US 3699005 A
Description (OCR text may contain errors)
United States Patent O 3,699,005 METHOD AND ARTICLE FOR DETECTING THE FERTILE PERIOD Raymond 0. Foster, 1202 Clara St., Joliet, II]. 60435 No Drawing. Continuation-impart of abandoned application Ser. No. 597,547, Nov. 29, 1966. This application Aug. 26, 1970, Ser. No. 67,258
Int. Cl. G01n 31/14 U.S. Cl. 195-103.5 R 6 Claims ABSTRACT OF THE DISCLOSURE A new and rapid test method is provided for predicting and detecting the fertile period of the female. The method involves a simple rapid test for alkaline phosphatase activity in the saliva of the female. In the method the female wets a novel test implement with her saliva .and the test implement will turn a detectable blue color in about one minute if an amount of alkaline phosphatase is present in the saliva sulficient to cause the color change. This amount of alkaline phosphatase is coincident with the onset of the fertile period of the female, and extends for the duration of the fertile period. The novel dry test implement consists of a dry bibulous material which has been impregnated before drying with an indoxyl phosphate and a buffer.
CROSS REFERENCE This application is a continuation-in-part of my earlier U.'S. application Ser. No. 597,547 which was filed Nov. 29, 1966 and now abandoned.
BACKGROUND OF THE INVENTION There has been much interest in the past as well as presently in being able to predict and detect the period of time during which the female is fertile, i.e., is capable of conceiving an offspring. The fertile period occurs during ovulation, when the ovum has departed the ovary and is still alive and in a position to be fertilized.
The importance is readily apparent of being able to predict and detect the fertile period, both to those who wish to take advantage of the fertile period and those who wish to avoid it. However, prior to the present invention, the methods and means available for such prediction and detection have failed to provide the degree of convenience and accuracy desired. Attempts to calculate said period by reference to the onset of menses are speculative and beset with inaccuracy because the human female, for instance, may ovulate at varying and unpredictable times. On the other hand, mechanical methods such as by taking and recording temperatures, while possibly more accurate, are distinctly inconvenient and may require considerable skill in observing and interpreting the data.
It has been found that alkaline phosphatase, an enzyme found in the saliva (or other body secretions, e.g., cervical mucus) varies in concentration during the female menstrual cycle, and has a relatively higher concentration during the first part of the females menstrual cycle and at the time of ovulation.
It is known in the chemical art that indigo, a strong "ice blue dye, is readily formed by the coupling of two indoxyl radicals. (Fieser and Fieser, Organic Chemistry, 1944, p. 872.) It is also known in the art that the presence of the enzyme, alkaline phosphatase, can be detected in tissue sections by a staining technique which depends upon deposition of indigo in crystalline form at the spot where the enzyme is localized in the tissue. (Holt, Proc. Roy. Soc., B 148, 1958, pp. -168.)
The procedure described by Holt is a tissue staining technique requiring not only specialized equipment to perform the procedure, but also highly trained personnel to conduct the test and read the results. Holts procedure is a non-quantitative laboratory method which requires prolonged incubation, the addition of an oxidizing agent such as a ferricyanide to increase the rate of depositions, and also must be graded microscopically.
In the face of the foregoing discouraging information in the prior art, I have, nevertheless, discovered a new and rapid test method for predicting and detecting the fertile period of the female quite accurately by a simple test which can be conducted by anyone regardless of technical skill. The test comprises contacting saliva of a female with a novel test implement which is a piece of dry, bibulous material which has been impregnated before drying with an indoxyl phosphate and a buffer. If alkaline phosphatase is present in the saliva in significant amount, as I have found it to be just prior to and during the fertile period, then the implement will turn a detectable blue color in about one minute after wetting with saliva. The implement will remain in its original colorless state if the female is not approaching nor within the fertile period.
SUMMARY OF THE INVENTION This invention relates to the discovery that the fertile period of the female may be predicted accurately by contacting the females saliva with a novel test implement, and to a novel analytical test implement useful in detecting the fertile period of the female which is coincident with an increase in certain enzyme activity.
Further in accordance with the present invention, there is now provided a test implement useful in detecting an increase in alkaline phosphatase activity which is coincident with the fertile period of the female which comprises a bibulous material impregnated with a non-toxic buffer capable of maintaining the pH at an alkaline level in the range of about 10.0 to about 10.3 and an indicator characterized by the formula R-O-PO wherein R is an indoxyl radical which upon cleavage of the ester bond by said alkaline phosphatase will form or develop a compound having a readily discernible color.
While any physiologically acceptable indoxyl compound having the above-specified formula and properties can be employed as the indicator, it has been found that indoxyl phosphate and five-bromo-indoxyl phosphate are particularly advantageous.
The cleavage of the ester bond in the aforesaid indoxyl compounds releases the indoxyl radical, or substituted indoxyl radical and, in the presence of oxygen or air two indoxyl radicals combine to form indigo, or a substituted indigo, a dark blue dye. In the practice of this invention this reaction normally takes place in a short period of time, such as about thirty seconds to about one minute. The degree of blue formed is relative to the amount of the alkaline phosphatase present.
The presence of phosphate ions in the saliva and those formed by the cleavage of the indicator member tends to inhibit the action of the enzyme, alkaline phosphatase. But, it has now been found that the action of the enzyme can be significantly increased by including in the test tape a compound capable of accepting and tying up phosphate ions. The compound should be inert with respect to the indicator and the buffer. Magnesium salts, e.g., the sulfate and chloride, have been found to be suitable.
The great advantage and convenience of this oral test is at once apparent. The female simply touches the test paper to her tongue to wet it, and waits a few minutes to see if a color change develops on the test paper. The re sult is easily observed, and does not require the recording and interpretation of data over a long period of time. Each test is complete per se. The test is reliable and simple as well as being convenient and relatively inexpensive.
This method and implement is also highly useful to the clinician in detecting pathological conditions because positive tests at a known non-fertile period are a clear indication of unusual and perhaps pathological conditions.
DESCRIPTION OF THE PREFERRED EMBODIMENT Describing in more detail the test implement or test paper, the test paper is preferably bibulous paper, for example, an absorbent and chemically pure grade of filter paper or the like. Cloth strips or porous and absorbent wood strips may also be employed. The test paper is impregnated with a solution of the active ingredients and is then subjected to drying to evaporate the solvents and leave the active ingredients on the paper.
In one representative formula, when indoxyl phosphate is the indicator, 1.5 grams of sodium alginate is dissolved in 100 cc. distilled water. Five grams of tris (hydroxy methyl) aminomethane, 2 grams of sodium carbonate and 500 milligrams of magnesium sulfate are dissolved in 100 cc. of distilled water. Two grams of indoxyl phosphate is dissolved in 40 cc. of distilled water.
The three solutions are combined with the resultant solution having a pH of about 10.0 to about 10.3. The large sheets of chemically pure filter paper are impregnated with the mixed solution and air dried.
Five bromo-indoxyl phosphate can be substituted for the indoxyl phosphate in the above representative formula to serve as the indicator. The concentration of the indicator can be varied within a range of 500 to 2500' milligrams depending upon the degree of final color desired.
It has been found on detailed study that a representative test paper, which is a piece of standard filter paper about inch wide by 1 inch long, will absorb on the average about 25 mg. of saliva on a standardized wetting time of about 5 seconds. In a normal female during her nonfertile period, this amount of saliva will contain an amount of alkaline phosphatase that is not significant and can be detected only with much difiiculty. Special techniques and formulations are needed and the development of a readable color takes so long as to be totally impractical. For example, time in excess of ten minutes is required and by then the test paper will have dried, shutting off the chemical reaction that develops the color. Thus, any amount of alkaline phosphatase lower than about 2.5 to 3.0 units of activity in the 25 mg. sample will not give an easily discernible color change. (A unit of alkaline phosphatase activity is defined as a microgram of phenol formed per ml. of sample in 69 minutes at 37 C.)
A typical test paper made as described above will have absorbed about 102 gamma of indoxyl phosphate after drying.
In one typical female it was found that a daily com- 4 parison of the basal body temperature with a chemical analysis of the amount of alkaline phosphatase ina 25 mg. sample of saliva showed excellent coordination between the drop in temperature normally associated with the immediate pre-ovulatory and early ovulatory stage and a rise in alkaline phosphatase activity to amount above the detectable level, for this patient, about 3.0 units of activity. Thus an activity high enough to be easily read on the subject test implement coincided with the immediate pre-ovulatory and ovulatory phase. This patient would know from a color change on my test implement that she was entering or was in her fertile period.
The test papers or test tapes so prepared have been used in clinical studies to determine the accuracy of the test tapes in detecting the fertile period as evidenced by color changes caused by the saliva of the female. When compared to the previous standard, namely, the (basal body temperature) thermal shift response, the foregoing test tapes give a significant correlation.
There is thus provided a convenient and accurate test method and test paper for detecting the fertile period by a simple test on saliva. The test papers above described, after contact with saliva during the fertile period, will detect an increase in alkaline phosphatase activity by developing a readily discernible color after but a short period of time. If there has been no significant increase in alkaline phosphatase above the amount normally present during a non-fertile period, the tape does not develop the readily discernible color upon contact with saliva.
While the foregoing test method and test tape are obviously of primary importance to the human female, it is apparent that the test is applicable to other female animals, and can be of considerable importance.
The response of the test implement may also be demonstrated by contacting other closely related body fluid, namely, cervical mucous with the test implement. The foregoing invention has been described in detail, but it will be apparent to those skilled in the art that certain substitutions can be made among the ingredients employed herein without, however, departing from the spirit of the invention. All such substitutions are considered to be included herein, provided they fall within the scope of the appended claims.
1. The method of detecting the fertile period of a female by use of a system which changes color coincident with the onset of the fertile period, which comprises wetting with saliva from a female, a dry bibulous test implement impregnated with an effective amount of an indicator selected from the group consisting of indoxyl phosphate and S-bromo-indoxyl phosphate, said amount being sufficient to produce a color change when the alkaline phosphatase concentration of the females saliva is at the elevated level coincident with the onset of the fertile period, and a non-toxic buffer which maintains the pH in the range from about 10.0 to about 10.3, observing the test implement for a color change within about one minute after wetting with the saliva, and predicting said fertile period based on the presence or absence of a color change.
2. The method of claim 1 wherein said indicator is indoxyl phosphate.
3. The method of claim 1 wherein said indicator is 5- bromo-indoxyl phosphate.
4. A test implement useful in detecting the fertile period of a female by use of a system which changes color coincident with the onset of the fertile period and adapted to detect the presence of an elevated level of alkaline phosphatase activity in saliva upon wetting said implement with saliva and observing the color change, which consists of a dry, bibulous material impregnated with an effective amount of an indicator selected from the group consisting of indoxyl phosphate and S-bromoindoxyl phosphate, said amount being sufficient to produce a color change when the alkaline phosphatase concentra- 6 tion is at the elevated level coincident with the onset of the fertile period, and a non-toxic bufier which maintains OTHER REFERENCES the pH in the range from about 10.0 to about 10.3. Holt, Nature, VOL 169, 271473 (1952) 5. The test implement of claim 4 wherein said indicator Holt Nature, Vol. 1 PP- 1012-1014 13 mdoxyl Phosphate- 5 Goldschmidt et al., Biological Abstracts, vol. 33,
6 The test implement of claim 4 wherein said indicaparagraph 40384 (1959). tor 1s S-bromo-mdoxyl phosphate.
ALVIN E. TANENHOLTZ, Primary Examiner References Cited UNITED STATES PATENTS 10 195 99 l- X-R- 2,905,594 9/1959 Morris 195-l03.5