drolysis of the di- and
triglycerides.
During digestion of dietary fats, pancreaticlipase has been
shown to attack triglycerides mostrapidly, diglycerides slightly
less rapidly, and tohave a sluggish effect on the
monoglyceridesformed (19). Furthermore, pancreatic lipase hasbeen
found to catalyze hydrolysis of the esterbonds at the outer,
primary (a and a') alcoholgroups of the glycerol, and to yield
/3-monoglycer-
1*+ HoocR'
I*+ HOOCR"'
192
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INTESTINAL MONOGLYCERIDELIPASE
ides as a most important product (20). Althoughisomerization of
the 8- to a-monoglycerides canoccur spontaneously, allowing
pancreatic lipaseto complete the hydrolysis, this third stage
pro-ceeds much more slowly, and considerable amountsof
monoglycerides are therefore available for in-testinal absorption.
This sequence is summarizedin Figure 4.
Evidence that monoglycerides may actually beabsorbed has been
provided by the double-labelstudies of monoglycerides and
triglycerides inwhich the glycerol portion and fatty acid
portionwere labeled with different isotopes (21, 22).Whereas some
of the absorbed monoglyceridesappeared in the lymph as
triglycerides after re-esterification by the intestinal mucosa,
consider-able amounts of the monoglycerides appeared tobe split, as
indicated by a fall in the ratio ofglycerol to fatty acid isotope
in the lymph tri-glycerides, compared to the original
monoglycerideisotopic ratios. Furthermore, in vitro splitting
ofmonoglycerides by intestinal epithelial cell prepa-rations has
recently been observed in the courseof experiments on
esterification of monoglycerides(23, 24).
In the present series of experiments, it washoped that methods
could be developed to demon-strate, under conditions reasonably
approximatingthose in the mammalian body, whether an intesti-nal
lipase distinct and different from pancreaticlipase exists.
Therefore, unphysiologic media,such as those containing synthetic
detergents orhigh degrees of alkalinity, were avoided, as
wereassays depending on such difficult-to-interprettechniques as
clearing of turbidity. Efforts weremade to exclude any
contamination by traces ofpancreatic lipase, insofar at least as
this could beaccomplished by washing the mucosa and thenthe
subcellular particles before assay. It mustbe admitted that some
pancreatic lipase couldadhere to the cell fractions and especially
to thebrush borders, but the latter contained a negligibleamount of
the total lipolytic activity of the gutepithelial cells.
Furthermore, the most highlywashed fractions, namely, microsomes
and mito-chondria, showed the highest specific activities andhad a
substrate specificity distinct from that ofpancreatic lipase.
Results by both of the methods used in thepresent experiments
were in agreement, and indi-
cated that indeed a lipase exists within the in-testinal
epithelial cells and has a great specificityfor monoglycerides.
Pancreatic lipase, on theother hand, has a maximal activity towards
tri-glycerides, somewhat less towards diglycerides,and is least
active with monoglycerides, especiallythe /8-monoglycerides as
substrates. If indeedpancreatic lipase acts strictly upon the
primaryalcohol ester bonds, this substrate specificity wouldbe
expected, for the triglycerides have two suchbonds, the
a-f3-diglycerides one, and f3-monoglycer-ides none. These isomers
of the lower glyceridesare those actually formed during digestion
of tri-glycerides by pancreatic lipase (20, 25).
The intracellular localization of maximal spe-cific activity of
the intestinal monoglyceride lipaseto the microsomes and
mitochondria was a con-sistent finding; whether or not the lower
level ofenzyme activity in the cell sap represented anartifactual
"solubilization" of the enzyme fromthe membranous structures during
preparationand differential centrifugation could not be
de-termined. The unimpressive degree of activity ofthe isolated
brush borders was another indicationof the difference between
digestion of fat and thatof carbohydrates and proteins. In the
latter, theproducts of intraluminal digestion are water-solu-ble,
and have been shown to undergo final diges-tion at the surface of
the intestinal cells by disac-charidases and oligopeptidases
localized in thebrush border (3, 6). The major products of
fatdigestion, however, free fatty acids and mono-glycerides, are
not water-soluble to any large ex-tent, and it is not surprising
that other mechanismsmay exist for the handling of these
substances.In this connection, it is pertinent that several ofthe
enzymes necessary for chemical transforma-tions of the absorbed
fatty acids and monoglycer-ides to higher glycerides seem to be
localized pri-marily in the microsomal fraction of the
intestinalepithelial cell, and to some extent in the mitochon-dria
(23, 24, 26).
Better characterization of the properties of theintestinal
monoglyceride lipase will perhaps bepossible when isolation and
purification of the en-zyme from the crude tissue fractions can be
ac-complished. The definition of this enzyme as alipase depends on
its substrate specificity, al-though it could be argued that in
view of the maxi-mal hydrolytic specificity for monocaprin, it
193
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JOHN R. SENIOR AND KURT J. ISSELBACHER
should be called an esterase. The truly water-soluble,
short-chain monoglycerides, however,were less rapidly split than
the intermediate-lengthmonoglycerides and the long-chain
unsaturatedmonoglycerides.
The data presented correlate with observationsof Tidwell and
Johnston (10) and with the veryrecent studies from the same
laboratory in whichPope, Askins, and McPherson (27) have de-scribed
a lipase in hamster intestinal homogenateswith a definite
monoglyceride specificity.
Whether the low activity of the intestinal lipasetowards tri-
and diglycerides is a function of thepoor solubility of these
compounds in aqueousmedia has not been resolved by the present
stud-ies. In contrast to our findings, DiNella, Meng,and Park (9)
found hog intestinal mucosal lipaseto hydrolyze ester bonds of tri-
and diolein asrapidly as mono-olein. Those studies, however,were
carried out under quite different conditions,i.e., with media
containing Span 20 11 at pH 9.0.The crucial questions yet to be
settled relate tothe importance of the physical state of the
sub-strate and the conditions existing in the medium.It is
noteworthy that pancreatic lipase, which hasbeen purified and
characterized (28) to a muchgreater degree than intestinal lipase,
shows higheractivity when its substrate is water-insoluble,seems to
act at lipid-aqueous interfaces, and infact is inhibited if its
substrate is put into truesolution.
Whereas the physiologic importance of pan-creatic lipase in
intraluminal fat digestion is wellestablished, the role and
significance of the in-testinal lipase is yet to be determined. It
seemsreasonable, however, that the presence of an en-zyme system in
the cell active in the hydrolysis ofmonoglycerides provides a
mechanism for com-pleting the digestion of glycerides after their
ab-sorption from the lumen of the intestine. In viewof the present
findings and the previous demon-stration of the ability of the
intestinal cell to con-vert monoglycerides to higher glycerides
(24), abalance appears to exist within the mucosal cell be-tween
monoglyceride hydrolysis and esterifica-tion to di- and
triglycerides. The mechanism(s)regulating these two pathways
remains to be elu-cidated.
11 Sorbitan monolaurate.
SUMMARY
An intestinal lipase has been demonstrated in ho-mogenates and
subcellular particles of rat intesti-nal epithelial cells. This
lipase exhibits prominentspecificity for monoglycerides, and is
much lessactive upon the di- or triglycerides of long-clhainfatty
acids. A maximal hydrolytic activity wasfound for monoglycerides of
intermediate chainlength, i.e., from 8 to 12 carbon atoms in
thesaturated fatty acyl chain. Unsaturated long-chain
monoglycerides were split at rates compara-ble to those for
saturated monoglycerides ofshorter chain length. A survey of the
cell frac-tions showed the monoglyceride lipase activity tobe
concentrated primarily in the microsomal andmitochondrial
fractions, with considerably lowerspecific activities present in
the cell sap and iso-lated brush borders. Fluoride and
EDTA(ethylenediamine tetraacetic acid) did not in-hibit the enzyme
activity.
The intestinal monoglyceride lipase was foundto be distinct and
different from pancreatic lipasein a) its specificity for
monoglycerides rather thantriglycerides, b) its ability to catalyze
hydrolysisof ester bonds on the ,B- as well as the a-hydroxylgroups
of glycerides, and c) its activity upon dis-solved rather than
suspended substrates. Otherdifferences appear to exist in the
effect of inhibi-tors, pH optima, and chain-length specificity,
butfurther studies are necessary when purer enzymepreparations are
obtained.
The physiologic importance of the intestinalmonoglyceride lipase
is not yet known. It may,however, function in the intracellular
completionof fat digestion before the synthesis of chylomi-crons by
the intestinal mucosa.
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