Neonatal urinary steroids in Smith-Lemli-Opitz syndrome associated with 7-dehydrocholesterol reductase deficiency.

The biosynthetic abnormality in Smith-Lemli-Opitz syndrome (SLOS) is a deficiency of 7-dehydrocholesterol (7DHC) reductase, the enzyme responsible for catalyzing the final step in the Kandutsch-Russell pathway for cholesterol synthesis. Because the disposition of 7DHC and 8-dehydrocholesterol [8DHC; cholesta-5,8(9)-dien-3beta-ol] produced in this syndrome is little understood, we have analyzed urine from three young infants by gas chromatography/mass spectrometry to characterize its steroid metabolites. All steroid metabolites of adrenal origin found in normal infant urine were also found in urine from the patients with SLOS but in reduced amount. Quantitatively, the major steroids in these SLOS patients were identified by mass spectrometry as homologs of normal neonatal steroids possessing an additional double bond. Generally, two forms of each steroid were present in a similar amount. Because of the markedly increased levels of 7DHC and 8DHC in SLOS, these almost certainly represented the 5,7 and 5,8(9) unsaturated forms of each metabolite. The most abundant steroids were tentatively identified as 3beta,16alpha-dihydroxy-5,7-pregnadien-20-one and 3beta,16alpha-dihydroxy-5,8(9)-pregnadien-20-one, although similar 21-hydroxylated steroids and homologs of 16alpha-hydroxy-DHEA were also found. This study shows that all enzymatic steps used by cholesterol in the DHEA synthetic pathway are also functional for 7DHC and 8DHC.

Determination of an aldosterone antagonist in urine by high-performance liquid chromatography using automated column switching.

An automated high-performance liquid chromatographic assay for the determination of an aldosterone antagonist (I) is described using column switching for direct injection of urine samples. After dilution with buffered internal standard solution, the sample was injected onto a clean-up column (17 X 4.6 mm I.D.), dry-packed with C18 reversed-phase material (particle size 30 micron). Polar urine components were removed by flushing the clean-up column with water. Retained substances, including I and the internal standard, were desorbed by backflush elution onto a 5-micron ODS-silica analytical column (125 X 4 mm I.D.), separated with water-methanol-tetrahydrofuran, and detected at 295 nm. After backflushing the analytical column and re-equilibrating the clean-up column, the system was ready for the next injection. The limit of quantification was ca. 100 ng/ml, using a 100-microliter specimen of diluted urine. The mean inter-assay precision of the method up to 25.6 micrograms/ml was 2%. Practicability and accuracy of the new method were demonstrated by the application to excretion studies performed with human volunteers.

Discovery of a natural spirolactone derivative in man and animal.

The presence of 6,7-dihydroxy-6,7-dihydrocanrenone (DHC) in man and in animal has been shown. Sodium loading results in a decrease of urinary DHC. On the contrary, sodium depletion increases its concentration.

Identification of some human urinary metabolites of orally administered potassium canrenoate by stable isotope-labeling techniques.

A single oral dose (200 mg) of an equimolar mixture of potassium canrenoate and its 20,20,21,21-tetradeutero analogue was administered to three healthy men. The steroids in urine collected for 24 hr after dosage were isolated on XAD-2 resin, and purified and fractionated into groups by lipophilic gel chromatography before and after hydrolysis of conjugates. GC/MS analysis of these fractions allowed the detection and identification of canrenone, canrenoic acid and its ester glucuronide, 3 beta-hydroxy-3-deoxocanrenone, 3beta-hydroxy-4,5alpha-dihydro-3-deoxocanrenone and a 3epsilon-hydroxy-4,5,6,7-tetrahydro-3-deoxocanrenone. In addition a number of di- and trihydroxy compounds formed by reduction and hydroxylation were partially identified from their E1 and C1 mass spectra. The results provide information on the metabolism of oral potassium canrenoate in man, and demonstrate the utility of combining stable isotope labeling, lipophilic gel chromatography, and GC/MS in studies of steroidal spirolactones.

Congenital adrenal hyperplasia caused by 11 beta-hydroxylase deficiency with onset of symptoms after one spontaneous pregnancy.

A case of adrenogenital syndrome due to 11beta-hydroxylase deficiency is described in a mother, 25 years of age, who had experienced a successful pregnancy 5 years previously. At that time no abnormality had been suspected and pregnancy was achieved without therapy. Subsequently the patient was examined because of secondary sterility. The menstrual cycles were anovulatory. Only slight virilization was observed and blood pressure was normal. Diagnosis was based on the observation of highly increased urinary excretion of 17-ketosteroids and 17-ketogenic steroids, with especially high excretion of tetrahydro-11-deoxycortisol. Following suppression with dexamethasone and adequate maintenance treatment, the patient conceived and had an uneventful pregnancy. This is apparently the first report of pregnancy in adrenogenital syndrome due to 11beta-hydroxylase deficiency.