Note: Descriptions are shown in the official language in which they were submitted.
~2();Z~69
METHOD FOR THE QUANTITATIV~ MEASUREMENT OF
PHOSPHATIDYL GLYCEROL
The present invention relates to a novel method for
the quantitative measurement of phosphatidyl glycerol.
Recently, in the management of newborn babies, the
respi~atory distress syndrome (RDS) in the perinatal period
constitutes a big problem and the deaths due to it occupy
a high percentage of total deaths. It is regarded that
RDS occurs due to the lack of pulmonary surfactant. This
material is important to prevent contraction of alveoli
pulmonum which expands immediately after the birth. The
lack of this pulmonary surfactant brings about atelectasis
in respiration and occurrence of RDS. Consequently, if the
occurrence of RDS can be estimated in an earlv ~eriod and
the treatment o~ the newborn baby can be starte~ early, th~
occurrence of RDS may be prevented or it may be restrained
to a mild case. Accordingly, the measurement of this pul-
monary surfactant is the means of necessity. The means are
roughly classified into the method for the measurement
wherein the physical property of pulmonary surfactant is
utili~ed and the one wherein compositive components of
pulmonary surfactant are biochemically resolved. The lat-
ter method has been practised by measuring the ratio of
lipids such as the ratio of lecithin to sphingomyelin, that
of phosphatidyl glycerol to phosphatidyl inositol or the
quantitative measurement of dipalmitoyl phosphatidyl cho-
line by means of the thin-layer chromatography ~Am. J.
Obstet. Gynecol., 440: 109 (1971); Am. J. Obstet. Gynecol.,
613: 125 (1976); Am. J. Obstet. ~ynecol., ~94: 133 (1979);
Obstet. & Gynecol., 295: 57 (1981); Am. J. Obstet. Gynecol.,
697: 138 (1980); etc.]. From the results of these various
inspections, it has been recently recognized that the ex-
istence of phosphatidyl glycerol is an important factor
~2V2869
for the occurrence of RDS. However, the quantitative
measurement of phosphatidyl glycerol was very difficult
because the quantity thereof in existence is as little as
about one tenth of the amount of lecithin. Heretofore, as
the method for the quantitative measurement of phosphatidyl
glycerol, there has been known the method of thin-layer
chromatography wherein, after the removal of cell components
by the centrifugal separation from the liquor amnii, lipid
components are extracted from the supernatant, the compo-
n~nts of this extract are separated by the thin-layer chro-
matography, each phospholipid is quantitatively measured,
the ratios of various phospholipids are obtained and the
quantity of phosphatidyl glycerol is obtained indirectly
from the total phospholipid value preliminarily obtained
by the quantitative measurement of phosphoric acid accord-
ing to the wet combustion method [Am. J. O~stet. Gynecol.,
613: 125 (1976); Am. J. Obstet. Gynecol., 1079: 135 (1979);
Am. J. Obstet. Gynecol., 899: 133 (1979); Am. J. Obstet.
Gynesol., 440: 109 (1971)], or the method for the quantita-
tive measurement wherein the high speed liquid chromato-
graphy is used [Journal of Chromatography, 277: 223 (1981)].
However, this method of thin-layer chromatography had dis-
advantages that it takes two to three days for the extrac-
tion of lipid components from liquor amnii, it requires
complicated operations and, further, a heating at a high
temperature is necessary because of the spot detection.
Also it had such other defects that it is an indirect method
for the measurement based on the ratios of relative quanti-
ties from spots and total phospholipid and it can not per-
form simultaneous treatment of many samples because itemploys thin-layer chromatography.
The present inventors carried out various studies
about the method whereby, in a liquid containing various
lipid components to be inspected such as liquor amnii, only
12(~;~869
phosphatidyl glycerol can be quantitatively measured simp-
ly, conveniently and accurately in a short time. As the
result, they found ~uite unexpectedly that phospholipase
C (phospholipase C: 3.1.4.3 phosphatidyl choline choline
phosphohydrolase), which has the enzymatic action to act
on lecithin and catalyze the reaction to produce 1.2-digly-
ceride and phosphatidyl choline acts on phosphatidyl
glycerol producing glycero-3-phosphate and diglyceride and
they achieved a satisfactory method for the quantitative
measurement of phosphatidyl glycerol only by quantitatively
measuring this glycero-3-phosphate produced by the reaction
and extricated. More preferably, they have achieved a
method whereby only phosphatidyl glycerol may be quantita-
tively measured highly satisfactorily by allowing phos-
pholipase C to act on the liquid to be inspected to produce
glycero-3-phosphate allowing glycerophosphate oxidase to
act on this glycero-3-phosphate and measuring the quantity
of oxygen consumed or hydrogen peroxide produced in the
reaction. Thus the present invention relates to a method
for the quantitative measurement of phosphatidyl glycerol
in the liquid to be inspected, which comprises liberating
glycero-3-phosphate by the action of an enzyme which plays
a role of the catalyst in the reaction to produce glycero-
3-phosphate and diglyceride from phosphatidyl ~lycerol and
water and then quantitatively measuring glycero-3-yhosphate
produced; preferably, it relates to a method for the quan-
titative measurement of phosphatidyl glycerol in the liquid
to be inspected, which comprises carrying out the quantita-
tive measurement by the combination of the following steps
~0 (a), (b) and (c):
(a) liberating glycero-3-phosphate by allowing phos-
pholipase C to act on phosphatidyl glycerol,
(b3 allowing glycerophosphate oxidase to act on
glycero-3-phosphate,and then
(c) measuring the quantity of oxygen consumed or
..,
869
-- 4
hydrogen peroxide produced in the reaction.
In the present invention, it is advantageous to pre-
pare each reagent in an adjusted kit for the quantitative
measurement and besides the operation for the quantitative
measurement can be conducted at the room temperature of
about 37C and it is a very simple operation and in addi-
tion, the time required for the reaction is very short.
Moreover, based on the present invention, phosphatidyl
glycerol can be accurately measured down to a markedly low
concentration and the value of phosphatidyl glycerol can
be directly measured revealing a great usefulness. Also,
since the measurement can be conducted simply and con-
veniently in a short time, many samples can be simultane-
ously measured. Thus, the present invention provides a
useful method for the quantitative measurement of phos-
phatidyl glycerol.
First, as an example of the enzyme which plays a role
of the catalyst in the reaction to produce glycero-3-phos-
phate and diglyceride from phosphatidyl glycerol and water
in the present invention, phospholipase C may be included.
This phospholipase C (E.C.3.1.4.3) has been known as an
enzyme which is a catalyst in the reaction to produce each
1 mole of diglyceride and phosphatidyl choline from each 1
mole of lecithin and water. As far as the substance can
be a catalyst in the enzyme reaction mentioned above, there
may be used any one such as the one obtained by the ext~ac-
tion of phospholipase C-containing cells or an enzyme re-
agent sold on the market, for example, a microorganism-
originated enzyme obtained from the culture of a phos-
pholipase C-producing microorganisms which belongs to a
Streptomyces genus such as Streptomyces hachijoensis A-
1143 strain ¦FERM-P No. 1329), Streptomyces albireticuli
IFO 12737, Streptomyces cinn~mnneum IFO 12852 (Strepto-
~20286g
-- 5 --
verticillium cinnamoneum subsp. cinnamoneum IFO 12852).
Streptomyces griseocarinensis IFO 12776 (Streptoverticil-
lium griseocarneum IFO 12776~ [among the bacteria belong-
ing to Streptomyces genus, those which form whirl in its
aerobacteria system are separated from Streptomyces genus
to be named Streptoverticillium genus (Baldacci; 1958)]
or Clostridium welchii and Bacillus cereus [Japanese Ex-
amined Published Patent Application No. 1356/1978, I,ipid
Metabolism P214 (1960)] or enzyme reagent of phospholipase
C sold on the market. The quantity of phospholipase C to
be used should be appropriately modified and designed ac-
cording to the time required for the measurement and the
concentration of phosphatidyl glycerol and no particular
limitation is to be imposed on it. For instance, per one
test, there may be used phospholipase C of usually not less
than 0.1 unit, preferably about 1 - 100 units. In addition,
this phospholipase C is preferably used after it is dis-
solved in a buffer such as weakly acidic to weakly alkaline
Tris-HCl buffer, citric acid buffer, boric acid buffer,
PIPES-NaOH buffer or imidazole buffer andl if necessary,
it may be adjusted by adding thereto a surfactant such as
sodium deoxycholate or serum albumin. Then, the enzyme
solution containing phospholipase C thus adjusted and the
liquid to be inspected are mixed and glycero-3-phosphate
is produced from phosphatidyl glycerol in the liquid to
be inspected with the consumption of water. The mixing
ratio of the both is not particularly limited, and they
may be mixed at such ratio that preferably about 1 - 100
units of phospholipase C is contained per one test of the
liquid to be inspected. The reaction temperature may be
about 37C and the reaction time may be what is sufficient
for the liberation of glycero-3-phosphate,usually not less
than 5 minutes, preferably not less than 10 minutes. Then,
glycero-3-phosphate liberated by the reaction is quantita-
tively measured. From this value of quan~itative ~easure-
., , ~ i,
2136~
~ 6 -
ment, the value of phosphatidyl glycerol in the liquid to
be inspected is obtained.
For the quantitative measurement of glycero-3-phos-
phate, various known methods for the quantitative chemicalmeasurement or those using enzymes may be employed. Pre-
ferably, an enzymatic method ~or the quantitative measure-
ment wherein one species or more of enzymes whose substrate
is glycero-3-phosphate is employed for action and the de-
tectable change of enzyme action in the reaction is quanti-
tatively measured is simple and convenient. For instance,
the GPO type method for quantitative measurement wherein
glycerophosphate oxidase (GPO) is allowed to act on the
liberated glycero-3-phosphate so that the oxygen in the re
action mixture is consumed to produce h~drogen peroxide is
particularly simple and convenient.
Further, in this GPO type method for the quantitative
measurement, the quantity of oxygen consumed in the reac-
tion liquid is measured with an oxygen electrode; thequantity of hydrogen peroxide produced is measured with a
hydrogen peroxide electrode as the electrical change. The
quantity of oxygen or h~drogen peroxide can be measured
by an enzyme electrode wherein an oxygen electrode or a
hydrogen peroxide electrode and immobilized enzyme are assembled.
Immobilized enzymes may be formed by various means such as
inclusion, adsorption and bonding; for instance, GPO or GPO
and phospholipase C are immobilized by a known immobiliza-
tion means such as a method wherein GPO or GPO and phos-
pholipase C are included and immobilized with acrylamide;or a method wherein GPO or GPO and phospholipase C are
blended with a protein such as albumin and the proteins are
cross linked with each other to cause immobilization; or a method
wherein GPO or GPO and phospholipase C are included with
collagen or fibroin or are covalent-bonded with them; or a
l~U2~;9
method wherein GPO or GPO and phospholipase C are immobiliz-
ed by the adsorption onto a porous organic polymer resin or
by the covalent bonding with the same, a method wherein GPO
or GPO and phospholipase C are included and immobilized us-
ing a photosetting resin. The form of the immobilizedemzyme may be the one wherein the immobilized enzyme is
processed into a membranelike, fibrous, granular or tubular
shape which is preferable in use for an enzyme electrode
to be assembled into an oxygen electrode or a hydrogen
peroxide electrode and the immobilized enzyme may be used
as an enzyme electrode wherein this immobilized enzyme is
incorporated into the detecting part of the electrode to be
used so that the amount of the effective enzyme used for the
quantitative measurement by an electrical means is markedly
small.
Further, the quantitative measurement of hydrogen per-
oxide may be carried out by using an indicator composition
which produces a detecta~le material by the reaction with
hydrogen peroxide. As the indicator composition,there usually
are used the compositions whose change may be quantitatively
- measured by a spectroscopic means, for instance, a colora-
tion reagent composition whose color change occurs in the
visible range, a fluorescence reagent composition which
fluoreSces by ultraviolet rays irradiation or a photogenic
reagent composition which gives forth light. For example,
as a coloration reagent composition, a material containing
a substance having peroxidase action and chromogen may be
used. As the substance having the peroxidase action, usual-
ly the peroxidase originated from horseradish is often usedand, as the chromogen, usually the combination of an elec-
tron acceptor and a phenolic compound is often used.
Further, as an electron acceptor, such compound is used as,
for example, 4-aminoantipyrine, 2~hydrazinobenzothiazole,
3-methyl-2-benzothiazolone hydrazine, 2-aminobenzothiazole
~,'
~ ~Z~Z869
. _ ~
or the like. As a phenolic compound, such compound is
used as, for example~ phenol, 3-methyl-N-ethyl~N-(B-
hydroxyethylJaniline , 3,5-xylenol, N,N-dimethylaniline,
N,N-diethylaniline or the like.
As luminous substrates in a fluorescence reagent com-
position or a photogenic reagent composition, there may be
illustrated various known ones, for example, bis(2,4,6-
trichlorophenol)oxalate, phenylthiohydantoin, homovanillic
acid, 4-hydroxyphenylacetic acid, vanillylamine, 3-methoxy-
tyramine, phloretic acid, hordenine, luminol monoanion,
lucigenine, wa~in and the like. Each of them may be, if
necessary, used together with an electron acceptor and/or
a substance having peroxidase action for the quantitative
measurement of hydrogen peroxide.
There is no particular limitation on the quantity of
the enzyme reagent or the chromogen used. For instance,
there may be used per one test usually not less than 0.05
unit, preferably 0.1 - 200 units of GPO and usually not
less than 0.05 unit, preferably 0.1 - 500 units of per-
oxidase. ~lso there may be used a solution so adjusted by
distilled water or weakly acidic or weakly alkaline
buffer solution that the concentration of an electron ac-
ceptor or a phenolic compound i5 usually not less than 0.1
mM. These reagents may be used separately from the enzyme
solution of phospholipase C mentioned above or may be
blended therewith, or further, these reagents may be formed
into such composition for quantitative measurement as a
freeze-dried composition or an integrate~ laminate by ap-
plying them onto filter papers, films or the li~e.
The GPO type method for the quantitative measurement
thus composed shows a high sensitivity to the glycero-3-
phosphate in the liquid to be inspected and, since
v
~ZV'~136~
g
it is not affected by the impurity in the liquid to be
inspected, it is an excellent method whereby an accurate
measurement can be carried out.
The GP0 used for this GPO type method may be any
enzymes as far as it plays the role of a catalyst in the
reaction to produce dihydroxyacetone phosphoric acid and
hydrogen peroxide from glycero-3-phosphate and oxy-
gen, for example, Streptococcus genus, Lactobacillus genus,
Lenconostoc genus, glycerophosphate oxidase producing
bacteria which belong to Pediococcus genus (Japanese
unexamined patent publication No. 7289211978), a enzyme
obtained from the culture of glycerophosphate oxidase-
producing bacteria which belongs to Aerococcus genus
(Aerococcus viridans IFO 12219 strain and IFO 12317 strain,
Japanese unexamined patent publication No. 15746/1980) and
enzymes sold on the market.
As another enzymatic method for the quantitative
measurement of glycero-3-phosphate, glycerophosphate
dihydrogenase which catalyses the reac~ion to produce
dihydroxyacetone phosphoric acid and reduced NAD
from glycero-3-phosphate and nicotine adenine
dinucleotide (NAD) is allowed to act on glycero-3-phosphate
liberated in the solution to be tested in the presence
of NA~ to produce dihydroxyacetone phosphoric acid and re-
duced NAD and then this reduced NAD is quan-
titatively measured so that the quantity of glycero-3-
phosphate may be determined. When the quantitative
measurement of this reduced NAD is conducted, such
method may be usually used as the measurement of absorbance
by a wave length of about 340 nm or, after it is aIlowed
to develop color by a water-soluble tetrazolium salt such
as 3-(4,5-dimethyl)-2-thiazolyl-2H-tetrazolium bromide,
2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-2H-tetrazolium
12~)Z8~9
-- 10 --
chloride, 3,3'-(3,3'-dimethoxy-4,4'-biphenylene)-bis[2-(p-
nitrophenyl-5-phenyl-2H-tetra~olium chloride)](Nitrotetra-
zolium Blue) or 2,6-dichlorophenolindophenol in the pre-
sence of diaphorase or phenazine methosulfate, the color
may be measured in accordance with absorbances using their
special absorption wave lengths.
In addition, as to these enzymes, reagents and the
like, it is simple and convenient to use those sold on the
market and the quantity to be used may be appropriately
designed. Also, if necessary, a surfactant or a stabilizer
may be used and they may ~e processed into various composi-
tions.
In addition, as the liquid to be inspected which is
the object in the present invention, any sample may be
used as far as it contains phosphatidyl glycerol, for in-
stance, body fluids such as amniotic fluid sampled.
When amniotic fluid is the liquid to be inspected, the
sample obtained may be used as it is or when the content of
phosphatidyl glycerol is remarkably small 7 the sample may
be concentrated for instance, 5 - 6 ml of liquor amniotic
fluid sampled is extracted with 15 - 18 ml of chloroform-
methanol (2:1), the chloroform layer is collected by cen-
trifuging at 2000 rpm and it is evaporated to dryness in
nitrogen gas to obtain the total lipid. Then, after dis-
solving this total lipid in a definite amount of 1%"Triton
X-lOO"solution, the obtained solution may be used as the
sample of amniotic fluid. In this way, each enzyme solu-
tion of phospholipase C and other enzymes such as used in
GPO type methods of quantitative measurement mentioned
above and other reagents may be allowed to act on the solu-
tion to be inspected successively or simultaneously. In
this case, there is no particular limitation on the use
* Trademark for octylphenoxy polyethoxy ethanol; it is a
nonionic surfactant.
12{)Z869
11 --
ratio of the liquld to be inspected to the enzyme reagent
and the like and usually, about 0.1 - 3 ml of the solution
of enzyme reagent or the like is used for 0.01 ml - 1 ml
of the liquid to be inspected. As the reaction condition,
it is preferable to conduct the reaction at about 37C and,
as the reaction time, any length may be selected as far as
the reaction is completely terminated; usually the reaction
is continued for not less than 5 minutes, preferably not
less than 10 minutes. Also as the reaction medium, water
or a weakly acidic to weakly alkaline buffer as a solvent
of each rea~ent and the like is used.
Thus, by the quantitative measurement of the liquid
to be inspected, phosphatidyl glycerol may be directly and
quantitatively measured in a very short time down to an
extremely low concentration. In addition, no complicated
operation is needed and the operation may be carried out
at the normal temperature. Consequently, this is a good
method for the quantitative measurement.
Also there is no particular limitation to the method
for the measurement of the activity of phospholipase C and
GPO used in the practical examples mentioned later in the
present invention and they may be illustrated by examples
as follows:
(a] Method for the measurement of the activity of
phospholipase C
0.1 ml of 4% phosphatidyl choline(separated from egg
yolk and purified) solution, 0.3 ml of 0.1 M Tris-hydro-
chloric acid buffer (pH 8.5) and 0.1 ml of 20 mM aqueous
solution of MgC12 are mixed together and to this is added
0.1 ml of enzyme solution containing phospholipase C.
The mixture is allowed to react at 37C for 15 minutes and
the reaction is suspended by adding 0.8 ml of 3.6%
~L2~;~8~9
- 12 -
trichloroacetic acid and 0.1 ml of 5.5~ serum albumin and
the reaction mixture is allowed to stand in water bath for
20 min.,then filtered by Toyo filter paper (No. 5B).
0.5 ml of the filtrate is taken into a Kjeldahl flask,
0.55 ml of 60% HC104 is added to decompose for 2 hours at
170GC, and liberated inorganic phosphor is allowed to develop
color by addition of 0.5 ml of amidol sulfite testing
solution, 0.25 ml of 3.3% ammonium molybdate and 3.2 ml of
water. After being left to stand at room temperature for
20 min.,the absorbance is measured at OD 650 m~. The
activity of enzyme which extricates 1 ~mole of inor~anic
phosphor per 15 minutes is defined as 1 unit (U).
(b3 Method for the measurement of the activity of
GPO
0.2 M Tris-hydrochloric acid buffer (pH 8.0) 0.2 ml
peroxidase (0.5 mg/ml, 45 U/ml) 0.1 ml
0.3% tW/V) 4-aminoantipyrine 0.1 ml
0.1 M DL-glycero-3-phosphate 0.1 ml
0.2% (V/V) N,N-dimethylaniline 0.2 ml
Distilled water 0.3 ml
One mililiter of the reaction liquid having the com-
position mentioned above is charged in a small test tube
and preheated at 37C for 3 minutes. To this is added 20
~1 of enzyme solution containing GPO and the mixture is
allowed to react for 10 minutes. Then, the reaction is
suspended by the addition of 2.0 ml of 0.25% (W/V) sodium
laurylbenzene sulfonate and the absorbance of the product
is measured at a wave length of 565 nm.
The activity of the enzyme is calculated in accordance
with the following equation:
Activity of enzyme (U/ml) = ( 6 o ) x ( 10 )
.~
, .
l~Z86g
- 13 -
wherein ~A shows the absorbance for 10 minutes at a wave
length of 565 nm.
The ~mbodiment of the present invention will be men-
S tioned in the following examples. However, the present in-
vention is not intended to be limited by them.
Fig. 1 and Fig. 2 indicate the quantitative measure-
ment curve of phosphatidyl glycerol.
Example 1
0.2 M dimethylglutarate-NaOH buffer (pH 7.5) 0.2 ml
10 mM CaC12 0.2 ml
10% sodium deoxycholate 0.05 ml
1% Bovine serum albumin 0.1 ml
Solution containing 40 U/ml phospholipase C
and 50 U/ml GPO 0.1 ml
45 U/ml peroxidase 0.1 ml
0.3% 4-aminoantipyrine 0.2 ml
0.3% 3-methyl-N-ethyl-N-~-hydroxyethyl~aniline 0.2 ml
Distilled water 0.~5 ml
Total 2.0 ml
2.0 ml of reaction composition for quantitative measure-
ment of phosphatidyl glycerol having the above compositionis prepared and thereto is added 50 ~1 of solution to be in-
spected which contains various phosphatidyl glycerol ~con-
tent of phosphatidyl glycerol being 20 n moles ~ 100 n moles~
and the mixture is reacted at 37C for 15 minutes and then
the color development of the pigment produced after reaction
is measured by absorbance at the wave length of 550 nm (OD
550).
The results are as shown by Fig. 1. A satisfactory
tendency of linear relation was obtained as against the
,
~;~0~8~g
- 14 -
content of phosphatidyl glycerol of the inspected solution.
Example 2
0.2 M dimethyl glutarate-NaOH buffer (pH 7-5) 0 2 ml
10% sodium deoxycholate 0.05 ml
Solution containing 20 U/ml phospholipase C
and 50 U/ml GPO 0.1 ml
~5 U/ml peroxidase 0.1 ml
0.3% 4-aminoantipyrine 0.2 ml
0.3~ phenol 0.2 ml
1% Bovine serum albumin 0.1 ml
Distilled water 1.05 ml
Total 2.0 ml
2.0 ml of reaction composition for quantitative
measurement of phosphatidyl glycerol having the aforesaid
composition is prepared, to such solution is added 50 ~l
of the solution to be inspected containing various phos-
phatidyl glycerol (content of phosphatidyl glycerol being10 n moles ~100 n moles), the mixture is allowed to react
for 15 minutes at 37C and the color shade of the pigment
produced after reaction was measured by absorbance at wave
length of 500 nm (OD 500).
The results are as shown in Fig. 2, where a quantita-
tive measurement curve having satisfactory linearity against
the content of phosphatidyl glycerol was obtained.
