Characterization of the cell-specific expression of parathyroid hormone-related protein in normal and neoplastic prostate tissue.

OBJECTIVES: Parathyroid hormone-related protein (PTHrP) is a primary factor in the pathogenesis of malignancy-associated hypercalcemia. By alternative splicing, the human PTHrP gene can generate three different species of mRNA that encode three initial translational isoforms of 139, 173, and 141 amino acids. We recently reported that PTHrP was present in normal prostatic neuroendocrine cells and was overexpressed in prostate cancer tissue as demonstrated by immunostaining. This study was undertaken to further clarify the complex expression of PTHrP gene in normal prostate tissue and prostate cancer. METHODS: PTHrP mRNA in samples prepared from normal prostate tissue, prostate cancer, and three prostate cancer cell lines, PC3, LNCaP, and DU145 was assessed using Northern hybridization. Expressed PTHrP isoforms were deduced from differential reverse transcription-polymerase chain reaction (RT-PCR) assays with exon-specific primers. Further localization of different species of PTHrP mRNA was performed using nonradioactive in situ hybridization with exon-specific probes on consecutive sections of normal and neoplastic prostate tissue. RESULTS: Northern hybridization showed that the PTHrP expression level was higher in prostate cancer than in normal prostate tissue. All three PTHrP isoforms could be detected in normal prostate tissues and prostate cancer with differential RT-PCR. Further analysis using in situ hybridization with exon-specific probes revealed that all three PTHrP isoforms were present in prostatic neuroendocrine cells and only PTHrP-1-139 isoform could be clearly detected in prostate cancer tissue. Two androgen-insensitive cell lines, PC3 and DU145, derived from a bone metastasis and a brain metastasis, respectively, expressed all three mRNA species encoding for the three isoforms, but DU145 cells expressed less than PC3 cells. Androgen-sensitive LNCaP cells exhibited a low level of expression of mRNA species encoding for PTHrP-1-139 and PTHrP-1-173, and no expression of PTHrP1-141 isoform. CONCLUSIONS: All three initial translational isoforms of PTHrP are produced by prostatic neuroendocrine cells. The mature products of PTHrP might exert their effects on other prostatic epithelial cells in a paracrine fashion and also participate in the homeostatic regulation of the ejaculate. In prostate cancer, differential expression of these three isoforms is evident and PTHrP-1-139 isoform is more abundant than the other two forms. These findings are valuable for designing future research studies to further elucidate the biological functions of PTHrP in normal prostatic glands and prostate cancer.

Cloning and characterization of the alpha 1-antichymotrypsin produced by human prostate tissue.

BACKGROUND: alpha 1-antichymotrypsin (ACT) forms stable complexes with prostate-specific antigen (PSA), a serine protease, and this complex is the major form of PSA in the blood circulation. alpha 1-antichymotrypsin occurs in the blood in approximately 10(5) molar excess to PSA, mainly due to hepatic production, but local prostatic production of ACT has been demonstrated by immunohistochemistry and in situ hybridization. The present study was performed to further characterize this prostate-produced ACT. METHODS: The nucleotide structure of the prostatic transcript was determined from ACT coding clones isolated from prostatic cDNA. The occurrence of a prostatic ACT transcript was analyzed by Northern blot. RT-PCR was used to detect ACT transcripts in cultured prostatic cancer cells. RESULTS: Screening of two prostatic cDNA libraries showed the frequency of ACT transcripts to be about 1 clone in 40,000. The cDNA sequence of prostatic ACT is identical to that of the previously published hepatic ACT. Northern blot analysis of mRNA extracted from prostatic tissue showed a single transcript of approximately 1.5 kb. RT-PCR analysis demonstrated an ACT transcript in cultured prostatic cancer cells. CONCLUSIONS: In this study we provide further evidence for a local, prostatic production of ACT. The cDNA sequence data suggest that the peptide backbone of prostatic ACT is identical to the protein derived from the liver, and thus may be functional as a protease inhibitor.

Suppression of the growth and invasiveness of human prostate cancer cells in vitro by neuropeptide antagonist substance P analogues.

Neuropeptides may be essential to the growth and progress of prostate cancer, particularly during androgen-independent cell development. To determine whether neuropeptide antagonists substance P analogues can be used as therapeutic agents in the treatment of prostate cancer, their effects on the growth and invasiveness of established human prostate cancer cell lines were examined. The effects of [d-Arg(1), d-phe(5), d-Trp(7,9), Leu(11)]substance P and [Arg(6), d-Trp(7,9), MePhe(8)]substance P(6-11) on two androgen-independent cell lines (PC-3 and DU-145) and an androgen-dependent cell line (LNCaP) were studied. The cytotoxicity of substance P analogues was assessed based on their effects on DNA synthesis and cell proliferation by [(3)H]thymidine incorporation and cell growth assays, respectively. Inhibition of the invasiveness of prostate cancer cells was estimated based on the extent of cell penetration of reconstituted basement membrane. Substance P analogues inhibited DNA synthesis and cell proliferation of prostate cancer cells dose dependently but complete recovery was achieved by the addition of bombesin or substance P. [d-Arg(1), d-phe, d-Trp(7,9), Leu(11)]substance P inhibited the invasiveness of PC-3 cells. Neuropeptide antagonists substance P analogues have been found useful as therapeutic agents for prostate cancer. Their action occurs primarily through the inhibition of Ca(2+) mobilizing neuropeptides such as bombesin and substance P.

Alteration of the hormonal bioactivity of parathyroid hormone-related protein (PTHrP) as a result of limited proteolysis by prostate-specific antigen.

OBJECTIVES: To discover whether the proteolytic activity of prostate-specific antigen (PSA) affects the structure and function of parathyroid hormone-related protein (PTHrP), as both are abundant components of human seminal plasma. METHODS: The ability of PTHrP to act as a substrate was studied by incubating a synthetic polypeptide, consisting of 34 amino acid residues of the amino-terminal domain of PTHrP, with purified PSA. The incubate was then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, high-pressure liquid chromatography separation, amino-terminal peptide sequencing, and mass spectrometry. The physiologic effect of the proteolytic activity of PSA on PTHrP was studied by measuring any alteration in PTHrP (1-34)-induced elevation of cyclic adenosine monophosphate (cAMP) production by UMR-106 rat osteosarcoma cells in culture. All cell culture experiments were performed with PSA and PTHrP (1-34) at physiologic concentrations. RESULTS. Our data show that PSA proteolytically cleaves PTHrP (1-34) after either residue 22 or 23, generating three peptide fragments. Both cleavages occur carboxy terminally of a phenylalanine residue. The cAMP production in rat osteosarcoma cells, induced by the amino-terminal portion of PTHrP (1-34), as a result of its structural similarity with parathyroid hormone (PTH), was abated by PSA in a dose- and time-dependent fashion. In contrast, heat-inactivated PSA had no effect on cAMP production. CONCLUSIONS: Our study demonstrates that PTHrP is a substrate for PSA. The cleavage of the amino-terminal portion of PTHrP completely disrupts its ability to interact with the PTH/PTHrP receptor and thus inhibits its PTH-like activity. The proteolytic processing of PTHrP by PSA may play an important role in the post-translational/post-secretional regulation of prostatic PTHrP activities, which are believed to include regulation of prostate growth and differentiation.

Calcitonin receptor mRNA expression in the human prostate.

OBJECTIVES: A subpopulation of prostate neuroendocrine (NE) cells contain calcitonin (CT). It has been postulated that CT-producing cells in the prostate account for the high CT level in the semen, and may be involved in the regulation of other epithelial cells via a paracrine mechanism. The presence of CT binding sites in the plasma membrane fraction of prostate tissue has been demonstrated by radioligand binding assay. In the present study, we investigated the CT receptor gene expression in the human prostate, a key component of the autocrine/paracrine loop in the CT functional pathway. METHODS: Reverse transcription polymerase chain reaction (RT-PCR) was carried out to evaluate the CT receptor mRNA expression in normal prostate tissue. Subsequent DNA sequencing was used to verify RT-PCR amplified products and to determine the isoform of the receptor. To define the location of the CT receptor expression, nonradioactive in situ hybridization was performed with a digoxigenin-labeled probe complementary to the coding region of the CT receptor mRNA. A polyclonal antibody against CT was used to reveal the CT-secreting cells in the prostate. RESULTS: CT receptor MRNA expression was detected in the prostate tissue. Further analysis of the DNA sequence showed that CT receptor expressed in the prostate was the isoform without a 16-amino acid insert in the first intracellular domain. In situ hybridization revealed that CT receptor was present in the prostate NE cells. Immunocytochemical staining of mirror image sections showed that some CT-secreting cells also expressed CT receptor. CONCLUSIONS: CT receptor expression in the prostate, a key component in the CT functional pathway, is located in subsets of dispersed NE cells (CT secreting and CT nonsecreting), which indicates that prostate CT may play an important role in the autocrine/paracrine regulation of the prostate NE system.

The characterization of chromosomal rearrangements by a combined micro-FISH approach in a patient with myelodysplastic syndrome.

We report the application of a combined strategy: chromosome microdissection, degenerate oligonucleotide primed-PCR, and reverse chromosome painting (micro-FISH), as well as forward chromosome painting, for the characterization of chromosomal rearrangements in a MDS patient with the karyotype 46,XX, -11, +r analyzed by G-banding. The karyotype was refined as 46,XX,der(2)t(2;11) (q35;?p13),der(11)dic r(11)(:p13-->q13::p13-->q13:). Our study demonstrated that the chromosome composition of a neoplasia can be identified more systematically and accurately using these combined molecular cytogenetic approaches. The DOP-PCR methodology modified is suitable for the practical application of micro-FISH on specimens prepared for routine banding analysis.

Immunohistochemical localization of parathyroid hormone-related protein in prostatic intraepithelial neoplasia.

Parathyroid hormone-related protein (PTHrP) is a regulatory protein hormone that has been associated with normal fetal growth and differentiation as well as fetal calcium regulation. Parathyroid hormone-related protein has been implicated in a variety of carcinomas as a major factor in the development of humoral hypercalcemia of malignancy and may also play a role as an autocrine growth factor. In a previous immunohistochemical study we found that all prostatic adenocarcinomas (CAP) express PTHrP. In the current study, we evaluated PTHrP in prostate intraepithelial neoplasia (PIN) in radical prostatectomy specimens. A validated mouse monoclonal antibody, 9H7, raised against fragment 109-141 of the carboxy-terminus of PTHrP was used for immunostaining. The results generally showed negative to weak staining of normal and hyperplastic tissue and strong staining in PIN. The staining intensity was further evaluated by computer based image analysis. The relative optical density in PIN (9.24 +/- 9.05) was significantly (P = .008) higher than that in normal gland (.00 +/- 3.6). These findings suggest that PTHrP may be involved in the pathogenesis of prostatic dysplasia, and its immunohistochemical evaluation may have diagnostic use in the evaluation of PIN.

Overexpression of human epidermal growth factor receptor and c-erbB-2 by neuroendocrine cells in normal prostatic tissue.

OBJECTIVE: It has been suggested that prostatic neuroendocrine (NE) cells play an important role in the growth and differentiation of the prostate by secreting various neuropeptides and serotonin. However, the mechanism by which NE cells themselves are regulated is virtually unknown. In the present study we evaluated the expression of the human epidermal growth factor receptor (EGFR) family (HER) in prostatic NE cells. METHODS: Formalin-fixed, paraffin-embedded tissue sections from twenty radical prostatectomy specimens were immunostained with validated rabbit polyclonal antibodies raised against human EGFR and c-erbB-2, using the streptavidin-peroxidase enzyme conjugate method. RESULTS: A strong immunoreactivity was observed with both antibodies in the cytosol of a few epithelial cells. These cells frequently had a dendritic appearance and were located in the acini and ducts. The EGFR-positive cells were predominant in most cases. Double immunostaining revealed the colocalization of both antigens with chromogranin A, a polypeptide that is expressed by most NE cells. Moreover, EGFR and c-erbB-2 appeared to be colocalized as well as independently expressed by different subpopulations of NE cells. CONCLUSIONS: The results suggest that prostatic NE cells might be regulated by the HER protein family, probably, in a ligand-specific fashion. This is the first report identifying a potential pathway regulating prostatic NE cells.

Relationship between serum testosterone and sex hormone binding globulin in menstruating and postmenopausal women.

The relationship between total testosterone (T), sex hormone binding globulin (SHBG) and calculated non-SHBG-bound testosterone (NST) was studied in randomly collected blood samples from healthy menstruating (n = 61) and postmenopausal (n = 65) women. In 12 of the menstruating women, blood samples were also collected more frequently during the menstrual cycle. Total T and SHBG were positively correlated in menstruating women in random samples as well as during different phases of the menstrual cycle, but not in postmenopausal women. Upper and lower limits of NST were independent of SHBG in menstruating but not in postmenopausal women. The data are at variance with the common concept about SHBG regulation and suggest a kind of compensatory mechanism in order to maintain a constant androgen homeostasis in menstruating but not in postmenopausal women. Consequently, supranormal total T or subnormal SHBG values do not necessarily indicate hyperandrogenicity in normally menstruating women.

The genes for SHBG/ABP and the SHBG-like region of vitamin K-dependent protein S have evolved from a common ancestral gene.

Sex hormone binding globulin (SHBG) is the most important sex steroid transport protein in human plasma. It is the product of the same single gene as the androgen binding protein (ABP) of testis. Protein S is another protein, which is an important cofactor in the anticoagulation system and, as far as is known today, functionally unrelated to SHBG/ABP. Protein S also has a role in the complement system. A comparison of the human genes for SHBG/ABP and protein S reveals a sequence similarity, which is of a low grade only, between the SHBG/ABP protein and a similar sized COOH-terminal domain of protein S. However, the intron-exon organization exhibits a striking similarity in the two genes, illustrating evolutionary events leading to the appearance of two functionally different proteins from common ancestral genetic elements.

2-Iodoestradiol binds with high affinity to human sex hormone binding globulin (SHBG).

Human sex hormone binding globulin (SHBG) binds a set of steroids that differ slightly from each other in structure. Dihydrotestosterone and testosterone are bound with high affinity by SHBG whereas estradiol is bound with a lower affinity. In this work we have studied the binding to human SHBG of the derivatives obtained by substituting iodine in the aromatic A-ring of estradiol. Three A-ring iodinated estradiol derivatives, 2-iodoestradiol, 4-iodoestradiol and 2,4-di-iodoestradiol, were obtained by treating 17 beta-estradiol with NaI and Chloramine T and separating the reaction products by HPLC. Their structures were confirmed by mass spectrometry and 1H-NMR. The corresponding radioactive compounds were obtained with use of Na[125I] in the same synthesizing procedure. Incubation of whole serum, serum albumin and purified SHBG with each of the three [125I]iodoestradiols followed by agarose gel electrophoresis showed only 2-iodoestradiol to have a strong binding to SHBG. This steroid was also bound to albumin, but with a lower affinity. Besides SHBG and albumin, there were no other binders of 2-iodoestradiol in human serum. The affinity constant for the binding of 2-iodoestradiol to purified human SHBG at 37 degrees C and physiological pH was determined by a dextran-coated charcoal method to be 2.4 x 10(9) M-1 (i.e. exceeding that of dihydrotestosterone). It was found that 0.9 mol of 2-iodoestradiol was bound per mol of SHBG dimer (93 kDa) at saturation, and that 2-iodoestradiol competed with dihydrotestosterone for the same binding site of SHBG. It was concluded that 2-iodoestradiol has a remarkably high affinity for human SHBG, and that its gamma-emitting 125I-analog is useful for binding studies of human SHBG.

Characterization of the human sex hormone binding globulin (SHBG) gene and demonstration of two transcripts in both liver and testis.

A genomic cosmid clone for human sex hormone binding globulin (SHBG), a liver-secreted plasma glycoprotein that binds sex steroids, was isolated with a previously characterized liver cDNA as probe. Southern blot analysis of genomic DNA indicated that only one SHBG gene is present in the human haploid genome. A 3.8 Kb Xba I-fragment of the clone containing the entire coding region of SHBG was sequenced. The SHBG gene has 8 exons. The 5'-end preceding the translation start site had no TATA box or CAAT box promoter elements. Screening of a human testis cDNA library resulted in the isolation of two distinct cDNA forms. One cDNA was identical with the previously characterized liver SHBG cDNA, thus suggesting that human SHBG and the androgen binding protein (ABP) produced by Sertoli cells are coded for by the same gene. The second cDNA differed from the first by having exon I exchanged with a completely different sequence and exon VII deleted. An exon coding for the 5'-end of this cDNA was found in the cosmid clone 1.5 kb upstream of the first SHBG exon. Primer extension experiments showed the alternatively spliced transcript corresponding to the second cDNA to be present in both liver and testis. From the primary structure of this putative SHBG-gene-related protein, it may be deduced that it is a protein very different from SHBG and probably without steroid binding activity.

Decreasing serum levels of sex hormone-binding globulin around the menopause and temporary relation to changing levels of ovarian steroids, as demonstrated in a longitudinal study.

Blood samples collected longitudinally in 17 women over a period of 3 years, starting 11/2 years before the menopause, were assessed for sex hormone-binding globulin (SHBG), 17 beta-estradiol (E2), progesterone, and total testosterone. A slight (7.2%) decrease in mean SHBG from 4.25 +/- 1.67 (standard deviation) mg/l to 3.95 +/- 1.61 mg/l was observed within the 6-month period encompassing the menopause. More specifically, the decrease appeared to commence at the menopause and to become clearly significant (P = 0.01) some 2 to 6 months later. During the subsequent year, a further decrease to 3.64 +/- 1.42 mg/l was observed, amounting to a total decrease in mean SHBG by 14.4% (P less than 0.001). Of the hormones, only E2 exhibited a marked decrease (P less than 0.01) within this same 6-month period. The changes in SHBG during the 6-month transition period from premenopause to postmenopause correlated significantly (P = 0.013) only with those of E2. It is concluded that decreasing E2 levels appear to play a significant role in the downward modulation of SHBG levels commencing at the menopause.

A cDNA coding for human sex hormone binding globulin. Homology to vitamin K-dependent protein S.

Affinity purified antibodies to human sex hormone binding globulin (SHBG) were used in screening a human liver cDNA library, constructed in the expression vector lambda gt11. One clone, identified as producing recombinant SHBG, carried a cDNA insert of 1.1 kb. The nucleotide sequence of the insert had an open reading frame coding for 356 amino acid residues. The coding sequence was followed by a short 3'-region of 19 non-translated nucleotides and a poly(A) tail. Confirmation that the cDNA clone represented human SHBG was obtained by the finding of a complete agreement in amino acid sequence with several peptide fragments generated from purified SHBG by proteolytic cleavage. The primary structure of SHBG shows a considerable homology to that of protein S, a vitamin K-dependent protein with functions in the coagulation system.

Subunits of human sex hormone binding globulin. Interindividual variation in size.

Sex hormone binding globulin (SHBG), a dimeric plasma glycoprotein with a molecular mass of about 90 kDa, was purified from healthy individuals by a rapid two-step procedure using immunoaffinity chromatography on a monoclonal antibody column followed by fast protein liquid chromatography. The individual SHBGs so isolated were pure by several criteria, and the overall yield was usually about 20% according to radioimmunoassay. The isolated SHBGs were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate which showed the SHBG isolated from most subjects to be composed of subunits of two different sizes (52 and 49 kDa) present in the approximate ratio of 10:1 (double-banded SHBG). The SHBG of the remaining subjects contained a third subunit with an estimated molecular mass of about 56 kDa (triple-banded SHBG). In this kind of SHBG, the two heavy subunits were present in approximately equal amounts, suggesting that individuals with triple-banded SHBG are heterozygotes for a genetic variant of the protein. The various subunits of SHBG were separated and individually subjected to amino-terminal amino acid sequence analysis. They all had a heterogeneous amino terminus, but since the sequences obtained seemed to be identical, the structural differences between the subunits would appear to reside in other parts of the molecules. On isoelectric focusing, both kinds of SHBG were resolved into about 10 components, all with steroid-binding activity. Differences were noted between double-banded and triple-banded SHBG, the latter having a greater abundance of acidic species. Screening of 121 healthy individuals by a procedure involving small-scale isolation of SHBG on an antibody column followed by Western blotting revealed that 20% of the subjects had triple-banded SHBG. This new variant of SHBG was found in persons of both sexes and in children as well as in adults.

Free testosterone and testosterone/SHBG index in hirsute women: a comparison of diagnostic accuracy.

Serum levels of sex hormone binding globulin (SHBG), total testosterone (T), free testosterone (fT), 4-androstene-3,17-dione (A-4) and dehydroepiandrosterone sulfate (DHAS) were determined in 30 patients with the polycystic ovary syndrome. Subnormal levels of SHBG were found in 60% of the patients. Elevated levels of T were found in 53%, of fT in 53%, of the T/SHBG ratio in 90%, of A-4 in 75% and of DHAS in 36% of the patients. The better diagnostic accuracy of the T/SHBG ratio compared to fT may be explained by the regulatory effect of SHBG binding upon the albumin-bound fraction of T, which may be another biologically active T fraction. It is concluded that the assay of fT does not offer any further diagnostic advance compared to conventional determinations of T. Assays of fT do not provide any information about the origin of the elevated levels of biologically active androgen.

Immunoradiometric assay of sex-hormone binding globulin with use of two different monoclonal antibodies.

Two monoclonal antibodies (MK1 and MK2) reacting with human sex-hormone binding globulin (SHBG) were obtained from mice hybridomas. The dissociation constants for the binding of SHBG to MK1 and MK2 were 7.5 and 75 pmol/L, respectively. MK1 was coupled to polyacrylamide beads with a yield of 37%, resulting in 15 mg of antibody per gram of beads. The maximal binding of SHBG by the MK1-beads was 16% of the theoretical capacity. The amount of 125l-labeled MK2 bound to MK1-beads was related to the amount of SHBG present. The system has been used for the immunoradiometric assay (IRMA) of SHBG in serum, and has been standardized with purified SHBG. Assay sensitivity is 3 micrograms/L; intra- and inter-assay (total variation) CVs were 5% and 10%, respectively. Values obtained with the assay for 100 patients' sera agreed well with those obtained with a conventional radioimmunoassay, and SHBG in a patient's serum subjected to gel chromatography eluted as a symmetrical peak with the expected retention when the effluent was analyzed with the present assay. Analytical recovery of SHBG added to serum from a man, a woman, and a pregnant woman ranged between 93% and 107%. The mean (and SD) concentrations of SHBG in sera from healthy women and men were 3.7 +/- 1.1 and 1.8 +/- 0.9 mg/L, respectively.

Radioimmunoassay of human sex hormone binding globulin: improved radioiodination procedure.

Sex hormone binding globulin (SHBG), purified by affinity chromatography from retroplacental blood plasma, was reacted with 3-(p-hydroxyphenyl) propionic acid N-hydroxysuccinimidyl ester (PHPPS, Bolton-Hunter reagent). The derivative of SHBG obtained (parahydroxyphenylpropionyl-SHBG; PHPP-SHBG) was stable and could, in contrast to underived SHBG, be efficiently 125I-iodinated with a lactoperoxidase technique. The PHPP-SHBG labelled with 125I had good antiserum binding and stability properties and was used for radioimmunoassay (RIA) of SHBG in serum. The RIA requires a total incubation time of 3 h. It has been standardized with purified SHBG and has a sensitivity of 5 micrograms/l, giving a lowest detectable concentration in the routine procedure (samples diluted 1:40) of about 0.2 mg/l. Variation within and between assay was 4.1% and 7.2%, respectively, for samples with values within the normal range. Values obtained by this RIA procedure correlate well with those obtained by a dihydrotestosterone binding method and by an electroimmunoassay technique. The mean serum concentration of SHBG in healthy, regularly menstruating women (n = 42) was 3.7 +/- 1.0 (SD, standard deviation) mg/l and in healthy men (n = 100) 2.0 +/- 0.9 mg/l.

Electroimmunoassay of sex hormone binding globulin. Enhanced sensitivity by autoradiography using A-ring 125I-17-beta-oestradiol.

Four different 125I-iodinated steroids were tested for their binding to human sex hormone binding globulin (SHBG) using an electrophoretic technique. 17-beta-oestradiol iodinated in its A-ring was found to bind with high affinity to SHBG. This radioactive steroid was used to increase the sensitivity of the electroimmunoassay of SHBG by adding the steroid to the samples before electroimmunoassay. The radioactive steroid incorporated into the immunoprecipitates could be observed by autoradiography. The sensitivity of the assay, which employed a rabbit antiserum against purified human SHBG and was standardized with pure SHBG, was about 0.2 mg/l. The precision of the method, calculated as the coefficient of variation within and between assays, was 2.4% and 2.6%, respectively, for values within the normal range. The mean SHBG concentration in healthy regularly menstruating women (n = 50) was 3.50 +/- 0.74 (SD) mg/l when measured in plasma, and 3.78 +/- 0.80 mg/l when measured in serum. The corresponding mean concentrations in healthy men (n = 28) were 2.26 +/- 0.45 mg/l in plasma and 2.44 +/- 0.49 mg/l in serum. The modified electroimmunoassay described in this paper is a simple modification, which increases the sensitivity sufficiently to permit reliable quantification of SHBG over the entire range of concentration which could be relevant in clinical practice.

Peripheral levels of dehydroepiandrosterone sulfate, dehydroepiandrosterone, androstenedione, and testosterone following different doses of danazol.

Peripheral serum levels of dehydroepiandrosterone sulfate (DHAS), dehydroepiandrosterone (DHA), androstenedione (A4) and testosterone (T) were measured in two groups of premenopausal women with fibrocystic breast disease, given 200 and 400 mg of danazol a day respectively for 6 months. During treatment, DHAS levels increased and DHA, DHA/DHAS ratio, A4, and T decreased. A tendency towards dose dependency was observed. The changes in DHAS, DHA and DHA/DHAS ratio were interpreted as resulting from inhibition of liver sulfatase activity by danazol. The decreased A4 and T levels probably reflect a suppression of ovarian steroidogenesis due to enzyme inhibition by danazol, and for the latter steroid also and increased metabolism due to interaction of the drug with sex hormone-binding globulin.