2. PROFILING URINARY STEROIDS BY GAS
2.4. Clinical Indications for Urinary Steroid Profiling
A wide range of clinical symptoms, such as ambiguous genitalia, sodium-loosing states, precocious pseudopuberty, hirsutism, virilization, arterial hypertension, hypokalemia, trunkal obesity, primary amenorrhea, and hypoglycemia, are the most important clinical indications for the use of urinary steroid profiling for diagnostic purposes (Table 4) [17]. Urinary steroid profiling is also useful to monitor treatment and compliance in CAH due to enzyme deficiencies and after surgery of adrenal or gonadal tumors. The advantages of the method are nonselec- tivity and independence of circadian rhythm of most steroid hormones secreted.
Another advantage, which bears special importance for pediatric endocrinology, is that the procedure is noninvasive, because the analytical sample can easily be obtained.
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(a)
(b)
Figure 3 (a) Steroid profile (FID) of a healthy female neonate at age 2 wk (free and glucuronide fraction). AD, SS, CB: internal standards. For abbreviations see Table 2. (b) Urinary steroid profile (FID) in a 3-wk-old male neonate with 21-hydroxylase deficiency.
15β-OH-PO: 5β-pregnane-3α,15β,17α-triol-20-one.
Clinical Steroid Analysis 319
(a)
(b)
Figure 4 Cortisol (a) and androgen (b) metabolite excretion rates (corrected for body size) from childhood to adulthood. Since no significant sex differences have been ob- served, data have been combined.
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Table 4 Clinical Indications For the Use of GC-MS Urinary Steroid Profiling
Indications Diagnostic of Not pathognomonic for
Ambiguous genitalia 21-Hydroxylase defect Mixed gonadal dysgenesis 11β-Hydroxylase defect True hermaphroditism 3β-Hydroxysteroid- XX male
dehydrogenase defect Leydig cell hypoplasia 17α-Hydroxylase defect Androgen receptor defects 5α-Reductase deficiency 17β-Hydroxysteroid- Lipoid adrenal hyperplasia dehydrogenase defect Sodium-loosing states 21-Hydroxylase defect Renal insufficiency
3β-hydroxysteroid- Hypoaldosteronism dehydrogenase defect
Lipoid adrenal hyperplasia 18-Hydroxylase defect 18-Hydroxysteroid-
dehydrogenase defect Adrenal insufficiency Pseudohypoaldosteronism
Precocious pseudo- 12-Hydroxylase defect (sim- McCune-Albright‘s syndrome puberty ple virilizing, late onset) Testotoxicosis
11β-Hydroxylase defect β-hCG Excess Adrenal tumors
Gonadal tumors
Hirsutism, virilization 21-Hydroxylase defect (sim- Idiopathic hirsutism ple virilizing, late onset)
11β-Hydroxylase defect 3β-Hydroxysteroid dehydro-
genase defect Adrenal tumors Gonadal tumors Polycystic ovaries Cortisone reductase defi-
ciency
Hypertension, hypo- 11β-Hydroxylase defect Renal hypertension kalemia 17α-Hydroxylase defect Phaeochromocytoma
Apparent mineralocorticoid excess (AME) Type I and II
Cushing’s disease Adrenal tumors
Hypoglycaemia Adrenal insufficiency Islet-cell hyperplasia Nesidioblastosis
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2.4.1. Gas Chromatography–Mass Spectrometry Urinary Steroid Profiling in Patients with Congenital Enzyme Defects of Steroid Biosynthesis
2.4.1.1. 21-Hydroxylase Deficiency. The most common cause of ambig- uous genitalia in the female newborn (46,XX) is CAH due to 21-hydroxylase deficiency. Decreased cortisol synthesis induces excess adrenocorticotropic hor- mone (ACTH) secretion and overproduction of 17α-hydroxyprogesterone and 21-deoxycortisol. The low aldosterone production causes hyperkalemia and hy- ponatremia (salt-loosing CAH). This salt-loosing state presents a major symptom in the affected male newborn, as well as hyperpigmentation of genitalia. Patients with simple virilizing and late-onset CAH have accelerated growth rate and bone age with precocious pseudopuberty in males and virilization in females. The uri- nary steroid profile in newborns with salt-loosing CAH is dominated by 17- hydroxpregnanolones, pregnanetriol, 11-oxo-pregnanetriol, and 15β-hydroxy- pregnanolone (see Fig. 3b). In simple virilizing and late-onset CAH, additional detectable or low amounts of cortisol metabolites are excreted [18,19].
2.4.1.2. 11β-Hydroxylase Deficiency. The clinical signs for 11β-hy- droxylase deficiency are clitoromegaly and ambiguous genitalia in genetically female newborns, and hyperpigmentation of the genital area in male newborns.
Postnatally, in both sexes rapid somatic growth, advanced bone age, progressive clitoral or penile enlargement, premature pubarche, and hypertension occur. The urinary steroid profile of newborns is characterized by excretion of 6-hydroxy- tetrahydro-11-deoxycortisol (6α-OH-THS) [20]. In older infants and adults, ex- cretion of metabolites of 11-deoxycortisol is increased, while excretion of cortisol metabolites is low or absent [21].
2.4.1.3. 3β-Hydroxysteroid Dehydrogenase Deficiency. The clinical spectrum of 3β-hydroxysteroid dehydrogenase (3β-HSD) deficiency at birth in- cludes both salt-loosing and non–salt-loosing forms, independent of the extent of genital ambiguity [22,23]. The urinary steroid profile in the salt-loosing form has a characteristic fingerprint: dehydroepiandrosterone (DHEA), 16α-hydroxy- DHEA, PT, and 17α-hydroxypregnenetriol are the major excreted steroids be- cause of the virtually absent or very low excretion of cortisol metabolites. In late-onset 3β-HSD deficiency, i.e., during adolescence or adulthood, varying de- grees of hypogonadism occur in males [24], and hirsutism, irregular menses, and polycystic ovaries occur in females. Patients with simultaneous elevation of post- ACTH serum 17α-hydroxypregnenolone to 17α-hydroxprogesterone ratio and elevated basal urinary 5-ene steroid excretion have mild 3β-HSD deficiency [25].
2.4.1.4. 17α-Hydroxylase/17,20-Lyase Deficiency. Male pseudoherma- phroditism or ambiguous genitalia in 46,XY-individuals, absent pubertal devel- opment in 46,XX-individuals with hypokalemia, and development of hyperten-
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sion are the signs for 17α-hydroxlase deficiency. Deficiency of 17α-hydroxylase leads to reduced production of both gonadal sex steroids and cortisol, with accom- panying overproduction of corticosterone and 11-deoxycorticosterone. The uri- nary steroid profile is dominated by metabolites of corticosterone and its precur- sors and lacking in cortisol metabolites [26,27].
2.4.1.5. 18-Hydroxylase Deficiency. The 18-hydroxylase deficiency can present as infection-triggered, life-threatening salt-loosing state with hyperka- lemia in newborns and young infants. It is characterized by aldosterone biosyn- thetic defects both in 18-hydroxlase deficiency (CMO I) and in 18-hydroxysteroid dehydrogenase deficiency (CMO II) [28]. The urinary steroid profile in 18-hy- droxylase deficiency is characterized by increased excretion of free corticosterone and metabolites of corticosterone, while 18-hydroxylated corticosterone metabo- lites are absent or very low. The excretion of cortisol metabolites is normal (Fig. 5).
2.4.1.6. 18-Hydroxysteroid Dehydrogenase Deficiency. In CMO II, the urinary steroid profile shows, in addition to high amounts of corticosterone metabolites, 18-hydroxylated corticosterone metabolites (18-OH-THA, 18-OH- THB) (Fig. 6) [29].
Pseudohypoaldosteronism, another condition associated with severe salt- wasting, can be detected by profiling urinary steroids [30].
2.4.1.7. Adrenal Insufficiency. Low excretion of glucocorticoid metabo- lites and adrenal androgen metabolites characterize the steroid profile. However, the different causes (Addison’s disease, lipoid adrenal hyperplasia, and congenital adrenal hypoplasia) cannot be differentiated using urinary steroid profiling.
2.4.1.8. 11β-Hydroxysteroid Dehydrogenase Deficiency. Failure to thrive, polyuria, polydipsia, hypertension, hypokalemia, and nephrocalcinosis are the symptoms for apparent mineralocorticoid excess due to 11β-hydroxysteroid dehydrogenase deficiency (cortisol oxidase deficiency) and/or a steroid ring A reductase defect [31]. In the urinary steroid profile, the excretion of THE is much too low compared with the high THF, 5α-THF and free cortisol excretion [32].
2.4.1.9. Cortisone Reductase Deficiency. In hirsutism and virilization in females, cortisone reductase deficiency has been described. Patients with this disorder convert all their cortisol into cortisone. This gives rise to an apparent cortisol deficiency. Adrenocorticotropic hormone increases and stimulates the adrenal steroid synthesis. The urinary steroid profile is characterized by a very high excretion of THE, cortolones, and adrenal androgens, and low excretion of THF and 5α-THF [33].
Clinical Steroid Analysis 323
Figure 5 Urinary steroid profile (FID) in 18-hydroxylase deficiency (CMO I). Male, age 7 wk.
2.4.1.10. 5α-Reductase Deficiency. In genetically male infants, ambig- uous genitalia at birth and pubertal virilization are the clinical symptoms in inher- ited 5α-reductase deficiency. In urine, extremely low excretion of 5α-THF in young infants, and additional low excretion of androsterone and 11β-hydroxy- androsterone in older children indicate 5α-reductase deficiency [34].
2.4.2. Further Pathological Conditions
2.4.2.1. Polycystic Ovary Syndrome. Polycystic ovary (PCO) syndrome is characterized by menstrual irregularity and hirsutism. It is a common cause of anovulatory infertility. The urinary steroid profile is dominated by adrenal
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Figure 6 Urinary steroid profile (FID) in 18-hydroxysteroid-dehydrogenase deficiency (CMO II). Male, age 5 yrs. 18-OH-THA: 17-hydroxy-tetrahydro-11-dehydrocorticoster- one. 18-OH-THB: 18-hydroxy-tetrahydrocorticosterone.
androgens and high excretion of androsterone and 5α-THF. In urinary steroid metabolites, the 5α/5β ratio is significantly increased (Fig. 7) [35].
2.4.2.2. Cushing’s Syndrome. Prolonged hypercortisolism leads to Cushing’s syndrome. The major symptoms are growth failure, obesity, predomi- nantly of the trunk and the neck, and a full moonlike face. Hyperglycemia, purple striae, hirsutism, osteoporosis, muscular weakness, and hypogonadism are the additional signs. The etiology of Cushing’s syndrome is frequently iatrogenic (long-term treatment with synthetic corticoids). In these cases, the urinary steroid profile shows absence or low excretion of adrenal steroids and excretion of metab- olites of the prescribed corticoid drug. In the remaining cases, pituitary adenoma, adrenal adenoma and carcinoma, primary adrenocortical nodular dysplasia, McCune–Albright’s syndrome, and ectopic ACTH- or corticotropin-releasing
Clinical Steroid Analysis 325
Figure 7 Chromatogram (FID) of urinary steroids in polycystic ovary syndrome Fe- male, age 14 yrs.
factor (CRF)-secreting tumors can all cause Cushing’s syndrome. Independent of the pathogenetic cause, the urinary steroid profile is characterized by increased excretion of THF and free cortisol, 6β-hydroxycortisol and 20α-dihydrocor- tisol [14].
2.4.2.3. Adrenocortical Tumors. Virilization, hypercortisolism, femini- zation, abdominal pain, palpable abdominal tumor, or combinations of these fea- tures are clinical symptoms of adrenocortical tumors. Both types of adrenocortical tumors (carcinoma and adenoma) can produce a wide variety of steroid hormones.
This is a consequence of multiple enzyme deficiencies in tumor tissues. The tu- mor cells are capable of synthesizing large amounts of steroid hormone precur- sors independent of ACTH stimulation. Excessively high amounts of DHEA and other 3β-hydroxy-5-ene steroids characterize the urinary steroid profile in chil- dren with adrenocortical carcinoma, but similar profiles can also be produced by adrenal adenomas. Elevated 11β-hydroxy-androsterone excretion alone or com- bined with high excretion of cortisol metabolites or 3β-hydroxy-5-ene steroids are characteristic of the urinary steroid profile for adrenocortical adenomas [36,37].
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In adults suffering from adrenocortical carcinoma, the elevated excretion of 3β-hydroxy-5-ene steroids and/or high amounts of THS and cortisol metabolites (THF) are the major characteristics of the urinary steroid profile [38].