Note: Descriptions are shown in the official language in which they were submitted.
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THIS invention relates to a novel method for the measure-
ment of polyunsaturated fat, i.e. fatty acid, levels in
body fluids such as serum or plasma and to a reagent for
use in this method.
The measurement of polyunsaturated fatty acid levels in
body fluids~ particularly serum and plasma, is a useful
procedure in the clinical pathology laboratory or
3 physician's rooms for monitoring the effects of diet and
treatments on body fluid polyunsaturated fatty acid
levels particularly in conditions associated with
atherosclerosis and hypercholesterolaemia. The lengthy
assay time of present established procedures ~or deter-
mining polyunsaturated fatty acid levels and the
special;zed equ;pment re~uired prevents routine analysis
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on a wide scale at the present time.
I.inoleic (9, 12 octadecadienoic~, linolenic (9, 12, 15
octadecatrienoic) and arachidonic (5, 8, 11, 14 eico-
satetraenolc) acids constitute the three main polyun-
saturated fatty acids present in serum or plasma together
with small amounts of pentaenoic and hexaenoic fatty
acids. A11 of these aclds contain the cis, cis-1,4-
pentadiene system. ` O~ these acids, linoleic acid is
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present in the greatest concentrations (usually up to
95%). Linoleic, linolenic and arachidonic acids are
sometimes referred to as the essential fatty acids, that
is, those fatty acids that cannot be biosynthesised or
are synthesised in inadequate amounts by animals that
require these nutrients for growth, maintenance and
proper functioning of many physical processes. Polyun-
saturated fa~ty acids constitute normally between 25 and
40% (w/v) of the total fatty acid content and are there-
~-J 10 fore presen~ in normal subjects in the range 0,75 to
2,00 g/litre.
Accordiny to the invention, there is provided a method of
determining the polyunsaturated fatty acid levels in a
body fluid including the steps of converting the polyun-
saturated fatty acld content of a~sample of body fluid
into free acid or salt -Form, taking a predetermined
volume of this body Fluid, oxidising the po1yunsaturated fatty
ac;ds or salts in the volume oF body fluid with molecular
oxygen in the presence of excess of an oxy~enase enzyme which
is specific for polyunsaturated fatty acids which contain a
cis,cis-1,4-pentadiene system in a suitable bu~Fer, and measuring
the am~unt of oxygen consumed by the volume o~ body
fluid by means of an oXygen electrode.
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An oxygen electrode is relatively inexpensive and enables
the amount of oxygen consumed to be deternlined rapidly
and in highly turbid or coloured solutions. The oxygen
electrode is a polargraphic device for measuring the con-
centration of oxygen dissolved in a given mediu~ and depends
on the electrolysis of dissolved oxygen at a weakly negative
electrode. The oxy~en electrode has been known since the
early par~ of this century. In 1956, Clark improved the
electrode considerably by using an oxygen permeable, nnn-
conducting membrane to isolate the electrolytic cell fi~om
the sample under measurement - Clark, L.C., Trans. Am. Soc.
Art. Ink. Org. 2, 41~ 1956. Oxygen electrodes are
com~ercially available. The oXygen electrode can be
coupled in known manner to a standard recorder for
following and recordin~ the rate and amount o-f oxygen
consumption.
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F~ The amount of oxygen consumed by the body fluid is directly
,~ proportional to the amount of polyunsaturated fatty acids
in the body fluid. Since a sample of body fluid of pre- ~
determined volume is used the concentrakion of polyun- ~ ;
saturated fatty~acids in`the body fluid can be readily
calculated. The oXygen electrode measures the amount of
oXYgen consumed and the amount of oxygen consumed is
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38153
determined when equilibrium conditions are reached.
Sufficient molecular oxygen must, of course, be present to
ensure that the content of polyunsaturated fatty acids or salts
in the predetermined vo1ume is oxidised. It is a simple matter
to ensure that sufficient molecular oxygen is present because
the likely concentrations of polyunsaturated fatty acids present
in body fluids is known. The source of molecular oxygen is
usually air saturated solutions.
The time it takes for equilibrium conditions to be reached is
10 ~ a function of the activity of the enzyme present. The greater
the activity the quicker will the equilibrium conditions be
reached. In all cases, however, there must be an excess of enzyme,
i.e. suffic;ent enzyme activity present to catalyse the reaction
and overcome any inhibiting effect of monounsaturated and saturated
fatty acids present 1n body fluids. The amoun1 of en y me necessary
is determinable without difficulty because the likely con-
centrations of fatty acids in body fluids are known.
Polyunsaturated~fatty acids are present in body fluids in the
form of esters. It is necessary to conver~ the esters into the
free acid or salt form, prior to oxidation. It is preferrecl
that the esters be converted into the salt form and this can
conveniently be achieved~by means of saponification.
Saponl ~ cation, as is known in the art, involves reacting an
ester, usually with heat~ with aqueous alkali, e.y. sodium or
potassium hydroxide, to form an alcohol and the salt of the
acid corresponding to the ester. Saponification is preferably
achieved by mea~s of a methanolic potassium hydroxide solution.
It is a surprising aspect and an advantage of the invention that
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the oxidation can be performed on the salts of the acids.
If desired, the esters can be converted into Free acid form.
This is conveniently achieved by means of saponification
followed by acidification, e.g. wit;h a mineral acid such as
hydrochloric acid, or by enzymic hydrolysis using for example
lipases, cholesterol esterases or phospholipases. By using
selected enzymes it is possible to determine the levels oF
polyunsaturated fatty acids esterified to cholesterol,
glycerol or phospholipids. The oxygenase enzyme is preferably
lipoxygenase (Linoleate: oxygen oxidoreduc-tase E.C. No. t.l3.11.12).
.
The preferred buffer is one having a pH of 7 to 10. A
particularly suitable buffer is a borate buffer of molarity
0,1 to 2,0 preferably 1,~, and a p~l in the abové range,
preferably 9. The invention includes within its scope an
oxygenase enzyme specific for polyunsaturated fa~ty acids
which contain a cis,cis-l,4-pentadiene system in a preferred
buffer as defined above.
.
The oxidation will generally take place at a temperature of
15 to 40C. The method of the invention has a number of
advantages over known methods of determining polyunsaturated
fatty acid levels in body fluids such as the gas chromatographic
.
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metho~. The method of the invention is very rapid and
utilises very small quantit;es of body fluids. Further-
more, as is mentioned above, it is not necessary to convert
the esters of the body fluids into the free acids as the
salts may be used.
- An example of the invention will now be described. The
~ollowing reagents were used:
S_ya Bean Lipoxygenase: This was purchased from Miles-
Seravac, Cape Town, with an activity of 50 000 unitsfmg.
One unit is defined by the manufacturers as the amount of
enzyme which causes an increase in absorbance at 234 nm,
due to the oxidation of linoleic acid, o~ 0,001 per minute
at 25C. 50 000 Miles-Seravac units = 6 International
Units~at 25C. This value was in fact obtained on
assaying the lipoxygenase in the oXygen electrode with
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linoleic acid as substrate. En7yme solutions were
prepared by dissolving approximately 50 mg lipoxygenase
in l,O ml of l,O M potassium borate buffer, pH 9,0.
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Linoleic acid solution: A standard solution was made by
d;ssolving 80 ~Q (72 mg~ linoleic acid in lO ml absolute
~ ~thanol (25,8 micromoles/ml).
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Buf~er System: 1,0 M potassium borate buffer, pll 9,0
was used as the buffer for all experiments. This was
prepared by dissolving 61,83 9 crystalline boric acid in
500 ml distilled water and adding 20~ (w/v) aqueous KOH
to bring the pH to 9,0. The volume of this solution was
then made to 1 litre by further addition of distilled
water.
? Methanolic-KOH: 14,3 g of potassium hydroxide were
dissolved in 100 ml of methanol.
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The oxygen consumption was measured using a commercially ~-
aYailable oxygen electr~de connected to a circulating
water bath. The electrode was covered by an 0,0005 incl
Teflon membrane, and the cell volume was maintained at
1,5 ml. The output signal was recorded by means of a
standard recorder. The recorder was calibrated
r using air saturated water. The oXygen concentrations in
,' air saturated solutions were calculated by the method of
Glasstone (Glasstone S, Elements Q~ Physical Chemistry,
1st Ed. pp 343-344, 1946, D. van Nostrand Co. Inc. Ne~
York).
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In order to confirm that one molecule o~ oxygen is consumed
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per molecule of polyunsaturated fatty acid, an experiment
was carried out to record the oXygen consumption obtained
on addition of varying amounts o~ llinoleic acid to a
solution containing lipoxygenase~ Linoleic acid was
chosen because it is the major constituent o~ polyunsaturated
fatty acids in body fluids.
. 1,5 ml of the po-tassium borate buffer were added to the
oXygen electrode cell together with varying volumes (1 to
( ) . 6 ~) of the standard linoleic acicl solution. After
: thermal equilibration of the mixture at 37C, 100 ~Q of
lipoxygenase solution were added to start the reaction.
E~uilibrium conditions were reached aFter two or three
minutes, and the amount of oxygen consumed after five
minutes was plotted against the amount of linoleic acid
: 15 (~moles) added. The results are shown in the attached
graph from which it can be seen that the stoichiometry of
. the reaction is 1:1. In the graph the amount of linoleic
O acid added in ~moles is plotted along the abscissa and
: the total Xygen consumed in ~moles is plotted.along
the ordinate.
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The method of the invention was then carried out on serum
and plasma. 50 ~Q of plasma or serum were pipetted into
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a small test-tube, 0,12 ml of the methanolic-KOH solution
added and the tube covered and placed in a water-bath at
60C for 10 minutes. The contents of the tubes after
cooling were made up to 0,25 ml with methanol. 25 ~Q
of the resulting solution ~ere then added to 1,5 ml of
the borate buffer in the reaction-chamber of the oxygen
electrode. After temperature equilibration had been
achieved at 37C9 the reaction was lnitiated by addit;on
of 100 ~Q of the lipoxygenase solution (~5 mg).
f ~ 10 Equilibrium conditions were attained within 5 minutes,
and the total oxygen consumed after 5 minutes was cal-
culated from the recorder reading. ,,Because of the direct
relationship between the oxygen consumed and content of
polyunsaturated fatty acids in the serum and plasma this
reading gave the number of micromoles of polyunsaturated
fatty acids present in the sample. Since the volume of
the sample is known, tne concentration of polyunsaturated '
fatty acids in the serum and plasma can be readily cal-
~-) culated.
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