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BACKGROUND OF THE INVENTION
Instruments capable of continuously indicating the chemical composition of
blood have proved to be useful in regulating operative and postoperative
managements of patients, and in teaching and research. At first, such
instruments were used with sensors mounted directly in the extracorporeal
blood circuit that is used for perfusion of open-heart surgery patients.
Later, continuous monitoring of both machine and patients was conducted by
means of continuous withdrawal of blood pumped into external cuvettes
equipped with appropriate sensors. Satisfactory systems are now provided
for a rapid and accurate measurement of blood composition such as pH,
pCO.sub.2 and pO.sub.2.
In addition to the analytical techniques mentioned above, oxygen and carbon
dioxide have been measured on the skin by virtue of their diffusing
through it. Recently, the continuous monitoring of blood oxygen by a
heated electrode positioned on hyperemic skin has been accomplished.
Substances such as halogenated organic compounds, particularly fluorinated
compounds, have also been found to diffuse through the skin and have been
measured. For instance, with reference to U.S. Pat. No. 3,911,138,
quantitative measurements have been made of skin-diffused fluorinated
compounds by gas chromatography and electron-capture detectors.
Other techniques have been employed for measuring biological substances in
the blood. For instance, ethanol is currently measured in blood, either
directly or by a breath sampling, by classical chemical, gas
chromatographic and enzyme methods. One of the alcohol enzyme methods
depends upon the polarographic measurement of hydrogen peroxide, while
others depend upon the consumption of oxygen. However, none of these
methods readily lend themselves to continuous monitoring.
In brief, while there are a variety of techniques available for the
measurement of blood gases and other substances, new methods are desired
which more readily lend themselves to continuous monitoring or enable the
measurement of key biological substances.
SUMMARY OF THE INVENTION
This invention is directed to a new method for cutaneously measuring
substances in the body. The method is conducted by contacting the
substrate through the skin of a mammal with an enzyme selective for the
substrate being analyzed, then reacting the substrate with the enzyme and
directly detecting a condition of the skin as a measure of the amount of
substrate. The procedure is completely non-invasive or is non-invasive
after one implant.
In a most preferred embodiment, the skin is arterialized and the enzyme is
reacted with the substrate in the blood at or near the skin surface. A
condition of the reaction is detected such as the amount of oxygen
consumed, or hydrogen peroxide or carbon dioxide by-products, as a measure
of the amount of substance. The skin capillaries may be arterialized by
heating or chemical treatment.
It has been discovered that biological substances which do not diffuse from
the blood through the skin may still be measured according to this
invention. For instance, one of these substances is glucose. In accordance
with one preferred technique of this invention, a substance such as
glucose under the skin may be measured by means of a skin-contact oxygen
electrode, particularly a heated electrode. This electrode is sometimes
referred to herein simply as a transcutaneous oxygen electrode or
tcpO.sub.2 electrode. The heat arterializes the capillaries in the skin,
that is to say, the blood in the skin is brought into equilibrium with the
blood in the arteries. Quantitative measurements may then be made. In this
method, glucose oxidase is placed just beneath the dermis where it
catalyzes the consumption of oxygen according to the amount of glucose
available, as expressed by the equation:
GLUCOSE+OXYGEN.fwdarw.GLUCONIC ACID+HYDROGEN PEROXIDE
The glucose diffuses to the implanted enzyme where it is oxidized and the
resultant decrease in oxygen is sensed by the electrode placed over or
near the enzyme site. The gluconic acid diffuses away from the site to be
picked up by the blood or the lymphatic stream. The hydrogen peroxide also
diffuses away, or may be decomposed by local catalase activity. Should
hydrogen peroxide be a problem, it can be destroyed by incoporating
catalase with the glucose oxidase. Thus, in accordance with this
embodiment, the skin condition being detected is a resultant decrease in
oxygen in the skin layer as a measure of the amount of glucose in the
blood under the skin.
In an alternative embodiment, the enzyme may react with a substance to
produce by-product hydrogen peroxide which may then be sensed by a
hydrogen peroxide sensitive electrode. For instance, an H.sub.2 O.sub.2
polarographic anode may be employed to detect subdermal components. Thus,
a transcutaneous tcpO.sub.2, tcpH.sub.2 O.sub.2, or even a tcpCO.sub.2
electrode may be employed as the skin condition analyzer.
In addition to positioning polarographic electrodes on hyperemic skin to
detect oxygen in a local subdermal oxygen sink or by-product hydrogen
peroxide, other procedures for quantitation of the substrate may be
employed. For instance, a colorimetric method may be used for detecting
amounts of hydrogen peroxide produced by enzymatic reaction. The amount of
hydrogen peroxide produced may be measured by a system which comprises a
chromogenic reagent or reagents capable of undergoing a color change in
the presence of hydrogen peroxide, the amount of hydrogen peroxide present
being measured by colorimetrically measuring the color change. One known
method of doing this is by means of a quadravalent-titanium and xylenol
orange which react to form a stable red color with hydrogen peroxide
(Taurnes & Nordschow, Amer. J. Clin. Path., 1968, 49, 613). Reference may
be had to this article for details or to U.S. Pat. No. 3,907,645 suitable
reactants. The amount of hydrogen peroxide produced is measured by the
intensity of the color.
Furthermore, an enzyme reactant may be tattooed in the skin. In this form
an enzyme or a detector of the enzyme reaction may be immobilized in the
skin and a color change or a condition of the skin may be visually
observed or measured.
The reaction of the enzyme with the substance being measured may also be
followed through the skin by measuring the electrons which are removed
during the enzymatic reaction and transferred, for instance, to a colored
dye. For example, lactic acid will undergo an enzymatic reaction with
lactic acid dehydrogenase. In this reaction, electrons are removed from
the acid and are available for transfer to a colored dye which intensifies
and the amount of the lactic acid is measured by intensity of the color.
Therefore, in its broader aspect, this invention is directed to the
cutaneous measurement of a corporeal substance by reacting an enzyme with
the substance anywhere across the layer of skin and detecting a condition
of the skin as a measure of the amount of the substance. The enzyme may be
placed on, in or under the skin in accordance with any particular
technique. In one particularly preferred form, the enzyme is implanted
below the skin. The implantation allows for continuous monitoring of the
substance under examination. For instance, it has been shown that a
subdermal glucose oxidase may be implanted and does in fact interact with
glucose to produce a local oxygen sink which is measurable with a
tcpO.sub.2 electrode. The intensity and extent of the oxygen sink in the
presence of a given flux of glucose is dependent upon the geometry of the
implant and the activity of the enzyme. Both of these can be controlled.
The exact nature of healing, fibrous tissue invasion and capillary new
growth following implantation cannot, of course, be controlled but, the
implantation can be regulated with satisfactory practical limits. The
tissue reaction to such implants in humans after many months has been
small and they are easily replaced and removed. The sensing of glucose via
oxygen in this way may be accomplished by relating the difference in the
polarographic oxygen current between the normal skin and the enzyme
modulated skin. For instance, reference may be had to my earlier patents,
namely U.S. Pat. Nos. 3,912,386; 3,380,905 and 3,539,455 for specific
electrode structures which may be used to detect oxygen and H.sub.2
O.sub.2. Using devices of the type mentioned in my patents, a dual
electrode system may be used to sense glucose by relating the difference
in polarographic oxygen current between the normal skin and the enzyme
modulated skin. In another form, a single electrode system can be
employed. For instance, polarographic anodes of the types described in my
U.S. Pat. No. 4,040,908 may be employed to measure hydrogen peroxide
by-product as a measure of the substrate.
In one form of procedure, the enzyme is dissolved in water and injected
just under, or into, the skin and a tcpO.sub.2 electrode is positioned on
the skin and secured just over the enzyme site. The temperature of the
skin is controlled at approximately 38.degree.-44.degree. C. In another
procedure, enzyme powder has been mixed with silicone or fluorocarbon oils
before subcutaneous injection. Enzyme has also been mixed with silicone
monomer and converted with suitable catalyst to a thin rubber-like polymer
sheet about half the size of a postage stamp which is then implanted
through an incision in the skin. Such implants heal rapidly and retain
enzyme activity for many days or weeks and probably much longer. Enzyme
implants have been made using a thin sheet of reinforced Silastic
(organosilicone polymers, Dow Corning subdermal implant No. 501-1.007 in.
thick) coated with enzyme, immobilized by treatment with glutaraldehyde
solution and drying in the cold. Enzyme, either free or immobilized has
also been trapped between two layers of cellophane, cuprophan or collagen
just before implantation.
Therefore, various modes of cutaneous treatment, including implantation,
and devices for achieving same, may be employed in accordance with the
principles of this invention. Skin implants may be very small, perhaps a
sphere with a minimum dimension of about 1 mm in diameter. Patients who
require continuous monitoring, such as diabetics may be provided with a
skin implant and their condition may be continuously monitored by any of
the aforementioned detection techniques. Furthermore, in another form, the
encapsulated enzyme is embodied below the surface of the skin in such a
way that it is visible. A dye may be added thereto such that a change in
color is effected when the glucose reaches a certain value. Redox dyes
directly coupled or indirectly coupled through an emzyme-glucose reaction
could be used. Such devices would give a warning signal to a diabetic.
Also, there are some substances which form in the body and enter the blood
when hypoxia is present. Hypoxanthine is one. Using a tcpO.sub.2 electrode
and xanthine oxidase EC1.2.3.2., one could have an hypoxia warning device
which fastens to the skin and warns of the presence of this substance by
virtue of .DELTA.pO.sub.2 over normal skin and skin with a xanthine
oxidase implant.
This invention and its numerous advantages along with other embodiments
will be exemplified with reference to the drawings and the following
experiments.
FIG. 1 illustrates a typical transcutaneous electrode arrangement for
detecting skin oxygen content as a measure of the substrate.
FIGS. 2 and 3 are charts illustrating measurements of glucose and ethanol
with a transcutaneous pO.sub.2 electrode.
I--EXPERIMENTS
An electrode of FIG. 1 was employed in these experiments in the measurement
of glucose. The overall arrangement of the circuit and polarographic cell
may be obtained with reference to my above mentioned patents. Such devices
are well known. Their structures or their operation per se need not be
detailed here.
The transcutaneous pO.sub.2 in air breathing cats was measured with the
electrode at about 38.degree.-44.degree. C. The measurement of oxygen
transcutaneously depends upon the fact that this gas readily leaves the
capillary blood and diffuses through the skin to the outside. By heating
the skin to approximately 38.degree.-44.degree. C., the capillary vessels
in the blood stream dilate, the skin becomes red and the amount of oxygen
that is diffused from the skin increases and in fact comes into
equilibrium with arterial blood. Hence, the pO.sub.2 of an air breathing
animal on the surface of the skin is on the order of magnitude of about 80
mm of Hg. (A of FIG. 2). Following the breathing of oxygen, this
transcutaneous pO.sub.2 may increase to the region of 150 mm or so shown.
This procedure was followed by insertion of glucose oxidase in different
forms underneath the surface of the cat's skin. With reference to FIG. 1,
any of the forms of injection or implantation discussed above may be used.
Then, the transcutaneous pO.sub.2 was remeasured. The difference or
.DELTA.-pO.sub.2 is a reflection of the blood glucose content.
With reference to FIG. 2, the effect of increasing and decreasing blood
glucose levels is illustrated. After returning from oxygen to air
breathing, there was a prompt fall in the tcpO.sub.2. Then, beginning at a
few seconds after the injection of glucose (B) (10 cc of 5% was given
intravenously), there was a further oxygen decrease. Breathing of oxygen a
few minutes after the injection results in an increased, but to a much
lower average tcpO.sub.2, than breathing oxygen before. Then, a glucose
injection (C) decreased the tcpO.sub.2. After the injection of glucose
oxidase (D) which converts the circulating glucose to gluconic acid, while
the cat was still breathing oxygen, the tcpO.sub.2 increased to the
highest point measured. On return to air breathing, the level of the
tcpO.sub.2 dropped. The tcpO.sub.2 response to oxygen was greatly
decreased when glucose was given. Both of the abrupt falls in tcpO.sub.2
following oxygen breathing were obtained after returning the animal to air
breathing.
In this series of experiments, no attempt was made to perfectly quantitate
the result but to demonstrate the principle of the skin sensing electrode,
namely increasing amounts of glucose in the blood are reflected by a
decreasing transcutaneous pO.sub.2. Further evidence that the initial
.DELTA.-pO.sub.2 was a reflection of glucose was found by injecting the
enzyme glucose oxidase directly into the bloodstream of the cat. When the
purified enzyme was injected this way, there was a prompt increase in the
tcpO.sub.2 leveling off at a certain value, thus demonstrating that the
initial reading was due to glucose since the enzyme when injected
intravenously converts all the glucose to gluconic acid.
II--EXPERIMENTS
In another set of experiments, glucose oxidase was mixed with a silicone
preparation and then a catalyst was added. The material was then pressed
between two glass slides to produce a thin film of silicone rubber having
glucose oxidase embedded in it. When this membrane was hardened, it was
placed subcutaneously in a cat and healed in a perfectly normal manner
after a few days. Immediately after implantation, there was a difference
detected by the electrode of FIG. 1 in the .DELTA.-tcpO.sub.2 between the
normal skin and the enzyme treated skin. It had previously been
demonstrated that glucose oxidase mixed with the polymerizable silicone is
active in the oxidation of glucose to gluconic acid. In another
preparation, glucose oxidase was mixed with silicone oil and this was
injected subcutaneously. Still in another form of implantation, the
glucose oxidase was mixed with fluorocarbon liquid and injected
subcutaneously. In still another form, a glucose oxidase was trapped
between a thin layer of Silastic reinforced (artificial skin) and a layer
of collagen. In each form, the method of this invention was established,
namely that the amount of glucose could be detected by measuring the
difference in .DELTA.-tcpO.sub.2.
III--EXPERIMENTS
In another set of experiments, an oxygen-consuming alcohol oxidase was
placed on the skin of an anesthetized cat. The animal was anesthetized
with sodium pentobarbital and maintained at 38.degree. C. with an infrared
heater modulator modulated by a rectal thermistor signal. The electrode
was fastened to the shaved skin just below the thorax. A few crystals of
the oxidase preparation in about 50 .mu.l of water (215 mg/50 .mu.l) was
placed on the skin and the electrode was set in place. After a stable
reading was obtained, alcohol solution was injected. The results are shown
in FIG. 3 and numbers referred to at the points of injection, namely 2, 4,
10 and 20, are the number of milliliters of 10% ethanol given
intravenously. The measurements of the circulating ethanol were employed
using a transcutaneous tcpO.sub.2 electrode of the type shown in FIG. 1.
With reference to FIG. 3, it is demonstrated that increasing amounts of
alcohol decreased the tcpO.sub.2 step-wise and that recovery toward the
initial value occurred over the following minutes. The less than expected
effect of the 20 milliliter dose was not understood, but may possibly be
due to a pharmacological affect on the skin or possibly an acute drop in
blood pressure.
Other means of performing the experiments of the above type involving
volatilizable components, such as alcohol, include the incorporation of
the enzyme in the electrode's electrolyte, immobilizing it on the
membrane, and the use of two cathodes, one coated with enzyme and one
uncoated. As mentioned above, one may also have a coated and an
enzyme-free spot on the skin and calibrate by measuring the pO.sub.2
versus blood or end tidal alcohol. The temperature control required for
the tcpO.sub.2 measurement is ideal for stabilizing enzyme activity.
Enzyme would be best dissolved in a buffer with suitable coenzymes and
stabilizing agents. There are several alcohol oxidases and dehydrogenases
with varying specificity toward alcohols of different chemical structures,
but all respond to ethanol for use in Experiments III. Of course, the
tcpO.sub.2 skin procedure above discussed with reference to alcohol can be
used for the continuous measurement of other volatile enzyme substrates
where oxygen depletion is utilized in their measurement.
In view of the above experiments, it is obvious that a number of other
enzymes can be used in order to detect and measure a substance
transcutaneously. The following Table is a listing of the enzymes, their
identifying number, source and typical substrates with which they may
react for measurement in accordance with the principles of this invention.
t,0130
Any enzyme may be used which, in the process of catalyzing the reaction
with its substrate or substrates directly or indirectly, consumes or
requires oxygen.
Using the international nomenclature of the enzyme commission (see for
example T. E. Barman Enzyme Handbook, Vol. 1, 2, and Supplement,
Springer-Verlag, New York 1969), classes of enzymes can be described which
will be useful in this invention. Since new enzymes are discovered each
year, examples of presently known enzymes can be used to illustrate the
principles involved. There are six main classes:
1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Lysases
5. Insomerases
6. Ligases
Most of the oxygen consuming enzymes are in Class 1. If such enzymes may
use molecular oxygen directly, they are then called oxygen
oxidoreductases, or if indirectly, through a "coenzyme" or "cofactor"
which is reduced by the enzyme and reoxidized by molecular oxygen, they
are simply called oxidoreductases.
Class 1, the oxidoreductases, are divided into subclasses, for example, 1.1
are those acting on the CH--OH group of donors. Class 1.1 is divided as
follows:
1.1.1. with NAD or NADP as acceptor
1.1.1. with cytochrome as acceptor
1.1.3. with oxygen as acceptor
1.1.99 with other acceptors
Glucose oxidase, an oxygen oxidoreductase acting on the CH-OH group of
donors is therefore 1.1.3. Glucose oxidase is a 1.1.3. enzyme and is key
numbered as 1.1.3.4., galactose oxidase is 1.1.3.9. If glucose is oxidized
by a "dehydrogenase" enzyme, it is called glucose dehydrogenase:
Glucose+NAD(P)=Gluconolactone+reduced NAD(P)
It is classed as 1.1.1.47.
Galactose dehydrogenase uses NAD, rather than NADP, as a cofactor:
Galactose+NAD=Galactolactone+reduced NAD (or "NADH")
These two dehydrogenases do not consume oxygen directly but via cofactors.
The dehyrogenase reaction stops when all the cofactor is used up by
reduction to NADH or NADPH. The NADH or NADPH can be reoxidized to NAD or
NADP by a number of means, including oxidation by another cofactor, by a
platinum anode, or by oxygen. Hence, the glucose substrate, the donor
molecule, is oxidized by oxygen, indirectly.
Other natural cofactors, such as cytochrome or synthetic substances, can
act as cofactors with the final result that a specific substrate is
oxidized with the stoichiometric consumption of molecular oxygen.
The other five main classes of enzymes can be used in conjunction with the
oxidoreductases, or oxygen-consuming dehydrogenases, to expand the range
of analysis. Examples could be found to illustrate a reaction for each of
the main classes of 2, 3, 4, 5 and 6.
A Class 2 example is dextransucrase (EC2.4.1.5) which catalyzes the
reaction of low molecular weight dextran with sucrose to give a larger
dextran polymer. It consumes sucrose and yields fructose. Hence, depending
on conditions, it could be used to measure dextran or sucrose.
A Class 3 example is sucrase, commonly found in yeast, which is a
beta-fructofuranoside and is a hydrolase. It is EC3.1.1.2. which splits
sucrose into fructose and glucose. With glucose oxidase, it could be used
to measure sucrose.
A Class 4 example is oxalate decarboxylase, EC4.1.1.3, and splits oxalate
into formate+CO.sub.2. This enzyme, found in wood fungus, could measure
oxalate by the release of CO.sub.2. A transcutaneous pCO.sub.2 electrode
may be used to measure the pCO.sub.2 which is related to the CO.sub.2
by-product and hence the oxalate concentration. This enzyme does not
require a co-factor.
Another Class 4 example is acetoacetate decarboxylase 4.1.1.10 which reacts
with acetoacetate to give glycine+CO.sub.2. This enzyme is found in liver.
The acetoacetate is found in diabetes which is not properly controlled.
There are many other CO.sub.2 producing enzymes such as:
Pyruvate decarboxylase EC4.1.1.1
Aspartate decarboxylase EC4.1.1.12
Glutamate decarboxylase EC4.1.1.13
Lysine decarboxylase EC4.1.1.18
Arginine decarboxylase EC4.1.1.19
In general, Class 5 enzymes could be used with oxygen oxidoreductases where
the D-form of an enzyme was more stable than the L-form. For example,
L-alamine could be converted to D-alamine so that it could be oxidized by
D-amino acid oxidase.
An example in Class 6 is an enzyme (EC6.4.1.4) which uses CO.sub.2 to
convert 3-methylcrotonoyl CoA to 3-methylglutaconyl-CoA. These CoA
compounds are panthethenic acid condensed with ADP and thioethanolamine
and they play key roles in animal metabolism.
In performing the techniques of this invention, it should be understood
that foreign or other proteins injected subcutaneously are absorbed
rapidly. If glucose oxidase is injected subcutaneously it is absorbed. If
the dose is high enough the animal may die because glucose is converted in
part to H.sub.2 O.sub.2 and this converts the hemoglobin to methemoglobin
which does not carry oxygen. Proteolytic enzymes may destroy the enzyme or
it may be picked up by Kupffer cells. If the immune system of the body
including the opsonins can contact the enzyme, it will be marked for
destruction. Some antibodies attach to enzymes (Freund's adjuvant is used
to mix with the enzyme before injection) and they are inactivated by
antibodies. In view of these observations, a preferred technique is to not
let the enzymes escape and to not let immune proteins or macrophages
contact the enzyme. The enzymes or co-enzymes could be placed in a
containter such as a plastic bag or encapsulated in particles so that a
substrate such as glucose can diffuse in, but protein molecules cannot
permeate. Peroxide could be destroyed in the bag with catalase or allowed
to diffuse out to be destroyed. Also, as developed above, the enzyme can
be immobilized on an inert substrate such as nylon or silver.
Glutaraldehyde treated tissues such as heart valves from other species
have been used as substitute heart valves in human beings. Glutaraldehyde
is also widely used to immobilize enzymes. Hence, glutaraldehyde can be
used to immobilize and affix enzymes to surfaces for implantation where
the probability of a rejection process would be very low. It is also
recognized that monochromatic, dichromatic or multiplechromatic light can
be transmitted through the earlobe and the light spectrum received on the
other side to reveal the oxygen saturation of the blood. A transparent
enzyme implant in the earlobe could be designed with an appropriate dye
such that substrate concentration would be reflected by transmitted light.
As a result of enzyme reactions, fluorescence and phosphorescence can
occur. Hence, by a suitable implant containing the enzyme and the
photoactivated substance, one could detect substrate concentration by
measuring the amount of light emitted to the skin by the phosphorescent
reaction.
In view of the above description, other details and operating parameters
will be obvious to a person of ordinary skill in this art.
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