Glucuronides of unconjugated 6-hydroxylated bile acids in urine of a patient with malabsorption

The excretion of bile acids in urine from a patient with chronic malabsorption was investigated. Bile acids were separated according to mode of conjugation using a lipophilic anion exchanger, diethylaminohydroxypropyl Sephadex LH-20. Following hydrolysis, individual bile acids were analyzed by computerized GC/MS. In addition, bile acid glucuronides were isolated and their methyl ester trimethylsilyl ether derivatives were directly analyzed by GC/MS. The patient had a normal or slightly increased excretion of bile acids in urine. Bile acids carrying a hydroxyl group at C-6 constituted about 40% of the total. Tetrahydroxylated bile acids were present which have not been found in healthy subjects. Glucuronides of otherwise unconjugated bile acids accounted for 20% of the total. About 90% of these conjugates were 6-hydroxylated, hyodeoxycholic acid being the major bile acid. It is suggested that a specific abnormality of bile acid metabolism is related to the disease in this patient.     

Interferences in the radioimmunological determination of urinary free cortisol

The magnitude of interferences arising in the radioimmunological assessment of urinary free cortisol is studied (a) by comparing cortisol immunoreactivities from crude urine, after organic solvent extraction of different selectivity and after additional chromatography by high pressure liquid chromatography (HPLC) and (b) by evaluation of the profile of immunoreactivity resulting from the fractions eluted by HPLC. Three antisera from different sources have been investigated. Values of cortisol-immunoreactivity in crude urine were about six times and values of a simple dichloromethane extract about three times higher than values obtained after HPLC. The main part of the interfering compounds arising in organic extracts have a polarity similar to cortisol, which cannot be easily eliminated by simple solvent extraction procedures. Specific estimation of urinary cortisol by radioimmunoassay requires a preceding Chromatographic technique of high efficiency, such as HPLC, which represents an adequate tool for the routine laboratory.     

Congenital adrenal hyperplasia. Plasma and urinary steroid conjugates in seven children with steroid 21-hydroxylase deficiency

Plasma neutral steroid sulphates and urinary neutral steroid sulphates and glucuronides from seven children (0.17–10.4 years) with steroid 21-hydroxylase deficiency were determined using gas-liquid chromatography and gas chromatography -mass spectrometry. Three of the patients were salt-losers. Large inter-individual variation in plasma concentration and urinary excretion of these compounds was observed. However, the group did have certain characteristic features. In plasma, the main compounds present were 3β-hydroxy-5-ene steroids. A progesterone metabolite, 5α-pregnane-3β,20α-diol, and a 17α-hydroxyprogesterone metabolite, 5β-pregnane-3α, 17α, 20α-triol, were present as sulphate conjugates and 3α, 17α-dihydroxy-5β-pregnan-20-one sulphate was identified for the first time in human peripheral plasma. In the urine, metabolites of 17α-hydroxyprogesterone were predominant and 5β-pregnane-3α, 17α,20α-triol, excreted mainly as a glucuronide, alone comprised about 50% of the neutral steroid excretion in these subjects. The next most abundant steroid was 3α,17α,20α-trihydroxy-5β-pregnan-11-one. The following compounds have not previously been found in the urine of patients with steroid 21-hydroxylase deficiency but were found in the present subjects: 5-androstene-3β, 17β-diol, 5α-pregnane-3β, 20α-diol and 3β,17α-dihydroxy-5β-pregnan-20-one. The pattern of the plasma and urinary steroids determined clearly differentiates the subjects with a steroid 21-hydroxylase defect from normal subjects and from patients with a 3β-hydroxysteroid dehydrogenase deficiency.     

The early recognition of the 21-hydroxylase deficiency variety of congenital adrenal hyperplasia

The urinary steroids excreted by three newborn infants with 21-hydroxylase deficiency and by 15 healthy newborns aged two days have been compared after analysis by gas liquid chromatography (GLC). The identity of each steroid was carefully checked by gas chromatography/mass spectroscopy (GC-MS). The enzyme deficiency leads to the elevated excretion of urinary precursor metabolites, mainly 3α,17α,20α-trihydroxy-5β-pregnan, 3α,17α,20α-trihydroxy-5β-pregnan-11-one and 3α,17α-dihydroxy-5β-pregnan-20-one.In the search for a quick and firm confirmation of suspected 21-hydroxylase deficiency in a newborn baby by means of a GLC-profile of urinary steroids, most attention has up to now been paid to 3α,17α,20α-trihydroxy-5β-pregnan. However, 3α,17α-dihydroxy-5β-pregnan-20-one is a better indicator, as it enables one to confirm the existence of this disease soon after birth directly from the GLC-profile without further analyses by GC-MS.     

The effect of ibuprofen on the excretion of steroid metabolites

An inexpensive gas Chromatographic method is described that allows simultaneous measurement in urine of androsterone (A), aetiocholanolone (E), 11-hydroxyandrosterone (11-OA), 11-hydroxyaetiocholanolone (11-OE), pregnanediol (PD), pregnanetriol (PT), tetrahydrocortisone (THE) and tetrahydrocortisol (THF). Dehydroepiandrosterone was also resolved by the column.Ibuprofen was administered to five healthy normal males at a dose used therapeutically in rheumatoid arthritis (RA). The above urinary steroids were measured weekly during a control period, during a four week period of drug treatment and for four weeks after drug treatment had ceased.The excretion of A fell to a mean of 63% of the control value (P < 0.02) and returned to the control value within two weeks. 11-OA, which showed a greater variability than A, fell to the same extent (P < 0.1). No other steroid measured showed a change that could be related to the drug.     

Capillary gas chromatographic determinations of urinary bile acids and bile alcohols in CTX patients proving the ineffectivity of ursodeoxycholic acid treatment

Urine samples and serum samples of a patient with cerebrotendinous xanthomatosis (CTX) were investigated by means of capillary gas chromatography, both before and during oral treatment with ursodeoxycholic acid (UDCA), and the results compared with those obtained during chenodeoxycholic acid (CDCA) therapy. The predominantly excreted bile alcohol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol and two abnormal bile acids, i.e. 23-norcholic acid and 23-hydroxycholic acid were determined. In addition, the serum cholestanol/cholesterol ratio was determined. Whereas previous experiments demonstrated that the urinary excretion of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol and the abnormal bile acids decreased within a few weeks during CDCA therapy, the present study shows that their urinary excretions remain essentially the same during UDCA treatment. In contrast to the decrease in the serum cholestanol/cholesterol ratio during CDCA therapy, this ratio remains essentially the same during UDCA therapy. It is therefore concluded that, in contrast to CDCA therapy, UDCA treatment is not effective in the treatment of CTX.     

Lipidex chromatography in the radioimmunoassay of serum and urinary cortisol.

A highly specific method for the determination of cortisol in human serum and urine is described. The sample is first extracted with diethyl ether/ethyl acetate (1 : 1, by vol.), then chromatographed on a highly lipophilic derivative of Sephadex (hydroxyalkoxypropyl Sephadex, Lipidex) in light petroleum/chloroform (1 : 1, by vol.), and finally cortisol is measured by radioimmunoassay using a cortisol-21-BSA antiserum. Bound and unbound radioactivities are separated using dextran-coated charcoal technique. The 8 a.m. values (mean +/- S.D.) of cortisol among 11 young females and 16 young males were 152 +/- 32 ng/ml (range 111-235) and 185 +/- 21 ng/ml (103-232), respectively. The respective values at 4 p.m. were 84 +/- 29 ng/ml (26-117) and 84 +/- 36 ng/ml (32-172). The importance of Lipidex chromatography was demonstrated with assays of serum samples from children with congenital adrenal hyperplasia; without chromatography cortisol values were 6 times those with chromatography. Specific cortisol assays from pregnancy serum also necessitated Lipidex chromatography. Among 11 young men and 9 young women the mean daily urinary cortisol excretion was 56 +/- 26 mug (32-109) and 60 +/- 24 mug (39-109), respectively. Specific urinary cortisol determination could not be achieved without Lipidex chromatography.     

Analysis of bile acids and their conjugates in jejunal juice by thin-layer chromatography and direct densitometry

A method for the separation and determination of bile acids and their conjugates from jejunal juice is described. The bile acids were separated by thin-layer chromatography on silica gel G, individual bile acids demonstrated on the chromatogram with a spray reagent and direct densitometry performed. The method separates unconjugated from conjugated acids and also separates conjugated trihydroxy and dihydroxy acids. The method has been evaluated by comparison with the specific 3-hydroxysteroid dehydrogenase technique.     

A semiautomated method for the determination of 11-deoxy-17-oxo-steroids in urine

A semiautomated method is described for the determination of total 11-deoxy-17-oxo-steroids (11-DOOS: androsterone, etiocholanolone plus dehydroepiandrosterone) in urine.

Urinary conjugates are manually extracted on a XAD-2 resin, hydrolysed by (β-glucuronidase and “solvolysed” in acid ethyl-acetate according to Burnstein-Lieberman. The free 11-DOOS are extracted automatically with iso-octane and estimated colorimetrically by the Zimmerman reaction in an Auto-Analyzer II system.

The method was evaluated by investigation of its precision, accuracy, sensitivity and specificity. It was found to be satisfactory for the rapid and reliable screening of large numbers of urine samples.     

Oxysterol concentrations are associated with cholesterol concentrations and anemia in pediatric patients with sickle cell disease

Sickle cell disease (SCD) causes anemia, oxidative stress, chronic inflammation, and lipid abnormalities. Oxysterols are oxidized derivatives of cholesterol and affect cholesterol metabolism and eryptosis. Our aim was to determine whether the plasma concentrations of 7-ketocholesterol (7-KC) and cholestane-3β,5α,6β-triol (C-triol) were associated with hemolysis and lipid profile in patients with SCD. A total of 32 steady-state pediatric patients with SCD (22 HbSS and 10 HbSß⁺) and 25 healthy controls were included in the study. Hemolysis parameters, ferritin, serum iron, lipids, 7-KC and C-triol concentrations of all subjects were measured. Oxysterols were quantified with N,N-dimethylglycine derivatization via LC-MS/MS. 7-KC and C-triol concentrations were found to be increased in SCD patients, while there was no difference between the HbSS and HbSß⁺ subgroups. 7-KC concentrations s were correlated negatively with hemoglobin and positively with lactate dehydrogenase concentrations, while C-triol concentrations were negatively correlated with HDL cholesterol. Furthermore, while 7-KC and C-triol concentrations were highly correlated among controls, there was no correlation in patients. The findings of our study suggest that 7-KC and C-triol may have a role in SCD pathophysiology. The lack of correlation in patients’ 7-KC and C-triol concentrations suggest alterations in oxysterol production in patients with SCD.     

The application of high pressure liquid chromatography to the analysis of bile salts in human bile.

Two high pressure liquid chromatography (HPLC) systems have been evaluated for the separation of conjugated bile salts from human bile. Partisil-10 ODS with mobile phase of methanol/water pH 2 (55 : 45) at 200 nm, 0.1 AUF UV detection gave only partial separation of bile salts. However, a mu Bondapak fatty acid analysis column using isopropanol/8.8 mmol/l potassium phosphate pH 2.5 (32 : 68) as the mobile phase and 193 nm, 0.1 AUF UV detection separated all the six conjugated bile salts in bile. The limit of detection ranged from 0.1 microgram for sodium taurocholate to 0.2 microgram for sodium glycodeoxycholate. The reproducibility and the application of the method to the analysis of conjugated bile salts was demonstrated using bile from five patients. Its application to the studies of hepato-biliary disease is discussed.     

Glucose-6 phosphate dehydrogenase deficiency: an easy and sensitive quantitative assay for the detection of female heterozygotes in red blood cells

Different methods for the quantitative determination of glucose-6-phosphate dehydrogenase (G-6-PD) were compared and one of them found to be highly precise. Maleimide inactivation of 6-phosphogluconate dehydrogenase (PGD) was investigated. It was shown that this inactivation is time-dependent and causes loss of assay precision. The most precise method was adapted to lysates of red blood cells from females, known to be heterozygote for G-6-PD deficiency and from non-deficient males and females. Heterozygote gene carriers were detected at a rate of 97.0%.     

Glutamate abnormalities in obsessive compulsive disorder: neurobiology, pathophysiology, and treatment

Obsessive compulsive disorder is prevalent, disabling, incompletely understood, and often resistant to current therapies. Established treatments consist of specialized cognitive-behavioral psychotherapy and pharmacotherapy with medications targeting serotonergic and dopaminergic neurotransmission. However, remission is rare, and more than a quarter of OCD sufferers receive little or no benefit from these approaches, even when they are optimally delivered. New insights into the disorder, and new treatment strategies, are urgently needed. Recent evidence suggests that the ubiquitous excitatory neurotransmitter glutamate is dysregulated in OCD, and that this dysregulation may contribute to the pathophysiology of the disorder. Here we review the current state of this evidence, including neuroimaging studies, genetics, neurochemical investigations, and insights from animal models. Finally, we review recent findings from small clinical trials of glutamate-modulating medications in treatment-refractory OCD. The precise role of glutamate dysregulation in OCD remains unclear, and we lack blinded, well-controlled studies demonstrating therapeutic benefit from glutamate-modulating agents. Nevertheless, the evidence supporting some important perturbation of glutamate in the disorder is increasingly strong. This new perspective on the pathophysiology of OCD, which complements the older focus on monoaminergic neurotransmission, constitutes an important focus of current research and a promising area for the ongoing development of new therapeutics.     

Oxycodone and morphine have distinctly different pharmacological profiles: radioligand binding and behavioural studies in two rat models of neuropathic pain

Previously, we reported that oxycodone is a putative kappa-opioid agonist based on studies where intracerebroventricular (i.c.v.) pre-treatment of rats with the kappa-selective opioid antagonist, nor-binaltorphimine (nor-BNI), abolished i.c.v. oxycodone but not morphine antinociception, whereas pretreatment with i.c.v. naloxonazine (mu-selective antagonist) produced the opposite effects. In the present study, we used behavioural experiments in rat models of mechanical and biochemical nerve injury together with radioligand binding to further examine the pharmacology of oxycodone. Following chronic constriction injury (CCI) of the sciatic nerve in rats, the antinociceptive effects of intrathecal (i.t.) oxycodone, but not i.t. morphine, were abolished by nor-BNI. Marked differences were found in the antinociceptive properties of oxycodone and morphine in streptozotocin (STZ)-diabetic rats. While the antinociceptive efficacy of morphine was abolished at 12 and 24 weeks post-STZ administration, the antinociceptive efficacy of s.c. oxycodone was maintained over 24 weeks, albeit with an approximately 3- to 4-fold decrease in potency. In rat brain membranes irreversibly depleted of mu- and delta-opioid binding sites, oxycodone displaced [(3)H]bremazocine (kappa(2)-selective in depleted membranes) binding with relatively high affinity whereas the selective mu- and delta-opioid ligands, CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2)) and DPDPE ([D-Pen(2,5)]-enkephalin), respectively, did not. In depleted brain membranes, the kappa(2b)-ligand, leu-enkephalin, prevented oxycodone's displacement of high-affinity [(3)H]bremazocine binding, suggesting the notion that oxycodone is a kappa(2b)-opioid ligand. Collectively, the present findings provide further support for the notion that oxycodone and morphine produce antinociception through distinctly different opioid receptor populations. Oxycodone appears to act as a kappa(2b)-opioid agonist with a relatively low affinity for mu-opioid receptors.     

Activity of liver enzymes in multiple sclerosis patients with Hot-nature diet and co-supplemented hemp seed,evening primrose oils intervention

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