U.S. patent number 3,864,084 [Application Number 05/277,810] was granted by the patent office on 1975-02-04 for method for measurement of oxygen content in blood and other liquids by oxygen inhibition of chemical reactions.
Invention is credited to M. Judah Folkman.
United States Patent |
3,864,084 |
Folkman |
February 4, 1975 |
Method for measurement of oxygen content in blood and other liquids
by oxygen inhibition of chemical reactions
Abstract
Method for measuring the oxygen content of blood. The blood
sample to be analyzed is mixed with a monomer solution and free
radical initiated polymerization is induced and timed. Oxygen in
the sample inhibits polymerization. The logarithm of the time
required to polymerize is linearly proportional to the oxygen
content of the sample. Liquids other than blood can be analyzed by
the disclosed method.
Inventors: |
Folkman; M. Judah (Brookline,
MA) |
Family
ID: |
23062448 |
Appl.
No.: |
05/277,810 |
Filed: |
August 3, 1972 |
Current U.S.
Class: |
436/68; 436/11;
526/229; 526/306; 436/136; 526/234 |
Current CPC
Class: |
G01N
31/00 (20130101); G01N 33/4925 (20130101); Y10T
436/207497 (20150115); Y10T 436/102499 (20150115) |
Current International
Class: |
G01N
33/49 (20060101); G01N 31/00 (20060101); C08f
003/90 (); G01n 033/16 () |
Field of
Search: |
;23/23B,23R
;260/89.7R |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Reese; Robert M.
Attorney, Agent or Firm: Toupal; John E.
Claims
What is claimed is:
1. A method of measuring the oxygen content of liwuids comprising
the steps of:
mixing a test liquid containing oxygen with an activator liquid to
produce a polymer by a free radical initiated polymerization
reaction that is inhibited by oxygen in said test liquid;
isolating said test liquid and said activator liquid from any gas
phase during said mixing step; and
timing said polymerization reaction to determine the amount of
oxygen initially present in said test liquid and consumed during
said reaction.
2. A method according to claim 1 including a degassing step for
removing gases from said activator liquid before said mixing
step.
3. A method according to claim 1 wherein the temperature of said
liquids is regulated during said mixing and timing steps.
4. A method according to claim 1 where said test liquid is
water.
5. A method according to claim wherein said test liquid comprises a
fluorocarbon.
6. A method according to claim 5 comprising an equilibrating step
to equilibrate said fluorocarbon with a gaseous atmosphere before
said mixing step.
7. A method according to claim 1 wherein said timing step comprises
a viscosity monitoring step.
8. A method according to claim 7 comprising the step of comparing
the measure of time yielded by said timing step to a standard of
comparison for determing the amount of oxygen in said test
liquid.
9. A method according to claim 8 wherein said polymerization
comprises gelation and said polymer is a gel.
10. A method according to claim 8 wherein said standard of
comparison is a graph comprising a curve with upper and lower end
points.
11. A method according to claim 10 wherein said upper end point is
located by oximetery of a fully oxygenated blood sample.
12. A method according to claim 10 wherein said lower end point is
located by oximetery of a physiological saline solution.
13. A method according to claim 8 wherein said test liquid
comprises a monomer solution and said activator liquid comprises
free radical ions.
14. A method according to claim 13 wherein said test liquid further
comprises blood.
15. A method according to claim 14 wherein said test liquid further
comprises an anticoagulant.
16. A method according to claim 15 wherein said test liquid further
comprises a buffer.
17. A method according to claim 8 wherein said mixing step
comprises mixing a reduction activator with said liquids for
producing sulfate radical ions in said activator liquid.
18. A method according to claim 17 wherein said reduction activator
comprises bisulfite and said monomer comprises a vinyl monomer and
said activator liquid comprises persulfate ions.
19. A method according to claim 18 wherein said reduction activator
comprises sodium bisulfite and said vinyl monomer is acrylamide and
said activator liquid comprises ammonium persulfate.
20. A method according to claim 18 wherein said polymerization
includea copolymerization.
21. A method according to claim 20 wherein said test liquid further
comprises bisacrylamide.
22. A method of measuring the oxygen content of liquids comprising
the steps of:
providing an oxygen containing test liquid and an activator
liquid;
degassifying said activator liquid;
mixing said test liquid with said activator liquid to produce a
polymer by a free radical initiated polymerization reaction that is
inhibited by oxygen in said test liquid; and
timing said polymerization reaction to determine the amount of
oxygen initially present in said test liquid and consumed during
said reaction.
23. A method of measuring the oxygen content of fluorocarbons
comprising the steps of:
mixing a fluorocarbon test liquid containing oxygen with an
activator liquid to produce a polymer by a free radical initiated
polymerization reaction that is inhibited by oxygen in said test
liquid; and
timing said polymerization reaction to determine the amount of
oxygen initially present in said fluorocarbon test liquid and
consumed during said reaction.
24. A method of measuring the oxygen content of water comprising
the steps of:
mixing a test sample of water with an activator liquid to produce a
polymer by a free radical initiated polymerization reaction that is
inhibited by oxygen in said test sample; and
timing said polymerization reaction to determine the amount of
oxygen initially present in said test sample and consumed during
said reaction.
25. A method according to claim 24 comprising the step of isolating
said liquids from any gas phase during said mixing step.
Description
BACKGROUND OF THE INVENTION
This invention relates to a method for measuring the oxygen content
in blood and other liquids and, more particularly, to measurement
by timing inhibition of free radical initiated polymerization
caused by oxygen.
Van Slyke and Neill introduced the manometric method for oxygen
analysis of blood which has remained the standard against which
other methods are judged. In essence, it involves liberation of
oxygen from a blood sample by a combination of hemolysis,
conversion of hemoglobin to methemoglobin, and vacuum extraction;
measurement of the pressure exerted by the liberated gas at a fixed
volume; then absorption of the oxygen with a reducing solution and
a second measurement of pressure. Since the volume is fixed and the
temperature can be measured, the pressure difference gives the
oxygen content by the ideal gas law.
This method is cumbersome, with several important disadvantages.
First, each determination is time-consuming, requiring about 15
minutes for completion. Second, the manipulations required are
numerous and exacting; there are many openings for error and a
great deal of training and experience is necessary if a technician
is to master the technique. Third, the ordinary sample size is
large, usually 1.0 ml, a significant problem in repetitive followup
studies in very small pediatric patients. Fourth, dissolved
anesthetic gases such as N.sub.2 O adversely affect the results
obtainable because of incomplete removal by the reducing
solution.
Gas chromatography has been applied to the problem. In this method,
the gas peak corresponding to oxygen is integrated and related to
the total gas volume and sample volume. The method is said to be
accurate and reproducible, but requires a large outlay of money for
the instrument and sophisticated training for its operator.
With the invention of the Clark oxygen electrode came the
possibility of polarographic methods. The Clark electrode gives a
rapid direct reading of oxygen tension (pO.sub.2) on the basis of
current developed at fixed voltage between a platinum electrode
exposed to the oxygen-containing solution through an
oxygen-permeable membrane, and a reference electrode. Oxygen
tension can be used to calculate oxygen saturation from a standard
oxyhemoglobin dissociation curve. The oxygen content can be derived
if the oxygen capacity of the blood in question is known (on the
basis of hemoglobin level: each gram of hemoglobin binds a constant
1.34 ml of oxygen). This method is adaptable to very small sample
volumes and the measurement can be made rapidly; however, there are
several major drawbacks. As an indirect method, it is vulnerable to
inaccuracies related to the intermediate steps in calculation. The
standard dissociation curve itself, for example, is now thought to
shift position in consonance with levels of 2,3-DPG and possibly
other factors, in response to hypoxia, anemia, stress, etc. The
values of factors which affect the curve, especially temperature
and pH, must be known accurately. Hemoglobin must be determined
independently. The number of ancillary values which must be
obtained makes this method somewhat complicated and open to
error.
A second polarographic approach has been to measure the increase in
pO.sub.2 resulting from liberation of bound oxygen from hemoglobin
in dilute solution when the hemoglobin is oxidized to
methemoglobin, and from this increase calculate the oxygen content.
In skilled hands this method is accurate and requires only
microliter samples. However, the apparatus is expensive and
sophisticated.
The object of this invention therefore is to provide a rapid method
of measuring oxygen content of blood that does not require highly
trained technicians or elabrate equipment, yet will provide
accurate, reproducible results.
SUMMARY OF THE INVENTION
This invention is characterized by a method for measuring the
oxygen content of liquids. A test liquid, containing oxygen, is
mixed with an activator liquid and reacts therewith to produce a
product at a rate dependent on the quantity of oxygen present. The
rate at which the product is produced is timed and thus provides an
indication of the quantity of oxygen in the test liquid. As
described more fully below, the reagents should be isolated from
any gaseous phase and temperatures should be regulated. In
addition, depending on the process and the accuracy desired, the
reagents may be degassed before use as is discussed below. Among
the advantages of this method is the small volume of test liquid
required. In addition, results are accurate and repeatable and up
to 20 tests per hour can be made. Furthermore, pH of the test
liquid does not affect the result if the reagents are properly
buffered. If the test liquid includes blood the results are
unaffected by pCO.sub.2 and drugs commonly given to postoperative
cardiac patients.
The timing step includes monitoring the viscosity of the product
and comparing the measure of time obtained to a standard of
comparison such as a chart or a graph. For example, disclosed
herein is a reagent system utilizing the oxygen inhibition of
polymerization of vinyl compounds. In this system, when
polymerization is complete, a gel is formed thus producing a
radical difference in viscosity and a clearly defined end point.
Therefore, comparison of the measured time quickly and accurately
provides a measure of the oxygen in the test liquid.
Although the subject method was intended to aid in the oximetry of
blood it has been found useful for other liquids such as
fluorocarbons and water. Thus, other uses for the method become
evident. For example, if a fluorocarbon is permitted to equilibrate
with a gaseous phase and oximetry is then performed on the
fluorocarbon in accord with the disclosed method, measure of the
oxygen in the gaseous phase is supplied.
DESCRIPTION OF THE DRAWINGS
These and other features and objects of the present invention will
become more apparent upon a perusal of the following description
taken in conjunction with the accompanying drawings wherein:
FIG. 1 shows a syringe being filled with reagent by a hypodermic
needle in accordance with the subject method;
FIG. 2 shows 2 syringes filled with the reagents and each
containing a glass bead wherein the reaction in the left syringe is
incomplete but the reaction in the right syringe has reached the
point of gelation and the glass bead therein has become
immobilized;
FIG. 3 is a graph of reaction time v. oxygen content that is a
typical standard of comparison;
FIG. 4 shows schematically apparatus for practicing the subject
method semiautomatically with electromagnetic monitoring apparatus;
and
FIG. 5 is an isometric view of a water bath apparatus utilized in
conjunction with the syringe mixing chamber shown in FIG. 4 to
practice the subject method under controlled temperature
conditions.
DESCRIPTION OF THE PREFERRED METHOD
The rate at which many chemical reactions proceed is affected
markedly by the presence or absence of oxygen. Thus many reactions
can potentially be used for performing oxygen assays in accordance
with the subject method. The copolymerization of two vinyl
monomers, specifically acrylamide and bisacrylamide, is discussed
in detail by way of example only. Other compounds that can be used
for oxygen assays will be mentioned below and still others will be
obvious to skilled chemists.
Polymerization of acrylamide is initiated by sulfate radical ions
(SO.sub.4 .sup.-.) generated by the scission of a weak 0--0 linkage
in persulfate ions (S.sub.2 O.sub.8 =) with the activation energy
advantage provided by a reduction activator, bisulfite (HSO.sub.3
.sup.-). The sulfate radical ion attacks the double bond of an
acrylamide molecule as follows to generate an acrylamide radical.
##SPC1##
In the absence of oxygen, polymerization would then occur through
propagation steps based on acrylamide radicals attacking fresh
acrylamide molecules to yield new radicals which would then attack
further fresh acrylamide molecules, and so forth in a chain
reaction, with the polymer solution becoming progressively more
viscous and ultimately gelling as polymerization proceeds.
##SPC2##
The firmness of the polymer thus formed is increased by adding a
certain proportion of N,N'-methylenebisacrylamide, which permits
cross-linking of polymer chains: ##SPC3##
In the presence of oxygen, the acrylamide radical reacts with
molecular oxygen in preference to another molecule of acrylamide to
yield a peroxy radical. ##SPC4##
This, in contradistinction to the acrylamide radical, is relatively
unreactive. Thus the acrylamide, rather than lengthening its
polymer chain, is tied up as long as oxygen is present. The oxygen
is used up, of course, as it is incorporated into acrylamide
molecules. Once its concentration in the medium becomes
sufficiently small, the peroxy radicals and acrylamide radicals
will again react with acrylamide, and the more reactive
acrylamide-ended radicals will react quickly in chain-lengthening
steps. (The peroxy radicals are, of course, slow to react. However,
once a peroxy radical does react with an acrylamide molecule, it
forms an acrylamide-ended radical and thus continues to react
rapidly.) The net result of all this is a lag period during the
course of the polymerization reaction, during which oxygen is used
up, followed by rapid polymerization. The transition between these
phases can be quite abrupt. The more oxygen present in relation to
acrylamide, the longer the lag period.
Three solutions are generally used in the acrylamide oxygen assay
in addition to the blood sample: a monomer solution, a persulfate
solution and a bisulfite solution.
1. Monomer Solution: This is prepared according to the formula:
Acrylamide 6.12g
Bisacrylamide 0.308 g
Distilled Water 20 ml
and degassed 8 min in 30 ml syringes with a rotary vacuum pump. If
the liquid to be assayed varies in pH, it is beneficial to buffer
the monomer solution by dissolving 0.477 g of HEPES
(N-2-hydroxyethylpiperacine-N-2-Ethanesulfonic acid (availabe from
Calbiochem, Los Angeles), or other buffer, in a portion of the
distilled water along with the acrylamide and bisacrylamide,
titrating to pH 7.4, adding the remainder of the distilled water
and degassing as described. The degassing step can be omitted if a
new standard curve is prepared daily as described below. The
acrylamide appears quite stable on storage. It should be kept
stoppered, refrigerated and in the dark to slow any spontaneous
polymerization. Before use the solution is rewarmed to room
temperature (23.degree. ).
2. Bisulfite Solution: For banked heparinized (anti-coagulated)
blood, 0.123 g sodium bisulfite is dissolved in 50 ml of degassed
distilled water and transferred to evacuated glass tubes. For fresh
heparinized blood, the amount of bisulfite is increased to 0.160 g.
Degassing of the water can be omitted as described below. The
solution is prepared fresh daily. These quantities were empirically
determined to be optimum.
3. Persulfate Solution: 0.154 g of ammonium persulfate is dissolved
in 50 ml distilled water and the solution is degassed. The
degassing can be omitted if a new standard curve is prepared daily.
The solution is prepared fresh daily.
Referring now to FIGS. 1 and 2, the acrylamide assay is run
manually in a 1 ml plastic tuberculin syringe mixing chamber 21,
calibrated in 0.01 ml divisions, containing a glass bead 22 that is
4 mm in diameter and serves to mix reactants and to detect the
formation of a polymer gel by monitoring viscosity The plunger 23
is moved to the 0.40 ml mark and monomer solution is injected to
the tip of the syringe orifice 24. The solution is injected from a
hypodermic needle 25 on another syringe 26 as shown in FIG. 1. Note
that once fluid is in the syringe 21 and a liquid-gas interface 27
appears, new fluids are inserted below that interface. The plunger
23 is then moved to the 0.60 ml mark and persulfate solution is
injected, again to the tip of the syringe orifice 24. The plunger
23 is moved to the 0.70 ml mark and 0.10 ml blood sample with
heparin is added, to the tip of the orifice. The present contents
of the syringe comprise the test liquid. The syringe 21 is capped
with a cap 28 and mixed by inversion 10-15 times. Hemoglobin is
liberated from red cells by this procedure. The cap 28 is removed,
the plunger lowered to below the 1.0 ml mark, and 0.30 ml of the
bisulfite activator solution, premeasured in a 1 ml syringe, is
rapidly injected to begin the reaction and production of the
product. Timing with a stopwatch begins as this injection is
concluded. The air bubble at the top is expressed, the syringe 21
capped and held as shown in FIG. 2 (the syringe 21 is held as shown
in FIG. 2 to avoid warming the solution by body heat) and mixed by
inversion while observing the glass bead 22. Timing stops when the
glass bead 22 becomes immobilized (as shown in the right syringe)
by an increase in viscosity showing the product has completed the
formation of a polymer gel. This is preceded by a 10 second warning
period of increasing viscosity.
A blank determination of a base reference time is performed
identically with 0.10 ml normal saline (physiological saline
solution) in place of blood. The polymerization time observed for
the blank is subtracted from each experimental polymerization
time.
The polymerization reaction is exothermic. With a thermal probe in
the reaction syringe, the temperature is observed to rise
4.degree.-5.degree.within the 15 seconds preceding gelation; after
gelation the temperature rises more rapidly to about 20.degree.
over the starting level. Thus, temperature could be used as in
indication of the gelation.
Oxygenated blood, initially bright red in the reaction syringe 21,
slowly darkens to a deep violet as the polymerization reaction
proceeds. The bright red color can be restored to the gel
subsequently by exposure to oxygen, suggesting that the initial
color change is due to deoxygenation of the oxyhemoglobin. Thus,
color is indicative of gelation.
Referring next to FIG. 3 there is typical standard of comparison
that is a graph 31 that is a curve 32 with an upper end point 33
and a lower end point 34. Comparison of results of oximetry by the
subject method and by conventional techniques has shown a plot of
the logarithm of time v. oxygen content is linear to 22 ml 0.sub.2
/100ml blood.
There was some day-to-day variation in the polymerization time --
oxygen content curves obtained with non-degassed acrylamide making
it desirable to find a rapid means to establish a daily standard
curve. This was most readily accomplished by computing the oxygen
content of a fully oxygenated blood sample from the hemoglobin
content (1.34 ml O.sub.2 / gm Hb) plus dissolved oxygen, and using
this sample to establish the upper end 33 of the curve 32, while
using a saline blank to establish the lower end 34 of the curve.
The lower end 34 of the curve 32 does not go through the origin
because saline is not a true blank. Only blood with no oxygen would
be. This is no problem, however, inasmuch as the curve 32 is
linear. Corrected polymerization time is sample polymerization time
minus blank polymerization time.
Use of manual mixing and timing and hand-held syringes 21 for the
oxygen assay, though it is simple and inexpensive, opens the method
to a substantial amount of deviation and error.
It was felt imperative to develop a semiautomatic system that would
eliminate subjective estimation of the end point of the reaction
fna minimize the variability of mixing vigor and temperature, so
more reproducible and accurate results could be obtained.
Accordingly, the mixing apparatus with an electromagnetic monitor
system, as shown in FIG. 4, was devised. The reaction mixing
chamber 41 consists of a 10 ml plastic syringe 42 containing a 1/2
inch plastic-coated magnetic stirring bar 43. The nozzle end of the
syringe barrel 42 is wrapped with a 12 turn coil 44 of insulated
electrical hookup wire within the magnetic field of the bar 43. The
ends of this coil 44 are connected to an amplifier 45. The syringe
nozzle access opening is plugged with a rubber injection port 46
after being filled with 1.2 ml of acrylamide, so that no air space
remains and the solution is isolated from the atmosphere.
Subsequently, all reagents are injected through this rubber port 46
so that no air is admitted to the chamber 41 at any time. The
syringe 42 is clamped horizontally over a magnetic stirrer motor 47
which is switched on and adjusted to low speed (about 75 rpm). The
amplifier 45 is connected to a monitor 48 that can be a chart
recorder. It should be emphasized that the monitor can also be a
digital clock responsive to the amplifier 45.
As the magnetic stirrer 47 causes the stirring bar 43 to turn, an
electromotive force is generated in the coil 44 and recorded by the
chart recorder. Measurement indicates this is about 1 mV peak
amplitude. Persulfate, blood and bisulfite are injected through the
injection port 46 in the usual sequence, with all volumes 3 times
that for the manual method. When a gel forms, the magnetic stirring
bar 43 is immobilized by the increased viscosity and the
alternating voltage drops markedly to a low level produced by the
magnetic field of the stirrer 47. The voltage drop occurs over
about 2 seconds. The time interval from the addition of bisulfite
can easily be read off the chart paper if the paper speed is
known.
To secure accurate temperature control, a jacketed water bath
assembly 51 has been designed and is shown in FIG. 5. This assembly
permits 10 ml syringes to be placed rapidly in position with
respect to a permanently installed coil 52 in a lucite tube 53 over
the stirrer motor 47. There are also jackets 54 to store
persulfate, bisulfite and blood and assure they are at the
appropriated temperature. A divider 55 separates the bath 51 into a
storage chamber 56 and a reaction chamber 57. Separate inlets 58
and outlets 59 are provided for each chamber 56, 57. Separate
chambers are supplied because the reaction is exothermic and thus
the reaction chamber 57 requires a greater water flow to stabilize.
A thermometer 61 indicates the temperature in the reaction chamber
57.
It should be noted that the syringe apparatus shown in FIG. 4 is
useful in carrying on any reaction requiring atmospheric isolation
and/or viscosity monitoring and the bath 51 shown in FIG. 5 is
useful in those reactions if the temperature must be
controlled.
Other reagent systems are suggested below. The final choice of any
one system must be made by the user of the method in light of his
preferences and the liquid to be assayed.
Bisulfite need not be used. Other reduction activators of
persulfate include ferrous ions and ferrocyanide.
Blood, too, is a weak reduction activator for persulfate.
Consequently, if time is not of the essence, a test solution of
blood and a monomer can be used and the addition of the activator
persulfate solution initiates a slow polymerization process that is
completed in several hours. The natural consequence of the above is
that if a reduction activator is used it must be added quickly
after the addition of the blood, and the time delay between
addition of the blood and the reduction activator should be uniform
from test to test.
Reduction activators can be eliminated. A test solution of blood,
or another liquid to be assayed, and a monomer can be tested with
an activator liquid comprising a compound such as benzoyl peroxide
that spontaneously generates free radicals and thus starts
polymerization.
Furthermore, other uses of the method are evident. A few other uses
are suggested below.
The oximetry of fluorocarbons has been a difficult process.
However, it has been found that fluorocarbons can be assayed
quickly by the subject method, and a plot of log time vs. oxygen
content is linear up to 22 ml. oxygen/100 ml. fluorocarbon. In
addition, the high oxygen solubility of fluorocarbons permits other
uses such as the one following.
Measurement of tracheal and atmospheric oxygen should be possible
if the gas is first equilibrated with blood or fluorocarbon and the
fluid then analyzed by the acrylamide method. Solubility data for
oxygen in the liquid in question would permit extrapolation back to
oxygen tension in the gas phase, most easily if the solution obeys
Henry's law of gas solubility as do fluorocarbon emulsions.
With suitable adjustments of activator and monomer concentrations
(to be determined empirically) oxygen content of water samples
could be determined rapidly and in the field if necessary.
With a suitable microassay based on the acrylamide principle, for
oxygen in aqueous solution, any enzymatic reaction which consumes
or produces oxygen could be assayed if the reaction chamber is
sealed and sampling can be performed anaerobically. Aliquots of the
enzyme-substrate mixture could be removed at intervals and assayed
for oxygen content to provide an index of the rate of reaction.
Obviously, many modifications and variations of the present
invention are possible in light of the above teachings. It is to be
understood, therefore, that the invention can be practiced
otherwise than as specifically described.
* * * * *