The early recognition of the 21-hydroxylase deficiency variety of congenital adrenal hyperplasia

The urinary steroids excreted by three newborn infants with 21-hydroxylase deficiency and by 15 healthy newborns aged two days have been compared after analysis by gas liquid chromatography (GLC). The identity of each steroid was carefully checked by gas chromatography/mass spectroscopy (GC-MS). The enzyme deficiency leads to the elevated excretion of urinary precursor metabolites, mainly 3α,17α,20α-trihydroxy-5β-pregnan, 3α,17α,20α-trihydroxy-5β-pregnan-11-one and 3α,17α-dihydroxy-5β-pregnan-20-one.In the search for a quick and firm confirmation of suspected 21-hydroxylase deficiency in a newborn baby by means of a GLC-profile of urinary steroids, most attention has up to now been paid to 3α,17α,20α-trihydroxy-5β-pregnan. However, 3α,17α-dihydroxy-5β-pregnan-20-one is a better indicator, as it enables one to confirm the existence of this disease soon after birth directly from the GLC-profile without further analyses by GC-MS.     

A gas chromatographic method for the determination of neutral steroid profiles in urine, including studies on the effect of oxytetracycline administration on these profiles in men

A capillary gas chromatographic method for the determination of 'total' metabolic profiles of urinary neutral steroids was developed. The method is based on anion exchange chromatographic separation and purification of monoglucuronide-, monosulphate- and double-conjugated neutral steroids on DEAE-Sephadex A-25 microcolumns and the final analysis of the individual steroids in these conjugate groups is carried out by capillary column gas-liquid chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The method was shown to provide a convenient and accurate determination of total metabolic profiles of neutral steroids in urine and thus, can be used for metabolic studies of steroids and for diagnostic purposes. In the present investigation the effect of a tetracycline antibiotic on the production and metabolism of neutral steroids in men was studied during a 5-day oral administration of oxytetracycline. The results showed that the influence of oxytetracycline on neutral steroids was minor and mainly restricted to the changes in urinary neutral steroid glucuronide excretion. Oxytetracycline decreased the mean daily excretion of total neutral steroid monoglucuronides by 20% and a statistically significant decrease was found in the urinary excretion of 3 alpha-hydroxy-5 beta-androstan-17-one-glucuronide (etiocholanolone, 31%, P less than 0.05), 5 beta-pregnan-3 alpha,20 alpha-diolglucuronide (pregnanediol, 32%, P less than 0.05) and corticosteroid glucuronides, including 3 alpha,11 beta,17 alpha,20 beta,21-pentahydroxy-5 beta-pregnan- and 3 alpha,17 alpha,20 beta,21-tetrahydroxy-5 beta-pregnan-11-one-glucuronides (beta-cortol and beta-cortolone, 36%, P less than 0.05). The reason for this effect is unknown, but may be partly due to inhibition of intestinal hydrolysis of biliary steroid conjugates, which previously was shown to result in an interruption of enterohepatic circulation of steroids and an increased excretion of steroid conjugates by the faecal route.     

Congenital adrenal hyperplasia. Plasma and urinary steroid conjugates in seven children with steroid 21-hydroxylase deficiency

Plasma neutral steroid sulphates and urinary neutral steroid sulphates and glucuronides from seven children (0.17–10.4 years) with steroid 21-hydroxylase deficiency were determined using gas-liquid chromatography and gas chromatography -mass spectrometry. Three of the patients were salt-losers. Large inter-individual variation in plasma concentration and urinary excretion of these compounds was observed. However, the group did have certain characteristic features. In plasma, the main compounds present were 3β-hydroxy-5-ene steroids. A progesterone metabolite, 5α-pregnane-3β,20α-diol, and a 17α-hydroxyprogesterone metabolite, 5β-pregnane-3α, 17α, 20α-triol, were present as sulphate conjugates and 3α, 17α-dihydroxy-5β-pregnan-20-one sulphate was identified for the first time in human peripheral plasma. In the urine, metabolites of 17α-hydroxyprogesterone were predominant and 5β-pregnane-3α, 17α,20α-triol, excreted mainly as a glucuronide, alone comprised about 50% of the neutral steroid excretion in these subjects. The next most abundant steroid was 3α,17α,20α-trihydroxy-5β-pregnan-11-one. The following compounds have not previously been found in the urine of patients with steroid 21-hydroxylase deficiency but were found in the present subjects: 5-androstene-3β, 17β-diol, 5α-pregnane-3β, 20α-diol and 3β,17α-dihydroxy-5β-pregnan-20-one. The pattern of the plasma and urinary steroids determined clearly differentiates the subjects with a steroid 21-hydroxylase defect from normal subjects and from patients with a 3β-hydroxysteroid dehydrogenase deficiency.     

High resolution gas chromatography of urinary steroids on glass capillary columns

A gas chromatographic method for the determination of urinary steroids on glass capillary columns is described. The steroids are separated as trimethylsilyl or methoxime-trimethylsilyl ether derivatives. In this way it is possible to obtain a profile of urinary steroids without any prepurification steps. Steroids of diagnostic importance (androsterone, etiocholanolone, dehydroepiandrosterone, 11-keto-etiocholanolone, pregnanediol, pregnanetriol, pregnenetriol, pregnanetriolone, tetrahydro S and tetrahydro E) can be determined quantitatively in the same chromatogram. Compared to other specific methods, this procedure allows a fast recognition of disorders of the steroid metabolism (e.g. the differentiation of various types of congenital adrenal hyperplasia).     

Quantitative alterations of steroid urinary profiles associated with diabetes mellitus

Capillary gas chromatography was used for the acquisition of quantitative metabolic profiles of urinary steroids from normal subjects and diabetic patients. Structures of 70 steroid metabolites have been assigned tentatively through mass spectrometry. Quantitative differences between the groups of normal and pathological samples (both males and females) are seen primarily for androsterone, etiocholanolone, dehydroepiandrosterone, 11-oxygenated 17-ketosteroids, corticosteroid metabolites, and a newly discovered metabolite, recently identified as a C26 pentol. The only significant difference between two different age groups of normal males was shown by androsterone.     

Congenital adrenal hyperplasia caused by defect in steroid 21-hydroxylase. Establishment of definitive urinary steroid excretion pattern during first weeks of life.

The steroid excretion of two female infants with congenital adrenal hyperplasia due to 21-hydroxylase deficiency has been studied during the first weeks of life. The techniques used were gas chromatography on an open-tubular column and combined gas chromatography-mass spectrometry using selected ion recording. During the first days of life 3beta-hydroxy-5-ene steroids predominate and the levels found were considerably greater than those found in normal infants. Selected ion recording mass spectrometry permitted detection of pregnanetriol and 11-oxo-pregnanetriol several days before these steroids could be determined with accuracy by conventional gas chromatography. Pregnanetriol and 11-oxo-pregnanetriol were first detected on the third day of life. The results of this investigation demonstrate that 21-hydroxylase deficiency may be indicated during the first week of life by an increased 3beta-hydroxy-5-ene steroid excretion, but the definitive excretion pattern required for firm diagnosis may not develop for several days. The amounts of the definitive steroids excreted may not be sufficient to be detected by the more usual methods for several weeks.     

Capillary gas chromatographic measurement of neutral urinary steroids: evaluation and comparison with alternative methodology

The major neutral urinary steroids have been quantitated by capillary gas chromatography and the reproducibility and accuracy of the method have been fully evaluated. Typical intra- and inter-assay coefficients of variation for individual steroids ranged from 4.9 to 10.1% and from 15.6 to 21.5%, respectively. Comparison of steroid values with alternate and commonly employed procedures for measuring urinary 17-oxosteroids, total 17-oxogenic steroids, and cortisol and plasma dehydroepiandrosterone-sulphate yielded correlation coefficients between 0.71 and 0.79. A definite relationship between serum cortisol and urinary trihydroxy-pregnanedione was also shown. Urinary androstenedione metabolites, however, could not be significantly (P greater than 0.05) correlated with the normalised androgen ratio in plasma.     

Stability of steroids in plasma over a 10-year period

In order to examine whether plasma samples may be used for steroid analysis after long periods of storage, cortisol, testosterone, oestrone and oestradiol were remeasured in samples, which had been analysed 1.3-10.8 years earlier. The method for the measurement of these steroids was unchanged over this period. The results demonstrate that at a temperature of -25 degrees C steroids remained stable. Only cortisol and testosterone concentrations showed a small, insignificant decrease (6-9%) after 3 to 4 years of storage. These differences are well within the range of the precision of the method (interassay variation), which over a period of 11 years was 9.4%, 8.0%, 10.0% and 9.5% for cortisol, testosterone, oestrone and oestradiol, respectively. It is concluded that steroid hormones in human plasma are stable in our laboratory, and that they might be analysed even after more than 10 years of storage at -25 degrees C.     

Method for screening urinary steroids by gas chromatography

Numerous methods are available for measuring urinary steroids in evaluating endocrine dysfunctions. Measurements of these particular steroids, or groups of them, usually involve tedious isolation methods, corrections for interferences and losses of the steroid, and (or) expensive reagents. We show how gas-liquid chromatography provides a rapid, sensitive, and direct method for several steroid metabolites in urine. Androstanol, androsterone, etiocholanolone, dehydroisoandrosterone, pregnanediol, pregnanetriol, 11-keto-17-ketosteroids, and the 11beta-hydroxy-17-ketosteroids can be identified and quantified.     

Rapid quantitative gas-chromatographic fractionation of the major urinary i7-oxosteroids and i7-oxogenic steroids

A rapid gas-chromatographic method for the fractionation of the major 17-oxosteroids and 17-oxogenic steroids in urine is presented. The method utilises borohydride reduction, periodate oxidation and gas-liquid chromatographic (GLC) separation of steroid formate esters on SE 30. A single estimation may take less than 4 h, providing estimates of the urinary excretion of pregnanetriol and THS (together); allopregnanetriol and allo-THS (together); aetiocholanolone; androsterone; THE, THF, cortols and cortolones (as a group); allo-THE, allo-THF, allo-cortols and -cortolones (as agroup); pregnanediol; 11-oxygenated aetiocholanolone; 11-oxygenated androsterone. The method has been evaluated by comparing 17-oxosteroid (17-OS) and 17-oxogenic steroid (17-OGS) values calculated from the individual GLC values with those obtained by group estimations on the same urine. The correlation coefficients between the GLC values and the group values did not differ significantly from 1.000 (17-OS, 0.979; 17-OGS, 0.976). Recovery experiments using reference steroids and available steroid glucuronides indicated quantitative recoveries. Steroid sulphates were not measured. Reproducibility was of the order of 10% (coefficient of variation). The specificity of the method was examined by trapping GLC peaks which were. subjected to mass spectroscopy and chromatographed in other GLC systems. In only one case so far examined has a GLC peak proved to be other than the expected steroid formate.     

Detection of 3 beta-hydroxysteroid dehydrogenase deficiency in a newborn by means of urinary steroid analysis

A urinary steroid excretion pattern of a 3-wk-old newborn, suffering from 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) deficiency, has been produced, employing capillary gas chromatography and subsequent mass spectrometric identification of the various excreted steroids. The diagnosis could be established, apart from the clinical symptoms, on the basis of a grossly elevated excretion of 16-OH-DHEA and 16-OH-pregnenolone, combined with mass spectrometric identification of the following steroids: 17-OH-preganolone, pregnanetriol, pregnanolone, pregnenetriol and 17-OH-pregnenolone.     

Hydrolysis of steroid glucuronides with beta-glucuronidase preparations from bovine liver, Helix pomatia, and E. coli.

We determined the enzymic activity of beta-glucuronidase preparations from bovine liver, Helix pomatia, and Escherischia coli with steroid glucuronides and nonsteroid glucuronides as substrates. We also studied the effect of Na2SO4 on the enzymic hydrolysis of several substrates with the three preparations of beta-glucuronidase. Na2SO4 increases the rate of hydrolysis of all substrates with beta-glucuronidase from bovine liver. Hydrolysis of a steroid glucoronide with beta-glucuronidase from Helix pomatia and E. coli is inhibited by Na2SO4. None of the three enzyme preparations gives complete hydrolysis of urinary steroid conjugates, because urine contains inhibitors, which can be removed by absorption chromatography of the urine on a column of neutral polystyrene resin Amberlite XAD-2. But when Amberlite XAD-2 is not used, hydrolysis of urinary glucuronides of androsterone, etiocholanolone, pregnanediol, estriol, and 17-hydroxycorticosteroids proves that, given an incubation time of 24 h, the beta-glucuronidase preparation from bovine liver, in the presence of Na2SO4, is suited for determining all of the above steroids except esriol; the preparation from Helix pomatia is good for determining estriol and 17-hydroxycorticosteroids; the preparation from E. coli is good for determining androsterone, 17-hydroxycorticosteroids, and especially estriol, the glucuronide, of which is maximally hydrolyzed in 2 h.     

Determination of steroid profiles in healthy and diseased states: identification and quantitation of a block of 17 alpha-hydroxylase

The steroid metabolic profile on a patient with a suspected block in steroid biosynthesis was analyzed by gas chromatography and gas chromatography-mass spectrometry. The results of this work led to an interpretation of a block at the 17 alpha-hydroxylase step. Although the steroid metabolic profile was complex, we could not detect any steroids with a hydroxy moiety at position C-17. The use of gas chromatography-mass spectrometry, in this case, was the method of choice for a final diagnosis because of the confusion that resulted from other, more classical, forms of analysis. Data from our patient's sample are reported and compared to normals and to other cases where a block in 17 alpha-hydroxylase have been reported.     

The urinary steroid profile in patients diagnosed with adrenal incidentaloma

Objective

The aim of this study was to investigate the possible urinary markers of hormonal activity in patients with non-functioning adrenal incidentalomas. In order to evaluate the endocrine activity of forementioned tumours, urinary steroid metabolite levels were analyzed in samples from patients and controls. Possible blocks in metabolic pathways of the examined hormones were determined by comparing selected urinary steroid metabolite sums and ratios in both groups of interest.

Design

Urine samples were collected from 20 patients with non-functioning adrenal incidentalomas and from 25 controls matched in terms of age, sex and BMI. Excretion of 19 major urinary steroid metabolites was analyzed by gas chromatography. The results were subjected to statistical analysis.

Results

In patients with adrenal incidentalomas sum of total urinary cortisol metabolites was significantly increased in respect to the control group. We also observed a shift towards tetrahydrocorticosterone, cortisol and etiocholanolone production in patients. No significant differences in production of other urinary steroid metabolites were noted in patients with adrenal incidentalomas in respect to control group.

Conclusions

Our data suggests that not only urinary free cortisol but also its metabolite such as tetrahydrocortisol and other steroids including etiocholanolone and corticosterone tetrahydrometabolite might be urinary markers for the endocrine activity of adrenal incidentalomas. Enhanced levels of these urinary steroid metabolites indicate an impairment of 11β-hydroxysteroid dehydrogenase activity and slightly increased activity of 5β-reductase in patients with adrenal incidentalomas.     

Low urinary oestrogen excretion during pregnancy, due to an impairment of fetal adrenocorticotrophic hormone secretion.

Four cases of newborn infants whose mothers had a low urinary oestrogen excretion during pregnancy are reported. Placental sulphatase deficiency in placental insufficiency were excluded by a dehydroepiandrosterone sulphate loading test. Postnatally, they developed a clinical picture characterized by an inappropriate secretion of cortisol which, by the results of an adrenocorticotrophic hormone stimulation test, appeared to be due to an impairment of adrenocorticotrophic hormone secretion. The prenatal and postnatal steroid metabolism is discussed.     

Mechanisms of interference of nonesterified fatty acids in radioimmunoassays of steroids.

Addition of nonesterified fatty acids caused an apparent increase in unbound steroid present in supernatants in radioimmunoassays for steroids utilizing dextran-coated charcoal. Nonesterified fatty acid interference occurred at the initial binding interaction between steroid and its antiserum, and also at the step separating bound from unbound steroid. It was determined that nonesterified fatty acids, which had been added to radioimmunoassays, formed micelles and trapped steroids. The extent of the entrapment was inversely related to the number of polar groups in the steroid molecule, that is, hydrophobic steroids more easily interacted with the nonesterified fatty acid micelles. However, if the charcoal concentration were increased, the nonesterified fatty acid effect was eliminated for assays of polar steroids and greatly reduced for non-polar steroid assays.     

The diagnosis of 21-hydroxylase deficiency in a prematurely born infant on the basis of the urinary steroid excretion pattern

The urine of a 6-day-old prematurely born female infant (birth weight 1060 g) suspected of having a 21-OH deficiency showed no steroid abnormalities on capillary GLC analysis. Using GC-MS tetrahydrocortisone (THE) and also 3, 17-dihydroxy-5β-pregnane-20-one (17-OH-Polone) were absent, but two androstanetriolone peaks were observed. In the urine collected on day 9 THE was absent, but a large amount of 3, 11β-dihydroxy-5-androstane-17-one (11-HA) was found by GC-MS to be contaminated by a small amount of 17-OH-Polone. The next urine specimen collected on the 22nd day while the child received cortisol therapeutically showed the characteristic steroid profile for the diagnosis 21-OH deficiency, large peaks of 17-OH-Polone, pregnanetriol (P₃) and 11-keto-pregnanetriol (11-keto-P₃). Over the next few weeks two other compounds were found to have been excreted in relatively large amounts, 3ξ, 16ξ, 17ξ, 20ξ-pregnanetetrol (16-OH-P₃) and surprisingly also a 21-hydroxylated compound, namely 3β, 20, 21-trihydroxy-5-pregnene. These same two compounds were also found in the urine of another infant with suspected 21-OH deficiency.

The urinary steroid excretion patterns characteristic for 21-OH deficiency are dependent on the maturity and age of .the infant. In the prematurely born infant androstanetriolones appear in the urine before 17-OH-Polone. The occurrence of these different steroid excretion patterns is tentatively explained.     

Comparison of chemical and radiochemical methods in determination of steroid hormones]

While in some cases steroids can be measured directly in serum or plasma by radioimmunoassay (RIA), in other cases, especially when analyses are carried out in urine, the samples must be processed before RIA can be performed. The operations involved in the preparation of urinary or blood extracts suitable for the RIA of steroid hormones are examined and compared in terms of practicability with the analytical procedures currently used for the chemical determination of the same steroids or their metabolites.     

Trends in the misuse of androgenic anabolic steroids among boys 16-17 years old in a primary health care area in Sweden.

OBJECTIVE: To evaluate the effects of an appearance programme in preventing the misuse of androgenic anabolic steroids among male adolescents in a primary health care area in Sweden. METHODS: Attitudes to steroid hormones among 16-17 years old male and female adolescents are discussed. A well-established anonymous multiple-choice questionnaire was answered by 921 adolescents and statistically analysed. RESULTS: The misuse of androgenic anabolic steroids tended to decrease after the intervention. CONCLUSIONS: The misuse of androgenic anabolic steroids did not increase, and even tended to decrease, after the intervention, indicating that drug-abuse among male adolescents can be decreased through discussions about appearance and attitudes. Repeat and prospective studies have to be done before this intervention programme can be generalised.     

Long-term effects of aminoglutethimide on steroid metabolism in congenital adrenal hyperplasia.

Five cases of congenital adrenal hyperplasia due to C21-hydroxylase defect were treated with a combination of aminoglutethimide and prednisolone. In the third year of treatment the urinary levels of 17-oxosteroids increased above normal values while the total 17-hydroxy-corticosteroids were normally low. Specifically, urinary pregnanetriol was normal in 3 cases. To determine the reasons for this disparity the adrenal metabolism of cholesterol, as judged by the urinary steroid metabolites, was studied. Fractionation of urinary steroid metabolites was by thin-layer chromatography (TLC) followed by gas-liquid chromatography (GLC). The results indicate that aminoglutethimide inhibits steroidogenesis less than prednisolone; that a pathway from cholesterol via 17 alpha, 20 alpha-dihydroxycholesterol to dehydroepiandrosterone is likely to operate after long-term aminoglutethimide therapy; that 11 beta-hydroxylase, at least for pregnenes may be inhibited by aminoglutethimide and that the metabolic breakdown of testosterone may be delayed by this drug.