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United States Patent (19) 11 Patent Number: 4,458,686 Clark, Jr

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United States Patent (19) 11 Patent Number: 4,458,686 Clark, Jr 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 enzyme whose activity is specific for a particular substance or substrate 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-product 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 oxygen 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 glucose 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 oxidase 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--HYDROGEN PEROXIDE 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.
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