Biomedical subjects

C H Shackleton

Publications and source records attributed to C H Shackleton.

185 records · Page 11Linked to original sources

PubMedDate not supplied

Late-onset 21-hydroxylase deficiency: reliable diagnosis by steroid analysis of random urine collections.

The feasibility of performing steroid analysis by capillary gas chromatography on random urine samples for the detection of mild late-onset 21-hydroxylase deficiency was evaluated. Comparisons were made of basal excretions of androgen and 17 alpha-hydroxyprogesterone metabolites with plasma levels (basal and stimulated) of 17 alpha-hydroxyprogesterone and testosterone in six patients with the disorder. The following steroid metabolite excretion ratios were determined for normal controls and affected individuals. 1) 17 alpha-hydroxypregnanolone/tetrahydrocortisone + tetrahydrocortisol + 5 alpha-tetrahydrocortisol (cortisol metabolites) (normal 0.017-0.10, affected 0.17-0.42); 2) pregnanetriol/cortisol metabolites (normal 0.03-0.15, affected 0.17-0.99); 3) pregnanetriolone/cortisol metabolites (normal 0.02-0.014, affected 0.08-0.20); 4) androsterone + etiocholanolone/cortisol metabolites (normal 0.26-1.02, affected 0.34-1.47). Among the 21-deoxy steroid ratios, there was no overlap between affected and unaffected individuals. Two of six affected individuals had androsterone + etiocholanolone/cortisol metabolite ratios in the normal range. This method provides excellent discrimination between normal and affected individuals, precluding the need for an ACTH-stimulation test. It is anticipated that it will be increasingly used for diagnosis of the condition.

Adolescent↗

PubMedDate not supplied

The 21-acetylation of corticosteroids by Clostridium sporogenes.

A strain of Clostridium sporogenes, an anaerobic bacterium, isolated from sewage in New York City synthesizes two constitutive enzymes with action on steroid molecules: (i) an enzyme capable of selectively acetylating the 21-hydroxyl function of certain steroids and (ii) the corresponding esterase. Under our experimental conditions the enzymes have a strict structural requirement for 3-keto-4-ene and C-20-keto or 20 alpha-hydroxyl group and convert their respective substrates to a mixture of free and acetylated products.

Acetylation↗

PubMedDate not supplied

Identification of 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one as the major ovarian steroid produced by the teleost Micropogonias undulatus during final oocyte maturation.

This study describes the identification of 17 alpha,20 beta, 21-trihydroxy-4-pregnen-3-one (20 beta-dihydro-11-deoxycortisol, 20 beta-S) as a major steroid product of the ovary of Atlantic croaker (Micropogonias undulatus) incubated in vitro. This is the first report which describes 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one as a major product of teleost steroidogenic tissue. The steroid was identified by a variety of methods, including HPLC, TLC, GC-MS, UV absorbance, and reactions with specific enzymes. Full grown oocytes, prior to final oocyte maturation (FOM), accumulated only small amounts of 20 beta-S. However, a substantial increase in 20 beta-S production occurred at the time of FOM. These results suggest that 20 beta-S is a maturational steroid in this species.

17-Hydroxycorticosteroids↗

PubMedDate not supplied

A paradox: elevated 21-hydroxypregnenolone production in newborns with 21-hydroxylase deficiency.

21-Hydroxypregnenolone and its metabolite 5-pregnene-3 beta, 20 alpha 21-triol have been measured in the sulfate fraction of neonatal urine. These two steroids are the major two 21-hydroxylated 5-pregnenes produced by neonates and are almost exclusively excreted as disulfates. The excretions of these steroids by normal infants and infants with 21-hydroxylase deficiency were compared. In addition to measurement of the absolute excretion, the excretion relative to the total 3 beta-hydroxy-5-ene output was also determined. The results show that 21-hydroxypregnenolone excretion is highly elevated in 21-hydroxylase deficiency (affected, mean 887 micrograms/24 h, range 453-1431 micrograms/24 h; normal, mean 117 micrograms/24 h, range 17-263 micrograms/24 h), but when compared to excretion of other delta 5 steroids the excretion is slightly low [(21-hydroxypregnenolone + 5-pregnene-3 beta, 20 alpha, 21-triol)/total 3-beta-hydroxy-5-ene steroids, 2.9% affected; 3.6% normal]. This difference was not statistically significant. There is thus no evidence that the 21-hydroxylase acting on pregnenolone is deficient in congenital adrenal hyperplasia. The explanation of the normal activity of "pregnenolone 21-hydroxylase," although not clearly defined, is probably associated with two recent findings by other workers: (a) that the human fetus has an active 21-hydroxylase distinct from the adrenal enzyme and (b) that a 21-hydroxylase structurally very different from the adrenal enzyme, with high activity towards pregnenolone (but no activity towards 17-hydroxyprogesterone), has been isolated from rabbit hepatic microsomes.

17-alpha-Hydroxypregnenolone↗

PubMedDate not supplied

Characterization of bacterially expressed rat estrogen receptor beta ligand binding domain by mass spectrometry: structural comparison with estrogen receptor alpha.

Functional rat estrogen receptor beta ligand binding domain (rER beta LBD, aa 210-485) and human estrogen receptor alpha ligand binding domain (hER alpha LBD, aa 301-553) were expressed in Escherichia coli. Hormone binding assays revealed that both ER beta and ER alpha LBDs bound the natural ligand estradiol (E2) with similar affinity (Kd approximately 100 pM). Competitive binding experiments were carried out with ICI 164384, 4-hydroxytamoxifen, 16 alpha-bromo-estradiol, and genistein employing [3H]E2 as a tracer. No significant differences in responses of ER alpha and ER beta LBDs to ICI 164384 and 4-hydroxytamoxifen were observed, 16 alpha-Bromo-estradiol and genistein discriminated between the ER subtypes and acted as ER alpha and ER beta selective ligands, respectively. Final purification of recombinant proteins was achieved on an E2 affinity column, where they were subjected to in situ carboxymethylation. The partially carboxymethylated proteins actively bound E2. The carboxymethylated rER beta LBD had a molecular mass of 32251.6 Da, equivalent to the calculated mass with the addition of three carboxymethyl groups. No other proteins (of lower or higher molecular mass) were detected, so the LBD was considered structurally authentic and pure. By using a combination of intact protein mass spectrometric fragmentation and trypsin proteolysis (98% sequence coverage), it was established that rER beta cysteine-289 and -354 were not carboxymethylated on the affinity column, suggesting that they were shielded from alkylation in the E2-bound conformational state. Concurrent analysis of hER alpha LBD showed that under the same experimental conditions, the two equivalent ER alpha cysteines were not alkylated (alpha C381 and alpha C447). These data support close structural relationship between the E2-bound ER alpha LBD and ER beta LBD proteins.

Alkylation↗