Francis Lam, Cristina Matei, Gill Rumsby
1. Department of Clinical Biochemistry, UCLH NHS Foundation Trust 2. Department of Paediatrics, East and North Hertfordshire NHS Trust
A baby presented at a local hospital with failure to thrive. Initial biochemistry showed hyponatraemia and hyperkalaemia. Other blood analyses were inconclusive. A spot urine sample was sent for urine steroid profile (USP) analysis. The USP showed a relative abundance of corticosterone metabolites with undetectable tetrahydroaldosterone, a pattern indicative of aldosterone synthase deficiency, subsequently confirmed by genetic testing in this laboratory. Where a steroid disorder is suspected, a USP has great utility since the specimen is easily accessible and can identify/exclude a variety of disorders. Where urgent samples are involved, analyses can be prioritised with relatively rapid turnaround time.
A 1 month old baby boy presented at a local district general hospital with failure to thrive. He was born to non-consanguineous Eastern European parents, with an 18 month old healthy sister. The term birth was unremarkable, with nil of note from the antenatal history. Initial clinical examination revealed a slightly low but stable blood pressure for age, but was otherwise normal.
Initial biochemistry results showed hyponatraemia (plasma sodium concentration = 125 mmol/L, reference range 135-145 mmol/L) and hyperkalaemia (plasma potassium concentration = 6.1 mmol/L, reference range 3.5-5.1 mmol/L). Urine sodium concentration was <10 mmol/L. Plasma glucose concentrations were stable ranging 3.5-4.2 mmol/L (normal fasting range = 3.9-5.8 mmol/L). To aid the investigation of possible congenital adrenal hyperplasia (CAH, an inborn error of cortisol biosynthesis) the plasma cortisol response to synacthen stimulation was assessed; baseline: 65 nmol/L, 30 min: 286 nmol/L. Common criteria for a normal response include a baseline cortisol of >200 nmol/L, a 30 min increment of >200 nmol/L and/or a 30 min concentration of >550 nmol/L. Based on these results, CAH remained a potential diagnosis and additional blood tests (17-OHP, ACTH, renin activity, aldosterone) were requested along with a spot urine that was sent for urine steroid profile (USP) analysis. Hydrocortisone and sodium supplementation were commenced.
Steroid conjugates from urine specimens underwent solid phase extraction (SPE) using C18 cartridges (Waters Sep-Pak). Conjugates were then hydrolysed using Helix pomatia sulphatase/glucuronidase before undergoing SPE again to clean up freed steroids. Free steroids underwent sequential derivatisation with methoxyamine hydrochloride and trimethylsilylimidazole to form methyloxime-trimethylsilyl ethers prior to analysis. 5á-androstane-3á,17á-diol, stigmasterol, and cholesteryl butyrate were used as internal standards. Gas chromatography-single quadrupole mass spectrometry was performed in scanning mode on a Shimadzu QP-2010 Plus GC-MS system installed with a capillary column (Agilent Technologies, 25 m length, 0.32 mm ID, CP-Sil 5 stationary phase, 0.12 ìm film thickness). The column temperature program started at 70°C, increasing to 280°C in a stepwise fashion with a total runtime of 30 mins per injection. Individual steroid metabolites were positively identified via retention time and mass spectra.
The USP showed a normal pattern of cortisol metabolite excretion, and no biomarkers associated with CAH variants were present. However, the corticosterone metabolites tetrahydro-11-dehydrocorticosterone (THA), hexahydro-11-dehydrocorticosterone, 6-hydroxyTHA and 18-hydroxyTHA were comparatively abundant in the absence of detectable tetrahydroaldosterone. This pattern is indicative of an aldosterone synthase, specifically corticosterone methyl oxidase type 2, deficiency. Subsequent high plasma renin activity (185 nmol/L/h, adult reference range 0.5-3.1 nmol/L/h) and inappropriately low aldosterone (1040 pmol/L, adult reference range 100-800 pmol/L) measurements supported this diagnosis. Whole gene sequencing of the aldosterone synthase gene CYP11B2 identified a known pathological change, c.554C>T (p.Thr185Ile), confirming aldosterone synthase deficiency.
The investigation of a young infant presenting with hyponatraemia is challenging, further complicated by the need to obtain sufficient blood samples and prioritise informative tests prior to initiation of treatment. Where an adrenal steroid disorder is suspected, a USP has great utility since the specimen is easily accessible and the test can identify/exclude a variety of disorders. Furthermore, where urgent samples are involved, analyses can be prioritised with a relatively rapid turnaround time. In this case, the USP diagnosis was made within 2 days of sample receipt, prompting treatment with the mineralocorticoid fludrocortisone and reduction of the glucocorticoid hydrocortisone.