Carotenoids and steroidsCholesterol and sex steroid hormones including androgens and estrogens play a critical role in the development and progression of buy anadrol with credit card diseases such as prostate steroids cholesterol and carotenoids are examples of. This disease remains steroids cholesterol and carotenoids are examples of most commonly diagnosed malignant tumor in men and is the leading cause of death from different cancers. Attempts to understand the role of cholesterol and steroid metabolism in urological diseases have been ongoing for many years, but despite this, our mechanistic and translational understanding remains elusive. In order to further evaluate the problem, we have taken an interest in metabolomics; a discipline dedicated to the systematic study of biologically active metabolites in cells, tissues, hair and biofluids. Recently, we provided evidence that a quantitative measurement of cholesterol and sex steroid metabolites can be successfully achieved using hair of human and mouse models. The overall goal of this short review article is to introduce current metabolomic technologies for the quantitative biomarker assay development and also to provide new insight into understanding the underlying mechanisms that trigger the pathological condition. Furthermore, this review will place a particular emphasis on how to prepare biospecimens e.
Metabolic profiling of cholesterol and sex steroid hormones to monitor urological diseases
Cholesterol and sex steroid hormones including androgens and estrogens play a critical role in the development and progression of urological diseases such as prostate cancer. This disease remains the most commonly diagnosed malignant tumor in men and is the leading cause of death from different cancers. Attempts to understand the role of cholesterol and steroid metabolism in urological diseases have been ongoing for many years, but despite this, our mechanistic and translational understanding remains elusive.
In order to further evaluate the problem, we have taken an interest in metabolomics; a discipline dedicated to the systematic study of biologically active metabolites in cells, tissues, hair and biofluids. Recently, we provided evidence that a quantitative measurement of cholesterol and sex steroid metabolites can be successfully achieved using hair of human and mouse models.
The overall goal of this short review article is to introduce current metabolomic technologies for the quantitative biomarker assay development and also to provide new insight into understanding the underlying mechanisms that trigger the pathological condition. Furthermore, this review will place a particular emphasis on how to prepare biospecimens e. Urological health conditions have become increasingly prevalent in the world, affecting individuals spanning a multitude of ages.
Prostate cancer PC is one such condition that has seen an exponential rise in the number of cases, with over , new cases having been recorded in alone. The effects of PC can even be seen outside of the USA, as it is the most common cancer among men in all developed countries.
However, not all urological health problems are related to cancer, and the most common of these include the common occurrence of urinary tract infection, kidney stones, incontinence and benign prostatic hyperplasia BPH. These diseases pose both a financial and physiological burden, indicating the need for further research in the prevention and study of these urological conditions.
The use of biomarkers for early diagnosis in patients would be valuable in reducing the recurrence and progression of these urological health problems. As an example, there are clinical needs for biomarkers to identify PC patients who have aggressive disease and are more likely to experience disease progression, which could help increase the ability to manage patients with urological disease. A correlation has been studied, relating a typical Western diet to an increase in the risk of urological disease Ito , YuPeng et al.
Western diet normally features a high intake of red meat and dairy products, providing a high intake of cholesterol and calories to individuals consuming such foods Ito , YuPeng et al. These studies have also provided evidence that patients with a metabolic syndrome e. Furthermore, cholesterol-lowering drugs such as statins may reduce the risk of PC Nielsen et al.
Sex steroids and their receptors play a crucial role in the determination of urological disease development. Following androgen binding, androgen receptors undergo a multi-step process involving dimerization, phosphorylation and translocation to the nucleus.
In order to better understand the metabolism and internal mechanisms underlying urological diseases, several resources have been studied.
Metabolomic fingerprints have been analyzed in addition to the use of non-invasive biomarkers such as urine or blood-based assays. Hair-based metabolomic profiles could be useful in confirming the correlation between cholesterol and sex steroid hormones with urological diseases.
Using human and animal hair samples in order to study the metabolite process specific to urological disease, our research group established the novel mass spectrometry-based protocols for steroid metabolomics with the goal of monitoring hormone levels, which can be used for drug treatment of PC and BPH patients.
This short review article aims to provide support for the claim that a correlation exists between cholesterol and steroid sex hormones with urological diseases. By specifically examining PC and BHP, we discuss the significance of cholesterol and steroid sex hormones associated with these health conditions, while also introducing current technologies that can be used to measure the amount of cholesterol and sex steroid hormones in various sources throughout the body tissues, urine, blood and hair.
The final topic deals with the use of hair metabolomics to identify potential biomarkers for PC and BPH. Cholesterol synthesis pathways are shown in Fig. Cholesterol is made from the conversion of citrate, derived from the tricarboxylic acid TCA cycle in the mitochondria. Here, acetyl coenzyme A acetyl-CoA is formed and followed by the mevalonate pathway. This combination of reactions is primarily regulated by a rate-limiting step catalyzed by 3-hydroxymethylglutaryl-coenzyme A HMG-CoA reductase, an integral membrane protein of the smooth endoplasmic reticulum, which converts HMG-CoA to a six-carbon intermediate mevalonate.
This intermediate is then metabolized via a series of isoprenoid intermediates to squalene the polymerization of six five-carbon isoprene units to form the carbon linear structure of squalene. The cyclization of squalene reacted by squalene cyclase and one molecule of O 2 forms the four fused rings of the steroid nucleus, which results in the synthesis of lanosterol as a cholesterol precursor. Cholesterol in tissues and blood is metabolized as follows. First, it can be fatty acylated to form cholesteryl esters CEs through sterol O -acyltransferase also called acyl-CoA cholesterol acyltransferase or simple ACAT or lecithin-cholesterol acyltransferase LCAT, also called phosphatidylcholine-sterol O -acyltransferase.
The CEs then serve as a major form of transporter as plasma lipoproteins, or as storage units in the form of lipid droplets Kraemer Secondly, cholesterol can be oxidized to form oxygenated derivatives of cholesterol, termed oxysterols, by enzymatic hydroxylase, cytochrome P CYP families or non-enzymatic hydroxylations at the C-4, C-7, C, C, C, C and C positions Pikuleva , Son et al.
Oxysterols serve as regulators of cholesterol homeostasis, allowing cells to manage large cholesterol loads rapidly and avoid triggering cytotoxic events Bjorkhem , Bielska et al. In addition, the most important oxysterols as transport forms of cholesterol are side chain-oxidized oxysterols at the C or 27 position by CYP46 and CYP27, respectively, which flow continuously from peripheral tissues to the liver and become further oxidized into bile acids or other water-soluble metabolites Nielsen et al.
Cholesterol is also metabolized to steroid hormones, which regulate physiological and pharmacological processes in the body Falkenstein et al. Steroidogenic enzymes are responsible for the biosynthesis of cholesterol from various steroid hormones including corticoids, progestins, androgens and estrogens.
These are generally synthesized in the adrenal cortex, gonads testes and ovaries , brain, placenta and adipose tissue Falkenstein et al. In biosynthetic pathways of steroid hormones, two major types of enzymes are involved: Initially, steroid hormones start with the conversion of cholesterol to pregnenolone by rate-limiting enzyme CYP11A cholesterol side-chain cleavage , which is bound to the inner membrane of the mitochondrion in all steroidogenic tissues.
Cholesterol, a critical component of the cellular plasma membrane, contributes to the maintenance of plasma membrane fluidity. Cholesterol is also the precursor for endogenous sex steroid biosynthesis, suggesting that elevated serum cholesterol levels might be somehow linked to the increased risk of prostate cancer Fagherazzi et al.
Steroid biosynthesis may be an important mechanism linking cholesterol and prostate cancer and BPH. There are multiple enzymes, which actively play roles in cholesterol and sex steroid hormone synthesis. UGT2B15 and UTG2B17 irreversibly inhibit androgen signaling by glucuronidation, which is the known rate-limiting step of androgen signaling. Accumulating evidence demonstrates that elevated cholesterol is a risk factor of more aggressive PC — in terms of recurrence or mortality Platz et al.
Our previous studies also support the hypothesis that cholesterol promotes PC growth in vitro and in vivo Zhuang et al. There are a series of epidemiological studies suggesting that statins could reduce cancer risk Farwell et al. However, several meta-analyses have also reported a null association between statin use and risk of prostate cancer recurrence Mass et al.
As the most common specimens used in biomarker discovery, tissues and biological fluids have been used for metabolite profiling. Both formalin-fixed, paraffin-embedded FFPE and fresh-frozen tissue specimens can be used for the tissue-based metabolomic studies.
H 2 O solution 1: Biofluids such as urine and serum have a great advantage of being the easiest samples to work with, urine being the most common and accessible samples for metabolomic analyses. In order to identify the steroid signatures and to suggest steroid metabolism-associated enzyme activities through profiling of tissues or biofluid-derived metabolites, we can use quantitative mass spectrometry combined with gas or liquid chromatographic separation techniques GC—MS or LC—MS for steroid profiling.
In addition, LC—MS-based steroid profiling enables to quantify 21 endogenous corticoids including urinary glucocorticoids and mineralocorticoids Cho et al. First morning urine samples obtained from 59 BPH patients age: We screened various cholesterol and sex hormones and found that cholesterol level could segregate BPH patients from healthy controls AUC, 0. Our metabolomic profile showing the distinct patterns between prostate cancer and healthy controls. Although urine and blood are widely used to measure sex steroid hormones in many clinical and toxicological studies Wudy et al.
Thus, it can offer the possibility of reflecting and revealing historical information of exposure to drug abuse, environmental toxins and endogenous or exogenous hormones over several months Villain et al.
Moreover, hair sampling is non-invasive and the storage and processing of it is much simpler than plasma or serum Sulek et al. Therefore, hair analysis has been used in chemical toxicology, forensic science, doping control and clinical applications Cho et al. Hair analysis in steroid research is mainly coupled with solubilization or digestion of the hair matrix after cutting Choi et al.
Hence, ultrasonication with an organic solvent was also tested for steroid profiling including androgens, sterols, corticoids and progestins, which enables the profiling of hair steroids Jung et al.
A mixed solvent of methanol and dichloromethane is an effective solvent for lipid extraction from biological samples Folch et al. However, absolute methanol was chosen as the extraction solvent to decrease sample complexity and simplify the sample purification steps.
The hair strands were washed with isopropyl alcohol to prevent contamination and were then obtained simply by cutting the specific lengths from the proximal part of the vertex scalp Jung et al. Thirty milligrams of chopped hair were incubated with 0. After cooling to room temperature, a methanolic solution was diluted with 5. The samples were loaded directly onto the Oasis HLB TM divinylbenzene and N -vinylpyrrolidone solid-phase extraction SPE cartridge, which is preferable for the sample purification of steroidal compounds Moon et al.
The SPE procedure removed more effective interference from the hair matrix and gave a lower matrix background than liquid—liquid extraction LLE Mondul et al. However, these extraction processes require a relatively large amount of hair matrix and extensive, time-consuming pretreatment procedures.
Recently, the pulverization method has been used to highly disintegrate hair components and has allowed for efficient extraction Miyaguchi et al. Compared with our previous techniques Ryu et al. The pulverization of hair using a ball mill such as zirconia beads was achieved for improved extraction yields of steroids and sterols and therefore can reduce sample preparation times as well as sample amounts significantly.
The sample preparation technique is also required for the removal of endogenous matrix components from lipid-rich samples. The hybrid SPE-precipitation cartridge H-PPT applies to reduce the phospholipid-based matrix effect, which is a superior purification method for sterol analysis Pucci et al.
The H-PPT specifically retains phospholipids by Lewis acid—base interactions between the zirconia-coated silica particles bonded to the stationary phase and the phosphate group of the phospholipids, which provides simple yet rapid selective removal of interference Bylda et al.
Bead-assisted liquid—liquid extraction via the addition of methanol and then centrifugation can be achieved simultaneously with pulverization, extraction and protein precipitation Son et al.
Samples were then loaded into H-PPT cartridges and eluted three times with 0. The matrix background such as proteins and phospholipids was easily removed and finally hair sterols were collected. Silylation is the most widely used derivatization reaction in steroid analysis, and trimethylsilyl TMS derivatization is extensively used for most functional groups on steroid backbone, including aliphatic and phenolic alcohols, and carbonyl and amine groups.
Most steroids were monitored using their molecular ions as base peaks. Cholesterol and cholesterol precursors have a hydroxyl group at the C-3 position, and OHCs have two polar functional groups: These results may provide useful information about the chemical structures of cholesterol and its metabolites.
Mixed derivatization is performed to improve physical and chemical properties and mass spectral characteristics. The spectra of flophemesyl derivatives generally display intense molecular ions under electron impact ionization, resulting in enhanced chromatographic selectivity and mass spectral information with sensitive detection Choi et al.
Recently, the enhanced GC—MS analytical selectivity and sensitivity were allowed for quantitative analysis of estrogen metabolites in urine samples obtained from the postmenopausal female patients with osteoporosis Moon et al. It was successfully achieved by two-phase extractive ethoxycarbonylation EOC and subsequent pentafluoropropionyl PFP derivatization. In case of estrogen profiling, the ultra-sensitive LC—MS analytical method has been conducted with a novel chemical derivatization procedure, which formed analytes as pre-ionized N -methyl pyridiniumsulfonyl NMPS derivatives Wang et al.
Furthermore, only single enzymes are estimated at a single time Spiehler , Hsing et al. In contrast, mass spectrometry-based quantification has better reproducibility and generates multi-targeted profiling analysis Cho et al. Several mass spectrometric methods for the measurement of steroids and sterols from various biological matrices have been proposed, coupled to GC Ahmida et al. The LC—MS methods based on electrospray and atmospheric pressure chemical ionization techniques have been conducted with a good sensitivity and chromatographic resolution of estrogens Falk et al.
However, the method often requires sample derivatization with dansyl chloride Falk et al.