Measurement of body substances.

0001This invention relates to a cutaneous method of analysing body substrates.

0002Instruments capable of continuously indicating the chemical composition of blood have proved to be useful in regulating operative and post-operative 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, C0₂ and ^p0₂.

0003In 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 example, United States Patent Specification No. 3 911 138 discloses a method of taking quantitative measurements of skin-diffused fluorinated compounds by gas chromatography and electron-capture dectectors.

0004Other techniques have been employed for measuring biological substances in the blood. For example, 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.

0005US-A-4 071 020 discloses a method for the in vivo measurement of substances on the epidermis by the application of a suitable reagent to the skin surface followed by detection of the reaction products. US-A-3 542 662 discloses an enzyme electrode which must be placed in direct contact with the body fluid or tissue for the enzyme substrate in the fluid or tissue to be measured by the detection of oxygen. US-A-4 020 830 also discloses an enzyme electrode which must be contacted with a solution containing the enzyme substrate. The device can be in the form of a needle for insertion into a tissue, when the method performed is invasive.

0006In summary, 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 non-invasive measurement of key non-gaseous biological substances.

0007Our copending European Patent Publication No. 24 155 discloses apparatus suitable for use in a cutaneous method of analysing body substrates comprising <ul id="ul0001" list-style="none"><li>(i) an enzyme implant which is adapted for insertion into the body of a mammal and contains an enzyme which is reactable with a non-gaseous substrate in the body, which substrate is to be measured, to produce a detectable change in the skin of the mammal, and</li><li>(ii) analysing means for analysing said change in the skin, without invading the body at the time of detection, as a measure of the amount of said non-gaseous substrate in the body.</li></ul>

0008According to the present invention there is provided a method of detecting the presence in a body of a non-gaseous substrate which is volatilizable through the skin, characterised in that the method comprises introducing at the surface of the skin an enzyme selected for the substrate to be detected for a sufficient time for any of said substrate present to react with said enzyme and detecting a condition of the skin as an indication of the presence of said substrate in the body.

0009The apparatus of the invention is used in a non-invasive method for cutaneously measuring non-gaseous 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.

0010In a most preferred method, the skin is arterialized and the enzyme is reacted with the substrate at 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 capilliaries may be arterialized by heating or chemical treatment.

0011It has been discovered that biological substances which do not diffuse from the blood through the skin may still be measured. For example, 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 tcp0₂.electrode. The heat arterializes the capilliaries 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: <ul id="ul0002" list-style="none"><li>Glucose + Oxygen- Gluconic Acid + Hydrogen Peroxide</li></ul>

0012The glucose diffuses to the implanted enzyme where it is oxidised 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 incorporating catalase with the glucose oxidase. Thus, in accordance with this method, 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.

0013In an alternative method, 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 example, an H₂0₂ polarographic anode may be employed to detect subdermal components. Thus, a transcutaneous tcp0₂, tcpH₂0₂, or even a tcpC0₂ electrode may be employed as the skin condition analyzer.

0014In 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 example, 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 colour change in the presence of hydrogen peroxide, the amount of hydrogen peroxide present being measured by colorimetrically measuring the colour change. One known method of such measurement is by means of a quadravalent-titanium and xylenol orange which react to form a stable red colour with hydrogen peroxide (Taurenes &amp; Nordschow, Amer. J. Clin. Path., 1968, 49, 613). Reference may be made to this article for details and to United States Patent Specification No. 3 907 645 for suitable reactants. The amount of hydrogen peroxide produced is measured by the intensity of the colour.

0015Furthermore, 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 colour change or a condition of the skin may be visually observed or measured.

0016The 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 example, to a coloured 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 coloured dye which intensifies and the amount of lactic acid is measured by intensity of the colour.

0017In a broader aspect, the method is directed to the cutaneous measurement of a corporeal substance by reacting an enzyme with the substance anywhere across the layer of the skin and detecting a condition of the skin as a measure of the amount of substance. The enzyme is placed on the skin in accordance with any particular tenchique. In one particularly preferred form in our copending European Patent Publication No. 24 155 the enzyme is implanted below the skin. The implantation allows for continuous monitoring of the substance under examination. For example, 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 tc_P0₂ 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 capilliary 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 ind 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 nodulated skin. For example, reference may be made to prior US-A-3 912 386, 3 380 905 and 3 539 455 for specific electrode structures which may be used to detect oxygen and H₂0₂. Using devices of this type, a iual electrode system may be used to sense glucose by relating the difference in polarographic oxygen current between the normal skin and the enzyme nodulated skin. In another form, a single electrode system can be employed. For example, polarographic anodes of the type described in US-A-4 040 908 may be employed to measure hydrogen peroxide by-product as a neasure of the substrate.

0018According to one procedure, the enzyme is iissolved in water and injected just under, or into, the skin and a tcpO₂ electrode is positioned on the skin and secured just over the enzyme site. The temperature of the skin is controlled at approximately 38 to 44^oC. In another procedure, enzyme powder has been mixed with silicone and 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 inplanted 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 prior to implantation.

0019Therefore, various modes of cutaneous treatment, including implantation, and devices for achieving same may be employed in the above described methods. Skin implants may be very small, e.g. 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 encapuslated 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 colour is effected when the glucose reaches a certain value. Redox dyes directly coupled or indirectly coupled through an enzyme-glucose reaction could be used. Such devices would give a warning signal to a diabetic.

0020Also, there are some substances, e.g. hypoxanthine, which form in the body and enter the blood when hypoxia is present. Using a tc_P0₂ electrode and xanthine oxidase EC1.2.3.2. an hypoxia warning device may be assembled which fastens to the skin and warns of the presence of this substance by virtue of Δ-pO₂ over normal skin and skin with a xanthine oxidase implant.

0021The invention will now be further described and illustrated with reference to the accompanying drawings and the following Experiments. <ul id="ul0003" list-style="none"><li>Figure 1 illustrates a typical transcutaneous electrode arrangement for detecting skin oxygen contact as a measure of the substrate, and</li><li>Figure 2 is a plot showing measurement of ethanol with a transcutaneous p0₂ electrode.</li></ul>

Experiments

0022In this series of experiments, the oxygen-consuming alcohol oxidase was placed on the skin of an anesthetized cat. The animal was anesthetized with sodium pentobarbital and maintained at 38⁰C 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 µl of water (215 mg/50 µl) 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 Figure 2 and numbers referred to at the points of injection, namely 2, 4, 10 and 20, are the number of millilitres of 10% ethanol given intraveneously. The measurements of the circulating ethanol were employed using a transcutaneous tc_P0₂ electrode of the type shown in Figure 1. With reference to Figure 2, it is demonstrated that increasing amounts of alcohol decreased the tcpO₂ step-wise and that recovery toward the initial value occurred over the following minutes. The less than expected effect of the 20 millilitre dose was not understood, but may possibly be due to a pharmacological affect on the skin or possibly an acute drop in blood pressure.

0023Other 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 p0₂ versus blood or end tidal alcohol. The temperature control required for the tc_P0₂ 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 the Experiments. The tc_P0₂ 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.

0024In 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. <tables id="tabl0001" num="0001"><img file="EP0154664A2_D0001.tif" /></tables><tables id="tabl0002" num="0002"><img file="EP0154664A2_D0002.tif" /></tables><tables id="tabl0003" num="0003"><img file="EP0154664A2_D0003.tif" /></tables><tables id="tabl0004" num="0004"><img file="EP0154664A2_D0004.tif" /></tables>

0025Any enzyme may be used which, in the process of catalyzing the reaction with its substrate or substrates directly or indirectly, consumes or requires oxygen.

0026Using 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 are useful in the above described method. Since new enzymes are regularly discovered, examples of present known enzymes can be used to illustrate the principles involved. There are six main classes: <ul id="ul0004" list-style="none"><li>1. Oxidoreductases</li><li>2. Transferases</li><li>3. Hydrolases</li><li>4. Lysases</li><li>5. Isomerases</li><li>6. Ligases.</li></ul>

0027Most 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.

0028Class 1, the oxidoreductases, are divided into sub-classes, for example, 1.1 are those acting on the CH-OH group of donors. Class 1.1 is divided as follows: <ul id="ul0005" list-style="none"><li>1.1.1. with NAD or NADP as acceptor</li><li>1.1.1. with cytochrome as acceptor</li><li>1.1.3. with oxygen as acceptor</li><li>1.1.99 with other acceptors</li></ul>

0029Glucose oxidase, as oxygen oxidoreductase icting on the CH-OH group of donors is therefore L.1.3. Glucose oxidase is a 1.1.3. enzyme and is key lumbered as 1.1.3.4., galactose oxidase is 1.1.3.9. If glucose is oxidized by a "dehydrogenase" enzyme, it Ls called glucose dehydrogenase: <ul id="ul0006" list-style="none"><li>Glucose + NAD(P) = Gluconolactone + reduced NAD(P) It is classed as 1.1.1.47.</li></ul>

0030Galactose dehydrogenase uses NAD, rather than NADP, as cofactor: <ul id="ul0007" list-style="none"><li>Galactose + NAD = Galactolactone + reduced NAD (or "NADH")</li></ul>

0031These two dehydrogenases do not consume oxygen iirectly but via cofactors. The dehydrogenase 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 plantinum anode, or by oxygen. Hence, the glucose substrate, the donor molecule, is oxidized by oxygen, indirectly.

0032Other natural cofactors, such as cytochrome or synthetic substances, can act as cofactors with the final result that a specific substrate is oxidised with the stoichiometric consumption of molecular oxygen.

0033The 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.

0034A 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.

0035A 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.

0036A Class 4 example is oxalate decarboxylase, EC4.1.1.3., and splits oxalate into formate + C0₂. This enzyme, found in wood fungus, could measure oxalate by the release of C0₂. A transcutaneous pC0₂ electrode made be used to measure the pC0₂ which is related to the C0₂ by-product and hence the oxalate concentration. This enzyme does not require a cofactor.

0037Another Class 4 example is acetoacetate decarboxylase 4.1.1.10 which reacts with acetoacetate to give glycine + C0₂. This enzyme is found in liver. The acetoacetate is found in diabetes which is not properly controlled.

0038There are many other C0₂ producing enzymes such as: <tables id="tabl0005" num="0005"><img file="EP0154664A2_D0005.tif" /></tables>

0039In 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.

0040An example in Class 6 is an enzyme (EC6.4.1.4.) which uses C0₂ 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.

0041In performing the techniques described above, 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 H₂0₂ 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 enzyme or coenzymes could be placed in a container 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. Glutaraladehyde 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 immobilise 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 recognised that monochromatic, dichromatic or multi-pleochromatic 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.

0042As 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.