Efficacy of single-voided urine metanephrine and normetanephrine assay for diagnosing pheochromocytoma.

Urinary catecholamines (CAs) and their metabolites are usually measured during the process of diagnosing pheochromocytoma (pheo), but a 24-hour urine collection is not convenient for outpatients. Since 1987 we have utilized "spot" urine metanephrine (MN) and normetanephrine (NMN) assays for management of patients with pheo or adrenal incidentaloma. MN and NMN were measured by radioimmunoassay in 82 patients with surgically proved pheo and 15 patients with incidentaloma. In 10 patients with pheo, MN and NMN were measured with fractional every-3-hour urine samples, which were accumulated and then measured as a 24-hour urinary specimen. Fractions of 3-hour MN and NMN excretion were constant (MN 98.5 +/- 9.6%, NMN 97.6 +/- 10.8%; 24-hour MN and NMN 100%). The average levels of MN and NMN in patients with pheo were 6801 ng/mg creatinine (Cr) (range 93-88,248, median 1426) and 5627 ng/mg Cr (range 219-31,528, mean 3190), whereas the MN and NMN levels in patients with incidentaloma were 123 ng/mg Cr (range 36-246, mean 133) and 251 ng/mg Cr (range 84-472, mean 220), respectively. When we selected a cutoff value for MN + NMN of 1000 ng/mg Cr, the sensitivity was 97.6% and the specificity 100% for diagnosing pheo. When the standard was set as > 500 ng/mg Cr for either MN or NMN, both the sensitivity and specificity were 100%. The assay for MN and NMN is simple and effective, not only for screening but for diagnosing pheo and managing incidentaloma.

Reduction of telomeric length and c-erbB-2 gene amplification in human breast cancer, fibroadenoma, and gynecomastia. Relationship to histologic grade and clinical parameters.

BACKGROUND: Telomeric deletions contribute to genetic instability and may represent an important mechanism of carcinogenesis. Amplification of the c-erbB-2 gene has been demonstrated in breast carcinoma. The clinical significance of telomeric deletions and c-erbB-2 gene amplification therefore was studied in patients with breast disorders. METHODS: The Southern blot analysis was used to measure telomeric length as well as the c-erbB-2 gene amplification of breast carcinomas, adjacent normal breast tissues, fibroadenomas, and cases of gynecomastia. RESULTS: Significant reductions in telomeric length and concentration were observed in all breast tissues when compared to placental DNA. Mean telomeric lengths were lowest in carcinomas and fibroadenomas. There were no significant differences, however, in the telomeric lengths among tissues from patients with breast carcinomas, fibroadenomas, or gynecomastia. The degree of telomeric deletion correlated significantly with histologic grade and was most notable in Grade 3 (scirrhous) breast carcinoma. The extent of telomeric deletion reflects the histologic aggressiveness of breast carcinoma, and telomeric reduction already can be seen in the adjacent normal breast tissues from patients with breast cancer. c-erbB-2 gene amplification was observed in 26.8% of the patients with breast carcinoma. c-erbB-2 gene amplification was not observed, however, in patients with fibroadenomas or gynecomastia. The degree of telomeric deletion did not correlate with c-erbB-2 gene amplification, tumor size, clinical stage, steroid receptors, or prognosis. Telomeric length was shorter in lymph node-negative tumors than in lymph node-positive tumors. CONCLUSIONS: These findings indicate that a shorter telomere length reflects growth advantage in breast cancer tissue, and telomeric reduction may promote cancer progression.

[Radioimmunoassay of synthetic steroid hormones].

The problem and recent progress of radioimmunoassay for various kinds of synthetic steroid hormones are reviewed. The specificity and sensitivity depend mostly on the position of synthetic antigen (hemisuccinate or oxime). Using good antibodies, a direct radioimmunoassay without extraction procedures of samples is available. A simplified method is required for the monitoring of synthetic steroids to know the effects and side effects.

Effect of hemodialysis on the concentration of the seven tumor markers carcinoembryonic antigen, alpha-fetoprotein, squamous cell carcinoma-related antigen, neuron-specific enolase, CA 125, CA 19-9 and CA 15-3 in uremic patients.

The incidence of the 7 tumor markers carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), squamous cell carcinoma-related antigen (SCC), neuron-specific enolase (NSE), CA 125, CA 19-9 and CA 15-3 was studied before and after hemodialysis (HD) in 144 uremic patients who had no malignancies. Before HD, of all tumor markers, the mean concentration of SCC only exceeded the normal value. The positive rate was highest in SCC (95.1%), and that of CEA and NSE was 25.7 and 10.6%, respectively. However, AFP was within the normal range in all cases. Among CA antigens, the positive rate of CA 125 was 7.6%, of CA 19-9 was 6.3% and of CA 15-3 was 3.5%. After HD, the incidence as well as the mean concentration of all tumor markers increased. A parallel increment of total protein was observed after HD. The membrane filter used in HD appears to be insufficient to remove tumor marker proteins during HD. It is necessary to consider the clinical interpretation of elevated tumor markers in patients with uremia.

[A clinical evaluation of the CA 72-4 serum level as a possible tumor marker].

The level of CA 72-4 in serum has been measured in various diseases by an immunoradiometric assay using CA 72-4 RIA Kit for an evaluation of its clinical significance as a possible tumor marker. CA 72-4 showed a high positive rate in patients with ovarian and bile duct cancers. In contrast, the false positive rate was low in benign gynecological and digestive diseases. Serum CA 72-4 was found to be increased in patients with a mucinous cystadenocarcinoma and in other histological types in the early stages of an ovarian cancer. Further, the serum CA 72-4 level changed in parallel with the clinical course of the ovarian cancer. Therefore, our results suggest that measurement of CA 72-4 be combined with measurement of CA 125, so as to provide a better sensitivity and specificity in monitoring an ovarian cancer.

[Clinical evaluation of various tumor markers in pleural effusion and in the serum].

Various tumor markers and enzymes in the pleural effusion and serum have been measured in 47 patients with carcinoma and in 43 patients with benign disease, by means of a radioimmunoassay and biochemical methods. CEA in the pleural effusion showed a high specificity and sensitivity for the detection of the malignancy. In patients with a lung cancer, measurement of the NSE in the serum was more useful than in the pleural effusion. Further, both CA19-9 and SCC in the pleural effusion showed a high specificity in a differential diagnosis of cancer and benign diseases. On the other hand, CA 125, TPA and IAP in the pleural effusion and in the serum showed a high sensitivity, but a low specificity for diagnosing the malignancy. The levels of ADA were significantly higher in tuberculous pleural effusions than in carcinomatous effusions. Therefore, this suggests that measurement of the various tumor markers in both the pleural effusion and in the serum is useful in achieving a differential diagnosis of malignant and benign diseases.

The changes in plasma cortisol and urinary free cortisol by an overnight dexamethasone suppression test in patients with Cushing's disease.

We studied the suppressibility of cortisol secretion in 15 patients with Cushing's disease by measuring morning plasma cortisol level as well as the 24-hour urinary free corisol (UFC) excretion following single doses of increasing amounts of dexamethasone (ranging from 0.5 to 32 mg) given at 11 p.m. The mean plasma cortisol level in patients with Cushing's disease was twice as high as in normal subjects, whereas the mean UFC in these patients was 6 times as high. Plasma cortisol in seven patients were suppressed by less than 4 mg of dexamethasone (in 2 cases, less than 0.5 mg; in 3 cases, less than 2 mg; and in 2 cases less than 4 mg). In these cases, basal plasma cortisol and UFC were less than 25 micrograms/dl and 350 micrograms/day, respectively. Among the other eight patients, plasma cortisol was partially suppressed in 5 cases and not suppressed in 3 cases by high doses of dexamethasone (16-32 mg). In these cases the basal plasma cortisol and UFC were more than 25 micrograms/dl and 350 micrograms/day, respectively. There was a significant correlation between the basal plasma cortisol and UFC (r = 0.687, p less than 0.01). These data suggest that the suppression by increasing amounts of dexamethasone in most cases with Cushing's disease was related to the severity of hypercortisolism.

Hypercortisolism and the resistance to dexamethasone suppression during gestation.

Maternal adrenocortical function was studied by measuring plasma cortisol and urinary free cortisol during gestation. Changes in suppressibility of pituitary-adrenocortical function were determined by dexamethasone administration. Urinary free cortisol as well as plasma cortisol increased during the course of gestation. The suppressibility by dexamethasone became less effective as pregnancy advanced. These results suggest that pregnant women have pituitary-adrenocortical hyperfunction and tissue refractoriness to glucocorticoid which increases during the course of gestation.

[Ovarian inhibin activity in rats with persistent estrus].

To investigate the significance of changes in ovarian inhibin in gonadal dysfunction with persistent estrus, female rats with persistent estrus were studied as animal model for polycystic ovaries (PCO). Newborn female Wistar rats treated with testosterone propionate s. c. (TP) and female rats raised under continuous light (LL) started at 9 weeks of age entered into continuous estrus 5 and 2 or 3 weeks after the treatment, respectively. Ovaries in both groups had many polycystic follicles Normal adult female rats with 4 day cycles were served as control. Ovarian inhibin content was measured by FSH suppressing activity of charcoal-treated ovarian homogenates using a rat anterior pituitary cell culture system. Blood samples were obtained at 11:00 a. m. and plasma levels of LH, FSH, PRL, estradiol (E2), progesterone (P) and testosterone were measured by RIA. Comparisons were made statistically between each experimental group and normal proestrus (PE). Both LH and FSH were similar to normal PE in TP LL, suggesting a lack of LH and FSH surges probably through hypothalamic dysfunction in TP and LL. Plasma PRL levels were variably elevated in both groups. Ovarian inhibin contents were comparably high to PE in both groups with parallel increases in plasma E2 and P. Plasma testosterone was elevated only in TP. In conclusion, female rats with persistent estrus induced by TP or LL exhibited polycystic ovarian changes similar to PCO. Increased inhibin content in TP and LL may reflect persistent follicular activity.

[A study of the clinical usefulness of the CA15-3 radioimmunoassay kit].

We have studied the clinical usefulness of the CA15-3 radioimmunoassay (RIA) kit that has been developed with two monoclonal antibodies (115D 8 and DF 3). Serum levels of CA15-3 exceeding the normal level were found in a high percentage of patients with ovarian cancer (80%), lung cancer (62.5%) and breast cancer (29.3%). In patients with breast cancer, the mean concentrations of CA15-3 before operation were higher than those after operation. Further, the levels of CA15-3 showed a good correlation with the extent and the stage of the breast cancer. These results suggest that CA15-3 levels can provide monitoring information in the follow-up management of patients with breast cancer.

[Biochemical diagnosis of pheochromocytoma by determining normetanephrine and metanephrine concentrations in single voided urine].

Diagnosis of pheochromocytoma has been made by the determination of urinary noradrenaline and adrenaline excretion for 24 hours. The assay procedure and the collection of urine for 24 hrs. are intricate. In the present study, we have ascertained the clinical significance of urinary normetanephrine (NM) and metanephrine (M), chemically stable metabolites of catecholamines, in single voided urine for a diagnosis of pheochromocytoma. Urine and plasma samples were collected from 361 normal subjects, 59 patients with essential hypertension, 22 patients with chronic renal failure and 22 patients with pheochromocytoma. Urinary NM and M concentrations were determined by radioimmunoassay with prior hydrolysis by acidification with 1N HCl. Plasma NM and M concentrations in normal subjects were 71.8 +/- 30.7 pg/ml and 41.5 +/- 8.61 pg/ml, respectively. Plasma NM was increased in 8 and plasma M was increased in 20 of 21 patients with pheochromocytoma, although many of these overlapped with those patients with chronic renal failure (NM, 285.9 +/- 175.1 pg/ml; M, 206.3 +/- 186.7 pg/ml) and essential hypertension (NM, 107.7 +/- 90.7 pg/ml; M, 46.7 +/- 20.2 pg/ml). Urinary NM and M concentrations did not show specific diurnal variation and there was significant correlations between the values in single voided urine and those in the 24 hour urine. Urinary NM and M concentrations in normal controls were 197.5 +/- 46.7 ng/mg Cr. and 125.3 +/- 37.1 ng/mg Cr., respectively. Urinary NM concentration was increased in 14 and urinary M concentration was increased in all of 17 patients with pheochromocytoma. In addition, urinary M concentration was higher in most of the 17 patients with pheochromocytoma than that in the patients with chronic renal failure and essential hypertension. However, the values in three patients with Sipple's syndrome with a small adrenal tumor or recurrent cases overlapped with those in other diseases. Relationships between urinary concentrations of NM and/or M and tumor size showed positive correlations. Urinary NM and M concentrations showed significant decreases after surgical removal of the tumors. These results suggest that NM and/or M concentrations in single voided urine could be a sensitive and specific diagnostic tool for pheochromocytoma.

Testosterone restores testicular inhibin content in cryptorchid rats.

The effects of testosterone administration on testicular inhibin content and histology were studied in bilaterally cryptorchid rats, in which a marked decrease in testicular inhibin content had been observed. Mature male Wistar rats weighing approximately 300 g were made bilaterally cryptorchid by placing the testes in the abdominal cavity. Testosterone in oil, 0.1, 1.0 or 10 mg, was given i.m. each week. Testicular inhibin and testosterone content, histology and plasma LH, FSH and testosterone were studied 2 weeks later. Abnormally decreased testicular inhibin in cryptorchidism was restored toward normal by testosterone in a dose dependent manner in 2 weeks after surgery. Sertoli cell structure also recovered toward normal with increasing amount of testosterone. Decreased testicular testosterone content and Leydig cell atrophy were observed with suppressed plasma LH and FSH after testosterone. These results showed that the increased plasma concentration of testosterone had a stimulatory effect on the Sertoli cell function in cryptorchidism, in which compensated Leydig cell failure was demonstrated.

Studies on pituitary-gonadal function in patients with Cushing's syndrome.

Basal levels of sex steroids, and the responses of LH and FSH to LH-RH were studied in twenty-five female patients with Cushing's syndrome (17 Cushing's disease and 8 adrenocortical adenoma). Only two patients had a regular menstrual cycle. Amenorrhea or oligomenorrhea had been of long duration in the other cases except for three postmenopausal patients. In patients with Cushing's disease, basal estradiol was low or below normal in 86%. Progesterone was normal in 83%, but testosterone was high in half of the cases. The response of LH to LH-RH in patients with Cushing's disease was normal in 35%, low in 35% and high in 29% of the cases. FSH response to LH-RH was normal in 23.5%, low in 23.5% and high in 53%. In patients with adrenocortical adenoma, basal of estradiol was low or below normal, but progesterone and testosterone were normal in all cases. The response of LH and FSH to LH-RH in all patients with adrenocortical adenoma was higher than normal. In three postmenopausal women, a higher response of LH and FSH to LH-RH was seen in two cases and suppressed in one case. These data suggest that the main site of suppression of the gonadal axis in patients with adrenocortical adenoma is the gonad rather than the pituitary gland or hypothalamus, though the mechanism of hypogonadism in patients with Cushing's disease is heterogeneous.

Effect of uni- and bilateral cryptorchidism on testicular inhibin and testosterone secretion in rats.

The effect of uni- and bilateral cryptorchidism on testicular inhibin and testosterone secretion and their relationships to gonadotropins were studied in rats. Mature Wistar male rats weighing approximately 300 g were made either uni- or bilaterally cryptorchid. Testicular inhibin and testosterone content and plasma levels of LH and FSH were examined 2 weeks later. A similar remarkable decrease in testicular inhibin content was found in uni- and bilaterally cryptorchid testes. On the other hand, the testicular testosterone content was significantly decreased only in unilaterally cryptorchid testis with an inverse increase in the contralateral testis. Plasma testosterone levels were normal and plasma LH and FSH increased significantly in both of the cryptorchid groups. These results showed that cryptorchidism impairs both Sertoli and Leydig cell functions. While testosterone production was compensated by increased LH for 2 weeks, neither inhibin secretion nor storage changed in cryptorchid or contralateral testes during the same period.

[A clinical evaluation of CA 125 levels in serum, ascites and pleural effusion].

CA 125 in serum, ascites and pleural effusion has been measured in various diseases by an immunoradiometric assay using ELISA CA 125 Kit for an evaluation of its clinical significance as a possible tumor marker. Serum CA 125 was found to be elevated in 90% of the cases involving ovarian cancer and especially in cases of serous cystadenocarcinoma in the early stages, though these serum levels decreased after effective treatment. The CA 125 also was elevated in 63% of the cases involving lung cancer, mostly in the advanced cases regardless of the histological type. CA 125 in ascites proved to be only elevated in malignant diseases, but it was equally high in pleural effusions both in benign and malignant diseases.

Short term treatments with prolactin, HMG or testosterone fail to affect testicular inhibin content in rats.

To investigate the effect of prolactin (PRL) on testicular function, especially on spermatogenesis, testicular inhibin content in male rats treated with PRL was compared with those treated with HMG and testosterone. Mature Wistar male rats were given 10 or 50 IU of ovine PRL, 10 IU of HMG and 5 mg of testosterone, i. m. for 5 consecutive days and testes were removed for assessing inhibin content. Inhibin content was measured by a FSH suppressing activity in cultured rat anterior pituitary cells using aquous extract of testes. Five days' treatment with PRL, HMG, or testosterone did not influence testicular inhibin content in male rats. The possibility that these treatments had transiently affected testicular inhibin content, or that inhibin content did not reflect inhibin production was not ruled out.

The significance of alpha-subunit as a tumor marker for gonadotropin-producing pituitary adenomas.

We studied gonadotropin hormone alpha-subunit and gonadotropin secretion in four patients with gonadotropin-producing pituitary adenomas. All four patients had elevated plasma alpha-subunit levels, ranging from 2.8-8.5 ng/ml (normal, less than 0.5 ng/ml). alpha-Subunit responses to LHRH were less than those in seven patients with primary gonadal failure. The relative proportions of the gonadotropin and alpha-subunit peaks in one patient were the same before and after LHRH administration, based on gel filtration studies of plasma. The alpha-subunit levels decreased little during testosterone treatment in the two adenoma patients so treated. Immunohistochemical study of the adenomas from two patients demonstrated definite staining with alpha-subunit and gonadotropin antisera. Elevated plasma levels of alpha-subunit and its relative unresponsiveness to LHRH stimulation or testosterone suppression suggest that the alpha-subunit originated in tumor tissue and that its measurement is useful for the diagnosis of a gonadotropin-producing tumor in patients with elevated plasma levels of LH and/or FSH.

Prolactin interacts with gonadotropin through a suppression of c-AMP production probably as a LH-sensitive adenylate cyclase inhibitor.

The interaction between gonadotropin and prolactin (PRL) on ovarian steroidogenesis as well as c-AMP production was studied in rat ovaries. Ovaries obtained from adult female Wistar rats in a morning of proestrus were chopped into 30-40 pieces and subjected to short term incubation studies using various buffers. HCG-stimulated c-AMP, estradiol (E2), progesterone (P) secretions were suppressed in a dose-dependent manner by ovine (o) PRL in a plain Gey-Gey (G-G) buffer. Addition of 3-isobutyl-1-methyl-xanthine (IBMX) increased c-AMP accumulation as well as E2 and P secretions. Deletion of Ca++ from the IBMX buffer stimulated c-AMP production, but suppressed steroid secretion. The inhibitory effect of PRL on E2 and P was not demonstrated in IBMX buffer at any Ca++ concentration examined despite suppression of c-AMP production. In conclusion, it was demonstrated that PRL inhibited gonadotropin-stimulated production of E2 and P by inhibiting c-AMP production. IBMX stimulated accumulation of c-AMP, E2 and P and counteracted with the antigonadal effect of PRL. Ca++ inhibited c-AMP accumulation but stimulated E2 and P secretions. The data suggested that PRL exerts its antigonadal effect through an inhibition of adenylate cyclase action in a manner similar to that of Ca++.