Methodological Aspects and Relevance of the Study of Vegetable Oil, Fat and Lipoprotein Oxidation Using Pancreatic Lipase and Arylesterase

M. NUS et al.: Study of Fat with Pancreatic Lipase and Arylesterase, Food Technol. Biotechnol. 44 (1) 1–15 (2006)
1
ISSN 1330-9862
review
(FTB-1467)
Methodological Aspects and Relevance of the Study
of Vegetable Oil, Fat and Lipoprotein Oxidation Using
Pancreatic Lipase and Arylesterase
Meritxell Nus2, Francisco J. Sánchez-Muniz2 and José M. Sánchez-Montero1*
1Biotransformations Group, Organic and Pharmaceutical Chemistry Department, Faculty of Pharmacy,
Complutense University, E-28040 Madrid, Spain
2Nutrition and Bromatology I (Nutrition) Department, Faculty of Pharmacy,
Complutense University, E-28040 Madrid, Spain
Received: July 4, 2005
Revised version: November 23, 2005
Accepted: November 29, 2005
Summary
Fats and oils as major dietary components are involved in the development of chronic
diseases. In this paper the physiological relevance and some methodological aspects re-
lated to the determination of two enzymes enrolled in metabolism of fat – pancreatic li-
pase and arylesterase – are discussed. Pancreatic lipase has been extensively used to study
the triacylglycerol fatty acid composition and the in vitro digestion of oils and fats. The ac-
tion of this enzyme may be coupled to analytical methods as GC, HPLC, HPSEC, TLC-
-FID, etc. as a useful tool for understanding the composition and digestion of thermal oxi-
dized oils. Pancreatic lipase hydrolysis occurs in the water/oil interface, and it presents a
behaviour that seems to be Michaelian, in which the apparent Km and the apparent Vmax of
the enzymatic process depend more on the type of oil tested than on the degree of alter-
ation. The kinetic behaviour of pancreatic lipase towards thermally oxidized oils also de-
pends on the presence of natural tensioactive compounds present in the oil and surfac-
tants formed during the frying. Arylesterase is an HDL binding enzyme that inhibits LDL
oxidation. Low serum concentration of this enzyme has been related to increased cardio-
vascular disease risk. In this paper the most widely used methods for the determination of
arylesterase activity are commented on. The importance of intrinsic factors (e.g. substrates,
cofactors) participating in the enzyme reaction is also discussed. Moreover, several sugges-
tions about further researches on the influence of extrinsic factors (e.g. diet, oxidative stress)
upon the enzyme activity are proposed.
Key words: arylesterase, fat, lipoproteins, LDL, oils, pancreatic lipase, thermal oxidized fats
Introduction
and oils used for cooking improve food taste and change
Lipids are main components of food. They contain
food composition (1,2).
different molecules such as fatty acids, cholesterol, etc.,
The excess of fat and saturated fatty acids in food
which play important physiological roles. Moreover, fats
has been related to some chronic diseases such as obe-
*Corresponding author; Phone: ++34 91 39 41 820; Fax: ++34 91 39 41 822; E-mail: jsanchez@farm.ucm.es;
Website: www.biotransformaciones.com

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M. NUS et al.: Study of Fat with Pancreatic Lipase and Arylesterase, Food Technol. Biotechnol. 44 (1) 1–15 (2006)
sity and cardiovascular disease (3). Thus, the study of
and lipids interact at the interface is still not entirely clear
fat and oil composition has gained importance through-
and is a subject of intense investigation (12).
out the last decades. Moreover, fats are susceptible to
Due to their wide-ranging significance, lipases remain
oxidation and hydrolysis (4) and the origin of athero-
a subject of intensive study (13,14). Research on lipases
sclerosis has been related to low density lipoprotein
is focussed particularly on structural characterization,
(LDL) peroxidation (5). Several methods have been pro-
elucidation of the mechanism of action, kinetics, sequenc-
posed for the study of fats, oils and plasma lipid carriers
ing and cloning of lipase genes, and general characteriza-
– the lipoproteins. Most of them are qualitative or semi-
tion of performance (13,14).
quantitative, while quantitative methods are less avail-
Lipases find promising applications in organic chemi-
able and common. Related to oxidation and thermal oxi-
cal processing, detergent formulations, synthesis of bio-
dation, many qualitative methods inform about the pre-
surfactants, the oleo chemical industry, the dairy industry,
sence or absence of fats alteration and the susceptibility
the agrochemical industry, paper manufacture, nutrition,
of LDL to oxidation, but there are less available quanti-
cosmetics, and pharmaceutical processing. Development
tative methods.
of lipase-based technologies for the synthesis of novel
This paper sums up some central aspects of fats and
compounds is rapidly expanding the uses of these en-
oils reviewing some major enzymes related to the study
zymes in these industries (15). Lower amounts of these
of the fat/oil metabolism. Among them, the pancreatic
enzymes are used in oleo chemical transformations (14).
lipase deserves special mentioning. Some in vivo and in
They can play an important role in the processing of g-
vitro related studies are reviewed. In addition, the aryl-
-linolenic acid, a polyunsaturated fatty acid (PUFA); as-
esterase, a PON1 (paraoxonase 1) related enzyme, has
taxanthine, a food colorant; methyl ketones, flavour mo-
demanded recent interest because it has been found
lecules characteristic of blue cheese; 4-hydroxydecanoic
bounded to high density lipoproteins (HDL) and seems
acid, used as a precursor of the fruit flavour g-decalac-
to play a protective antioxidant (and thus, protective car-
tone; dicarboxylic acids, for use as prepolymers; cheaper
diovascular risk) effect. The relevance and some meth-
glycerides, through interesterification to more valuable
odological aspects of this enzyme will also be reviewed.
forms (e.g. cocoa butter replacements for use in choco-
late manufacture) (16). Lipases can also be used to modify
vegetable oils at position 2 of the triacylglycerol (TG), to
Physiological Importance of Enzymes
obtain fats similar to human milk fat for use in baby
feeds (17); to synthesize lipid esters (18), including iso-
Pancreatic lipase
propyl myristate, for use in cosmetics; and to produce
monoglycerides to use as emulsifiers in food and phar-
Lipases (triacylglycerol acylhydrolases, E.C. 3.1.1.3)
maceutical applications.
are ubiquitous enzymes of considerable physiological sig-
The increasing awareness of the importance of chi-
nificance and industrial potential. In eukaryotes, lipases
rality in the context of biological activity has stimulated
are involved in various stages of lipid metabolism in-
a growing demand for efficient methods for industrial
cluding fat digestion, absorption and reconstitution, and
synthesis of pure enantiomers, including chiral anti-in-
also in lipoprotein metabolism. In plants, lipases are found
flammatory drugs such as naproxen (19) and ibuprofen
in energy reserve tissues.
(20–24); antihypertensive agents such as angiotensin-con-
The major human lipases include the gastric, pan-
verting enzyme (ACE) inhibitors (e.g. captopril, enalapril,
creatic and bile salt-stimulated lipases which aid in the
ceranopril, zofenapril, and lisinopril); and the calcium
digestion and assimilation of dietary fats, and the he-
channel-blocking drugs such as diltiazem. Lipases are used
patic, lipoprotein and endothelial lipases that function
in synthesis of these drugs (25).
in the metabolism of lipoproteins (6). The pancreatic, he-
patic, lipoprotein and endothelial lipases are members
Arylesterase
of the lipase gene family. Pancreatic lipase is produced
Another group of enzymes related to fat and choles-
by the pancreatic acinar cells, and it is one of the exocrine
terol metabolism are the esterases, which do not act at
enzymes of pancreatic juice that is essential for digestion
interfaces but rather in a homogeneous polar phase. One
of dietary fats in the intestinal lumen. The substrate of
example of an esterase is arylesterase (E.C. 3.1.1.2), which
this enzyme is not a single molecule but a non-aqueous
is supposed to be one of the three activities that can show
phase of aggregated lipids made up of aggregates of es-
the PON1 (26). In recent studies, it has been hypothe-
ter molecules, micelles or monolayers interfacing with an
sized that arylesterase is a HDL-bound enzyme located
aqueous medium (7,8). It requires colipase as cofactor
at the same place as apolipoprotein (apo) A1 and apo J
for its enzymatic activity. Colipase relieves phosphatidyl
or chlusterin (27,28). Its native substrate is still unknown
choline-mediated inhibition of the interfacial lipase-sub-
but it hydrolyses aromatic esters such as phenylacetate
strate complex, helps anchor the lipase to the surface and
(PA) (Scheme 1):
stabilizes it in the »open« active conformation (9,10).
In contrast to esterases, lipases are activated only
OH
when adsorbed to an oil–water interface (11) and do not
O
hydrolyze dissolved substrates in the bulk fluid. A true
O
Arylesterase
+
lipase will split emulsified esters of glycerol and long-
OH
-chain fatty acids such as triolein and tripalmitin. Lipases
O
are serine hydrolases and display little activity in aque-
Scheme 1. Scheme of phenylacetate hydrolysis by arylesterase.
ous solutions containing soluble substrates. How lipases
Phenol, which absorbs at 270 nm, and acetic acid are obtained

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M. NUS et al.: Study of Fat with Pancreatic Lipase and Arylesterase, Food Technol. Biotechnol. 44 (1) 1–15 (2006)
3
Arylesterase protects LDL and HDL-cholesterol from
the recent adherence to diet and the nutritional status
oxidation and also facilitates the cholesterol reverse trans-
(e.g. the trienic to tetraenic ratio (20:3/20:4) is a marker
port (29,30). This protection is probably related to the
of malnutrition).
ability of arylesterase to hydrolyze some oxidized phos-
Differences in the distribution of fatty acids among
pholipids (31) and cholesteryl linoleate hydroperoxides
the three possible positions of the glycerol moiety in TG
(30), which are present in oxidized-LDL.
from natural fats and oils were first demonstrated sys-
We must distinguish between two different actions of
tematically using enzymatic hydrolysis (42). Pancreatic
this enzyme: antioxidant and arylesterase. For its ability
lipase specifically permits analysis of the fatty acids at
to protect LDL-cholesterol against oxidation, it needs the
position sn-2 (Scheme 2). More complex stereospecific hy-
sulphydryl group on cysteine-284 to be free, as shown
drolysis procedures have been developed to determine
in the study of Aviram et al. (32), which used sulphydryl
fully the positional distributions of the fatty acids (42).
blocking agents like p-hydroxymercurybenzoate (PHMB).
However, this cysteine-284 is not necessary for the aryl-
esterase activity because the substitution of this amino
OOCR1
OH
Pancreatic
acid by serine or alanine does not eliminate its aryleste-
R2COO
+ H2O
R2COO
+ R1COOH + R3COOH
rase activity. On the other hand, calcium is required for
lipase
OOCR3
OH
its arylesterase activity but not for its ability to protect
LDL-cholesterol against oxidation.
Scheme 2. Schematic reaction of pancreatic lipase hydrolysis on
The arylesterase activity is rather variable among
triacylglycerols
subjects. This variability has been related to mutations
in the gene that codes for the production of PON1. The
Lipases are among the most important enzymes used
PON1 gene is located on the long arm of chromosome 7
in the oil and fat industries. These enzymes catalyze the
(33). The major polymorphisms of PON1 include the re-
hydrolysis of TG and acyl and aryl esters (42,43) and
placement of glutamine (Q or A) by arginine (R or B) at
many are also capable of catalyzing organic reactions in
position 192, and that of leucine (L) by methionine (M)
nonaqueous media (44–46).
at position 55. There is no discrimination between the
Pancreatic lipase hydrolyzes or synthesizes TG with
allozymes for the phenylacetate substrate, but this gene
positional and fatty acid specificities, and is extensively
polymorphism may affect the antioxidant action of PON1.
used as gastrointestinal tract lipase in experimental sys-
Which allozyme is more protective against LDL-choles-
tems since it is inexpensive and easily available. Human
terol oxidation is still under discussion.
pancreatic lipase seems the »ideal« lipase to study the
Antioxidant and arylesterase activities are both af-
hydrolysis and digestion of fat with nutritional implica-
fected by lifestyle and some living factors such as diet
tions to human being. Nonetheless, the porcine pancre-
(34,35) and smoking (36). Furthermore, arylesterase ac-
atic lipase displays 86 % homology with human pancre-
tivity is reduced in hypercholesterolemia, type 2 diabe-
atic lipase (47), and therefore it is used instead of it.
tes and cardiovascular diseases (37,38).
During substrate hydrolysis by lipases, three stages
Arylesterase requires free calcium to work (39). Its
can be defined: (i) interface enzyme adsorption, (ii) in-
activity is also enhanced when chloride is in the medium
terfacial activation and (iii) catalysis. The enzyme, pres-
and inhibited when a high concentration of bicarbonate
ent in aqueous medium, is capable of penetrating into
is available (39).
the interface between the lipophilic substrate and water.
Only when both the substrate and the enzyme are at the
Methods for the Assessment of the
interface, the enzyme-substrate complex occurs, and this
Fat Composition and Quality
leads to catalytic product formation and regeneration of
Plasma TG can be studied quantitatively and quali-
the enzyme (43,48).
tatively. Most recent methods used in clinics and research
The pancreatic lipase hydrolyzes TG molecules that
measure total TG amount in plasma using sequential ac-
contain short-chain fatty acids more rapidly than mole-
tion of lipase, glicerol quinase, glicerol phosphate oxidase
cules containing only long-chain fatty acids. Moreover,
and peroxidase (40). The enzyme method is associated
ester bonds of some very long polyunsaturated fatty ac-
with the Trinder colour reaction (41). Measurements can
ids such as docosahexaenoic acid and of some trans fatty
be performed in a few minutes. Moderate hypertriglyce-
acids such as trans-3-hexadecenoic, and the phytanic acid
ridemia is achieved for TG levels between 150–200 mg/
ester of glycerol are hydrolyzed more slowly, probably as
dL but present recommendations suggest <110 mg/dL
a result of steric hindrance caused by the proximity of
(1.24 mmol/L) for children and 130 mg/dL (1.46 mmol/L)
substituent groups to these ester bonds (43,49). TGs con-
for adults as desirable values.
taining behenic acid are hydrolyzed with difficulty by
TGs are usually molecules composed of glycerol es-
pancreatic lipase, a fact of interest for food industry
terified with long-chain fatty acids. The fatty acids differ
when obtaining food with less available energy (50).
in chain length (short, medium, large), ramification (bran-
The structural analysis of TG depends on certain im-
ched and unbranched), unsaturation (saturated, mono-
portant conditions: the use of calcium ions is essential,
unsaturated, and polyunsaturated), position of double
bile salts are necessary for the reaction, and TG must be
bounds (D for relative position of double bounds to the
well dispersed by previous vigorous shaking, as the en-
acid end; n or w for the relative position to the methyl
zyme attacks only the micellar form of fats. For this rea-
end), and geometric isomerization (cis or trans). The fat-
son, methyl oleate (51) or hexane (52) are sometimes ad-
ty acid composition of plasma TG gives information of
ded as carriers to increase the solubility of saturated fats

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M. NUS et al.: Study of Fat with Pancreatic Lipase and Arylesterase, Food Technol. Biotechnol. 44 (1) 1–15 (2006)
with high melting points. Preincubation at 42 °C has also
degradation of the corresponding TG and derivatized into
been recommended instead of the use of carriers (53). The
3,5-dinitrophenylurethanes (DNPU), 3,5-dinitrobenzoates,
use of isooctane and cyclohexane use has been recom-
nicotinates, 1- and 2-anthrylurethanes, have successfully
mended for microbial lipases (54,55). Concentrations of
been identified by electrospray ionization mass spectro-
various cations, bile salts and the enzyme, the buffer pH
metry (ESI-MS), chiral-phase HPLC and GC (62,63).
and temperature should be adjusted to their optima so
According to Christie (42), by using small variations
that an appreciable degree of hydrolysis (50–60 % is suf-
of the classical method of Brockerhoff (64), it is possible
ficient) occurs in a short time (56).
to obtain phospholipid derivatives (e.g. phosphatidylcho-
The semi-micro method developed by Luddy et al.
lines) from diacylglycerols after pancreatic lipase hydro-
(57) has been recommended as the best practical proce-
lysis. These compounds can be further hydrolyzed by
dure for the structural analysis of TG (58). Facts of this
phospholipases. Analysis of the resulting diacylglycerols
method are commented below:
can determine the structure of the original TG.
Tris(hydroxymethyl)methylamine (Tris) buffer (1 M,
pH=8, 1 mL), calcium chloride solution (2.2 %, 0.1 mL)
Enzymatic Methods to Study Oxidized Oils
and a solution of bile salts (0.05 %, 0.25 mL) are added
and Lipid Peroxidation
to the TG (up to 5 mg) in a stoppered test tube, and the
whole is allowed to equilibrate at 40 °C in a water-bath
Fats and oils are susceptible to structural changes
for 1 min before the pancreatic lipase preparation (1 mg)
due to hydrolysis and autooxidation. These changes are
is added. The mixture is shaken vigorously at this tem-
increased at high temperatures, producing a higher
perature by means of a mechanical shaker for 2–4 min
amount of thermal oxidation compounds (2). Unsatura-
until the desired degree of hydrolysis is attained, when
tion increases the fat oxidation susceptibility (e.g. lino-
the reaction is stopped by the addition of ethanol (1 mL)
leic acid is 40 times more oxidizing than oleic acid) (65).
followed by 6 M hydrochloric acid (1 mL). The solution
Thermally oxidized oils, such as those produced by re-
is extracted three times with diethyl ether (10-mL por-
peated frying, contain a complex mixture of products such
tions), with centrifugation if necessary to break any
as oxidized TG monomers, TG dimers, and TG polymers.
emulsions, the solvent layer is washed twice with dis-
These products are mainly associated with changes in
tilled water (5-mL portions) and dried over anhydrous
the physicochemical properties of fats (66–68). Consequen-
sodium sulphate. On removal of the solvent, the prod-
tly, the oil/water interface could vary and the lipolytic
ucts obtained after the hydrolysis can be studied by dif-
activity of the lipase could be altered. Moreover, no stud-
ferent methods such as thin layer chromatography (TLC),
ies on the hydrolysis of oxidized or polymerized oils have
or high performance liquid chromatography (HPLC). For
been carried out with lipases other than pancreatic li-
its determination by GC after the removal of the solvent,
pase. Two groups of studies related to oxidized oils, in
it is necessary to resuspend the hydrolysis products
vitro and in vivo techniques are reviewed in this section.
with N-methyl-N-trimethylsilylheptafluorbutyramide
(MSHFBA) to convert the free fatty acids into the corre-
In vitro techniques
sponding sylil ethers (59).
The 2-monoacyl-sn-glycerols formed during the pro-
As is mentioned before, pancreatic lipase displays
cedure are generally isolated by TLC and then transeste-
greater reactivity towards TGs, which contain more hy-
rified to fatty acid methyl esters prior to gas chromato-
drophilic fatty acids, such as C10 and C12, than towards
graphic analysis. However, the presence in altered oils
those containing C16 and C18 (69). Linoleic and linole-
of many different compounds with repartion factor (R
nic acids are normal constituents of fats and oils. When
f)
values similar to those of the 2-monoacyl-sn-glycerols
prooxidant conditions are present they are converted to
makes the TLC separation rather inadvisable, so HPLC
their respective hydroperoxydes. Studies of oxidized oils
should be preferred.
employing pancreatic lipase have suggested that altered
oils are less hydrolyzed than unaltered oils (4). How-
A commonly and rapidly used procedure for inves-
ever, some »paradoxical« results have been published and
tigating esterase and lipase activity employs p-nitrophenyl
will also be commented on. The preferential hydrolysis
esters with aliphatic acyl chains of various lengts: short
of hydroperoxy linoleoyl and linolenoyl groups by pan-
chains (acetate or butyrate) to measure esterases activ-
creatic lipase may therefore be due to the presence of
ity, and long chains (palmitate or oleate) to measure li-
hydrophilic hydroperoxy groups that are more suscepti-
pases activity. The release of p-nitrophenol is measured
ble to lipase hydrolysis. Miyashita et al. (70) suggested
spectrophotometrically at 410 nm. But this method has
that hydroperoxides do not inhibit lipase activity, although
many limitations, as it must be performed at neutral or
lipid peroxidation products react with enzyme protein,
alkaline pH where some lipases cannot work (60).
reducing its biological activity. The loss of enzyme ac-
No lipolytic enzyme has yet been isolated that is ca-
tivity could be due to structural changes in the proteins,
pable of distinguishing between the positions 1 and 3 of
caused mainly by protein-centered free radical interme-
a triacyl-sn-glycerol. In order to determine the composi-
diates (71–73).
tions of positions sn-1, sn-2 and sn-3 a number of inge-
Yoshida and Alexander (74) studied the enzymatic
nious stereospecific analytic procedures have been de-
hydrolysis of acylglycerol products obtained from ther-
veloped and reviewed (42,61).
mally oxidized corn, sunflower and soybean oils. These
Regioisomers (reverse isomers) of 1,2-diacyl-sn-gly-
oils were heated at 180 °C for 50, 70 and 100 h, with aer-
cerols with various pairs of saturated and unsaturated
ation, and then three fractions were eluted from the oils:
acyl groups, which were prepared by partial Grignard
the non-polar fraction contained monomeric compounds,

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M. NUS et al.: Study of Fat with Pancreatic Lipase and Arylesterase, Food Technol. Biotechnol. 44 (1) 1–15 (2006)
5
the slightly polar fraction contained dimeric compounds
thermally oxidized frying oils, and (c) oils used in frying
and the polar fraction included polymeric compounds.
but without any preliminary separation of the non-oxi-
The monomers in the non-polar fraction were hydrolyz-
dized TG fractions.
ed by pancreatic lipase as rapidly as those in the corre-
Hydrolysis of polar fractions produced similar re-
sponding unheated oils. The dimers in the slightly polar
sults in terms of the relative hydrolysis rate. Oxidized
fraction were hydrolyzed much more slowly, and the poly-
TG monomers were the type of altered compounds most
mers in the polar fraction were barely hydrolyzed at all.
extensively hydrolyzed (80–89 %) after 20 min. Polymers
The source of the oil had no effect: pancreatic lipase hy-
displayed low hydrolysis values (11–42 %), while dimers
drolyzed heated corn, sunflower and soybean oils to a
gave intermediate values. Hydrolysis of oils used in frying
similar degree. Taking into account the studies of Ohfuji
revealed that the most degraded oils produce a signifi-
et al. (75) with thermally oxidized dimers, Yoshida and
cantly reduced level of total hydrolytic products (diacyl-
Alexander (74) explain the lower hydrolysis of dimers in
glycerols, monoacylglycerols and fatty acid monomers).
relation to monomers. This is further discussed in the in
An increase in the amount of polymers and dimers af-
vivo techniques section.
fects the hydrolysis rate of non-oxidized TG to a remark-
Márquez-Ruiz et al. (76) tested the in vitro action of
able degree: their percentage of hydrolysis in slightly de-
pancreatic lipase on complex glycerides from thermally
graded oils is about 95 %, while this percentage falls to
oxidized oils. Pure commercial olive oil was heated at
as low as 52 % in degraded oils, containing 47.6 % of
180 °C for 150 h. Samples of non-heated olive oil, heated
dimers plus polymers.
olive oil, a 1:1 mix of both and the polar fraction iso-
The position of the oxygenated group in the TG could
lated from olive oil heated for 150 h were subjected to
modulate its enzymatic hydrolysis, which has physiologi-
enzymatic hydrolysis with pancreatic lipase under stan-
cal importance. The classical method of Brockerhoff (64)
dardized conditions for 2 min and for 30 min. As ex-
would be useful to differentiate between oxidized acyls
pected, the percentage of the released free fatty acids in-
in position 1 and 3 in the triacyl-sn-glycerol.
creased substantially with hydrolysis time but decreased
In vitro hydrolysis of thermally oxidized TG by pan-
in proportion to the degree of oil alteration. A much high-
creatic lipase has also been investigated (78–80). Two differ-
er degree of hydrolysis by pancreatic lipase was found
ent aspects have been studied: total hydrolysis of thermally
for dimers of TG than for polymers in the olive oil heat-
oxidized oils (final point technique), and enzymatic hydro-
ed for 150 h. Similar results were obtained when the polar
lysis kinetics of altered oils (dinamic hydrolysis technique).
fraction of the olive oil heated for 150 h was hydrolyzed.
Hydrolysis produced no further significant triacylglyce-
Final point technique
rol polymer loss and fatty acid release after a 15-minute
The final point technique includes the study and
reaction period, demonstrating the difficulty involved in
quantitation of the compounds obtained after hydrolyz-
the hydrolysis of these high molecular mass products.
ing altered oils for a given time. Our group has used
The same group, Márquez-Ruiz et al. (77), has stud-
this method extensively for the study of unused and used
ied two aspects of the in vitro hydrolysis of abused oils
oils. Thus, the enzymatic hydrolysis of palm olein that
using pancreatic lipase: (i) the susceptibility to hydroly-
had been used 60 or 90 times to fry potatoes was com-
sis of altered compounds present in the used frying oils,
pared to that of unused palm olein. Porcine pancreatic
(ii) the influence of the degradation level of used frying
lipase was used for the hydrolysis reaction. Five hun-
fats on the hydrolysis of non-oxidized TG. Frying oils
dred-gram sets of potato slices were fried in 3 L of olein
ranging from 3.1 to 61.4 % in polar compound content
without any replenishment with unused oil. The food to
were studied using a combination of adsorption chro-
oil ratio in the fryers was kept at 500 g per 3 L by emp-
matography, high-performance size exclusion chromato-
tying the content of one fryer into the others after each
graphy (HPSEC), TLC-FID and in vitro hydrolysis for 2
10 uses (78–80).
min and for 20 min with pancreatic lipase. Three types
This successive frying of potatoes significantly in-
of substrates were subjected to enzymatic hydrolysis: (a)
creased the total polar content in the palm olein from
polar fractions isolated from trilinolein samples subject-
(9.3±0.1) mg/100 mg of oil to (26.4±0.3) mg/100 mg of
ed to heating at 180 °C, (b) polar fractions isolated from
oil after 90 uses (Table 1). Using a combination of col-
Table 1. Composition of palm olein and sunflower oil before and after potato frying
w(total polar
w(TG
w(TG
w(oxidized
w(non-oxidized
Number of
w(DG)
content)
polymers)
dimers)
TG)
TG)
fryings
mg/100 mg of oil
Palm olein
0
9.3±0.1
0.1±0.0
1.0±0.3
1.1±0.2
6.7±0.2
93.7±0.1
60
18.2±0.2
1.6±0.0
5.0±0.1
5.3±0.1
6.1±0.1
82.8±0.5
90
26.4±0.3
3.7±0.0
7.9±0.1
8.3±0.1
6.2±0.1
73.6±0.3
Sunflower oil
0
4.0±0.2
0.1±0.0
0.5±0.0
1.9±0.1
1.0±0.1
96.0±0.8
30
18.9±0.3
2.2±0.1
7.8±0.2
7.0±0.2
1.4±0.1
91.1±0.3
60
27.7±0.3
5.6±0.2
11.0±0.2
8.9±0.2
1.7±0.1
72.3±0.3
Data are the mean of three samples±s.d., TG: triacylglycerols, DG: diacylglycerols

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M. NUS et al.: Study of Fat with Pancreatic Lipase and Arylesterase, Food Technol. Biotechnol. 44 (1) 1–15 (2006)
umn chromatography and HPSEC (81), it was found that
might decrease hydrolysis of intact TG, as Márquez-Ruiz
polymer, dimer, and oxidized triacylglycerol contents in
et al. (77) have demonstrated. All these data clearly indi-
palm olein also increased several times (Table 1).
cate that the lipolytic hydrolysis of palm olein is greater
Unused palm olein and palm oleins from the 60th
when the alteration of the substrate is lower.
and 90th frying, as well as polar fractions from the un-
The amount of polymers hydrolyzed from palm ole-
used and used palm oleins separated by the column chro-
ins and from their corresponding polar fractions is shown
matographic method of Waltking and Wessels (82) were
in Table 3. The percentage of polymers remaining non-
hydrolyzed by porcine pancreatic lipase (E.C. 3.1.1.3) at
hydrolyzed was much higher in the polar fractions than
37 °C for 20 min following a slight variation of the Luddy
in their corresponding oleins, suggesting that some com-
et al. method (57).
pounds in the oils must play a role in the hydrolytic ac-
The degree of hydrolysis of the unused olein was
tion of pancreatic lipase.
similar to that of the olein used 40 times for frying pota-
toes (Table 2). However, hydrolytic compounds from
Dynamic hydrolysis technique
palm olein used 90 times were significantly lower.
The titrimetric assay was used to determine lipolytic
These results could be explained by the presence of
activity (80). The acid released during the hydrolysis was
non-oxidized TG in the samples, which are the natural
continuously titrated at 37 °C with NaOH solutions at
substrates for pancreatic lipase. The palm olein that had
pH=8.3 with the aid of pH stat and pH meter. The reac-
been used 90 times contained a lower amount of unal-
tion time was 10 min. In all cases, lipase activity was
tered TG than the unused olein or that used 40 times
measured as initial reaction rates in order to avoid the
(Table 1).
possible inhibition that might take place due to the ap-
The net hydrolysis of TG was much higher in un-
pearance of reaction products. Specific lipase activity was
used palm olein (83.8 %), and in palm olein from the
defined as the mmol of free fatty acids released per min
40th frying (87.1 %) than in palm olein from the 90th
and per mg of crude enzyme.
frying (65.8 %). This means that pancreatic lipase hydro-
Substrate concentration in the reactor was determin-
lysis of TG is maintained or increased when palm olein
ed taking into account its molecular mass, which was
is moderately altered, but that it decreases when a high
calculated by HPSEC (84), extrapolating oil sample Rf in
degree of alteration exists.
the calibration curves performed using acylglycerol stan-
In a previous study (83), the existence of a balance
dards and polyethylene glycol in different states of mat-
between factors that improve or impair the pancreatic
ter (83).
lipase hydrolysis was suggested. This balance will be
The kinetics of the enzymatic hydrolysis of vegeta-
discussed in the next section. Although non-oxidized and
ble oils that had been used in frying and that presented
oxidized TG were not tested separately, it also seems pos-
different degrees of alteration were also investigated us-
sible that the increased proportion of oligomers in samples
ing porcine pancreatic lipase (83). The aim of the study
Table 2. Concentration of diacylglycerols, monoacylglycerols and free fatty acids in unused palm olein and in palm olein used 40
and 90 times for frying potatoes after a 20-minute enzymatic hydrolysis by pancreatic lipase
Hydrolyzed oil
Number of fryings
w(MG)
w(DG)
w(FFA)
mg/100 mg of sample
Palm olein
0
24.82±1.10
24.95±0.20
34.99±1.50
Palm olein
60
25.37±1.70
24.33±1.60
36.95±1.50
Palm olein
90
13.41±0.10
26.49±0.30
23.70±0.30
Data are the mean of three samples±s.d., MG: monoacylglycerols, DG: diacylglycerols, FFA: free fatty acids
Table 3. In vitro enzymatic hydrolysis of polymers from palm olein and from the polar fraction of unused palm olein and that used
40 and 90 times for frying potatoes
w(polymers)
Initial
After hydrolysis
Hydrolyzed
mg/100 mg of sample
%
Palm olein (40th frying)
0.70±0.02
0.30±0.02
57.10
Polar fraction from palm olein (40th frying)
5.92±0.50
4.67±0.20
21.10
Palm olein (90th frying)
4.12±0.02
2.65±0.10
35.70
Polar fraction from palm olein (90th frying)
13.94±0.00
13.93±0.10
00.00
Data are the mean of three samples±s.d.

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M. NUS et al.: Study of Fat with Pancreatic Lipase and Arylesterase, Food Technol. Biotechnol. 44 (1) 1–15 (2006)
7
was to understand the effect of the thermally oxidized
data was fitted to the Michaelis-Menten equation by non-
products that appear during frying on in vitro lipolysis
linear regression, and the corresponding apparent Vmax
by pancreatic lipase. To better understand lipase hydro-
and apparent Km were estimated.
lysis of thermally oxidized oils, the effect of thermally
Comparison of the activity of pancreatic lipase on
oxidized acylglycerol models upon pancreatic lipase ac-
palm olein and sunflower oil, both with approximately
tivity was also studied (79).
18 mg polar content/100 mg of oil (Table 1, Fig. 1), clearly
Successive uses in potato frying, with no replenish-
suggests that the hydrolytic efficiency of pancreatic li-
ment of unused oil, significantly increased total polar com-
pase is greater in palm olein than in sunflower oil.
pound content of palm olein to 26 mg/100 mg of oil and
As can be seen from Table 4, kinetic parameters for
that of sunflower oil to 28 mg/100 mg of oil after 90 and
palm olein and sunflower oil did not indicate a clear re-
60 frying operations, respectively. TG polymers, TG dimers,
lationship with the number of frying operations, and
and oxidized TG increased 37.0, 7.9, and 7.5 times re-
therefore with the content of altered compounds in the
spectively in palm olein, and 56.0, 22.0, and 4.7 times in
oils. However, looking at the apparent Km or Vmax of
sunflower oil (Table 1).
palm olein or sunflower oil samples (unused or used in
The activity of pancreatic lipase can be modulated
frying), it can be concluded that palm olein is more eas-
not only by the presence of complex compounds (e.g. di-
ily hydrolyzed by pancreatic lipase than sunflower oil.
mers and polymers of TG) in the substrate but also by
The difference in the kinetic behaviour of the enzyme
altered compounds arising during the hydrolysis step (e.g.
with unused and used oils must be related to their dif-
altered fatty acids). With this objective in mind, velocity
ferent fatty acid composition and degree of alteration.
vs substrate concentration relationships were measured
Palm olein has a higher content of palmitic and oleic ac-
and the corresponding kinetic parameters were calculated:
ids but a lower content of linoleic acid than sunflower
v refers to the initial velocity in mmol/min×mg of enzy-
oil; oleic acid is known to be released by pancreatic li-
me; and S to the substrate concentration in mM.
pase with the highest relative speed (85). Comparison of
Fig. 1 shows the activity of pancreatic porcine lipase
both oils, whose polar compound content was similar,
toward palm olein and sunflower oil, both with approx-
shows that sunflower oil has a higher amount of poly-
imately 18 mg of polar compounds/100 mg of oil. Both
mers and dimers, and a lower amount of diacylglycerols
oils were hydrolyzed by pancreatic lipase following Mi-
than palm olein (Table 1). Another aspect to be taken
chaelis–Menten saturation kinetic behaviour. Each set of
into account is the presence of minor compounds that
0.16
0.06
)
)
E
E
0.12
. mg
. mg
i
n
i
n
Sunflower Oil
l/m 0.04
l/m
Palm Olein
mo
mo
(µ
(µ 0.08
v/
v/
0.02
0.04
0.00
1.00
2.00
0.00
1.00
2.00
3.00
c(substrate)/mM
c(substrate)/mM
(A)
(B)
Fig. 1. Activity of pancreatic porcine lipase toward (A) palm olein and (B) sunflower oil (both with approximately 18 % in polar
content). Experiment conditions in the text. Solid line corresponds to computer fitting of the data to the Michaelis-Menten equation,
and error bars refer to 95 % confidence limits using the Student’s t test
Table 4. Composition of non polar triacylglycerols of palm olein (NPTPO) before and after thermal oxidation at 180 °C
Heated time
w(total polar content)
w(TG dimers)
w(TG polymers)
w(oxidized TG)
h
mg/100 mg of sample
NPTPO
0
0
0
0
0
1
16.28
5.16±0.02
1.30±0.01
9.82±0.04
4
60.48
14.80±0.03
16.60±0.12
29.10±0.10
Data are the mean of three samples±s.d., TG: triacylglycerol

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8
M. NUS et al.: Study of Fat with Pancreatic Lipase and Arylesterase, Food Technol. Biotechnol. 44 (1) 1–15 (2006)
can affect the enzyme reaction. Palm olein has a high
apparent Km and apparent Vmax after 2 h can be related
amount of tocotrienols, while sunflower oil is rich in to-
to the disappearance of natural emulsifier in NPTPO,
copherols. The amount of some antioxidants (e.g. poly-
while increased production of polar surfactants would
phenols) and tensioactive substances is also quite differ-
explain the increase in the Km after 4 h, in relation to the
ent in both oils (86,87), explaining the highest hydrolysis
NPTPO samples heated for 2 h.
of palm olein. All these facts affect the enzyme activity
and thus the reaction rate, which will be discussed be-
In vivo studies
low.
Digestion of TG implies complicated physicochemi-
Changes in the apparent Km and apparent Vmax were
cal reactions and numerous interactions between lipoly-
not related to the degree of alteration of the oils. Thus,
tic products, phospholipids, bile salts, proteins, and car-
enzymatic hydrolysis catalyzed by porcine pancreatic li-
bohydrates. The process can be summarized as follows:
pase is not deeply affected by compounds of high mo-
alimentary canal enzymes, mostly pancreatic lipase, pre-
lecular mass and high polarity. However, these data must
pare fatty acids for absorption and transport through the
be evaluated in light of the process itself, because dyna-
enterocyte membrane by converting water-insoluble TG
mic hydrolysis data account for what occurs in the oil-
into more hydrophilic molecules, diacylglycerols, mono-
-water interface. Thus, it should be indicated that there
acylglycerols and free fatty acids (92).
exist factors that interfere with (e.g. complexity of sam-
ple, losses of antioxidants) and factors that enhance the
Few systematic studies investigating the major steps
enzymatic reaction (interface substrates, sample polarity,
in fat digestion and absorption of thermally oxidized
surfactant concentration, etc.). Regardless of the complex-
and polymerized oils (e.g. micelle formation, micelle-cell
ity of the sample, the preferential interface substrates for
membrane transport) have been carried out. Although
pancreatic lipase are always the same: TG and diacyl-
there is a consensus in that heated fats present reduced
glycerols, which are in much higher proportion than oli-
digestion and absorption (93), other authors (94–96) did
gomers. It is also known (88,89) that the concentration
not find that frying had any significant effect on the di-
of tocopherols decreases significantly as a consequence
gestion of different oils.
of heating.
The main reason reported (97) for the lower diges-
Yoshida and Alexander, and Ohfuji et al. (74,75) re-
tion of diets that included linseed oil heated to 275 °C
ported that the lower the degree of enzymatic hydrolysis,
was the presence of dimers of fatty acids in this polymer-
the higher the molecular mass of the acylglycerol sam-
ized fat. Deuel (98) considered that the extent to which
ples. As previously mentioned, Miyashita et al. (70) de-
frying fats were polymerized was one of the principal
monstrated that porcine pancreatic lipase is not inacti-
factors affecting their digestibility. Potteau et al. (99,100)
vated in the presence of monohydroperoxides and the
reported that as polymerization of oil increases, its diges-
hydrolysis preferentially takes place in molecules with
tive use declines, as part of the polymers are eliminated
esterified monohydroperoxide fatty acids rather than in
in the faeces.
those containing non-oxidized fatty acids. Henderson et
On the other hand, polar dimers and polymers of
al. (90) concluded that the high molecular mass polymers
TG (101) appear to be better hydrolyzed and their prod-
of TG present in oxidized fish oils can be hydrolyzed by
ucts absorbed than non-polar dimers in lymph cannu-
pancreatic lipase in vitro. The polymers formed in oils
lation studies. Márquez-Ruiz et al. (102) found high di-
during frying display surfactant properties (91). Emul-
gestibility values for oxidized dimers and polymers. Bot-
sions with higher surfactant content are more stable and
tino et al. (49) reported apparent digestibility of dimers
their interface surface is larger, suggesting that the pres-
between 30–70 %, but Kajimoto and Mukai (103) ques-
ence of surfactants enhances the enzymatic hydrolysis.
tioned such high values. Ohfuji et al. (75) found that di-
The efficiency of pancreatic lipase in hydrolyzing
meric compounds from thermally oxidized oils are ab-
palm olein (expressed by the V
sorbed by rats. However, 2 out of 3 of the ester bonds in
max) tends to increase with
the alteration level of the substrate (Table 4). However,
TG monomers are readily hydrolyzed whereas in dimers,
the enzymatic affinity for this substrate (expressed by
which are larger, due to a number of different chemical
the apparent K
entities and C-C linkages, internal ester groups are not
m) does not change. The kinetic parame-
ter apparent V
available for hydrolysis (104). According to Paulose and
max / apparent Km in unused oils shows a
tendency to increase in oils with about 18 % in polar
Chang (105), as a result of heating, much of the dimeric
content, and to decrease in oils containing about 27 % of
structure is complex because intramolecular and inter-
polar compounds. The hydrolytic behaviour of non-po-
molecular linkages can coexist. This would interfere with
lar TG of palm oleins (NPTPO) subjected to 180 °C for 1
the hydrolysis of dimers and polymers even more, re-
to 4 h also suggests that this balance between factors ex-
sulting in lower hydrolysis for heated oils. Ohfuji and
ists (Table 2) (79). Comparison between kinetic behav-
Kaneda (104) obtained no appreciable enzymatic hydro-
iour of unheated NPTPO and unused palm olein clearly
lysis of polymeric compounds in oxidized oils.
indicates than the latter is a better substrate for pancrea-
Studies on altered fat digestion often use radiolabel-
tic lipase than NPTPO. Both samples differ in major and
led markers, which do not always correspond to the same
minor compounds because NPTPO contains only isolat-
composition as that of the altered compounds present in
ed non-oxidized TG, while unused palm olein contains,
heated or used frying fats. In addition, these markers are
in addition to those compounds, other molecules with
potentially harmful and rather expensive. During the last
higher polarity (oligomers, oxidized TG, diacylglycerols
few years the in vivo digestibility and absorption coeffi-
and free fatty acids). These differences can affect the hy-
cients of some oils have been studied by our group with-
drolytic behaviour of pancreatic lipase. A decrease in the
out using radiolabelled materials (106,107). True digest-

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M. NUS et al.: Study of Fat with Pancreatic Lipase and Arylesterase, Food Technol. Biotechnol. 44 (1) 1–15 (2006)
9
ibility of unheated olive oil was tested 2, 4, 6 and 7 h
olein increased, the digestibility ratio decreased (12 and
after administrating 1 g of olive oil/100 g of body mass
37 % lower after the 40th and 90th frying operation, re-
to young adult Wistar rats by means of esophageal pro-
spectively). These results suggest that under the experi-
bes. Control rats were administered isotonic saline solu-
mental conditions applied, hydrolysis of non-oxidized TG
tion at the rate of 1 mL/100 g of body mass. Afterwards,
by pancreatic lipase is inhibited, or at least retarded, by
50 mL of isotonic saline solution were slowly passed
the presence of thermally oxidized compounds. Hender-
from the distal esophagus to the distal ileum in order to
son et al. (90) reported that in the case of oils containing
obtain the luminal fat. Remaining gastrointestinal lumi-
low amounts (less than 4 %) of polymers as substrates
nal fat showed a linear but inverse relationship (r=–0.99;
both TG and polymers were almost completely hydro-
p<0.001) with the duration of the experiment. A 4-hour
lyzed by pancreatic lipase after 1 hour in vitro, but when
test was found to be adequate, because after this period
highly oxidized oils (containing 20 or 30 % of TG poly-
half of the oil administered remained in the lumen, mak-
mers) were used, some TG remained intact.
ing it possible to accurately determine the different non-
Data related to oxidized TG digestibility coefficient
-digested and/or non-absorbed thermally oxidized com-
are difficult to be explained as it depends on a complex
pounds (108).
balance between their formation from polymers and di-
The in vivo digestibility and absorption coefficients
mers and their disappearance by hydrolysis (Fig. 2). Rel-
of palm oleins submitted to potato frying were related
atively low digestibility of these compounds contrasts
to their thermal oxidation level (106). The palm oleins
with the data of other authors (70,111) who suggest that
studied were the same ones that were discussed in the
oxidized TG are well-absorbed and appear to be ade-
in vitro studies section (Table 1). When these used palm
quately hydrolyzed by pancreatic lipase because of the
oleins were administered by esophageal probe at the ra-
higher polarity and similar molecular mass of oxidized
te of 1 g/100 g of body mass to young adult Wistar rats
TG and non-altered TG. According to Carey et al. (109),
which had fasted overnight, a significant decrease was
during the first stage of fat digestion, absorption depends
noted in the true digestibility and true absorption coeffi-
on lipolytic enzyme activity. Afterwards, molecular po-
cients after the 4-hour experiment. However, no changes
larity greatly influences the entry of lipidic products in-
in the intracellular gastrointestinal fat content were
to the micellar phase and finally, molecular mass further
found. Modifications in fat digestibility and absorption
limits luminal uptake of fat.
were highly correlated (p<0.001) with total alteration and
As the number of uses of the palm olein for frying
the presence of TG polymers, TG dimers, and oxidized
increased, luminal fat tended to present a lower percent-
TG.
age and amount of monoacylglycerols and free fatty ac-
These results concur with those of Carey et al. (109)
ids. This could be attributed to lower pancreatic lipase
and suggest that the most critical events under the ex-
activity. However, monoacylglycerols and free fatty ac-
perimental conditions applied take place in the intesti-
ids from highly altered palm oleins are more polar than
nal lumen, because clearance of the intracellular gastro-
those from less altered palm olein and thus may under-
intestinal fat does not change significantly. True digest-
go increased absorption, contributing to the lower per-
ibility and absorption coefficients were positive and sig-
centages found in the remaining luminal fat.
nificantly correlated with hydrolytic alteration products
Similar results were found when studying short-term
administered. Results suggest that palm olein with a high
digestion of olive oil and sunflower oil both heated at
level of thermally oxidized products was digested less
180 °C for 50 h (107,108). Heating increased significantly
efficiently than unused palm oleins but the level of ther-
the polar material of both oils. After 4 h, the remaining
mally oxidized products did not affect the clearance of
lumen fat (non-hydrolyzed and/or non-absorbed) was
intracellular gastrointestinal fat.
higher in the heated samples, due to the lower hydroly-
A 4-hour experiment was also designed to investi-
gate how thermally oxidized and polymerized compounds
present in palm oleins used repeatedly for frying of po-
0.8
tatoes were hydrolyzed in vivo (110). A combination of
0.7
in vivo short-term fat digestion, column chromatography
and HPSEC techniques was employed. The possible in-
0.6
hibitory role of some altered compounds, such as oligo-
0.5
mers, on fat digestibility of non-thermally oxidized TG
C 0.4
Unused
was also tested by comparing the hydrolysis of palm ole-
TD
40 fryings
ins with a different composition in altered compounds.
0.3
After the administration of palm olein used in frying,
90 fryings
0.2
the percentage of oligomers and oxidized TG in the po-
lar fraction of the luminal fat markedly increased, while
0.1
the percentage of diacylglycerols and free fatty acids de-
0
creased. This is in part due to the high content of ther-
–0.1
mally oxidized compounds in these oils and in part to
PTG
DTG
OTG
NOTG
the reduced hydrolytic activity of pancreatic lipase for
such compounds.
Fig. 2. True digestibility coefficient (TDC) of triacylglycerol po-
lyers (PTG), triacylglycerol dimers (DTG), oxidized triacylgly-
True digestibility of non-oxidized TG was greater
cerols (OTG) and non-oxidized triacylglycerols (NOTG) from
than that of oligomers. However, as the number of uses,
unused palm olein and from palm olein used 40 and 90 times
and thus the content of polar compounds of the palm
for frying of potatoes after 4-hour in vivo experiment

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10
M. NUS et al.: Study of Fat with Pancreatic Lipase and Arylesterase, Food Technol. Biotechnol. 44 (1) 1–15 (2006)
sis and absorption of the polymers. In conclusion, after
of the coupled reaction of the tiophenyl acetate hydroly-
a 4-hour experiment, true digestibility coefficients of al-
sis catalysed by the arylesterase.
tered oils, measured by fat disappearance from intesti-
Many studies have established the relationship be-
nal lumen, were significantly lower than those of the
tween arylesterase and HDL and LDL. In this way, the
unused oils. True digestibility of polymers and dimers
group of La Du has postulated the role of arylesterase in
was quite high but decreased as the alteration of the oil
the prevention of LDL and HDL oxidation (30), and in
increased. Non-oxidized TG hydrolysis was negatively
further studies they demonstrate the inhibition of this
affected by the presence of large amounts of thermally
enzyme by the oxidized LDL (115).
oxidized compounds.
Many factors such as diet can affect arylesterase ac-
tivity. Sarandol et al. (34) studied the effect of red wine
consumption on this activity and concluded that taking
Studies with Arylesterase
0.375 g alcohol/kg of body mass diminishes the aryles-
terase activity because of the positive antioxidant status
The other group of enzymes studied in this paper is
produced by the moderate consumption of red wine.
the esterases, arylesterase particularly. The interest in aryl-
Wallace et al. (35) studied the effect of a diet rich in ol-
esterase has grown much in the last few decades, as we
ive oil on a population of women with type 2 diabetes,
have previously commented on. Nonetheless, the avail-
and demonstrated the antiatherogenic role of oleic acid
able information about the uses of this enzyme is much
by increasing arylesterase activity.
reduced in comparison with that of pancreatic lipase. This
enzyme is used to predict cardiovascular risk and gives
Our group is now studying the effect of some di-
information about the anti-oxygen-free radical action of
etary compounds on the arylesterase activity. With this
HDL (29,30). Arylesterase has just been purified and ad-
aim, a restructured beef meat containing 20 % of nut mass
ded to the Protein Data Bank (112), but systems and
was given to a population of men and women with at
spectrophotometric methods to determine its enzymatic
least two cardiovascular disease risk factors. The design
activity had already been developed (89,113).
of the study was cross-over controlled by placebo. The
study had two different 5-week experimental periods (in-
Eckerson first proposed a method based on the spec-
tervention and control) and a washout period of 1 month
trophotometric measure of the appearance rate of phenol
and a half between each of them. During the interven-
as the product of the reaction catalyzed by the aryleste-
tion period, volunteers were given restructured beef steak
rase (Scheme 1). The initial rates of hydrolysis were de-
containing nuts 4 times a week and 2 restructured beef
termined at 270 nm. The assay mixture included 1.0 mM
phenyl acetate and 0.9 mM CaCl2 in 20 mM Tris/HCl,
pH=8.0 at 25 °C (89). The E
) 2.0
270 for the reaction is 1295
–3
M–1 cm–1 and one unit of arylesterase activity is equal to
10
1 mol of PA hydrolyzed per L per minute. This seems to
×
Buffer
be the elective method for many authors, although mod-
µL 1.6
Tris/HCl
SBF
ifications such the addition of NaCl as an effective sti-
min/
mulator for the reaction have been made to the buffer
/
(114).
1.2
Due to the low reproducibility and sensitivity of the
Eckerson et al. method (89), our group has developed a
ty/(mol
vi 0.8
new method using simulated body fluid (SBF) as buffer
ti
ac
instead of Tris/HCl, at 37 °C and pH=7.34–7.4. Fewer
errors and higher precision were achieved using Nus et
0.4
al. method (39) rather than the Eckerson et al. (89).
Experiments using the method by Eckerson et al. as
r
ylesterase
well as that by Nus et al. (39) show that arylesterase ex-
A 0.0
hibits Michaelis-Menten kinetics (Fig. 3), with the appar-
0
1
2
3
4
5
ent K
–4
m and Vmax values shown in Table 5. As this en-
c(phenylacetate) / (M×10 )
zyme has not been commercialized, the results are given
per µL of serum diluted in ratio 1:40 used for the essay.
Fig. 3. Michaelis-Menten kinetic adjustment of human serum
arylesterase using simulated body fluid (SBF) and Tris/HCl.
Another method has recently been proposed by Lo-
Curves were obtained changing phenylacetate (PA) concentra-
rentz et al. (113). It is a mechanized essay based on the
tion (1×10–4 to 5×10–4 M) and maintaining a fixed serum volume
diminution of the hexacyanoferrate-III as a consequence
(50 mL)
Table 5. Apparent kinetic parameters of arylesterase obtained using Tris/HCl according to Eckerson et al. (89) and using simulated
body fluid (SBF) buffer according to the Nus et al. (39)
Buffer
Vmax / U
Vmax 95 % CL / U
Km / M
Km 95 % CL / M
Tris/HCl
5.299 × 10–5
4.18 × 10–5 – 6.42 × 10–5
2.765 × 10–4
1.62 × 10–4 – 3.91 × 10–4
SBF
6.305 × 10–5
4.62 × 10–5 – 7.99 × 10–5
2.643 × 10–4
1.13 × 10–4 – 4.16 × 10–4
CL: confidence limits

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