U.S. patent number 3,869,349 [Application Number 05/454,659] was granted by the patent office on 1975-03-04 for method for the enzymatic hydrolysis of cholesterol esters.
This patent grant is currently assigned to Eastman Kodak Company. Invention is credited to Charles T. Goodhue, Hugh A. Risley.
United States Patent |
3,869,349 |
Goodhue , et al. |
March 4, 1975 |
Method for the enzymatic hydrolysis of cholesterol esters
Abstract
A totally enzymatic method for the hydrolysis of cholesterol
esters using a lipase having cholesterol esterase activity and a
protease and a method for quantitative determination of total
cholesterol in compositions containing both free and esterified
cholesterol comprising enzymatically hydrolyzing the cholesterol
esters with a lipase having cholesterol esterase activity and a
protease and determining total cholesterol by gas-liquid
chromatography or some other suitable technique are described.
Inventors: |
Goodhue; Charles T. (Rochester,
NY), Risley; Hugh A. (Rochester, NY) |
Assignee: |
Eastman Kodak Company
(Rochester, NY)
|
Family
ID: |
23805542 |
Appl.
No.: |
05/454,659 |
Filed: |
March 25, 1974 |
Current U.S.
Class: |
435/11; 435/921;
435/19; 435/197; 435/222; 435/225; 435/897; 435/23; 435/220;
435/839; 435/918 |
Current CPC
Class: |
C12Q
1/60 (20130101); Y10S 435/839 (20130101); Y10S
435/918 (20130101); Y10S 435/897 (20130101); Y10S
435/921 (20130101) |
Current International
Class: |
C12Q
1/60 (20060101); G01n 031/14 () |
Field of
Search: |
;195/13.5R,4,30 |
Foreign Patent Documents
Other References
J Hyun et al., "The J. of Biol. Chem.," 244, No. 7, pp 1937-1945,
1969..
|
Primary Examiner: Tanenholtz; Alvin E.
Assistant Examiner: Fan; C. A.
Attorney, Agent or Firm: Girard; A. L.
Claims
What we claim is:
1. In a process of assaying an aqueous proteinaceous liquid
containing cholesterol esters for total cholesterol content in
which said ester is hydrolyzed to liberate all of said cholesterol
followed by determining the amount of cholesterol, the improvement
comprising effecting said hydrolysis by treating said aqueous
proteinaceous liquid with both a lipase having cholesterol esterase
activity and a protease.
2. The improved method of claim 1 wherein said lipase having
cholesterol esterase activity releases at least 25 mg% cholesterol
(in 2 hours at 37.degree.C under nitrogen) when 50 mg of a
preparation of said lipase in 5 ml 0.1 M phosphate buffer, pH 7.0,
is used to treat a dispersion of cholesteryl linoleate prepared by
dispersing 200 mg cholesteryl linoleate in 5 ml of ethyl ether and
100 ml boiling water containing 430 mg of sodium cholate.
3. The improved method of claim 1 wherein said aqueous liquid is
serum and said treating step is accomplished with a mixture
comprising from about 600 to about 1500 units of said lipase having
cholesterol esterase activity and from about 50 to about 500 units
of said protease per ml of serum.
4. The method of clain 1 wherein said treatment is carried out at a
temperature of between about 25.degree. and 55.degree.C and at a pH
of between about 6.5 and about 9.5.
5. The improved method of claim 1 wherein said lipase having
esterase activity is a microbial lipase.
6. The improved method of claim 5 wherein said microbial lipase is
the lipase from Candida cylindracca.
7. The improved method of claim 1 wherein said lipase is selected
from the group consisting of wheat germ lipase, pancreatic lipases
and the lipase from Candida cylindracca.
8. The improved method of claim 1 wherein said protease is selected
from the group consisting of .alpha.-chymotrypsin, papain,
bromelain, Bacillus subtilis protease, Aspergillus oryzae protease,
Streptomyces griseus protease and mixtures thereof.
9. The improved method of claim 8 wherein said lipase is selected
from the group consisting of wheat germ lipase, pancreatic lipases
and the lipase from Candida cylindracca.
10. The method of claim 9 wherein said treatment is carried out at
a temperature of between about 25 and 55.degree.C and at a pH of
between about 6.5 and about 9.5.
Description
FIELD OF THE INVENTION
This invention relates to an enzymatic method for hydrolyzing
cholesterol esters in complex aqueous solutions which may contain
both free and esterified cholesterol, for example, blood serum and
in particular to a process comprising enzymatically hydrolyzing
cholesterol esters using a lipase having cholesterol esterase
activity and a protease.
BACKGROUND OF THE INVENTION
The most common clinical estimations of cholesterol in blood serum
are for "total cholesterol." This value is a measure of cholesterol
and cholesterol esters present in the serum and anything else such
as cholesterol precursors that respond indiscriminately to the
usual tests which are based on reactions involving "free"
cholesterol and require prior conversion of cholesterol esters to
"free" cholesterol.
In a well-known conventional procedure, serum is extracted with an
organic solvent, the extract is saponified with alcoholic KOH and
the liberated cholesterol is isolated and assayed. These methods
require the handling of corrosive chemicals and are tedious,
time-consuming, and not readily automated.
German, Offenlegungsschrift No. 2,246,695 published Mar. 29, 1973,
describes an enzymatic assay for free cholesterol using a
cholesterol oxidase, however, this technique requires hydrolysis of
the blood serum cholesterol esters using the cumbersome techniques
of the prior art prior to application of the enzymatic assay.
G. Bucolo, and H. David, Clin. Chem., 19 476 (1973) describe a
lipase-protease system for hydrolyzing serum triglycerides;
however, it is specifically stated that cholesterol esters are not
hydrolyzed in this system.
OBJECTS OF THE INVENTION
It is therefore an object of the present invention to provide a
simplified and improved technique for hydrolyzing cholesterol
esters.
It is another object of the present invention to provide a
reproducible enzymatic process for the quantitative hydrolysis of
cholesterol esters in blood serum, useful as an initial step in the
quantitative determination of total cholesterol in blood serum.
SUMMARY OF THE INVENTION
The foregoing objects are accomplished by an improved process for
hydrolyzing cholesterol esters which comprises treating a sample of
an aqueous solution containing cholesterol esters, for example
blood serum, with a mixture of a lipase having esterase activity
and a protease to release free cholesterol. Subsequently, the free
cholesterol can be assayed using any suitable technique including
gas-liquid chromatography, cholesterol oxidase assay, or any of a
number of other well known techniques for the assay of "free"
cholesterol.
DETAILED DESCRIPTION OF THE INVENTION
According to a preferred embodiment of the present invention,
hydrolysis of cholesterol esters in complex aqueous solutions
(referred to hereinafter generally as blood serum) is achieved by
treating the blood serum with a mixture comprising per ml of serum
from about 20 to about 50 mg of a lipase having cholesterol
esterase activity and from about 5 to about 50 mg of a protease at
a temperature of from about 25.degree. to about 55.degree.C and a
pH of from about 6.5 to about 9.5 for about 5 to about 15 minutes,
preferably with agitation and in an inert atmosphere. When the
foregoing concentrations of lipase and protease are utilized, the
lipase should contain at least about 30 international units per mg
and the protease at least about 10 units per mg. One unit of lipase
is defined as the amount of the enzyme which will liberate 1 micro
mole of fatty acid in a given time at a given pH and temperature
using a substrate containing esterified fatty acid. For the
preferred lipase materials described hereinbelow the conditions are
1 minute at pH 7 and 37.degree.C with olive oil as substrate. One
unit of protease will hydrolyze casein to produce color equivalent
to 1 micro mole (181 .mu.g) tyrosine per minute at pH 7.5 and a
temperature of 37.degree.C. (Color per Folin-Ciocalteu Reagent). It
should, of course, be clear that as the level of enzyme activity
per unit by weight of preparation increases or decreases, so also
will the quantity of enzyme preparation added vary. Most
preferably, the ratio of lipase to protease on an activity basis
should range from about 3 to about 10 and at least about 1000 units
of lipase should be used per ml of serum. Relative lipase activity
to esterase activity is usually about 10 to about 50.
Generally speaking and in accordance with illustrative embodiments
of our invention, we contact an aqueous medium containing the
cholesterol ester, preferably blood serum, which contains both
esterified and free cholesterol, with a mixture of a lipase which
demonstrates cholesterol esterase activity as defined in Example 3
and a protease. The lipase may be of plant or animal origin, but we
prefer and find best a microbial lipase such as the lipase from
Candida cylindracca. Lipases from Chromobacterium viscosum, variant
paralipolyticum, crude or purified, the lipase from Rhizopus
delemar, purified, for example as noted in Fukumoto et al, J. Gen.
Appli. Microbiol, 10, 257-265 (1964), and lipases having similar
activity, which are those described in the aforementioned Bucolo
and Davis publication, do not demonstrate the required cholesterol
esterase activity. Specifically preferred commercial lipase
preparations include wheat germ lipase supplied by Miles
Laboratories of Elkhart, Ind., Lipase 3000 supplied by Wilson
Laboratories, Chicago, Ill., Steapsin supplied by Sigma Chemical
Co., St. Louis, Mo., (both of the last two enzymes are pancreatic
enzymes) and Lipase M (from Candida cylindracca) supplied by Enzyme
Development Corporation, New York, N.Y. Screening of lipases for
this purpose to determine their cholesterol esterase activity may
be accomplished using the technique described in Example (3) below.
Using this technique, any lipase which demonstrates a cholesterol
esterase activity which releases above about 25 mg% cholesterol in
the screening procedure of Example 3 should be considered useful in
the successful practice of the present invention.
Proteases in general may be used in the successful practice of this
invention. These include by way of example, chymotrypsin,
Streptomyces griseus protease (commercially available under the
registered trademark "Pronase"), Aspergillus oryzae protease,
Bacillus subtilis protease, elastas, papain and bromelain. Mixtures
of such enzymes, of course, may also be employed, at times, with
advantageous results as demonstrated in the examples below.
The protease utilized as described above, is only necessary where
the cholesterol ester is present in a protein containing solution
the most notable and important of which is, of course, blood serum.
In the case where a simple non protenaceous solution is being
assayed, it is possible, as demonstrated in Example 3 below, to
obtain ester hydrolysis using only the lipase. As also demonstrated
by the Examples, however, in a protein containing solution such as
serum, the presence of the protease is essential to useful
results.
As will be further elaborated below, the free cholesterol liberated
by the action of the foregoing enzyme mixture may be assayed in a
number of ways. According to a preferred embodiment of the instant
invention, this assay is performed using gas-liquid
chromatography.
According to a preferred embodiment of this "free" cholesterol
assay technique, an aliquot generally from about 0.5 to about 5 ml
of the hydrolyzed aqueous composition to be tested, hereinafter
blood serum, is mixed with from about 0.5 to about 2 ml of heptane
or some other suitable organic solvent, for example, isooctane,
containing from about 25 mg to about 50 mg weight percent
octacosane or some other organic suitable for use as an internal
standard. The heptane may, of course, be replaced with any other
solvents suitable for gas-liquid chromatography, for example,
isooctane. The solvent mixture is then extracted with water
according to conventional techniques, preferably using from about 3
to about 10 ml of water per ml of solvent solution. The water
extracted solvent solution is then reacted with a silylating agent,
for example, (N,O-bis(trimethylsilyl) trifluoroacetanide with 1%
trimethylchlorosilane or a mixture of equal volumes of
trimethylchlorosilane and 1,1,1,3,3,3,-hexamethyldisilazane for a
period of from about 2 to about 15 minutes. The silylated solvent
solution is then passed through a conventional gas-liquid
chromatograph to determine the total cholesterol in the sample
under examination. This method for determining cholesterol
concentration is an adaptation of the technique described in detail
in J. L. Driscoll, D. Aubuchon, M. Descoteaux and R. F. Martin,
Anal. Chem., 43, 1196 (1971).
One of the most significant advantages of the instant enzymatic
hydrolysis technique involves the requirement for dilution of blood
serum for hydrolysis and assay. Surprisingly, using the techniques
described herein, undiluted serum is hydrolyzed as rapidly and
readily as diluted serum. This is quite surprising in view of the
uniform requirements for serum dilution described in the prior
art.
The following examples serve to illustrate particular embodiments
of the present invention.
"Validate," a reconstituted serum standard produced by the
Warner-Lambert Company, was used in the examples below. The total
cholesterol content of "Validate" (lot 2560121) was checked by
saponifying an aliquot according to the method of Driscoll et al,
and analyzing the heptane extract by both glc and the
Liebermann-Burchard method. Values of 160 and 162 mg% respectively
were obtained. These are well within the range of values quoted by
the supplier (148-192 mg%).
EXAMPLE 1 HYDROLYSIS OF CHOLESTEROL ESTER IN SERUM
A mixture of 1 ml "Validate" (a serum cholesterol standard sold by
Warner-Lambert and containing 148-192 mg% cholesterol), 40 mg
Lipase M, 40 mg papain, 8 mg .alpha.-chymotrypsin, and 0.1 M tris
buffer to 3 ml total volume (pH 7.2) is incubated in a 25 ml flask
under nitrogen at 50.degree.C and 250 rpm for 10 min.
The hydrolysis is performed in an atmosphere of nitrogen in order
to minimize artifacts introduced by autooxidation of cholesterol
and its esters; of course, proper correction for such autooxidation
factors will permit hydrolysis to be performed in a normal
atmosphere.
EXAMPLE 2
QUANTITATIVE ESTIMATION OF TOTAL CHOLESTEROL BY GAS-LIQUID
CHROMATOGRAPHY (GLC)
One ml samples of serum or reconstituted serum standards
("Validate," Warner-Lambert) containing up to 150 mg% cholesterol
are mixed with 5 ml ethanol and shaken 3 minutes with 1 ml heptane
containing 50 mg% octacosane. Five ml of water is added and the
mixture is shaken again for 3 minutes. When the layers separate,
equal portions of the heptane layer and N,O-bis(trimethylsilyl)
trifluoroacetamide with 1% trimethylchlorosilane are mixed. After 5
minutes reaction 1 .mu.l samples are injected into a Hewlett
Packard F and M 810 chromatograph with a single stainless steel
column (1/8 inch .times. 4 feet) packed with 3% SE30 on ov 1. Gas
flow rate 20 ml/min, oven temperature 250.degree.C, injection port
260.degree.C, flame detector 265.degree.C, range 10.sup.2,
attenuation .times. 1, chart speed 1/2 inch/min. Octacosane
retention time is about 1 min. Cholesterol retention time is about
21/2 min. Runs are complete in about 4 min. Under these conditions
the amount of cholesterol in the sample is proportional to the peak
height ratios of cholesterol to octacosane. This method is adapted
from Driscoll et al referred to above.
EXAMPLE 3
SCREENING OF LIPASES FOR CHOLESTEROL ESTERASE ACTIVITY
Tests were conducted with cholesteryl linoleate as the substrate
because it is the major ester component of human serum and because
it gives relatively stable emulsions compared to saturated esters
such as the palmitate.
A solution of 200 mg redistilled cholesteryl linoleate in 5 ml
ethyl ether was mixed with rapid stirring into 100 ml boiling water
containing 430 mg sodium cholate. Five ml of this suspension was
added to a solution of 50 mg of lipase preparation in 5 ml 0.1 M
phosphate, pH 7.0. This mixture was incubated 2 hours at
37.degree.C, 400 rpm under N.sub.2. Cholesterol esters remaining
after this treatment were determined by the hydroxylamine method of
J. Vonhoeffmayr and R. Fried, Z. Klin. Chem. u Klin., Biochem., 8,
134 (1970) which involves quantitative conversion of esters to
hydroxamic acids. The results are shown in Table 1.
Table 1 ______________________________________ Hydroxylamine Assay
with Cholesteryl Linoleate Suspension
______________________________________ Cholesterol Enzyme released
(mg%) ______________________________________ Lipase (Miles) 30
Lipase 3000 (Wilson) 42 Wheat Germ Lipase (Miles) 59 Steapsin
(Sigma) 59 Lipase M (Enzyme Development Corp.) 68
______________________________________
All of the enzymes show esterase activity. However, Lipase M is
preferred because of its significantly greater esterase activity
and also because it is a relatively inexpensive commercial enzyme.
As purchased, the preparation is about 80% lactose, so on a protein
basis its activity is about five times its activity on a weight
basis.
EXAMPLE 4
ACTIVITY OF LIPASE WITH SERUM CHOLESTEROL ESTERS
Mixtures containing 40 mg. of lipase in 1 ml "Validate" were
incubated 10 min. at 50.degree.C under nitrogen in 25 ml flasks at
250 rpm. The mixture was extracted and cholesterol was estimated as
in Example 2. The enzymes tested and the results are given in Table
II.
Table II ______________________________________ Esterase Activity
with Serum as Substrate ______________________________________
Enzyme Cholesterol (mg%) ______________________________________
None 23 Lipase 3000 26 Lipase M 36
______________________________________
Lipase M and lipase 3000 while exhibiting considerable esterase
activity on cholesteryl linoleate emulsions show very little
activity with serum esters.
EXAMPLE 5
ESTERASE ACTIVITY OF LIPASE-PROTEASE COMBINATIONS ON SERUM
CHOLESTEROL ESTERS
Combinations of Lipase M and various proteases were tested in the
same manner as in Example 4. Proteases, except for
.alpha.-chymotrypsin, were added directly to serum at 40 mg per ml;
.alpha.-chymotrypsin was added at 8 mg per ml. The amount of
cholesterol (mg%) released by each combination is shown in Table
III.
Table III ______________________________________ Enzymes
Cholesterol (mg%) ______________________________________ None 23
Lipase M 36 .alpha.-Chymotrypsin (Sigma, Type 11 3X crystallized)
37 Chymotrypsinalpha. 129 Papain (Sigma, Grade-Crude Type 11) 29
Lipase M + papain 115 chymotrypsin + papain 114
______________________________________
It is seen that in the presence of proteases such as
.alpha.-chymotrypsin or papain the cholesterol esterase activity of
Lipase M was enhanced nearly four fold. The proteases themselves
may have a slight esterase activity but their major effect probably
is to increase the availability of cholesterol esters to the lipase
by breaking up ester-lipoprotein complexes in serum. Most
cholesterol esters in serum are bound to lipoproteins.
Thus, for Lipase M to be optimally effective on serum cholesterol
esters, a protease must also be present.
EXAMPLE 6
A series of commercially prepared proteases were tested for their
ability to enhance esterase activity of Lipase M in serum. Protease
was added directly to serum in the amounts shown in Table IV.
Lipase M concentration was 40 mg per ml serum. Otherwise assay and
conditions are the same as in Example 4. The results are contained
in Table IV.
Table IV ______________________________________ Amount added
Cholesterol Protease (mg/ml) (mg%)
______________________________________ 1. Aspergillus oryzae 20 118
(Sigma Type II) 40 123 80 145 2. Streptomyces griseus 5 147 (Sigma
Type VI) 10 144 20 140 3. Bacillus subtilis 5 140 (Sigma Type VIII)
10 118 20 135 4. Bromelain 5 114 (Sigma Grade II) 10 127 20 132 5.
Protease 30 20 80 (Rohm and Haas) 40 127 80 161 6. Pronase
(Calbiochem, Grade A) 5 133 7. Subtilisin BPN (Sigma Type VII) 5 87
8. Protease, bacterial (Calbiochem, Grade B) 10 142 9. Lipase M 36
______________________________________
All of the proteases tested appear to enhance the activity of
Lipase M somewhat more than .alpha.-chymotrypsin and papain.
However, it is difficult to compare the activities of these
proteases on the basis of units given by suppliers since several
different assays are used. In general, the enzymes judged less pure
were used in the higher amounts.
The effect of pH value on the esterase activity was tested with
different buffers and enzymes as described in Examples 7 and 8
below.
EXAMPLE 7
Four different buffers were tested in a system containing three
enzymes. Each sample consists of 1 ml "Validate," 40 mg Lipase M,
40 mg papain, 8 mg .alpha.-chymotrypsin and 0.1 M buffer to 3 ml
total volume. The mixtures were incubated and tested as in Example
4. The results are depicted in FIG. 1.
The measurement of pH optimum in this assay with serum as a
substrate may be somewhat ambiguous because two enzymes (lipase and
protease) are necessary. It is possible that the pH optimum of each
enzyme may not coincide. In this study tris buffer
(tris(hydroxymethyl)aminomethane) at pH 7.2 was superior.
EXAMPLE 8
Studies with tris buffer were conducted on combinations of Lipase M
with the series of proteases described in Example 6. The amount of
each protease used was that which gave the best result in the
experiment shown in Table IV. Three pH values between 7 and 9 were
tested. The assay was the same as described in Example 6 except
that time was reduced to 5 minutes so that it would be expected to
find cholesterol values well below 160 mg%, the maximum available
in the "Validate" substrate.
Each incubation mixture contained 0.5 ml "Validate," 0.5 ml 0.2M
tris buffer at pH shown, 40 mg lipase M and the protease in the
amounts shown. Incubation was for 5 minutes under N.sub.2 at
50.degree.C, 250 rpm. Samples were analyzed by glc as in Example 2.
The results are indicated in Table V.
Table V ______________________________________ Protease Amt
Cholesterol mg% (mg) pH pH pH 7.2 8.1 9.0
______________________________________ 1. A. oryzae 80 66 86 72 2.
Thermolysin (Calbiochem, Grade A) 10 58 74 52 3. Bromelain 20 66
100 72 4. B. subtilis 5 54 65 70 5. S. griseus 5 56 58 30 6.
Pronase 5 67 74 40 7. Subtilisin 5 56 66 56 8. .alpha.-chymotrypsin
8 84 56 22 9. Protease, bacterial (Calbiochem) 10 86 100 102
______________________________________
Most of these proteases show an optimum within the three values
tested. The best results were obtained with bromelain at pH 8.1,
Calbiochem bacterial protease at pH values from 8 to 9, A. oryzae
protease at pH 8.1 and .alpha.-chymotrypsin at pH 7.2.
EXAMPLE 9
Undiluted serum (1 ml "Validate" ) was incubated with 40 mg Lipase
M, protease in amounts indicated in Table VI, and 12.1 mg of
"Trisma Base" or Tris buffer at pH 7.2. The incubations were run at
50.degree.C for 10 min. under nitrogen and at 250 rpm. Cholesterol
was analyzed as before by glc. The results are shown in Table
VI.
Table VI
__________________________________________________________________________
Hydrolysis of Cholesterol Esters in Undiluted Serum with Lipase M
and Various Proteases
__________________________________________________________________________
No Additions "Trizma Base".sup.a Tris Buffer pH 7.2.sup.b Amt
Cholesterol Cholesterol Cholesterol Protease (mg) pH Range (mg%) pH
Range (mg%) pH Range (mg%)
__________________________________________________________________________
1. B. subtilis 5 7.10-6.89.sup.c 104 8.61-7.85 84 6.98-6.64
208.sup.d 2. Protease (Calbiochem) 5 7.03-6.52 116 8.52-7.71 100
7.01-6.82 206 3. A. oryzae 80 6.90-6.27 90 7.60-7.25 103 6.72-6.70
174 4. Bromelain 20 6.78-6.56 119 8.25-7.93 52 6.82-6.45 165 5.
.alpha.-Chymotrypsin 8 7.08-6.93 91 8.65-8.19 51 7.00-6.94 162 6.
Pronase 5 6.82-6.66 134 8.72-7.99 64 7.01-6.89 160 7. S. griseus 5
6.88-6.48 96 8.60-7.94 63 6.89-6.68 119
__________________________________________________________________________
.sup.a "Trizma Base" (Sigma) powder was added directly to serum.
.sup.b Tris-HCl buffer, pH 7.2 freeze dried, then added as powder
directl to serum. .sup.c pH value at start of incubation and pH
value at end. .sup.d Some values higher than 160 mg% occasionally
obtained from same lo of "Validate" calibrated at 160 mg%.
Although according to a preferred embodiment of the present
invention quantitative estimation of total cholesterol is achieved
using gas-liquid chromatography, any of the well known conventional
techniques for the analysis of total "free" cholesterol (after
cholesterol ester hydrolysis has been achieved) may be used. These
include the Pearson, Stern and McGarack, Carr and Drecker, and Zak
methods described at pages 355-361 of Fundamentals of Clinical
Chemistry, TIETZ, N. W., W. B. SAUNDERS CO. (1970) as well as the
well known Lieberman-Burchard technique and the cholesterol oxidase
method described in German Offenlegungschrifft No. 2,246,695
referred to hereinabove which is incorporated herein by reference
insofar as it describes a technique for quantitatively determining
total free cholesterol by treating a cholesterol solution with
cholesterol oxidase and measuring the quantity of one or more of
the products of this oxidation. Furthermore, hydrolysis as
described herein may be used as an integral part of a single
solution assay using cholesterol oxidase as is described in
concurrently filed U.S. Pat. application Ser. No. 454,622 filed
Mar. 25, 1974, in the names of Goodhue, Risley and Snoke entitled
"Method and Composition for Blood Serum Cholesterol Analysis,"
which is incorporated herein by reference insofar as it describes
another useful application of the novel hydrolysis described
herein.
While the invention has been described in detail with particular
reference to preferred embodiments thereof, it will be understood
that variations and modifications can be effected within the spirit
and scope of the invention.
* * * * *