United States Patent (19) 11 Patent Number: 4,458,686 Clark, Jr. 45) Date of Patent: Jul. 10, 1984 (54), CUTANEOUS METHODS OF MEASURING 4,269,516 5/1981 Lubbers et al...... 356/427 BODY SUBSTANCES 4,306,877 12/1981 Lubbers ...... 128/633 Primary Examiner-Benjamin R. Padgett 75) Inventor: Leland C. Clark, Jr., Cincinnati, Ohio Assistant Examiner-T. J. Wallen 73. Assignee: Children's Hospital Medical Center, Attorney, Agent, or Firm-Wood, Herron & Evans Cincinnati, Ohio 57 ABSTRACT (21) Appl. No.: 491,402 Cutaneous methods for measurement of substrates in 22 Filed: May 4, 1983 mammalian subjects are disclosed. A condition of the skin is used to measure a number of important sub - Related U.S. Application Data stances which diffuse through the skin or are present (62) Division of Ser. No. 63,159, Aug. 2, 1979, Pat. No. underneath the skin in the blood or tissue. According to 4,401,122. the technique, an whose activity is specific for a particular substance or is placed on, in or 51). Int. Cl...... A61B5/00 under the skin for reaction. The condition of the skin is 52). U.S. Cl...... 128/635; 128/636; then detected by suitable means as a measure of the 436/11 amount of the substrate in the body. For instance, the (58), Field of Search ...... 128/632, 635, 636, 637, enzymatic reaction or by- of the reaction is

128/633; 356/41, 417, 427; 422/68; 23/230 B; detected directly through the skin as a measure of the 436/11 amount of substrate. Polarographic electrodes or en 56) References Cited zyme electrodes are employed as skin-contact analyzers in the transcutaneous measurement of or hydro U.S. PATENT DOCUMENTS gen peroxide to quantitatively determine blood sub 3,795,239 3/1974 Eberhard et al...... 128/635 stances such as and alcohol. In a preferred 3,958,560 5/1976 March ...... 128/633 quantitative technique, the skin is arterialized, i.e., 3,960,753 6/1976 Larrabee ...... 23/230 LC heated or otherwise treated to arterialize the skin capil 4,003,707 1/1977 Lubbers et al...... 128/633 laries when the measurements are made. Colorimetric 4,034,756 7/1977 Hiquchi et al...... 128/260 4,215,940 8/1980 Lubbers et al...... 356/41 detection methods are also employed. 4,240,438 12/1980 Updike et al...... 128/635 4,255,053. 3/1981 Lubbers et al...... 356/417 23 Claims, 3 Drawing Figures

ENZYMATIC TRANSCUTANEOUS GLUCOSE MEASUREMENT

PLEXGLAS LUCIT

TEFLON RING

ELECTROLYTE CHAMBER

S K N OXYGEN GEL WITH PERMEABLE (OR) GLUCOSE MEMBRANE OXDASE U.S. Patent Jul. 10, 1984 4,458,686

ENZYMATIC TRANSCUTANEOUS GLUCOSE MEASUREMENT

PLEXGLAS LUCT

e Ag t Pt 5urn

GLASS 8mm woma s TEFLON RING ELECTROLYTE 3. CHAMBER

TIME-> O 2O 4O 6O 8O IOO TME/MINUTES 4,458,686 1 2 It has been discovered that biological substances CUTANEOUS METHODS OF MEASURING BODY which do not diffuse from the blood through the skin SUBSTANCES may still be measured according to this invention. For instance, one of these substances is glucose. In accor This is a division of application Ser. No. 63,159, filed 5 dance with one preferred technique of this invention, a Aug. 2, 1979, now U.S. Pat. No. 4,401,122. substance such as glucose under the skin may be mea sured by means of a skin-contact oxygen electrode, BACKGROUND OF THE INVENTION particularly a heated electrode. This electrode is some Instruments capable of continuously indicating the times referred to herein simply as a transcutaneous oxy chemical composition of blood have proved to be useful O gen electrode or tcpC2 electrode. The heat arterializes in regulating operative and postoperative managements the capillaries in the skin, that is to say, the blood in the of patients, and in teaching and research. At first, such skin is brought into equilibrium with the blood in the instruments were used with sensors mounted directly in arteries. Quantitative measurements may then be made. the extracorporeal blood circuit that is used for perfu In this method, glucose is placed just beneath sion of open-heart surgery patients. Later, continuous 15 the dermis where it catalyzes the consumption of oxy monitoring of both machine and patients was conducted gen according to the amount of glucose available, as by means of continuous withdrawal of blood pumped expressed by the equation: into external cuvettes equipped with appropriate sen sors, Satisfactory systems are now provided for a rapid GLUCOSE--OXYGENGLUCONIC and accurate measurement of blood composition such as 20 ACID-- pH, CO2 and pC). The glucose diffuses to the implanted enzyme where In addition to the analytical techniques mentioned it is oxidized and the resultant decrease in oxygen is above, oxygen and carbon dioxide have been measured sensed by the electrode placed over or near the enzyme on the skin by virtue of their diffusing through it. Re 25 site. The gluconic acid diffuses away from the site to be cently, the continuous monitoring of blood oxygen by a picked up by the blood or the lymphatic stream. The heated electrode positioned on hyperemic skin has been hydrogen peroxide also diffuses away, or may be de accomplished. Substances such as halogenated organic composed by local catalase activity. Should hydrogen compounds, particularly fluorinated compounds, have peroxide be a problem, it can be destroyed by incorpo also been found to diffuse through the skin and have 30 rating catalase with the glucose oxidase. Thus, in accor been measured. For instance, with reference to U.S. dance with this embodiment, the skin condition being Pat. No. 3,911,138, quantitative measurements have detected is a resultant decrease in oxygen in the skin been made of skin-diffused fluorinated compounds by layer as a measure of the amount of glucose in the blood gas chromatography and electroncapture detectors. under the skin. Other techniques have been employed for measuring 35 In an alternative embodiment, the enzyme may react biological substances in the blood. For instance, ethanol with a substance to produce by-product hydrogen per is currently measured in blood, either directly or by a oxide which may then be sensed by a hydrogen perox breath sampling, by classical chemical, gas chromato ide sensitive electrode. For instance, an H2O2 polaro graphic and enzyme methods. One of the alcohol en graphic anode may be employed to detect subdermal zyme methods depends upon the polarographic mea 40 surement of hydrogen peroxide, while others depend components. Thus, a transcutaneous tcpo2, tcpH2O2, or even a topCO2 electrode may be employed as the skin upon the consumption of oxygen. However, none of condition analyzer. these methods, readily lend themselves to continuous In addition to positioning polarographic electrodes monitoring... . on hyperemic skin to detect oxygen in a local subdermal In brief, while there are a variety of techniques avail 45 oxygen sink or by-product hydrogen peroxide, other able for the measurement of blood gases and other sub procedures for quantitation of the substrate may be stances, new, methods are desired which more readily employed. For instance, a colorimetric method may be iend themselves to continuous monitoring or enable the used for detecting amounts of hydrogen peroxide pro measurement of key biological substances. duced by enzymatic reaction. The amount of hydrogen SUMMARY OF THE INVENTION 50 peroxide produced may be measured by a system which This invention is directed to a new method for cuta comprises a chromogenic reagent or reagents capable of neously measuring substances in the body. The method undergoing a color change in the presence of hydrogen is conducted by contacting the substrate through the peroxide, the amount of hydrogen peroxide present skin of a mammal with an enzyme selective for the 55 being measured by colorimetrically measuring the color substrate being analyzed, then reacting the substrate change. One known method of doing this is by means of with the enzyme and directly detecting a condition of a quadravalent-titanium and xylenol orange which react the skin as a measure of the amount of substrate. The to form a stable red color with hydrogen peroxide procedure is completely non-invasive or is non-invasive (Taurnes & Nordschow, Amer. J. Clin. Path, 1968, 49, after one implant. 60 613). Reference may be had to this article for details or In a most preferred embodiment, he skin is arterial to U.S. Pat. No. 3,907,645 suitable reactants. The ized and the enzyme is reacted with the substrate in the amount of hydrogen peroxide produced is measured by blood at or near the skin surface. A condition of the the intensity of the color. reaction is detected such as the amount of oxygen con Furthermore, an enzyme reactant may be tattooed in sumed, or hydrogen peroxide or carbon dioxide by-pro 65 the skin. In this form an enzyme or a detector of the ducts, as a measure of the amount of substance. The skin enzyme reaction may be immobilized in the skin and a capillaries may be arterialized by heating or chemical color change or a condition of the skin may be visually treatent. ;:... observed or measured. 4,458,686 3. 4 The reaction of the enzyme with the substance being layers of cellophane, cuprophan or collagen just before measured may also be followed through the skin by implantation. measuring the electrons which are removed during the Therefore, various modes of cutaneous treatment, enzymatic reaction and transferred, for instance, to a including implantation, and devices for achieving same, colored dye. For example, lactic acid will undergo an 5 may be employed in accordance with the principles of enzymatic reaction with lactic acid dehydrogenase. In this invention. Skin implants may be very small, perhaps this reaction, electrons are removed from the acid and a sphere with a minimum dimension of about 1 mm in are available for transfer to a colored dye which intensi diameter. Patients who require continuous monitoring, fies and the amount of the lactic acid is measured by such as diabetics may be provided with a skin implant intensity of the color. 10 and their condition may be continuously monitored by Therefore, in its broader aspect, this invention is any of the aforementioned detection techniques. Fur directed to the cutaneous measurement of a corporeal thermore, in another form, the encapsulated enzyme is substance by reacting an enzyme with the substance embodied below the surface of the skin in such a way anywhere across the layer of skin and detecting a condi that it is visible. A dye may be added thereto such that tion of the skin as a measure of the amount of the sub- 15. a change in color is effected when the glucose reaches stance. The enzyme may be placed on, in or under the a certain value. dyes directly coupled or indi skin in accordance with any particular technique. In one rectly coupled through an enzyme-glucose reaction particularly preferred form, the enzyme is implanted could be used. Such devices would give a warning below the skin. The implantation allows for continuous signal to a diabetic. ... monitoring of the substance under examination. For 20 Also, there are some substances which form in the instance, it has been shown that a subdermal glucose body and enter the blood when hypoxia is present. oxidase may be implanted and does in fact interact with Hypoxanthine is one. Using a topO2 electrode and xan glucose to produce a local oxygen sink which is meas thine oxidase EC1.2.3.2., one could have an hypoxia ureable with a topO2 electrode. The intensity and extent warning device which fastens to the skin and warns of of the oxygen sink in the presence of a give flux of 25. the presence of this substance by virtue of ApO2 over glucose is dependent upon the geometry of the implant normal skin and skin with a implant. and the activity of the enzyme. Both of these can be This invention and its numerous advantages along controlled. The exact nature of healing, fibrous tissue with other embodiments will be exemplified with refer invasion and capillary new growth following implanta ence to the drawings and the following experiments. tion cannot, of course, be controlled but, the implanta- 30 FIG. 1 illustrates a typical transcutaneous electrode tion can be regulated with satisfactory practical limits. arrangement for detecting skin oxygen content as a The tissue reaction to such implants in humans after measure of the substrate. many months has been small and they are easily re FIGS. 2 and 3 are charts illustrating measurements of placed and removed. The sensing of glucose via oxygen glucose and ethanol with a transcutaneous pO2 elec in this way may be accomplished by relating the differ-35 trode. ence in the polarographic oxygen current between the normal skin and the enzyme modulated skin. For, in I-EXPERIMENTS stance, reference may be had to my earlier patents, An electrode of FIG. 1 was employed in these experi namely U.S. Pat. Nos. 3,912,386; 3,380,905 and ments in the measurement of glucose. The overall ar 3,539,455 for specific electrode structures which may be 40 rangement of the circuit and polarographic cell may be used to detect oxygen and H2O2. Using devices of the obtained with reference to my above mentioned pa type mentioned in my patents, a dual electrode system tents. Such devices are well known. Their structures or may be used to sense glucose by relating the difference their operation per se need not be detailed here. in polarographic oxygen current between the normal The transcutaneous p02 in air breathing cats was skin and the enzyme modulated skin. In another form, a 45 measured with the electrode at about 38-44 C. The single electrode system can be employed. For instance, measurement of oxygen transcutaneously depends upon polarographic anodes of the types described in my U.S. the fact that this gas readily leaves the capillary blood Pat. No. 4,040,908 may be employed to measure hydro and diffuses through the skin to the outside. By heating gen peroxide by-product as a measure of the substrate. the skin to approximately 38-44 C., the capillary ves In one form of procedure, the enzyme is dissolved in 50 sels in the blood stream dilate, the skin becomes red and water and injected just under, or into, the skin and a the amount of oxygen that is diffused from the skin tepO2 electrode is positioned on the skin and secured increases and in fact comes into equilibrium with arte just over the enzyme site. The temperature of the skin is rial blood. Hence, the pop of an air breathing animal on controlled at approximately 38-44 C. In another pro the surface of the skin is on the order of magnitude of cedure, enzyme powder has been mixed with silicone or 55 about 80 mm of Hg. (A of FIG. 2). Following the fluorocarbon oils before subcutaneous injection. En breathing of oxygen, this transcutaneous p02 may in zyme has also been mixed with silicone monomer and crease to the region of 150 mm or so shown. This proce converted with suitable catalyst to a thin rubber-like dure was followed by insertion of glucose oxidase in polymer sheet about half the size of a postage stamp different forms underneath the surface of the cat's skin. which is then implanted through an incision in the skin. 60 With reference to FIG. 1, any of the forms of injection Such implants heal rapidly and retain enzyme activity or implantation discussed above may be used. Then, the for many days or weeks and probably much longer. transcutaneous p02 was remeasured. The difference or Enzyme implants have been made using a thin sheet of A-pO2 is a reflection of the blood glucose content. reinforced Silastic (organosilicone polymers, Dow Cor With reference to FIG. 2, the effect of increasing and ning subdermal implant No. 501-1.007 in. thick) coated 65 decreasing blood glucose levels is illustrated. After with enzyme, immobilized by treatment with glutaral returning from oxygen to air breathing, there was a dehyde solution and drying in the cold. Enzyme, either prompt fall in the topO2. Then, beginning at a few sec free or immobilized has also been trapped between two onds after the injection of glucose (B) (10 cc of 5% was 4,458,686 5 6 given intravenously), there was a further oxygen de crease. Breathing of oxygen a few minutes after the III-EXPERIMENTS injection results in an increased, but to a much lower In another set of experiments, an oxygen-consuming average topO2, than breathing oxygen before. Then, a was placed on the skin of an anesthe glucose injection (C) decreased the tcpO2. After the 5 tized cat. The animal was anesthetized with sodium injection of glucose oxidase (D) which converts the pentobarbital and maintained at 38 C. with an infrared circulating glucose to gluconic acid, while the cat was heater modulator modulated by a rectal thermistor sig still breathing oxygen, the tcpC2 increased to the high nal. The electrode was fastened to the shaved skin just est point measured. On return to air breathing, the level below the thorax. A few crystals of the oxidase prepara of the tcpo2 dropped. The tcpC2 response to oxygen 10 tion in about 50 pull of water (215 mg/50 ul) was placed was greatly decreased when glucose was given. Both of on the skin and the electrode was set in place. After a the abrupt falls in tcpo2 following oxygen breathing stable reading was obtained, alcohol solution was in were obtained after returning the animal to air breath jected. The results are shown in FIG. 3 and numbers 1ng. referred to at the points of injection, namely 2, 4, 10 and In this series of experiments, no attempt was made to 15 20, are the number of milliliters of 10% ethanol given perfectly quantitate the result but to demonstrate the intravenously. The measurements of the circulating principle of the skin sensing electrode, namely increas ethanol were employed using a transcutaneous tcpC)2 ing amounts of glucose in the blood are reflected by a electrode of the type shown in FIG. 1. With reference decreasing transcutaneous p02. Further evidence that to FIG. 3, it is demonstrated that increasing amounts of the initial A-pO2 was a reflection of glucose was found 20 alcohol decreased the tcpC2 step-wise and that recov by injecting the enzyme glucose oxidase directly into ery toward the initial value occurred over the following the bloodstream of the cat. When the purified enzyme minutes. The less than expected effect of the 20 milliliter was injected this way, there was a prompt increase in dose was not understood, but may possibly be due to a the tcpC2 leveling off at a certain value, thus demon pharmacological affect on the skin or possibly an acute strating that the initial reading was due to glucose since 25 drop in blood pressure. the enzyme when injected intravenously converts all Other means of performing the experiments of the the glucose to gluconic acid. above type involving volatilizable components, such as alcohol, include the incorporation of the enzyme in the II-EXPERIMENTS electrode's electrolyte, immobilizing it on the men In another set of experiments, glucose oxidase was 30 brane, and the use of two cathodes, one coated with mixed with a silicone preparation and then a catalyst enzyme and one uncoated. As mentioned above, one was added. The material was then pressed between two may also have a coated and an enzyme-free spot on the glass slides to produce a thin film of silicone rubber skin and calibrate by measuring the poversus blood or having glucose oxidase embedded in it. When this mem end tidal alcohol. The temperature control required for brane was hardened, it was placed subcutaneously in a 35 the tcpC)2 measurement is ideal for stabilizing enzyme cat and healed in a perfectly normal manner after a few activity. Enzyme would be best dissolved in a buffer days. Immediately after implantation, there was a differ with suitable coenzymes and stabilizing agents. There ence detected by the electrode of FIG. 1 in the A-tcpO2 are several alcohol and dehydrogenases with between the normal skin and the enzyme treated skin. It varying specificity toward alcohols of different chemi had previously been demonstrated that glucose oxidase cal structures, but all respond to ethanol for use in Ex mixed with the polymerizable silicone is active in the periments III. Of course, the tcpO2 skin procedure oxidation of glucose to gluconic acid. In another prepa above discussed with reference to alcohol can be used ration, glucose oxidase was mixed with silicone oil and for the continuous measurement of other volatile en this was injected subcutaneously. Still in another form zyme substrates where oxygen depletion is utilized in of implantation, the glucose oxidase was mixed with 45 their measurement. fluorocarbon liquid and injected subcutaneously. In still In view of the above experiments, it is obvious that a another, form, a glucose oxidase was trapped between a number of other can be used in order to detect thin layer of Silastic reinforced (artificial skin) and a and measure a substance transcutaneously. The follow layer of collagen. In each form, the method of this in ing Table is a listing of the enzymes, their identifying vention was established, namely that the amount of 50 number, source and typical substrates with which they glucose could be detected by measuring the difference may react for measurement in accordance with the in A-tcpC2. principles of this invention. TABLE Enzyme Number Source Typical Substrates Glycollate oxidase 1.1.3.1 spinach glycollate rat liver L-lactate D-lactate (+)-mandalate Lactate oxidase 1.1.3.2 M. phlei L-lactate Glucose oxidase 1.1.3.4 g-D-glucose Penicillium amagasakienses 2-dioxy-D-glucose (bee) 6-dioxy-6-fluoro-D-glucose Penicilium notatum 6-methyl-D-glucose ..3.5 B-D-glucose D-galactose D-nannose 1.3.8 at liver L-gulono-M-lactone L-Gulonolactone oxidase L-galactonolactono D-manonolactone D-altronolactone 4,458,686

TABLE-continued Enzyme Number Source : Typical Substrates. . . . Hemma-mma 1.1.3.9 Dactylium dendroides D-galactose - Polyporus circinatus stachyose lactose L-2-Hydroxyacid oxidase 1.1.3.a hog renal cortex L-2-hydroxyacid 1.2.3. rabbit liver formaldehyde pig liver acetaldehyde Xanthine oxidase 1.2.3.2 bovine milk : purine porcine liver hypoxanthine. benzaldehyde xanthine Pyruvate oxidase 1.2.3.3 ... pyruvate . . . requires thiamine phosphate Oxalate oxidase 1.2.3.4 oxalate Dihydro-orotate-dehydrogenase 1.3.3.1 Zymobacterium oroticum L-4, 5-dihydro-orotate NAD D-Aspartate oxidase 1.4.3.1 rabbit kidney D-aspartate D-glutamate L-Amino-acid oxidase 4.3.2 diamond rattlesnake L-methionine. cotton mouth moccasin L-phenylalanine " | rat kidney 2-hydroxy acids L-lactate D-Amino acid oxidase 1.4.3.3 hog kidney D-alanineD-valine 1 . D-proline Monoamine oxidase 1.4.3.4. beef plasma monoamine placenta benzylamine octylamine Pyridoxamine phosphate oxidase 1.4.3.5 rabbit liver pyridoxamine phosphate Diarmine oxidase 4.3.6 bovine plasma diamines . . . pea seedlings spermidine procine plasma tyramine 1.5.3.1 Macaca mulatta sarcosine rat liver metochondria N-Methyl-L-amino acid oxidase 1.5.3.2 N-methyl-L-amino acids oxidase 1.5.3.3 Neisseria perflava spermine Serratia marcescens spermidine Nitroethane oxidase 1.7.3.1

nitroethane aliphatic nitro compounds Urate oxidase 1.7.3.3 hog liver urates: . . . . ox kidney 18.3.1 beef liver sulfite. Alcohol oxidase Basidiomycetes ethanol and methanol oxidase Basidiomycetes D-glucose " . Polyporus obtusus D-glucopyranose D-xylopyranose 1-sorbose 8-D-gluconolactone NADH oxidase beefheart - NADH mitochondria 1.1.3.2 L-malate 1.1.3.6 cholesterol N-Acetylindoxyl oxidase 1.7.3.2 N-acetylindoxyl Thiol oxidase 1.8.3.2 R: CR-SH Ascorbate oxidase 1.10.3.3 squas L-ascorbate 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. 50 reduced by the enzyme and reoxidized by molecular Using the international nomenclature of the enzyme oxygen, they are simply called . commission (see for example T. E. Barman Enzyme Class 1, the oxidoreductases, are divided into sub Handbook, Vol. 1, 2, and Supplement, Springer-Verlag, classes, for example, 1.1 are those acting on the CH-OH New York 1969), classes of enzymes can be described group of donors. Class 1.1 is divided as follows: which will be useful in this invention. Since new en- 55 ... 1.1.1. with NAD or NADP as acceptor zymes are discovered each year, examples of presently 1.1.1. with as acceptor known enzymes can be used to illustrate the principles 1.1.3. with oxygen as acceptor involved. There are six main classes: 1.1.99 with other acceptors 1. Oxidoreductases Glucose oxidase, an oxygen acting on 2. 60 the CH-OH group of donors is therefore 1.1.3. Glucose 3. oxidase is a 1.1.3. enzyme and is key numbered as 4. Lysases 1.1.3.4., galactose oxidase is 1.1.3.9. If glucose is oxi 5. dized by a "dehydrogenase' enzyme, it is called glucose 6. dehydrogenase: Most of the oxygen consuming enzymes are in Class 65 1. If such enzymes may use molecular oxygen directly, Glucose--NAD(P)=Gluconolactone-reduced they are then called oxygen oxidoreductases, or if indi NAD(P) rectly, through a "coenzyme" or "' which is It is classed as 1.1.1.47. 4,458,686 9 10 Galactose dehydrogenase uses. NAD, rather than dose is high enough the animal may die because glucose NADP, as a cofactor: is converted in part to H2O2 and this converts the hemo Galactose--NADe Galactolactone-reduced. NAD globin to methemoglobin which does not carry oxygen. (or “NADH') Proteolytic enzymes may destroy the enzyme or it may These two dehydrogenases do not consume oxygen be picked up by Kupffer cells. If the immune system of directly but via cofactors. The dehyrogenase reaction the body including the opsonins can contact the en stops when all the cofactor is used up by reduction to zyme, it will be marked for destruction. Some antibod NADH or NADPH. The NADH or NADPH can be ies attach to enzymes (Freund's adjuvant is used to mix reoxidized to NAD or NADP by a number of means, with the enzyme before injection) and they are inacti including oxidation by another cofactor, by a platinum 10 vated by antibodies. In view of these observations, a anode, or by oxygen. Hence, the glucose substrate, the preferred technique is to not let the enzymes escape and donor molecule, is oxidized by oxygen, indirectly. to not let immune proteins or macrophages contact the Other natural cofactors, such as cytochrome or syn enzyme. The enzymes or co-enzymes could be placed in thetic substances, can act as cofactors with the final a container such as a plastic bag or encapsulated in result that a specific substrate is oxidized with the stoi 15 chiometric consumption of molecular oxygen. particles so that a substrate such as glucose can diffuse The other five main classes of enzymes can be used in in, but protein molecules cannot permeate. Peroxide conjunction with the oxidoreductases, or oxygen-con could be destroyed in the bag with catalase or allowed suming dehydrogenases, to expand the range of analy to diffuse out to be destroyed. Also, as developed sis. Examples could be found to illustrate a reaction for above, the enzyme can be immobilized on an inert sub each of the main classes of 2, 3, 4, 5 and 6. strate such as nylon or silver. Glutaraldehyde treated A Class 2. example is dextranslucrase (EC2.4.1.5) tissues such as heart valves from other species have which catalyzes the reaction of low molecular weight been used as substitute heart valves in human beings. dextran with sucrose, to give a larger dextran polymer. Glutaraldehyde is also widely used to immobilize en It consumes sucrose and yields fructose. Hence, de 25 zymes. Hence, glutaraldehyde can be used to immobil pending on conditions, it could be used to measure ize and affix enzymes to surfaces for implantation where dextran or sucrose. the probability of a rejection process would be very A Class 3 example is sucrase, commonly found in low. It is also recognized that monochromatic, dichro yeast, which is a beta-fructofuranoside and is a hydro matic or multiplechromatic light can be transmitted lase. It is EC3.1.1.2. which splits sucrose into fructose 30 through the earlobe and the light spectrum received on and glucose. With glucose oxidase, it could be.used to the other side to reveal the oxygen saturation of the ease SCOSe. blood. A transparent enzyme implant in the earlobe A Class 4 example is oxalate decarboxylase, could be designed with an appropriate dye such that EC4.1.1.3, and splits oxalate into formate--CO2. This substrate concentration would be reflected by transmit enzyme, found in wood fungus, could measure oxalate 35 ted light. by the release of CO2. A transcutaneous pCO2 electrode As a result of enzyme reactions, fluorescence and may be used to measure the pCO2 which is related to the phosphorescence can occur. Hence, by a suitable im CO2 by-product and hence the oxalate concentration. plant containing the enzyme and the photoactivated This enzyme does not require a co-factor. substance, one could detect substrate concentration by Another Class 4 example is acetoacetate decarboxyl measuring the amount of light emitted to the skin by the ase 4.1.1.10 which reacts with acetoacetate to give phosphorescent reaction. glycine-CO2. This enzyme is found in liver. The aceto In view of the above description, other details and acetate is found in diabetes which is not properly con operating parameters will be obvious to a person of trolled ordinary skill in this art. There are many other CO2 producing enzymes such 45 I claim: aS 1. A non-invasive cutaneous method of analyzing body substrates in the body which comprises Pyruvate decarboxylase EC4.1.1.1 contacting outside of the body an amount of a non Aspartate decarboxylase EC4.1.1.12 Glutamate decarboxylase EC4.1.1.13 50 gaseous body substrate which has volatilized Lysine decarboxylase EC4.1.1.18 through the skin with an enzyme selected for the Arginine decarboxylase EC4.1.1.19 substrate being analyzed, reacting the substrate with the enzyme, and detecting a condition of the skin as a measure of the In general, Class 5 enzymes could be used with oxy amount of substrate in the body. gen oxidoreductases where the D-form of an enzyme 55 was more stable than the L-form. For example, L-ala 2. The method of claim 1 comprising reacting the mine could be converted to D-alamine so that it could substrate with an enzyme to produce by-product hydro be oxidized by D-amino acid oxidase. gen peroxide and detecting said hydrogen peroxide at An example in Class 6 is an enzyme (EC6.4.1.4) the skin as a measure of the amount of substrate. which uses CO2 to convert 3-methylcrotonoyl CoA to 60 3. The method of claim 2 comprising contacting the 3-methylglutaconylCoA. These CoA compounds are skin with a hydrogen peroxide sensitive electrode. panthethenic acid condensed with ADP and trioe 4. The method of claim 3 wherein the electrode is a thanolamine and they play key roles in animal metabo polarographic anode. lism. 5. The method of claim 1 comprising reacting the In performing the techniques of this invention, it 65 substrate with an enzyme to produce hydrogen perox should be understood that foreign or other proteins ide by-product and detecting said by-product at the skin injected subcutaneously are absorbed rapidly. If glucose colorimetrically as a measure of the amount of the Sub oxidase is injected subcutaneously it is absorbed. If the strate. 4,458,686 11 12 6. The method of claim 1 comprising contacting the arterializing the skin, and skin surface with the enzyme and detecting a condition detecting a condition of the skin as a measure of the of the enzymatic reaction at the skin as a measure of the amount of substrate in the blood. s amount of substrate. 15. The method of claim 14 comprising the further 7. The method of claim 1 comprising arterializing the 5 steps of contacting the skin with the enzyme and heat skin. ing the skin. 8. The method of claim 7 wherein the skin is arterial 16. The method of claim 15 comprising the further ized by heating. step of detecting hydrogen peroxide by-product with a 9. The method of claim 8 wherein the skin is warmed polarographic electrode as a measure of the amount of to a temperature of about 38-44 C. 10 substrate. 10. The method of claim 7 wherein the skin is arterial 17. The method of claim 15 wherein the enzyme is ized by chemical treatment. encapsulated in a material permeable to the substrate. 11. The method of claim 1 wherein said detection is 18. The method of claim 14 comprising detecting the substrate colorimetrically. W by means of an electrode. 15 19. The method of claim 14 comprising detecting 12. The method of claim 11 wherein said enzyme is hydrogen peroxide by-product with an extracorporeal included in the electrolyte of said electrode. electrode as a measure of the amount of substrate. 13. The method of claim 11 wherein said electrode 20. The method of claim 14 comprising the further has a membrane associated with it and said enzyme is step of vaporizing the substrate and extracorporeally immobilized on said membrane. reacting the enzyme with the vaporized substrate. 14. A non-invasive method of extracorporeally ana 21. The method of claim 20 wherein the substrate is lyzing blood substrates in the body of mammalian sub an alcohol. jects which comprises 22. The method of claim 21 wherein the enzyme is transcutaneously contacting outside of the body a selected from the group consisting of alcohol oxidase non-gaseous blood substrate which has volatilized 25 and . through the skin with an enzyme selected for the 23. The method of claim 20 wherein the substrate is substrate being analyzed, S. ethanol. reacting the substrate with the enzyme, it it is

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