CA1081589A - Composition and method for the quantitative determination of phospholipids - Google Patents
Composition and method for the quantitative determination of phospholipidsInfo
- Publication number
- CA1081589A CA1081589A CA284,986A CA284986A CA1081589A CA 1081589 A CA1081589 A CA 1081589A CA 284986 A CA284986 A CA 284986A CA 1081589 A CA1081589 A CA 1081589A
- Authority
- CA
- Canada
- Prior art keywords
- choline
- reaction
- phospholipase
- lecithin
- composition
- 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
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
-
- 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
- C12Q2326/00—Chromogens for determinations of oxidoreductase enzymes
- C12Q2326/90—Developer
- C12Q2326/92—Nitro blue tetrazolium chloride, i.e. NBT
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/906—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
- G01N2405/04—Phospholipids, i.e. phosphoglycerides
Abstract
ABSTRACT OF THE DISCLOSURE
Phospholipid content in a sample is quantitatively determined by enzymatically converting the phospholipids into choline and then determining the amount of the choline.
Phospholipid content in a sample is quantitatively determined by enzymatically converting the phospholipids into choline and then determining the amount of the choline.
Description
~(~8~S~
I sACKGROUND OF THE INVENTION
I sACKGROUND OF THE INVENTION
2 The present invention rela-tes generally to an improved
3 composition and method for the quantitative determination of
4 phospholipids in a sample. More specifically, the invention is concerned with a diagnostic agent useful in the quantitative 6 determination of phospholipids in blood serum.
7 Phospholipids are found in serum, egg, meat, vegetables 8 and the like. It is important to determine the phospholipid g content in such substances for diagnosing disease, dietetics, lo etc. In this regard, medical science has recognized the useful-ll ness of determining the content of phospholipids in blood serum 12 as an aid in diagnosing disease such as hypercholesteremia, l3 liver disease and the like.
~4 A number of tests, techniques and methods have been IS proposed and are being used to measure or estimate the amount of ~6 phospholipids in the blood. Among the known methods, more 7 widely used conventional methods are colorimetric procedures 8 utilizing molybdate. Some of these procedures depend upon the conversion of phospholipid to inorganic phosphorous by incinerat-ing a sample; reacting the phosphorous with molybdate to form a 21 - phosphomolybdic acid; reducing the phosphomolybdic acid with a 22 reducing agent to form molybdenum blue; and measuring the u absorption color of the molybdenum blue. [J. Lah. Clin. Med.
24 Vol. 35, 155 (1950); J. Biol. Chem. Vol. 234, 466 (1959); Shinryo Vol. 16, 677 ~1963); Rinsho Byorl Vol. 10, 194 (1962), ibid. Vol.
26 15, 853 (1967)].
27 More recently, a method has been proposed which contem-28 '~plates the enzymatic conversion of the phospholipids present in 29 the blood specimen to phosphoric acid and the measurement of the ~color of the reaction solution obtained by reacting the phosphoric 31 acid with molybdate and then reducing the resultant mixture - 2 - ~
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1 (Yatron Document RM 137-K, Yatron Co., Ltd.).
2 The known methods ~or the ~uantitative determination of 3 phospholipids suffer from one or more of the disadvantages of 4 requiring highly skilled laboratory techniques, requiring a great deal of time to perform, not giving consistently precise 6 results and requiring blank measurements owing to interference 7 of the phosphorous contained in the sample and vessels.
9 The present invention provides a simple, practical and 0 economical diagnostic colorimetric method for detecting the presence of phospholipids in serum and making a precise auanti-2 tative determination thereof. The method offers the advantages 3 of extremely rapid color development and maintenance of 4 color value for a long period of time. Further, this method may be conducted by personnel unskilled in laboratory techniques.
6 FurthermOre~lthe method of the present invention 7 provides an all-enzymatic determination of the phospholipids and, 18 therefore, a decided improvement of routine medical diagnosis 19 ~ plus an adaptability of the method for automatic analysis apparatus.
21 The present invention is based upon the following 22 combination of enzymatic reactions:
23 ~ Phospholipids such as lecithin, sphingomyelin, 74 ~lisolecithin, cephalin, phosphatidylserine, etc. are present in blood serum. Among these phospholipids in serum, the total 26 ;amount of lecithin, sphingomyelin and lysolecithin corresponds 27 to about 95% of the amount of total phospholipids in various 28 samples of serum. These three phospholipids are converted to 29 choline by an enzymatic reaction.
The amount of the reduced form of acceptor produced in 31 ~ the enzymatic conversion of choline to glycine betaine aldehyde . . , ,1 , ~08~
1 in the presence of choline dehydrogenase (hereinafter referred 2 to as CLDH) and hydrogen acceptor is directly proportional 3 to the amount of phospholipid in the blood serum.
4 Further, the amount of the reduced form of nicotin-amide adenine dinucleotide (hereinafter referred to as NADH) 6 produced in the enzymatic conversion of glycine betaine 7 aldehyde to betaine aldehyde in the presence of betaine 8 aldehyde dehydrogenase (hereinafter referred to as BADH) and g nicotinamide adenine dinucleotide (hereinafter referred to as NAD) or consumed NAD in the enzymatic reaction is also 11 directly proportional to the amount of phospholipid in the l2 blood seru~.
13 The reduced form of acceptor thus produced can then be 1~ detected by measuring the absorption of the color of the reaction solution at a wave length of from 400 to 700 nm.
6 The NADH thus produced may also be detected by measuring 7 the absorption of the reaction solution at a wave length of 340 nm.
18 The amount of consumed NAD can be calculated by 19 subtracting the remaining NAD from the added NAD. The remaining NAD in the reaction solution can be detected by 21 measuring the absorption of the reaction solution at a wave 22 length of from 260 to 280 nm.
23 These reactions are known individually. That is, 2~ lecithin catalytically reacts with phospholipase D (hereinafter referred to as PLD) to form choline [Reaction I, J. Biol. Chem.
26 Vol. 231, 703 (1958), ibid, Vol. 172, 191 (1948~]. Sphingomyelin 27 and lecithin catalytically react with phospholipase C (hereinafter 28 referred to as PLC) to form phosphoryl choline [Reaction II, 29 Biochem. J. Vol. 35, 884 (l946); Biochem. Biophys. Acta. Vol.
59, 103 (1962)], the phosphoryl choline catalytically reacts 31 with phosphatase to form choline [Reaction III, J. Biol. Chem.
,., : , . :.
- :, ~ . . .,, ~ , . . :
~8~S~9 1 Vol. 244, 308 (1969)]. Lysolecithin catalytically reacts 2 with phospholipase B (hereinafter referred to as PLB) to 3 form glycerophosphorylcholine [Reaction IV, "Biochemist's 4 Handbook" E. & F. N. Spou Ltd. 282 (1961); Nature Vol. 169, 29 (1952); siochem. J. Vol. 71, 615 (1959)], the glycero-6 phosphorylcholine catalytically reacts with glycerophosphoryl-7 choline dièsterase (hereinafter referred to as GPD) to form 8 choline [Reaction V, J. Biol. Chem. Vol. 206, 647 (1954);
g Biochem. J. Vol. 62, 689 (1956)].
IQ The choline formed in the reaction described above, catalytically reacts with CLDH in the presence of a hydrogen 12 acceptor to form glycine betaine aldehyde and reduced form of 13 acceptor [Reaction VI, Agr. Biol. Chem. Vol. 39, 1513 (1975)].
l~ The glycine betaine aldehyde catalytically reacts with BADH
in the presence of NAD to form glycine betaine and NADH
16 [Reaction VII, J. Biol. Chem. Vol. 209, 511 (1954)].
17 The enzymatic reactions described above are schematically 18 represented as follows:
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- 51 =C
o O o~ . ~~ ~ ~ o p~ ~
O F~
m ~ Z a~
o=~-~o ~ ~3 ~
~-~ -U I ~
~ rl X
~ ~1 0 o~ ~~ Z
~ ~ I I~
a+ ~ a~ O ~ o ~ u :r=tC =O-- ~ ~ p~
O H
~ O
rl +
a P~
~ I
~ ~ a O ~ ~ ,, __ _ , _ ,, _ ~" ,1 o~ 0=~--O O
O O
O
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1~ 9 Cholin~
~ CLDH
[Reaction VI] ~ - Hydrogen acceptor +\ ~ (electron transmitter) OHC-CH N(CH ) OH ~ Reduced form of 2 3 3 acceptor Glycine betaine aldehyde Detection ~ BADH
[Reaction VII~
~ NAD
+ NADH
Glycine betaine Detection where Rl, R2 and R3 represent hydrocarbon groups.
~8~5~9 ' :`
1 : The quantitative determination of lecithin content 2 in a sample is carried out according to the process consisting 3 of Reactions I and VI (hereinafter referred to as Process D) 4 or Reactions I, VI and VII (hereinafter referred to as ProcesS
D').
6 The quantitati~e determination of total content of 7 sphin~omyelin and lecithin in a sample is carried out aecording 8 to the process consisting of Reactions II, III and VI (herein~
9 a~ter referred to as Process C) or Reactions II, III, VI and VII (hereinafter referred to as Process C').
11 The quantitative determination of lysolecithin lt content in a sample is carried out according to the process 13 consisting of Reaetions IV, V and VI (hereinafter referred to 14 as Process B) or Reactions IV, V, VI and VII (hereinafter referred to as Process B').
16 The quantitative determination of total content of 17 ;leeithin, shingomyelin and lysolecithin is earried out aecording 18 to Proeesses C and B, Processes C and B', Processes C' and B, 19 or Processes C' and B'.
DETAILED DESCRIPTION OF THE INVENTION
21 Broadly, the present invention comprises the combination 22 of a system for convertin~ phospholipids in a sample to choline 23 ~ and a system for detecting the so-produced choline.
24 ; The system for converting the phospholipids to eholine ~ comprises one or more enzymes selected from the group eonsisting 26 of PLD, PLC and phosphatase, and PLB and GPD.
27 The eholine detecting system comprises CLDH and a 28 hydrogen acceptor or further comprises BADH and NAD and if 29 necessary, comprises an electron transmitter.
i lS89 ;`
Additional subject matter of the invention is a 2 novel reagent composition for converting of phospholipid 3 in a sample to choline which comprises, as a first component, ,~l 4 one or more enzymes selected from the group consisting of PLD,~
s PLC and phosphatase, and PLB and GPD; and a second component , 6 comprising a choline detecting reagent.
7 According to the present invention, the quantitative 8 determination of phospholipids may be performed by conducti~g g the individual reactions stepwise. That is, the enzyme reac-tions are divided into any desired group and after completlon 11 of the reaction of the preceding s~p, the successive enzymes are added thereto, and the reactio~-~f the next step is allowed 13 to proceed. After completion of Reaction VI, the absorption 14 of the visible part of the colored_reaction solution by 15 ` formation of the reduced form of acceptor is measured or after 16 completion of Reaction VII,the absorption of the ultraviolet part of the reaction solution is measured for detecting NADH
formed or the remaining NAD. The absorption values obtained 19 by one or more of the above-s~eps are compared with a calibration curve obtained by carring out the above-steps on the standard 21 ~ compound, whereby the content of the phospholipids in a sample æ is determined.
23 According to the present invention, the presently 2~ preferred method for the quantitative determination of phos-pholipids in a sample is carried out by subjecting 26 a sample to reaction with a reagent consisting of enzymes~for 27 converting phospholipids to choline, CLDH and a hydrogen 2~ acceptor; and, if necessary, BADH, NAD, an electron tran~mitter, 29 surfactant, etc. in a buffer solution.
Enzymes used in the present invention are known and 31 those obtained from various sources may be used.
~08~S~ `
1 Any PLD may be used so long as it acts only on 2 lecithin among phospholipids. Suitable PLD are obtained 3 from cabbage or the like by extraction and purification 4 and are commercially available from such sources as Sigma Co., U.S.A. and Boehringer Mannheim Co., West Germany.
6 Any PLC may be used so long as it acts only on 7 sphingomyelin and lecithin among phospholipids. Suitable 8 PLC are obtained from cell bodies of microorganism belonging g to Escherichia coli by extraction and purification and are o commercially available from such sources as Worthington Co., Il U.S.A. as Catalog No. 5130 and Baehringer Mannheim Co., 12 West Germany, as Catalog No. 15429.
l3 Phosphatase obtained from cell bodies of micro-l~ organisms belonging to the species Clostridium perfringens or Clostridium welchii or the like by extraction and purification 6 may be used and are commercially available from such sources as Worthington Co. as Catalog No. 5640 and Sigma Co., U.S.A. as l8 Catalog No. P 7633, etc.
Ig Any PLB may be used so long as it acts only on lysolecithin among phospholipids. Suitable PLB may be obtained 21 rom cell bodies of microorganisms belonging to the species 22 Penicillium notatum by extraction [Biochem. J. Vol. 70, 559 23 (1958)]. Those extracted from mucous membranes of livers and 2~ intestines of rats, oxen, and pigs, etc. are also appropriate.
Suitable GPD may be obtained from cell bodies of 26 microorganisms belonging to the species Serratia plymuthicum 27 by extraction [J. Biol. Chem. Vol. 206, 647 (1954)]. Those 2~ extracted from livers of rats, oxen, and pigs [Biochem. J., 29 vol. 62, 689 (1956)], etc. are also appropriate.
Suitable CLDH may be obtained from cell bodles of 3I microorganisms belonging to the species Pseudomonas ~
. -- 10 --: .: : ,.;:,, ,, . . : ,. .
:~8~LS8g - -`
I [Agr. Biol. Chem. Vol. 39, 1513 - 151~ (1975)]. Those 2 extracted from mitochondria of rats [J. Biol. Chem. Vol. 234, 3 1605 (1959)], etc. are also appropriate.
4 Suitable sADH may be extracted from cell bodies of microorganisms belonging to the species Pseudomonas aeruginosa 6 [Agr. Biol. Chem. Vol. 39, 1513 - 1514 (1975)]. Those purified 7 from a supernatant of homogenate of rat livers [J. Biol. Chem.
8 Vol. 209, 511 (1954)], etc. are also appropriate.
9 An amount of enzvmes to be used to that of phospholipid 0 is determined in such a range that the detecting operation can be Il exactly carried out. That is, the following amount of enzymes l2 may be used to one ~g of phospholipid;
l3 0.005 - 1 IU for PLD, 0.005 - 1 IU for PLC, 0.005 -l~ 1 IU for PLB, 0.005 - 50 IU for phosphatase, 0.005 - 1 IU for s GPD, 0.1 - 50 IU for CLDH, 0.1 - 100 IU for BADH.
The foregoing enzymes are used in an appropriate buffer solution. Preferable concentrations of the enzymes in the buffer solution are 0.01 - 1 IU/mQ for PLD; 0.01 -19 1 IU/mQ for PLC; 0.01 - 1 IU/mQ for PLB; 0.1 - 50 IU/mQ for phosphatase; 0.01 - 1 IU/mQ for GPD; 0.1 - 50 IU/mQ for CLDH;
2l and 0.1 - 100 IU/mQ for BADH. IU is an international unit of 22 enzyme factor; and the factor capable of decomposing 1 ~ mole 23 of substrate in one minute is defined as 1 IU, i.e. 1 IU =
2~ mol/min.
2s It is desirable to provide the buffer solution at a 26 pH in the range of about from 5.0 to 10.0, preferably from 27 6.5 to 7.5. For this purpose, buffers which may be used 28 include phosphate, succinate, citrate, borate, acetate, 29 glycylglycinate, tris-malonate as well as other buffers which are generally effective within the pH range of from 5.0 to 10Ø
3I The concentration of the buffer is not critical.
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1 However, it is preferred to use a relatively dilute buffer 2 solution and for this purpose a 0.l - 0.2 mol/Q solution is 3 recommended.
g Examples of the hydrogen acceptor are 3,3'-(3,3'-dimethoxy-4,~'-biphenylene)-bis~[2-(p-nitrophenyl)-5-phenyl-6 2H-tetrazolium chloride] (hereinafter referred to as nitro-7 Ts), 3,3'-(3,3'-dimethoxy-4,4'-biphenylene-bis [2,5-bis 8 (p-nitrophenyl)-2H-tetrazolium chloride] (hereinafter referred 9 to as TNTB), 3-(p-indophenyl-2-(p-nitrophenyl)-5-phenyl-2H-0 tetrazolium chloride (hereinafter referred to as INT), 2,6-11 dichlorophenyl-indophenol (hereinafter referred to as DCPIP) 12 and NAD.
13 When DCPIP is used, it is used with an electron 1~ transmitter such as phenazine methosulfate and the like which s accepts an electron from choline and donates it to DCPIP.
Since NAD is liable to be converted to NADH by 7 enzymes, the coenzyme of which is NAD, there is sometimes 8 cause for an error when there are such enzymes in the reaction 19 system. It is, therefore, preferable to avoid such combinations.
Preferable concentration of the hydrogen acceptor in 21 the buffer solution is 0.l - 500 mmol/Q.
22 Preferable concentration of the electron transmitter 23 is 0.l - 50 mmol/Q.
24 Typical samples in which the amount of phospholipids would be determined, include living components such as serum, 26 liver, etc. and various foods such as vegetable oil, fish oil, 27 vegetables, etc. When the sample is solid it should be ground 28 and then subjected to extraction with water or a small amount 29 of organic solvent such as alcohol, chloroform or the like.
On the other hand, when the sample is an oil, a surfactant may 31 be added to the reagent solution to improve the affinity with - , : ,: , ..
, . . , , ,-:. ~
~lS~
1 the reagent solution. Suitable surfactants include such higher 2 alcohols as polyethyleneglycol, etc. Preferably, the concen-3 tration of the surfactant in the reagent solution is 0.01 -4 5 g/Q.
The enzymatic reactions are carried out at 20 to 45C, 6 preferably 30 to 40C, at pH 5 to lO~for 4 to 60 minutes.
7 After completion of the enzymatic reactions, the 8 absorption of the reaction solution is measured by utilizing g a spectrophotometer, or the like.
o When the amount of the formation of reduced form of Il acceptor is determined, the absorption of visible part of the 12 reaction solution is measured at a wave length in a range of 13 400 to 700 nm.
14 When NAD is used as a hydrogen acceptor in Reaction Is VI, the quantitative determination of reduced form of acceptor, l6 i.e. NAD~ formed in Reaction VI is carried out by measuring l7 the absorption of the reaction solution at a wave length of 18 340 nm.
19 When the amount of NADH is determined, the absorption of ultraviolet part of the reaction solution is measured at 340 21 nm and when the amount of remaining NAD is determined, the 22 absorption is measured at 260 - 280 nm.
23 The colors developed in the reaction by using nitro-TB, 2~ TNTB, INT and DCPIP as the hydrogen acceptor are blue, red-brown, red (or violet) and red-blue, respectively.
26 When NAD is used as a hydrogen acceptor in Reaction VI
27 and the quantitative determination of phospholipid is carried 28 out by measuring the amount of NADH produced after completion 29 of Reaction VII, the quantitative value of NADH determined represents total sum of NADH formed in Reactions VI and VII, 31 and therefore the amount of NADH formed in Reaction VI is taken ~ - 13 -~8~L5~3~
I into consideration. Reaction VII usually proceeds to 100%
2 and thus the amount of NADH formed in Reaction VII can be 3 presumed to be 1/2 of said sum value.
4 The composition of the present invention may be used in various forms. For example, the ingredients may be 6 mixed in liquid form or in powder form. The liquid formulation 7 may be readily reconstituted for later use simply by the 8 addition of water or buffer solution. The powders, if desired, 9 may be tableted for convenience in use.
o Practice of certain specific embodiments of the invention is illustrated by the following representative l2 examples.
I3 Example 1 l~ Quantitative determination of content of lecithin, sphingomyelin and lysolecithin in normal human blood serum.
In this example, 20 ~Q of blood serum is added to 3 mQ of 0.15 mol/Q phosphate buffer solution (pH 7.2) containing 0.04 IU of PLC, 5 IU of phosphatase, 0.014 IU of PLB, 0.014 IU
l9 of GPD, 5 IU of CLDH and 0.6 mmol of TNTB.
The enzymatic reaction is allowed to proceed for 2l 15 min. at 37C. After completion of the reaction, the absorp-22 tion of the reaction solution at 550 millimicrons is measured 23 with a spectrophotometer to determine the content of reduced 2~ form of acceptor formed in the solution.
2s The content of the phospholipids is obtained by 26 comparing the absorption value with a standard curve obtained 27 by applying the method described above on a standard sample o~
28 lecithin (produced by Sigma Co., Ltd., Catalogue No. L-2004).
29 As a control, the same serum is subjected to extraction using a chloroform-methanol solution according to the Folch 31 extraction method. The resulting extract is oxidized by using ~o~3~s89 perchloric acid to form an inorganic phosphorous and a 2 molybdate; and, thereafter, hydrazine sulfate is added to the 3 resulting solution to form a colored solution. Absorption of 4 the color developed at a wave length of 700 nm is measured with a spectrophotometer [Rinsho syori, Vol. 10, 194 (1962)].
6 The samples are measured 10 times for each method 7 described above. The results are given in Table 1.
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Table 1 Content of phospholipids (mg/dl) 3 ~ Test run No.
Present method Prior method 1 185.3 189.3 6 2 187.2 195.2 7 3 184.4 199.4 ~ 4 186.7 190O8 9 5 185.1 187.2 lo 6 181.9 193.6 Il 7 188.2 195.1 12 8 187.5 188.8 13 9 186.3 192.3 1~ 10 184.6 190.5 lS Averaqe 185.7 192.2 16 Standard deviation 1.85 3.68 17 Coefficient of variation 0.99 % 1.91 ~
19 As is apparent from Table 1, the standard deviation and coefficient of variation in the present method are less 21 than those of the known method.
22 To make a comparison with the prior known method 23 ` as to the content of total phospholipids, multiplication by a 24 factor of 1.0416 gives an average of the content of total ~phospholipids, 193.4 mg/dl~ almost the same numercial value as 26 that of the prior method.
27 ;Example 2 28 " Quantitative determination of lecithin, sphingomyelin 29 ., and lysolecithin in patient serum (II b type).
i In this example, 20 ~1 of patient serum is added to 31 ; 3 ml of 0.15 mol/l tris-buffer solution (pH 7.2) containing , .
'I - 16 ~
1 D181S~
0.04 IU of PLC~ 5 IU of phosphatase, o. 014 IU Of~ PLB~ O.014 IU
2 of GPDr 5 IU of CLDHr 5 IU of BADH and 0.09 mmol of NAD.
3 . The enzymatic reaction is allowed to proceed for 4 15 min. at 37C. After completion of the reaction, the absorption of the reaction solution at 340 nm millimicrons is measured at 6 various time intervals using a spectrophotometer to determine the 7 content of NADH formed in the solution. The values are given in 8 Table 2.
lo Table 2 Il 12 Content of total phospholipid 13 (mg/dl) Is30 seconds 305.2 161 minute 312.1 175 minutes 314.3 lô15 minutes 313.0 As is apparent from the values set forth in Table 2, 21 the quantitative determination of total phospholipid according 22 to the present method can be carried out in a short period of time.
23 ; Example 3 24 ~ Quantitative determination of content of total ` phospholipid in serum.
26 In this example, the procedures described in I ~"
27 Example 1 are repeated except 0.6 mmol of INT is used instead 28 of TNTB, and normal human serum and patient serums (IIa, IIb and 29 IV types) are used.
~ The results are given in Table 3.
31 The content of total phospholipid, according to the -- 17 ~
., I
;, . .:
" , I
- . :., , :... ~ : ~ , . . . .
3~8~5~9 present method, is obtained by multiplication b~ a value of 2 1.0416.
4 Table 3 6 The content of total phospholipid(mg/dl) Serum 7 Present method Prior method 8 Normal human194.2 + 14.0 201.5 + 32.3 9 II a type275.3 + 15.4 278.7 + 40.2 lo II b type341.0 + 19.5 348.2 + 51.2 Il IV type 273.5 + 12.3 263.2 + 52.3 ~2 l3 Example 4 14 Quantitative determination of lysolecithin content in patient blood serum.
lo In this example, 100 ~1 of blood serum is added to 17 3 ml of 0.15 mol/l phosphate buffer solution (pH 7.2) containing 18 0.014 IU of PLB, 0.014 IU of GPD, 5 IU of CLDH and 0.6 mmol l9 of TNTB.
The enzymatic reaction is allowed to proceed for 21 15 minutes at 37C. Ater completion of the reaction, the 22 absorption of the reaction solution is measured with a spectro-23 photometer at a wave length of 550 nm. The red-brown color of 24 the reaction solution is compared with a calibration curve obtained by using a standard compound.
26 As a control (known method), the same serum is 27 developed by thin layer chromatography using silica gel. The 28 resultant sample is incinerated and a color is developed by 29 using a molybdic acid reagent. The color is measured with a densitometer. The results are given in Tab]e 4.
i ~!
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~0~S~9 - Table 4 Content of lysolecithin (mg/dl) 3 Test run No.
Present methodPrior Method 1 35.36 35.45 6 2 35.82 36.85 7 3 35.14 34.67 8 4 35.38 35.59 9 5 34.98 36.98 o 6 35.67 35.56 7 35.28 36.02 2 8 35.43 35.95 13 935 33 34.38 14 10 35.87 35.21 ls Average 35.42 35.66 16 Standard deviation 0.28 0.83 17 Coefficient of variation0.79% 2.32%
19 Example 5 ~ In this example, 100 ~1 of normal human blood serum is 21 added to 3 ml of 0.15 mol/l glycylglycine buffer solution (pH 7.2) 22 containing 0.04 IU of PLB, 0.04 IU of GPD, 5 IU of CLDH, 5 IU
~3 of BADH and 0.09 mmol of NAD. The enzymatic reaction is allowed 24 to proceed for 15 minutes at 37C.
After completion of the reaction, the absorption of the 26 reaction solution at 340 nm is measured with a spectrophotometer 27 to determine the content of NADH formed in the solution.
18 The content of lysolecithin is obtained by comparing 29 the absoprtion value with a standard curve obtained by applying the above method on a known quantity of lycolecithin ~made by 31 Sigma Co., Ltd., Catalogue No. L 6626).
.
i, , ~al8~S~
The results are given in Table 5.
3 Table 5 Content of lysolecithin (mg/dl) Sample No.
6 30 sec. 1 min. 5 min. 15 min.
.
7 1 13.25 17.66 17.69 17.58 8 2 11.65 15.35 15.36 15.32 9 3 15.23 20.10 20.11 20.08 IO 4 12.51 16.83 16.86 16.75 1l 5 11.23 14.92 14.95 14.89 13~xample 6 14 Quantitative determination of lysolecithin content in various serums.
16 In this example, the procedure described in 7 Example 4 is repeated except 0.6 mmol of INT is used instead of TNTB and 10 samples each o~ normal human blood serum and patient 19 serums (IIa, IIb and IV types) are used.
~ The results are given in Table 6.
22 Table 6 24 Content of lysolecithin (mg/dl) Kind of serum Present method Prior method 26 Normal human serum 17.63 + 4.66 18.43 + 5.42 27 Patient serum IIa type 25.25 + 5.70 26.26 + 7.41 28 " IIb type 35.36 ~ 9.23 35.97 + 11.45 29 " IV type 27.08 + 8.64 26.84 + 11.77 . , .
- 20 ~
l i l ~ Example 7 2 Quantitative determination of lecithin content in 3 patient blood serum.
4 In this example, 20 ~1 of blood serum is added to 3 ml of 0.15 mol/l phosphate buffer (pH 7.2) containing 0.014 IU of PLD, 5 6 IU of CLDH and 0.6 mmol of TNTB. The enzymatic reaction is al-7 lowed to proceed for 15 minutes at 37C. After completion of the 8 reaction, the absorption of the reaction solution at 550 nm is mea-9 sured with a spectrophotometer. The color of the reaction solution lo is compared with a calibrated color chart obtained according to 11 the method described above using lecithin as a standard compound.
12 As a control, the procedure o~ the prior method 13 described in Example 4 is repeated on the same serum. The 1~ results are given below in Table 7.
lS
Table 7 . ~
7 Content of Lecithin (mg/dl) Test run No. ----~--------- ----18 Present method Prior method _ . _ . . . _ . . .
9 1 126.88 126O88 2 128.47 130.50 21 3 126.48 138.45 22 ~ 124.08 117.50 23 5 123.88 118.50 2~ 6 128.67 113.51 25 ~ 7 127.87 141.24 26 8 126.08 114.71 27 9 12~.28 124.48 28 10 129.07 134.46 . ~
29 Average 126.57 126.02 Standard deviation1.97 9.96 3I Coefficient of variation 1.55 7.90 . , .
, ~ ~
s~ :
Example 8 2 Quantitative determination of lecithin content in 3 normal human blood serum.
4 In this example, 20 ~1 of blood serum is added to 3 ml of 0.15 mol/l glycylglycine buffer solution (pH 7.2) containing 6 0.04 IU of PLD, 5 IU of CLDH and 0.09 mmol of NAD and the en-7 zymatic reaction is allowed to proceed for 15 minutes at 37~C.
8 The content of NAD~ formed in the reaction solution is 9 determined by using a spectrophotometer. An increase in absorbance at 340 nm is monitored for 15 minutes.
1I The results are given in Table 8.
13 Table 8 IS Content of lecithin (mg/dl) Sample No.
16 3 sec. 1 min. 5 min. 15 min.
1 124.6 124.8 128.3 126.61 18 2 126.8 127.7 130.1 128.1 19 3 125.2 126.0 128.6 127.2 4 123.8 123.7 124.4 123.8 21 5 128.6 128.4 132.0 130.7 23 ~ Example 9 :
24 In this example, the procedure described in Example 7 is repeated except 0.6 mmol oE INT is used instead of 26 TNTB and 10 samples each of normal human blood serum and patient 27 blood serums (IIa, IIb and IV types) are used.
28 The results (average data) are given in Table 9.
.
.
. .~
s~ ~
Table 9 3 Content of lecithin (mg/ml) Kind of serum 4 Present method Prior method Normal human 128.3 + 4.8 128.85 + 8.75 6 IIa type 175.18 + 9.8 173.68 + 12.34 7 IIb type 219.90 + 10.8 226.05 + 12.84 8 IV type 174.88 + 11.04 173.69 + 16.72 lo Example 10 Quantitative determination of sphingomyelin content 2 in blood serum l3 (1) Determination of total content of lecithin and sphingomyelin 14 in blood serum In this example, 20 ~1 of blood serum is added to 3 ml 6 (pH 7.2) of 0.15 mol/l phosphate buffer solution containing 7 0.04 IU of PLC, 5 IU of phosphatase, 5 IU of CLDH and 0.6 mmol of 18 TNTB. The enzymatic reaction is allowed to proceed for 15 min.
9 at 37C. After completion of the reaction, the absorption of the reaction solution at 550 nm is measured and the absorption value is compared with a calibrated color chart 22 obtained by the determination according to the method described 23 above using a known quantity of L-~-lecithin as a standard 24 compound.
As a result, the total content of lecithin and 26 ~ sphin~omyelin is obtained.
27 (2) Determination of lecithin content in serum 28 ` In this example, the procedure described in the 29 above determination of total content of lecithin and sphingomyelin ~ is repeated except using 0.04 IU of PLD in place of PLC and 31 phosphatase.
; - 23 --: , . .. . .
~lSWI
_ As a result, the content of lecithin in blood serum 2iS obtained.
3The content of sphingomyelin is calculated by 4subtracting the content of lecithin from the total value.
5As a control, the procedure of the known method 6described in Example 4 is repeated on the same serum.
7The results are given in Table 10.
1I Table 10 13 Present method (mg/dl) Lecithin Sphingo-Test run No + Lecithin myelin Prior Sphingo- method myelin (mg/dl) 16 ---~----1 167.20 126.88 40.32 41.69 8 2 171.35 128.47 42.88 46.21 ~9 3 165.56 126.46 39.08 37.85 4 163.85 124.08 39.77 42.32 21 5 169.53 125.88 43.65 37.01 22 6 170.20 128.67 41.53 38.35 23 ~7 168.35 127.87 40.48 43.32 24 8 171.12 128.08 43.04 41.51 9 165.35 124.28 41.07 40.21 26 ~ 10 168.82 129.07 39.75 39.35 28 Average 168.13 126.97 41.15 40.78 29 Standard deviation 2.57 1.78 1.57 2.80 Coefficient of 1.53 1.40 3.81 6.86 31 variation :` :
i :: : : . : : . -: .
s~ ~
. Example ll 2 Quantitative determination of sphingomyelin content 3 in blood serum.
4 (l) Determination of the total content of lecithin and sphingo-myelin 6 In this example, 20 ~l of blood serum is added to 3 ml 7 (pH 7.2) of 0.15 mol/l of glycyl-glycine buffer solution containing 8 0.04 IU of PLC, 5 IU of phosphatase, 5 IU of CLDH, 5 IU of BADH
9 and 0.09 mmol of NAD.
The enzymatic reaction is allowed to proceed for 11 15 minutes at 37C. After completion of the reaction, the ab-2 sorption of the reaction solution at 340 nm is measured to deter-13 mine NADH formed in the reaction solution with a spectrophotometer.
14 The calibration curve described in Example lO is used as a ~5 calibration curve.
(2) Determination of lecithin content 12 In this example, the procedure described in the 8 above determination of total content of lecithin and sphingomyelin 19 is repeated except using 0.04 IU of PLD in place of PLC and phosphatase.
21 The content of sphingomyelin is calculated in the 22 same manner as in Example lO.
23 The results are given in Table ll.
2s `-.
!
Il .
,, . ' .:
.: ' . .
~o~lsss~ ~
~ Table 11 t 3 Present method (mg/dl) 4 Lecithin S~hingo-Test run No Leci-thin myelin Prior ' Sphingo- method myelin (mg/ml) 6 -- _ 7 1 213.6 161.3 52.3 56.3 8 2 215.4 159.6 55.8 51.7 9 3 209.5 162.1 47.4 49.6 lo 4 210.3 161.2 49.1 55.8 Il 5 213.2 160.8 52.4 52.7 12 6 211.2 163.0 48.2 48.6 ~3 7 208.9 159.4 49.5 43.8 14 8 214.3 156.3 58.0 52.8 9 215.0 162.1 53.9 49.6 16 10 214.2 159.6 54.8 58.4 17 _ - Average 212.5 160.5 52.1 51.9 Standard deviation 2.37 1.91 3.53 4.28 Coefficient of 1.1 1.2 6.7 8.2 ~ variation 23 ~, Example 12 24In this example, the procedures described in 25Examples 10 and 11 are repeated on 10 samples of serum. When the 26 ~uantitative determination of sphingomyelin is calculated, the 27 correlation by combination of the determination of reduced form 28 of acceptor (absorption of visible part) and -that of NADH
29 (absorption of ultraviolet part) is studied.
The results are given in Tables 12 and 13.
~ - 26 ~
. ~ , Il,` ~ I
~a~8~ii8~a Table 12 2 Method according to Method according to 3 Example 10 Exmaple 11 _ _ 4 P Q P' Q' .
Sample Leci~hine Lecithine No. +
sphingo- Lecithin sphingo-Lecithin 6 myelin myelin 1 167.2 128.4 166.7 127.9 2 213.3 164.1214.5 165.3 9 ,~,.
3 269.8 204.2267.3 208.3 4 233.6 175.2233.8 174.5 214.5 162.4212.3 159.9 l2 6 267.9 202.3268.3 203.1 l3 7 283.2 219.9281.9 220.1 8 201.0 159.3200.3 261.1 9 226.7 174.9224.3 173.8 172.4 132.1173.1 133.0 l8 Table 13 Content of sphingomyelin (mg/ml) _ _ .
21 p _ Q p, _ Q, p _ O, p, _ Q
~ = . . . . _ . .. . . _ 1 38.8 38.8 39.1 38.8 2 49.2 49.2 48.0 50.4 3 65.6 59.0 61.5 63.1 2s 4 58.4 59.3 59.1 58.6 27 52.1 52.4 54.6 49.9 6 65.5 65.2 64.8 66.0 7 63.3 61.8 63.1 62.0 8 41.7 39.2 39.9 41.0 9 51.8 50.5 52.9 49.4 40.3 40.1 39.4 41.0 . .
; - 27 -.
~81S19~3 correlation between P and P' Coefficient of correlation y = 1.00, Y = O.99X + 1.54 3 Correlation between Q and Q' 4 Coefficient of correlation y = 1.00, Y = 1.02X - 3.03 Correlation between P-Q and P'-Q' 6 Coefficient of correlation ~ = 0.98, Y = 0.93X + 2.56 7 Correlation between P-Q' and P'-Q
8 Coefficient of correlation y = 0.97, Y = 0.96X + 1.82 'i ,
7 Phospholipids are found in serum, egg, meat, vegetables 8 and the like. It is important to determine the phospholipid g content in such substances for diagnosing disease, dietetics, lo etc. In this regard, medical science has recognized the useful-ll ness of determining the content of phospholipids in blood serum 12 as an aid in diagnosing disease such as hypercholesteremia, l3 liver disease and the like.
~4 A number of tests, techniques and methods have been IS proposed and are being used to measure or estimate the amount of ~6 phospholipids in the blood. Among the known methods, more 7 widely used conventional methods are colorimetric procedures 8 utilizing molybdate. Some of these procedures depend upon the conversion of phospholipid to inorganic phosphorous by incinerat-ing a sample; reacting the phosphorous with molybdate to form a 21 - phosphomolybdic acid; reducing the phosphomolybdic acid with a 22 reducing agent to form molybdenum blue; and measuring the u absorption color of the molybdenum blue. [J. Lah. Clin. Med.
24 Vol. 35, 155 (1950); J. Biol. Chem. Vol. 234, 466 (1959); Shinryo Vol. 16, 677 ~1963); Rinsho Byorl Vol. 10, 194 (1962), ibid. Vol.
26 15, 853 (1967)].
27 More recently, a method has been proposed which contem-28 '~plates the enzymatic conversion of the phospholipids present in 29 the blood specimen to phosphoric acid and the measurement of the ~color of the reaction solution obtained by reacting the phosphoric 31 acid with molybdate and then reducing the resultant mixture - 2 - ~
~ .. . ... .. .. .. .
, ... ~ .. . ..... . ... . ...
. . .
, . . . ~ .. .
" . .
~081~
1 (Yatron Document RM 137-K, Yatron Co., Ltd.).
2 The known methods ~or the ~uantitative determination of 3 phospholipids suffer from one or more of the disadvantages of 4 requiring highly skilled laboratory techniques, requiring a great deal of time to perform, not giving consistently precise 6 results and requiring blank measurements owing to interference 7 of the phosphorous contained in the sample and vessels.
9 The present invention provides a simple, practical and 0 economical diagnostic colorimetric method for detecting the presence of phospholipids in serum and making a precise auanti-2 tative determination thereof. The method offers the advantages 3 of extremely rapid color development and maintenance of 4 color value for a long period of time. Further, this method may be conducted by personnel unskilled in laboratory techniques.
6 FurthermOre~lthe method of the present invention 7 provides an all-enzymatic determination of the phospholipids and, 18 therefore, a decided improvement of routine medical diagnosis 19 ~ plus an adaptability of the method for automatic analysis apparatus.
21 The present invention is based upon the following 22 combination of enzymatic reactions:
23 ~ Phospholipids such as lecithin, sphingomyelin, 74 ~lisolecithin, cephalin, phosphatidylserine, etc. are present in blood serum. Among these phospholipids in serum, the total 26 ;amount of lecithin, sphingomyelin and lysolecithin corresponds 27 to about 95% of the amount of total phospholipids in various 28 samples of serum. These three phospholipids are converted to 29 choline by an enzymatic reaction.
The amount of the reduced form of acceptor produced in 31 ~ the enzymatic conversion of choline to glycine betaine aldehyde . . , ,1 , ~08~
1 in the presence of choline dehydrogenase (hereinafter referred 2 to as CLDH) and hydrogen acceptor is directly proportional 3 to the amount of phospholipid in the blood serum.
4 Further, the amount of the reduced form of nicotin-amide adenine dinucleotide (hereinafter referred to as NADH) 6 produced in the enzymatic conversion of glycine betaine 7 aldehyde to betaine aldehyde in the presence of betaine 8 aldehyde dehydrogenase (hereinafter referred to as BADH) and g nicotinamide adenine dinucleotide (hereinafter referred to as NAD) or consumed NAD in the enzymatic reaction is also 11 directly proportional to the amount of phospholipid in the l2 blood seru~.
13 The reduced form of acceptor thus produced can then be 1~ detected by measuring the absorption of the color of the reaction solution at a wave length of from 400 to 700 nm.
6 The NADH thus produced may also be detected by measuring 7 the absorption of the reaction solution at a wave length of 340 nm.
18 The amount of consumed NAD can be calculated by 19 subtracting the remaining NAD from the added NAD. The remaining NAD in the reaction solution can be detected by 21 measuring the absorption of the reaction solution at a wave 22 length of from 260 to 280 nm.
23 These reactions are known individually. That is, 2~ lecithin catalytically reacts with phospholipase D (hereinafter referred to as PLD) to form choline [Reaction I, J. Biol. Chem.
26 Vol. 231, 703 (1958), ibid, Vol. 172, 191 (1948~]. Sphingomyelin 27 and lecithin catalytically react with phospholipase C (hereinafter 28 referred to as PLC) to form phosphoryl choline [Reaction II, 29 Biochem. J. Vol. 35, 884 (l946); Biochem. Biophys. Acta. Vol.
59, 103 (1962)], the phosphoryl choline catalytically reacts 31 with phosphatase to form choline [Reaction III, J. Biol. Chem.
,., : , . :.
- :, ~ . . .,, ~ , . . :
~8~S~9 1 Vol. 244, 308 (1969)]. Lysolecithin catalytically reacts 2 with phospholipase B (hereinafter referred to as PLB) to 3 form glycerophosphorylcholine [Reaction IV, "Biochemist's 4 Handbook" E. & F. N. Spou Ltd. 282 (1961); Nature Vol. 169, 29 (1952); siochem. J. Vol. 71, 615 (1959)], the glycero-6 phosphorylcholine catalytically reacts with glycerophosphoryl-7 choline dièsterase (hereinafter referred to as GPD) to form 8 choline [Reaction V, J. Biol. Chem. Vol. 206, 647 (1954);
g Biochem. J. Vol. 62, 689 (1956)].
IQ The choline formed in the reaction described above, catalytically reacts with CLDH in the presence of a hydrogen 12 acceptor to form glycine betaine aldehyde and reduced form of 13 acceptor [Reaction VI, Agr. Biol. Chem. Vol. 39, 1513 (1975)].
l~ The glycine betaine aldehyde catalytically reacts with BADH
in the presence of NAD to form glycine betaine and NADH
16 [Reaction VII, J. Biol. Chem. Vol. 209, 511 (1954)].
17 The enzymatic reactions described above are schematically 18 represented as follows:
2~
~8~S8S~ , o~ ~ o --~ ~ m c~ u ~ p, + Z;
- 51 =C
o O o~ . ~~ ~ ~ o p~ ~
O F~
m ~ Z a~
o=~-~o ~ ~3 ~
~-~ -U I ~
~ rl X
~ ~1 0 o~ ~~ Z
~ ~ I I~
a+ ~ a~ O ~ o ~ u :r=tC =O-- ~ ~ p~
O H
~ O
rl +
a P~
~ I
~ ~ a O ~ ~ ,, __ _ , _ ,, _ ~" ,1 o~ 0=~--O O
O O
O
. .
1~ 9 Cholin~
~ CLDH
[Reaction VI] ~ - Hydrogen acceptor +\ ~ (electron transmitter) OHC-CH N(CH ) OH ~ Reduced form of 2 3 3 acceptor Glycine betaine aldehyde Detection ~ BADH
[Reaction VII~
~ NAD
+ NADH
Glycine betaine Detection where Rl, R2 and R3 represent hydrocarbon groups.
~8~5~9 ' :`
1 : The quantitative determination of lecithin content 2 in a sample is carried out according to the process consisting 3 of Reactions I and VI (hereinafter referred to as Process D) 4 or Reactions I, VI and VII (hereinafter referred to as ProcesS
D').
6 The quantitati~e determination of total content of 7 sphin~omyelin and lecithin in a sample is carried out aecording 8 to the process consisting of Reactions II, III and VI (herein~
9 a~ter referred to as Process C) or Reactions II, III, VI and VII (hereinafter referred to as Process C').
11 The quantitative determination of lysolecithin lt content in a sample is carried out according to the process 13 consisting of Reaetions IV, V and VI (hereinafter referred to 14 as Process B) or Reactions IV, V, VI and VII (hereinafter referred to as Process B').
16 The quantitative determination of total content of 17 ;leeithin, shingomyelin and lysolecithin is earried out aecording 18 to Proeesses C and B, Processes C and B', Processes C' and B, 19 or Processes C' and B'.
DETAILED DESCRIPTION OF THE INVENTION
21 Broadly, the present invention comprises the combination 22 of a system for convertin~ phospholipids in a sample to choline 23 ~ and a system for detecting the so-produced choline.
24 ; The system for converting the phospholipids to eholine ~ comprises one or more enzymes selected from the group eonsisting 26 of PLD, PLC and phosphatase, and PLB and GPD.
27 The eholine detecting system comprises CLDH and a 28 hydrogen acceptor or further comprises BADH and NAD and if 29 necessary, comprises an electron transmitter.
i lS89 ;`
Additional subject matter of the invention is a 2 novel reagent composition for converting of phospholipid 3 in a sample to choline which comprises, as a first component, ,~l 4 one or more enzymes selected from the group consisting of PLD,~
s PLC and phosphatase, and PLB and GPD; and a second component , 6 comprising a choline detecting reagent.
7 According to the present invention, the quantitative 8 determination of phospholipids may be performed by conducti~g g the individual reactions stepwise. That is, the enzyme reac-tions are divided into any desired group and after completlon 11 of the reaction of the preceding s~p, the successive enzymes are added thereto, and the reactio~-~f the next step is allowed 13 to proceed. After completion of Reaction VI, the absorption 14 of the visible part of the colored_reaction solution by 15 ` formation of the reduced form of acceptor is measured or after 16 completion of Reaction VII,the absorption of the ultraviolet part of the reaction solution is measured for detecting NADH
formed or the remaining NAD. The absorption values obtained 19 by one or more of the above-s~eps are compared with a calibration curve obtained by carring out the above-steps on the standard 21 ~ compound, whereby the content of the phospholipids in a sample æ is determined.
23 According to the present invention, the presently 2~ preferred method for the quantitative determination of phos-pholipids in a sample is carried out by subjecting 26 a sample to reaction with a reagent consisting of enzymes~for 27 converting phospholipids to choline, CLDH and a hydrogen 2~ acceptor; and, if necessary, BADH, NAD, an electron tran~mitter, 29 surfactant, etc. in a buffer solution.
Enzymes used in the present invention are known and 31 those obtained from various sources may be used.
~08~S~ `
1 Any PLD may be used so long as it acts only on 2 lecithin among phospholipids. Suitable PLD are obtained 3 from cabbage or the like by extraction and purification 4 and are commercially available from such sources as Sigma Co., U.S.A. and Boehringer Mannheim Co., West Germany.
6 Any PLC may be used so long as it acts only on 7 sphingomyelin and lecithin among phospholipids. Suitable 8 PLC are obtained from cell bodies of microorganism belonging g to Escherichia coli by extraction and purification and are o commercially available from such sources as Worthington Co., Il U.S.A. as Catalog No. 5130 and Baehringer Mannheim Co., 12 West Germany, as Catalog No. 15429.
l3 Phosphatase obtained from cell bodies of micro-l~ organisms belonging to the species Clostridium perfringens or Clostridium welchii or the like by extraction and purification 6 may be used and are commercially available from such sources as Worthington Co. as Catalog No. 5640 and Sigma Co., U.S.A. as l8 Catalog No. P 7633, etc.
Ig Any PLB may be used so long as it acts only on lysolecithin among phospholipids. Suitable PLB may be obtained 21 rom cell bodies of microorganisms belonging to the species 22 Penicillium notatum by extraction [Biochem. J. Vol. 70, 559 23 (1958)]. Those extracted from mucous membranes of livers and 2~ intestines of rats, oxen, and pigs, etc. are also appropriate.
Suitable GPD may be obtained from cell bodies of 26 microorganisms belonging to the species Serratia plymuthicum 27 by extraction [J. Biol. Chem. Vol. 206, 647 (1954)]. Those 2~ extracted from livers of rats, oxen, and pigs [Biochem. J., 29 vol. 62, 689 (1956)], etc. are also appropriate.
Suitable CLDH may be obtained from cell bodles of 3I microorganisms belonging to the species Pseudomonas ~
. -- 10 --: .: : ,.;:,, ,, . . : ,. .
:~8~LS8g - -`
I [Agr. Biol. Chem. Vol. 39, 1513 - 151~ (1975)]. Those 2 extracted from mitochondria of rats [J. Biol. Chem. Vol. 234, 3 1605 (1959)], etc. are also appropriate.
4 Suitable sADH may be extracted from cell bodies of microorganisms belonging to the species Pseudomonas aeruginosa 6 [Agr. Biol. Chem. Vol. 39, 1513 - 1514 (1975)]. Those purified 7 from a supernatant of homogenate of rat livers [J. Biol. Chem.
8 Vol. 209, 511 (1954)], etc. are also appropriate.
9 An amount of enzvmes to be used to that of phospholipid 0 is determined in such a range that the detecting operation can be Il exactly carried out. That is, the following amount of enzymes l2 may be used to one ~g of phospholipid;
l3 0.005 - 1 IU for PLD, 0.005 - 1 IU for PLC, 0.005 -l~ 1 IU for PLB, 0.005 - 50 IU for phosphatase, 0.005 - 1 IU for s GPD, 0.1 - 50 IU for CLDH, 0.1 - 100 IU for BADH.
The foregoing enzymes are used in an appropriate buffer solution. Preferable concentrations of the enzymes in the buffer solution are 0.01 - 1 IU/mQ for PLD; 0.01 -19 1 IU/mQ for PLC; 0.01 - 1 IU/mQ for PLB; 0.1 - 50 IU/mQ for phosphatase; 0.01 - 1 IU/mQ for GPD; 0.1 - 50 IU/mQ for CLDH;
2l and 0.1 - 100 IU/mQ for BADH. IU is an international unit of 22 enzyme factor; and the factor capable of decomposing 1 ~ mole 23 of substrate in one minute is defined as 1 IU, i.e. 1 IU =
2~ mol/min.
2s It is desirable to provide the buffer solution at a 26 pH in the range of about from 5.0 to 10.0, preferably from 27 6.5 to 7.5. For this purpose, buffers which may be used 28 include phosphate, succinate, citrate, borate, acetate, 29 glycylglycinate, tris-malonate as well as other buffers which are generally effective within the pH range of from 5.0 to 10Ø
3I The concentration of the buffer is not critical.
.. . .
s~s ..
1 However, it is preferred to use a relatively dilute buffer 2 solution and for this purpose a 0.l - 0.2 mol/Q solution is 3 recommended.
g Examples of the hydrogen acceptor are 3,3'-(3,3'-dimethoxy-4,~'-biphenylene)-bis~[2-(p-nitrophenyl)-5-phenyl-6 2H-tetrazolium chloride] (hereinafter referred to as nitro-7 Ts), 3,3'-(3,3'-dimethoxy-4,4'-biphenylene-bis [2,5-bis 8 (p-nitrophenyl)-2H-tetrazolium chloride] (hereinafter referred 9 to as TNTB), 3-(p-indophenyl-2-(p-nitrophenyl)-5-phenyl-2H-0 tetrazolium chloride (hereinafter referred to as INT), 2,6-11 dichlorophenyl-indophenol (hereinafter referred to as DCPIP) 12 and NAD.
13 When DCPIP is used, it is used with an electron 1~ transmitter such as phenazine methosulfate and the like which s accepts an electron from choline and donates it to DCPIP.
Since NAD is liable to be converted to NADH by 7 enzymes, the coenzyme of which is NAD, there is sometimes 8 cause for an error when there are such enzymes in the reaction 19 system. It is, therefore, preferable to avoid such combinations.
Preferable concentration of the hydrogen acceptor in 21 the buffer solution is 0.l - 500 mmol/Q.
22 Preferable concentration of the electron transmitter 23 is 0.l - 50 mmol/Q.
24 Typical samples in which the amount of phospholipids would be determined, include living components such as serum, 26 liver, etc. and various foods such as vegetable oil, fish oil, 27 vegetables, etc. When the sample is solid it should be ground 28 and then subjected to extraction with water or a small amount 29 of organic solvent such as alcohol, chloroform or the like.
On the other hand, when the sample is an oil, a surfactant may 31 be added to the reagent solution to improve the affinity with - , : ,: , ..
, . . , , ,-:. ~
~lS~
1 the reagent solution. Suitable surfactants include such higher 2 alcohols as polyethyleneglycol, etc. Preferably, the concen-3 tration of the surfactant in the reagent solution is 0.01 -4 5 g/Q.
The enzymatic reactions are carried out at 20 to 45C, 6 preferably 30 to 40C, at pH 5 to lO~for 4 to 60 minutes.
7 After completion of the enzymatic reactions, the 8 absorption of the reaction solution is measured by utilizing g a spectrophotometer, or the like.
o When the amount of the formation of reduced form of Il acceptor is determined, the absorption of visible part of the 12 reaction solution is measured at a wave length in a range of 13 400 to 700 nm.
14 When NAD is used as a hydrogen acceptor in Reaction Is VI, the quantitative determination of reduced form of acceptor, l6 i.e. NAD~ formed in Reaction VI is carried out by measuring l7 the absorption of the reaction solution at a wave length of 18 340 nm.
19 When the amount of NADH is determined, the absorption of ultraviolet part of the reaction solution is measured at 340 21 nm and when the amount of remaining NAD is determined, the 22 absorption is measured at 260 - 280 nm.
23 The colors developed in the reaction by using nitro-TB, 2~ TNTB, INT and DCPIP as the hydrogen acceptor are blue, red-brown, red (or violet) and red-blue, respectively.
26 When NAD is used as a hydrogen acceptor in Reaction VI
27 and the quantitative determination of phospholipid is carried 28 out by measuring the amount of NADH produced after completion 29 of Reaction VII, the quantitative value of NADH determined represents total sum of NADH formed in Reactions VI and VII, 31 and therefore the amount of NADH formed in Reaction VI is taken ~ - 13 -~8~L5~3~
I into consideration. Reaction VII usually proceeds to 100%
2 and thus the amount of NADH formed in Reaction VII can be 3 presumed to be 1/2 of said sum value.
4 The composition of the present invention may be used in various forms. For example, the ingredients may be 6 mixed in liquid form or in powder form. The liquid formulation 7 may be readily reconstituted for later use simply by the 8 addition of water or buffer solution. The powders, if desired, 9 may be tableted for convenience in use.
o Practice of certain specific embodiments of the invention is illustrated by the following representative l2 examples.
I3 Example 1 l~ Quantitative determination of content of lecithin, sphingomyelin and lysolecithin in normal human blood serum.
In this example, 20 ~Q of blood serum is added to 3 mQ of 0.15 mol/Q phosphate buffer solution (pH 7.2) containing 0.04 IU of PLC, 5 IU of phosphatase, 0.014 IU of PLB, 0.014 IU
l9 of GPD, 5 IU of CLDH and 0.6 mmol of TNTB.
The enzymatic reaction is allowed to proceed for 2l 15 min. at 37C. After completion of the reaction, the absorp-22 tion of the reaction solution at 550 millimicrons is measured 23 with a spectrophotometer to determine the content of reduced 2~ form of acceptor formed in the solution.
2s The content of the phospholipids is obtained by 26 comparing the absorption value with a standard curve obtained 27 by applying the method described above on a standard sample o~
28 lecithin (produced by Sigma Co., Ltd., Catalogue No. L-2004).
29 As a control, the same serum is subjected to extraction using a chloroform-methanol solution according to the Folch 31 extraction method. The resulting extract is oxidized by using ~o~3~s89 perchloric acid to form an inorganic phosphorous and a 2 molybdate; and, thereafter, hydrazine sulfate is added to the 3 resulting solution to form a colored solution. Absorption of 4 the color developed at a wave length of 700 nm is measured with a spectrophotometer [Rinsho syori, Vol. 10, 194 (1962)].
6 The samples are measured 10 times for each method 7 described above. The results are given in Table 1.
g _ /
/
IJ
~7 IB
2-~ /
29 / .. _ /
~ , .
~13lSI~ `
Table 1 Content of phospholipids (mg/dl) 3 ~ Test run No.
Present method Prior method 1 185.3 189.3 6 2 187.2 195.2 7 3 184.4 199.4 ~ 4 186.7 190O8 9 5 185.1 187.2 lo 6 181.9 193.6 Il 7 188.2 195.1 12 8 187.5 188.8 13 9 186.3 192.3 1~ 10 184.6 190.5 lS Averaqe 185.7 192.2 16 Standard deviation 1.85 3.68 17 Coefficient of variation 0.99 % 1.91 ~
19 As is apparent from Table 1, the standard deviation and coefficient of variation in the present method are less 21 than those of the known method.
22 To make a comparison with the prior known method 23 ` as to the content of total phospholipids, multiplication by a 24 factor of 1.0416 gives an average of the content of total ~phospholipids, 193.4 mg/dl~ almost the same numercial value as 26 that of the prior method.
27 ;Example 2 28 " Quantitative determination of lecithin, sphingomyelin 29 ., and lysolecithin in patient serum (II b type).
i In this example, 20 ~1 of patient serum is added to 31 ; 3 ml of 0.15 mol/l tris-buffer solution (pH 7.2) containing , .
'I - 16 ~
1 D181S~
0.04 IU of PLC~ 5 IU of phosphatase, o. 014 IU Of~ PLB~ O.014 IU
2 of GPDr 5 IU of CLDHr 5 IU of BADH and 0.09 mmol of NAD.
3 . The enzymatic reaction is allowed to proceed for 4 15 min. at 37C. After completion of the reaction, the absorption of the reaction solution at 340 nm millimicrons is measured at 6 various time intervals using a spectrophotometer to determine the 7 content of NADH formed in the solution. The values are given in 8 Table 2.
lo Table 2 Il 12 Content of total phospholipid 13 (mg/dl) Is30 seconds 305.2 161 minute 312.1 175 minutes 314.3 lô15 minutes 313.0 As is apparent from the values set forth in Table 2, 21 the quantitative determination of total phospholipid according 22 to the present method can be carried out in a short period of time.
23 ; Example 3 24 ~ Quantitative determination of content of total ` phospholipid in serum.
26 In this example, the procedures described in I ~"
27 Example 1 are repeated except 0.6 mmol of INT is used instead 28 of TNTB, and normal human serum and patient serums (IIa, IIb and 29 IV types) are used.
~ The results are given in Table 3.
31 The content of total phospholipid, according to the -- 17 ~
., I
;, . .:
" , I
- . :., , :... ~ : ~ , . . . .
3~8~5~9 present method, is obtained by multiplication b~ a value of 2 1.0416.
4 Table 3 6 The content of total phospholipid(mg/dl) Serum 7 Present method Prior method 8 Normal human194.2 + 14.0 201.5 + 32.3 9 II a type275.3 + 15.4 278.7 + 40.2 lo II b type341.0 + 19.5 348.2 + 51.2 Il IV type 273.5 + 12.3 263.2 + 52.3 ~2 l3 Example 4 14 Quantitative determination of lysolecithin content in patient blood serum.
lo In this example, 100 ~1 of blood serum is added to 17 3 ml of 0.15 mol/l phosphate buffer solution (pH 7.2) containing 18 0.014 IU of PLB, 0.014 IU of GPD, 5 IU of CLDH and 0.6 mmol l9 of TNTB.
The enzymatic reaction is allowed to proceed for 21 15 minutes at 37C. Ater completion of the reaction, the 22 absorption of the reaction solution is measured with a spectro-23 photometer at a wave length of 550 nm. The red-brown color of 24 the reaction solution is compared with a calibration curve obtained by using a standard compound.
26 As a control (known method), the same serum is 27 developed by thin layer chromatography using silica gel. The 28 resultant sample is incinerated and a color is developed by 29 using a molybdic acid reagent. The color is measured with a densitometer. The results are given in Tab]e 4.
i ~!
.. ..... .... .. . ....... .
~0~S~9 - Table 4 Content of lysolecithin (mg/dl) 3 Test run No.
Present methodPrior Method 1 35.36 35.45 6 2 35.82 36.85 7 3 35.14 34.67 8 4 35.38 35.59 9 5 34.98 36.98 o 6 35.67 35.56 7 35.28 36.02 2 8 35.43 35.95 13 935 33 34.38 14 10 35.87 35.21 ls Average 35.42 35.66 16 Standard deviation 0.28 0.83 17 Coefficient of variation0.79% 2.32%
19 Example 5 ~ In this example, 100 ~1 of normal human blood serum is 21 added to 3 ml of 0.15 mol/l glycylglycine buffer solution (pH 7.2) 22 containing 0.04 IU of PLB, 0.04 IU of GPD, 5 IU of CLDH, 5 IU
~3 of BADH and 0.09 mmol of NAD. The enzymatic reaction is allowed 24 to proceed for 15 minutes at 37C.
After completion of the reaction, the absorption of the 26 reaction solution at 340 nm is measured with a spectrophotometer 27 to determine the content of NADH formed in the solution.
18 The content of lysolecithin is obtained by comparing 29 the absoprtion value with a standard curve obtained by applying the above method on a known quantity of lycolecithin ~made by 31 Sigma Co., Ltd., Catalogue No. L 6626).
.
i, , ~al8~S~
The results are given in Table 5.
3 Table 5 Content of lysolecithin (mg/dl) Sample No.
6 30 sec. 1 min. 5 min. 15 min.
.
7 1 13.25 17.66 17.69 17.58 8 2 11.65 15.35 15.36 15.32 9 3 15.23 20.10 20.11 20.08 IO 4 12.51 16.83 16.86 16.75 1l 5 11.23 14.92 14.95 14.89 13~xample 6 14 Quantitative determination of lysolecithin content in various serums.
16 In this example, the procedure described in 7 Example 4 is repeated except 0.6 mmol of INT is used instead of TNTB and 10 samples each o~ normal human blood serum and patient 19 serums (IIa, IIb and IV types) are used.
~ The results are given in Table 6.
22 Table 6 24 Content of lysolecithin (mg/dl) Kind of serum Present method Prior method 26 Normal human serum 17.63 + 4.66 18.43 + 5.42 27 Patient serum IIa type 25.25 + 5.70 26.26 + 7.41 28 " IIb type 35.36 ~ 9.23 35.97 + 11.45 29 " IV type 27.08 + 8.64 26.84 + 11.77 . , .
- 20 ~
l i l ~ Example 7 2 Quantitative determination of lecithin content in 3 patient blood serum.
4 In this example, 20 ~1 of blood serum is added to 3 ml of 0.15 mol/l phosphate buffer (pH 7.2) containing 0.014 IU of PLD, 5 6 IU of CLDH and 0.6 mmol of TNTB. The enzymatic reaction is al-7 lowed to proceed for 15 minutes at 37C. After completion of the 8 reaction, the absorption of the reaction solution at 550 nm is mea-9 sured with a spectrophotometer. The color of the reaction solution lo is compared with a calibrated color chart obtained according to 11 the method described above using lecithin as a standard compound.
12 As a control, the procedure o~ the prior method 13 described in Example 4 is repeated on the same serum. The 1~ results are given below in Table 7.
lS
Table 7 . ~
7 Content of Lecithin (mg/dl) Test run No. ----~--------- ----18 Present method Prior method _ . _ . . . _ . . .
9 1 126.88 126O88 2 128.47 130.50 21 3 126.48 138.45 22 ~ 124.08 117.50 23 5 123.88 118.50 2~ 6 128.67 113.51 25 ~ 7 127.87 141.24 26 8 126.08 114.71 27 9 12~.28 124.48 28 10 129.07 134.46 . ~
29 Average 126.57 126.02 Standard deviation1.97 9.96 3I Coefficient of variation 1.55 7.90 . , .
, ~ ~
s~ :
Example 8 2 Quantitative determination of lecithin content in 3 normal human blood serum.
4 In this example, 20 ~1 of blood serum is added to 3 ml of 0.15 mol/l glycylglycine buffer solution (pH 7.2) containing 6 0.04 IU of PLD, 5 IU of CLDH and 0.09 mmol of NAD and the en-7 zymatic reaction is allowed to proceed for 15 minutes at 37~C.
8 The content of NAD~ formed in the reaction solution is 9 determined by using a spectrophotometer. An increase in absorbance at 340 nm is monitored for 15 minutes.
1I The results are given in Table 8.
13 Table 8 IS Content of lecithin (mg/dl) Sample No.
16 3 sec. 1 min. 5 min. 15 min.
1 124.6 124.8 128.3 126.61 18 2 126.8 127.7 130.1 128.1 19 3 125.2 126.0 128.6 127.2 4 123.8 123.7 124.4 123.8 21 5 128.6 128.4 132.0 130.7 23 ~ Example 9 :
24 In this example, the procedure described in Example 7 is repeated except 0.6 mmol oE INT is used instead of 26 TNTB and 10 samples each of normal human blood serum and patient 27 blood serums (IIa, IIb and IV types) are used.
28 The results (average data) are given in Table 9.
.
.
. .~
s~ ~
Table 9 3 Content of lecithin (mg/ml) Kind of serum 4 Present method Prior method Normal human 128.3 + 4.8 128.85 + 8.75 6 IIa type 175.18 + 9.8 173.68 + 12.34 7 IIb type 219.90 + 10.8 226.05 + 12.84 8 IV type 174.88 + 11.04 173.69 + 16.72 lo Example 10 Quantitative determination of sphingomyelin content 2 in blood serum l3 (1) Determination of total content of lecithin and sphingomyelin 14 in blood serum In this example, 20 ~1 of blood serum is added to 3 ml 6 (pH 7.2) of 0.15 mol/l phosphate buffer solution containing 7 0.04 IU of PLC, 5 IU of phosphatase, 5 IU of CLDH and 0.6 mmol of 18 TNTB. The enzymatic reaction is allowed to proceed for 15 min.
9 at 37C. After completion of the reaction, the absorption of the reaction solution at 550 nm is measured and the absorption value is compared with a calibrated color chart 22 obtained by the determination according to the method described 23 above using a known quantity of L-~-lecithin as a standard 24 compound.
As a result, the total content of lecithin and 26 ~ sphin~omyelin is obtained.
27 (2) Determination of lecithin content in serum 28 ` In this example, the procedure described in the 29 above determination of total content of lecithin and sphingomyelin ~ is repeated except using 0.04 IU of PLD in place of PLC and 31 phosphatase.
; - 23 --: , . .. . .
~lSWI
_ As a result, the content of lecithin in blood serum 2iS obtained.
3The content of sphingomyelin is calculated by 4subtracting the content of lecithin from the total value.
5As a control, the procedure of the known method 6described in Example 4 is repeated on the same serum.
7The results are given in Table 10.
1I Table 10 13 Present method (mg/dl) Lecithin Sphingo-Test run No + Lecithin myelin Prior Sphingo- method myelin (mg/dl) 16 ---~----1 167.20 126.88 40.32 41.69 8 2 171.35 128.47 42.88 46.21 ~9 3 165.56 126.46 39.08 37.85 4 163.85 124.08 39.77 42.32 21 5 169.53 125.88 43.65 37.01 22 6 170.20 128.67 41.53 38.35 23 ~7 168.35 127.87 40.48 43.32 24 8 171.12 128.08 43.04 41.51 9 165.35 124.28 41.07 40.21 26 ~ 10 168.82 129.07 39.75 39.35 28 Average 168.13 126.97 41.15 40.78 29 Standard deviation 2.57 1.78 1.57 2.80 Coefficient of 1.53 1.40 3.81 6.86 31 variation :` :
i :: : : . : : . -: .
s~ ~
. Example ll 2 Quantitative determination of sphingomyelin content 3 in blood serum.
4 (l) Determination of the total content of lecithin and sphingo-myelin 6 In this example, 20 ~l of blood serum is added to 3 ml 7 (pH 7.2) of 0.15 mol/l of glycyl-glycine buffer solution containing 8 0.04 IU of PLC, 5 IU of phosphatase, 5 IU of CLDH, 5 IU of BADH
9 and 0.09 mmol of NAD.
The enzymatic reaction is allowed to proceed for 11 15 minutes at 37C. After completion of the reaction, the ab-2 sorption of the reaction solution at 340 nm is measured to deter-13 mine NADH formed in the reaction solution with a spectrophotometer.
14 The calibration curve described in Example lO is used as a ~5 calibration curve.
(2) Determination of lecithin content 12 In this example, the procedure described in the 8 above determination of total content of lecithin and sphingomyelin 19 is repeated except using 0.04 IU of PLD in place of PLC and phosphatase.
21 The content of sphingomyelin is calculated in the 22 same manner as in Example lO.
23 The results are given in Table ll.
2s `-.
!
Il .
,, . ' .:
.: ' . .
~o~lsss~ ~
~ Table 11 t 3 Present method (mg/dl) 4 Lecithin S~hingo-Test run No Leci-thin myelin Prior ' Sphingo- method myelin (mg/ml) 6 -- _ 7 1 213.6 161.3 52.3 56.3 8 2 215.4 159.6 55.8 51.7 9 3 209.5 162.1 47.4 49.6 lo 4 210.3 161.2 49.1 55.8 Il 5 213.2 160.8 52.4 52.7 12 6 211.2 163.0 48.2 48.6 ~3 7 208.9 159.4 49.5 43.8 14 8 214.3 156.3 58.0 52.8 9 215.0 162.1 53.9 49.6 16 10 214.2 159.6 54.8 58.4 17 _ - Average 212.5 160.5 52.1 51.9 Standard deviation 2.37 1.91 3.53 4.28 Coefficient of 1.1 1.2 6.7 8.2 ~ variation 23 ~, Example 12 24In this example, the procedures described in 25Examples 10 and 11 are repeated on 10 samples of serum. When the 26 ~uantitative determination of sphingomyelin is calculated, the 27 correlation by combination of the determination of reduced form 28 of acceptor (absorption of visible part) and -that of NADH
29 (absorption of ultraviolet part) is studied.
The results are given in Tables 12 and 13.
~ - 26 ~
. ~ , Il,` ~ I
~a~8~ii8~a Table 12 2 Method according to Method according to 3 Example 10 Exmaple 11 _ _ 4 P Q P' Q' .
Sample Leci~hine Lecithine No. +
sphingo- Lecithin sphingo-Lecithin 6 myelin myelin 1 167.2 128.4 166.7 127.9 2 213.3 164.1214.5 165.3 9 ,~,.
3 269.8 204.2267.3 208.3 4 233.6 175.2233.8 174.5 214.5 162.4212.3 159.9 l2 6 267.9 202.3268.3 203.1 l3 7 283.2 219.9281.9 220.1 8 201.0 159.3200.3 261.1 9 226.7 174.9224.3 173.8 172.4 132.1173.1 133.0 l8 Table 13 Content of sphingomyelin (mg/ml) _ _ .
21 p _ Q p, _ Q, p _ O, p, _ Q
~ = . . . . _ . .. . . _ 1 38.8 38.8 39.1 38.8 2 49.2 49.2 48.0 50.4 3 65.6 59.0 61.5 63.1 2s 4 58.4 59.3 59.1 58.6 27 52.1 52.4 54.6 49.9 6 65.5 65.2 64.8 66.0 7 63.3 61.8 63.1 62.0 8 41.7 39.2 39.9 41.0 9 51.8 50.5 52.9 49.4 40.3 40.1 39.4 41.0 . .
; - 27 -.
~81S19~3 correlation between P and P' Coefficient of correlation y = 1.00, Y = O.99X + 1.54 3 Correlation between Q and Q' 4 Coefficient of correlation y = 1.00, Y = 1.02X - 3.03 Correlation between P-Q and P'-Q' 6 Coefficient of correlation ~ = 0.98, Y = 0.93X + 2.56 7 Correlation between P-Q' and P'-Q
8 Coefficient of correlation y = 0.97, Y = 0.96X + 1.82 'i ,
Claims (9)
1. A composition for the determination of phospholipids in a sample comprising a first component comprising at least one enzyme selected from the group consisting of phospholipase D, phospholipase C and phosphatase and phospholipase B and glycerophosphorylcholine diesterase; and a second component comprising a choline detecting reagent.
2. The composition of Claim 1 wherein said choline detecting reagent is choline dehydrogenase and hydrogen acceptor.
3. The composition of Claim 2 further comprising betaine aldehyde dehydrogenase and nicotinamide adenine dinu-cleotide.
4. The composition of Claim 1 wherein said phospholipid is lecithin, and said first component is phospho-lipase D.
5. The composition of Claim 1 wherein said phospholipid is lysolecithin, and said first component is phospholipase B and glycerophosphorylcholine diesterase.
6. The composition of Claim 1 wherein said phospholipid comprises lecithin and sphingomyelin, and said first component is phospholipase C and phosphatase.
7. The composition of Claim 1 wherein said phospholipid comprises lecithin, sphingomyelin and lysolecithin, and said first component comprises phospholipase B, phospho-lipase C, phosphatase, and glycerophosphorylcholine diesterase.
8. A method for detecting phospholipid in a sample which comprises reacting said sample with a choline forming system comprising, one or more enzymes selected from the group consisting of phospholipase D, phospholipase C and phosphatase, and phospholipase B and glycerophosphorylcholine diesterase, and thereafter reacting the resulting solution with a choline detecting system comprising a choline dehydrogenase and a hydrogen acceptor.
9. The method according to Claim 7 wherein said choline detecting system further comprises betaine aldehyde dehydrogenase and nicotinamide adenine dinucleotide.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP98233/76 | 1976-08-19 | ||
| JP9823376A JPS5324888A (en) | 1976-08-19 | 1976-08-19 | Composition for quantitating fats containing phosphorus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1081589A true CA1081589A (en) | 1980-07-15 |
Family
ID=14214230
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA284,986A Expired CA1081589A (en) | 1976-08-19 | 1977-08-18 | Composition and method for the quantitative determination of phospholipids |
Country Status (4)
| Country | Link |
|---|---|
| JP (1) | JPS5324888A (en) |
| CA (1) | CA1081589A (en) |
| DE (1) | DE2737513B2 (en) |
| FR (1) | FR2362394A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5937737B2 (en) * | 1978-05-25 | 1984-09-11 | 新日本製鐵株式会社 | Steel materials for hot forging |
| CA1215903A (en) * | 1981-06-25 | 1986-12-30 | Hideo Misaki | Assay method for component relating to lipids, composition for assay and process for production of enzyme used therefor |
| DE3128480A1 (en) * | 1981-07-18 | 1983-01-27 | Boehringer Mannheim Gmbh, 6800 Mannheim | METHOD FOR DETERMINING LECITHIN IN FRUIT WATER AND REAGENT FOR CARRYING OUT THIS METHOD |
| JPS58107199A (en) * | 1981-12-16 | 1983-06-25 | Toyo Jozo Co Ltd | Qualitative analysis of phosphatidyl glycerol |
| DE4409987C1 (en) * | 1994-03-23 | 1995-07-06 | Wolfgang Dr Bernhard | High pressure liquid chromatography (HPLC) method for phospholipid |
| US20030017523A1 (en) * | 2000-02-08 | 2003-01-23 | Osamu Hotta | Method of measuring lipid components and method of examining of renal failure |
-
1976
- 1976-08-19 JP JP9823376A patent/JPS5324888A/en active Pending
-
1977
- 1977-08-18 CA CA284,986A patent/CA1081589A/en not_active Expired
- 1977-08-18 FR FR7725345A patent/FR2362394A1/en active Granted
- 1977-08-19 DE DE19772737513 patent/DE2737513B2/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5324888A (en) | 1978-03-08 |
| DE2737513A1 (en) | 1978-02-23 |
| FR2362394B1 (en) | 1981-08-28 |
| DE2737513B2 (en) | 1980-02-14 |
| FR2362394A1 (en) | 1978-03-17 |
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