CA1095388A - Determination of polyunsaturated fat levels in body fluids - Google Patents
Determination of polyunsaturated fat levels in body fluidsInfo
- Publication number
- CA1095388A CA1095388A CA280,926A CA280926A CA1095388A CA 1095388 A CA1095388 A CA 1095388A CA 280926 A CA280926 A CA 280926A CA 1095388 A CA1095388 A CA 1095388A
- Authority
- CA
- Canada
- Prior art keywords
- body fluid
- polyunsaturated fatty
- cis
- fatty acids
- oxygen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
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-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
- C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE.
A method of determining the polyunsaturated fatty acid levels in a body fluid such as serum or plasma includes the steps of converting the polyunsaturated fatty acid content of a sample of body fluid into free acid or salt form, taking a predetermined volume of this body fluid, oxidising the polyunsaturated fatty acids or salts in the volume of body fluid with molecular oxygen in the presence of excess of an oxygenase enzyme which is specific for polyunsaturated fatty acids which contain a cis,cis-1,4-pentadiene system in a suitable buffer, and measuring the amount of oxygen consumed by the volume of body fluid by means of an oxygen electrode.
A reagent for use in the above method comprises a borate buffer of molarity 0,1 to 2 molar and pH 7 to 10 and contains an oxygenase enzyme which is specific for polyunsaturated fatty acids containing a cis,cis-1,4-pentadiene system.
A method of determining the polyunsaturated fatty acid levels in a body fluid such as serum or plasma includes the steps of converting the polyunsaturated fatty acid content of a sample of body fluid into free acid or salt form, taking a predetermined volume of this body fluid, oxidising the polyunsaturated fatty acids or salts in the volume of body fluid with molecular oxygen in the presence of excess of an oxygenase enzyme which is specific for polyunsaturated fatty acids which contain a cis,cis-1,4-pentadiene system in a suitable buffer, and measuring the amount of oxygen consumed by the volume of body fluid by means of an oxygen electrode.
A reagent for use in the above method comprises a borate buffer of molarity 0,1 to 2 molar and pH 7 to 10 and contains an oxygenase enzyme which is specific for polyunsaturated fatty acids containing a cis,cis-1,4-pentadiene system.
Description
` ~Oft~3~3 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 :, . .
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 :
, - , - - ~ .. . . ~..... . .
j3~8 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
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 :, . .
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 :
, - , - - ~ .. . . ~..... . .
j3~8 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.
: ` ' . ~ - . . : . . - - .
1095i38B
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.
--. "~;
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 - 4 ~
.:
. ~' ~' .
. ;;:
~`:
: . ~ . .- . `
., .
., .. . .
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 -- ia~9 s;~8 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 .
.
, ,. .. , . . :. ; . .. ::
,, ''' ~ ' ', -'' , '",' ', ' ".'' ': ~ ' " ~. `;: ,
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.
: ` ' . ~ - . . : . . - - .
1095i38B
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.
--. "~;
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 - 4 ~
.:
. ~' ~' .
. ;;:
~`:
: . ~ . .- . `
., .
., .. . .
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 -- ia~9 s;~8 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 .
.
, ,. .. , . . :. ; . .. ::
,, ''' ~ ' ', -'' , '",' ', ' ".'' ': ~ ' " ~. `;: ,
3~ 3~3~
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 :: ", 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.
;
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).
:
.
, . ;~
' 3~
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.
~ , 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).
~, In order to confirm that one molecule o~ oxygen is consumed . `~;
':
::
. .
s~
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.
:
The method of the invention was then carried out on serum and plasma. 50 ~Q of plasma or serum were pipetted into .
, ~ . . ,, ~ , . . .
,--~ 3IC~S 3~
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.
~: :
, ~ , ~
- 10 - .
, , . ~: ,, , . i . . , . ... ~,. :: , . .
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 :: ", 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.
;
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).
:
.
, . ;~
' 3~
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.
~ , 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).
~, In order to confirm that one molecule o~ oxygen is consumed . `~;
':
::
. .
s~
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.
:
The method of the invention was then carried out on serum and plasma. 50 ~Q of plasma or serum were pipetted into .
, ~ . . ,, ~ , . . .
,--~ 3IC~S 3~
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.
~: :
, ~ , ~
- 10 - .
, , . ~: ,, , . i . . , . ... ~,. :: , . .
Claims (11)
1. A method of determining the polyunsaturated fatty acid levels in a body fluid including the steps of converting the polyunsaturated fatty acid content of a sample of body fluid into free acid or salt form, taking a predetermined volume of this body fluid, oxidising the polyunsaturated fatty acids or salts in the volume of body fluid with molecular oxygen in the presence of excess of an oxygenase enzyme which is specific for polyunsaturat-ed fatty acids which contain a cis, cis-l, 4 pentadiene system in a suitable buffer, and measuring the amount of oxygen consumed by the volume of body fluid by means of an oxygen electrode.
2. A method according to claim 1 wherein the body fluid is serum or plasma.
3. A method according to claim 1 or claim 2 wherein the oxygenase enzyme is lipoxygenase.
4. A method according to claim 1 wherein the buffer is one having a pH of 7 to 10.
5. A method according to claim 1 wherein the buffer is a borate buffer having a molarity of 0,1 to 2 molar.
6. A method according to claim 1 wherein the buffer is a borate buffer having a molarity of 1,0 and a pH of 9.
7. A method according to claim 1 wherein the polyunsatu-rated fatty acid content of the body fluid is converted into salt form by saponification,
8. A method according to claim 7 wherein the saponification is achieved by means of a methanolic potassium hydroxide solution.
9. A reagent for use in the method of claim 1 comprising a borate buffer of molarity 0,1 to 2 molar and pH 7 to 10 and con-taining an oxygenase enzyme which is specific for polyunsaturated fatty acids containing a cis, cis-l, 4-pentadiene system.
10. A reagent according to claim 9 having a molarity of 1,0 and a pH of 9.
11. A reagent according to claim 9 wherein the oxygenase enzyme is lipoxygenase.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ZA76/3930 | 1976-07-01 | ||
| ZA00763930A ZA763930B (en) | 1976-07-01 | 1976-07-01 | Determination of polyunsaturated fat levels in body fluids |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1095388A true CA1095388A (en) | 1981-02-10 |
Family
ID=25570462
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA280,926A Expired CA1095388A (en) | 1976-07-01 | 1977-06-20 | Determination of polyunsaturated fat levels in body fluids |
Country Status (11)
| Country | Link |
|---|---|
| JP (1) | JPS5310494A (en) |
| AU (1) | AU506806B2 (en) |
| BE (1) | BE856304A (en) |
| CA (1) | CA1095388A (en) |
| DE (1) | DE2728987A1 (en) |
| FR (1) | FR2356940A1 (en) |
| GB (1) | GB1523270A (en) |
| IE (1) | IE45352B1 (en) |
| NL (1) | NL7707292A (en) |
| SE (1) | SE7707620L (en) |
| ZA (1) | ZA763930B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS58126798A (en) * | 1982-01-21 | 1983-07-28 | Toyo Jozo Co Ltd | Novel method for determination of unsaturated fatty acid |
| WO2005040410A1 (en) * | 2003-10-29 | 2005-05-06 | Novozymes A/S | Screening for lipolytic enzyme or amidase activity |
-
1976
- 1976-07-01 ZA ZA00763930A patent/ZA763930B/en unknown
-
1977
- 1977-06-20 CA CA280,926A patent/CA1095388A/en not_active Expired
- 1977-06-22 AU AU26316/77A patent/AU506806B2/en not_active Expired
- 1977-06-27 GB GB26785/77A patent/GB1523270A/en not_active Expired
- 1977-06-28 DE DE19772728987 patent/DE2728987A1/en not_active Withdrawn
- 1977-06-30 NL NL7707292A patent/NL7707292A/en not_active Application Discontinuation
- 1977-06-30 BE BE178941A patent/BE856304A/en unknown
- 1977-06-30 SE SE7707620A patent/SE7707620L/en unknown
- 1977-07-01 JP JP7896677A patent/JPS5310494A/en active Pending
- 1977-07-01 FR FR7720346A patent/FR2356940A1/en active Pending
- 1977-07-01 IE IE1374/77A patent/IE45352B1/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| IE45352L (en) | 1978-01-01 |
| FR2356940A1 (en) | 1978-01-27 |
| AU2631677A (en) | 1979-01-04 |
| NL7707292A (en) | 1978-01-03 |
| BE856304A (en) | 1977-12-30 |
| AU506806B2 (en) | 1980-01-24 |
| DE2728987A1 (en) | 1978-01-05 |
| JPS5310494A (en) | 1978-01-30 |
| ZA763930B (en) | 1978-02-22 |
| SE7707620L (en) | 1978-01-02 |
| IE45352B1 (en) | 1982-08-11 |
| GB1523270A (en) | 1978-08-31 |
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| Date | Code | Title | Description |
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| MKEX | Expiry |