Profiling urinary steroid

PROFILING URINARY STEROIDS BY GAS CHROMATOGRAPHY-MASS SPECTROMETRY... 

Pseudohypoaldosteronism, another condition associated with severe salt-wasting, can be detected by profiling urinary steroids [30]. 

Shackleton CH (1986) Profiling steroid hormones and urinary steroids. J Chromatogr 379 91-... 

Shackleton CH, Taylor NF, Honour JW (1980b) An Atlas of Gas Chromatographic Profiles of Neutral Urinary Steroids in Health in Disease. Packard-Becker, Delft... 

Detection and quantitation of steroids. The confirmation of the steroid content of a biological sample is a routine procedure in many GC-MS laboratories. The development of the human urinary steroid profiles ... 

Wudy SA, Homoki J, Wachter UA, Teller WM. Diagnosis of the adrenogenital syndrome caused by llp-hydroxylase deficiency using gas chromatographic-mass spectrometric analysis of the urinary steroid profile. Dtsch Med Wochenschr 1997 122 3-11. 

Buiarelli et al. (2004) extended the above analytical approach to many more related steroids when they published a method for the direct analysis of 15 urinary anabolic steroids in a single run, namely T, epitestosterone, dehydroepiandrosterone (DHEA), androsterone, etiocholanolone, their sulfates and their glucuronides (Figure 2,2), They extracted 2 mL of human urine by solid-phase extraction with methanol elution and reconstituted the residue in aqueous methanol in the presence of deuterated internal standards (da-epitestosterone glucuronide, [16,16,17-"H3 testosterone sulfate and [16,16,17-2H3]testosterone), then monitored, for example, mJz. 289-97 and 109 for T and epitestosterone, miz 367-97 for their sulfates, and m/z 463-113 and 287 for their glucuronides. The method does not achieve quantitation, but it allows the estimation of ratios, which makes it possible to monitor the urinary steroid profile, which is useful for monitoring the abuse of anabolic steroids. 

Whereas the earlier work used GC for the estimation of only selected urinary steroid constituents, most current work favors the multicomponent (metabolic profiling) approach. The preparation of mixed derivatives , such as, for example, the methoxime-trimethylsilyl derivatives of Gardiner and Homing [291], in principle facilitates conversion of all metabolites containing hydroxy and carbonyl groups. Through the introduction of capillary columns to steroid analysis [12,292,293], it became possible to separate the complex mixtures of such derivatives. 

Fig. 3.18. Representative urinary steroid profiles of a normal male versus a diabetic male. Reproduced from [268].

Although the present use of urinary steroid profiling techniques appears to be largely confined to biomedical research, their gradual acceptance in clinical diagnosis and preventive medicine (to establish the risk of biochemical endocrine disorders)... 

Figure 1 GC-MS Profile analysis. Total ion chromatogram profiles produced by scanning methoxy-trimethylsilyl derivatives of urinary steroids. (A) Profile from a patient with 11 -hydroxylase deficiency in which the principal steroids are metabolites of IIjS-deoxycortisol (substance S) such as tetrahydrosubstance S (THS) and hexahydrosubstance S epimers (HHS). (B) Profile from the patient s father this profile is essentially normal. Some of the major metabolites are C19 steroids such as androsterone (An), et-iocholanone (Etio), pregnanetriol (PT), and cortisol metabolites (THE, tetrahydrocortisone THF, tetrahydrocortisol and 5a-THF, 5a-tetrahydrocortisol). Peaks 1-3 are internal standards. (Reprinted from Shackleton CHL, Merdinck J, and Lawson AM (1990) In McEwan CN and Larsen BS (eds.) Mass Spectrometry of Biological Materials, pp. 297-377. New York Dekker courtesy of Marcel Dekker Inc.)...

Disease Cause Change in urinary steroid profile... 

Mozzarino, M. Rossi, F. GiacomeUi, L. Botre, F. Effect of the systemic versus inhalatory administration of synthetic glucocorticoids on the urinary steroid profile as studied by gas chromatography-mass spectrometry. Anal. Chim. Acta 2006,559, 30-36. 

Benzyloxime Derivatives of Steroids. New Metabolic Profile Procedure for Human Urinary Steroids Anal. Lett. 4(3) 151-160 (1971) CA 75 31050u... 

Comparison of Chromatographic Techniques for Determination of Neutral Urinary Steroid Profiles Ergeb. Exp. Med. 20(Fortschr. Chrom-atogr. Methoden Ihre Anwend. Klin. Biochem.) 51-54 (1975) CA 85 43180f... 

Sex Differences in Human Urinary Steroid Metabolic Profiles Determined by Gas Chromatography Anal. Biochem. 80(2) 329-343 (1977) CA 87 19468b... 

The interpretation of the numerical values of urinary metabolites obtained via gaschromatographic urinary steroid profiles is extremely complex since several parameters, e.g. bone age, pubertal stadium etc., must be taken into consideration... 

In this chapter, urinary steroid profiling is discussed first, followed by a description of the state of the art concerning clinical profiling of steroid hormones in plasma by stable isotope dilution (ID) GC-MS. The reason is merely a historical one the art of urinary steroid profiling matured earlier and has found more widespread use. The early attempts to measure plasma steroids by GC-MS were rapidly surpassed by the introduction of immunoassay techniques with which MS could not compete, especially with respect to analytical run time and cost. However, it was not until recently that it was realized that the lack of specificity of immunoassays might initiate a renaissance for clinical MS techniques in steroid analysis. 

Characteristics of Urinary Steroid Profiling by Gas Chromatography-Mass Spectrometry... 

During early infancy (first 3 mo of life) 3p hydroxy-5-ene steroid sulfates and free or glucuronidated metabolites of cortisol predominate in the urinary steroid profile (Table 2) [8,9]. In infants up to 3 mo of age, separation of steroid glucuronides and free steroids from steroid monosulfates by LH-20 chromatography is necessary prior to quantitative steroid profiling [3]. 

Quantitative urinary steroid profiling by GC-MS using selective ion monitoring (SIM) provides much higher sensitivity, but is a more selective approach. It enables one to further determine aldosterone and 18-hydroxylated cortisol metabolites in hypertension research [10], and in different steroid enzyme deficiencies [3]. 

The urinary steroid excretion changes fundamentally between 4 and 7 mo of age. The Sp hydroxy 5 ene steroids disappear and adult type metabolites of cortisol (5a-THF, THF) become the predominant urinary steroids. At age 6 to 8 yr, the urinary steroid profile is changing again. Increasing amounts of adrenal androgens are excreted in girls and in boys, too. Later, at age 14 to 16 yr, the urinary steroid profile resembles the adult pattern in both sexes [10,13]. The ratio of adrenal androgens to cortisol metabolites is normally <0.8 [14]. 

Table 4 Clinical Indications For the Use of GC-MS Urinary Steroid Profiling...

Gas Chromatography-Mass Spectrometry Urinary Steroid Profiling in Patients with Congenital Enzyme Defects of Steroid Biosynthesis... 

Hydroxylase DeGcIency. The 18-hydroj lase deficiency can present as infection triggered, life threatening salt-loosing state with hyperka lemia in newborns and young infants, ft is characterized by aldosterone biosyn thetic defects both in 18 hydroxlase deficiency (CMO I) and in 18 hydroxysteroid dehydrogenase deficiency (CMO If) [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). 

HydroxysteroidDehydrogenase DeGcIency. In CMO II, the urinary steroid profile shows, in addition to high amounts of corticosterone metabolites, 18 hydrojQ lated corticosterone metabolites (18-OH-THA, 18-OH-... 

AdrenalInsufGclency. Low excretion of glucocorticoid metabolites 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. 

Hydroxysteroid Dehydrogenase DeGciency. Failure to thrive, polyuria, polydipsia, hypertension, hypokalemia, and nephrocalcinosis are the symptoms for apparent mineralocorticoid excess due to lip 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, 5a-THF and free cortisol excretion [32]. 

Cortisone Reductase DeGciency. 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 5a-THF [33]. 

Figure 5 Urinary steroid profile (FID) in 18 hydroxylase deficiency (CMO I). Male, age 7 wk.

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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