Heat-map visualization of gas chromatography-mass spectrometry based quantitative signatures on steroid metabolism

Abstract

Abnormalities in steroid hormones are responsible for the development and prevention of endocrine diseases. Due to their biochemical roles in endocrine system, the quantitative evaluation of steroid hormones is needed to elucidate altered expression of steroids. Gas chromatographic-mass spectrometric (GC-MS) profiling of 70 urinary steroids, containing 22 androgens, 18 estrogens, 15 corticoids, 13 progestins, and 2 sterols, were validated and its quantitative data were visualized using hierarchically clustered heat maps to allow "steroid signatures". The devised method provided a good linearity (r 2 Ͼ 0.994) with the exception of cholesterol (r 2 ϭ 0.983). Precisions (% CV) and accuracies (% bias) ranged from 0.9% to 11.2% and from 92% to 119%, respectively, for most steroids tested. To evaluate metabolic changes, this method was applied to urine samples obtained from 59 patients with benign prostatic hyperplasia (BPH) versus 41 healthy male subjects. Altered concentrations of urinary steroids found and heat maps produced during this 70-compound study showed also differences between the ratios of steroid precursors and their metabolites (representing enzyme activity). Heat maps showed that oxidoreductases clustered (5␣-reductase, 3␣-HSD, 3␤-HSD, and 17␤-HSD, except for 20␣-HSD). These results support that data transformation is valid, since 5␣-reductase is a marker of BPH and 17␤-HSD is positively expressed in prostate cells. Multitargeted profiling analysis of steroids generated quantitative results that help to explain correlations between enzyme activities. The data transformation and visualization described may to be found in the integration with the mining biomarkers of hormone-dependent diseases. Many naturally occurring steroids with similar chemical structures could yield biological information [7]. Endogenous steroids are divided into five groups, namely, androgens, estrogens, corticoids, progestins, and sterols, which are generally synthesized from cholesterol in the adrenal cortex, ovaries, and testes (Scheme 1). In biosynthetic pathways of steroid hormone, two major types of enzymes are involved, cytochrome P450 and steroid oxidoreductase. Abnormalities of these enzymes often lead to hormonal imbalances that have serious consequences, and which are responsible for the development of hormone-dependent diseases (see Supplementary Table 1, which can be found in the electronic version of this article). For example, concentrations of corticoids and their metabolic ratios provide diagnostic evidence of apparent mineralocorticoid excesses caused by 11␤-HSD deficiency [8] and congenital adrenal hyperplasia, which are caused by deficiencies of enzymes like hydroxylase (at C-11, 17, and 21) or 3␤-HSD [9]. In addition, enhanced androgen activity generated by the conversion of testosterone to dihydrotestosterone (DHT) by 5␣-reductase was utilized to allow early therapeutic intervention in young men [10].Enzyme activity profiles can be used to describe the functional diversities of biological systems, which are d...

A novel GC-MS method in urinary estrogen analysis from postmenopausal women with osteoporosis

“…Using the EOC-PFP derivatization, 16 ␣ -OH-E1 was separated completely from 16-keto-E2, whereas they were coeluted in both TMS and EOC-TMS experiments ( 15 ). Both compounds have similar molecular weight and mass fragments.…”

“…spectrometry (GC-MS)-based estrogen methods. These have been developed to measure the endogenous estrogen metabolites in biological specimens using RIA or enzyme immunoassay (EIA) ( 13,14 ), and GC-MS ( 4,(15)(16)(17)(18) or LC-MS (19)(20)(21). Although immunoassays have limited applicability due to an overestimation by cross reactions (22)(23)(24)(25), LC-MS-based profi ling is a proven technique with high sensitivity and selectivity in estrogen analysis combined with chemical derivatization for the phenolic hydroxyl groups of estrogens as follows: a ) pentafl uorobenzyl (PFB) derivatization with ECAPCI-MS ( 21 ), b ) dansyl ( 20 ) and picolinoyl ( 19 ) derivatizations with LC-ESI/MS, and c ) N-methyl-2-pyridyl ( 26 ), 1-(2,4-dinitro-5-fl uorphenyl)-4,4,-dimethylpiperazine ( 27 ), or N-methyl-nicotinyl derivatizations with LC-ESI/MS ( 28 ).…”

“…In high-resolution GC-MS analysis, estrogen analysis is essential in blocking active hydrogens in both phenolic and aliphatic hydroxy groups. For this purpose, perfl uoroacylation and trimethylsilylation are mainly performed ( 4,(15)(16)(17)(18). As a different approach, alkoxycarbonylation, which is carried out with highly reacting agents for amino-, thiol-, imidazole-or phenolic hydroxy groups, can be suitable for direct reactions in aqueous media ( 17,(30)(31)(32)(33)(34).…”

“…When high-resolution capillary GC-MS is used for estrogen analysis (15)(16)(17)(18), it is essential to block the active hydrogens in these polar functional groups. Although TMS derivatives are commonly used in GC-MSbased estrogen analysis ( 15,16 ), TMS derivatives are not sensitive enough for quantifi cation of urinary estrogen metabolites and the derivatization process is time consuming. As a different approach, the direct derivatization in aqueous solution to make estrogen extractable by nonpolar organic solvents was more preferred.…”

Diagnostic Evaluation of Enzyme Activity Related to Steroid Metabolism by Mass Spectrometry-Based Steroid Profiling

“…The biochemical expression of steroid hormones depends on the rate of metabolism of cholesterol (Figure 1), 15 which is biosynthesized via the mevalonate pathway. The mevalonate pathway leads to the production of lanosterol, which can then be converted to cholesterol through either the Bloch pathway, via desmosterol, or the KandutschRussell pathway, via 7-dehydrocholesterol ( Figure 2).…”