Fluid and Semen. S. H. Ying, M.D., E. Day, M.D., W. F. Whitmore, Jr., M.D., and H. J. Tagnon, M.D.*

Transcription

1 Fibrinolytic Activity in Human Prostatic Fluid and Semen S. H. Ying, M.D., E. Day, M.D., W. F. Whitmore, Jr., M.D., and H. J. Tagnon, M.D.* IT HAS BEEN KNOWN since the work of Huggins and Neall that human prostatic fluid contains an enzyme or enzymes able to digest fibrin. This activity has also been found in the blood of some patients with prostatic carcinoma.4, 5, 6 Recent experiments, to be published elsewhere, have shown that the prostatic enzyme or enzymes are proteolytic on a substrate of casein as well as of fibrin. 7 In this study the fibrinolytic activity of prostatic fluid and/or semen was measured in 74 men over 5 years of age. The purposes of the study were, first, to establish normal values for prostaticfluid and semen fibrinolytic activities in patients of this age group and, second, to determine the existence of any correlations between such enzymatic activities and the clinical status of the prostate. MATERIALS AND METHODS Prostatic-fluid specimens and semen specimens were obtained from a group of men past the age of 5 years. The donors were, in general, healthy men who appeared at the Strang Cancer Prevention Clinic of Memorial Center for a general cancer-detection examination. The cases were un- From the Division of Preventive Medicine of Sloan-Kettering Institute, the Urologic Service of the Department of Surgery, and the Department of Medicine, Memorial Center, New York, N. Y. This investigation was supported in part by Grant DRG No. 28 from the Damon Runyon Memorial Fund for Cancer Research. * Present address: Institut Jules Bordet, Brussels, Belgium. 8

2 Vol. 7, No. I, 1956 SEMINAL AND PROSTATIC FIBRINOLYSIN 81 selected to the extent that all individuals in the above category who were willing to submit to the necessary examinations were included. Prostatic Fluid Prostatic fluid was obtained by digital rectal massage of the prostate and collected directly in a dry test tube. The volume of prostatic fluid obtained by massage varied between.5 cc. and 2 cc., with the average volume being approximately.25 cc. The specimen was immediately placed in dry ice and later stored in the deep freeze at -2 C. until the determinations were made. For the quantitative assay of fibrinolytic activity,.5 cc. of prostatic fluid was diluted to a volume of 1 cc. with O.OlM phosphate buffer at ph 7.4, and.1 cc. of the diluted fluid was employed for the determination. Thus, the actual volume of prostatic fluid employed in the assay was.5 cc. In the 3 patients in whom prostatic-fluid fibrinolytic activity was reassayed, 8 days, 7 months, and 8 months, respectively, elapsed between the successive determinations. oj Semen Semen specimens were obtained by artificial maneuver, the entire ejaculate being collected in a clean, dry, wide-mouthed test tube. The patients were instructed to abstain from intercourse for 3 days before the specimens were obtained. The specimens were placed in dry ice within 1 minutes after collection and later stored at -2 C. until the determinations were made. For the quantitative fibrinolysin asssay.5 cc. of semen was diluted to a volume of 1 cc. with O.OlM phosphate buffer, and.2 cc. of the diluted fluid was used in the determination. Thus, the actual volume of semen employed in the assay was.1 cc. In the 4 patients in whom both prostaticfluid and semen fibrinolytic activities were determined, four weeks or more elapsed between the collection of the respective specimens... Fibrinolysin Determination The method of fibrinolysin assay employed was that described by Shulman and Tagnon,S wherein the fibrinolytic activity of the unknown specimen is compared to that of a standard plasmin. The unknown and 25 /Lg. of a standard plasmin preparation are each incubated with a substrate of fibrinogen tagged with FSI to which thrombin has been added to form a fibrin clot. After incubation, the counts per minute (CPM) of FSI in the

3 82 YING ET AL. Fertility & Sterility TABLE 1. Fibrinolytic Activity in the Prostatic Fluid Fibrinolytic activity CPM sample CPM 25 p.g. Interval between Clinical status No. Name Age oj prostate plasmin Quotient 2 specimens M.G. 62 / B.R / J.N. 65 / E.S / E.A / W.H / J.w. 8 I.R / J.O / / R.M / M.F / nd determination 76/ mo., 2 da. after 1st. 12 H.W / nd determination 51 88/ da. after 1st 13 A.M / T.R / E.M / W.F / E.H. 18 J.S / W.F / / L.F / S.S / O.K / J.H / R.C / H.O / J.C / S.G / / E.w / E.E S.K / G.S / W.D. 6 86/ R.L / J.M / J.S / M.T / F.N / C.W / L.G /

4 ...," Vol. 7, No.1, 1956 SEMINAL AND PROSTATIC FIBRINOLYSIN 83 TABLE 1. (Continued) ~ ~ _- Fibrinolytic activity CPM sample No. Name Age Clinical status CPM 25J.Lg. Interval between oj prostate plasmin Quotient 2 specimens ~ ~ r 4 LH / C.L / E.R / A.M / R.A / C.S / L.E / W.N / A.B / S.C / M.T / nd determination 448/ mo. after 1st 51 W.F / E.H / L.L / S.A / M.K / H.E / A.F / H.F / P.P / H.S / respective ltrates give a measure of the amount of dissolution of the fibrin clots. The CPM of the unknown is then compared with the CPM of the plasmin standard, thus providing a measure of the fibrinolytic activity of the unknown. Although values for fibrinolytic activity in the unknown specimens which extend beyond the standard reference curve based on the activity of 25 flg. of standard plasmin may not be strictly quantitative, they are significant in illustrating the relative levels of fibrinolytic activity. Clinical estimates regarding the size of the prostate were based on digital.. rectal examination, although such estimates are known to be subject to error. The designation "" refers to a prostate thought to be within normal limits of size; "+" refers to a prostate thought to be mildly enlarged; "+ +" to a prostate thought to be moderately enlarged; and "+++" to a prostate thought to be markedly enlarged. In none was carcinoma suspected.

5 84 YING ET At. Fertility & Sterility RESULTS In Table 1 the data are arranged in order of increasing fibrinolytic activity of the prostatic fluid. The fibrinolytic activities are expressed as the ratio of counts per minute (CPM) of the unknown prostatic fluid to CPM of the plasmin standard and also as the quotient of this ratio. Since all these determinations were not performed at the same time, the CPM of the plasmin controls varied from one assay group to the next but remained constant within anyone group of determinations. The age of the patient and the estimated size of the prostate are indicated. In Table 2 the data are arranged in order of increasing fibrinolytic activity of semen. These data include 4 patients listed in Table 1 in whom semen fibrinolytic activity as well as prostatic-fluid fibrinolytic activity was determined. In the prostatic fluid the mean value with the standard deviation of the TABLE 2. Fibrinolytic Activity in the Semen No. Name Age F.S. 6 2 F.B M.L S.w. 5 W.H E.e H.G S.W. 6 9 H.S I.S L.H n.r H.B S.K B.R N.S A.B J.1. 6 Clinical status oj prostate Fibrinolytic activity oj semen Volume CPM specimen oj semen (cc.) plasmin Quotienta CPM 25,..g. 4. / / / / / / / / / / / / / / / / / / Fibrinolytic activity oj prostatic fluid CPM specimen CPM 25,..g. plasmin Quotient 1197/ / / / Interval between 2 specimens 29 da. 3 mo. 1 mo. & 13 da. 1 mo. & 12 da. a Semen quotient must be divided by 2 for comparison with prostatic-fluid quotient (see next page, first paragraph).

6 Vol. 7. No. I SEMINAL AND PROSTATIC FIBRINOLYSIN B5 mean for the quotient of the fibrinolytic activity was for all 6 cases; for 29 cases without prostatic enlargement; and for 31 cases with benign enlargement of the prostate. In the semen the mean value with the standard deviation of the mean for the quotient of the fibrinolytic activity was for all 18 cases; for 12 cases without prostatic enlargement; and for 6 cases with benign enlargement of the prostate. However, the volume of prostatic fluid used in the assay was.5 cc. and the volume of semen used was.l cc. DISCUSSION The wide range of prostatic-fluid fibrinolytic activity observed in the 29 patients with prostates of normal size precludes the possibility of establishing any "normar value, even within the relatively narrow confines of the age group under consideration. It is possible that inherent limitations in the technic of obtaining prostatic fluid account in part for the wide variations observed. Usually the volume of prostatic fluid obtained by massage is small, so that the relative importance of small volumes of "contaminants" from the seminal vesicles or the urethra, including urine, is great. In the 3 patients who had repeated examinations of prostatic fluid, 2 had quite marked differences in the fibrinolytic activities of successive specimens, suggesting that the fibrinolytic activity of the prostatic fluid of the individual patient may be different at different times. The fibrinolytic activity of semen is derived from the prostatic-fluid component, 1,2 and this activity accounts for the prompt dissolution of the seminal clot. The fibrinolytic activity of the semen of the 12 patients with prostates of normal size varied over a wide range. Four patients had determinations of both prostatic-fluid and semen fibrinolytic activities. The respective specimens were obtained at intervals of at least 4 weeks. Two of the 4 patients showed higher fibrinolytic activity in their semen than in the prostatic fluid. This apparent paradox may be explained by assuming different levels of fibrinolytic activity in the prostatic fluid of the same individual at different times. Moreover, the semen may contain substances which act as inhibitors or accelerators of fibrinolytic activity. Hormonal Influences Evidence has been presented elsewhere that the circulating prostatic

7 86 YING ET Ai. Fertility & Sterility fibrinolysin detected in certain patients with prostatic cancer may be depressed by estrogen therapy.4, 5 The authors have limited data suggesting that estrogen administration may depress prostatic-fluid fibrinolytic activity in individuals without prostatic cancer. 8 It is possible that hormonal alterations of as yet undefined nature play an important role in the etiology of human benign prostatic enlargement. In the present study, it was postulated that any endocrine alteration etiologically connected with human benign prostatic enlargement might reveal itself through measurable changes in the fibrinolytic activity of prostatic fluid. The data presented fail to provide any definite evidence thereof. Volumetric Contribution of Prostatic Fluid The mean values of the quotients for prostatic-fluid and semen fibrinolytic activities among patients without prostatic enlargement, 2.79 and 2.74, respectively, taken in conjunction with the dilution factors employed in the assay, suggest that the volumetric contribution of prostatic fluid to the total semen volume is of the order of 5 per cent. The mean values of the quotients for prostatic-fluid and semen fibrinolytic activities among patients with benignly enlarged prostates, 2.52 and 3.33, respectively, are not significantly different from the corresponding values of patients without prostatic enlargement. It seems possible that obtaining a specimen of prostatic fluid and semen from the same individual within the interval of a few hours and determining the fibrinolytic activity of each specimen may provide a quantitative means of measuring the volumetric contribution of prostatic fluid to the total ejaculate, provided that prostatic fluid is the source of all semen fibrinolytic activity and that accelerators or inhibitors of such activity do not exist in the semen. Although semen and prostatic-fluid fibrinolytic activities in the same patient show a lack of correlation in our limited data, this discrepancy may be due to the fact that the respective specimens were collected at intervals of several weeks. CONCLUSIONS The fibrinolytic activity of human prostatic fluid and semen may be quantitatively determined by measuring the liberation of soluble p31_ containing substances from a substrate consisting of 1 l3l -tagged fibrin. The variations in fibrinolytic activity under the conditions described are wide

8 I... Vol. 7, No.1, 1956 SEMINAL AND PROSTATIC FIBRINOLYSIN 87 and preclude the possibility of establishing a "normal" value from present data. However, the mean values suggest that the prostate contributed about 5 per cent of the volume of the whole ejaculate of semen in the cases studied. r REFERENCES 1. HUGGINs, c., and NEAL, W. Coagulation and liquefaction of semen: Proteolytic enzymes and citrate in prostatic fluid. ]. Exper. Med. 76:527, HUGGINs, c., and VAIL, V. C. Plasma coagulation and fibrinogenolysis by prostatic fluid and trypsin. Am. J. Physiol. 139: 129, SHULMAN, N. R., and TAGNON, H. J. Proteolytic activity determined with a substrate tagged with radioactive iodine. ]. Biol. Chern. 186:69, TAGNON, H. J., SCHULMAN, P., WHITMORE, W. F., JR., and LEONE, L. A. Prostatic fibrinolysin: Study of a case illustrating role in hemorrhagic diathesis of cancer of the prostate. Am.]. Med. 15:875, TAGNON, H. J. Unpublished data. 6. TAGNON, H. J., WHITMORE, W. F., JR., and SHULMAN, N. R. Fibrinolysis in metastatic cancer of the prostate. Cancer 5:9, TAGNON, H. J., WHITMORE, W. F., JR., SCHULMAN, P., and KRAVITZ, S. C. The significance of fibrinolysis occurring in patients with metastatic cancer of the prostate. Cancer 6:63, TAGNON, H. J., YING, S. H., WHITMORE, W. F., JR., and DAY, E. Unpublished data. Sterility and Fertility Society / Great Britain The Sterility and Fertility Society of Great Britain will hold its annual conference on Thursday and Friday, June 14 and 15, The Conference will take place on Thursday at the Meeting House of the Zoological Society of London and on Friday at the National Institute for Medical Research, Mill Hill, London. Members of the American Society for the Study of Sterility who wish to attend the meeting or participate in the program may obtain further information from the Secretary of the Society, MR. H. H. FOURACRE BARNS, 31 Weymouth Street, Portland Place, W. 1, London.