SOME OBSERVATIONS ON THE DISTRIBUTION OF RADIOACTIVE ZINC IN PROSTATIC AND OTHER HUMAN TISSUES

By OWEN DANIEL, F.R.C.S.,' FARID HADDAD, M.D., GEORGE PROUT, M.D., and WILLET F. WHITMORE, M.D., F.A.C.S.

From the Urologic Service of the Departmenr of Surgery, with the co-operarion of the Division of Clinical Investigalion, Sloan Kettering Institute, Memorial Center for Cancer

and Allied Diseases, New York, .V.Y.

IT has been reported that the prostate glands and testicles of man and other animals contain relatively much more zinc than any other tissues, and postulated that zinc plays an important part in general and reproductive physiology (Bertrand and Vladesco, 1921). More recently it has been shown that zinc occurs in high concentration in human semen and that spermatozoa contain the highest concentration of zinc in any human material yet examined (Mawson and Fischer, 1953).

With the aim of amplifying knowledge of the natural distribution of zinc and investigating the possible use of radioactive zinc in the diagnosis and treatment of prostatic disease, we have given tracer doses of the radioactive isotope (j5Zn to fifteen patients suffering from various types of malignant disease, who in addition required prostatic surgery.

METHODS

The isotope was obtained as a solution of zinc chloride in dilute hydrochloric acid containing 88 pc. of radioactivity and 50 pg. of carrier zinc per millilitre. One or in some cases 2 ml. of this solution was diluted to 5 ml. with physiological saline and injected slowly into a forearm vein of each subject. According to the degree of decay the injected radioactivity varied from 50 to 100 pc. and the amount of carrier zinc was 50 or 100 pg. At operation twenty to ninety-six hours later specimens of blood and tissues were taken and the radioactivity of the samples was measured in a well-type scintillation counter. Satisfactory counting rates were obtained from 1 ml. of a 1 in 10,000 dilution of the injected fluid, 1 ml. of blood, 0.25 ml. of prostatic secretion, and 200 to 500 mg. of tissue. Results are calculated in terms of the percentage of the total injected radioactivity contained in 1 ml. of blood, 1 ml. of prostatic secretion, or I g. of wet tissue and expressed as the 65Zn organ/blood ratio. There was no deliberate attempt to remove blood or tissue fluids from any of the tissue samples and no correction has been made for possible variation due to difference in the blood content of various samples.

RESULTS

Data concerning the fifteen patients, most of whom were in their sixth or seventh decade, are grouped according to prostatic histology (Table I). In addition to various forms of malignant disease elsewhere, ten patients suffered from benign prostatic enlargement and of these, five were found to have superimposed histological evidence of chronic prostatitis. One man (C. L.), aged 37 years, suffered from advanced carcinoma of the bladder, but had an apparently normal prostate, and four patients suffered from carcinoma of the prostate. The general condition of each patient is indicated in Table I.

Present address : University Department of Surgery, The Royal infirmary, Sheffield, 6. The b5Zn was supplied by the U.S. Atomic Energy Commission, Oak Ridge, Tennessee.

27 1

B R I T I S H J O U R N A L O F U R O L O G Y

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DISTRIBUTION OF RADIOACTIVE ZINC IN PROSTATIC AND OTHER HUMAN TISSUES 213

Prostatic tissue was obtained from six patients by transurethral electro-resection, from four by transvesical or retropubic enucleation, from four by perineal biopsy, and from one by total cystoprostatectomy. There was no indication for prostatic surgery on one patient suffering from carcinoma of the prostate, so that in his case tissue was not obtained from the primary tumour but from metastases in the inguinal lymph nodes.

The radioactive content of 1 ml. of whole blood, at the time of operation as indicated in Table I, varied from 0.6 to 3.9 by !O-3 per cent. of the total injected dose, the highest level occurring in the blood of the man (C. L.) suffering from carcinoma of the bladder. From a few patients blood samples were taken at intervals over periods of twelve to fourteen days. In these samples the radioactive content gradually doubled from the end of the first to the end of the sixth or eighth day after injection, then decreased at a rather slower rate. Blood samples were taken from one patient at fifteen minutes, two hours, six hours, twenty-four hours, and three, eight, and fifteen days after injection. In whole blood the highest concentration of radioactivity was found after fifteen minutes. At two hours the concentration fell to the lowest level, and after twenty-four hours rose gradually as described in the other patients (Fig. 1). In plasma separated

FIG. 1 The radioactive content of 1 ml. of whole blood (broken line) and plasma (continuous line) as a percentage of the total injected 65Zn by

1 0 - 3 at various times after intravenous injection.

from the corresponding blood samples the radioactive content fluctuated in an inverse fashion (see Fig. 1).

At the time intervals indicated in Table I the 65Zn organ/blood ratio for skin varied in different subjects from 0.1 to 1.9, fat from 0.2 to 0-8, testis from 0.7 to 2.5, and epididymis from 2.3 to 2.5.

The 65Zn organ/blood ratio, henceforward called the ratio, of adenomatous prostate tissue varied from 2.5 to 27-3. When chronic prostatitis was superimposed upon benign prostatic enlargement the ratio was uniformly low, ranging from 3.6 to 5-3. The ratio for the prostate of the man (C. L.) suffering from carcinoma of the bladder was 0.6. The ratio for carcinomatous prostatic tissue, in two patients (H. A. and P. O.), was 4.5. One of these (H. A.) had taken 15 mg. of stilbcestrol daily during the preceding two years and the other had had no previous treatment. In another patient with untreated carcinoma of the prostate (P. E.), perineal biopsy was performed and the carcinomatous tissue found to have a ratio of 40. Orchidectomy was carried out at the same time and histological examination showed atrophic testes. Two patients with untreated carcinoma of the prostate (P. 0. and S. A.) had widespread metastases and the ratio of the

274

Mean Zinc Content, p,g./g. Dry Weight.

Tissue.

B R I T I S H J O U R N A L O F U R O L O G Y

Zinc Organ/Blood Ratio. man aged 18 .

Death due to carcinoma

i Lung . 1 Spleen . ~ Heart .

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100 135 I86 82

226 245 ... ... 62

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FIG. 2 Photomicrograph of unstained autoradiograph prepared from prostatic adenoma from patient (B. 1.) using Kodak stripping film and an exposure of sixty-one days. Patient received 90 CIC. of 85Zn

forty-eight hours before prostatectomy. Blackening indicates radioactivity. x 220.

DISTRIBUTION OF RADIOACTIVE ZINC IN PROSTATIC AND OTHER HUMAN TISSUES 275

metastatic tissue was 6 and 2.5 respectively. The ratio of the primary tumour of the first of these was 4.5.

A few of the patients were subjected to prostatic massage and from two with benign prostatic enlargement (B. R. and S. H.) sufficient expressed secretion was obtained for radioactive counts to be made. The ratios of the secretions were 4.2 and 5, whilst the ratios for the corresponding prostatic tissues were 2.5 and 5.3 respectively. Attempts to obtain prostatic secretion from. patients with carcinoma of the prostate were unsuccessful.

65Zn in Post-mortem Specimens.-The patient (H. A.) who had been taking 15 mg. of stilbcestrol daily for two years was admitted in a terminal condition due to carcinoma of the prostate with widespread metastases and died of his disease eight days after receiving a tracer dose of 65Zn. Portions of various organs were obtained at autopsy and the ‘j5Zn ratio of these tissues has been compared with the zinc organ/blood ratios calculated from data reported by other workers from chemical determinations on autopsy and surgical specimens (Table TI).

Autoradiography of Prostatic Tissue.-Autoradiographs were prepared from prostatic tissue removed by retropubic enucleation from the patient (B. 1.). Surgery was performed thirty-six hours after the injection of 100 pc. of ‘j5Zn and the tissue immediately fixed in formalin. A few days later sections 5 p thick were cut and left in contact with autoradiographic stripping film for sixty-one days before development. A photomicrograph of one of the unstained sections (Fig. 2) shows blackening due to radioactivity localised to the cells lining some of the alveoli of a prostatic adenoma.

Most of the radioactivity in prostatic tissue is lost during fixation in water-based fixatives. Thus, when pieces of prostatic tissue each weighing approximately 250 mg. were transferred successively over a period of forty-eight hours through three vials each containing 2 ml. of formalin or Bouin’s solution, 70 per cent. of the initial radioactivity was lost into the solutions. However, there was no appreciable loss of radioactivity from tissues similarly bathed in alcohol.

DISCUSSION

The element zinc is present in small amounts in practically all animal and vegetable matter (Scholl, 1939). It was first thought that the metal was no more than a contaminant; animaI experiments have shown, however, that deprivation of zinc leads to failure of growth, sterility, premature greying of hair, liver necrosis, and eventually death (Todd et al., 1934 ; Stirn et al., 1935; Day and Skidmore, 1947; Nishimura, 1953). Ingestion of excessive amounts of zinc is also harmful, leading to microcytic and hypochromic anzmia, subnormal growth, reproductive failure, and liver atrophy in young rats (Smith and Larson, 1945; Sadasivan, 1951 a ; Hoch, 1952). The normal dietary intake of zinc in man is 15 mg. daily, of which 0.3 mg. is excreted in the urine and the remainder in the freces. The urinary excretion of zinc is relatively constant and independent of intake, whereas fzcal elimination corresponds more closely to intake (McCance and Widdowson, 1942 ; Tibble and Scoular, 1954).

Comparatively little is known of the physiological role of zinc, except that the metal is localised mainly in cell nuclei and plays a part in a wide variety of enzyme activities (Miyake, 1933 ; Koga, 1934 ; Rosenfeld and Tobias, 1951). Zinc is the prosthetic element of the enzyme carbonic anhydrase (Keilin and Mann, 1940), but seminal plasma contains negligible amounts of carbonic anhydrase and only 0.05 per cent. of the zinc in seminal fluid can be accounted for by this enzyme (Mawson and Fischer, 1952).

Zinc i s present in relatively large quantities in normal pancreatic tissue and is found in the purest preparations of insulin (Montgomery et al., 1943 ; Sheline et al., 1943 ; Sadasivan, 1951 h).

Manufactured by Kodak Ltd., London (Eng.).

276 B R I T I S H J O U R N A L OF U R O L O G Y

In alloxan-induced diabetes the concentration of 65Zn in the pancreas is considerably reduced (Lowry et al., 1954; Davidson, 1942).

Zinc is also found in urease and alkaline phosphatase (Banks, 1950). The ionic concentration of zinc determines the pH of optimal activity of certain phosphatases (Sadasivan, 1952). I n rabbit blood, zinc is confined to mononuclear cells and is positively correlated statistically to acid phosphatase (Mager and Lionetti, 1954). Reports by Roche et al. (1946) that zinc sulphate activates the alkaline phosphatase in the blood of normal patients and inhibits that in patients with carcinoma have not been substantiated by Bodansky and Bluemenfeld (1947) and Ellerbrook (1951).

Zinc is also said to enhance the action of the follicle stimulating hormone (FSH) and of the luteinising hormone (LH) of the anterior pituitary (Hawk et a/., 1954).

Most of the published work on zinc is based upon chemical or spectroscopic analyses. In attempting to evaluate the results of the radioactive zinc determinations it is instructiLe

to consider the zinc organlblood ratios calculated from data reported by other workers from chemical determinations on autopsy and surgical specimens (see Table 11). In these calculations we have assumed that 1 ml. of whole blood contains 10 pg. of zinc (Vallee, 1948), and hake converted the zinc content of dry tissue (Eggleton, 1940; Mawson and Fischer, 1953) into that of wet tissue by dividing by two (i.e., assumed that the small tissue samples used had a uniform water content of 50 per cent.). This type of calculation is open to obvious objections but may suffice for rough comparisons. The quoted workers make no mention of special measures to rid the tissue samples of blood, and as the samples used were of approximately the same size as those used by us there is no reason to believe that significant differences have been introduced through variation in the quantity of contained blood.

The chemically determined zinc ratios (see Table 11) are generally of the same order as the GjZn ratios, except for muscle, liver, adrenal, and most samples of prostatic tissue. The mean 6jZn ratio for muscle from nine of our patients is some twenty times less, and for liver (patient H. A,) nearly twice as great as the calculated zinc ratios. The disparity between the chemical and radioactive zinc ratios raises the possibility that uptake of radioactive zinc reflects the rate of turnover of zinc in the tissues rather than the concentrations of the naturally occurring metal. A further difficulty in interpretation arises from paucity of information regarding the rate of equilibration of intravenously injected zinc. Vallee et al. (1949) state that the ratio of 65Zn to total zinc is fairly constant in various organs but do not state the time interval during which this relationship is valid, although it would seem likely that such a relationship, if valid, must to some extent be a function of time. It should also be noted that Vallee and his co-workers report, in the same publication, observations on a series of puppies born of a dog receiving fi5Zn six days before delivery. All of the puppies were radioactive and analysis of organs of animals sacrificed at intervals up to seven days after birth showed a tendency for the concentration of radioactive zinc to increase in the liver and spleen and to decrease in the extremities.

The blood and liver of patients suffering from various forms of malignant disease have been reported to contain increased amounts of zinc (Addink, 1950). It has also been suggested that the livers of cachectic subjects have an increased concentration of zinc due to the relatively greater withdrawal of fat and carbohydrate than of the protein with which the metal is in association (Eggleton, 1940). The subject on whom we determined the fi5Zn liver ratio (H. A.) was cachectic.

The testicles of six patients examined by us had a 65Zn ratio varying from 0.7 to 2.5 with a mean of 1.5. The highest ratio occurred in the patient who had received a course of stilbastrol. It should be noted, however, that the age of the six patients ranged from 57 to 79 years, and that while two showed moderate spermatogenesis the other four had histological evidence of atrophy of the testes. It would be of interest to extend these observations to other age groups and to testicular tumour tissue.

The G5Zn ratio for prostatic tissue obtained by us was generally lower than the zinc ratio calculated from the chemical determinations of Mawson and Fischer (I 952). The lowest ratio

DISTRIBUTION OF RADIOACTIVE ZINC I N PROSTATIC AND OTHER HUMAN TISSUES 277

of all was 0.6, in the case of the man (C. L.) suffering from carcinoma of the bladder. It is noteworthy that this man’s prostate was normal on histological examination and that there was no clinical abnormality of the genital system, although his general condition could be described only as fair. This patient had, however, received a full course of deep X-ray therapy to his bladder two years before, and it is possible that this had depressed the zinc metabolism of the prostate, either directly or indirectly through the testicular damage.

Our observation that chronic prostatitis is accompanied by a low prostatic ‘j5Zn ratio conforms with the findings of Mawson and Fischer (1952) that such glands have a relatively low total zinc content (see Table 11).

Carcinomatous prostatic tissue has been reported to have a significantly low total zinc content (Mawson and Fischer, 1952). Two of our patients with carcinoma of the prostate, one untreated and the other having received a course of stilbcestrol, had prostatic tumour ‘j5Zn ratios of 4.5, which are of the same order as those found by us in some cases of benign enlargement with or without chronic prostatitis. Another patient with untreated carcinoma of the prostate had a 65Zn tumour ratio of 40, which is twice as much as the highest ratio found in any other tissue. The observations of Mawson and Fischer (1952) were based upon chemical determinations on tissues from five patients with carcinoma of the prostate, but it is not stated whether or not these patients had undergone orchidectomy or received hormone therapy. It is also possible that the zinc content of carcinomatous prostatic tissue is related to the degree of differentiation of the tumour or to the proportion of malignant cells to stroma.

Some neoplasms, such as carcinoma of the rectum, have a much higher zinc content than the corresponding normal tissue. Others, such as human bronchial carcinoma and methylcholanthrene-induced epidermoid carcinoma in rats, have a reduced zinc content as compared with the corresponding normal tissue (Carruthers and Suntzeff, 1945 ; Heath, 1949 ; Tupper et al., 1955). It would seem worth while to gain further information about the zinc content of carcinomatous prostate tissue, especially as our limited data indicate that the zinc content of metastatic tissue is of the same order as that of the primary tumour.

SUMMARY

Intravenous injections of 65Zn containing 50 to 100 pc. of radioactivity and 50 to 100 pg. of carrier zinc have been given to fifteen patients suffering from various forms of malignant disease and the radioactive content of blood and other tissues estimated twenty to ninety-six hours later. Results expressed as ‘j5Zn organ/blood ratios have been compared with zinc organ/blood ratios calculated from data reported by other workers from chemical determinations on similar tissues. The radioactive and chemical ratios are generally of the same order with the exception of muscle which had a much lower, and liver and certain samples of carcinomatous prostatic tissue which had a much higher ‘j5Zn ratio than Zn ratio. Carcinomatous prostatic tissue from one patient had a ‘j5Zn tissue/blood ratio of 40, which is twice as much as the highest ratio found in any other tissue.

This study was supported by the New York Cancer Committee and the Lerner Fund. The writers gratefully acknowledge the assistance of Miss Eva Simmel in preparing the autoradiographs.

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