Lipophilic Gel Research Articles

Serum unconjugated bile acids: Qualitative and quantitative profiles in ileal resection and bacterial overgrowth

Qualitative and quantitative profiles of unconjugated bile acids in the serum obtained over a 24-h period from three patients with ileal resections and one with a bacterial overgrowth are described. Unconjugated serum bile acids were determined using the high sensitivity and resolution of capillary column gas liquid chromatography after their rapid extraction and isolation using reverse phase octadecylsilane bonded silica cartridges and the lipophilic gel Lipidex 1000. Unconjugated serum bile acid concentrations were elevated throughout the day in both ileum resected patients and in conditions involving bacterial overgrowth when compared to healthy subjects. Total conjugated cholic acid concentrations were expectedly low in both intestinal disorders and were without the postprandial increases generally observed in healthy subjects. Qualitative gas Chromatographic profiles of serum unconjugated bile acids in bacterial overgrowth distinctly revealed a predominance of deoxycholic acid and other secondary bile acids in all samples, while, in conditions of an impaired enterohepatic circulation, deoxycholic acid was absent or present in only trace amounts. The potential significance of measuring serum unconjugated bile acids in intestinal disorders is discussed.

Studies on Steroids: CCXVII. Separation and characterization of bile acid 3-glucuronides in human urine by high-performance liquid chromatography

The separation and characterization of unconjugated and conjugated bile acid 3-glucuronides in biological fluids without prior deconjugation by high-performance liquid chromatography (HPLC) are described. A urine sample from a patient with obstructive jaundice was passed through a Sep-Pak C 18 cartridge and was separated into groups by ionexchange chromatography on a lipophilic gel, piperidinohydroxypropyl Sephadex LH-20, providing the glucuronide fraction. Subsequent resolution into individual 3-glucuronides was attained by HPLC on μBondapak C 18 and Shodex ODS Pak F-411 columns. The 3-glucuronides of cholate, deoxycholate, chenodeoxycholate, glycocholate, glycochenodeoxycholate and taurochenodeoxycholate were identified on the basis of their behaviour in HPLC using mobile phases of different pH. The enzymatic hydrolysis of these glucuronides and derivatization of deconjugated bile acids with 1-anthroyl nitrile followed by chromatographic separation on a Cosmosil 5C 18 column with fluorescence detection were carried out for unequivocal characterization. The ratio of unconjugated, glyco- and tauro-conjugated bile acid 3-glucuronides excreted in urine was found to be ca. 2:3:1.

The Ratio of Interlamellarly Fixed Water to Bulk Water Used as Quality Criterion in O/W Creams

AbstractO/W creams with crystalline gel structures are supposed to be four phase systems. Their dominant structural elements are the hydrophilic and lipophilic gel phases. The hydrophilic gel phases have — depending on the hydrophilic character of their polar groups — a very strong swelling capacity. Further, a dynamic equilibrium is maintained between the water interlamellarly inserted into the hydrophilic gel phase and the bulk water phase. The latter is mainly fixed mechanically by the hydrophilic gel phase. — The lipophilic gel phase, however, which immobilize the dispersed phase, have no or only an extremely weak swelling capacity. By means of thermogravimetry (TG) a quantitative differentiation between water interlamellarly fixed in the hydrophilic gel phase and bulk water is possible. — It is concluded that the ratio of interlamellarly fixed water and bulk water is an important criterion for the properties of O/W creams and that TG can be suitably applied not only as in process control during product manufacturing but also as a useful tool in the development of O/W creams with a controlled water release. During physical ageing of O/W creams the ratio of interlamellarly fixed water and bulk water is shifted towards the bulk water phase.

Colloidal structures of O/W creams.

O/W creams with crystalline gel structures (Water Containing Hydrophilic Ointment DAB 8, Non-ionic Hydrophilic Ointment DAC and a stearate cream) were investigated by means of small angle X-ray diffraction, differential scanning calorimetry, thermogravimetry and freeze fracture technique in combination with Transmission Electron Microscopy (TEM). It was found that these bases are building up two different colloidal gel structures, a hydrophilic gel phase and a lipophilic gel phase, each of them forming a coherent network. The hydrophilic gel phase is characterized by an interlamellar water layer; this interlamellarly fixed water is in a dynamic equilibrium with the bulk water phase. The lipophilic gel phase immobilizes mainly the inner (dispersed) phase. So the O/W creams presented here are supposed to be four-phase systems. The ratio of interlamellarly fixed water and bulk water, which may be determined easily by means of thermogravimetry, is found to be an important quality criterion for these O/W creams. The desired properties of water release of such a cream may be chosen by shifting the ratio of interlamellarly fixed water and bulk water.

Studies on steroids : CLXXXVII. Determination of serum bile acids by high-performance liquid chromatography with fluorescence labeling

A method for the simultaneous determination of bile acids in serum by high-performance liquid chromatography (HPLC) with fluorescence labeling is described. The bile acid fraction was obtained from a serum specimen by passing it through a BondElut cartridge. Bile acids were derivatized quantitatively into the fluorescent compounds through the hydroxyl group at C-3 by treatment with 1-anthroyl nitrile in the presence of quinuclidine in acetonitrile. These derivatives were separated into the free, glycine- and taurine-conjugate fractions by ion-exchange chromatography on a lipophilic gel, piperidinohydroxypropyl Sephadex LH-20. Subsequent resolution of each fraction into cholate, ursodeoxycholate, chenodeoxycholate, deoxycholate and lithocholate was attained by HPLC on a Cosmosil 5C 18 column using 0.3% potassium phosphate buffer (pH 6.0)—methanol (1:5) and 0.1% potassium phosphate buffer (pH 6.0)—methanol (1:8) as mobile phases. The anthroyl bile acids were monitored by fluorescence detection (excitation wavelength 370 nm; emission wavelength 470 nm), the limit of detection being 20 fmol. The proposed method proved to be applicable to the quantitation of bile acids in serum with satisfactory reliability and sensitivity.

Studies on steroids : CLXX. Separation and determination of bile acid 3-sulfates in human bile by high-performance liquid chromatography

A method for the simultaneous determination of sulfated bile acids in human bile without prior hydrolysis and solvolysis is described. The sulfate fraction was obtained from a bile specimen by passing it through a Sep-Pak C 18 cartridge, followed by group separation by ion-exchange chromatography on a lipophilic gel, piperidinohydroxypropyl Sephadex LH-20. Subsequent resolution into the 3-sulfates of unconjugated, glycine- and taurine-conjugated ursodeoxycholate, cholate, chenodeoxycholate, deoxycholate and lithocholate was attained by high-performance liquid chromatography (HPLC) on an SC-02 column. Separation of these sulfates was effected when acetonitrile—0.5% ammonium carbonate (8:31, 8:26 and 8:23, v/v) was used as mobile phase. The sulfated bile acids in human bile were unequivocally identified on the basis of their behaviour in HPLC using mobile phases of various pH values. The present method proved to be applicable to the characterization and quantitation of sulfated bile acids in human bile.

Fractionation of polar organic constituents in environmental samples using the lipophilic dextran gels Sephadex LH-20 and Sephasorb HP Ultrafine : Application to a weathered Ekofisk crude oil

A method has been developed for the enrichment and fractionation of trace amounts of polar organic constituents in environmental samples. The procedure involves two steps using the lipophilic gel filtration gels Sephadex LH-20 and Sephasorb HP Ultrafine in two different modes: (1) separation and isolation of alcohols, phenols, hydroperoxides, acids and highly polar components as a result of retardation of compounds containing hydroxyl groups on Sephadex LH-20 using stepwise gradients of chloroform, methanol and tetrahydrofuran as the mobile phase; (2) liphophilic-hydrophilic partitioning using Sephasorb HP Ultrafine swollen in methanol-water and eluted with stepwise gradients of n-hexane and benzene, which separates non-hydroxyl-containing constituents. The method leads to essentially quantitative recoveries of the different compounds, and is sufficiently rapid and convenient for use in routine analysis. Minimal degradation of the sample components occurs as this is a pure chromatographic procedure on highly inert gel matrices with no catalytic activity, avoiding the acid/alkali extractions and chromatography on more labile gel matrices that often accompany other procedures and usually alter the sample to some extent. The application of the method to a weathered Ekofisk crude oil is described.

Separation and Determination of Bile Acids in Human Bile by High-Performance Liquid Chromatography

Abstract A method for simultaneous determination of major bile acids in human bile is described. The unconjugated, glycine- and taurine-conjugated bile acids are extracted with Sep-pak C18 and separated into groups by ion-exchange chromatography on a lipophilic gel. Subsequently, resolution of each group into ursodeoxycholate, cholate, chenodeoxycholate, deoxycholate and lithocholate is attained into two stages by high-performance liquid chromatography on a Radial-PAK A column. First, 0.3% ammonium phosphate (pH 7.7)/acetonitrile (19:8, v/v) is used for separation of the latter three as a mobile phase. Ursodeoxycholate and cholate are efficiently separated in 0.3% ammonium phosphate (pH 7.7)/acetonitrile (23:8, v/v). The present method is applicable to quantitation of bile acids in human bile with satisfactory accuracy and precision.

Steroid bioconversion in water‐insoluble organic solvents: Δ1‐Dehydrogenation by free microbial cells and by cells entrapped in hydrophilic or lipophilic gels

AbstractA cell suspension in a water‐insoluble organic solvent (benzene: n‐heptane, 1 : 1 by volume) of Nocardia rhodocrous (previously induced to synthesize steroid Δ1dehydrogenase) rapidly catalyzed the stoichiometric oxidation of 4‐androstene‐3,17‐dione (4‐AD) to androst‐l,4‐diene‐3,17‐dione (ADD) in the presence of phenazine methosulfate (PMS). High levels of 4‐AD or PMS reduced the conversion rates. No appreciable decrease in the conversion rate was observed on adding aqueous buffer solution to the thawed ceils (up to 9.4 g water/g dry cell). The whole cells were immobilized by entrapment in a hydrophilic gel (H‐gel) or a lipophilic gel (L‐gel) by use of a water‐soluble or water‐insoluble photocrosslinkable prepolymer. The reticula of H‐ and L‐gel matrices were impregnated with water and organic solvent, respectively. Both the H‐ and L‐gels could convert 4‐AD to ADD in the presence of PMS, the L‐gel showing a slightly higher conversion rate. Various lines of evidence indicate that the limiting factor is the penetration rate of 4‐AD into gel particles for the H‐gel, and the penetration rate of PMS for the L‐gel. The catalytic activities decreased considerably after several successive runs with the free cell suspension system, while the immobilized cells were more stable, the stability of H‐gel and L‐gel being almost the same.

Steroids and hypertension. 1--Gas-liquid chromatography and gas chromatography mass spectrometry of 3,15- and 3, 16-dihydroxy-17-oxosteroids.

In order to analyse and quantitate the urinary 16-oxysteroids known or thought to be associated with hypertension, we have established for six 16-oxy-C19 reference steroids the following parameters: elution volume on lipophilic gel columns, gas chromatographic retention data expressed as methylene unit values of trimethylsilyl ether and O-methoxime trimethylsilyl ether derivatives on OV-1 and OV-17 packed columns and on SE-30 capillary column, and mass spectra of these compounds. These reference steroids were: 3 alpha, 16 alpha-dihydroxy-5 alpha-androstan-17-one, 3 alpha, 16 alpha-dihydroxy-5 beta-androstan-17-one, 3 beta, 16 alpha-dihydroxy-5 alpha-androstan-17-one, 3 beta, 16 alpha-dihydroxy-5-androsten-17-one, 3 beta, 16 beta-dihydroxy-5-androsten-17-one, 3 beta, 17 beta-dihydroxy-5-androsten-16-one and 3 alpha, 15 alpha-dihydroxy-5 beta-androstan-17-one. The proposed method was shown to be applicable to the specific analysis of 16-oxy-C19-steroids in biological samples since it achieved the selective isolation of these compounds from other steroids and their quantitative elution in a single fraction. The analysis of the urinary steroids of two patients with arterial hypertension demonstrated an elevated rate of 3 beta, 16 alpha-dihydroxy-5-androsten-17-one.

Lipophilic gel chromatography of steroids : Structure-standard elution volume relationships

Lipophilic gel chromatography of steroids : Structure-standard elution volume relationships

A new method for simultaneous determination of bile acids in human bile without hydrolysis

A method for simultaneous determination of major bile acids in human bile without prior hydrolysis is described. The unconjugated, glycine- and taurine-conjugated bile acids are separated into groups by ion-exchange chromatography on a newly developed lipophilic gel, piperidinohydroxypropyi Sephadex LH-20 (PHP-LH-20). Subsequently, resolution of each group into cholate, ursodeoxycholate, chenodeoxycholate, deoxycholate and lithocholate is attained into two stages by high-performance liquid chromatography on a μ-Bondapak C 18 column. First, 0.3% ammonium carbonate/acetonitrile (9 : 4, v/v) is used for separation of the latter three as a mobile phase. Cholate and ursodeoxycholate are separated in 0.3% ammonium carbonate/acetonitrile (11 : 4, v/v). The present method is applicable to quantitation of free and conjugated bile acids in human bile with satisfactory accuracy and precision.

The multicomponent analysis of conjugates of neutral steroids in urine by lipophilic ion exchange chromatography and computerised gas chromatography-mass spectrometry

A method for the separation and analysis of conjugates of neutral steroids in urine is described. Following Amberlite XAD-2 extraction of steroids, separation into neutral, glucuronide, monosulphate and disulphate conjugate groups was achieved by ion exchange chromatography on the lipophilic gel, diethylaminohydroxypropyl Sephadex LH-20 (DEAP-LH-20). Enzymatic and solvolytic procedures were used to hydrolyse the conjugate moiety and the steroids were analysed by gas-chromatography using open-tubular glass capillary columns after the preparation of the O-methyloxime-trimethylsilyl ether derivative. Characterization of the steroids was made by computerised GC-MS following repetitive magnetic scanning? Examples of the application of the method to the analysis of urine from normal male subjects, and a patient with Cushing's syndrome are presented.

Identification and quantitation of 16α-hydroxy c 21 steroid sulphates in plasma from pregnant women

Plasma steroids from 11 women in the last trimester of pregnancy were separated into conjugate class by chromatography on Sephadex LH-20. Fractions corresponding to mono- and disulphate conjugates were solvolyzed and the free steroids were separated by lipophilic gel chromatography into groups containing mono-, di- and trihydroxy steroids, respectively. Trimethylsilyl ether derivatives were prepared and repetitive-scanning gas chromatography-mass spectrometry (GC-MS) was used to characterize and quantitate the following 16α-hydroxy steroids in the monosulphate fraction: 3α,16α:-dihydroxy-5α-pregnan-20-one (range 8–80 ng/ml), 3β,16α-dihydroxy-5α-pregnan-20-one (6–55 ng/ml), 5α-pregnane-3α,16α,20α-triol (9–31 ng/ml) and 5α-pregnane-3β,16α,20α-triol (11–77 ng/ml). These compounds were also present as disulphates although at 5–50 times lower concentrations.

Isolation by affinity chromatography of a cholinergic proteolipid from [formula omitted

A cholinergic proteolipid fraction (i.e. a hydrophobic lipoprotein) was separated from the n. caudatus of the cow, using affinity chromatography with the lipophilic gel Sephadex LH-20 and p-phenyltrimethylamonium as the active group. High affinity binding studies showed that only the specific fraction, desorbed after an acetylcholine (or acid) pulse, and corresponding to 0,72% of the proteolipids, is the one that binds the cholinergic ligands. The binding of ( 3H)atropine and ( 14C)d-tubocurarine demonstrated that there are 814 picomoles/g fresh tissue of muscarinic sites and only 76 picomoles/g of nicotinic sites. The specific radioactivity for ( 3H)atropine is 10,000 nmoles/g protein, suggesting a high degree of purification of the specific cholinergic proteolipid.

Separation of C 19 and C 21 dihydroxysteroids by open-hole tubular glass columns and lipophilic gel chromatography

The combination of lipophilic gel chromatography and high-resolution glass capillary chromatography has been shown to be an important means of identification and quantification of steroid urinary metabolites. Some problems in the use of capillary columns, such as coating with polar and apolar stationary phases, column performance, thermal stability and injection systems, have been investigated and this type of chromatography has been used in the analysis of 33 dihydroxysteroids. Gel chromatography is a method complementary to high-efficiency gas chromatography. The steroids are eluted according to the number of hydroxyl groups in the molecules.

Purification by affinity chromatography of nicotinic and muscarinic hydrophobic proteins separated by Sephadex LH2O

Summary Hydrophobic protein fractions, having nicotinic or muscarinic binding properties were separated respectively from skeletal muscle and smooth intestinal muscle using the lipophilic gel Sephadex LH2O. Further purification of the specific fractions was carried out by a cholinergic affinity column, using elution with organic solvents, followed by a pulse of 10−3M acetylcholine . The results obtained confirm the validity of the Sephadex LH20 technique for the isolation and binding of cholinergic proteins proposed by De Robertis et al (4) and discard the criticisms raised by Levinson and Keynes (7).

Steroids in the human newborn; lipophilic gel separation and gas phase analysis.

GLC separations of newborn urinary steroids were carried out after isolation of sulfate and glucuronide fractions by differential hydrolysis. GLC and GC-MS data allowed the characterization of the major components in these complex mixtures. It was found that the sulfo-conjugated metabolites were typical of the new born steroid production whereas steroids excreted as glucuronides in the first days of life might have been mainly of maternal origin. Further study of individual steroid metabolites was undertaken using a preliminary fractionation of the extracts with a straight-phase lipophilic gel column. This type of separation appeared ideally suited as a complementary technique to GLC and GC-MS in the study of biological mixtures, since it provided clean and clear cut fractions with high recovery and offered a versatile type of separation yielding structural information. A diol-one fraction obtained by this method was studied by GC-MS with single ion monitoring which yielded evidence for the presence of 15 α-hydroxy-dehydroepiandrosterone (15 α-OH-DHA) in the sulfo-conjugated urinary steroids of a normal newborn infant.

Lipophilic gel and gas-phase analysis of steroid hormones: Application to the human newborn

Steroid extracts from the uring of newborn infants can be separated into two fractions according to their mode of conjugation: metabolites excreted in the sulphate and glucuronide fractions appeared to be of different biological significance. Both fractions still represent complex mixtures of structurally related compounds which are not completely resolved by gas-liquid chromatography (GLC). Methods are presented for the fractionation of the extracts by lipophilic gel chromatography on a highly substituted hydroxyalkoxypropyl Sephadex derivative. A straight-phase system eluted with benzene permits the separation of groups of steroids according to the number of hydroxyl groups in the molecule. A reversed-phase system eluted with methanol- n-heptane (9:1) permits the rapid isolation of the polar steroids. Both systems provide additional structural information of the chromatographed steroids and form the basis of useful separation and purification procedures prior to GLC and gas chromatography—mass spectrometry (GC-MS). An example is given of the application of the straight-phase system to the fractionation of a urinary extract for GC-MS and mass fragmentography.

Preparation and evaluation of a hydroxycyclohexyl derivative of sephadex LH-20

Sephadex LH-20 has been substituted with hydroxycyclohexyl residues to give a stable gel for use in lipophilic gel chromatography. The gel can be used for straight-phase and reversed-phase systems. Separations of a model set of steroidal compounds are described. Hydroxysteroids epimeric at C-3, C-7, C-11, C-17 or C-20 have been separated as the free alcohols in a straight-phase system. Five ▶ 5-steroids have been separated from the corresponding dihydrosteroids of the 5α-series.