GB2215045A

GB2215045A - Process and apparatus for determining the oxygen content of a substance

Info

Publication number: GB2215045A
Application number: GB8825485A
Authority: GB
Inventors: Gary D Steinman
Original assignee: David Diagnostics Inc
Current assignee: David Diagnostics Inc
Priority date: 1988-02-12
Filing date: 1988-11-01
Publication date: 1989-09-13
Also published as: GB8825485D0; JPH0223887A; DE3821077A1

Description

1 A 2 2 1 r., 0 4 5 PROCESS AND APPARATUS FOR DETERMINING THE OXYGEN

CONTENT OF A SUBSTANCE.

The present invention relates to a process for determining the oxygen content of a substance. More particularly, the present invention provides a method for determining the oxygen content in biological fluids, such as blood, and includes an apparatus for carrying out the method of the invention in a dry chemistry procedure.

It is well known in the biological and related sciences that oxygen is essential to drive a cell's mitochondrial process of oxidative phosphorylation whereby energy is stored as adenosine triphosphate (ATP). Oxygen levels in the blood vary according to the supply received from the lungs and by demand of the tissues. The majority of the o:,ygen in the bloodstream is carried by hemoglobin of the erythrocytes. Anything affecting one or more of these parameters car, alter the level of blood oxygen. For example, if blood flow through the lungs is impeded, such as in the case in a pulmonary embolus, the oxygen content of the blood, as measured by its partial pressure, can be correspondingly diminished. Thus, the determination of blood oxygen levels (as well as pH and carbon dioxide) can give a meaningful picture of the body's metabolic/respiratory status.

With present technologies, blood gases are most commonly determined by a laboratory-based polarographic instrument which utilizes a specific membrane electrode. Such a method, while being quite accurate when properly employed, has the drawback of requiring a rapid specimen transport in ice to a laboratory for analysis by a highly skilled technician, carefully repeated calibrations, and a high cost for the instrumentation.

fc 1 Inasmuch as tests for arterial blood oxygen and the like are frequently ordered in emergency situations, a critical need exists in the art for a simple, accurate and inexpensive method for the determination of oxygen levels in blood, and other biological fluids, which would be applicable for use at the bedside of a patient, with a minimum of manipulations to achieve at least a semi-quantitative value, pending more detailed testing in the laboratory at a later time.

Such a method should preferably be capable of being carried out in solution, as well as in a dry phase, a dry phase procedure being particularly preferred as being economical and expedient by allowing for the use of minute amounts of premeasured reagents. The method should preferably also allow for an oxygen level determination via visual means and be readily adaptable to existing instrumentation, thereby affording further economy and convenience of use. The method of the present invention which employs at least one enzyme (E) of a group of approximately 55 enzymes (which are a part of a larger group of enzymes known as oxidoreductases), presently known to use oxygen as a hydrogen acceptor in the following reaction system which results in the production of hydrogen peroxide:

AH 2 + 0 2 ------ 2 ------- A + H 2 0 2 Examples of potentially suitable enzymes include glucose oxidase, ceruloplasmin, oxalate oxidase and L-amino acid oxidase.

Although apparently unknown to the prior art, one car, apply the foregoing reaction system to determine the level of 0 2 in a given sample by initially fixing the amounts of t 1 oxidase and reactant. An added advantage of such a reaction system is that the peroxide product can be measured by the well-known peroxidase- mediated Trinder reaction, which utilizes a quinone-imine chromogen. The intensity of the visible colour produced is proportional to the amount of hydrogen peroxide present. This value, in the case of an oxidase reaction, would, in turn, be a direct measure of the amount of oxygen present under the reaction conditions noted above. It is known that the concentration of dissolved oxygen is proportional to its partial pressure.

Although many oxidases might serve this purpose, it is desirable to choose one which is commercially available and whose reactant is usually in very low concentration in the blood so that unrelated physiological processes would not alter the basic oxygen-measuring purpose of the test system. In addition, one or more of the reaction products should be quantifiable by a coupled reaction yielding a visible colour in proportion to the primary reaction rate.

An illustrative of the testing process of the present invention, an example of a typical analytical system using sarcosine oxidase (EC 1.5.3.1) is more fully described.

It, however, should be noted that the following example is given by way of illustration only, and is not intended - invention, as a limitation on the scope of the present since many similar oxidases could be employed having a similar utility:

(1) CH 3 -NH-CH 2 -COOH + H 2 0 + 0 2 ---------------------- > Sarcosine Oxidase H 2 0 2 + NH 2 -CH 2-COOH + HCHO 1 I- 11 While the chemistry of this and other oxidases has been known for some time, there appears to have been no attempt to utilize their oxygen dependencies in a quantitative analytical fashion, let alone exploit this property to measure oxygen levels in biological systems. It has been the general practice of biochemists and other persons skilled in the art to carry out the reaction of oxidases from various sources in room air. Thus, the prior art, by directing the use of saturating amounts of oxygen effectively teaches away from the methodology of the present invention, and still less does it disclose or suggest application of the foregoing principles to a quantitative determination of oxygen in biological fluids, such as whole blood, blood plasma, blood serum, cerebrospinal fluid, urine, saliva, tears, gastro-intestinal fluid, etc.

As discussed above, the peroxide by-product from Reaction (1) can be quantitated by using peroxidase and an indicator (In), according to the following reaction system:

(2) H 2 0 2 + In -------------------- > Colour Peroxidase Alternatively, one can choose to analyze for the formaldehyde (HCHO) product of Reaction (1) with the aid of formaldehyde dehydrogenase (EC 1.2.1.46) in the presence of nicotinamide adenine dinucleotide (NAD). The quantity of the reduced NAD product (NADH) is determined by a coupled reaction system involving the reduction of nitrotetrazolium blue (NTB) or iodonitrotetrazolium violet (INT) in the presence of phenazine methosulfate (PMS) or diaphorase (EC 1.6.4.3). The amount of coloured formazan produced is measured visually or colorimetrically, and is proportional to the amount of oxygen present in the original test sample:

1 M 4 11 (3) HCHO + H 2 0 + NAD form. dehydrogenase HCOOH + NADH (4) NADH + INT ------ --------- Colour As further illustrative of the analytical method of the present invention, the following reagent system is described. It should, of course, be understood that the present invention is not limited in scope solely to the 10 following system.

Example

The following reagent solutions are prepared:

1) Sarcosine oxidase (from Arthrobacter species; with K gluconate and EDTA) - 25 units/1 ml water.

2) Peroxidase (from horseradish; EC 1.11.1.7) 320 units/ml water.

3) 4-aminoantipyrine - 6 mg/4 ml 0.2M Tris buffer (pH 7.4) + 1 drop N, Ndimethylaniline.

4) Sarcosine (free base) - 5 mg/2 ml water.

I Fifty microliter aliquots of solutions #1, #2 and #3, after deoxygenating in an aierobic environment, are mixed with 25 microliters of test serum, followed by 50 microliters of solution #4. After incubation at room temperature for one minute, 3 ml of 0.1 N HCI is added and the optical density is read at 565 nm. Results are correlated with test samples of known concentration run in parallel.

That the application of an oxidase reaction for the measurement of oxygen is not restricted to sarcosine oxidase can be demonstrated by parallel reaction systems using pyruvate oxidase (EC 1.2. 3.3), choline oxidase (EC1.1.3.17) and diamine oxidase EC 1.4.3.6) for example.

As part of the present invention, an apparatus is provided for running the foregoing reaction test system in a dry chemistry procedure. The drawing figure illustrates a preferred embodiment of such an apparatus.

Turning to a consideration of the drawing in detail, filter paper B is impregnated with the mixture of reagents #1, #2 and #3, and air-dried. To prevent premature reaction, the sarcosine solution #4 is prepared, preferably in a non-aqueous solvent rather than water, and is then used to impregnate the same filter paper, which is again air- dried. Alternatively, an aqueous solution of sarcosine can be used, but the impregnated paper must be dried in an oxygen-free environment.

The bottom of filter paper or pad B is adherent to a clear stiff support layer of thin plastic. It should be noted that the filter paper or pad, B, may be any suitable bibulous material, e.g., a strip or swab made of paper, a cellulose derivative, spun glass, cotton, wool, or some other synthetic material. The size and absorptivity of the pad would determine the volume of specimen being tested, thus avoiding a pipetting step. A solution of ethyl cellulose in benzene is applied to the top of pad B and is air-dried so as to provide a semi- permeable barrier which allows the passage of plasma and gases but not blood cells. Such a barrier is disclosed in U.S. Patent No. 3,298,789, the pertinent portions of which are hereby incorporated by reference herein. The treated filter paper is then enclosed in a clear impermeable plastic or glass cube (A) containing an anerobic gas, such as nitrogen, hydrogen or helium.

A top of enclosed container A is a luer-lock receptacle (C). A syringe containing the test blood sample is applied to C. When screwed down, the projection of the syringe tip pierces or displaces barrier D, which is made 1 f Z c of a thin plastic film. This allows the test sample to pass onto pad B while avoiding contact with room air. After incubation at room temperature, the colour passing through the lower transparent layer of pad B is quantitated by the application of a reflectance photo- meter or by visual comparison to a standard colour chart. The unit is intended to be discarded after testing has been completed.

Alternatively, the reaction chamber could be in the form of a thin cylindrical column, with the treated pad at the top of the column and an inert absorbent in the body of the column to retain and exhibit the resultant colour exiting the reaction pad.

Finally, in a preferred embodiment of the present inventive paper phase method, one may remove red blood cells and large macromolecules from test samples by dipping a bibulous material into a solution of ethyl cellulose or cellulose acetate in benzene, or similar organic solvent. This is followed by the removal of the bibulous material from the foregoing solution and, then air-drying the bibulous material before commencing the testing procedure.

It is also evident that the methodology revealed in the present invention could be applied to any situation where the measurement of oxygen content is important, such as food processing or metal treatment. Furthermore, one could simultaneously determine pH and CO 2 with adjacent reaction pads which utilize colorimetric systems already known for these analytes, thus making a complete arterial blood gas determination possible.

C 1

Claims

A process for measuring the oxygen content of a substance, comprising the steps of:

(a) supplying to a test sample of the substance being tested a predetermined amount of a reduced reactant (AH 2), an oxidase enzyme (E) and a buffer in a reaction mixture of enzymatic Reaction I, AH + 0 ---------------------) A + H 0 (1) 2 2 2 2 (b) measuring a rate of appearance of a reaction product in Reaction I, wherein the rate of appearance of said reaction product is directly proportional to a concentration of oxygen in said test sample, and (c) comparing the rate of appearance of said reaction product in Reaction I against a reaction rate for the appearance of the same in at least one standard of known oxygen concentration thereby determining 4Che oxygen concentration of said test sample.
2. A process according to claim 1, wherein the buffer employed is a Tris buffer.
3. A process according to Claim 1 or Claim 2, wherein said enzyme is sarcosine oxidase and said reactant is sarcosine.
4. A process according to any preceding claim wherein the reaction product being measured is hydrogen peroxide.
5. A process according to Claim 4, wherein steps (b) and (c) are carried out by a comparison of the optical density of a resultant colour in coupled Reaction II, H 2 0 2 + In Peroxidase --- Colour, (II) 1 i wherein In is a quinone-imine chromogen indicator.
6. A process according to any one of claims 1 to 3 wherein the reaction product being measured is formaldehyde.
7. A process according to Claim 6, wherein steps (b) and (c) are carried out by a comparison of the optical density of a resultant colour in coupled Reactions III and IV, HCHO + H 2 0 + NAD -----------------------;> form. dehydrogenase HCOOH + NADH (III) NADH + INT ------------------ Colour (IV), PMS 15. wherein, NAD is nicotinamide adenine dinucleotide; NADH is reduced NAD; INT is iodonitrotetrazolium violet; and, PMS is phenazine methosulfate.
8. A process according to Claim 5 or Claim 7, wherein the resultant colour is measured by transmission spectrophotometry.
9. A process according to Claim 5 or Claim 7, wherein the resultant colour is measured by visual comparison with a standardized colour chart.
10. A process according to any preceding claim wherein 30 the substance being tested is a biological fluid.
11. A process according to claim 10, wherein said biological fluid being tested is whole blood, blood plasma or blood serum.
12. A process for measuring the oxygen concentration of a substance by means of a dry chemistry procedure, comprising the steps of: (a) supplying pre-determined amounts of an oxidase and a buffer in an aqueous solution; (b) adding an indicator system for quantitative colorimetric analysis of said aqueous solution;.c) dipping a bibulous material into said aqueous solution; (d) removing said bibulous material from said aqueous solution; (e) air-drying said bibulous material after removal from said aqueous solution; (f) dipping said bibulous material into an anhydrous organic solution of a reduced reactant; (g) removing said bibulous material from said anhydrous organic solution of said reduced reactant; (h) air-drying said bibulous material following said step (g); (i) applying to said bibulous material said substance being tested so as to initiate an enzymatic Reaction (I):

AH 2 + 0 2 ------------------ > A + H 2 0 2; (I) E -(j) measuring a rate of appearance of at least one of the products of Reaction (I) by a quantity of colour produced in a quantitative colorimetric analysis; and (k) comparing the quantity of said colour produced to at least one standard of a known oxygen concentration whereby the quantity of colour produced is directly proportional to the oxygen concentration of the substance 30 being tested.
13. A process according to claim 12 wherein said bibulous material is a strip or swab made of paper.
14. A process according to Claim 12, wherein said bibulous material is made of a cellulose derivative or synthetic material.

R D t,
15. A process according to Claim 12, wherein said bibulous material is spun glass.
16. A process according to Claim12, wherein said bibulous material is made of cotton.
17. A process according to Claim 12, wherein said bibulous material is made of wool.
18. A process according to any one of claims 12 to 18, wherein the substance being tested is a biological fluid.
19. A process according to Claim 18, wherein said biological fluid being tested is whole blood, blood plasma or blood serum.
20. A process according to Claim 18 or Claim 19, further comprising the steps, preceding step (i), of: removing red cells and large macromolecules from said biological fluid when said biological fluid is blood by dipping sr- aid bibulous material into a solution selected from the group consisting of ethyl cellulose in benzene and cellulose acetate in benzene; removing said bibulous material from said solution; and air-drying said bibulous material.
21. A process according to any one of Claims 12 to 20, wherein the intensity of the colour produced is quantitated 30 by reflectance spectrophotometry.
22. A process according to any one of claims 12 to 20, wherein the intensity of said colour produced is quantitated by visual comparison with a standardized colour chart.

f i 1 1 "X 1
23. Apparatus for measuring the oxygen concentration of a substance by means of a dry chemistry procedure, comprising: a cube or column having an enclosed internal anerobic environment; a bibulous material contained within said cube or column, said bibulous material impregnated with pre determined amounts of an oxidase, a buffer, a reduced reactant, and an indicator for colorimetric analysis, said bibulous material having a covering of cellulose or a cellulose derivative; a luer-lock receptacle located atop of said cube or column, said luer-lock receptacle being capable of receiving a syringe containing a sample of said substance to be tested; a barrier, capable of being pierced or displaced by said syringe, said barrier located between said luer-lock receptacle atop said cube or column and said cube or column; wherein after applying to said bibulous material said substance being tested so as to initiate an enzymatic Reaction (I):

AH 2 + 0 2 ---- ------------- > A + H 2 0 2 (1) E the rate of appearance of at least one of the products of Reaction (I) is measured by the intensity of colour produced in a quantitative colorimetric analysis and the quantity of said colour produced is compared to at least one standard of a known oxygen concentration whereby the quantity of colour produced is directly proportional to the oxygen concentration of said biological fluid being tested.

i% i i 4
24. Apparatus according to Claim 23, wherein said cube or column is made of clear impermeable plastics or glass.
25. Apparatus according to Claim 23 or Claim 24, wherein the cube or column contains a gas selected from nitrogen, hydrogen, helium and mixtures thereof.
26. Apparatus according to any one of Claims 23 to 25, wherein said bibulous material is impregnated with a reaction mixture consisting of sarcosine oxidase, peroxidase, tetramethylbenzidine, and 0. 2 M Tris buffer (pH 7.4), then dried, then impregnated with sancosine in a non-aqueous solvent and then dried again.
27. Apparatus according to any one of Claims 23 to 25 wherein said bibulous material is impregnated with a reaction mixture consisting of sarcosine oxidase, formaldehyde dehydrogenase, nicotinamide adenine dinucleotide, iodonitrotetrazolium violet, phenazine methosulfate and 0.2 M Tris buffer (PH 7.4), then dried, then impregnated with sarcosine in a non-aqueous solvent, and then dried again.
28. Apparatus according to any one of Claims 23 to 27, wherein said bibulous material is filter paper.
29. Apparatus according to any one of Claims 23 to 28, wherein said barrier is made of a thin plastic film.
30. Apparatus according to any one of claims 23 to 29, wherein the substance tested is a biological fluid.
31. Apparatus according to claim 30 wherein said biological fluid is whole blood, blood plasma or 35 blood serum.

1 t A - i 1
32. A process according to Claim 1 substantially as herein described and exemplified.
33. Apparatus according to Claim 1 substantially as herein described with reference to the accompanying drawing.

Published 1989 atThe Patent Office. State House, 65'71 HighHolborn,Lo ndorlWClR4TP. Further copies maybe obtained from The Patent Office Sales Branch. St Marv Cray. Orpington. Kent BFZ 3RD, printed by Multiplex techniques ltd. St Mary Cray. Kent, Con. 1187 i