Direct quantitative digital autoradiography—thin-layer chromatography of 3α,3β- and 5α-reduced and 17β-dehydrogenated androgens derived from testosterone metabolism

Vingler, Philippe; Kermici, M.; Krien, Paul

doi:10.1016/0378-4347(91)80435-f

Cited by 9 publications

(3 citation statements)

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“…Determination of cortisol in human plasma after derivatization with isonicotinic acid hydrazide was performed to improve sensitivity of TLC method [108]. Direct quantitative determination of eight major androgens including testosterone, epiandrosterone was performed by Vingler [109]. Zarzycki et al proposed optimization process for fractionation of selected steroids using micro thin-layer chromatography [110,111].…”

Section: Thin-layer Chromatography Tlcmentioning

confidence: 99%

“…Nevertheless, the immunoassay methods continue to be employed for steroid determination in the clinical laboratories due to their automation attained thanks to commercially available kits. Steroids can be analyzed and determined using different chromatographic techniques, including thin layer chromatography (TLC) [105][106][107][108][109][110][111][112][113], gas chromatography (GC) [114][115][116] and high performance liquid chromatography (HPLC) . The electromigration techniques make use of many methods, that is: capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), capillary gel electrophoresis (CGE), capillary isotachophoresis (CITP), micellar electrokinetic chromatography (MEKC) (also called micellar electrocinetic capillary chromatography (MECC), and capillary electrochromatography (CEC).…”

Section: Separation Techniquesmentioning

confidence: 99%

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Plasma Steroid Level Measured Using Modern Separation Techniques as Biomarkers in Biological Diagnostics

Konieczna¹,

Bączek

2010

CPA

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During the last decade there has been an increased focus on the application of steroids as biomarkers in biomedical practice. The analysis of steroid hormones in biological samples of plasma or serum is currently routinely used in clinical diagnosis being an essential source of information on not only metabolic pathways, but also disorders of the metabolism. More importantly, the steroid hormones may reduce cancer development on the endocrinal basis, reveal abuse of anabolic substances, or even depression incidences. In the case of biomedical research, quantitative determination of steroids in serum or plasma creates an opportunity to diagnose diseases efficiently in their early stages and to monitor a patient for a possible recurrence of a disease after therapy. Therefore, an accurate measurement of steroids in the plasma or serum has become important for the contemporary medicine, even if troublesome especially in view of their low concentration in biological samples.The most common methods of steroid quantification in clinical practice nowadays include immunoassays such as radioimmunoassay or enzyme immunoassay. However, the main disadvantage of the immunoassay techniques consists in cross reactivity of the antibodies used in the assay with the related hormones. On the other hand, there is a number of applications where the analysis is performed with the use of modern separation techniques.The aim of this review is to present the development and applicability of the various analytical methods, all based on separation techniques and used for determination of steroids in the clinical laboratory, taking into account the recent progress in those areas. The review shows the advantages of high-performance liquid chromatography applied to determine non-volatile and thermally labile steroids and steroid conjugates. It also demonstrates that the currently used mass spectrometry offers practically useful structural information on the composition of steroids. A number of techniques and methods are compared and critically discussed pondering finally their specific analytical requirements like sensitivity, specificity, simplicity, limit of detection, and quantification. Nonetheless, plasma or serum sampling seems to remain one of the most convenient methods for population screening purposes.

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Section: Thin-layer Chromatography Tlcmentioning

confidence: 99%

Section: Separation Techniquesmentioning

confidence: 99%

Plasma Steroid Level Measured Using Modern Separation Techniques as Biomarkers in Biological Diagnostics

Konieczna¹,

Bączek

2010

CPA

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“…20 pi of the rcdissolvcd mixture were applied to silica gel thin-layer chromatography (TLC) plates which were then developed with two ascents of a benzene:ethanol (9:1 vol/ vol) solvent system [17], After the plates had been run, the reference steroids were visualized with iodine vapour, and the radioactivity was detected using an automatic TLC linear analyzer (Berthold) and quantified as a percentage of total radioactivity by integration of the peaks using a multichannel analyser (Berthold) [24], Aliquots of the rcdissolvcd mixture were also analysed using a liquid scintillation counter (Wallac): counting efficiency and counting time were 37% and 5 min, respectively. In a number of plates corresponding to sev eral experiments, the areas of one radioactive peak, not coincidental with the reference steroids used, were scraped off and eluted with chloroformimethanol (2:1 vol/vol).…”

Section: Metabolism O F 3p-diolmentioning

confidence: 99%

Metabolism of 5α-Androstane-3β, 17β-diol in Bald and HairyAreas of the Scalp

Caballero¹

1994

Horm Res

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In androgen target tissues, testosterone undergoes extensive biotransformation to 5α-dihydrotestosterone which in turn is metabolized into 5α-androstane-3α, 17β-diol and 5α-androstane-3β, 17β-diol. From studies mainly in the prostate, it has been shown that 5α-androstane-3β, 17β-diol can be further converted to 5α-androstane-3β,6α(β)/7α(β), 17β-triols. The presentstudies show that among the metabolites of 5α-androstane-3β, 17β-diol in scalp homogenates, three were isolated by thin-layer chromatography and identified against standard steroids: 5α-dihydrotestosterone, epiandrosterone, and 5α-androstanedione. Another peak was observed near the origin with a greater polarity than 5α-androstane-3β, 17β-diol. The chromatograms obtained by incubating 5α-androstane-3β,17β-diol with scalp and prostate homogenates are similar, and it is known that the polar metabolites in the prostate are 5α-androstane-3β,6α(β)/7α(β),17β-triols, suggesting that these steroids are formed in both tissues. In addition, the similarity of chromatograms and mass spectra, obtained when 3β-diol polar metabolite(s) and authentic 5α-androstane-3β,7β,17β-triol were subjected to GC/MS, confirms this hypothesis. The dependence of the formation of polar metabolites on the substrate (5α-androstane-3β,17β-diol) concentration (50 nM-1.3 μM) was studied and resulted in saturation and linear Lineweaver plots from both bald and hairy areas of the scalp. The mean Michaelis constant (K_m) was 1 μM for both areas, and the maximal metabolic rates (V_max) were 10.9 ± 6.2 pmol/mg protein/h (n = 9) in bald scalp and 8.2 ± 0.1 pmol/mg protein/h (n = 3) in hairy scalp. The other metabolic rates of formation were: 25.8 ± 19.5-38.4 ± 17.9 pmol/mg protein/h for epiandrosterone, 45.8 ± 40.9-8.4 ± 2.2 pmol/mg protein/h for 5α-dihydrotestosterone, and 8.3 ± 2.6-9.2 ± 1.0 pmol/mg protein/h for androstanedione in bald and hairy scalp, respectively. As 5α-androstane-3β,6α(β)/7α(β),17β-triols do not possess androgenic properties, they are generally considered to be final metabolites, so that their production rate could be an important factor controlling the amount of 5α-androstane-3β, 17β-diol inside the target cell.

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Planar Chromatography in Clinical Chemistry

Jain

Sherma

2000

Encyclopedia of Analytical Chemistry

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Planar chromatography (PC) like any other chromatographic technique is a multistage distribution process. It is a form of liquid chromatography in which the stationary phase is supported on a planar surface rather than a column. Separations in PC occur because of differential migration velocities through the sorbent layer in a fixed separation time. The sample zones are fixed in space at the completion of the separation, allowing off‐line monitoring. There are two planar techniques: paper chromatography and thin‐layer chromatography (TLC). In recent years, TLC has superseded paper chromatography owing to greater speed, versatility, and reproducibility. Improvements in techniques and equipment as well as the ready availability of high‐quality plates with uniform layers have extended and increased the use of TLC in clinical chemistry and other disciplines, offering quantitative as well as qualitative analysis. High‐performance thin‐layer chromatography (HPTLC), in particular, has developed to the extent that separation and quantitation can provide results that are comparable with other analytical methods such as high‐pressure liquid chromatography (HPLC). The widespread application of TLC is also due to the fact that it is simple, robust, and flexible, and has a large sample throughput. Furthermore, coupled spectroscopic methods (TLC/mass spectrometry (TLC/MS), TLC/Fourier transform infrared spectrometry (TLC/FTIR) etc.) have considerably enhanced the reproducibility of TLC. TLC is often critically compared with column chromatography such as HPLC. This is a matter of endless dispute. Each technique has its own advantages and limitations. TLC is only inexpensive when qualitative results are needed. When reliable quantitative data are required, TLC is as expensive as column chromatography. In the authors' opinion, complementary rather than competitive chromatography offers the best solution to analytical requirements.

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Direct quantitative digital autoradiography—thin-layer chromatography of 3α,3β- and 5α-reduced and 17β-dehydrogenated androgens derived from testosterone metabolism

Cited by 9 publications

References 25 publications

Plasma Steroid Level Measured Using Modern Separation Techniques as Biomarkers in Biological Diagnostics

Plasma Steroid Level Measured Using Modern Separation Techniques as Biomarkers in Biological Diagnostics

Metabolism of 5α-Androstane-3β, 17β-diol in Bald and HairyAreas of the Scalp

Planar Chromatography in Clinical Chemistry

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Direct quantitative digital autoradiography—thin-layer chromatography of 3α,3β- and 5α-reduced and 17β-dehydrogenated androgens derived from testosterone metabolism

Cited by 9 publications

References 25 publications

Plasma Steroid Level Measured Using Modern Separation Techniques as Biomarkers in Biological Diagnostics

Plasma Steroid Level Measured Using Modern Separation Techniques as Biomarkers in Biological Diagnostics

Metabolism of 5&alpha;-Androstane-3&beta;, 17&beta;-diol in Bald and HairyAreas of the Scalp

Planar Chromatography in Clinical Chemistry

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Metabolism of 5α-Androstane-3β, 17β-diol in Bald and HairyAreas of the Scalp