Note: Descriptions are shown in the official language in which they were submitted.
il~OQ23
The present invention relates *o the analysis of
aqueous liquids for triglyceride and/or glycerol content and
more specifically to improved methods and compositions for
the assay of blood serum triglycerides.
The determination of serum triglyceride levels is
becoming increasingly important in the diagnosis of several
types of hyperlipemia and atherosc~erotic heart disease
~Kahlke, W. Med. Wscht. 91, I!. 26 (1966), Kuo, P.T. and
Basset, D.R., Amer. Intern. Med., ~, p. 465 (1963). Con-
lû ventional procedures for serum triglyceride determination
involve hydrolyzing the triglyceride to liberate glycerol and
treating the glycerol with various reagents to produce a
compound that can be quantitated spectrophotometrically.
Generally hydrolysis is achieved using a base, however, U.S.
Patent Nos. 3,703,591 to Bucolo et al and 3,759,793 to Stork
et al describe enzymatic techniques using a lipase alone ('793)
or in combination with a protease ('591) to achieve hydrolysis.
Other non-enzymatic hydrolysis techniques are described
~n German Patent Nos. 2,229~849 and 2,323,609.
Currently three enzymatic methods are conventionally
used for the determination of glycerol from. whatever source.
These are as follows:
1100023
~ .
'~
~, & E~
S ~ s~
9~, G ~4 C ~ V ~ c~ ^ 0
01 \ 't ~ 0 X o ~?
Ct) ~1~d o '~ -1 0 ~7 O O
~ O~ bO ~ ~ O S: c S
o ~. c~ ~ i` ¦ c j o ¦ s
. ~5 ~ ~0 ~ 0 ~ C ~ ~ O
a~ ~ ~ ~ ~ ~ c~ ~,
:~, ha5 S a~ :~, a~ ~ ~ S
S:: rl~ qD ~ ~
~ CO ~
E-l S ~ ~ ~ ~ S :;';
6,~ + P~+ ~ 1 ~0
O --I S a) ~
O ~ o O
23
Modifications of the method of (a) are also described
in German Patent No. 2,665,556, British Patent No. 1,322,462
and U.S. Patent No. 3,759,793. In all cases NADH production
or disappearance is measured at 340 nm in a U.V. spectrophoto-
meter. Method (a), utilized in many commercial "kits," is a
three enzyme sequence and NADH disappearance is measured.
Method (b) involves a two enzyme sequence in which NADH pro-
duction is measured as is the case with the single enzyme
glycerol dehydrogenase reaction (method (c)). The latter two
procedures are extremely pH-sensitive and subject to error if
strict pH control is not maintained. Also, in all three methods
(especially method (a)) stability of not only the diagnostic
enzymes but also the cofactor, NADH, is a major concern.
Errors in current enzymatic methods are discussed in greater
detail in Chen, H.P. and El-Mequid, S.S., Biochemical Medicine,
7, p. 460 (1973)-
Another method for triglyceride analysis is describedln German Patent No, 2,139,163. The method of this patent
involves hydrolysis of the triglycerides~ oxidation of
the resulting glycerol to formaldehyde and reaction of the
formaldehyde with ammonia and a stable, water- and alcohol-
soluble, colorless metal complex of acetylacetone to produce
a colored compound.
It is an ob~ect of the present invention to provide
improved methods and compositions for the quantitative determlnation of
glycerol and triglycerides, especially serum triglycerides,
which methods and composltions are relatively free of any
requirement for strict and narrow pH control and major concerns
~or reagent stability.
~lOt~Q23
According to the present invention, there is provided
a novel method for the detection of glycerol (as either a free
glycerol or glycerol formed by hydrolysis of fatty acid esters
of glycerol, e.g., triglycerides) in aqueous liquids compris-
ing the steps of:
(I) contacting in the presence of an electron acceptor:
(1) a sample of the aqueous liquid and
(2) enzymes and other reagents which effect an ordered
sequence of reactions, preferably quantitative,
wherein fatty acid esters of glycerol, if present,
are enzymatically hydrolyzed to glycerol, and glyc-
erol whether present in the free form initially or
liberated by hydrolysis of the esters is converted to
L-a-glycerophosphate which in turn is enzymatically
oxidized, producing a detectable change, and
(II) detecting the occurrence of the detectable change.
According to a preferred embodiment, the electron acceptor is
oxygen and an indicator composition which produces a detecta-
ble product on contact with hydrogen peroxide is included as a
reagent. The detectable product is generally a colored mate-
rial which, according to a highly preferred embodiment, is
quantifiable.
According to a further preferred embodiment, triglyc-
erides present in the aqueous solution are hydrolyzed using a
lipase.
According to yet another preferred emkodiment, glyc-
erol is converted to L-~-glycerophosphate using glycerol
kinase and the oxidation of L-~-glycerophosphate takes place
in the presence of L-~-glycerophosphate oxidase.
11~0~P23
A most preferred embodiment utilizes an indicator
composition comprising substance having peroxidative activity
and a dye precursor, the dye precursor comprising either (1) a
compound which forms a dye in the presence of hydrogen perox-
ide and substance having peroxidative activity or (2) a com-
pound or series of compounds which undergo no detectable
change in the visible range in the presence of hydrogen perox-
ide and substance having peroxidative activity but which
interact with another compound or series of compounds to pro-
duce a quantifiable product proportional to the glycerol or
triglyceride content of the sample under analysis.
Brief Description of the Drawings
A detailed pH-activity profile of the a-glycerol-
phosphate oxidase from strain 12755 is shown in Fig 1. Also
shown in Fig l is the apparent inhibition of the enzyme by
certain buffers.
A glycerol response curve for an element of the
invention is shown in Fig 2. For comparison, calibration data
for hydrogen peroxide are also shown in Fig 2.
The method of this invention represents an improve-
ment over prior-art methods and compositions in that the
instant method and compositions do not rely on the production
or disappearance of NADH with its attendant disadvantages
which are well-recognized and documented in the art.
In the present invention, triglycerides are prefera-
bly determined quantitatively by the following series of reac-
tions:
~ 00023
`
o ~
- l c
~ o
0 _1
~ 0
0 h
0 ~ ~ C
0 ~ ~0
S 0
~ 'I '~5 ~
O C) ~ 0
S '/ 0 0
) h
C C ~ C ~D !
0 C O
h 0--I 0
h O S
3 ~ ~ s o
+ .~ ~, ~ o o
,
0 s ~ C
~o ~ a S ~, u
~ E S ~
,~ ~ h ,~ ~, X ~C.~ C
h ~ ~ O ~ ~1 0
J o _ ~ .c
O ~ 3
bD a) x "S ~ ~ o
0 S~ ~ 0 X
~ ~ c c o a~ ~ o
~ S~ O ~ .
~: a) 0 ~ x ,D
~r1 O ~ h h
' ~ ~ h
C~ ~ ~ C ~ .
:C ~
~ ~ O Q~
E~ .C S C
0 `~ ~ X
O ~ ~ O
0 ~ 'C C~--
h 0 _I 0 C'~l X
c~ o ~ C a~
_I ~ ~ b~ C
bD C~ I ~, N
,1 7~ o ON
S
_I CU ~ S ~ t
23
In the combined reactions of the preferred composition,
formation of the detectable species is proportional to glycerol
and/or triglyceride concentration. This system has potential
use in many clinical applications, in particular, the deter-
mination of serum triglycerides.
The procedure of this invention has many inherent
adrantages over conventional methods. First, any leuco dye that
peroxidase will utilize as ~n electron donor is potentially
useful in the indicator composition; thus one can measure
the reaction at one of several wavelengths in the visible
region of the spectrum; depending upon dye selection. Secondly,
measurements made in the visible region are less subject to
interferences than those taken at 340 nm. Third, in addition
to dyes,substantially any means for detecting hydrogen peroxide
can be used. ~ourth, sta~ility of NAD+ or NADH is not a
concern since 2 is the cofactor in the a-glycerophosphate
oxidase reaction. Fifth, serum components that utilize NAD+
or NADH (for example, lactate plus lactate dehydrogenase)
which might interfere with prior art reaction sequences, do
not interfere with the instant procedure. Sixth, any means
w~ich n~as ~ s 2 consumption can be used as a detection means
when 2 is used as the electron acceptor. Finally, the
enz~mes used in th~ proposed sequence are active over a
relatively wide pH range, thus stringent pH control is not
necessary.
Although the discussion hereinafter will center
pr~marily around solutions and solution methods for quantifying
glycerol and triglycerides, it should be readily apparent to
the skilled artisan that all of the reagents may be provided
3 in dry or lyophilized form and reconstituted with water
ll~Q~23
immediately prior to use. Compositions of this type are
clearly contemplated hereby.
Hydrolysis:
In its most sophisticated embodiment the method of
the present invention is utilized to assay a~ueous liquids,
for example blood serum, for triglyceride content. According
to this embodiment triglycerides are hydrclyzed to free glycerol
by means of any of the well known techniques described in the
art. Enzymatic techniques are preferred.
These generally involve treatment of the serum sample with a
lipase, either in combination ~ith an
effector such as a protease or a surfactant or alone depending
upon the nature of the triglyceride. Detailed discussions of
such techniques and useful compositions for their performance
are contained in U.S. Patent No. 3,703,591 to Bucolo et al
issued November 21, 1972 and U.S. Patent ~o. 3,759,793 to
Stork et al issued September 18, 1973. Bucolo et al uses
a lipase preferably from Rhizopus arrhizus (var. delemar) and
similar materials in combination with a protease to achieve
hydrolysls of serum triglycerides while Stork et al dis-
closes the use of lipase from Rhizopus arrhizus alone to
achieve hydrolysis.
A rurther method ~nvolves
the hydrolysis of serum triglycerides using a compatible
mixture of a lipase which normally, of itself~ is not capable
of hydrolyzing protein associated triglycerides as found in
serum and, as an effector, a compatible surfactant.
, _
llO~CZ3
A compatible surfactant is one which stimulates triglyceride
hydrolysis by the lipase as described in the test below.
Thus, such a surfactant will not inhibit the activity of the
lipase, but actually enhance it. The lipase is preferably
from Candida cylindracea (Cand da ru~osa).
Useful lipases for triglyceride hydrolysis according
to any of the foregoing techniques may be of plant or animal
origin but we prefer, and find best, microbial lipases, such
as the lipase from Candida cylindracea, when the lipase is
used in combination with a surfactant as described below.
Lipases froim Chromobacterium viscosum, variant paralipolyti-
cum crude or purified, the lipase from Rhizopus arrhizus
(variant delemar), purified, for example, as noted by Fukumoto
et al, J Gen Appli Microbiol, 10, 257-265 (1964), and lipase
preparations having similar activity are also useful.
Other useful lipases and methods for their prepara-
tion are described in the following US patents:
2,888,385 to Grandel issued May 26, 1959
3~168,448 to Melcer et al issued February 2, 1965
3,189,529 to Yamada et al issued June 15, 1969
3,262,863 to Fukumoto et al issued July 26, 1966
3,513,073 to Mauvernay et al issued May 19, 1970
Because the lipases are readily available in lyophi-
lized form, they are easily incorporated into either dry mix-
tures for reconstitution with water or provided as stable
solutions of reagent which can be combined with other such
solutions to provide reaction mixtures for contact with sam-
ples for analysis.
Specifically preferred commercial lipases include
wheat-germ lipase supplied by Miles Laboratories, Elkhart,
Indiana, Lipase 3000~ supplied by Wilson Laboratories,
Steapsin~ supplied by Sigma Chemical Company (the last two
11q)~23
being pancreatic enzymes), and Lipase M~ (from Candida cyl-
indracea (Candida rugosa)) supplied by Enzyme Development Com-
pany.
Nonionic and anionic surfactants have been found use-
ful in combination with lipase preparations which of them-
selves are incapable of hydrolyzing serum triglycerides. Most
preferred from among such materials are the octyl and nonyl
phenoxy polyethoxy ethanols, such as those commercially
available from Rohm and Haas Company under the Triton trade-
mark. Best results are obtained with such surfactants when
the HLB number (hydrophile-lipophile balance) is below about
15 and the number of polyoxyethylene units in the polyoxyeth-
ylene chain is less than about 20.
Compatible compositions of lipase and surfactant are
readily defined by the following test:
The surfactant of the compositior. under evaluation
is added to unbuffered reconstituted serum (specifically
ValidateT~, a serum standard available from General Diagnos-
tics Division of Warner Lambert Company, Morris Plains, New
Jersey) at varying concentrations of between about 0 and 10
percent by weight and the solution incubated for about 5 min-
utes at 37 C. At this time, a sample of the proposed lip-
ase preparation is added and incubation continued for a
period of about 20 minutes. Aliguots (~0.2 ml) of this
solution are then diluted to 1.6 ml with water (contain-
ing 1.3 mM CaC12 to aid precipitate formation), placed
in a boiling water bath for 10 minutes and centrifuged
-11-
Z3
to clarify (4C, 37,000 Xg, 10 minutes). Glycerol in a 0.4 ml
aliquot of the clear supernatant is quantitatively determined in a total
volume of 1.2 ml by the method described by Garland, P.B. and
Randle, P.J., Nature, 196, 987-988 ti962). Any composition
which effects release Or at least about 50~ of the theoretical
concentration of available glycerol is considered useful and
within the scope of the present invention. When performing
the foregoing test it is most desirable to run a blank which
contains all of the components of the mixture but the lipase
preparation so that any reaction which may be due to free
glycerol or other components of the serum can be subtracted.
. . . _ . _ . . . _ _ . .
The preferred compositions accomplish at least 75% hydrolysis
Or the available triglyceride to glycerol in less than 10
minutes and most preferred are those which achieve subst2ntially
complete hydrolysis of the available triglyceride to glycerol,
i.e., a~ove abou~ 90%, in less than about 10 minutes. Examples
Or such preferred compositions are shown in Table II below.
When, for one reason or another, the pr~tease-lipase
combination of the prior art is used for hydrolysis, proteases
in general may be used. These include by way of example,
chymotrypsin, StrePtomyces griseus protease (commercially avail-
able under the registered trademark "Pronase"),'proteases from
Asper~illus oryzae and Bacillus subtilis~ elastase, papain, and
bromelain. Mixtures of such enzymes may,of course,be employed.
The useful concentrations of lipase and other
effectors such as surfactants, protease, etc. will vary broadly
depending upon the time limitations imposed on the assay, etc.
and these are readily determined by the skilled artisan. Typical
nonlimiting examples of useful concentrations are described in
the exam~les below. _ __
.
-12-
110~23
Hydrolysls of triglycerides can also be achieved
using any of the well known prior art "non-enzymaticl' techniques
~or obtaining the free glycerol prior to assay,
lncludlng treatment with a strong base.
Caution must be exercised, however, to insure that the glycerol
is delivered to the enzymatic glycerol assay composition in a
medium which does not contain materials which would inhibit
the enzymes of the glycerol assay system or otherwise interfere
with the reactions necessary to achieve an accurate glycerol
10 determination.
Glycerol A~say
Once triglyceride hydrolysis has been achieved,
the novel enzymatic ~lycerol assay of the
present invention can be implemented.
As shown in the reaction above, the first enzyme
used in the glycerol assay is glycerol kinase which catalyzes
the conversion of clycerol to L-~-glycerophosphate in the presence
of adenosine triphosphate (ATP). Generally, any glycerol
kinase is useful in the successful practice of the present
20 invention although those obtained from E. coli and Candida mycoderm
are preferred. Other glycerol kinase enzymes are well known in
the art. A complete discussion of such materials and further
references to their preparation and reactivity may be found in
T. E. Barman, Enzyme Handbook, I, Springer-Verlag, N.Y. (1969)
pgs. 401-402. Glycerol kinase from Worthington Biochemical Company
provides a satisfactory commercial source of the enzyme.
The next step in the reaction sequence involves the
oxidation of L-a-glycerophosphate in the presence of L-a-
glycerophosphate oxidase and an electron acceptor to produce
30 a detectable change. The detectable change is preferably a
11(~4~@Z3
color change or color formation which, in the preferred case,
is quantitatively related to the glycerol contained in the
liquid sample. Other detectable changes such as oxygen con-
sumption may also be monitored to detect the analytical result.
Any electron acceptor which will permit oxidation
of the a-glycerophosphate in the presence of the oxid se en~ with the
concomit2nt production of a detectable change is a suitable
candidate for use in this reaction. Particularly preferred
as electron acceptors are materials which provide, directly
or indirectly, a radiometrically detectable, preferably
colored product. m e utility of any particular electron
acceptor can be determined by experimentation with
potentially useful electron acceptors.
A highly preferred electron acceptor is oxygen
which will oxidize the L-x-glycerophosphate in the presence
of th~ oxidase to dihydroxyacetone phosphate and hydrogen
peroxide. Methods for determining hydro~en peroxide and
measuring the consumption of oxygen in reactions of this
type are, of course, well known. An alternative preferred
embodiment uses,as electron acceptor,material colored or
uncolored which undergoes a change in or the production of
color directly upon reduction in the presence of the enzyme
and the substrate. As described above, such materials can
be selected by testing in a specific use environment.
Such an environment is described in Example 5 below. Using
this method certain indolphenols~ potassium ferricyanide
and certain tetrazolium salts have been found to be useful
electron acceptors. Specifically, 2,6-dichlorophenolindol-
phenol alone or in com~ination with phenazine methosulfate
and 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-2H-tetrazoli~m
chloride either alone or in combination ~lth phenazine
~ )l
23
methosulfate have been found useful as electron acceptors
in this reaction.
The detectable change may also be determined using
potentiometric techniques, for example, by measuring oxygen
consumption using an oxygen electrode.
L-~-glycerophosphate oxidase is a microbial enzyme
which can be derived from a variety of sources. The properties
of enzyme from certain sources are more desirable than those
from others as will be elaborated below. ~enerally, the enzyme
may be obtained from Streptococcaceae, Lactobacillaceae and
Pediococcus. The enzyme from cultures of strePtococcus
faecalis, specific strains of which are obtainable from the
American Type Culture Collection, are specifically preferred.
~ic~rly usef~ and prefe ~ d en~s ~ obtained from st~ns ATCC 11700,
ATCC 19634 and ATCC 12755 identified on the basis of their
deposit in that collection. As will be described and demon-
~trated by example below, the enzyme from ATCC 12755 demonstrates
activity over a somewhat broader pH range than enzymes derived
from the other two strains and for this reason is most pre-
ferred.
The following two references describe both the enzymeand useful techniques for its preparation and extraction
Koditsche~ L.K. and Umbreit, W.W. ~-Glycerophosphate Oxidase
in Streptococcus faecium, F 24,"Journ~l of Bacteriology,
Vol. 98~ No. 3, p. 1063-1068 (1969) and Jacobs, N.J. and
Van Demark, P.J. "The Purification and Properties of the
a-Glycerophosphate Oxidizing Enzyme of Streptococcus faecalis,
10 Cl." Enzymes prepared according to the methods described
in either of these publications are useful in the successful
practice of the inven~ion. When any enzyme preparation of
unknown total composition is used, care should be exercised
1100@23
to extract any contaminants which may interfere with assay
results. For ex2mple, certain preparations of L-~-glycero-
phosphate ~xidase, derived as described belo.~, contained
sufficiently high concentrations Or impurities that the
crude pre~aration had to be purified using conventional
fractionation and column separation techniques before assays
of blood serum triglycerides free from unwanted interferences
could be achieved.
Detection of glycerol in aqueous solutions cont2iliing
glycerol and/or triglycerides, for example blood serul:~, is
preferably achieved using an indicator composition which
! detects the level of hydrogen peroxide produced in the
oxidation of L-a-glycerophosphate in the presence of oxygen.
Indicator compositions for the detection of enzymatically
~enerated hydrogen peroxide are well known in the art,
particularly as indicator compositions in the enzymatic
detection of glucose and uric acid. U.S. Patent Nos. 3,092,465
and 2,981,606 among many others describe such useful indicator
compositions.
The hydrogen peroxide indicator composition generally
comprises a substance having peroxidative activity, preferably
peroxidase, and a dye precursor which undergoes a color formation
or change in the presence of hydrogen peroxide and the substance
having peroxidative activity. Alternatively, the dye precursor
may be one or more substances which undergo no substantial
color change upon oxidation in the presence of H202 and
substance having peroxidative activity, but which in their
oxidized form react with a color-forming or -changing substance
(e.g., a coupler) to give visible evidence of chemical reaction.
~. S. Pat~nt No. 2,981,606 in particular provides a detailed
ll~ Z~
description Or such indlcator compositlons. The latter
dye precursor, i.e., one which produces color by virtue Or
a coupling reaction, is preferred ~n the practice of the
present lnvention.
A peroxidase is an enzyme whlch wlll catalyze a reactlon
wherein hydrogen peroxlde or other peroxlde oxidizes another substance.
The peroxidases are generally conjugated proteins containing
~ron porphyrin. Peroxidase occurs in horseradish, potatoes,
figtree sap and turnips (plant peroxidase); in milk (lacto
peroxidase); and in white blood corpuscles (verdo peroxidase~;
also it occurs in microorganisms. Certain synthetic peroxidases,
such as disclosed by Theorell and Maehly in Acta Chem. Scand.,
Vol. 4, pages 422 - 434 (1950), are also satisfactory. Less
~atisfactory are such substances as hemin, methemoglobin,
oxyhemoglobin, hemoglobin, hemochromogen, alkaline hematin,
hemin derivatives, and certain other substances which have
peroxidative activity.
Other substances which are not enzymes but which
h ve peroxidative activity are: iron sulfocyanate,
iron tannate, ferrous ferrocyanide, chromic salts (such as
potassium chromic sulfate) absorbed in silica gel, etc. These
substances are not as satisfactory as peroxidase~per se~but
are similarly useful.
Dye precursors whlch produce a color formation in the
presence of hydrogen peroxide and a substance having peroxidatlve
activity lnclude the following substances, with a coupler where
necessary:
-17-
~QC~23
(1~ Monoamines, such as aniline and its derivatives,
ortho-toluidine, para-toluidine, etc.;
(2) Diamines, such as ortho-phenylenediamine, N,N'-
dimethyl-para-phenylenediamine, N,N'-diethyl phenylenediamine,
benzidine, dianisidine, etc~;
(3) Phenols, such as phenol per se, thymol, ortho-,
meta- and para-cresols, alpha-naphthol, beta-naphthol, etc.;
(4) Polyphenols, such as catechol, guaiacol,
orcinol, pyrogallol, p,p-dihydroxydiphenyl and phloroglucinol;
(5) Aromatic acids, such as salicyclic, pyrocatechuic
and gallic acids;
(6) Leuco dyes, such as leucomalachite green and
leucophenolphthalein;
(7) Colored dyes, such as 2,6-dichlorophenolindo-
phenol;
(8~ Various biological substances, such as epinephrine,
the flavones, tyrosine, dihydroxyphenylalanine and tryptophane;
(9) Other substances, such as gum guaiac, guaiaconic
acid, potassium, sodium, and other water soluble iodides; and
bilirubin; and
(10) Such particular dyes as 2,2'-azine-di(3-ethyl-
benzothiazoline-(6)-sulfonic acid) and 3,3'-diaminobenzidine.
- 18 -
23
Otherlndicator composltions that are oxidizable bY Per
oxldes in the presence of peroxidase and can provide a radio-
metrically detectable species include certain dye-providing
compositions. In one aspect indicator compositions can include
a compound that, when o~dized in the presence o~ peroxidase, can couple
with itself or with its reduced form to provide a dye. Such
autocoupiing compounds include a variety of hydroxylated com-
pounds such as orthoaminophenols, 4-alkoxynaphthols, 4-amino-5-
pyrazolones, cresols, pyrogallol, ~uaiacol, orcinol, catechol
phloroglu~inol, p,p-dihydrox~ydiphenyl, gallic acid, pyrocatechuic
acid, salicylic acid, etc. Compounds of this type are well known
and described in the literature, such as in The Theory of the
Photographic Process, Mees and James Ed~ (1966), especially at
Chapter 17. In another aspect, the detectable change can be
provided by oxidation of a leuco dye in the presence of peroxidase to
provide the corresponding dyestuff form. ~epresentative leuco
dyes include such compounds as leucomalachite green and
leucophenolphthalein. Other leuco dyes, termed oxichromic
compounds, are described in U.S. Patent No. 3,880,658 and it
is further described that such compounds can be diffusible
with appropriate substituent groups thereon. The non-stabilized
oxichromic compounds described i-n U.S. Patent No. 3,880,65~
are considered preferable in the practice of this invention.
In yet another aspect, the detectable change can be pro~riàed
by indicator compositions that include a compound oxidizable ir t~e
presence o~ peroxidase and capable o~ undergGin~ oxidative conAen~t
with couplers,such as those containin~ phenolic ~roups or
activated methylene groups.
Representative such oxidizable compounds include such compounds
3 as benzidine and its homolo~s, p-phenylenediamines, p-aminophenols~
--19-
023
4-aminoantipyrine, etc. A wide range of such couplers, including
a number of autocoupling compounds, is described in the
literature, such as in Mees and James (supra) and in Kosar,
Light-Sensitive Systems, 1965, pages 215-249.
The indicator composition of the present
in~ention preferably comprises 4-methoxy-1-naphthol which
undergoes self coupling in its oxidized state or a combination
Or 1,7-dihydroxynaphthalene and 4-aminoantipyrine (~Cl). In
the latter composition the oxidized pyrine compound couples with
the dihydroxynaphthalene. The concentrations of the components
of the various indicator comp~sitions useful in the
elements described herein are dependent to a large extent
upon the concentration of glycerol in the sample, the
sophistication of the detection apparatus, the dye produced,
etc., and are readily determinable by the skilled artisan.
Typical values are shown in the examples below.
Of course, other means for detecting hydrogen
peroxide may also be used in the successful practice of the
present invention. For example, enzymes and other reagents
as described herein can be incorporated into membranes of
oxygen sensitive polarographic electrodes as described in
Rawls, Rebecca ~.~ "Electrodes Hold Promise in Biomedical
Uses," Chemical and Engineering News, January 5, 1976, p. 19.
As a further alternative, instead of measuring the
hydrsgen peroxide produced, it is also possible to measure oxygen
consumption using an oxygen sensitive electrode and thereby
determine the quantity of glycerol produced in above-described
reaction (1) of Table I which would result in the consumption
of that quantity of oxygen in reaction (3) in Table I.
-2~-
0~3
me concentration of the other components of the
no~el assay compositions described herein may also Yary
broadly depending upon the solution under assay (i.e. blood
serum, diluted or undiluted, or other complex aqueous solution
of glycerol and/or triglycerides). Table II below provides a
ready reference for the generally useful and preferred
concentration ranges of the various components of the novel
assay compositions described herein.
Table I~
Generally useful Preferred
Enzyme range U/ml level U/ml
Lipase (when used) 20-160 80
Glycerol kinase .05-1 0.2
Glycerophosphate 1-10 4
oxidase
Protease (when used) -30-2400 1200.0
Peroxidase 0.2-1.4 0.7
-
g/ml g/ml
Surfactant (when used) .01-.05 .02
Of course,useful results may be obtained outside of
these ranges-
.
In the foregoing Table II, one international unit of
enzyme is defined as that quantity of enzyme which results in
the conversion of one micromole of substrate in one minute at
37C and pH 7
As is well recognized in the àrt, each of the enzymes
possesses a pH-activity profile, i.e., the activity of the
23
enzyme varies with pH. These data are described ln detall for
~-glycerophosphate oxidase in the Examples. As demonstrated
by that data~ the pH activity profile of L-~-glycerophosphate
oxidase peaks at between about pH 5 and 8.5. The pH range
over which each of the enzymes in the novel reaction sequence
i8 most active is shown in Table III.
Table III
pH-~alue
Lipase 5_9
Glycerol kinase 7_9
L-~-Glycerophosphate oxidase 6.3-8.0
Peroxidase 6-8
From the foregoing table, it is readily apparent
that it is most desirable to buffer the assay compositions
describ~d herein at a pH of between about 6.o and about 8.o
and most preferably between about 7.0 and about 8Ø Techniques
for achieving this type of buffering are well kno~n in the art
and involve dissolving, dispersing, or otherwise distributing,
suitable concentrations of buffer materials in the reagent
20 composition or, alternatively, providing them in dry form when
a reconstitutable mixture is provided. Suitable buffers for
buffering to the aforementioned pH levels are described in
detail by Gvod in Biochemistry 5, 467 (1966). Particularly
preferred buffers are the phosphates, such as potassium phosphate.
The concentration of detectable species produced can,
Or course9 be detected using any of the well known methods.
For example, by comparison to a standard color chart, spectro-
photometrically, etc.
22
llOQQ23
The following enzyme-preparation techniques and stan-
dardized procedures and compositions were used in the examples
which follow.
Standard Solutions - Exact concentrations of glycerol standard
solutions were determined by the method of Garland and Randle,
Nature, 196, 987-988 (1962). Hydrogen peroxide solutions were
standardized by measuring the A240 (optical absorbance at
240 nm) and using E240=43.6 for pertinent calculations.
Serum samples were analyzed for triglyceride concentration by
the semiautomated fluorometric method of Kessler and Lederer,
"Fluorometric Measurement of Triglycerides, Automation in Ana-
lytical Chemistry", Technican Symposia, L T Sheggs, Jr, Ed,
Medical, Inc, NY, NY, 341 (1966).
Glycerol and Tri~lyceride Quantitation by the ~-GP Oxidase
Method - Incubation mixtures for glycerol detection contained
in a total volume of 1.0 ml: 200 ~moles potassium phosphate
buffer, pH 8.0; 4.2 purpurogallin units horseradish peroxi-
dase; 2.5 ~moles MgSO4; 2.4 ~moles ATP; 10 mg Triton X-
100~; 96 ~g 4-aminoantipyrene hydrochloride; 32 ~g 1,7-
dihydroxynaphthalene (added as an 0.8 percent solution in
ethanol); and 4 units of ~-GP oxidase. (Excess glycerol
kinase was present in the ~-GP oxidase preparation.) For
triglyceride guantitation, incubation mixtures contained 10 mg
(8 units/mg) lipase from Candida rugosa in addition to the
above components. All components were equilibrated at 37 C
for 5 minutes and A4go (initial) was determined. Reactions
were initiated by addition of either a glycerol standard (5-
100 nmoles) or serum ~20 ~1) and allowed to proceed for 20
to 30 minutes. The A490 (final) was then measured. Varia-
tions of this standard system are indicated where necessary.
l~Q1~23
Calculation of Triglyceride Concentration_ - Triglyceride
glycerol concentrations of unknown samples were determined in
the following way: The aA490 (A490 (f~nal) minus A490 (initial))
for samples incubated in the presence of the standard glycerol
detection system was subtracted from the aA490 of the same
samples incubated in the presence of Lipase M and the standard
glycerol detection system. Triglyceride concentrations were
determined from this Lipase M depen~ent change in absorbance by
use of a calibration curve with either glycerol or pre-analyzed 1,
10 serum samples as standards.
Growth of S ~ecalis
S. faecalis (species designated in Table IV below)
was maintained on slants containing 0.1% glucose, 1% tryptone,
1% yeast extract, o.65~ K2HP04 and 1.5% agar. Water suspensions
of the slant colonies (0.2 ml of 1.0 ml suspension per flask)
were used to inocu'ate flasks filled with 25 ml of media each.
These were shaken at 120 rpm (2 inch throw) in a New Brunswick
Psycrotherm Incubator Shaker at 30C for 22 hours.
Preparation of Cell-Free Extracts
The cells from 10~ ml of media were harvested by
centrifugation (4C, 10,000 X g, 10 min), washed with 40 ml
of c~ld 0.05 M potassium phosphate buffer, pH 7.0, centrifuged
again, and suspended in 10 ml of buffer. Cells then were
disrupted by sonication (Branson J-17A sonifier operating at
a setting of 40) in a Rosett cooling cell for 7 minutes; the
temperature was maintained below 8C~ The supernatant from a
centrifugation at lO,OOOX g for 10 minutes was used as enzyme
source. In all cases the amount of soluble protein had reached
23
a maximum during the indicated sonication period. Using
bovine serum albumin as standard, protein concentration was
determined by the method of Lowry et al (Lowry, D.H.,
Roseborough, N.S., Farr, A.L. and Randall, R.J., J. Biol.
Chem. 193, 265 tl951).
.
Isolation of a-Glycerophosphate Oxidases from
Streptococcus faecalis
The results in Table IV compare ~-glycerophosphate
oxidases isolated from three strains of Streptococcus faecalis.
In each case the organism was cultured aerobically at 30C for
22 hours in a glucose medium, cells were collected by centrifug-
ation and then disrupted by sonication. Routinely,the super-
natant from a 10,000 X g centrifugation was used as enzyme
~ource (crude extract). However, the oxidases remained in
solution even after centrifugation at 100,000 X g fcr 1 hour.
In all cases the rate of decrease in dissolved oxygen was
proportional to the amount of crude extract and was absolutely
dependent on both D, L-~-glycerophosphate and the extract. As
can be seen, all three strains displayed oxygen-linked activity.
The enzyme from strain ATCC 11700 reportedly has a pH optimum
of 5.8 and the oxidase from strain ATCC 19534 displays a
maximum at pH 7Ø This trend is also seen in Table IV.
By analogy the activity from strain 12755 was similar to the
one from strain ATCC 19634.
23
Table IV
solation of ~-Glycerophosphate Oxidases
from Three Strains of
. strePtococcus faecalis
Reactions were carried out at 21 in 0.05 M
potassium phosphate buffer at the pH indicated
with 0.13 M DL-~-glycerophosphate as substrate.
pH of
S. faecalis Incubation Decrease in %
cultureMixture Dissolved 2
_
~O/min/mg
ATCC 11700 6.o 4.52
7.3 1.4
ATCC 19634 6.1 3.94
7.5 ~.90
ATCC 12755 5.~ 4.93
6. 7.10
110~23
Oxygen Electrode Assay of a-Glycerophosphate Oxidase
L-a-Glycerophosphate oxidase was assayed by measuring
the decrease in dissolved oxygen with a New Brunswick D.O.
Analyzer. The oxygen electrode was calibrated against both
N2 and air saturated water with constant agitation provided
by a magnetic stirrer. Incubation mixtures containing buffer
and D,L-a-gl~cerophosphate in a total volume of 7.5 ml were
allowed to equilibrate at 21C. Then reaction was initiated
by enzyme addition and the rate of decrease in dissolved
oxygen was calculated from the linear portion of the curve.
Exact conditions and concentrations for each experiment are
given where appropriate.
Spectrophotometric Assay of a-Glycerophosphate Oxidase
Q-GP oxidase was assayed with a reagent containing
in a total volume of 1.0 ml: 100 ~moles pot~ssium phosphate
buffer, pH 7.0, 66 ~g o-dianisidine, 25 ~g horseradish peroxidase
(4.6 purpurogallin units) and 200 ~moles D,L-c-glycerophosphate
(at pH 7.0). The re~gent was equilibrated at 37, and the
reaction was initiated by the addition of an aliquot of enzyme.
Activity was calculated from the initial linear slope of the
reaction trace at 430 nm, with ~ = 1.08 x 104.
Properties of the a-Glycerophosph~te Oxidase in Crude
Fxtracts
A detailed pH-activity profile of the a-glyceroph3s-
phate oxidase from strain 12755 is shown in Figure 1. Op~imum
activity was observed over the broad pH range of 6.3 to 7.~;
~elow pH 6.o and above pH ~.0 activity decreased rapidly. Also
shown in Figure 1 is +he apparent inhibition of the en~yme by
either tris-HCl ~ or glycine-KGH (X) buffers. At pH 7./
3~ ~he activity in 0.1 M potassium phosphate buffer was 4- times
ll(~Q~P23
that observed in 0.1 M glycine-KOH. However, when an incubation
was carried out in the presence of both 0.1 M glycine-KOH and
0.07 M potassium phosphate buffer, pH 7.7, 82~ of the original
activit~ (in presence of 0.1 M potassium phosphate buffer) was
restored. This suggests that tris-HCl and glycine-KOH were not
inhibitors, but rather that potassium phosphate buffer activated
the enzyme. Sodium acetate buffer also must stimulate the
enzyme (Figure 1), since at pH 6.5 activity in sodium acetate
buffer was at least 90~ that obser~ed in potassium phosphate
buffer.
L-a-Glycerophosphate Oxidase Purification
Preliminary investigations of the a-glycerophosphate
oxidase-glycerol detection system indicated that the crude
a-glycerophosphate (~-GP) oxidase preparation contained
impurities. Some of these impurities apparently prevented
the use of the crude enzyme in serum studies since substrates
for these enzymes were apparently present in the serum at
concentrations comparable to normal triglyceride levels.
~ Certain of these impurities also acted on substrates present
in serum to produce hydrogen peroxide which, of course,
lnterfered with the preferred detection technique. The
results of purification using protein fractionation techniques
are shown in Table V.
ll~Q(~i23
~,' g o a:~ ~ $
_,
_,
~ ~,
_, o o~ ~Ct ~o
~ N Xo O
U~ C~ C~ ~
O C~ t O ~ ~ C~l 0
E~ E-' 1:~ ~
~ 1 ~ ~D E~ O O H t~ ~
~1 ~ '~ 1
O V~ ~
~; ~ td ~ O O O ~ ~ ~1
O O ~ a) L~ o o o
H ~; O ~ ~ lS~ ~ O O
V ~ E~ O _
~ a o ~
~ X
~; O o 0
* r~
c~ i~ ~
i~ Xo ~
~ p I 8 01 ) a~ 8 0 r~
~ ~ L~ ~O c\J 0
~ ~ ~ ~ L~
O . N r~
a) o
s~
.. O
0 ~ ~
Q) O ~ 0 ~ ~1
~ O s ~
tH O U:) r~ o ~r5 E
~ ~ O
':;2 ~ ~ ~ o^ ~ o~ a) o ~ ~ il
L~ ~ i_ ~ ~ ~ O t) ~ m i~
~:1~ ~d ~ ~ Lr~ ~ ~ 00 1 ~) ~,
V'C5 ~ ~ t~ O O C~ I i~ t~ _I O
O ~ ~ O ~ E~ cd O ~
iX; ~ X S~ ~ O ~ ~ 0 ~5
P~ ~ ¢ ~ ~
*
--2~3-
23
Stability of ~-glycerophosphate oxidase
The enzyme solution was completely stable for at least
four months when stored frozen at -20 C. Repeated freezing
and thawing did not denature the enzyme. Also the enzyme was not
inhibited by Triton X-100~ even at surfactant concentrations as
high as 2 percent.
The following examples serve to illustrate particular
embodiments of the present invention.
Example 1 Calibration curve for glycerol and hydrogen peroxide
A glycerol response curve is shown in Fig 2. Mixtures
were prepared as described above under Glycerol and Triglyceride
Quantitation by the a-GP Oxidase Method. Reactions were initi-
ated by substrate addition and were essentially complete in 15
min at 37 C. A good relationship between glycerol concentra-
tions (.) and dye formation (x) was observed for coupled reac-
tions 2, 3 and 4. For comparison, calibration data for hydrogen
peroxide are also shown in Fig 2. Both sets of data were used to
construct a single curve indicating essentially complete produc-
tion of hydrogen peroxide from a stoichiometric amount of glyc-
erol.
Example 2 Quantitative determination of a triglyceride sub-
strate
A triglyceride emulsion was prepared by sonicating olive
oil (3.6 ~moles/ml) in 0.4% Triton X-1001~ (octyl phenoxy
polyethoxyethanol -available from Rohm and Haas Company) in an
ice bath for 10 min.
Quantitative determination of the triglyceride substrate by
coupling reactions 1, 2, 3 and 4 was compared with quantitative
determination by the method of Garland and Randle. Sufficient lip-
ase from Candida rugosa was added to catalyze rapid (less than 1 min)
Q~23
and complete hydrolysis of the triglyceride. Triglyceride
glycerol was determined by comparing the ~A430 after a 30 minute
incubation to the glycerol concentration response curve similar to
Figure 2. The results, shown in Table VI.demonstrate good
agreement between the two methods. Triglyceride values determined
with the ~-glycerophosphate oxidase method were slightly higher
in all cases but the difference was greater than 10% only in
sample 2.
Table VI_
Triglyceride Concentration
Garland and~-Glycerophosphate Oxidase
Sample Randle Method
1 18.4 ig.o
2 36.8 42.0
3 73.6 78.o
4 110.0 114.0
Exam~le 3 ~titative ~eter~nation of Serum Triglycerides by the
a-Glycerophosphate Oxi~se ~ethod __
By means of the preferred buffer system of 0.2 M
potassium phosphate buffer at pH 8.o, ten serum samples were
assayed for triglyceride glycerol in concentrations ranging
from 0.50 to 6.50 mM.
Control mixtures contained only the standard components
for glycerol detection; sample mixtures contained lipase from
C~ndida rugosa plus the standard components for glycerol
detection. All mixtures were equilibrated at 37C for 5 minutes
and the initial ~490 was determined, Reactions were initiated by
addition of 20 ~1 of each serum sample and after 20 minutes incuba-
tion, the final A490 was measured. Triglyceride glycerol concen-
trations were determined from an aqueous glycerol calibrationcurve such as in Fig. 2 after the ~A490 of the controls were
6ubtracted from the ~A4~o of the samples. Results of comparison
110~23
to the reference method of Kessler and Lederer are shown in
Table VII. Good agreement was observed between the two
methods.
-31-
llOQ~23
U
~ s
~ ~ Q~ ~ O L~ O O O O U~
~? O cd O ~ 0 Ir~ J ~ Ir\ 15~
_I ~ S S ~ O ~ J --1 ~) N ~ ~ O
~ ~ ~ bD ~ ~ O ~ ~ U~ ~1 0
r1 ¢ S E "~ J ~ ~r) ~J ~1
~ ~ ~ O ~ ~ t- O O O
C~ cn O s~ Q,3 ~ J ~ O ~ a~
O t~d ~7o ~ O O ~1 0
:~ S l E O ¦ a
v ~ S
a~ ~ O ~ O O O O u~ o u~ o
UJ ~1 ,S ~ ~ ~ ~D U~ O (~ 1 ~ N O
~ ~ Q) ~ ~ O ~ O~ O O ~ O O ~
a) ~ ~ ~: bD ~D O ~ ~ ~ J (~ Lr~ 0 0
O ~: au E Lr~ J ~) c~
O ~ ~ ~ ~ ~ D O O ~ ~ o
O S E-~ Gt ~ 15~ ) ~ ~ O ~ O lS~ N O
~ ~ ~ ~O ~ ~ ~ C`J ~ O
h O ~;
a~ R
~ S~
C~
~ a~
t ~ H N ~ J ~ ~ 0 a~ O
u~
--3~--
~l~C~23
ExamPle 4
The precision of the method described herein was
tested by repetitive assay of two different pooled serum samples;
one contained a normal and one contained a high triglyceride
level. The results are shown in Table VIII. Coefficients of
. variation of 5.1% and 2.6% were calculated for the normal and
abnormal sera respectively.
TABLE VIII
Reproducibility of the a-GP Oxidase ~ystem for Triglyceride
Quantitative Determination
_ Triglyceride Concentration
nL~
_
Normal Serum Level High Serum Level
1.60 4-9
1.66 4.82
1.61 4.80
1.42 5.25
1.62 4 76
1.55 5 00
1.54 4.78
1.70 4.92
1.66 4 q6
1.54 4 ~3
4.91
4.79
4.90
4.80
mean 1.59 4.89
S.D. ~ 0.081 0.13
.CQY 5.10 2.60
Example 5 Alternate Electron Acce~tors
To illustrate electron acceptors other than
oxygen, reaction mixtures containing the follo~ing ingre-
dlents were prepared:
0.1 M potassium phosphate buffer to pH 7;
O.2 M D,T.-a-glycerophosphate;
-33-
110~23
electron acceptor as and at the level specified
in Table IX.
In each instance the mixture was equilibrated at 37C and
the reaction was initiated by enzyme addition. The activity
of the enzyme was calculated as described.hereinabove using
E6oo=16 x 103 for 2,6-dichlorophenolindolphenol, E400=1 x 103 for
K3Fe(CN6) and E505=18-5 x 103 for 2-(p-iodophenyl)_3_(p_
nitrophenyl)-5-phenyl-2M-tetrazolium chloride (INT).
The results are shown in Table IX.
-34-
.
.. .
l~){!Q23
0
tr;
OJ ~ O J
~ N~` O
~ .. . . . .
+' ~ OO O O O O ~
,1
. S~
a) o
~1
O bD
e> C
0 ~ ,~
~s; ~ O
S~
v~ N
0
S~
0~ 0 1
X X . ,
O O O ~O ~ ~ O C\J ~, C~l -
tX)t_ c~J~1 ~i a
C~ U C~
S~ ~o (- ,C,
o a) o
X ~ ~ S~ I
H O
~3 S~ ~ Q)
o
~; ~ C
E~ a~
c)
V ~ ~ P~
O ~ ~ . t
S. O O O X ~:
O O
O 0~ ~ ~ ~2~
O S~ ~ C S`--
.-1 o O a~ ~ ~ ~ ~' I
~)
F~ ~ ~ ~ ~ 3
a~
) O O ~ ~ --
0 C.
S~ C~ ~ S
s~ ~ ~ O o o ~ a)
a~ ~ ~ ~I S S S S
O O O ~ +' ~ O Q.
~ s~ ~ C a) ^ ~ ~ o
cc ~
C~ S S ~ ~ ^ ~
o ~ ,~ a) H
o o s~
O O N N~ N ~`--
C ^ ~ ~ ~ o c~J
~-1 ~ V VS --OS 11
bD I I a) Q) * ~ E~
X ~ (~ (~) Z ~ ~ H
O CU C~J ~ X H H ~ *
--35 -
ll~)QQ23
The method described herein can of course be used
to quantify any one of the various reagents and enzymes used
~n the total reagent system. For example, ATP can be deter-
mined with a composition which includes all of the reagents
except ATP which would be introduced by the sample for assay.
Similarly, glycerol kinase, lipase and ~-glycerophosphate
can be determined using compositions which include all of
the other required materials but that under assay.
The assay compositions described herein may, of
course, be incorporated into a matrix of absorbent material
of the type well known in the art by impregnation or otherwise
to yield test compositions suitable for qualitative or semi-
quantitative assay of glycerol or triglycerides. Typical
such materials and elements produced therewith which can be
adapted for the assay of glycerol or triglycerides are those
described, for example, in the following U.S. Patents:
3,092,465, 3,418,0~9, 3,418,083, 2,893,843, 2,893,844,
2,912,309, 3,oo8,879, 3,802,-842, 3,798,o64, 3,298,739,
3,~15,647, 3,917,453, 3,933,594, 3,936,357, etc.
The invention has been described in detail with
particular reference to certain preferred embodiments thereof,
but ~t will be understood that variations and modifications
can be effected within the spirit and scope of the invention.
-36-
