Marco A. Rivarola

Percentage binding of testosterone, androstenedione and dehydroisoandrosterone in human plasma

Abstract Equilibrium dialysis, ultrafiltration by centrifugation and by vacuum gave similar results for the percentage binding of testosterone (T), androstenedione (Δ) and dehydroisoandrosterone (D) when using plasma samples diluted 1:5 at 37°C. The percentage binding of T is significantly lower in male plasma than in female plasma. Pregnancy and estrogen treatment in males increase T binding, whereas ProveraRtreatment in males decreases it. In addition to T itself, 5α-androstan-3α, 17β-diol, estradiol-17β and to a smaller extent estrone can compete for T binding. The percentage binding of Δ was similar in male and female plasma. Pregnancy and estrogen or ProveraR treatment in males had no effect. In range of concentration used, there was no binding competition by the steroid tested. Although the percentage binding of D was not influenced by sex, it was increased in pregnant women and estrogen-treated men. There was also a slight competition by T.

Expression of Aromatase, Estrogen Receptor α and β, Androgen Receptor, and Cytochrome P-450scc in the Human Early Prepubertal Testis

The expression of aromatase, estrogen receptor α (ERα) and β (ERβ), androgen receptor (AR), and cytochrome P-450 side chain cleavage enzyme (cP450scc) was studied in prepubertal testis. Samples were divided in three age groups (GRs): GR1, newborns (1- to 21-d-old neonates, n = 5); GR2, postnatal activation stage (1- to 7-mo-old infants, n = 6); GR3, childhood (12- to 60-mo-old boys, n = 4). Absent or very poor detection of ERα by immunohistochemistry in all cells and by mRNA expression was observed. Leydig cells (LCs) of GR1 and GR2 showed strong immunostaining of aromatase and cP450scc but weak staining of ERβ and AR. Interstitial cells (ICs) and Sertoli cells (SCs) expressed ERβ, particularly in GR1 and GR2. Strong expression of AR was found in peritubular cells (PCs). For all markers, expression in GR3 was the weakest. In germ cells (GCs), i.e. gonocytes and spermatogonia, aromatase and ERβ were immunoexpressed strongly whereas no expression of ERα, AR, or cP450scc was detected. It is proposed that in newborn and infantile testis, testosterone acting on PCs might modulate infant LC differentiation, whereas the absence of AR in SCs prevents development of spermatogenesis. The role of estrogen is less clear, but it could modulate the preservation of an adequate pool of precursor LCs and GCs.

Unexpected Peripheral Markers of Thyroid Function in a Patient with a Novel Mutation of the MCT8 Thyroid Hormone Transporter Gene

The specific thyroid hormone transporter, MCT8, located on the X chromosome, has led to the identification a novel syndrome. The objective is to relate phenotype with several tissue-specific thyroid functions. A 1-year-old boy, who had severe psychological damage and low serum T4, had received l-T4 for 3 months. At admission, body length was normal but weight was low. Off therapy, serum TSH was mildly elevated, serum T4 and free T4 were low, and serum T3 and free T3 were high. Direct sequencing of the MCT8 gene revealed a single nucleotide change that resulted in a novel nonsense mutation at codon 261 (Q261X) in exon 3. Since serum T3 was high, peripheral markers of hyperthyroidism were looked for. Bone age was advanced, despite the presence of malnutrition and low T4. Serum SHBG, a marker of thyroid hormone action in liver, was markedly elevated. Markers of skeletal muscle catabolism, ammonemia and lactic acid, were found to be elevated. The phenotype of MCT 8 mutation might be explained by differences in the entry of thyroid hormones into different cells. In the presence of an inactive MCT8 transporter, the high blood T3 levels might not be enough to prevent brain damage early in life, while they seem to be able to induce a postnatal state of peripheral hyperthyroidism in other tissues, such as liver, bone and skeletal muscle.

Determination of testosterone and androst-4-ene-3,17-dione concentration in human plasma

Abstract A double isotope dilution method is presented for the determination of testosterone and andros T-4-ene-3, 17-dione concentration in human plasma consisting of the main following steps: 1/ ether extraction and Florisil column chromatography; 2/ paper chromatography (System 1) for separation of testosterone and androst-4-ene-3, 17-dione; 3/ NaBH 4 reduction of andros T-4-ene-3, 17-dione to testosterone and paper chromatography (System 11); 4/ acetylation of testosterone with C 14 -acetic anhydride; 5/ bidimensional thin layer chromatography; 6/ paper chromatography (System 111); 7/ NaBH 4 reduction of testosterone acetate to 3β-hydroxy-androst-4-ene-17β-acetate; 8/ paper chromatography (System 1). After correction for the blank, mean testosterone and androst-4-ene-3,17-dione plasma concentrations were found to be 55 4 and 99 mμg/100 ml for normal adult males and 41 and 180 mμg/100 ml for normal adult females.

Genetic and clinical spectrum of aromatase deficiency in infancy, childhood and adolescence.

Introduction: cP450aromatase deficiency provides clues for the understanding of the role of aromatase in prepubertal and pubertal human health and disease. Placental aromatization of androgens protects the female fetus against the virilizing action of fetal androgens. After birth, the dual effect of aromatase deficiency, excessive androgens, and insufficient estrogens is responsible for a variable clinical picture. Nineteen cases of aromatase gene (CYP19) deficiency have been reported. Phenotype: Phenotype is dependent on sex and age. In newborns, aromatase deficiency should be considered in the etiology of 46,XX DSD, after ruling out congenital adrenal hyperplasia. In prepubertal aromatase deficient girls, high levels of ovarian androgens and gonadotropins facilitate the formation of ovarian cysts. Bone mineralization can be affected and bone aging is delayed. In pubertal girls, there is poor sexual development and abnormal virilization. The phenotype may be variable according to enzyme activity level. Insulin sensitivity may be abnormal in both men and women. Finally, aromatase might also play a role in the regulation of testicular cell mass in the newborn testis. Conclusion: Adequate interpretation of clinical data should lead to the analysis of the CYP19 gene for diagnostic confirmation and implementation of appropriate management.

Mutations of the steroid 21-hydroxylase gene in an Argentinian population of 36 patients with classical congenital adrenal hyperplasia.

In several studies carried out in USA and Europe, gene deletions, large gene conversions and six point mutations accounted for over 90% of the mutated alleles reported in classical congenital hyperplasia (CAH). In order to know the relative frequencies of mutations in a Latin-American population, the CYP21 active gene was analyzed in 42 patients with CAH belonging to 36 families attending two Argentinian clinics. The salt wasting form was diagnosed in 24 index cases and the simple virilized form in 12. When available, parents were also studied. DNA was extracted from peripheral blood leukocytes and specific PCR amplification of four different fragments of the CYP21 gene was carried out, followed by electrophoresis of the amplified product. The four fragments include segments of the gene containing the six most frequently reported abnormalities in classical CAH: IN2, EX3, R356W, cluster EX6 and I172N. Point mutations were studied by allelic specific oligonucleotide hybridization; Q318X was studied by digestion of the PCR product with PsT1 restriction enzyme and electrophoresis on 6% non-denaturing polyacrylamide gels. Deletions and macroconversions as well as confirmation of homozygote point mutations were studied by Southern blotting. Percentage distribution of abnormalities was as follows: deletion/macroconversion 18, IN2 18, I172N 15.3, Q318X 13.8, R356W 5.5, EX3 2.7, cluster EX6 0, not characterized 26.7. The complete genotype could be determined in 20 families while in 12 additional ones, the mutation was detected in one allele. Deletion/macroconversion, IN2, EX3 and Q318X were detected more frequently in salt wasting patients while I172N and R356W were found in simple virilized patients. However, genotype was not always concordant with phenotype. It is concluded that there are differences in the frequency of several gene mutations and in that of deletion/macroconversion between this Latin-American population and several reported American and European populations. In particular the percentage of deletion/macroconversion, IN2, EX3 and cluster EX6 was lower while I172N was higher in our Latin-American population. Furthermore the frequency of mutations not characterized was larger. This information is useful to delineate appropriate strategies for prenatal diagnosis in this particular population.

Splanchnic Extraction and Interconversion of Testosterone and Androstenedione in Man

A constant infusion of (3)H-testosterone and (14)C-androstenedione was administered to four human subjects, two males and two females, until the concentrations of radioactive testosterone and androstenedione in systemic plasma became constant. At that time the concentrations of radioactive testosterone and androstenedione in hepatic vein plasma were determined. Splanchnic extraction of testosterone and androstenedione and the contribution of the splanchnic system to the blood interconversion of testosterone and androstenedione were calculated. Androstenedione is extracted by the splanchnic system more efficiently than testosterone since 82.3% of androstenedione and 44% of testosterone were removed from the plasma after one passage. The fraction of testosterone entering the splanchnic system that is transferred to blood androstenedione was 0.011 and the maximum possible transfer due to recirculation was 0.026. This was 28% of the total blood transfer from testosterone to androstenedione. The fraction of androstenedione entering the splanchnic system that is transferred to blood testosterone after one passage was 0.005, whereas the maximum possible transfer in this system was 0.006. This represented only 16% of the total transfer from androstenedione to testosterone. Therefore, a large fraction of the interconversion of testosterone and androstenedione in vivo occurs outside the splanchnic system.

Adrenarche: postnatal adrenal zonation and hormonal and metabolic regulation.

Adrenarche is the direct consequence of the organogenesis of the zona reticularis (ZR). Proliferation of cortical cells could take place in the outermost layers of the adrenal cortex. Cells could then migrate to differentiate the zona glomerulosa (ZG) and zona fasciculata (ZF) during fetal life, and the ZR during postnatal life. After adrenarche, there are detectable increases in circulating DHEA and DHEA-S. Adrenarche could result from an increase in 17,20-lyase activity of P450c17 secondary to high levels of cytochrome b5 expression, and from a decrease in 3βHSD2 expression along with an increase in the expression of SULT2A1 in the ZR. The GH-IGF system and insulin, among other factors, might also modulate adrenal androgen production. Furthermore, high concentrations of estradiol enhance basal and ACTH-stimulated DHEA-S production, while aromatase expression was observed in the human adrenal medulla but not in the ZR, suggesting that estrogens produced in the adrenal medulla might be involved in the regulation of androgen production in the ZR. Premature adrenarche might be associated with ovarian hyperandrogenism and polycystic ovarian syndrome in females, as well as with insulin resistance in both sexes. However, many questions remain, transforming adrenal androgens into markers of diseases important for human health.

Evaluation of gonadal function following long-term treatment for acute lymphoblastic leukemia in girls

Twenty-four girls were studied following long-term treatment (mean: 50 months) for acute lymphoblastic leukemia; 14 were prepubertal and 10 pubertal. Follow-up during endocrine studies ranged from 2 months to 6.7 years (mean: 2.3 years). Five of 14 prepubertal patients started clinical pubertal development at a normal age and were reevaluated during puberty, increasing the pubertal group to 15 patients. Thirteen of 15 pubertal patients had received cranial radiotherapy. Ten of 15 pubertal patients started menses during the endocrine study. Although age of menarche was normal, in nine patients it was below the normal mean. Except for the remaining patient, all had received cranial cobalt therapy. In 6 of 19 patients bone age was significantly accelerated. Serum gonadotrophin response to LH-RH was normal in 13 prepubertal patients and in 10 pubertal patients. In 3 of 10 pubertal patients follicle-stimulating hormone (FSH) values were temporarily elevated. Only one pubertal patient had oligoamenorrhea. Five patients were studied by measuring serum progesterone on days 19-22 of the cycle to determine corpus luteum function. Three of them showed progesterone levels compatible with adequate corpus luteum function (6, 19, and 12 ng/ml, respectively) and two presented low progesterone levels (2 ng/ml), probably because of their short gynecological age (0.24 and 0.3 years, respectively). This study suggests that neither the disease nor the long-term antileukemia therapy seems to injure gonadal function in girls. A tendency to early sexual development was observed, which may be related to cranial cobalt therapy.

Three New SF-1 (NR5A1) Gene Mutations in Two Unrelated Families with Multiple Affected Members: Within-Family Variability in 46,XY Subjects and Low Ovarian Reserve in Fertile 46,XX Subjects

Background: Three novel heterozygous SF-1 gene mutations affecting multiple members of two unrelated families with a history of 46,XY disorders of sex development (DSD) and 46,XX ovarian insufficiency are described. Methods: Clinical and mutational analysis of the SF-1 gene in 9 subjects of two families. Results: Family 1 had 2 affected 46,XY DSD subjects. One, born with severe perineal hypospadias, was raised as a male, and presented normal adolescence. The other, born with ambiguous genitalia, uterus, and mild testicular dysgenesis, was raised as a female. A W279X heterozygous mutation and an intronic deletion (g3314-3317delTCTC (IVS 4 + 8) was found in the SF-1 gene. In family 2, 4/6 affected siblings had 46,XY DSD or hypospadias. An affected 46,XX sister had normal sexual development but increased FSH levels. The 37-year-old affected mother had entered menopause. An Y183X heterozygous mutation was detected. Conclusion: An extreme within-family phenotypic variability, ranging from severe prenatal undervirilization to normal pubertal development, was observed in 46,XY-affected siblings, indicating that other unknown factors might be involved in the phenotype. Low ovarian reserve and preserved fertility in 46,XX subjects can be observed in heterozygous SF-1 gene mutations.