A fast UPLC-HILIC method for an accurate quantification of dendrogenin A in human tissues.

Dendrogenin A (DDA) is a newly-discovered steroidal alkaloid, which remains to date the first ever found in mammals. DDA is a cholesterol metabolites that induces cancer cell differentiation and death in vitro and in vivo, and thus behave like a tumor suppressor metabolite. Preliminary studies performed on 10 patients with estrogen receptor positive breast cancers (ER(+)BC) showed a strong decrease in DDA levels between normal matched tissue and tumors. This suggests that a deregulation on DDA metabolism is associated with breast carcinogenesis. To further investigate DDA metabolism on large cohorts of patients we have developed an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS) procedure for the quantification of DDA in liquid and in solid tissues. This method enabled the identification of DDA analogues such as its geometric isomer C17 and dendrogenin B (C26) in human samples showing that other 5,6α-epoxycholesterol conjugation products with biogenic amines exist as endogenous metabolites . We report here the first complete method of quantification of DDA in liquid and solid tissues using hydrophilic interaction liquid chromatography (HILIC). Two different methods of extraction using either a Bligh and Dyer organic extraction or protein precipitation were successfully applied to quantify DDA in solid and liquid tissues. The protein precipitation method was the fastest. The fact that this method is automatable opens up possibilities to study DDA metabolism in large cohorts of patients.

Developing an Enzyme-Assisted Derivatization Method for Analysis of C27 Bile Alcohols and Acids by Electrospray Ionization-Mass Spectrometry.

Enzyme-assisted derivatization for sterol analysis (EADSA) is a technology designed to enhance sensitivity and specificity for sterol analysis using electrospray ionization⁻mass spectrometry. To date it has only been exploited on sterols with a 3ß-hydroxy-5-ene or 3ß-hydroxy-5α-hydrogen structure, using bacterial cholesterol oxidase enzyme to convert the 3ß-hydroxy group to a 3-oxo group for subsequent derivatization with the positively charged Girard hydrazine reagents, or on substrates with a native oxo group. Here we describe an extension of the technology by substituting 3α-hydroxysteroid dehydrogenase (3α-HSD) for cholesterol oxidase, making the method applicable to sterols with a 3α-hydroxy-5ß-hydrogen structure. The 3α-HSD enzyme works efficiently on bile alcohols and bile acids with this stereochemistry. However, as found by others, derivatization of the resultant 3-oxo group with a hydrazine reagent does not go to completion in the absence of a conjugating double bond in the sterol structure. Nevertheless, Girard P derivatives of bile alcohols and C27 acids give an intense molecular ion ([M]⁺) upon electrospray ionization and informative fragmentation spectra. The method shows promise for analysis of bile alcohols and 3α-hydroxy-5ß-C27-acids, enhancing the range of sterols that can be analyzed at high sensitivity in sterolomic studies.

Quantitative analysis of the tumor suppressor dendrogenin A using liquid chromatography tandem mass spectrometry.

Dendrogenin A (DDA) was recently identified as a mammalian cholesterol metabolite that displays tumor suppressor and neurostimulating properties at low doses. In breast tumors, DDA levels were found to be decreased compared to normal tissues, evidencing a metabolic deregulation of DDA production in cancers. DDA is an amino-oxysterol that contains three protonatable nitrogen atoms. This makes it physico-chemically different from other oxysterols and it therefore requires specific analytical methods We have previously used a two-step method for the quantification of DDA in biological samples: 1) DDA purification from a Bligh and Dyer extract by RP-HPLC using a 250×4.6mm column, followed by 2) nano-electrospray ionization mass spectrometry (MS) fragmentation to analyze the HPLC fraction of interest. We report here the development a liquid chromatography tandem mass spectrometry method for the analysis of DDA and its analogues. This new method is fast (10min), resolving (peak width <4s) and has a weak carryover (<0.01%). We show that this technique efficiently separates DDA from its C17 isomer and other steroidal alkaloids from the same family establishing a proof of concept for the analysis of this family of amino-oxysterols.

Improvement of 5,6α-epoxycholesterol, 5,6ß-epoxycholesterol, cholestane-3ß,5α,6ß-triol and 6-oxo-cholestan-3ß,5α-diol recovery for quantification by GC/MS.

5,6α-epoxycholesterol (5,6α-EC) and 5,6ß-epoxycholesterol (5,6ß-EC) are oxysterols involved in the anticancer pharmacology of the widely used antitumor drug tamoxifen. They are both metabolized into cholestane-3ß,5α,6ß-triol (CT) by the cholesterol-5,6-epoxide hydrolase (ChEH) enzyme, and CT is metabolized by an as-yet uncharacterized enzyme into 6-oxo-cholestan-3ß,5α-diol (OCDO). A recent feasibility study showed that the 5,6-ECs may represent surrogate markers of tamoxifen activity in breast cancer patients undergoing endocrine therapy, thus there is a growing interest in their accurate quantification. These oxysterols are usually quantified by gas-liquid chromatography coupled to mass spectrometry (GC/MS), using an isotope dilution methodology with the corresponding deuterated oxysterol. This method is considered to be relative quantitative since all of the standards used are deuterated oxysterols, however it is not known whether the preparation of each oxysterol is affected in the same way by the extraction, pre-purification by solid phase extraction (SPE) and trimethylsilylation steps, particularly when using biological samples that contain many other reactive compounds. Thus, in this study we investigated the yield of the 5,6-ECs, CT and OCDO recovery from patient serum samples at different stages of their work-up and trimethylsilylation prior to GC/MS analysis, using [14C]-labeled analogs to follow these oxysterols at each step. We measured a 40 to 60% loss of material for the 5,6-ECs and OCDO, however we also describe the conditions that improved their recovery. Our data also show that the use of deuterated 5,6α-EC, 5,6ß-EC, CT and OCDO is an absolute requirement for their accurate quantification.

Sterol ratios as a tool for sewage pollution assessment of river sediments in Serbia.

In this work, source pollution tracing of the sediments of the Danube River and its tributaries in Serbia was performed using sterol ratios. Improved liquid chromatography-tandem mass spectrometry method, which enabled complete chromatographic separation of four analytes with identical fragmentation reactions (epicoprostanol, coprostanol, epicholestanol and cholestanol), was applied for the determination of steroid compounds (hormones, human/animal and plant sterols). A widespread occurrence of sterols was identified in all analyzed samples, whereas the only detected hormones were mestranol and 17α-estradiol. A human-sourced sewage marker coprostanol was detected at the highest concentration (up to 1939 ng g(-1)). The ratios between the key sterol biomarkers, as well as the percentage of coprostanol relative to the total sterol amount, were applied with the aim of selecting the most reliable for distinction between human-sourced pollution and the sterols originated from the natural sources in river sediments. The coprostanol/(cholesterol + cholestanol) and coprostanol/epicoprostanol ratios do not distinguish between human and natural sources of sterols in the river sediments in Serbia. The most reliable sterol ratios for the sewage pollution assessment of river sediments in the studied area were found to be coprostanol/(coprostanol + cholestanol), coprostanol/cholesterol and epicoprostanol/coprostanol. For the majority of sediments, human-derived pollution was determined. Two sediment samples were identified as influenced by a combination of human and natural biogenic sources.

Food poisonings by ingestion of cyprinid fish.

Raw or dried gallbladders of cyprinid fish have long been ingested as a traditional medicine in the Asian countries, particularly in China, for ameliorating visual acuity, rheumatism, and general health; however, sporadic poisoning incidences have occurred after their ingestion. The poisoning causes complex symptoms in patients, including acute renal failure, liver dysfunction, paralysis, and convulsions of limbs. The causative substance for the poisoning was isolated, and its basic properties were examined. The purified toxin revealed a minimum lethal dose of 2.6 mg/20 g in mouse, when injected intraperitoneally. The main symptoms were paralysis and convulsions of the hind legs, along with other neurological signs. Liver biopsy of the euthanized mice clearly exhibited hepatocytes necrosis and infiltration of neutrophils and lymphocytes, suggesting the acute dysfunction of the liver. Blood tests disclosed the characteristics of acute renal failure and liver injury. Infrared (IR) spectrometry, fast atom bombardment (FAB) mass spectrometry, and 1H- and 13C-nuclear magnetic resonance (NMR) analysis indicated, a molecular formula of C27H48O8S, containing a sulfate ester group for the toxin. Thus, we concluded that the structure of carp toxin to be 5α-cyprinol sulfate (5α-cholestane-3α, 7α, 12α, 26, 27-pentol 26-sulfate). This indicated that carp toxin is a nephro- and hepato- toxin, which could be the responsible toxin for carp bile poisoning in humans.

Technical note: Hapten synthesis, antibody production and development of an enzyme-linked immunosorbent assay for detection of the natural steroidal alkaloid Dendrogenin A.

We have recently discovered the existence of 5α-Hydroxy-6ß-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3ß-ol, called Dendrogenin A (DDA), as the first endogenous steroidal alkaloid ever described in mammals. We found that the DDA content of tumors and cancer cell lines was low or absent compared with normal cells showing that a deregulation in DDA biosynthesis was associated with cancer and therefore suggesting that DDA could represent a metabolomic cancer biomarker. This prompted us to produce antibodies that selectively recognize DDA. For this purpose, the hapten 5α-hydroxy-6ß-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3ß-o-hemisuccinate with a carboxylic spacer arm attached to the 3ß-hydroxyl group of DDA was synthesized. The hapten was coupled to bovine serum albumin and keyhole limpet hemocyanin for antibody production to develop an enzyme-linked immunosorbent assay (ELISA). The protein conjugates were injected into BALB/c mice to raise antibodies. The monoclonal antibodies that were secreted from the hybridoma cell lines established were assessed with indirect ELISA by competitive assays using dilutions of a DDA standard. The antibodies from the selected hybridomas had an IC(50) value ranging from 0.8 to 425 ng/ml. Three antibodies showed no cross-reactivity with structurally related compounds including histamine, cholesterol, ring B oxysterols and a regio-isomer of DDA. In this study, high-affinity and selective antibodies against DDA were produced for the first time, and a competitive indirect ELISA was developed.

Suppression of Chlorella vulgaris growth by cadmium, lead, and copper stress and its restoration by endogenous brassinolide.

Brassinosteroids play a significant role in the amelioration of various abiotic and biotic stresses. In order to elaborate their roles in plants subjected to heavy metals stress, Chlorella vulgaris cultures treated with 10(-8) M brassinolide (BL) were exposed to 10(-6)-10(-4) M heavy metals (cadmium, lead and copper) application. Under heavy metals stress, the growth and chemical composition (chlorophyll, monosaccharides, and protein content) have been decreased during the first 48 h of cultivation. The inhibitory effect of heavy metals on C. vulgaris cultures was arranged in the following order: copper > lead > cadmium. C. vulgaris cultures treated with BL in the absence or presence of heavy metals showed no differences in the endogenous level of BL. On the other hand, treatment with heavy metals results in BL level very similar to that of control cell cultures. These results suggest that the activation of brassinosteroids biosynthesis, via an increase of endogenous BL, is not essential for the growth and development of C. vulgaris cells in response to heavy metals stress. Simultaneously, BL enhanced the content of indole-3-acetic acid, zeatin, and abscisic acid in cultures treated with heavy metals. Levels per cell of chlorophylls, protein, and monosaccharides are all increased by BL treatment when compared to nontreated control cells. Application of BL to C. vulgaris cultures reduced the accumulation of heavy metals stress on growth, prevented chlorophyll, monosaccharides, and protein loss, and increased phytochelatins content. The arrested growth of C. vulgaris cells treated with heavy metals was restored by the coapplication of BL. It suggested that BL overcame the inhibitory effect of heavy metals. From these results, it can be concluded that BL plays the positive role in the alleviation of heavy metals stress.

Determination of fecal sterols by gas chromatography-mass spectrometry with solid-phase extraction and injection-port derivatization.

Injection-port derivatization combined with solid-phase extraction (SPE) was developed and applied for the first time to determine five types of fecal sterols (coprostanol, cholestanol, epicholestanol, epicoprostanol and cholesterol) with gas chromatography-mass spectrometry (GC-MS). In this method, silylation of fecal sterols was performed with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) at GC injection-port. The factors influential to this technique such as injection-port temperature, purge-off time, derivatization reagent (BSTFA) volume, and the type of organic solvent were investigated. In addition, the conditions of SPE (including the type of SPE cartridge, the type of elution organic solvent) were also studied. After SPE followed by injection-port silylation by GC-MS, good linearity of analytes was achieved in the range of 0.02-10ng/mL with coefficients of determination, R(2)>0.995. Good reproducibility was obtained with relative standard deviation less than 19.6%. The limits of detection ranged from 1.3ng/mL to 15ng/mL (S/N=3) in environmental water samples. Compared with traditional off-line silylation of fecal sterols performed with water bath (60 degrees C, 30min), this injection-port silylation method is much simpler and convenient. The developed method has been successfully applied for the analysis of fecal sterols from real environmental water samples.

Multivariate logistic regression for predicting total culturable virus presence at the intake of a potable-water treatment plant: novel application of the atypical coliform/total coliform ratio.

Predicting the presence of enteric viruses in surface waters is a complex modeling problem. Multiple water quality parameters that indicate the presence of human fecal material, the load of fecal material, and the amount of time fecal material has been in the environment are needed. This paper presents the results of a multiyear study of raw-water quality at the inlet of a potable-water plant that related 17 physical, chemical, and biological indices to the presence of enteric viruses as indicated by cytopathic changes in cell cultures. It was found that several simple, multivariate logistic regression models that could reliably identify observations of the presence or absence of total culturable virus could be fitted. The best models developed combined a fecal age indicator (the atypical coliform [AC]/total coliform [TC] ratio), the detectable presence of a human-associated sterol (epicoprostanol) to indicate the fecal source, and one of several fecal load indicators (the levels of Giardia species cysts, coliform bacteria, and coprostanol). The best fit to the data was found when the AC/TC ratio, the presence of epicoprostanol, and the density of fecal coliform bacteria were input into a simple, multivariate logistic regression equation, resulting in 84.5% and 78.6% accuracies for the identification of the presence and absence of total culturable virus, respectively. The AC/TC ratio was the most influential input variable in all of the models generated, but producing the best prediction required additional input related to the fecal source and the fecal load. The potential for replacing microbial indicators of fecal load with levels of coprostanol was proposed and evaluated by multivariate logistic regression modeling for the presence and absence of virus.

Isotopomer spectral analysis of intermediates of cholesterol synthesis in patients with cerebrotendinous xanthomatosis.

Four patients with cerebrotendinous xanthomatosis (CTX) and 2 healthy controls received a constant proximal intraduodenal infusion of 1- 13 C-acetate as a stable-isotope-labeled marker of sterol synthesis. One patient was treated with pravastatin (20 mg twice daily) and another patient with chenodeoxycholic acid (250 mg tid). Every hour, venous blood and duodenal samples were obtained. Stable-isotope enrichment of neutral and polar sterols in serum and bile was assessed by gas chromatography/mass spectrometry. Isotopomer spectral analysis was performed on cholesterol, lathosterol, Delta-8-cholesterol, methylsterol, and lanosterol. Stable-isotope labeling of cholestanol, bile acids, and bile alcohols was analyzed by assessing the change over time of the ratio of M + 3 to M + 0. Eleven hours after marker infusion, we found up to 50% newly synthesized lathosterol in serum and up to 80% in bile, with similar results for other cholesterol precursors. In cholesterol, stable-isotope labeling could be demonstrated in all study subjects with a more prominent labeling in bile than in serum. No stable-isotope labeling was detected in cholestanol. Only minor stable-isotope incorporation was detectable in polar sterols in some subjects. Therapy with pravastatin did not have any effect on fractional or absolute synthesis rates or on the concentrations of cholestanol or cholesterol precursors compared to untreated patients with CTX. In contrast, therapy with chenodeoxycholic acid markedly lowered the concentrations of cholestanol and cholesterol precursors, led to a disappearance of bile alcohols, and reduced absolute synthesis rates of lathosterol. Isotopomer spectral analysis proved to be a powerful method to assess the endogenous synthesis of cholesterol precursors in patients with CTX. Higher fractional synthesis in bile than in serum may be due to the size of the pools in bile vs serum. Cholestanol exhibits no marker uptake and is therefore probably synthesized from preformed cholesterol. Biliary cholesterol secretion in patients with CTX is decreased compared to healthy controls.

High-performance liquid chromatographic separation of ultraviolet-absorbing bile alcohol derivatives.

This paper describes the high-performance liquid chromatographic separation of UV-absorbing bile alcohol derivatives. Bile alcohols were treated with 3 alpha-hydroxysteroid dehydrogenase to form the corresponding 3-keto bile alcohols. The 3-keto bile alcohols produced were converted to the 2,4-dinitrophenylhydrazone derivatives, separated using a Nova-Pak Phenyl column, and monitored at 364 nm. The separation of stereoisomers related to the configuration of hydroxyl groups on the side chain of the bile alcohols, which was not achieved by gas chromatography, could also be accomplished.

Cerebrotendinous xanthomatosis.

A case report on a 23-year-old female patient with cerebrotendinous xanthomatosis (CTX) is presented. From 8 years of age, the patient clinically showed multiple xanthoma masses on both knees, both heels, and the nasal bridge, juvenile cataracts, multiple abnormal neurologic dysfunctions, and dementia. The level of cholestanol in urine, serum, and xanthoma mass tissues was increased, as determined by capillary gas chromatography.

Use of positive ion fast atom bombardment mass spectrometry for rapid identification of a bile alcohol glucuronide isolated from cerebrotendinous xanthomatosis patients.

The identification of a major biliary and plasma bile alcohol glucuronide, 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 25-tetrol-3-0-beta-D-glucuronide, present in cerebrotendinous xanthomatosis (CTX) patients, was investigated by positive ion fast atom bombardment mass spectrometry (FAB-MS). The spectrum was characterized by abundant ions formed by attachment of a proton, [M + H]+, or of alkali ions, [M + Na]+ and [M + 39K]+, to the glucuronide salt. These ions allowed an unambiguous deduction of the molecular weight of the sample. It is suggested that FAB-MS could be used in the rapid diagnosis of CTX.

[Gas-chromatographic analysis of hypercholestanol in the bile and its role in the pathogenesis of cholelithiasis]. / Gazozhidkostno-khromatograficheskoe opredelenie giperkholestanol- kholii i ee znacheniev patogeneze kholelitiaza.

The cholestanol/cholesterol ratio in the bladder and liver bile was estimated by capillary gas-liquid chromatography in patients with dyskinesia of the gallbladder, chronic acalculous cholecystitis, and chronic calculous cholecystitis. Cholesterol biliary calculi and their surface and intermediate layers and the core were examined either. The authors came to a conclusion that cholestanol/cholesterol ratio in the bladder and liver bile reflected the progress of liver cholesterol metabolism starting from the early stages of cholelithiasis to the formation of cholesterol biliary calculi. Therefore hypercholestanolcholia should be considered as an indicator of the risk of cholesterol biliary calculi development.

Analysis of cholesterol, cholesterol-5,6-epoxides and cholestane-3 beta,5 alpha,6 beta-triol in nipple aspirates of human breast fluid by gas chromatography/mass spectrometry.

A method has been developed for the quantitative determination of cholesterol and three of its major oxidative metabolites (the 5 alpha,6 alpha-epoxide, the 3 beta,5 alpha,6 beta-triol, and the 5 beta,6 beta-epoxide) in a single sample of human breast fluid (2-50 microliters), using gas chromatography/mass spectrometry with selected ion monitoring. High specificity and reliable quantification is achieved by the use of the inverse stable isotope dilution method, employing deuterium-labeled variants of the compounds as internal standards.

Xanthogranulomatosis in an adult: lipid analysis of xanthomas and plasma.

Xanthomatosis in the absence of hyperlipidemia is unusual but has been associated with compositional abnormalities of lipoprotein particles. An adult who developed juvenile xanthogranulomatosis in association with oral contraceptive ingestion is reported. Plasma lipids and lipoprotein electrophoresis were normal, as in a few other patients reported with this disorder. However, analysis of cutaneous xanthoma and plasma by thin-layer and gas-liquid chromatography revealed that cholesterol was the principal lipid in xanthoma and that there were no unusual sterols in plasma or tissue. Possible mechanisms of xanthoma formation are discussed. Thus juvenile xanthogranulomatosis should be considered in adults with normolipemic xanthomatosis.

PIP: This article reports the case of a 23-year-old woman with juvenile xanthogranulomatosis, an unusual normolipemic xanthomatosis most often seen in young children. Chromatographic techniques were used to analyze this patient's plasma and xanthomatous tissue for beta-sitosterol, cholestanol, and other sterols that might be present in unusual quantities. The woman had normal fasting levels of plasma cholesterol and triglyceride. The lipoprotein electrophoresis was also normal, and levels of unusual sterols, such as cholestanol and beta-sitosterol, were not increased in plasma or in the xanthomas. Analysis of xanthoma tissue revealed that the predominant lipid was cholesterol. The only medication this patient reported using was a combination oral contraceptive (OC) containing 1 mg of norethindrone and 0.035 mg of ethinyl estradiol. OC use was initiated 1 month before the onset of cutaneous symptoms. The patient refused to discontinue OC use. Since it was not possible to withdraw the drug and observe the patient for regression of the lesions, a causal association of juvenile xanthogranulomatosis with OC use can not be asserted. This case suggests that juvenile xanthogranulomatosis should be considered in adults with normolipemic xanthomatosis. Possible mechanisms for cutaneous xanthoma formation include a defect in local lipid synthesis, an abnormality in local tissue uptake and degradation of lipoproteins that may or may not be coupled with an abnormality in circulating lipoproteins, or local invasion of histiocytes that then accumulate large amounts of cholesterol because of an intrinsic cellular abnormality.

Cerebrotendinous xanthomatosis (CTX): a clinical survey of the patient population in The Netherlands.

The clinical features and additional investigations of 20 Dutch patients suffering from cerebrotendinous xanthomatosis (CTX), an inborn error of metabolism in bile acid synthesis, are described. The onset was in the second or third decade. The clinical picture at the time of examination consisted of a combination of two or more of the following signs: cataract, xanthoma of a tendon, mental deterioration, pyramidal tract signs, cerebellar signs and epilepsy. Mental retardation was reported in patients. CT-scanning showed cerebellar hypodensity in 8 out of 16 patients but this feature did not correlate well with cerebellar signs. The EEG was abnormal in all but one patient. Treatment with chenodeoxycholic acid resulted in a normalization of EEG and biochemical abnormalities but not of the clinical signs. Cholic acid was equally effective but had much less side effects. The importance of a diagnosis in early life is stressed as well as the examination of clinically unaffected heterozygous relatives.

HepG2. A human hepatoblastoma cell line exhibiting defects in bile acid synthesis and conjugation.

We used capillary gas chromatography/mass spectrometry to demonstrate that a cell line derived from a well differentiated human hepatoblastoma, HepG2, synthesized and secreted the following bile acids (ng/10(7) cells/h): chenodeoxycholic acid (131.4), cholic acid (3.3), 3 alpha, 7 alpha-dihydroxy-5 beta-cholestan-26-oic acid (DHCA; 4.5), and 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestan-26-oic acid (THCA; 32.0). Deuterium from [7 beta-2H]7 alpha-hydroxycholesterol, which was added to the media, was incorporated into newly synthesized chenodeoxycholic acid, DHCA, and THCA, but not into cholic acid. Since THCA is a known precursor of cholic acid, these data suggest that HepG2 is specifically deficient in the side chain cleavage that transforms THCA into cholic acid. Greater than 90% of the bile acids synthesized and secreted by HepG2 were unconjugated. Conjugation could not be stimulated by the addition of glycine or taurine to the media. Approximately 30% of newly synthesized DHCA and THCA were sulfated. Chenodeoxycholic acid and cholic acid were not appreciably sulfated. In summary, cultured HepG2 cells synthesize bile acid, but in a pattern distinct from that of adult human liver. This cell line may be a model for studying pathways of human bile acid synthesis, conjugation, and sulfation.