U.S. patent number 3,616,254 [Application Number 04/641,733] was granted by the patent office on 1971-10-26 for screening procedure for enzyme deficiencies.
Invention is credited to Ernest Beutler.
United States Patent |
3,616,254 |
Beutler |
October 26, 1971 |
SCREENING PROCEDURE FOR ENZYME DEFICIENCIES
Abstract
This screening method detects enzyme deficiencies in red blood
cells by use of a reaction mixture containing a pyridine
nucleotide, the reduced form of which fluoresces upon activation by
long wave ultraviolet light but not the oxidized form, thereby
detecting the presence or absence of enzyme deficiencies.
Inventors: |
Beutler; Ernest (Arcadia,
CA) |
Family
ID: |
24573630 |
Appl.
No.: |
04/641,733 |
Filed: |
May 8, 1967 |
Current U.S.
Class: |
435/15; 435/4;
435/25; 435/26 |
Current CPC
Class: |
C12Q
1/48 (20130101) |
Current International
Class: |
C12Q
1/48 (20060101); G01n 031/14 () |
Field of
Search: |
;424/94 |
Other References
"Chemical Abstracts," Vol. 53:9441c (1959) Radley et al.,
Fluorescence Analysis in U.V. Light, pp. 123, 152-154, 270,
273-274, 341, 412 and 415. .
Lowry et al., "Journal of Biological Chemistry," 224:1047-64, 1957
.
Conn et al., Outlines of Biochemistry, pg. 158, 2nd Ed., 1966 .
Marks, "Science," 127:1338-9 (1958) .
Beutler, "Journal of Clinical Investigation," 43(6):1302
(1964).
|
Primary Examiner: Monacell; A. Louis
Assistant Examiner: Hensley; Max D.
Claims
I claim:
1. A method for the detection of glucose-6-phosphate dehydrogenase
deficiency in red blood cells of a blood sample, consisting
essentially of:
adding one part of the blood sample to 10 parts of an aqueous
solution made up of 1 part of 0.01 molar aqueous solution of
glucose-6-phosphate, 1 part of 0.0075 molar aqueous solution of
oxidized triphosphopyridine nucleotide, 2 parts of an aqueous
solution of a red blood cell lysing agent, 3 parts of 0.25 molar
aqueous solution of potassium phosphate buffer at a pH of 7.4, and
3 parts of water;
allowing the resulting test mixture to react;
making a spot from the resulting test mixture on filter paper;
subjecting said spot after drying to longwave ultraviolet light
having a wavelength of from about 340 to about 370 millimicrons
whereby visible fluorescence of said spot is produced by said
ultraviolet light when said spot originates from a blood sample
having red blood cells containing glucose-6-phosphate dehydrogenase
and no fluorescence of said spot is produced by said ultraviolet
light when said spot originates from a blood sample having red
blood cells deficient in glucose-6-phosphate dehydrogenase.
2. A method according to claim 1 in which the aqueous solution of a
red blood cell lysing agent is a saturated solution of
digitonin.
3. A method according to claim 1 in which the aqueous solution of a
red blood cell lysing agent is a 1 percent solution of saponin.
4. A method for the detection of glucose-6-phosphate dehydrogenase
deficiency in red blood cells of a blood sample, consisting
essentially of:
adding one part of the blood sample to 10 parts of an aqueous
solution made up of 1 part of an aqueous solution of
ghucose-6-phosphate having a molarity of from 0.002 to 0.3, 1 part
of an aqueous solution of oxidized triphosphopyridine nucleotide
having a molarity of from 0.001875 to 0.075, 2 parts of an aqueous
solution of a red blood cell lysing agent, 3 parts of potassium
phosphate buffer having a molarity of from 0.025 to 1.25 and a pH
of from 6.5 to 8, and 3 parts of water;
allowing the resulting test mixture to react at room
temperature;
making a spot from the resulting test mixture on filter paper;
and
subjecting said spot after drying to longwave ultraviolet light of
from about 340 to about 370 millimicrons wavelength whereby visible
fluorescence of said spot is produced by said ultraviolet light
when said spot originates from a blood sample having red blood
cells containing glucose-6-phosphate dehydrogenase and no
fluorescence of said spot is produced by said ultraviolet light
when said spot originates from a blood sample having red blood
cells deficient in glucose-6-phosphate dehydrogenase.
5. A method according to claim 4 in which the aqueous solution of a
red blood cell lysing agent is an aqueous solution of digitonin
ranging from a one-tenth saturated to a saturated solution.
6. A method according to claim 4 in which the aqueous solution of a
red blood cell lysing agent is an aqueous solution of saponin
having a concentration ranging from a 1/10 percent to a 20 percent
solution.
7. A method for the detection of galactose-1-phosphate uridyl
transferase deficiency in red blood cells of a blood sample,
consisting essentially of:
adding one part of the blood sample to 10 parts of an aqueous
solution made up of one-thirtieth parts of an aqueous solution of
uridine diphosphoglucose having a molarity of 9.5.times.
10.sup..sup.-3 , one-fifteenth parts of an aqueous solution of
alpha-galactose-1-phosphate having a molarity of 2.7.times.
10.sup..sup.-2 , one-tenth parts of an aqueous solution of oxidized
triphosphopyridine nucleotide having a molarity of 6.6.times.
10.sup..sup.-3 , one-third part of an aqueous solution of
tris-acetate buffer having a pH of 8.0 and a molarity of 0.75,
two-fifteenths parts of an aqueous solution of a red blood cell
lysing agent, and one-third parts of water;
allowing the resulting test mixture to react;
making a spot from the resulting test mixture on filter paper;
subjecting said spot after drying to longwave ultraviolet light at
a wavelength of from about 340 to about 370 millimicrons whereby
visible fluorescence of said spot is produced by said ultraviolet
light when said spot originates from a blood sample having red
blood cells containing galactose-1-phosphate uridyl transferase and
no fluorescence of said spot is produced by said ultraviolet light
when said spot originates from a blood sample having red blood
cells deficient in galactose-1-phosphate uridyl transferase.
8. A method according to claim 7 in which the aqueous solution of a
red blood cell lysing agent is a saturated solution of
digitonin.
9. A method according to claim 7 in which the aqueous solution of a
red blood cell lysing agent is a 1 percent solution of saponin.
10. A method for the detection of galactose-1-phosphate uridyl
transferase deficiency in red blood cells of a blood sample,
consisting essentially of:
adding one part of the blood sample to from 5 to 20 parts of an
aqueous solution made up of 1/30 parts of an aqueous solution of
uridine diphosphoglucose having a molarity of from 2.times.
10.sup..sup.-3 to 9.5.times. 10.sup..sup.-2, one-fifteenth part of
an aqueous solution of alpha-galactose-1-phosphate having a
molarity of from 0.7.times. 10.sup..sup.-2 to 2.7.times.
10.sup..sup.-1 , one-tenth parts of an aqueous solution of oxidized
triphosphopyridine nucleotide having a molarity of from 1.6.times.
10.sup..sup.-3, to 6.6.times. 10.sup..sup.-2, 1/3 parts of an
aqueous solution of tris-acetate buffer having a pH of from 6.2 to
9.2 and a molarity of from 0.075 to 7.2, two-fifteenths part of an
aqueous solution of a red blood cell lysing agent, and one-third
parts of water;
allowing the resulting test mixture to react;
making a spot from the resulting test mixture on filter paper;
and
subjecting said spot after drying to ultraviolet light of from
about 340 to about 370 millimicrons wavelength whereby visible
fluorescence of said spot is produced by said ultraviolet light
when said spot originates from a blood sample having red blood
cells containing galactose-1-phosphate uridyl transferase and no
fluorescence of said spot is produced by said ultraviolet light
when said spot originates from a blood sample having red blood
cells deficient in galactose-1-phosphate uridyl transferase.
11. A method according to claim 10 in which the aqueous solution of
red blood cell lysing agent is an aqueous solution of digitonin
ranging from a one-tenth saturated to a saturated solution.
12. A method according to claim 10 in which the aqueous solution of
a red blood cell lysing agent is an aqueous solution of saponin
having a concentration ranging from a 1/10 percent to a 20 percent
solution.
Description
BACKGROUND OF THE INVENTION
This invention relates to a new type of mass screening procedure
for the detection of enzyme deficiencies affecting red blood cells,
and more particularly concerns the presence or absence of
fluoresence in test samples upon being subjected to long range
ultraviolet light, depending upon whether reduced or oxidized
pyridine nucleotide is present in the test sample.
PRIOR ART
Applicant knows of no prior art making use of fluorescence of a
pyridine nucleotide under long wave ultraviolet light to screen for
enzyme abnormalities in red blood cells. Generally, various dyes
have been used in the past to detect such enzyme deficiencies.
Detection of glucose-6-phosphate dehydrogenase deficiency through
dye decolorization techniques requires anaerobic conditions, which
are difficult to maintain and unsuitable for mass screening. Other
tests require fresh blood samples or separation of the blood
hemoglobin from the enzyme or special paper.
Where the enzyme deficiency is lack of galactose-1-phosphate uridyl
transferase, methylene blue has been used as a receptor dye but
requires anaerobic conditions after gassing with carbon monoxide.
The reaction is somewhat light sensitive, and it is necessary to
use an illuminated water bath for the test.
With respect to screening for glutathione reductase deficiency,
applicant knows of no method for screening for this enzyme
deficiency in red blood cells.
The procedure described herein is extremely simple, does not
require anaerobic conditions, requires only a minute quantity of a
blood sample, can be carried out at room temperature or at
37.degree. C., the blood sample need not be freshly obtained, and
is very easy to interpret.
SUMMARY OF THE INVENTION
The invention comprises a new type of screening procedure for the
detection of various enzymatic deficiencies of red blood cells. The
sample to be tested, either of whole blood or of the red cells
alone, is added to a reaction mixture containing pyridine
nucleotide, a buffer, a red cell hemolyzing agent, and an enzyme
substrate.
Depending upon the nature of the enzyme deficiency to be detected,
the pyridine nucleotide in the reaction mixture is either in
reduced or oxidized form.
The spots from the resulting test mixture are made on ordinary
filter paper, immediately as a baseline or control spot, and after
varying periods of time. The resulting test mixture may be kept at
room temperature or incubated at 37.degree. C. After the spots have
dried on the filter paper, the spots are subjected to longwave
ultraviolet light preferably at a wavelength of from 340 to 370
millimicrons.
Spots containing reduced pyridine nucleotide will persistently
fluoresce under longwave ultraviolet light. Such fluorescence
remains stable for several days at room temperature.
Where the reaction mixture uses reduced pyridine nucleotide and
where the reduced pyridine nucleotide is oxidized in the resulting
test mixture, there is loss of fluorescence since oxidized pyridine
nucleotide does not fluoresce upon subjection to longwave
ultraviolet light.
In particular, my invention will detect deficiencies in red blood
cells including glucose-6-phosphate dehydrogenase, hereinafter
referred to as G-6-PD; pyruvate kinase, hereinafter referred to as
PK; glutathione reductase, hereinafter referred to as GSSG-R; and
glactose-1-phosphate uridyl transferase, hereinafter referred to as
transferase.
In general, my invention is readily adaptable to the screening for
many other enzymatic abnormalities, such as phosphogluconic
dehydrogenase deficiency and triose phosphate isomerase
deficiency.
Theoretically, any enzyme deficiency which results either in the
reduction of pyridine nucleotide or oxidation of reduced pyridine
nucleotide in a test mixture, can be detected by my new
procedure.
Precise diagnosis of disorders resulting from such enzyme
deficiencies is of great medical value, both from the viewpoint of
genetic counseling and also from that of medical therapy. To assay
all of these enzymes by known procedures hitherto, is costly, time
consuming, and requires expensive and specialized laboratory
facilities.
Accordingly, there is a great need for simple and rapid procedures
which will differentiate the more common enzyme deficiencies in red
blood cells from one another.
It is, therefore, an object of this invention to provide a rapid
and relatively simple procedure for the detection of a G-6-PD
deficiency in red blood cells.
Another object of this invention is to provide a rapid and
relatively simple procedure for the detection of a GSSG-R
deficiency in red blood cells.
Still another object of this invention is to provide a rapid and
relatively simple procedure for the detection of a PK deficiency in
red blood cells.
Yet another object of this invention is to provide a rapid and
relatively simple procedure for the detection of transferase
deficiency in red blood cells.
A yet further objection of this invention is to provide a rapid and
relatively simple procedure for the detection of various enzyme
deficiencies in red blood cells.
These and other objects will be more readily understood by
reference to the following specification and claims.
DESCRIPTION OF PREFERRED EMBODIMENTS
The same general procedure is used for all screening procedures. In
general, one part of whole blood or red cell suspension, usually
0.020 ml. is added to 10 parts of the reaction mixture, usually
0.200 ml. Immediately after this addition, a spot of the resulting
test mixture may be made on filter paper, as a control baseline
spot. After specified periods of incubation of the test mixture,
additional spots from the test mixture are made on the filter
paper. After the spots have dried, the filter paper is inspected in
a darkened room under a convenient source of longwave ultraviolet
light.
The detection of enzyme deficiency is based upon the fact that
relatively minute quantities of reduced pyridine nucleotides will
fluoresce intensely upon activation by longwave ultraviolet light
at a wavelength of from about 340 to about 370 millimicrons.
In the case of the test for G-6-PD deficiency or transferase
deficiency, the tests are based upon the reduction of pyridine
nucleotide.
In the case of the test for PK deficiency or GSSG-R deficiency, the
tests are based upon the oxidation of a reduced pyridine
nucleotide.
Although there is some quenching of fluorescence in the presence of
hemoglobin in the blood, two factors minimize this quenching. In
the first place, the exciting ultraviolet wavelength used in my new
procedure is below the maximum absorption band of hemoglobin, which
is the Soret region. Further, the emission maximum for my new
procedure is located at 465 millimicrons which is near an
absorption minimum of hemoglobin.
Secondly, when the resulting test mixture is spotted on ordinary
filter paper, there is some chromatographic separation of the blood
hemoglobin from the pyridine nucleotides, and consequently,
considerable intensification of fluorescence results.
In all of these test procedures, the volume of the blood sample to
be tested in relation to the reaction mixture may vary over a range
from a minimum of one part of blood sample to 20 parts of reaction
mixture, up to a maximum of one part of blood sample to 5 parts of
reaction mixture.
Where an incubator is not immediately available, such as in a field
screening program, the test reaction may be allowed to proceed at
room temperatures.
The following is an example for the detection of G-6-PD deficiency
in red blood cells, illustrating the use of my new screening
procedure.
EXAMPLE 1
A blood sample suitable for testing may be collected in acid
citrate dextrose (ACD), or in heparin, or in disodium
ethylenediamine tetra-acetic acid (EDTA) or other suitable blood
anticoagulant. The blood sample need not be fresh. Even several
week old blood samples stored in acid citrate dextrose at 4.degree.
C. are suitable for testing.
A suitable reaction mixture for testing for the presence of G-6-PD
comprises a mixture of the following:
---------------------------------------------------------------------------
Ingredient in Water solution Concentration Amount in ml.
__________________________________________________________________________
Glucose-6-phosphate 0.01 M 0.10 Triphosphopyridine nucleotide
0.0075 M 0.10 Digitonin, saturated solution 0.20 Potassium
phosphate buffer, 0.25 M 0.30 pH 7.4 Water Total 0.30 1.00 ml.
__________________________________________________________________________
The basis for the test is that when G-6-PD is present in the blood
sample, glucose-6-phosphate is oxidized during the reaction
sequence to 6-phosphogluconate, and the triphosphopyridine
nucleotide, hereinafter referred to as TPN, is reduced to reduced
triphosphopyridine nucleotide, hereinafter referred to as TPNH.
Since the blood sample also contains 6-phosphogluconic
dehydrogenase, the 6-phosphogluconate formed in the reaction is
oxidized by 6-phosphogluconic dehydrogenase, thereby reducing
additional TPN to TPNH. When activated by longwave ultraviolet
light, the TPNH fluoresces brightly.
In the test procedure, one volume of whole blood, usually 0.02 ml.
in amount, is added to 10 volumes of the reaction mixture, usually
0.20 ml., and the resulting test mixture is incubated for 5 to 10
minutes at 37.degree. C.. A spot is made from the resulting test
mixture on any suitable filter paper, such as Whatman No. 1. If
normal G-6-PD activity is present, the spot on the filter paper
will fluoresce brightly under activation by longwave ultraviolet
light. Where there is G-6-PD deficient blood, no appreciable
fluorescence appears upon subjecting the spot to longwave
ultraviolet light of from about 340 to about 370 millimicrons.
Once a test spot has been made on filter paper, and dried,
fluorescence is easily detectable for several days thereafter.
The test procedure for the presence of G-6-PD may be varied in many
ways without adversely affecting the results.
For example, instead of incubating the resulting test mixture at
37.degree. C., the reaction may be allowed to proceed at room
temperatures with equivalent results after a period of 10 to 15
minutes.
Instead of filter paper, any suitable absorbent material may be
used to make spots from the test mixture, so long as the material
permits substantial drying of the spots to halt the test
reaction.
Instead of disodium ethylene diamine tetra-acetic acid, the free
acid as well as other alkali salts of this acid may be used as an
anticoagulant.
Since the reaction proceeds rapidly even at 0.degree. C., the
reaction time allotted before the commencement of spot testing may
vary from a minimum of 2 minutes to a maximum of 10 to 15 minutes
at 37.degree. C. These reaction times should be increased by a
factor of about 1.5 where the reaction takes place at room
temperatures.
For example, instead of the 0.20 ml. of a saturated solution of
digitonin, 0.20 ml. of a 1 percent solution of saponin may be
substituted as an alternative ingredient in the reaction mixture.
When this substitution is made, the resulting test mixture need be
incubated only about 5 minutes at 37.degree. C.
The concentrations of TPN and glucose-6-phosphate may be varied
without substantially altering the results of this test procedure.
For example, the concentration of glucose-6-phosphate in the
reaction mixture may be varied over a range beginning with a low of
one-fifth to a high of 30 times the concentration given in example
1, with a corresponding range of TPN concentration varying over a
low of one-fourth to a high of 10 times the concentration given in
example 1.
The potassium phosphate buffer may vary in pH from a low of 6.5 to
a high of 8 and in a concentration range from a low of 1/10 to a
high of 5 times the concentration given in example 1. Suitable
phosphate buffers other than potassium may be used.
The digitonin solution need not be saturated so long as there is
sufficient digitonin present in the reaction mixture to effectively
lyse the red blood cells. The concentration of digitonin may vary
from a one-tenth saturated solution to a fully saturated solution.
Where saponin is used, the concentration may range from a 1/10
percent solution to a 20 percent solution. Other suitable lysing
agents may be used so long as no interference occurs with the test
reaction.
The following is an example for the detection of the absence of the
enzyme, galactose-1-phosphate uridyl transferase. The lack of this
enzyme in the blood causes galactosemia, which if untreated,
results in cirrhosis of the liver, blindness, and mental
retardation. If this enzyme deficiency is detected early, a
galactose-free diet will prevent these abnormalities.
EXAMPLE 2
A blood sample for testing is collected in heparin. The reaction
mixture for this test comprises the following:
---------------------------------------------------------------------------
Concentration edient in wat mount in ml.
__________________________________________________________________________
9.5.times.10.sup.-.sup.3 M glucose 2.7.times.10.sup.-.sup.2 M
6.6.times.10.sup.-.sup.3 M hate 0.4 0.6 Tris-acetate buffer, pH 8.0
0.75 M 2.0 Digitonin, saturated solution 0.8 EDTA
2.7.times.10.sup.-.sup.2 M Water 0.03 1.97 Total 6.00
__________________________________________________________________________
The basis for detection is that when whole blood is added to the
above reaction mixture, uridine diphosphoglucose (UDPG) and alpha
galactose-1-phosphate (Gal-1-P) react to form
alpha-glucose-1-phosphate in the presence of transferase. The
alpha-glucose-1 phosphate is transformed by phosphoglucomutase,
which is present in the hemolysate, to alpha-glucose-6-phosphate,
which in turn mutarotates spontaneously and with the help of
phosphohexose isomerase to beta-glucose-6-phosphate.
Glucose-6-phosphate dehydrogenase, present in the hemolysate,
oxidizes beta-glucose-6 phosphate to 6-phosphogluconate which in
turn is oxidized to ribulose-5-phosphate. Both of these steps
result in the reduction of TPN to TPNH, which fluoresces under
longwave ultraviolet light. Substantially no fluorescence occurs in
tests derived from transferase deficient blood samples.
In the test procedure, one volume of heparinized whole blood,
usually 0.02 ml., is added to 10 volumes, usually 0.2 ml. of
reaction mixture. The pipette used to add the blood to the reaction
mixture is left in the tube, and the resulting test mixture is
incubated aerobically at 37.degree. C. At the end of two hours, a
spot, a few microliters of test solution, is made on Whatman No. 1
filter paper and is allowed to dry at room temperature. Such drying
usually takes about 5 minutes. The spot is examined within 24 hours
after drying under longwave ultraviolet light of from about 340 to
about 370 millimicrons.
Under such longwave ultraviolet light, spots resulting from normal
blood fluoresce brightly, while spots from blood deficient in
transferase have no appreciable fluorescence. After drying, the
spots are relatively stable up to one week at room
temperatures.
The test procedure may be varied in several ways without adversely
affecting the results.
Streaks of capillary blood may be dried on filter paper or other
suitable absorbent material and placed into a suitable container
holding an appropriate volume, (about 10 times the blood volume) of
the reaction mixture. While the filter paper is immersed in the
reaction mixture, the reaction mixture is incubated for 2 to 3
hours at 37.degree. C. After incubation, a spot is made from the
resulting test mixture on filter paper or other suitable absorbent
material that permits drying of spots. The spot so made is allowed
to dry at room temperature, which usually takes about 5 minutes.
Thereafter, the resulting spot is examined under longwave
ultraviolet light for fluorescence in the same manner as
before.
So long as it has not coagulated, fresh whole blood may be used
instead of heparinized blood for the test.
The test may be carried out at room temperatures, with a
corresponding decrease in reaction rate of from one-half to
one-third the rate at 37.degree. C.
Any suitable absorbent material may be used instead of filter paper
so long as it permits drying of the spot made on it.
The time of incubation at 37.degree. C. may vary from 1 to 3 hours
without adversely affecting the results.
Instead of whole blood, a 50 percent suspension of red cells in
0.9% NaCl solution may be used with results similar to that using
whole blood.
EDTA may be omitted as an ingredient in the reaction mixture since
it only helps enhance the development of fluorescence in the test
mixture. Where blood for testing has been collected in EDTA to
prevent coagulation, the reaction mixture must omit EDTA.
Blood samples collected in heparin may be stored as long as one
week at room temperature and may still be used in this test
procedure with results equivalent to fresh blood samples.
The digitonin solution concentration may be varied from a low of
1/10 saturation up to saturation, so long as there is sufficient
digitonin to effectively lyse the red blood cells. Instead of
digitonin, a 1 percent solution of saponin may be used with
equivalent results. The concentration of saponin may vary from 1/10
to 20 percent without adverse effect on the test procedure. Other
suitable red blood cell lysing agents may also be used, so long as
no interference occurs with the test reaction.
The concentration of UDPG, Gal-1-P, and TPN in the reaction mixture
may be varied without substantially altering the results of the
test procedure. Thus, each of the corresponding concentrations of
UDPG, Gal-1-P, and TPN, may be varied over a range from a low of
one-fourth to a high of 10 times the concentrations given in
example 2.
The tris-acetate buffer may vary over a pH range of 6.2 to 9.2, and
a concentration range from a low of one-tenth to a high of 3 times
the concentration given in example 2.
The following is an example for the detection of a pyruvate kinase
deficiency in red blood cells.
EXAMPLE 3
Anticoagulants such as heparin, ACD, or EDTA, or other suitable
anticoagulants, may be used in the preparation of the blood sample
to be tested. The anticoagulated blood sample is then centrifuged
and the plasma and buffy coat are removed with careful aspiration
or other suitable means. The white blood cells are removed since
white cell pyruvate kinase activity may be normal even though the
red blood cells are deficient in pyruvate kinase.
Four volumes of physiologic saline solution are added to the red
blood cells to produce a 20 percent suspension of red blood cells.
The resulting suspension of red blood cells is now ready for
testing.
The following is a suitable reaction mixture for the text
procedure:
---------------------------------------------------------------------------
Ingredient in water solution Concentration Amount in ml.
__________________________________________________________________________
Phospho (enol) pyruvate 0.15 M (neutralized) 0.03
(tricyclohexylammonium salt) Adenosine diphosphate (ADP) 0.03 M
(neutralized) 0.10 DPNH 0.015 M (neutralized) 0.10 Magnesium
sulfate 0.08 M 0.10 Potassium phosphate buffer pH 7.4 0.25 M 0.05
Water Total 0.62 1.00
__________________________________________________________________________
The basis of this test procedure is that where pyruvate kinase is
present in the blood sample, a phosphate group from phospho (enol)
pyruvate is transferred to adenosine diphosphate (ADP), forming
pyruvate and adenosine triphosphate (ATP). Lactate dehydrogenase in
the hemolysate catalyzes the reduction of pyruvate to lactate with
the oxidation of the reduced diphosphopyridine nucleotide (DPNH) in
the reaction mixture to diphosphopyridine nucleotide (DPN). Since
DPNH fluoresces under longwave ultraviolet light and DPN does not,
there is a gradual loss of the fluorescence in a test sample
derived from normal blood.
In the test procedure, one volume of the red blood cell suspension,
usually 0.02 ml., is added to 10 volumes of the reaction mixture,
usually 0.20 ml., and the resulting test mixture is incubated at
37.degree. C. for 30 minutes, after which spots from the test
mixture are made on filter paper as previously described in
preceding examples, dried, and examined under longwave ultraviolet
light. Where the original blood sample contained red blood cells
deficient in pyruvate kinase, bright fluorescence of spots derived
from such a blood sample will persist, while spots derived from
normal blood samples will have no appreciable fluorescence, after
incubation.
The neutralized ingredients of the reaction mixture of example 3
may be neutralized to a pH of 7-8 with pH paper using approximately
0.2 N NaOH.
Hypotonic lysis is used to release enzyme from the red blood cells
rather than digitonin or saponin to prevent any substantial release
of enzyme from white cells which may remain in the treated blood
sample. Any suitable lysing agent may be used so long as it causes
no interference with the test reaction.
The concentration of the ingredients of the reaction mixture of
example 3 may be varied without adverse effect on the screening
procedure. The concentration of phospho (enol) pyruvate
(tricyclohexylammonium salt) may range from a low of 1/4 to a high
of 3 times the concentration given in example 3, with a
corresponding range in concentration of
ADP from one-third to 10 times, and with a corresponding range of
DPNH from one-fourth to 2 times, the concentrations given in
example 3.
The concentration of magnesium sulfate may range from a low of
one-fourth to a high of 5 times the concentration given in example
3, along with the corresponding ranges for the other ingredients in
the reaction mixture.
The potassium phosphate buffer may range from a pH of 6.5 to 7.5
and from a low of one-tenth to a high of 5 times the concentration
given in example 3.
The physiologic saline solution for the suspension of the red blood
cells may vary over a range of about one-fourth less to about
one-fourth more than isotonic strength, so long as lysing of the
red blood cells occurs in the resulting test mixture.
Instead of incubation of the test mixture at 37.degree. C. the
reaction may be allowed to proceed at room temperatures, with a
corresponding increase in reaction time of from 11/2 to 2
times.
The reaction time alloted before commencement of spot testing may
vary from a minimum of about 10 minutes to a maximum of about 30
minutes at 37.degree. C.
Other water soluble magnesium salts may be used instead of
magnesium sulfate so long as no interference occurs with the
reaction.
Various water soluble potassium phosphate salts may be used as a
buffer.
Fresh blood may be used so long as it has not coagulated.
Instead of disodium ethylene diamine tetra-acetic acid, the free
acid as well as other alkali salts of ethylene diamine tetra-acetic
acid may be used as an anticoagulant.
The following is an example for the detection of a GSSG-R
deficiency in red blood cells.
EXAMPLE 4
A blood sample suitable for testing may be collected in ACD, EDTA,
or heparin. Such samples may be stored for at least 3 weeks without
adverse effects on the test procedure.
A suitable reaction mixture for the detection of GSSG-R deficiency
is the following
---------------------------------------------------------------------------
Concentration edient in wat r mount in ml.
__________________________________________________________________________
Glutathione (oxidized) 0.033 M 0.1 TPNH 0.015 0.1 Potassium
phosphate buffer pH 7.4 0.25 M 0.6 Digitonin, saturated solution
0.2 Total 1 ml.
__________________________________________________________________________
The basis for this test procedure is that where glutathione
reductase is present in the blood hemolysate, oxidized glutathione
(GSSG) is reduced to glutathione (GSH) and TPNH is oxidized to TPN.
Since TPNH fluoresces when activated with longwave ultraviolet
light and TPN does not so fluoresce, a gradual loss of fluorescence
takes place as the reaction proceeds, where the blood sample is
normal.
In the test procedure, one volume of blood, usually 0.02 ml., is
added to 10 parts of reaction mixture, usually 0.20 ml. Spots from
the resulting test mixture are made on suitable filter paper every
15 minutes and the disappearance of fluorescence is compared with a
control sample over a suitable period of time up to 90 minutes,
under longwave ultraviolet light of from about 340 to about 370
millimicrons. A GSSG-R deficiency is detected by the persistence of
fluorescence in a spot.
The reaction may be carried out at 37.degree. C. or at room
temperatures. Where room temperatures are used, the reaction time
may be from 11/2 to 2 times the reaction time at 37.degree. C.
The concentrations of the ingredients of the reaction mixture of
example 4 may be varied without adversely affecting the test
results. The concentration of GSSG may range from a low of
one-fourth to a high of 20 times the concentration given in example
4, while the concentration of TPNH may vary over a corresponding
range of a low of one-half to a high of 2 times the concentration
given in example 4.
As in examples 1 and 2, saponin may be substituted for digitonin,
and the same ranges of concentrations may be used in example 4 as
indicated for digitonin and saponin in examples 1 and 2. The
concentration of phosphate buffer may vary from a low of 1/10 to a
high of 10 times the concentration given in example 4, and from a
pH OF 6.5 to a pH of 9.5 corresponding to the range in
concentration.
Other suitable lysing agents may be used instead of digitonin and
saponin provided that no interference results with the test
procedure.
Fresh blood may be used so long as it is not coagulated.
Various water soluble potassium phosphate salts may be used as a
buffer.
Instead of disodium ethylene diamine tetra-acetic acid, the free
acid as well as other alkali salts of ethylene diamine tetra-acetic
acid may be used as an anticoagulant.
Instead of filter paper, any suitable absorbent material may be
used to make spots from the test mixture, so long as the material
permits substantial drying of the spots to halt the test
reaction.
Although I have described preferred embodiments of my invention, it
is understood that the scope of the invention is not limited
thereby, but numerous variations in reagents and procedures are
possible without departing from the spirit and scope of the
invention as claimed hereinafter.
For example, any water soluble salt may be used as a buffer
provided that no interference occurs with the test reaction.
Fresh, uncoagulated blood, may be used in each of the test
procedures.
The screening procedures disclosed herein are applicable to
screening for the presence or absence of various other enzymes
associated with red blood cells by the use of the proper substrates
for the particular enzymes sought, along with any required
cofactors or coenzymes, where the presence or absence of any such
enzyme results in the oxidation or reduction of a pyridine
nucleotide, directly or indirectly.
* * * * *