Note: Descriptions are shown in the official language in which they were submitted.
~(~8~S~
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 - ~
~ .. . ... .. .. .. .
, ... ~ .. . ..... . ... . ...
. . .
, . . . ~ .. .
" . .
~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.
8 SUMMARY OF THE INVENTION
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:
19
21
22
23
2~
26
27
28
29
31
~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.
31
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.
-- 11 --
.. . .
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
- 12 -
- , : ,: , ..
, . . , , ,-
:. ~
~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
- 14 -
~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 _ /
/
12
13
IJ
16
~7
IB
19
21
22
23
2-~ /
26
27
28
29 / .. _
/
31
-- 1 5 --
~ , .
~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)
14
Is30 seconds 305.2
161 minute 312.1
175 minutes 314.3
lô15 minutes 313.0
19
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.
31
- 18 -
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%
18
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).
-- 19 --
.
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
12
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.
21
22 Table 6
23
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
. , .
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, ~ ~
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.
12
13 Table 8
14
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
22
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.
29
31
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.
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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
12
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
27
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.
24
2s `-
26
27
28
29
31
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!
Il .
,, . ' .:
.: '
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~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
21
22
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.
31
~ - 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
11
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
14
8 201.0 159.3200.3 261.1
9 226.7 174.9224.3 173.8
172.4 132.1173.1 133.0
17
l8
Table 13
Content of sphingomyelin (mg/ml)
_ _ .
21 p _ Q p, _ Q, p _ O, p, _ Q
~ = . . . . _ . .. . . _
22
1 38.8 38.8 39.1 38.8
23
2 49.2 49.2 48.0 50.4
24
3 65.6 59.0 61.5 63.1
2s
4 58.4 59.3 59.1 58.6
26
27 52.1 52.4 54.6 49.9
6 65.5 65.2 64.8 66.0
28
7 63.3 61.8 63.1 62.0
29
8 41.7 39.2 39.9 41.0
9 51.8 50.5 52.9 49.4
31
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
11
I3
14
16
17
18
19
21
22
23
24
26
27
28
29
31
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