Neutral Steroids Research Articles

Dietary Cholest-4-en-3-one, a Cholesterol Metabolite of Gut Microbiota, Alleviates Hyperlipidemia, Hepatic Cholesterol Accumulation, and Hyperinsulinemia in Obese, Diabetic db/db Mice.

Previous studies have shown that dietary cholest-4-en-3-one (4-cholestenone, 4-STN) exerts anti-obesity and lipid-lowering effects in mice. However, its underlying mechanisms are not fully understood. In the present study, we evaluated whether 4-STN supplementation would protect obese diabetic db/db mice from obesity-related metabolic disorders. After four weeks of feeding of a 0.25% 4-STN-containing diet, dietary 4-STN was found to have significantly alleviated hyperlipidemia, hepatic cholesterol accumulation, and hyperinsulinemia; however, the effect was not sufficient to improve hepatic triglyceride accumulation or obesity. Further analysis demonstrated that dietary 4-STN significantly increased the content of free fatty acids and neutral steroids in the feces of db/db mice, indicating that the alleviation of hyperlipidemia by 4-STN was due to an increase in lipid excretion. In addition, dietary 4-STN significantly reduced the levels of desmosterol, a cholesterol precursor, in the plasma but not in the liver, suggesting that normalization of cholesterol metabolism by 4-STN is partly attributable to the suppression of cholesterol synthesis in extrahepatic tissues. In addition, dietary 4-STN increased the plasma and hepatic levels of 4-STN metabolites cholestanol (5α-cholestan-3β-ol) and coprostanol (5β-cholestan-3β-ol). Our results show that dietary 4-STN alleviates obesity-related metabolic disorders, such as hyperlipidemia, hepatic cholesterol accumulation, and hyperinsulinemia, in db/db mice.

Promiscuity and Selectivity of Hydroxysteroid Dehydrogenases in the Biocatalyzed Reduction of 1,2‐Diketones

AbstractHydroxysteroid dehydrogenases (HSDHs) are NAD(P)H‐dependent alcohol dehydrogenases (ADHs) known for their exceptional stereo‐ and regioselectivity when acting on natural substrates, including neutral steroids, bile acids, and various steroid derivatives. Notably, in recent studies this specific subfamily of oxidoreductases has displayed intriguing substrate promiscuity, exhibiting the capacity to accommodate a diverse array of substrates, such as sterically hindered ketones and even α‐keto esters. Herein, the promiscuous nature of HSDHs was further explored by investigating their catalytic activity with representative 1,2‐diketones. This set encompasses symmetric aliphatic/aromatic diketones – namely, 3,4‐hexandione and benzil – as well as the asymmetric synthon 1‐phenyl‐1,2‐propanedione. In the case of 3,4‐hexandione, substrate conversion and selectivity closely resembled that previously observed with aliphatic α‐keto esters. On the contrary, a more heterogeneous behavior was observed in the case of aromatic substrates, with diverse performances in terms of conversions and stereo‐ or regioselectivity. Additionally, docking studies were carried out to get a deeper insight in the stereochemistry of 1,2‐diketones reduction catalyzed by the broad substrate scope and steroid‐active ketoreductase Is2‐SDR.

Fabrication of mixed-mode amphoteric β-cyclodextrin polymer for the simultaneous extraction of acidic and alkaline drugs

A multifunctional mixed-mode β-CD polymer adsorbent PNVCD-SIM containing amphoteric ions and multiple functional groups was prepared. Its physicochemical properties were characterized by scanning electron microscope (SEM), 1H nuclear magnetic resonance (1H NMR), 13C NMR, Fourier transform infrared spectroscopy (FT-IR), nitrogen adsorption–desorption test, elemental analysis (EA), and thermogravimetry and differential scanning calorimetry (TGA-DSC). The acidic indomethacin and alkaline promethazine were selected to evaluate the adsorption properties of PNVCD-SIM at different pH. Under optimal neutral pH conditions, the adsorption performance of PNVCD-SIM on acidic NSAIDs, alkaline phenothiazine, and neutral steroid hormone drugs was investigated. The results showed that PNVCD-SIM exhibited excellent diversified adsorption properties for acidic, alkaline, and neutral mixed drug components through the synergistic effects of β-CD inclusion, electrostatic interaction, hydrogen bond, π-π conjugation, and π-cation effect. The adsorption mechanism was consistent with the pseudo-second-order kinetic model and Langmuir isotherm model. Under the optimized conditions, a dispersive solid phase extraction method combined with high-performance liquid chromatography (dSPE-HPLC) using PNVCD-SIM as the adsorbent was established. The simultaneous extraction and detection of acidic-alkaline multi-component drugs in actual urine and lake water samples were successfully realized. This study provides new insight into the design and preparation of mixed-mode adsorption materials suitable for the rapid analysis and detection of multiple analytes.

Effective sample preparation procedure for the analysis of free neutral steroids, free steroid acids and sterol sulfates in different tissues by GC–MS

Steroids play an important role in cell regulation and homeostasis. Many diseases like Alzheimer’s disease or Smith-Lemli-Opitz syndrome are known to be associated with deviations in the steroid profile. Most published methods only allow the analysis of small subgroups of steroids and cannot give an overview of the total steroid profile. We developed and validated a method that allows the analysis of free neutral steroids, including intermediates of cholesterol biosynthesis, free oxysterols, C19 and C21 steroids, free steroid acids, including bile acids, and sterol sulfates using gas chromatography-mass spectrometry. Samples were analyzed in scan mode for screening purposes and in dynamic multiple reaction monitoring mode for highly sensitive quantitative analysis. The method was validated for mouse brain and liver tissue and consists of sample homogenization, lipid extraction, steroid group separation, deconjugation, derivatization and gas chromatography-mass spectrometry analysis. We applied the method on brain and liver samples of mice (10 months and 3 weeks old) and cultured N2a cells and report the endogenous concentrations of 29 physiological steroids.

Oligonol, a Low-molecular Weight Polyphenol Extracted from Lychee Fruit, Modulates Cholesterol Metabolism in Rats within a Short Period.

There is growing research interest in the hypocholesterolemic effect of various food components such as polyphenols. In this study, we examined the effects of oligonol-a low-molecular weight polyphenol extracted from lychee fruit-on cholesterol metabolism in rats under short-term administration. Administration of oligonol for 3 days significantly increased cecum weight and decreased cecal n-butyric acid concentrations in rats. Oligonol also significantly lowered the levels of hepatic cholesterol and increased the levels of total neutral steroids excreted in the feces. It also increased fecal β-muricholic acid significantly, whereas the levels of total acidic steroids remained unchanged. Gene expression of hepatic CYP7A1 (cytochrome P450 family 7 subfamily A member 1) significantly increased following the administration of oligonol. This increase could be ascribed to changes in the expression of farnesoid X receptor, small heterodimer partner, and fibroblast growth factor 15 in ileum. Our data suggest that oligonol induces hypocholesterolemic effects through the inhibition of biliary cholesterol absorption from the intestine and the upregulation of cholesterol catabolism in rats even following short-term administration. Therefore, oligonol may be an important food component for reducing cholesterol level.

Combined Effect of Diosgenin Along with Ezetimibe or Atorvastatin on the Fate of Labelled Bile Acid and Cholesterol in Hypercholesterolemic Rats.

We analyzed the effect of diosgenin, administered with atorvastatin or ezetimibe, on the fate of 3H(G)-taurocholic acid or 26-14C-cholesterol in hypercholesterolemic rats. Male Wistar rats received a hypercholesterolemic diet (HD), HD + atorvastatin (HD+ATV), HD + ezetimibe (HD+EZT), HD + diosgenin (HD+DG), HD+ATV+EZT, or HD+ATV+DG for 40 days. We also included a control normal group (ND). The labelled compounds were administered on day 30. The animals were placed in metabolic cages for daily feces collection. At day 40 the rats were sacrificed. Lipid extracts from blood, liver, spinal cord, testicles, kidneys, epididymis, intestine, and feces were analyzed for radioactivity. Cholesterol activity was the highest in the liver in HD rats. DG diminished one half of this activity in HD+DG and HD+ATV+DG groups in comparison with the HD group. HD+ATV rats showed four to almost ten-fold cholesterol activity in the spinal cord compared with the ND or HD rats. Fecal elimination of neutral steroids was approximately two-fold higher in the HD+DG and HD+ATV+DG groups. Taurocholic acid activity was four to ten-fold higher in HD+DG intestine as compared to the other experimental groups. Taurocholic activity in the liver of HD and HD+DG groups was two and a half higher than in ND. Our results show that the combination of DG and ATV induced the highest cholesterol reduction in the liver and other tissues.

Lactoferrin interacts with bile acids and increases fecal cholesterol excretion in rats.

Lactoferrin (LF) is a multifunctional cationic protein (pI 8.2-8.9) in mammalian milk. We previously reported that enteric-LF prevented hypercholesterolemia and atherosclerosis in a diet-induced atherosclerosis model using Microminipig, although the underlying mechanisms remain unclear. Because LF is assumed to electrostatically interact with bile acids to inhibit intestinal cholesterol absorption, LF could promote cholesterol excretion. In this study, we assessed the interaction between LF and taurocholate in vitro, and the effect of LF on cholesterol excretion in rats. The binding rate of taurocholate to LF was significantly higher than that to transferrin (pI 5.2-6.3). When rats were administered a high-cholesterol diet (HCD) containing 5% LF, LF was detected using ELISA in the upper small intestine from 7.5 to 60 min after the administration. Rats were fed one of the following diets: control, HCD, or HCD + 5% LF for 21 days. Fecal neutral steroids and hepatic cholesterol levels in the HCD group were significantly higher than those in the control group. The addition of LF to a HCD significantly increased fecal neutral steroids levels (22% increase, p < 0.05) and reduced hepatic cholesterol levels (17% decrease, p < 0.05). These parameters were inversely correlated (R = -0.63, p < 0.05). These results suggest that LF promotes cholesterol excretion via interactions with bile acids.

Hypolipidemic potential of squid homogenate irrespective of a relatively high content of cholesterol.

BackgroundOur previous study has shown that regardless of a relatively high amount of cholesterol, squid homogenate lowers serum and hepatic cholesterol in animals. Since this work, we have developed a new method to inhibit autolysis of squid proteins with sodium citrate. This study aims to investigate how squid homogenate prepared with sodium citrate affects lipid metabolism in Sprague–Dawley rats at the molecular level.MethodsWe prepared squid homogenate with sodium citrate to inhibit autolysis of squid protein. In Experiment 1 (Exp. 1), rats were given a cholesterol-free control diet or a squid diet, with squid homogenate added at the level of 5% as dietary protein for 4 weeks. Blood, the liver and adipose tissue were taken after 6 hours fasting. Serum and hepatic lipids and activities of enzymes related to lipid metabolism were measured. In Experiment 2 (Exp. 2), the above-mentioned diets had cholesterol added at the level of 0.1% and given to rats. Lipid parameters, enzyme activities, and gene expression of proteins involved in lipid metabolism in the liver and the small intestine were determined. In addition, feces were collected for two days at the end of Exp. 2 to measure fecal excretion of steroids.ResultsIn Exp.1, serum triglyceride and cholesterol were ~50% and ~20% lower, respectively, in the squid diet-fed rats than in the control diet-fed animals while hepatic cholesterol was ~290% higher in the squid diet-fed rats. When cholesterol was included into the diets (Exp. 2), serum lipids were significantly lower in the squid group while no difference of hepatic lipid was seen between two groups. Activities of hepatic lipogenic enzymes were significantly lower in rats on the squid diet while the enzyme responsible for fatty acid oxidation was not modified (Expt. 1 and 2). Hepatic level of mRNA of microsomal triglyceride transfer protein was significantly lower in the squid group. In the small intestine, the squid diet exhibited significantly lower gene expression of proteins involved in fatty acid transport and cholesterol absorption. Fecal secretion of acidic steroids, but not neutral steroids, was higher in rats fed the squid diet than in those fed the control diet.ConclusionThese results imply that newly-developed squid homogenate has hypolipidemic potential primarily through decreased absorption of bile acids in the small intestine and suppressed lipogenesis in the liver.

Facile Fabrication of Polymeric Ionic Liquid Grafted Porous Polymer Monolith for Mixed‐Mode High Performance Liquid Chromatography

AbstractA novel polymeric ionic liquid grafted porous polymer monolith has been facilely fabricated for mixed‐mode chromatography. The column is prepared from poly (glycidyl methacrylate‐co‐ethylene dimethacrylate) monolith through hydrolyzation of the epoxy moieties into hydroxyl groups, followed by "grafting from" polymerization of ionic liquid of 1‐vinyl‐3‐butylimidazolium chloride. Successful modification is characterized by scanning electron microscope, infrared spectroscopy, elemental analysis and mercury intrusion porosimetry. The HPLC performance of developed column is evaluated by separating acidic vitamin B analytes, neutral steroids and basic aromatic amines in mixed‐mode chromatography on a single column, respectively. The ionic liquid affords the monolith with both enhanced separation ability and improved column efficiency.

Stacking and Separation of Neutral and Cationic Analytes in Interface-Free Two-Dimensional Heart-Cutting Capillary Electrophoresis

Interface-free two-dimensional heart-cutting capillary electrophoresis is typically used to purify/concentrate and separate a single target fraction. Here, we demonstrate the simultaneous stacking and orthogonal separation of target fractions that belong to two different classes of compounds. The cationic species were first analyzed by capillary zone electrophoresis using an electrolyte of low pH in a fused-silica capillary. The neutrals that remained inside the capillary after the first dimension analysis were then separated by micellar electrokinetic chromatography using sodium dodecyl sulfate micelles that were electrophoretically introduced into the capillary. The suppression of electro-osmotic flow using low pH was essential in shifting the separation selectivity from electrophoresis to electrokinetic chromatography. Two approaches to stacking based on the solubility of the species present were developed. Stacking was achieved during the first dimension analysis via sweeping and/or analyte focusing by micelle collapse. Two artificial mixtures (eight cationic drugs/five neutral steroids, and three quaternary ammonium/three neutral organophosphate pesticides) were successfully analyzed with sensitivity enhancement factors from 15 to 100. Some analytical parameters and application to a spiked real water sample were also studied.

Au nanoparticle grafted thiol modified magnetic nanoparticle solid phase extraction coupled with high performance liquid chromatography for determination of steroid hormones in human plasma and urine

A new, simple, rapid and sensitive preconcentration method was developed for separation of two neutral steroids (progesterone and testosterone) through an off-line extraction/preconcentration procedure using Au nanoparticles grafted on a 3-(trimethoxysilyl)-1-propanethiol modified magnetic nanoparticle adsorbent prior to their determination by HPLC-UV. The preconcentration step was optimized by a comprehensive study on the main factors affecting the extraction/preconcentration efficiency of the steroids such as pH value, amount of surfactant, amount of magnetic adsorbent, sample volume, desorption conditions and ionic strength. The validity of the method was investigated and good analytical performance was obtained including a wide dynamic range of 0.1–200 ng mL−1, low detection limits of 0.05 and 0.07 ng mL−1, and good precision (as RSD%) lower than 3.90 and 4.19% for progesterone and testosterone, respectively. The method was applied to determine the steroidal hormones in human plasma and urine samples. The levels were found to be 0.8 and 9.2 ng mL−1 for progesterone and 7.9 and 97.5 ng mL−1 for testosterone in plasma and urine samples, respectively, which are within the normal range reported in the literature.

Medium-Chain Fatty Acids Enhanced the Excretion of Fecal Cholesterol and Cholic Acid in C57BL/6J Mice Fed a Cholesterol-Rich Diet

The objective of the present study was to investigate the cholesterol-reducing effect of medium-chain fatty acids (MCFAs) completed by elevated excretion of fecal neutral steroids and/or bile acids. Blood and liver lipid profiles, fecal neutral steroids, bile acids, and mRNA and protein expression of the genes relevant to cholesterol homeostasis were measured and analyzed in C57BL/6J mice fed a cholesterol-rich diet with 2% caprylic acid or capric acid for 12 weeks. Blood total cholesterol and low-density lipoprotein cholesterol (LDL-c) levels were reduced significantly as compared to diet with palmitic acid or stearic acid. Caprylic acid promoted the excretion of fecal neutral steroids, especially cholesterol. The excretion of fecal bile acids, mainly in the form of cholic acid was enhanced and accompanied by elevated expression of mRNA and the protein of hepatic cholesterol 7α-hydroxylase (CYP7A1). These results indicate that MCFAs can reduce blood cholesterol by promoting the excretion of fecal cholesterol and cholic acid.

Inulin-Type Fructans with Different Degrees of Polymerization Improve Lipid Metabolism but Not Glucose Metabolism in Rats Fed a High-Fat Diet Under Energy Restriction

Inulin-type fructan ameliorates metabolic diseases associated with obesity in animals. However, relatively little information is available on the comparative effects of inulins with different degree of polymerization (DP) on the lipid or glucose metabolism. The objective of this study was to investigate the effect of inulins with various DP on metabolic disorders associated with obesity in rats fed a high-fat diet under food restriction. Rats were fed a high-fat diet supplemented with 5 % inulin-GR (Raftiline GR), inulin-Tokachi (Tokachi), or inulin-HP (Raftiline HP) without cellulose for 28 days at normal energy intakes or 14.5 % energy restriction. Under food restriction, the dietary inulin-Tokachi (mean DP 15) and -HP (mean DP 24), but not -GR (mean DP 10), reduced (p < 0.05) the serum cholesterol and triglyceride levels, and liver triglyceride concentration in rats, compared to the control diet. The cecal neutral steroid, bile acid, and propionate concentrations in the Tokachi and HP groups were higher (p < 0.05) than in the CONT group, and the cecal Bifidobacterium count in the Tokachi group was higher (p < 0.05) than in the other groups. Findings suggest that, depending on DP, dietary supplementation with inulin (DP 15 or DP 24) in rats fed a high-fat diet, regardless of food intake, positively modulates lipid metabolism and fecal microbiota but not glucose metabolism.

In-capillary formation of polymer/surfactant complexes–assisted reversed-migration micellar electrokinetic chromatography for facile analysis of neutral steroids

In this study we developed a novel approach, using in-capillary formation of polymer/surfactant complexes (IPSC)–assisted reversed-migration MEKC (RM-MEKC), for the analysis of neutral steroids. This process involved two sequential events: in-capillary polymer/surfactant complexes formation during sample preconcentration, followed by IPSC separation. The procedure began with a polymer-filled capillary. Initially, on-line preconcentration of the sample was performed at the sample plug. Meanwhile, free surfactants migrated to interact with polymers, forming polymer–surfactant complexes. Analytes were then kinetically partitioned between the mixed phases (micelles and polymer–SDS complexes). Sodium dodecyl sulfate (SDS) and poly(N-isopropylacrylamide) (PNIPAAm) were employed as pseudo-stationary phases (PSPs). This system allowed the successful separation of five steroids (testosterone, hydrocortisone 21-acetate, dexamethasone, prednisolone, hydrocortisone) in acetate buffer and the determination of urinary free hydrocortisone; it also exhibited excellent performance for sample on-line concentration. The limit of detection for hydrocortisone was 20.98ng/mL (R2=0.9995). The polymer size, concentrations, end-group charges, and SDS concentrations were evaluated. This IPSC/RM-MEKC system, which can be adopted in commercial CE instruments, is easy to operate, suitable for combination with several sample preconcentration options, sensitive, robust, and environmentally sustainable. We suspect that such systems might have potential applications in clinical analyses and in microanalytical devices.

Effect of Cheonggukjang made with germinated soybean on lipid contents and fecal excretion of neutral steroids in rats fed a high cholesterol diet

This study was conducted to investigate the effect of cheonggukjang made with soybean germinated under dark and light conditions on lipid content and fecal neutral steroids in rats fed with a high cholesterol diet. Rats were divided into 4 groups: high cholesterol diet (HC); high cholesterol diet containing normal cheonggukjang (HNC); high cholesterol diet containing cheonggukjang made with soybean germinated under dark condition (HDC); high cholesterol diet containing cheonggukjang made with soybean germinated under light condition (HLC). Dietary supplementation with light-reacted cheonggukjang decreased hepatic triglycerides, compared with the other groups. Fecal excretion of total lipids and triglycerides increased when fed with cheonggukjang made with soybean germinated under light condition. Fecal excretion of coprostanol, cholesterol, and coprostanone tended to increase in the cheonggukjang-supplemented groups. These results suggest that dietary supplementation with cheonggukjang made with germinated soybean might prevent hyperlipidemia by improving the lipid metabolism through decreasing the liver triglycerides contents and increasing fecal excretion of triglycerides.

Dietary Combination of Fish Oil and Hemoglobin Hydrolysates Alters Serum and Liver Lipid Contents in Rat

Fish oil has been shown to have anti-inflammatory and anti-cancer effects, and to reduce serum triacylglycerol (TAG) levels by stimulating lipid oxidation and inhibiting lipogenesis in the liver. A small number of studies have demonstrated the synergistic effect of fish oil and other bioactive components. This study examined the effect of fish oil in combination with porcine hemoglobin (Hb) hydrolysates on serum and liver lipid contents in rats. Male Wistar rats were divided into four groups; one group was fed a casein and soybean oil-based semi-purified basal diet and other three groups were fed the basal diet supplemented with 2% fish oil, 0.175% Hb hydrolysates, and 2% fish oil plus 0.175% Hb hydrolysates, respectively, for 4 weeks. The fish oil diet decreased serum and liver TAG contents but did not change serum and liver cholesterol levels. The dietary combination of fish oil and Hb hydrolysates decreased serum and liver TAG and cholesterol contents owing to the additive effect of both compounds, and this diet reduced the serum non-high-density lipoprotein cholesterol content as a result of a synergistic effect. This hypolipidemic effect was in part caused by enhanced excretion of fecal fatty acids, neutral steroids, and acidic steroids. The results of this study suggest that the combined intake of fish oil and Hb hydrolysates may play beneficial roles in the prevention of cardiovascular disease as compared with fish oil alone.

Total Cholesterol and Triglyceride Levels in Patients with Breast Cancer

Total Cholesterol and Triglyceride Levels in Patients with Breast Cancer

Plasma low-density lipoprotein immobilized silica as stationary phase in nano-liquid chromatography

The feasibility to attach plasma low-density lipoprotein (LDL) particles covalently onto 5μm silica particles and use them as stationary phase in nano-liquid chromatography (nano-LC) for interaction and oxidation studies was clarified. Before the immobilization, both epoxy silica and aldehyde-activated particles were synthesized, LDL was immobilized to the particles via its protein component and capillary columns were packed with LDL-modified silica materials. The performance of the capillary columns was tested with neutral steroids, and the column with LDL immobilized to aldehyde-activated silica was selected for further studies due to its stronger retention toward steroids. The retention factors of the steroids were used as indicators of the column stability, and the RSDs from 0.8 to 5.7% (n=12) for 168 successive runs within 14 days carried out in the same capillary column and from 0.8 to 3.6% (n=6) in three different capillaries demonstrated that the capillary column was stable and that the capillary column-to-capillary column reproducibility was good. The lifetime of LDL-modified silica stationary phase was around 14 days. The applicability of the column for the separation of steroids and β-blockers was based mainly on the hydrophobic interactions with lipids of LDL in the stationary phase. The LDL immobilized silica column was successfully exploited also in the copper-mediated in situ oxidation of LDL. The results achieved demonstrated that nano-LC with plasma LDL immobilized silica phase can be exploited as nano-biomimicking tool for interaction studies and as a microreactor for oxidation studies with low consumption of reagents and human materials.

LC-ESI-MS/MS Analysis of Testosterone at Sub-Picogram Levels Using a Novel Derivatization Reagent

Testosterone analysis by LC-MS/MS is becoming the analytical method of choice over immunoassays due to its specificity and accuracy. However, neutral steroid hormones possess poor ionization efficiency in MS/MS, resulting in insufficient sensitivity for analyzing samples with trace concentrations of the hormones. The method presented here utilizes a derivatization step involving a novel, permanently charged, quaternary aminooxy (QAO) reagent or MS-tag that reacts to the ketone functionality of testosterone and significantly enhances its ESI-MS/MS sensitivity. This derivatization method enabled quantitation of total testosterone in human serum (200 μL) with a lower limit of quantitation (LLOQ) of 1 pg/mL (3.47 pmol/L), total testosterone in dried blood spots (8-10 μL) with a LLOQ of 40 pg/mL, and free testosterone in serum ultrafiltrate (400 μL) with a LLOQ of 0.5 pg/mL. The linearity of each of the high sensitivity applications was maintained over a broad dynamic range of 1-5000 pg/mL for the serum samples and 40-10,000 pg/mL for the dried blood spots (DBS) with R(2) >0.998. The %CV at the LLOQ was <15 for all applications. The QAO derivatization and sample preparation workflows are quick, simple, and robust. Comparison of the derivatization method with an LC-ESI-MS/MS nonderivatization method yielded high correlation and agreement. The derivatization reagent is universal and reacts with other compounds containing ketone or aldehyde functionality.

Multiple mechanisms of ligand interaction with the human organic cation transporter, OCT2

OCT2 is the entry step for organic cation (OC) secretion by renal proximal tubules. Although many drugs inhibit OCT2 activity, neither the mechanistic basis of their inhibition nor their transport status is generally known. Using representatives of several structural classes of OCT2-inhibitory ligands described recently (Kido Y, Matsson P, Giacomini KM. J Med Chem 54: 4548-4558, 2011), we determined the kinetic basis of their inhibition of 1-methyl-4-phenylpyridinium (MPP) transport into Chinese hamster ovary cells that stably expressed hOCT2. The "cluster II" inhibitors (which contain known OCT2 substrates) metformin and cimetidine interacted competitively with MPP. However, other cluster II compounds, including tetraethylammonium (TEA), diphenidol and phenyltoloxamine, were mixed-type inhibitors of MPP transport (i.e., decreasing J(max) and increasing K(t)). A cluster III (neutral steroid) representative, adrenosterone, and a cluster I (large, flexible cation) representative, carvedilol, displayed noncompetitive inhibitory profiles. Competitive counterflow (CCF) was used to determine whether the inhibitory ligands served as substrates of hOCT2. Carvedilol (cluster I) and adrenosterone (cluster III) did not support CCF, consistent with the prediction that members of these structural classes are likely to be nontransported inhibitors of OCT2. The cluster II representatives MPP, metformin, cimetidine, and TEA all supported CCF, consistent with independent assessments of their OCT2-mediated transport. However, the other cluster II representatives, diphenidol and phenyltoloxamine, failed to support CCF, suggesting that neither compound is transported by OCT2. An independent assessment of diphenidol transport (using liquid chromatography with tandem mass spectroscopy) confirmed this observation. The results underscore the caution required for development of predictive models of ligand interaction with multidrug transporters.