US 3813222 A
Description (OCR text may contain errors)
Un ted Sta es Pa e 3,813,222 Patented May 28, 1974 METHOD OF DETERMINING OVULATION TIME IN URINE AND FERTILITY IN FEMALES I. R. La Vietes, Belle Harbor, N.Y., assignor to La Mar Laboratories, Inc. Filed Oct. 14, 1971, Ser. No. 189,249 Int. Cl. Gtlln 31/22, 33/16; G07c 87/48 US. Cl. 23-230 B Claims ABSTRACT OF THE DISCLOSURE The presence of anterior pituitary hormone in mammalian urine, blood, serum or plasma is determined by mixing with the urine, blood, serum or plasma a test quantity of a stable solution of 2-4, dinitrophenylhydrazine and an aqueous solution of an alkali metal hydroxide and then comparing the color of the solution with a control color.
The present invention relates to the preparation of a reagent that is used to detect the presence of small amounts (20-50 micrograms) of anterior pituitary hormone (estrogen) in mammalian urine, blood, serum or plasma.
Another object of the invention is the utilization of said reagent to determine the time, during a female menstrual or oestrus cycle, when ovulation takes place.
Determination of estrogen levels is of interest to physicians in elucidating atypical hormonal patterns in order to provide proper treatment for a variety of disease conditions such as abortion, hypergonadism and hypogonadism. Determination of ovulation time and commensurately, whether, ovulation takes'place at all is an important physiological consideration. Information about this function is of interest to women who find it diflicult to conceive because of atypical menstrual cycles; to individuals who wish to regulate family size but because of a variety of considerations do not choose to use contraceptives; to the physician in assisting him in the diagnosis of several abnormal conditions such as abortion and sterility, and to the veterinarian in determining times of fecundity in household pets and animal stock.
Heretofore, the determination of ovulation time was a hit and miss affair which employed, in the case of humans, the taking of vaginal temperatures over a period of months, testing the vaginal mucosa for glucose which is difficult to perform and inaccurate or determining estrogen levels by means of a long and costly chromatographic procedure.
This product covered by this invention was required to fulfill two criteria; sensitivity to the low levels of estrogen present in normal non-gravid females and stability over a range of room temperatures (40 degrees-90 degrees F.) over long period of time (6-9 months).
The compound 2,4 dinitrophenylhydrazine (DNPH) is characterized by the presence of both hydrophilic and hydrophobic moieties which accounts for its well known limited solubility in almost all solvents and its limited stability once in solution. To'provide a means for increasing the solubility of the compound to the point where it would be effective as a reagent, solution was effected in.
hot hydrochloric acid. Similar results with varying degrees of solubility can also be ,obtained with other mineral acids, water and concentrated or dilute alcohols. To provide a means of stabilizing the compound once in solution a polyhydric alcohol such as polyethylene glycol was added. Similar results with varying degrees of stability can also be obtained with other polyhydric alcohols such as propylene glycol and glycerin.
The reagent can be typically prepared in the following manner:
A stock solution is made by dissolving 1 gram of DNPH and 0.1 gm. of methyl red in 175 ml. of concentrated hydrochloric acid and 50 ml. of polyethylene glycol with the aid of heat. The solution is then allowed to cool. Varying amounts of polyethylene glycol are then added to obtain a standardized solution. The solution of DNPH and the polyhydric alcohol should be 4.5-6.5 N, preferably about 5.5 N. No fixed amount can be set on the volume of diluent (polyethylene glycol) to be added for commercially prepared DNPH varies considerably from lot to lot, with respect to strength and purity. Standardization therefore, takes place by colorimetrically matching the solution against known standards.
The methyl red serves as a pH indicator and to intensify the colors obtained when the reaction has gone to completion. Other indicators of a similar type may be used such as methyl orange or methyl purple, or the indicator itself may be eliminated from the reagent completely.
The reagent described above (Reagent A) is used in conjunction with a second reagent (Reagent B) which is an aqueous solution of an alkali metal hydroxidesuch as potassium or sodium hydroxide. The second reagent solution should be 59.7 N, preferably 6.4 N. It is made in accordance with known procedures and of sufficient strength to give a final pH of 9-115 when used in the described procedure.
Urine was collected on a daily basis over a period of several months, from women who had a history of regular menstrual cycles. These women were instructed to take their basal temperature daily so that test results could be correlated through a corroborative method. The urine was tested daily by the described process.
There are provided 2 like vessels, as for example tubes or vials of approximately diameter and 2" height, these vessels can be calibrated for convenience, as for example at the 7 ml. level or a measuring device such as a. pipette can be used to dispense the reagents and/or urine. The vessels can be marked appropriately to aid in distinguishing between them. A specific volume of Reagent A is added to one of the vessels (Tube 1) to which is also added urine to the volume mark on the vessel or by means of a calibrated dropping device. The contents are shaken to eifect mixture and the vessel is set aside. A predetermined time period such as seconds is allowed to elapse after which a specific volume of Reagent B is added to Tube 1 and contents mixed. Tube 1 is set aside.
A specific volume of Reagent A is added to he remaining vessel (Tube 2) to which is also added a specific volume of urine from hte urine sample as used in Tube 1 by means of a calibrated dispensing device or to a pre-calibrated level marked on the vessel. A specific amount of Reagent B is added to Tube 2 immediately. The vessel is shaken to mix the contents and set aside. Tube 2 is meant to be a control against which the colors resulting from the chemical reaction in Tube 1 is compared. Tube 2 contains all the components found in Tube 1 except that the reaction has not gone to completion. The pH of the final solution in both tubes should be about 9-1 1.5.
After a time period, as for example, 3 minutes, Tube 1 and Tube 2 are compared visually or optically by means of a photometer. If the color of the liquid in Tube 1 is the same color or slightly lighter or darker than the color of the liquid in Tube 2, the reaction indicates that ovulation is taking place or has taken place within the last 24 hours, or will take place within the next 24 hours. If the color of the liquid in Tube 1 is much darker and in marked contrast to the liquid in Tube 2, then the reaction indicates that ovulation is not taking place.
A time element is involved with the test. The sensitivity of the test can be decreased by increasing the time element between the addition of Reagent A and Reagent B. This is an advantage as it gives an alternative method of increasing or decreasing the sensitivity of the test. The
test may be performed in a single vessel (Tube 1) without recourse to the use of a control sample. The color of the liquid in said Tube 1 may be compared to standards prepared previously that have been made on or in paper, glass, plastic and other like material or to previously prepared liquid samples of known concentration. The final colors may also be described verbally so that no standard or control need be employed as a means of comparison there being a marked difference in the color and lightnesses of the liquid resulting from a reaction indicating ovulation and a reaction indicating no ovulation.
What I claim is:
1. A method for detecting the presence of anterior pituitary hormone in urine, blood, serum or plasma of non-pregnant female mammals which comprises, in the following sequence:
Placing the urine, blood, serum or plasma in a test container;
Adding thereto a specific amount of a solution of 2,4- dinitrophenylhydrazine in a solvent, the solution having been stabilized by the addition of a polyhydric alcohol;
Allowing the mixture to stand for about 90 seconds thereby forming a color;
Adding a specific amount of an aqueous solution of alkali metal hydroxide whereby a fin-al solution having a pH of 9-1 1.5 is formed; and
Measuring the intensity of color of the final solution after a second time period, thereby determining whether ovulation has taken place.
2. A process for the preparation of a stable solution of 2,4-dinitrophenylhydrazine which comprises dissolving the 2,4-dinitrophenylhydrazine in a solvent and adding a polyhydric alcohol thereto, said polyhydric alcohol being a stabilizer.
3. The process as recited in claim 2 wherein the solvent is water.
4. The process as recited in claim 2 wherein the polyhydric alcohol is polyethylene glycol.
5. The process as recited in claim 2 in which the solvent is selected from the group consisting of alcohol and a mixture of water and alcohol.
6. The process of claim 2 in which the polyhydn'c alcohol is selected from the group consisting of propylene glycol and glycerine.
7. The process as recited in claim 2 wherein the solvent is a mineral acid.
8. The process as recited in claim 7 wherein the mineral acid is hydrochloric acid.
9. The process as recited in claim 2 wherein a pH indicator is added to the stable solution.
10. The process of claim 9 in which the pH indicator is selected from the group consisting of methyl red, methyl orange, and methyl purple.
References Cited UNITED STATES PATENTS 3,434,801 3/ 1969 Scherr 252408 X 3,436,186 4/1969 McSweeney et al. 23230 B 3,226,196 12/1965 La Vietes 23230 B OTHER REFERENCES L. Kahlenberg, Science 46, No. 1578, 344 (1925). Machbeth et al., Chem. Abstr. 29, 750 (1935). Roduta et al., Chem. Abstr. 31, 98 5 (1937). Reich et al., Chem. Abstr. 50, 395 c (1956).
L. Legradi, Chem. Abstr. 63, 12296 g (1965).
ROBERT M. REESE, Primary Examiner U.S. Cl. X.R.