21-hydroxylase deficiency is the commonest enzyme defect in congenital adrenal hyperplasia resulting in low circulating cortisol and, in severe cases, low aldosterone which may lead to cardiac arrest in the neonate. As the low levels of coitisol· lead to raised levels of adrenocorticotrophic hormone large concentrations of androgens are formed in utero which may lead to cliteromegaly and consequent confusion of the sexes at birth. High concentrations of the androgens give rise to rapid growth in early childhood and early fusion of the epiphyses results in short stature of the adult. 17-alphahydroxyprogesterone (170HP) is raised in the blood and saliva of children with 21-hydroxylase deficiency. 170HP is commonly measured by radioimmunoassay which limits the assay to being performed by laboratories equipped with beta or gamma counters. The aim of this project was to develop an enzymeimmunoassay which could be carried out using the minimum amount of equipment. Horseradish peroxidase was conjugated to 170HP-O-carboxymethyloxime using a mixed anhydride reaction. Separation of free and bound label was achieved using a second antibody linked to magnetisable particle solid phase. Measurement of the bound enzyme activity yielded colours which could be compared by eye, with known standards, to give a semi-quantitative assay. Alternatively the absorbance could be measured using a spectrophotometer for full quantitative results. The developed assays were evaluated, comparing the results with radioimmunoassay, and then used for measuring 170HP in blood and saliva in various clinical situations.

A Dissertation Submitted to the Faculty oC Arts University of the Witwatersrand, Johannesburg for the Degree of Master of Arts Johannesburg, 1974 / The relationship between amygdaloid damage and the intmperitoneal injection of betamethasone was studied with respect to the exploratory behaviour of male hooded rats. Amygdalaetomy produced attenuated longterm habituation of stimulus-specific and general exploratory behaviour. This habituation deficit was uninfluenced by betamethasone. Betamethasone also failed to influence long-tori. habituation in the intact animals. Although amygdalectomy did not alter short-term habituation, betamethasone accelerated the short-term habituation of general exploratory behaviour in both amygdalectomised and intact animals. On replication of the experiment betamethasone administration failed to influence exploration. It was tentatively concluded that amygdalectomy and betamethasone influence the habituation of exploratory activity under mutually exclusive circumstances. However, the failure to replicate the amygdalectomy- and betamethasone- induced changes in habituation could not be inteipreted within the context of the present experiment.

The adrenal gland is a highly dynamic organ with the ability to respond to changes in its environment with rapid changes in hormone production, and dramatic remodelling of its structure. Our understanding of the role of the melanocortin peptides in this process has been mostly restricted to the anterior pituitary hormone ACTH, via the melanocortin 2 receptor MC2R. The presence of additional melanocortin receptors and their antagonists has been demonstrated in rat and bovine adrenals, but the role of these in humans has not been well explored. An intensive clinical and biochemical work-up of a single patient with ACTH-independent Cushing's syndrome and a gastrointestinal stromal tumour (GIST) was performed. The data presented are consistent with the possibility that her disease was caused by release of some bioactive molecule released from the GIST. We propose that alpha-MSH is a possible candidate for this molecule, on the basis that the GIST immuno-stained for alpha- MSH but not ACTH, that alpha-MSH but not ACTH was present in supernatant from a primary culture, and that alpha-MSH has the potential to stimulate cortisol production from adrenal cells. The precise mechanism for alpha-MSH secretion from the tumour is not fully elucidated, and further work is required to corroborate this hypothesis. The patient had both pigmented skin and pigmented adrenal nodules, and we further demonstrated the presence of the alpha-MSH receptor MC1R was demonstrated with her excised adrenal gland. The pigment was identified as melanin, and we went on to show that same pattern in primary pigmented nodular adrenal disease, and to demonstrate that the zona reticularis in normal adrenal gland contains melanin, and has additional features in common with melanocytic tissues elsewhere in the body. The role of alpha-MSH in normal adrenal function, and the possibility that melanin is also playing an important role, perhaps for its antioxidant properties, is an exciting area for future study.

A Hiperplasia Adrenal Congênita (HAC) compreende um grupo de doenças autossômicas recessivas causadas por um defeito em uma das enzimas envolvidas na biossíntese do cortisol. Em 90% dos casos, a HAC é devido a deficiência na enzima 21-hidroxilase. Programas de triagem de recém-nascidos detectam a HAC através do teste bioquímico do hormônio 17-hidroxiprogesterona (17OHP), substrato da 21-HIDROXILASE, realizado em uma amostra de sangue seco em papel filtro. Entretanto, os valores do 17OHP podem variar devido a diferentes fatores, causando resultado falso positivos ou falso negativos. Com o intuito de elucidar esses casos, ensaios de biologia molecular têm sido empregados. Sendo assim, o presente trabalho teve como objetivo empregar metodologias moleculares para a análise de crianças com suspeita de HAC no Estado do Rio Grande do Sul. Três testes moleculares, minisequenciamento multiplex, sequenciamento e multiplex ligation-dependent probe amplification (MLPA) foram padronizados e/ou validados. Essas metodologias foram aplicadas para determinar o genótipo de 166 crianças com suspeita de HAC. A técnica de minisequenciamento multiplex foi utilizada para detectar doze mutações (p.Gln318Ter, p.Arg356Trp, p.Leu306PhefsX6, p.Val237Glu, IVS2-13A/C>G, p.Ile172Ans, p.Pro30Leu, p.Pro453Ser, p.Val281Leu, p.Gly110ValfsX21 e p.His62Leu) e o sequenciamento foi empregado quando necessária a análise de outras mutações pontuais. A identificação de grandes deleções, rearranjos e conversões foi realizada através do kit de MLPA. Como resultado, foi possível identificar 84 alelos patogênicos em 48 pacientes, sendo que as mutações mais frequentes encontradas foram p.Val281Leu (27,1%) e IVS2-13A/C>G (20,8%). Portanto, as metodologias empregadas neste trabalho foram eficientes para detectar as mutações mais frequentes descritas no Brasil e podem ser utilizadas em programas de triagem de recém-nascidos para ajudar a distinguir entre doentes e casos falsos positivos. / Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders caused by a defect in one of the enzymes involved in cortisol biosynthesis. In 90% of cases, CAH is due to steroid 21-hydroxylase deficiency. Newborn screening program detect CAH through 17-hydroxyprogesterone (17OHP) biochemistry test done in dried blood spotted on filter paper, which is the 21-hydroxylase substrate. However, the levels of 17OHP can vary due to different factors, causing false positives or false negatives results. To clarify and assist these cases, molecular biology assays have been used. Therefore, this work aimed to employ molecular assays to detect mutations in CYP21A2 gene of children suspected of CAH from Rio Grande do Sul state. Three molecular tests, multiplex minisequencing, direct sequencing and multiplex ligation-dependent probe amplification (MLPA) were standardized and/or validated. These assays were used to determine the genotype of 166 children suspected CAH. Multiplex minisequencing technique was performed to detect twelve point mutations (p.Gln318Ter, p.Arg356Trp, p.Leu306PhefsX6, p.Val237Glu, IVS2-13A/C>G, p.Ile172Ans, p.Pro30Leu, p.Pro453Ser, p.Val281Leu, p.Gly110ValfsX21 and p.His62Leu) and the direct sequencing was used when other point mutations needed to be analyzed. Large deletions, rearrangements and conversions was investigated by MLPA assay. As a result, it we were able to identify 84 pathogenic alleles in 48 patients, being p.Val281Leu (27.1%) and IVS2-13A/C>G (20.8%) the most frequent mutations. In conclusion, the methods used in this work were efficient to detect the most frequently mutations described in Brazil and can be used in newborn screening program to help distinguishing between affected and false positive cases.

Thesis (M. Sc.)--University of Hong Kong, 1972. / Typewritten.

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During the past decade or so, histochemists have paid increasing attention to the study of the adrenal gland. The majority of these studies have been on mammalian adrenals. The more noteworthy among them are those of Allen (1956, 1958), Arvy (1960, 196la), Deane et al., (1946, 1948), Erinko and his coworkers (1952, 1959, 1962, 1962a) Hillarp and Hokfelt (1954, 1955) and Wood (1963). These studies are correlated with the intensive biochemical investigations (reviews by Fieser and Fieser, 1959, Krayer, 1959, and Sourkes, 1962) and the recognition that one portion of the gland, namely the cortex, is essential for vertebrate life. [...]

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