Chromic acid degradation of natural (-)-stercobilin (1) yields 2(R)-methyl-3(R)-ethylsuccinimide (+2), whereby the absolute configuration of 1 at the chiral centers C-1, C-2, C-7, and C-8 is established. The substituted oxo-tetrahydrodipyrromethane precursor, 5, for the total synthesis of (-)-stercobilins 3 and 4, in which the relative configuration between the asymmetric centers is known, yields 2(S)-methyl-3(S)-ethylsuccinimide (-2) under the same conditions of degradation. Nuclear magnetic resonance studies of 1 and 3 show that in 1 the hydrogen atoms at C-2 and C-2', as well as those at C-7 and C-7', are trans relative to one another. Accordingly, natural (-)-stercobilin possesses the 2'(S), 7'(S) configuration, and has the configuration formula 6(1 (R), 2(R), 2'(S), 7'(S), 7(R), 8(R)). These results, coupled with those of earlier studies, also establish the absolute configuration of the (+)-urobilin 7 and of the phycobilin 8 at C-7'.

Bilin tetrapyrroles including stercobilin are unique to mammalian waste; they have been used as markers of source water contamination and may have important diagnostic value in human health conditions. Unfortunately, commercial isotopomers for bilins are not available. Thus, there is a need for isotopomer standards of stercobilin and other bilins for quantification in environmental and clinical diagnostic applications.

A procedure is described here using H2 (18) O to label the carboxylic acid groups of bilin tetrapyrroles. Reaction conditions as a function of temperature and reagent volume were found to produce a mixture of isotopomers, as assessed by electrospray ionization and Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). Stability as a function of storage time and temperature and in conjunction with solid-phase extraction (SPE) was assessed.

The highest labeling efficiency was achieved at 70 °C for 8 h, while a stable ratio of the isotopmers could be produced at 60 °C for 4 h. The stability of the isotopic distribution was maintained under storage (room temperature or frozen) for 20 days. It was also stable throughout SPE. The high mass accuracy and resolving power of FTICRMS enables clear distinction between (18) O-labeled bilins from other unlabeled bilins present, avoiding a potential interference in quantitation.

A procedure was developed to label bilins with (18) O. The final ratio of the (18) O-labeled bilin isotopomers was reproducible and highly stable for at least 20 days under storage. This ratio was not changed in any statistically significant way even after SPE. Thus a reliable method for producing stable isotopomer ratios for bilins has been achieved. Copyright © 2016 John Wiley & Sons, Ltd.

UNASSIGNED

Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders lacking a clinical biomarker for diagnosis. Emerging evidence shows that intestinal microflora from ASD subjects can be distinguished from controls, suggesting metabolite differences due to the action of intestinal microbes may provide a means for identifying potential biomarkers for ASD.

UNASSIGNED

The aim of this study was to determine if quantitative differences in levels of stercobilin and stercobilinogen, metabolites produced by biological action of intestinal microflora, exist in the fecal matter between an ASD mouse model population and controls.

UNASSIGNED

Pairs of fecal samples were collected from two mouse groups, an ASD model group with Timothy syndrome 2 (TS2-NEO) and a gender-matched control group. After centrifugation, supernatant was spiked with an 18O-labeled stercobilin isotopomer and subjected to solid phase extraction for processing. Extracted samples were spotted on a stainless steel plate and subjected to matrix-assisted laser desorption and ionization mass spectrometry using dihydroxybenzoic acid as the matrix (n = 5). Peak areas for bilins and 18O-stercobilin isotopomers were determined in each fecal sample.

UNASSIGNED

A 40-45% depletion in stercobilin in TS2-NEO fecal samples compared with controls was observed with p < 0.05; a less dramatic depletion was observed for stercobilinogen.

UNASSIGNED

The results show that stercobilin depletion in feces is observed for an ASD mouse model vs. controls. This may help to explain recent observations of a less diverse microbiome in humans with ASD and may prove helpful in developing a clinical ASD biomarker.

The degradation of haemoglobin haeme of senescent red blood cells - involving NADPH-dependent haeme oxygenase and biliverdin reductase - in the reticuloendothelial cells of the spleen, bone marrow and liver accounts for 80 to 90% of the 250 to 300 mg of bilirubin formed in 24 hours. The remaining 10 to 20% derive from catabolism of other haemoproteins and from the destruction of maturing red blood cells in the marrow. In studies with isotopically-labelled metabolic precursors of haeme this fraction can be found in the early-labelled peak. In plasma virtually all the bilirubin is tightly bound to plasma proteins, largely albumin, because it is only sparingly soluble in aqueous solutions at physiological pH. In the sinusoids unconjugated bilirubin dissocates from albumin, enters the liver cells across the cell membrane through non-ionic diffusion and is bound by the two cytoplasmic proteins Y (or ligandin) and Z. Little is known about the transfer of unconjugated bilirubin from these binding proteins to the smooth endoplasmatic reticulum, where it is converted to a water-soluble ester glucuronide by bilirubin UDP-glucuronyl transferase. The physiological significance of non-glucoronide conjugates (sulphate, disaccharides) is only of minor importance. Following conjugation, bilirubin is transferred rapidly across the canalicular membrane into the bile canaliculi. This process is energy-dependent and occurs against a concentration gradient. The epithelial lining of the intestine and of the gall bladder, which can easily reabsorb lipid-soluble unconjugated bilirubin, is virtually impermeable to organic anions of the size and charge of conjugated bilirubin, thereby ensuring efficient excretion of this pigment. In the intestinal tract bilirubin is reduced to urobilinogen, which is subsequently reabsorbed to some extent into the enterohepatic circulation, removed from plasma by the liver and excreted unchanged in the bile. This rapid bacterial reduction of bilirubin makes it unlikely that unconjugated bilirubin is formed and absorbed to an appreciable degree. The residual part of urobilinogen is further reduced to urobilin, stercobilin and dipyrrolmethenes and excreted in the faeces.

The detailed analysis of faecal bile pigments by high-performance liquid chromatography is described. Non-aqueous reversed-phase systems with acetonitrile-dimethyl sulphoxide or acetonitrile-dimethyl sulphoxide-methanol as the mobile phase on C1, C8 or C18-bonded silica are used for the group separation of verdinoids, violinoids and urobilinoids. A silica column, with acetonitrile-water-tetraethylene-pentamine as mobile phase, separates the laevorotatory stercobilin (C33H46N4O6) and half-stercobilin (C33H44N4O6) from the optically inactive urobilin (C33H42N4O6). The diastereoisomers are resolved by converting the urobilinoids into their dimethyl esters before chromatography on a silica column with n-heptane-methyl acetate-methanol containing 1% of diethylamine as the solvent system.

Vibrational circular dichroism (VCD) and IR spectra of dichloromethane solutions of l-stercobilin and d-urobilin hydrochlorides have been recorded in the mid-IR region. The spectra are best interpreted by combining molecular dynamics (MD) calculations and DFT calculations, within the QM/MM ONIOM-type framework, and the combined predicted results are better and more informative than the more standard analysis provided by DFT calculations. The same approach also sheds light on the Cotton effect sign inversion of room temperature versus low temperature electronic CD (ECD) spectra of the same compounds in methanol-glycerol solution. Finally, circularly polarized luminescence (CPL) spectra for l-stercobilin in chloroform solution provide some information on the excited state geometry of this molecule.

Germfree animals exhibit specific physiological signs which have been labeled germfree animal characteristics. Such characteristics have been used in animals and in man to evaluate the influence of antibiotics on the intestinal flora. In order to evaluate the feasibility of using germfree animal characteristics in the evaluation of the intestinal flora in man, out-patients and hospitalized patients without gastrointestinal disorders were investigated. All patients were on a normal diet and had not been treated with antibiotics the year prior to the study. Samples of faeces were obtained at routine controls or after 10-14 days in hospital and analyzed for coprostanol to cholesterol ratios, mucin and stercobilin contents and proteolytic and tryptic activity. No differences were observed between out-patients and hospitalized patients. The germfree animal characteristics was of the same order of magnitude in man as in conventional animals.

The kinetics of unconjugated 3H-bilirubin are described in 25 healthy dogs and 35 dogs with spontaneous hepatobiliary or haemolytic disease, using a two-compartment model. The bilirubin production rates from erythrocyte degradation (PE), ineffective erythropoiesis (PI) and catabolism of hepatic haemoproteins (PL), were derived from the incorporation of 14C-glycine into haemoglobin and stercobilin. These combined measurements permitted an integral survey of bilirubin metabolism in health and disease. The concentration of unconjugated bilirubin in plasma and its fraction of total bilirubin levels were similar in hepatic and haemolytic disorders. This was explained by the highly increased bilirubin production rates in both types of disease. In addition, the hepatic bilirubin clearance was severely impaired in fulminant hepatitis and in cirrhosis, and moderately decreased in the other hepatobiliary diseases and in primary haemolysis. The erythrocyte lifespan was reduced in all animals but one. In addition to haemolysis, the contribution of PI and PL was variable, and in two dogs PL was the principle source of highly increased bilirubin production rates. These data indicate that the concentration of unconjugated bilirubin in plasma or its fraction of total pigments is unreliable in the discrimination of canine hepatobiliary disease from haemolytic disorders.

In this quantitative assay, urinary urobilinogen is oxidized to urobilin with iodate in an acid medium, the pH is adjusted to 6 with sodium acetate, and the mixture is reacted with alcoholic HgCl2 solution, extracted with CHCl3, the measured spectrophotometrically at 513 nm. The artificial standards of previous methods have been replaced with crystalline stercobilin IX (commercially available), a urobilin closely related to the urinary urobilins. The reproducibility of the method, as assessed from 10 replicates of a single urine specimen to which urobilinogen was added, gave a coefficient of variation of 3.9%. Analytical recovery of urobilinogen added to urines was 90.4% (SD 14.5%). Bilirubin, biliverdin, mesobilirubin, coproporphyrin I, uroporphyrin I, and porphobilinogen do not interfere.