CA1144051A - Method for the analysis of triglycerides - Google Patents
Method for the analysis of triglyceridesInfo
- Publication number
- CA1144051A CA1144051A CA000374028A CA374028A CA1144051A CA 1144051 A CA1144051 A CA 1144051A CA 000374028 A CA000374028 A CA 000374028A CA 374028 A CA374028 A CA 374028A CA 1144051 A CA1144051 A CA 1144051A
- Authority
- CA
- Canada
- Prior art keywords
- poe
- enzyme
- glycerol
- kinase
- pyruvate
- 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
Links
Classifications
-
- 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
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
-
- 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
- C12Q1/61—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving triglycerides
Abstract
ABSTRACT
A method for the analysis of triglycerides in a biological fluid which comprises; (a) treating the bio-logical fluid to remove endogenous glycerol and pyruvate by combining with the fluid, as a first component, adenosine triphosphate (ATP), enzyme glycerol kinase (GK) and a metal cation activator for the GK, as a second component, enzyme pyruvate kinase (PK) in the presence of substrate phosphoenol pyruvate (PEP) and an enzyme PK activator, and as a third component, glutamate and enzyme alanine aminotransferase (GPT); (b) enzymatically hydrolyzing the triglycerides in the resulting fluid free of glycerol and pyruvate by com-bining the fluid with a mixture comprised of a microbial lipase, a surfactant selected from polyoxyethylene (POE) alkylamides, POE esters of fatty acids, POE mercaptans, POE
alkylamines, POE polyol esters, POE acetylenic glycols and POE phosphate esters, and an alkaline earth metal cation, in the form of a salt; and (c) determining the amount of gly-cerol produced from the enzymatic hydrolysis. The method disclosed obviates the need for parallel blank correction.
A method for the analysis of triglycerides in a biological fluid which comprises; (a) treating the bio-logical fluid to remove endogenous glycerol and pyruvate by combining with the fluid, as a first component, adenosine triphosphate (ATP), enzyme glycerol kinase (GK) and a metal cation activator for the GK, as a second component, enzyme pyruvate kinase (PK) in the presence of substrate phosphoenol pyruvate (PEP) and an enzyme PK activator, and as a third component, glutamate and enzyme alanine aminotransferase (GPT); (b) enzymatically hydrolyzing the triglycerides in the resulting fluid free of glycerol and pyruvate by com-bining the fluid with a mixture comprised of a microbial lipase, a surfactant selected from polyoxyethylene (POE) alkylamides, POE esters of fatty acids, POE mercaptans, POE
alkylamines, POE polyol esters, POE acetylenic glycols and POE phosphate esters, and an alkaline earth metal cation, in the form of a salt; and (c) determining the amount of gly-cerol produced from the enzymatic hydrolysis. The method disclosed obviates the need for parallel blank correction.
Description
o~
Background Of The Invention This invention relates generally to an analytical enzymatic determination of serum triglycerldes. More specif-ically, it relates to a method which incorporates a sequential and/or simultaneous sample pretreatment which eliminates en-dogenous glycerol and pyruvate interferences and the need for blank correction and a triglyceride assay which employs im-mobilized enzymes for the detection reaction.
Summary Of The Invention In accordance with an aspect of this invention, there is disclosed a method for the analysis of triglycerides in a biological fluid by pretreating the biological fluid to remove interferences arising from endogenous glycerol and pyruvate, enzymatically hydrolyzing the triglycerides in the resulting fluid and determining the amount of glycerol pro-duced by the enzymatic hydrolysis, the improvement which com-prises enzymatically hydrolyzing the triglycerides in the resulting fluid free of glycerol and pyruvate by combining the fluid with a mixture comprised of a microbial lipase, a surfactant selected from polyoxyethylene (POE) alkylamides, POE esters of fatty acids, POE mercaptans, POE alkylamines, POE polyol esters, POE acetylenic glycols and POE phosphate esters, and an alkaline earth metal cation, in the form of a salt.
In accordance with a more specific aspect of this invention there is disclosed a method for the analysis of triglycerides in a biological fluid which comprises: (a) treating the biological fluid to remove endogenous glycerol and pyruvate by combining with the fluid, as a first component, adenosine triphosphate (ATP), enzyme glycerol kinase (GX) and ' mab/C
;
a metal cation activator for the GK,' as a second component, enzyme pyruvate kinase (PK) in the presence of substrate phosphoenol pyruvate (PEP) and an enzyme PK activator, and as a third component, glutamate and enzyme alanine aminotrans- -ferase (GPT); (b) enzymatically hydrolyzing the triglycerides in the resulting fluid free of glycerol and pyruvate by com-bining the fluid with a mixture comprised of a microbial li-pase, a surfactant selected from -polyoxyethylene (POE) alkyl-amides, POE esters of fatty acids, POE mercaptans, POE alkyl-amines, POE polyol esters, POE acetylenic glycols and POE
phosphate esters, and an alkaline earth metal cation, in the form of a salt; and (c) determining the amount of glycerol produced from the enzymatic hydrolysis.
Detailed Description of the Invention The method disclosed and claimed herein is a pro-cedure for the analysis of triglycerides in biological fluids, especially body fluids such as serum.
In general, the procedure involves pretreating a -serum sample to remove interferences arising from endogenous glycerol and pyruvate, quantifying the glycerol produced by enzymatic hydrolysis by employment of an immobilized enzyme coil.
The novel procedure claimed herein advantageously eliminates the need for parallel blank correction, the latter step required in prior art methods.
In the method 2S claimed herein, the removal of glycerol and pyruvate interferences endogenous to the biological fluid is accomplished pursuant to the following r~action
Background Of The Invention This invention relates generally to an analytical enzymatic determination of serum triglycerldes. More specif-ically, it relates to a method which incorporates a sequential and/or simultaneous sample pretreatment which eliminates en-dogenous glycerol and pyruvate interferences and the need for blank correction and a triglyceride assay which employs im-mobilized enzymes for the detection reaction.
Summary Of The Invention In accordance with an aspect of this invention, there is disclosed a method for the analysis of triglycerides in a biological fluid by pretreating the biological fluid to remove interferences arising from endogenous glycerol and pyruvate, enzymatically hydrolyzing the triglycerides in the resulting fluid and determining the amount of glycerol pro-duced by the enzymatic hydrolysis, the improvement which com-prises enzymatically hydrolyzing the triglycerides in the resulting fluid free of glycerol and pyruvate by combining the fluid with a mixture comprised of a microbial lipase, a surfactant selected from polyoxyethylene (POE) alkylamides, POE esters of fatty acids, POE mercaptans, POE alkylamines, POE polyol esters, POE acetylenic glycols and POE phosphate esters, and an alkaline earth metal cation, in the form of a salt.
In accordance with a more specific aspect of this invention there is disclosed a method for the analysis of triglycerides in a biological fluid which comprises: (a) treating the biological fluid to remove endogenous glycerol and pyruvate by combining with the fluid, as a first component, adenosine triphosphate (ATP), enzyme glycerol kinase (GX) and ' mab/C
;
a metal cation activator for the GK,' as a second component, enzyme pyruvate kinase (PK) in the presence of substrate phosphoenol pyruvate (PEP) and an enzyme PK activator, and as a third component, glutamate and enzyme alanine aminotrans- -ferase (GPT); (b) enzymatically hydrolyzing the triglycerides in the resulting fluid free of glycerol and pyruvate by com-bining the fluid with a mixture comprised of a microbial li-pase, a surfactant selected from -polyoxyethylene (POE) alkyl-amides, POE esters of fatty acids, POE mercaptans, POE alkyl-amines, POE polyol esters, POE acetylenic glycols and POE
phosphate esters, and an alkaline earth metal cation, in the form of a salt; and (c) determining the amount of glycerol produced from the enzymatic hydrolysis.
Detailed Description of the Invention The method disclosed and claimed herein is a pro-cedure for the analysis of triglycerides in biological fluids, especially body fluids such as serum.
In general, the procedure involves pretreating a -serum sample to remove interferences arising from endogenous glycerol and pyruvate, quantifying the glycerol produced by enzymatic hydrolysis by employment of an immobilized enzyme coil.
The novel procedure claimed herein advantageously eliminates the need for parallel blank correction, the latter step required in prior art methods.
In the method 2S claimed herein, the removal of glycerol and pyruvate interferences endogenous to the biological fluid is accomplished pursuant to the following r~action
- 2 -mab/ c b ~ . ~
.
':
5~
scheme:
GK
glycerol + ATP > glycerol phosphate (endogenous) ~ Mn ~ ADP
/ K+
~ PEP
pyruvate +
endogenous pyruvate GPT ~ alanine glutamate a-ketoglutarate In the above given reaction-scheme, ATP is adenosine tri-phosphate, ADP is adenosine diphosphate, PEP is phosphoenol pyruvate.
In the above given reactions, the amount of endogenic glycerol is com-.~
pletely converted to the phosphorilated form by the action of a -speci-fic kinase, e.g., glycerol kinase (GK~. The phosphorilated glycerol is a non-interference in the new method. The ADP produced is recycled back to ATP under the influence of a specific kinase, e.g. pyruv~te kinase (PK), and in the presence of PEP. This latter reaction is needed to eliminate the ADP which will interfere in the accurate quantitation of triglycerides (TG). In the above given reaction-scheme, the endogenic pyruvate plus the pyruvate produced in the previous reac~ion and in the presence of glutamate are transformed to alanine and a-ketoglutarate produced in this reaction as non-interferences in the TG quantitation. In the above given reaction scheme, it is necessary to stop the enzyme activity of the specific
.
':
5~
scheme:
GK
glycerol + ATP > glycerol phosphate (endogenous) ~ Mn ~ ADP
/ K+
~ PEP
pyruvate +
endogenous pyruvate GPT ~ alanine glutamate a-ketoglutarate In the above given reaction-scheme, ATP is adenosine tri-phosphate, ADP is adenosine diphosphate, PEP is phosphoenol pyruvate.
In the above given reactions, the amount of endogenic glycerol is com-.~
pletely converted to the phosphorilated form by the action of a -speci-fic kinase, e.g., glycerol kinase (GK~. The phosphorilated glycerol is a non-interference in the new method. The ADP produced is recycled back to ATP under the influence of a specific kinase, e.g. pyruv~te kinase (PK), and in the presence of PEP. This latter reaction is needed to eliminate the ADP which will interfere in the accurate quantitation of triglycerides (TG). In the above given reaction-scheme, the endogenic pyruvate plus the pyruvate produced in the previous reac~ion and in the presence of glutamate are transformed to alanine and a-ketoglutarate produced in this reaction as non-interferences in the TG quantitation. In the above given reaction scheme, it is necessary to stop the enzyme activity of the specific
- 3 -mab/ ~_ ; ~" ' ' , ,~
,,~, '' ' ' ?S~
kinase (1) acting ~pon endogenous glycerol. This kinase reaction is stopped after all the endogenous glycerol has been transformed to glycerol-phosphate. Failure to stop the specific kinase ~1) activity will result in the conversion of glycerol (produced by lipolysis) to glycerol-l-phosphate with concomitant production of ADP. There are two general approaches to stop the specific kinase (1) activity:
(a) A metal chelating compound is used to sequestrate the specific metal ion used to activate the spe-ific kinase tl)-. Once the metal ion is chelated (e.g., by EDTA) the kinase (1) is completely rendered inactive.
~ b) In the above given reaction scheme all three speci-fic enzymes [kinase (1), kinase (2j and other specific enzymes] can be either free in solution or immobilized form.
In the case where the enzymes are immobilized there is no need to chemically stop the kinase (1) activity. This enzyme is physically attached to the inner wall of a plastic (nylon) coil and thus the enzyme does not enter into the lipolysis reaction.
For the above given reaction scheme the preferred formu-lations are as follows:
Reagent No. 1 This reagent contains glycerol kinase, pyruvate kinase,alanine aminotransferase and ATP. The reagent also contains a buffer system and required enzyme activators. Other reagent formulations were investigated and were found to give equivalent results. Table No. 1 illustrates the enzymes and substrates which could be used as replacements for the specific kinase (2) reaction.
,~ .
m:lh / (~
'' :
~, TABLE NO. 1 Enzyme Substrate Acetyl Kinase Acetyl Phosphate Creatine Kinase Creatine Phosphate 3-Phosphoglycerate Kinase Glycerate 1,3 Diphosphate Reagent No. 2 This reagent is used to stop the activity of the kinase (1) as discussed in approach (2) above.
The reagent responsible for the enzymatic hydrolysis of triglycerides comprises a coactive mixture of microbial lipase (e.g., Candida Cylindracea) a sur~actant (poIyoxyethylene alkanolamides) and CaC12. All three components are essential for complete hydrolysis.
More specifically, the microbial lipase is obtained from a culture broad of Candida Cylindracea. Such Candida lipase should have an activity within the range of 40,000 u/l.-l,OOO,OOO u/l. (reaction mix).
Other Candida lipases were found to give equivalent results when used in combination with the surfactant and CaC12.
As indicated above a surfactant is an essential component of the triglycerides-hydrolysis mixture. While we preferred to use a surfactant belonging to the nonionic group of polyoxyethylene alkanolamides, surfactants pertaining to the following nonionic groups were also found to give equivalent results:
1. Polyoxyethylene (POE) ester of fatty acids 2. PO~ mercaptans 3. POE Alkylamides mab/ ~
`'''` '' , ' `
' `4. POE Alkylamines 5. POE Polyol esters 6. POE Acetylenic glycols 7. POE Phosphate esters The general reaction sequences for the quantitation of glycerol produced by lipolysis is summarized by the following reaction scheme:
Glycerol + ATP Glycerol kinase ~ glycerol-l-phosphate + ADP
ADP + PEP Pyruvate kinase ~ ATP + Pyruvate K
Pyruvate LDH ~ Lactate /~~ .
NADH NAD+
In the above given scheme the glycerol produced by lipoly-sis is dialyzed into a buffered reagent containing PEP, ATP Mg and NADH. The glycerol kinase, pyruvate kinase and lactate dehydro-genase (LDH) reactions take place inside the immobilized enzyme coil (see flow diagram above) where the three latter enzymes are bound to the inner wall of a nylon tubing. Also, inside the immobili~ed enzyme coil NADH is oxidized to NAD. The decrease in absorbance of NADH to NAD is directly proportional to the amount of glycerol pro-duced in the lipolysis step.
mab/
-Brief Description of the Drawing Figure 1 represents a schematic flow sheet of the con-tinuous system or apparatus according to the present invention for the analysis of triglycerides in a biological ~luid.
Detailed Description of the Drawing To illustrate one embodiment of the present invention for the analysis of triglycerides in a biological fluid, reference is made to Fig. l. Fig. l illustrates a continuous system for the analysis of triglycerides.
An aqueous solution 1 which contains GK, PK, GPT, ATP, glutamate, PEP, Mn and K is pumped into conduit 3 where air 5 is added followed by sample 7. The mixed solution is conveyed via line 9 and passed through coil 11 in heating bath 13 which is maintained at a temperature of about between 30C and 50C. Coil 11 may be fabricated of a plastic material such as nylon, polyethylene, poly-vinyl chloride, polystyrene, etc. or aluminum, glass, stainless steel or silicon rubber. Nylon is preferred. As the solution passes through coil 11~ removal of endogenous-glycerol and pyruvate occurs.
The treated solution is conveyed via line 15 where solution 17 containing EDTA and surfactant are pumped via line 19 and combines with the solution which then flows through coil 21 which is at abmient temperature. On passing through coil 21, enzyme GK is inactivated.
After the solution passes through coil 21 it is conveyed via line 23 and is combined with a solution 25 containing microbial lipases and a surfactant at 27. The combined solution is then passed through coil 29 in heating bath 13 where enzymatic hydrolysis occurs. The .~ - 7 -mab/ ~
,,~, '' ' ' ?S~
kinase (1) acting ~pon endogenous glycerol. This kinase reaction is stopped after all the endogenous glycerol has been transformed to glycerol-phosphate. Failure to stop the specific kinase ~1) activity will result in the conversion of glycerol (produced by lipolysis) to glycerol-l-phosphate with concomitant production of ADP. There are two general approaches to stop the specific kinase (1) activity:
(a) A metal chelating compound is used to sequestrate the specific metal ion used to activate the spe-ific kinase tl)-. Once the metal ion is chelated (e.g., by EDTA) the kinase (1) is completely rendered inactive.
~ b) In the above given reaction scheme all three speci-fic enzymes [kinase (1), kinase (2j and other specific enzymes] can be either free in solution or immobilized form.
In the case where the enzymes are immobilized there is no need to chemically stop the kinase (1) activity. This enzyme is physically attached to the inner wall of a plastic (nylon) coil and thus the enzyme does not enter into the lipolysis reaction.
For the above given reaction scheme the preferred formu-lations are as follows:
Reagent No. 1 This reagent contains glycerol kinase, pyruvate kinase,alanine aminotransferase and ATP. The reagent also contains a buffer system and required enzyme activators. Other reagent formulations were investigated and were found to give equivalent results. Table No. 1 illustrates the enzymes and substrates which could be used as replacements for the specific kinase (2) reaction.
,~ .
m:lh / (~
'' :
~, TABLE NO. 1 Enzyme Substrate Acetyl Kinase Acetyl Phosphate Creatine Kinase Creatine Phosphate 3-Phosphoglycerate Kinase Glycerate 1,3 Diphosphate Reagent No. 2 This reagent is used to stop the activity of the kinase (1) as discussed in approach (2) above.
The reagent responsible for the enzymatic hydrolysis of triglycerides comprises a coactive mixture of microbial lipase (e.g., Candida Cylindracea) a sur~actant (poIyoxyethylene alkanolamides) and CaC12. All three components are essential for complete hydrolysis.
More specifically, the microbial lipase is obtained from a culture broad of Candida Cylindracea. Such Candida lipase should have an activity within the range of 40,000 u/l.-l,OOO,OOO u/l. (reaction mix).
Other Candida lipases were found to give equivalent results when used in combination with the surfactant and CaC12.
As indicated above a surfactant is an essential component of the triglycerides-hydrolysis mixture. While we preferred to use a surfactant belonging to the nonionic group of polyoxyethylene alkanolamides, surfactants pertaining to the following nonionic groups were also found to give equivalent results:
1. Polyoxyethylene (POE) ester of fatty acids 2. PO~ mercaptans 3. POE Alkylamides mab/ ~
`'''` '' , ' `
' `4. POE Alkylamines 5. POE Polyol esters 6. POE Acetylenic glycols 7. POE Phosphate esters The general reaction sequences for the quantitation of glycerol produced by lipolysis is summarized by the following reaction scheme:
Glycerol + ATP Glycerol kinase ~ glycerol-l-phosphate + ADP
ADP + PEP Pyruvate kinase ~ ATP + Pyruvate K
Pyruvate LDH ~ Lactate /~~ .
NADH NAD+
In the above given scheme the glycerol produced by lipoly-sis is dialyzed into a buffered reagent containing PEP, ATP Mg and NADH. The glycerol kinase, pyruvate kinase and lactate dehydro-genase (LDH) reactions take place inside the immobilized enzyme coil (see flow diagram above) where the three latter enzymes are bound to the inner wall of a nylon tubing. Also, inside the immobili~ed enzyme coil NADH is oxidized to NAD. The decrease in absorbance of NADH to NAD is directly proportional to the amount of glycerol pro-duced in the lipolysis step.
mab/
-Brief Description of the Drawing Figure 1 represents a schematic flow sheet of the con-tinuous system or apparatus according to the present invention for the analysis of triglycerides in a biological ~luid.
Detailed Description of the Drawing To illustrate one embodiment of the present invention for the analysis of triglycerides in a biological fluid, reference is made to Fig. l. Fig. l illustrates a continuous system for the analysis of triglycerides.
An aqueous solution 1 which contains GK, PK, GPT, ATP, glutamate, PEP, Mn and K is pumped into conduit 3 where air 5 is added followed by sample 7. The mixed solution is conveyed via line 9 and passed through coil 11 in heating bath 13 which is maintained at a temperature of about between 30C and 50C. Coil 11 may be fabricated of a plastic material such as nylon, polyethylene, poly-vinyl chloride, polystyrene, etc. or aluminum, glass, stainless steel or silicon rubber. Nylon is preferred. As the solution passes through coil 11~ removal of endogenous-glycerol and pyruvate occurs.
The treated solution is conveyed via line 15 where solution 17 containing EDTA and surfactant are pumped via line 19 and combines with the solution which then flows through coil 21 which is at abmient temperature. On passing through coil 21, enzyme GK is inactivated.
After the solution passes through coil 21 it is conveyed via line 23 and is combined with a solution 25 containing microbial lipases and a surfactant at 27. The combined solution is then passed through coil 29 in heating bath 13 where enzymatic hydrolysis occurs. The .~ - 7 -mab/ ~
4~1 resulting solution is conveyed via line 31 to dialyzer 33 where the dialyzed solution is conveyed to the bottom portion of the dia-lyzer and combined with solution 35 which contains NADH and Mg in solution via line 37 to which air 38 is also conveyed. The resulting solution is conveyed via line 39 to coil 41 where enzymes glycerol kinase (GK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) are immobilized. After passing through coil 41, the resulting solu-tion is photometrically scanned at 340 nm in flowcell 43.
In another embodiment of this invention, the above system is modified so that coil 11 contains immobilized enzyme GK.
In this embodiment, no EDTA is necessary. As to other embodiments, coil 11 may also contain immobilized enzyme PK and/or immobilized enzyme GPT.
EXAMPLE I
The following procedure illustrates ~ne manner of car-rying out this invention in a continuoùs-manner as in the Technicon AutoAnalyzertm biochemical analyzer as described in U.S. 2,797,149.
The human serum sample (0.020-0.028 ml.) is combined with reagent ~o. 1 which contains glycerol kinase (50-200 units/dl.), ATP (5-16.5 mM.), MnC12 (1-20 mM.), pyruvate kinase (50-1000 units/dl.)~
PEP (5-16 mM.), CaC12 (30-60 mM.), GPT (50-500 units/dl.), glutamate (50-30 mU.) aDd trls buffer (50-100 mM, p3 7.6~.
" .
;~-~ - 8 -mab/-~ ~
, : ~ , . . .
- ' ': - , , `
After mixing for 1/2 to 2 minutes at approximately 30 to 50C, to the resultant solution is added reagent ~o. 2 which contains EDTA (40-80 mM.), 5-POE-lauricamide (7-15 g./l.) and tris buffer (50-100 mM., pH 7.5). After mixing for 10-60 seconds at room temperature, to the resulting solution is added reagent No. 3 which contains lipase (100,000-2,000,000 units/l.), bovine serum al- -bumin (.1-2 g/dl.), CaC12 (40-200 mM), and maleate buffer (50-100 ~M, pH 6).
After mixing for 1-10 minutes at approximately 30-50C, the resulting solution is dialyzed across a dialyzer block equipped with a semi-permeable membrane.
~he dialyzed glycerol is combined with an aqueous buffer solution of pH 7.8 which contains reduced nicotinamide-adeninedinuc-leotide (NADH) (70-140 mg/l.), MgS04 (2-6 ~M) and KCl (20-60 mM) and this solution is passed ~hrough an immobilized enzyme coil to which is physically bound GK, PK and LDH. After passing through the coil, the decrease of the NADH absorbance is monitored at 340 nm.
.. .... ..
; EXAMPLE II
The procedure of Example I is repeated except that only enzyme GK is immobilized. In this procedure no EDTA is used.
EXAMPLE III
The procedure of Example I is repeated except that MgC12 is used in place of MnC12.
~ .~ _ g mab/ ~i - : ~
' :, :: :
$~/L
EXAMPLE IV
The procedure of Example II i9 repeated wherein enzyme PK ~s employed along with GK in immobilized nylon coil form.
EXAMPLE V
The procedure of Example II is repeated except that enzyme GPT is employed along with enzyme GK in immobilized nylon coil form.
.
EXAMPLE VI
The procedure of Example I is repeated except that the following surfactants are used in place of POE-5 lauric acid:
POE-5 cocoamide 15-POE cocoamine ' EX~IPLE VII
: . The procedure of Example I is repeated except that the following enzymes are used in place of GK:
glycerol dehydrogenase glycerol oxidase - In this procedure, the above enzymes together with : enzyme GPT are in immobilized form in the same coil. No enzyme PK is necessary.
mab/' h ., . . . ~ i : .
:
'~ t EXAMPLE VIII
The procedure of Example II is repeated wherein in place of t.he PEP-PK system, the following substrate-enzyme systems as a second component are used:
acetyl phosphate-acetyl kinase creatine phosphate-creatine kinase glycerate 1,3-diphosphate-3-phosphoglycerate kinase It should be understood by those skilled in the art that various modifications may be made in the present invention without departing from the spirit and scope thereof as described in the speci-fication and defined in the appended claims.
mab/
. , ,, . -.
In another embodiment of this invention, the above system is modified so that coil 11 contains immobilized enzyme GK.
In this embodiment, no EDTA is necessary. As to other embodiments, coil 11 may also contain immobilized enzyme PK and/or immobilized enzyme GPT.
EXAMPLE I
The following procedure illustrates ~ne manner of car-rying out this invention in a continuoùs-manner as in the Technicon AutoAnalyzertm biochemical analyzer as described in U.S. 2,797,149.
The human serum sample (0.020-0.028 ml.) is combined with reagent ~o. 1 which contains glycerol kinase (50-200 units/dl.), ATP (5-16.5 mM.), MnC12 (1-20 mM.), pyruvate kinase (50-1000 units/dl.)~
PEP (5-16 mM.), CaC12 (30-60 mM.), GPT (50-500 units/dl.), glutamate (50-30 mU.) aDd trls buffer (50-100 mM, p3 7.6~.
" .
;~-~ - 8 -mab/-~ ~
, : ~ , . . .
- ' ': - , , `
After mixing for 1/2 to 2 minutes at approximately 30 to 50C, to the resultant solution is added reagent ~o. 2 which contains EDTA (40-80 mM.), 5-POE-lauricamide (7-15 g./l.) and tris buffer (50-100 mM., pH 7.5). After mixing for 10-60 seconds at room temperature, to the resulting solution is added reagent No. 3 which contains lipase (100,000-2,000,000 units/l.), bovine serum al- -bumin (.1-2 g/dl.), CaC12 (40-200 mM), and maleate buffer (50-100 ~M, pH 6).
After mixing for 1-10 minutes at approximately 30-50C, the resulting solution is dialyzed across a dialyzer block equipped with a semi-permeable membrane.
~he dialyzed glycerol is combined with an aqueous buffer solution of pH 7.8 which contains reduced nicotinamide-adeninedinuc-leotide (NADH) (70-140 mg/l.), MgS04 (2-6 ~M) and KCl (20-60 mM) and this solution is passed ~hrough an immobilized enzyme coil to which is physically bound GK, PK and LDH. After passing through the coil, the decrease of the NADH absorbance is monitored at 340 nm.
.. .... ..
; EXAMPLE II
The procedure of Example I is repeated except that only enzyme GK is immobilized. In this procedure no EDTA is used.
EXAMPLE III
The procedure of Example I is repeated except that MgC12 is used in place of MnC12.
~ .~ _ g mab/ ~i - : ~
' :, :: :
$~/L
EXAMPLE IV
The procedure of Example II i9 repeated wherein enzyme PK ~s employed along with GK in immobilized nylon coil form.
EXAMPLE V
The procedure of Example II is repeated except that enzyme GPT is employed along with enzyme GK in immobilized nylon coil form.
.
EXAMPLE VI
The procedure of Example I is repeated except that the following surfactants are used in place of POE-5 lauric acid:
POE-5 cocoamide 15-POE cocoamine ' EX~IPLE VII
: . The procedure of Example I is repeated except that the following enzymes are used in place of GK:
glycerol dehydrogenase glycerol oxidase - In this procedure, the above enzymes together with : enzyme GPT are in immobilized form in the same coil. No enzyme PK is necessary.
mab/' h ., . . . ~ i : .
:
'~ t EXAMPLE VIII
The procedure of Example II is repeated wherein in place of t.he PEP-PK system, the following substrate-enzyme systems as a second component are used:
acetyl phosphate-acetyl kinase creatine phosphate-creatine kinase glycerate 1,3-diphosphate-3-phosphoglycerate kinase It should be understood by those skilled in the art that various modifications may be made in the present invention without departing from the spirit and scope thereof as described in the speci-fication and defined in the appended claims.
mab/
. , ,, . -.
Claims (28)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method for the analysis of triglycerides in a biological fluid which comprises:
(a) treating said biological fluid to remove en-dogenous glycerol and pyruvate by combining with said fluid, as a first component, adenosine triphosphate (ATP), enzyme glycerol kinase (GK) and a metal cation activator for said GK, as a second component, enzyme pyruvate kinase (PK) in the presence of substrate phospnoenol pyruvate (PEP) and an enzyme PK activator, and as a third component, gluta-mate and enzyme alanine aminotransferase (GPT);
(b) enzymatically hydrolyzing the triglycerides in the resulting fluid free of glycerol and pyruvate by com-bining said fluid with a mixture comprised of a microbial lipase, a surfactant selected from polyoxyethylene (POE) alkylamides, POE esters of fatty acids, POE mercaptans, POE
alkylamines, POE polyol esters, POE acetylenic glycols and POE phosphate esters, and an alkaline earth metal cation, in the form of a salt; and (c) determining the amount of glycerol produced from said enzymatic hydrolysis.
(a) treating said biological fluid to remove en-dogenous glycerol and pyruvate by combining with said fluid, as a first component, adenosine triphosphate (ATP), enzyme glycerol kinase (GK) and a metal cation activator for said GK, as a second component, enzyme pyruvate kinase (PK) in the presence of substrate phospnoenol pyruvate (PEP) and an enzyme PK activator, and as a third component, gluta-mate and enzyme alanine aminotransferase (GPT);
(b) enzymatically hydrolyzing the triglycerides in the resulting fluid free of glycerol and pyruvate by com-bining said fluid with a mixture comprised of a microbial lipase, a surfactant selected from polyoxyethylene (POE) alkylamides, POE esters of fatty acids, POE mercaptans, POE
alkylamines, POE polyol esters, POE acetylenic glycols and POE phosphate esters, and an alkaline earth metal cation, in the form of a salt; and (c) determining the amount of glycerol produced from said enzymatic hydrolysis.
2. The method of claim 1 wherein said metal cation activator for said enzyme GK is selected from Mn++ and Mg++ provided in the form of a salt.
3. The method of claim 2 wherein said metal cation is Mn++.
4. The method of claim 1 wherein said enzyme PK
activator is potassium ion (K+) provided in the form of a salt.
activator is potassium ion (K+) provided in the form of a salt.
5. The method of claim 1 wherein said alkaline earth metal cation is calcium.
6. The method of claim 1 wherein said enzymes GK, PK and GPT are employed free in solution and further comprising the step subsequent to step (a) of deactivating said GK metal cation activator by adding a metal chelating compound.
7. The method of claim 6 wherein said metal chelating compound is ethylenediamine tetraacetic acid (EDTA).
8. The method of claim 1 wherein said enzyme GK
is employed in immobilized form in which said enzyme is physically attached to the inner wall of a coil through which the fluid passes and contacts, and said other enzymes PK
and GPT are optionally immobilized on the same coil.
is employed in immobilized form in which said enzyme is physically attached to the inner wall of a coil through which the fluid passes and contacts, and said other enzymes PK
and GPT are optionally immobilized on the same coil.
9. The method of claim 8 wherein said coil is composed of a material selected from the group consisting of a plastic selected from nylon, polyethylene, polyvinyl chloride and polystyrene; aluminum, glass, stainless steel and silicon rubber.
10. The method of claim 9 wherein said coil is made of nylon.
11. The method of claim 1 wherein said microbial lipase is a Candida lipase.
12. The method of claim 11 wherein said Candida lipase is Candida Cylindracea having an activity in the range of 40,000 u/l to 1,000,000 u/l.
13. The method of claim 1 wherein said surfactant is POE alkylamide.
14. The method of claim 13 wherein said surfac-tant is selected from POE-5-lauricamide and POE-5-cocoamide.
15. The method of claim 1 wherein said glycerol determination comprises dialyzing the reaction product from said enzymatic hydrolysis into a buffered reagent comprising PEP, ATP, magnesium ion in the form of a salt, and NADH and passing said dialyzed solution through an immobilized enzyme coil to which is physically bound enzymes glycerol kinase (GK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) and photometrically measuring at 340 nm the decrease in absorbance of NADH and NAD.
16. The method of claim 15 wherein said coil is made of nylon.
17. The method of claim 8 wherein said glycerol determination comprises dialyzing the reaction product from said enzymatic hydrolysis into a buffered reagent comprising PEP, ATP, magnesium ion in the form of a salt, and NADH
and passing said dialyzed solution through an immobilized enzyme coil to which is physically bound enzymes glycerol kinase (GK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) and photometrically measuring at 340 nm the decrease in absorbance of NADH to NAD.
and passing said dialyzed solution through an immobilized enzyme coil to which is physically bound enzymes glycerol kinase (GK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) and photometrically measuring at 340 nm the decrease in absorbance of NADH to NAD.
18. The method of claim 17 wherein said coil and said immobilized enzyme coil are made of nylon.
19. In a method for the analysis of triglycer-ides in a biological fluid by pretreating said biological fluid to remove interferences arising from endogenous gly-cerol and pyruvate, enzymatically hydrolyzing the trigly-cerides in the resulting fluid and determining the amount of glycerol produced by said enzymatic hydrolysis, the im-provement which comprises enzymatically hydrolyzing the tri-glycerides in the resulting fluid free of glycerol and py-ruvate by combining said fluid with a mixture comprised of a microbial lipase, a surfactant selected from polyoxyethylene (POE) alkylamides, POE esters of fatty acids, POE mercap-tans, POE alkylamines, POE polyol esters, POE acetylenic glycols and POE esters, and an alkaline earth metal cation, in the form of a salt.
20. The method of claim 19 wherein said microbial lipase is a Candida lipase.
21. The method of claim 20 wherein said Candida lipase is Candida Cylindracea having an activity in the range of 40,000 u/l to l,000,000 u/l.
22. The method of claim 19 wherein said surfac-tant is a POE alkylamide.
23. The method of claim 22 wherein said surfac-tant is selected from POE-5 lauricamide and POE-5-cocoamide.
24. The method of claim 19 wherein said alkaline earth metal cation is calcium.
25. The method of claim 19 further comprising the improvement of determining the amount of glycerol pro-duced by said enzymatic hydrolysis by measuring the decrease in absorbance resulting from the oxidative conversion of NADH to NAD.
26. The method of claim 25 wherein said oxida-tive conversion is effected by dialyzing the reaction pro-duct from the enzymatic hydrolysis into a buffered reagent comprising PEP, ATP, magnesium ion in the form of a salt, and NADH and passing said dialyzed solution through an im-mobilized enzyme coil to which is physically bound enzymes glycerol kinase (GK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) and photometrically measuring at 340 nm the decrease in absorbance.
27. The method of claim 19 wherein said pretreat-ment employs a substrate-enzyme system selected from acetyl phosphateacetyl kinase, creatine phosphate-creatine kinase and glycerate 1,3-diphosphate-3-phosphoglycerate kinase.
28. The method of claim 19 wherein said pretreat-ment employs an enzyme selected from glycerol dehydrogenase and glycerol oxidase.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US06/171,112 US4338395A (en) | 1980-07-21 | 1980-07-21 | Method for the analysis of triglycerides |
| US171,112 | 1980-07-21 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1144051A true CA1144051A (en) | 1983-04-05 |
Family
ID=22622577
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000374028A Expired CA1144051A (en) | 1980-07-21 | 1981-03-27 | Method for the analysis of triglycerides |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US4338395A (en) |
| EP (1) | EP0044615B1 (en) |
| JP (1) | JPS57155998A (en) |
| AU (1) | AU539052B2 (en) |
| CA (1) | CA1144051A (en) |
| DE (1) | DE3163858D1 (en) |
Families Citing this family (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4547461A (en) * | 1983-01-18 | 1985-10-15 | Eastman Kodak Company | Composition, analytical element and method for the quantification of creatine kinase |
| JPS59183698A (en) * | 1983-04-02 | 1984-10-18 | Wako Pure Chem Ind Ltd | Quantitative determination of substrate |
| DE3340709A1 (en) * | 1983-11-10 | 1985-05-23 | Boehringer Mannheim Gmbh, 6800 Mannheim | COMPETITIVE INHIBITOR FOR GK |
| US4626511A (en) * | 1985-05-13 | 1986-12-02 | Wayne State University | Composition for reducing turbidity in samples of biological fluids |
| US5310679A (en) * | 1985-05-13 | 1994-05-10 | Artiss Joseph D | Composition for reducing turbidity in samples of biological fluids |
| US6312902B1 (en) | 1998-03-13 | 2001-11-06 | Promega Corporation | Nucleic acid detection |
| US6270974B1 (en) | 1998-03-13 | 2001-08-07 | Promega Corporation | Exogenous nucleic acid detection |
| US6235480B1 (en) | 1998-03-13 | 2001-05-22 | Promega Corporation | Detection of nucleic acid hybrids |
| US6268146B1 (en) | 1998-03-13 | 2001-07-31 | Promega Corporation | Analytical methods and materials for nucleic acid detection |
| US6270973B1 (en) | 1998-03-13 | 2001-08-07 | Promega Corporation | Multiplex method for nucleic acid detection |
| US6703211B1 (en) | 1998-03-13 | 2004-03-09 | Promega Corporation | Cellular detection by providing high energy phosphate donor other than ADP to produce ATP |
| US6391551B1 (en) | 1998-03-13 | 2002-05-21 | Promega Corporation | Detection of nucleic acid hybrids |
| US6335162B1 (en) | 1998-03-13 | 2002-01-01 | Promega Corporation | Nucleic acid detection |
| US7090975B2 (en) | 1998-03-13 | 2006-08-15 | Promega Corporation | Pyrophosphorolysis and incorporation of nucleotide method for nucleic acid detection |
| JP6047778B2 (en) * | 2012-01-12 | 2016-12-21 | 株式会社シノテスト | Method and reagent for measuring substance to be measured, and method for avoiding influence derived from bilirubin |
Family Cites Families (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4061141A (en) * | 1969-03-21 | 1977-12-06 | Viktor Holger Hyden | Apparatus and method for selectively separating amino acids and defined proteins in blood |
| CA921850A (en) * | 1969-08-15 | 1973-02-27 | The Procter & Gamble Company | Detergent composition containing an oxidizing bleach and proteolytic enzyme derived from thermophilic streptomyces rectus variety proteolyticus |
| DE2101796A1 (en) * | 1970-01-21 | 1971-08-05 | Baxter Laboratories Inc | Method for the determination of glycene in blood serum |
| US3703591A (en) * | 1970-12-16 | 1972-11-21 | Calbiochem | Triglyceride hydrolysis and assay |
| AT339505B (en) * | 1974-03-14 | 1977-10-25 | Boehringer Mannheim Gmbh | ENZYMATIC ANALYSIS PROCEDURE |
| US3926736A (en) * | 1974-08-30 | 1975-12-16 | Calbiochem | Enzymatic ethanol assay |
| JPS5211987A (en) * | 1975-07-15 | 1977-01-29 | American Hospital Supply Corp | Rapid hydrolysis method of triglyceride by enzyme and composite reagents for carrying out the method |
| GB1515195A (en) * | 1975-08-05 | 1978-06-21 | Hycel Inc | Triglyceride assay |
| AT354406B (en) * | 1975-08-12 | 1979-01-10 | Boehringer Mannheim Gmbh | METHOD AND REAGENT FOR DETERMINATION OF TRIGLYCERIDES |
| US4056442A (en) * | 1976-06-01 | 1977-11-01 | The Dow Chemical Company | Lipase composition for glycerol ester determination |
| US4080263A (en) * | 1976-08-11 | 1978-03-21 | Boehringer Mannheim Gmbh | Process and reagent for the rapid quantitative determination of lactate or alanine |
| US4179334A (en) * | 1976-08-19 | 1979-12-18 | Eastman Kodak Company | Hydrolysis of protein-bound triglycerides |
| CA1114269A (en) * | 1976-08-19 | 1981-12-15 | Charles D. Warburton | Integral element for the detection of glycerol or triglycerides |
| CA1100023A (en) * | 1976-08-19 | 1981-04-28 | Charles T. Goodhue | Process and composition for the quantification of glycerol and triglycerides |
| JPS5389492A (en) * | 1977-01-13 | 1978-08-07 | Uni Arabama | Method and reagent for determination of carxylic esterhydrolase |
| US4242446A (en) * | 1978-07-26 | 1980-12-30 | Coulter Electronics, Inc. | Method for determining a substance in a biological fluid and reagent combination for use in the method |
| DE2834705B1 (en) * | 1978-08-08 | 1980-01-24 | Boehringer Mannheim Gmbh | Method and reagent for carrying out enzymatic determinations |
| US4259440A (en) * | 1979-05-21 | 1981-03-31 | Miles Laboratories, Inc. | Hydrolysis and assay of triglycerides |
-
1980
- 1980-07-21 US US06/171,112 patent/US4338395A/en not_active Expired - Lifetime
-
1981
- 1981-03-27 CA CA000374028A patent/CA1144051A/en not_active Expired
- 1981-04-13 AU AU69457/81A patent/AU539052B2/en not_active Ceased
- 1981-04-27 JP JP56062672A patent/JPS57155998A/en active Pending
- 1981-06-15 DE DE8181302661T patent/DE3163858D1/en not_active Expired
- 1981-06-15 EP EP81302661A patent/EP0044615B1/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| AU6945781A (en) | 1982-01-28 |
| DE3163858D1 (en) | 1984-07-05 |
| EP0044615A3 (en) | 1982-02-03 |
| JPS57155998A (en) | 1982-09-27 |
| EP0044615A2 (en) | 1982-01-27 |
| US4338395A (en) | 1982-07-06 |
| EP0044615B1 (en) | 1984-05-30 |
| AU539052B2 (en) | 1984-09-06 |
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