U.S. patent number 3,898,130 [Application Number 05/451,735] was granted by the patent office on 1975-08-05 for rapid enzymatic hydrolysis of triglycerides.
This patent grant is currently assigned to American Hospital Supply Corporation. Invention is credited to Stanley K. Komatsu.
United States Patent |
3,898,130 |
Komatsu |
August 5, 1975 |
Rapid enzymatic hydrolysis of triglycerides
Abstract
A process and product for rapidly liberating glycerol from its
esterified form as a fatty acid ester, for example, when present in
an aqueous media such as serum, wherein a combination of a
pancreatic lipase and a microbial lipase, particularly Candida
lipase, are mixed with the fatty acid ester in the presence of a
bile salt.
Inventors: |
Komatsu; Stanley K. (Laguna
Hill, CA) |
Assignee: |
American Hospital Supply
Corporation (Evanston, IL)
|
Family
ID: |
26218995 |
Appl.
No.: |
05/451,735 |
Filed: |
March 18, 1974 |
Current U.S.
Class: |
435/159; 435/19;
435/198; 435/921 |
Current CPC
Class: |
C11C
1/045 (20130101); C11C 1/04 (20130101); C12Q
1/44 (20130101); Y10S 435/921 (20130101); G01N
2333/40 (20130101) |
Current International
Class: |
C12Q
1/44 (20060101); C11C 1/04 (20060101); C11C
1/00 (20060101); C12d 013/02 () |
Field of
Search: |
;195/30,63 |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
3703591 |
November 1972 |
Bucolo et al. |
|
Other References
Alford et al., Journal of Lipid Research, Vol. 5, pp. 390-394, July
1964..
|
Primary Examiner: Tanenholtz; Alvin E.
Attorney, Agent or Firm: Dawson, Tilton, Fallon &
Lungmus
Claims
I claim:
1. An enzymatic process for rapidly liberating glycerol from its
esterified form as a fatty acid ester in an aqueous fluid,
comprising the step of mixing said fluid with a combination of
Candida lipase, pancreatic lipase, and a bile salt selected from
the group consisting of the alkali metal salts of taurodeoxycholic,
taurocholic, taurochenodeoxycholic, and taurodehydrocholic
acids.
2. The process of claim 1 in which said acid is taurocholic
acid.
3. The process of claim 1 in which said acid is taurodeoxycholic
acid.
4. The process of claim 1 in which said acid is
taurochenodeoxycholic acid.
5. The process of claim 1 in which said acid is taurodehydrocholic
acid.
6. The process of claim 1 in which said bile salt is a sodium
salt.
7. The process of claim 1 in which said Candida lipase is obtained
from a culture of Candida cylindracea.
8. The process of claim 1 in which said Candida lipase in said
combination provides at least 0.28 units of lipase activity for
each microliter of aqueous fluid having a triglyceride value of 0
to 500 mg percent.
9. The process of claim 8 in which said Candida lipase provides at
least 0.54 units of lipase activity for each microliter of aqueous
fluid having a triglyceride value of 0 to 500 mg percent.
10. The process of claim 1 in which said pancreatic lipase in said
combination provides at least 0.14 units of lipase activity for
each microliter of aqueous fluid having a triglyceride value of 0
to 500 mg percent.
11. The process of claim 10 in which said pancreatic lipase
provides at least 1.2 units of lipase activity for each microliter
of aqueous fluid having a triglyceride value of 0 to 500 mg
percent.
12. The process of claim 1 in which said combination includes at
least 0.002 milligrams of said bile salt for each microliter of
aqueous fluid having a triglyceride value of 0 to 500 mg
percent.
13. The process of claim 12 in which said combination includes at
least 0.02 milligrams of said bile salt for each microliter of
aqueous fluid having a triglyceride value of 0 to 500 mg
percent.
14. A reagent combination for the rapid hydrolysis of fatty acid
esters to liberate glycerol therefrom, comprising a mixture of
Candida lipase, pancreatic lipase, and a bile salt selected from
the group consisting of the alkali metal salts of taurodeoxycholic,
taurocholic, taurochenodeoxycholic, and taurodehydrocholic
acids.
15. The combination of claim 14 in which said acid is taurocholic
acid.
16. The combination of claim 14 in which said acid is
taurodeoxycholic acid.
17. The combination of claim 14 in which said acid is
taurochenodeoxycholic acid.
18. The combination of claim 14 in which said acid is
taurodehydrocholic acid.
19. The combination of claim 14 in which said bile salt is a sodium
salt.
20. The combination of claim 14 in which said Candida lipase is
obtained from a culture of Candida cylindracea.
21. The combination of claim 14 in which said Candida lipase has at
least 14 lipase units for each milliliter of reagent
combination.
22. The combination of claim 21 in which said Candida lipase has at
least 27 lipase units for each milliliter of reagent
combination.
23. The combination of claim 14 in which said pancreatic lipase has
at least 7 lipase units for each milliliter of reagent
combination.
24. The combination of claim 23 in which said pancreatic lipase has
at least 60 lipase units for each milliliter of reagent
combination.
25. The combination of claim 14 in which at least 0.1 milligrams of
bile salt are provided in each milliliter of reagent
combination.
26. The combination of claim 25 in which at least 1.0 milligrams of
bile salt are provided for each milliliter of reagent
combination.
27. The reagent combination of claim 14 in which said pancreatic
lipase has an activity of about 10 to 100 lipase units per
milligram.
28. The combination of claim 27 in which said pancreatic lipase has
an activity of approximately 20 to 80 lipase units per
milligram.
29. The reagent combination of claim 14 in which said Candida
lipase has an activity of approximately 30 to 800 lipase units per
milligram.
30. The reagent combination of claim 29 in which said Candida
lipase has an activity of approximately 200 to 800 lipase units per
milligram.
Description
BACKGROUND AND SUMMARY
An effective enzymatic test for the determination of serum
triglycerides turns mainly on the development of a rapid and
complete process for the hydrolysis of the triglycerides, since
various procedures for measuring the glycerol formed by such a
reaction are well known. Thus, as described by F. H. Kreutz in
Klin. Wochshr. 40:362 (1962), glycerol may be readily determined by
combining the following reactions: ##EQU1##
In this combination of reactions, 1 mole of NADH is oxidized for
each mole of glycerol phosphorylized; therefore, a change in the
optical density at 340 nm is a direct measure of the amount of
glycerol in the assay.
U.S. Pat. No. 3,703,591 discloses that the preliminary step, that
of hydrolyzing the triglycerides to form glycerol, may be carried
out enzymatically using a mixture of a lipase and a protease. While
it has long been known that certain lipases may alone be used to
hydrolyze triglycerides, and that the enzymatic activity might
possibly be enhanced by the presence of a bile salt, prior
procedures using only lipases (with or without bile salts) have
been unsatisfactory because of the long duration of the hydrolysis
step and because even then incomplete hydrolysis is the usual
result.
Therefore, an important aspect of this invention lies in the
discovery that a complete and surprisingly rapid hydrolysis of
triglycerides may be achieved by using a combination consisting
only of certain lipases along with a bile salt, thereby dispensing
with the requirement of including a proteolytic enzyme such as
chymotrypsin as one of the reactants. More specifically, 100
percent hydrolysis may be accomplished in 3 to 5 minutes using a
combination of Candida lipase and pancreatic lipase with a bile
salt such as sodium taurodeoxycholate. The glycerol so produced may
then be assayed by any of a number of known methods, one such
method having already been described above.
Other objects and advantages of the invention will be apparent as
the specification proceeds. Additional references disclosing the
state of the art are: R. G. H. Morgan and N. E. Hoffman, Biochim.
Biophys. Acta, 248:143 (1971); R. L. Ory, J. Kiser and P. A.
Pradel, Lipids, 4:261 (1968); H. Brockerhoff, J. Bio. Chem.
246:5828 (1971); P. Desnuelle, Enzymes, 7:575 (1972); patent
2,527,305.
DESCRIPTION
It is preferred that the enzymatic hydrolysis of triglycerides by
the coactive lipases be undertaken in the presence of the
components of the three additional reaction systems represented in
the equations given above so that all of such reactions may be
undertaken simultaneously in a single operative procedure or, if
desired, in a two step procedure in which one of the components
needed for the conversion of glycerol, such as the glycerol kinase,
is added to all of the other components after hydrolysis has
occurred and an initial reading of optical density has been made.
Since the various components necessary for the enzymatic conversion
of glycerol have already been indicated and are well known in the
prior art, and since the proportions of such components are also
well known, a detailed discussion herein is believed unnecessary.
It is believed sufficient to state that the combination of lipases
and bile salt may be part of an assay mixture which includes one or
more of those components known for use in the enzymatic conversion
of glycerol and the concurrent oxidation of NADH in a colorimetric
or spectrophotometric test.
The system responsible for the enzymatic hydrolysis of
triglycerides comprises a coactive mixture of pancreatic lipase, a
microbial lipase, and a bile salt. All three components are
essential for effective hydrolysis. A variety of pancreatic lipases
having activity within the range of about 10 to 100 lipase units
per milligram (mg), and preferably within the range of 20 to 80
units per mg, are believed suitable, an example being the
pancreatic lipase sold under the designation "PL3" by Worthington
Chemical Company, of Freehold, New Jersey. The microbial lipase is
more specifically a Candida lipase which may, for example, be
obtained from the cultured broth of Candida cylindracea. Such
Candida lipase should have activity within the range of 30 to 800
lipase units per mg, and preferably within the range of 200 to 800
lipase units per mg. Other Candida lipases are believed to be
equally effective when used in combination with the pancreatic
lipase and bile salt of the triglyceride-hydrolyzing system.
While both pancreatic and Candida lipases must be present, it has
been found that the proportional amounts of those constituents,
measured in terms of lipase units, may be varied considerably in
accordance with selected time requirements for completion of
hydrolysis. One lipase unit of activity is the amount sufficient to
release one micromole of acid per minute at 25.degree.C. from an
olive oil emulsion containing gum acacia and 15 mg per milliliter
(ml) sodium taurocholate at a pH of about 8.0. Such definition of a
lipase unit, and the procedure on which it is based, are well
established and are disclosed more fully in Worthington Enzyme
Manual, p. 63 (1972).
More specifically, the amount of pancreatic lipase in the reaction
mixture should be at least 0.14 lipase units for each microliter of
body fluid (blood serum or plasma) having a triglyceride value
within the range of 0 to 500 mg per 100 ml (mg percent) in order to
achieve complete hydrolysis within 12 minutes. On the same basis,
the amount of Candida lipase in the mixture should be at least 0.28
units, and the amount of bile salt should be at least 0.002 mg, for
each microliter of body fluid. Where shorter reaction times are
required or desired, the amounts of such constituents must be
increased. Thus, for complete hydrolysis within 3 to 5 minutes, at
least 1.2 pancreatic lipase units, 0.54 Candida lipase units, and
0.02 mg of bile salt, are required for each microliter of body
fluid. Stated differently, in a reagent combination having a volume
of 1 milliliter, the values (for hydrolyzing 50 microliters of
serum or plasma having a triglyceride value of 0 - 500 mg percent
within 12 minutes) should be at least 7 pancreatic lipase units, 14
Candida lipase units, and 0.1 mg bile salt; or at least 60
pancreatic lipase units, 27 Candida lipase units, and 1.0 mg bile
salt (for the hydrolysis of 50 microliters of such serum or plasma
within 3 to 5 minutes).
Since speed in completing an assay may be important, particularly
in connection with clinical diagnostic tests, a maximum time period
for completion of hydrolysis in a clinical test has been
arbitrarily set at approximately 12 minutes. It is to be
understood, however, that in other tests where longer time periods
are more acceptable, lower concentrations or amounts of the
respective lipases and bile salt may be used.
As previously indicated, a bile salt is an essential component of
the system. While alkali metal salts of taurocholic,
taurochenodeoxycholic or taurodehydrocholic acid may be used,
particularly effective results have been achieved with alkali metal
salts of taurodeoxycholic acid. The salt of taurodeoxycholic acid,
in admixture with Candida lipase and pancreatic lipase under
optimum conditions, has been found to produce faster results at
lower concentrations than the other bile salts.
As indicated above, and as illustrated more fully by the examples
set forth hereinafter, the combination of pancreatic and Candida
lipases, in admixture with a bile salt, preferably the salt of
taurodeoxycholic acid, reacts with the triglycerides in body fluids
to produce complete hydrolysis in periods as short as 3 minutes.
Such a combination of reactants may be used in any test requiring
the rapid and complete hydrolysis of triglycerides. The process and
product of this invention may, for example, be used in conjunction
with a complete triglyceride assay including the three
glycerol-determining reactions described at the beginning of this
application and well known in the prior art. All of the components
required for the complete colorimetric determination of
triglycerides in body fluids may be premixed and lyophilized to
provide a stable reagent set for clinical use.
EXAMPLE 1
A reagent suitable for practicing this invention may be prepared by
making the following 3 ml reaction mixture:
Pancreatic lipase [Worthington PL3] , 180 lipase units
Candida lipase [Worthington], 80 lipase units
Sodium taurodeoxycholic acid, 3 mg
LDH, 10 International Units (IU)
Pyruvate kinase, 10 IU
NADH, 0.75 micromoles
Phosphoenolpyruvate, 1.5 micromoles
ATP disodium, 0.5 micromoles
Magnesium chloride, 0.0067 M
Potassium phosphate buffer, 0.1 M, pH 7.0
The assay is carried out by adding an aliquot of liquid containing
the triglyceride to be assayed, such as 50 microliters of serum or
plasma with triglyceride values of 0 to 500 mg percent, to the
above reaction mixture. Following incubation for approximately 5
minutes at a temperature between 25.degree.C. to 37.degree.C., the
optical density is measured at 340 nm. Thereafter, 10 units of
glycerol kinase is added and the mixture is again incubated at
25.degree.C. to 37.degree.C. for another 5 minutes. The optical
density is again determined at 340 nm, and the difference in
optical densities is proportional to the triglyceride content after
appropriate adjustment, using conventional clinical laboratory
procedures, for whatever blank reaction is produced.
EXAMPLE 2
The procedure of Example 1 was performed using the same reactants,
proportions, and conditions, except that a purified lipase obtained
from the cultured broth of Candida cylindracea nov. sp. was
substituted for the Candida lipase of the first example. Complete
hydrolysis of the triglycerides of the sample were obtained within
5 minutes in the same manner as set forth in Example 1.
EXAMPLE 3
Several reagent combinations were prepared in accordance with
Example 1 except that a variety of bile salts were used. The
results were tabulated below. Each reagent combination contained
0.1 mg Candida lipase (activity of 80 units), 3.0 mg pancreatic
lipase (activity 180 units), and the amount of bile salt indicated.
Times are given in minutes for completion (100 percent) of
hydrolysis of serum samples having triglycerides values of 100 mg
percent (i.e., 100 mg per 100 ml water) and 280 mg percent. TIMES
REQUIRED FOR COMPLETION OF HYDROLYSIS WITH ENZYME COMBINATIONS
INCLUDING DIFFERENT BILE SALTS Bile Salt (Na) Amount (mg) Time
(min.) Time (min.) for 100 mg% for 280 mg% Sample Sample
__________________________________________________________________________
(1) Taurocholic 6.0 20 -- 12.0 14 -- 18.0 10 -- 24.0 5 12 30.0 5 10
(2) Taurodeoxycholic 0.3 12 -- 1.0 7 7 1.5 5 6 3.0 4 5 (3)
Taurochenodeoxycholic 1.5 -- 14 3.0 -- 12 6.0 3 10 (4)
Taurodehydrocholic 6.0 13 -- 24.0 13 -- 30.0 10 --
__________________________________________________________________________
While in the foregoing an embodiment of the invention has been
disclosed in considerable detail for purposes of illustration, it
will be understood that many of those details may be varied without
departing from the spirit and scope of the invention.
* * * * *