Pyrroles as a Potential Biomarker for Oxidative Stress Disorders.

Redox imbalance or oxidative stress that results from both environmental and genetic factors is observed in patients with schizophrenia. Therefore, identifying markers of oxidative stress in the early stages of psychosis and using antioxidant treatments as an adjuvant to antipsychotics has important implications. The reaction of p-N,N-dimethylaminobenzaldehyde (DMAB) with pyrrole moieties has been well studied for well over a century for use as a marker of oxidative stress dysregulation. Throughout this time, pyrroles have been investigated with varying veracity in urine extracts to identify elevated levels in patients diagnosed with schizophrenia. Since the 1960's, various claims have been made with respect to what causes the colour change when DMAB is added to urine extracts. Whilst the substances from this reaction have not been fully elucidated, an objective look at most studies indicates that urobilinogen is likely to be one them. Urobilinogen has also been identified as a major interferent in our results. Both pyrroles and urobilinogen condense the DMAB reaction system (form condensation products) and are quite different. The urobilinogen detected in urine forms when gut microflora chemically reduces the bilirubin content of bile acids. In comparison, evidence suggests that the pyrrole fraction originates from the fragmentation of regulatory haem by reactive oxygen species (ROS) such as hydrogen peroxide and super and nitrous oxides. Clinical studies in our laboratories have established that pyrroles as a urine biomarker have specificity in detecting schizophrenia; however, caution must be applied as the readings are subject to interference by other DMAB active compounds that are present, such as urobilinogen. This review highlights the initial chemistry in isolating pyrroles and provides recommendations for standardised laboratory testing to ensure pyrroles are correctly measured and distinguished from other by-products.

Feasibility of Voltage-Applied SERS Measurement of Bile Juice as an Effective Analytical Scheme to Enhance Discrimination between Gall Bladder (GB) Polyp and GB Cancer.

A unique surface-enhanced Raman scattering (SERS) measurement scheme to discriminate gall bladder (GB) polyp and GB cancer by analysis of bile juice is proposed. Along with the high sensitivity of SERS, external voltage application during SERS measurement was incorporated to improve sample discriminability. For this purpose, Au nanodendrites were constructed on a screen-printed electrode (referred to as AuND@SPE), and Raman spectra of extracted aqueous phases from raw bile juice samples were acquired using the AuND@SPE at voltages from -300 to 300 mV. The sample spectra resembled that of bilirubin, possessing an open chain tetrapyrrole, showing that bilirubin derivatives in bile juice were mainly responsible for the observed peaks. Discrimination of GB polyp and GB cancer using just the normal SERS spectra was not achieved but became apparent when the spectra were acquired at a voltage of -100 mV. When voltage-applied SERS spectra of bilirubin and urobilinogen (one of bilirubin's derivatives) were examined, a sudden intensity elevation occurring at -100 mV was observed for urobilinogen but not bilirubin. Based on examination of corresponding cyclic voltammograms, the potential-driven strong adsorption of urobilinogen (no faradaic charge transfer) on AuND occurring at -100 mV induced a substantial increase in SERS intensity. It was presumed that the content of urobilinogen in the bile juice of a GB cancer patient would be higher than that of a GB polyp patient, and the contained urobilinogen was sensitively highlighted by applying -100 mV during SERS measurement, allowing clear discrimination of GB cancer against GB polyp.

Paper-Plastic Hybrid Microfluidic Device for Smartphone-Based Colorimetric Analysis of Urine.

In this work, a disposable paper-plastic hybrid microfluidic lab-on-a-chip (LOC) has been developed and successfully applied for the colorimetric measurement of urine by the smartphone-based optical platform using a "UrineAnalysis" Android app. The developed device was cost-effectively implemented as a stand-alone hybrid LOC by incorporating the paper-based conventional reagent test strip inside the plastic-based LOC microchannel. The LOC device quantitatively investigated the small volume (40 µL) of urine analytes for the colorimetric reaction of glucose, protein, pH, and red blood cell (RBC) in integration with the finger-actuating micropump. On the basis of our experiments, the conventional urine strip showed large deviation as the reaction time goes by, because dipping the strip sensor in a bottle of urine could not control the reaction volume. By integrating the strip sensor in the LOC device for urine analysis, our device significantly improves the time-dependent inconstancy of the conventional dipstick-based urine strip, and the smartphone app used for image analysis enhances the visual assessment of the test strip, which is a major user concern for the colorimetric analysis in point-of-care (POC) applications. As a result, the user-friendly LOC, which is successfully implemented in a disposable format with the smartphone-based optical platform, may be applicable as an effective tool for rapid and qualitative POC urinalysis.

Non-clinical safety assessment of Hwangryunhaedok-tang: 13-week toxicity in Crl:CD Sprague Dawley rats.

Hwangryunhaedok-tang (Huang-Lian-Jie-Du-Tang in Chinese, Oren-gedoku-to in Japanese) is a traditional herbal medicine with a long history of use for anti-inflammatory purposes. In this study, subchronic toxicity of daily oral administration of a Hwangryunhaedok-tang water extract (HHT) at 0, 250, 750, and 2000mg/kg for 13weeks was examined in rats. Mortality, clinical signs, and changes in body weight, food consumption, clinical signs, ophthalmological examination, urinalysis, hematology, serum biochemistry, gross observation, organ weight, and histopathology were monitored in accordance with Good Laboratory Practice and OECD guidelines. We found no mortality or abnormality in clinical signs, body weight, serum biochemistry, or organ weight in HHT-treated groups in either sex. However, there were significant changes in glucose, bilirubin, urobilinogen, protein (only male) in urine after 2000mg/kg/day HHT treatment for both sexes. In hematological examinations, we found a significant decreased number of red blood cells (RBC), whereas, an increased the mean corpuscular volume, number of platelets, and rate of reticulocyte (RET) after 2000mg/kg/day HHT treatment of male rats. In male and female rats, 750 and 2000mg/kg/day HHT treatment decreased the number of RBC and increased RET. Histopathological examinations revealed stomach mucosal erosion in female rats (2000mg/kg/day). No-observed-adverse-effect levels were established for 750mg/kg HHT in rats under the conditions of this study. However, other toxicological studies are necessary to evaluate the safety of HHT fully.

BilR is a gut microbial enzyme that reduces bilirubin to urobilinogen.

Metabolism of haem by-products such as bilirubin by humans and their gut microbiota is essential to human health, as excess serum bilirubin can cause jaundice and even neurological damage. The bacterial enzymes that reduce bilirubin to urobilinogen, a key step in this pathway, have remained unidentified. Here we used biochemical analyses and comparative genomics to identify BilR as a gut-microbiota-derived bilirubin reductase that reduces bilirubin to urobilinogen. We delineated the BilR sequences from similar reductases through the identification of key residues critical for bilirubin reduction and found that BilR is predominantly encoded by Firmicutes species. Analysis of human gut metagenomes revealed that BilR is nearly ubiquitous in healthy adults, but prevalence is decreased in neonates and individuals with inflammatory bowel disease. This discovery sheds light on the role of the gut microbiome in bilirubin metabolism and highlights the significance of the gut-liver axis in maintaining bilirubin homeostasis.

Assessment of urine metabolite biomarkers for the detection of S. haematobium infection in pre-school aged children in a rural community in Zimbabwe.

BACKGROUND: Early diagnosis of urogenital schistosomiasis is key to its control and elimination. The current gold standard microscopic examination techniques lack sensitivity in detecting light Schistosomiasis infections in pre-school aged children thus it is urgent to develop diagnostic tools that may be integrated into control programs. In this study, we evaluated the diagnostic performance of urine metabolite biomarkers using a chemical reagent strip in the detection of S. haematobium infection in pre-school aged children. METHODS: A case-control study was conducted involving 82 pre-school aged children that were age and sex matched. Urine samples were collected for 3 consecutive days and were evaluated using urine filtration gold techniques as the gold standard method. The samples were simultaneously measured for metabolite biomarkers specifically haematuria, proteins, ketones, nitrites, glucose, bilirubin and urobilinogen using chemical reagent strips. Pearson correlation test was used to measure the relationship between S. haematobium infection and the urine metabolite biomarkers. RESULTS: The diagnostic performance of urine biomarkers were correlated with the microscopic examination urine filtration technique. Haematuria (r = 0.592, p = 0.0001) and proteinuria (r = 0.448, p = 0.0001) were correlated to S. haematobium infection. Negative correlations with p > 0.05 were recorded for ketones and urobilinogen. Highest sensitivity was 65.9 % (CI, 49.4 - 79.9) for haematuria whilst protein (albumin) biomarker had a lower specificity value of 43.9 % (28.5 - 60.3). Inversely, highest sensitivity was 87.8 % (73.8 - 95.9) for proteinuria whilst haematuria had a lower sensitivity value of 82.9 % (67.9 - 92.8). The positive predictive values ranged from 57.7 % (41.6 - 72.2) to 79.4 % (65.5 - 88.7) whereas negative predictive values ranged from 70.8 % (60.8 - 79.2) to 52.0 % (48.7 - 55.3). With respect to diagnostic efficiency, haematuria had a fair diagnostic performance with an area under the curve of 0.76 followed by proteinuria with proteinuria whilst the remaining metabolites fail discriminating ability with an area under the curve of <0.5. CONCLUSION: Although haematuria and protein biomarkers in urine are moderately sensitive and specific, they are important morbidity indicators of urogenital schistosomiasis in pre-school aged that may be utilised during screening in schistosomiasis control programs. We recommend comprehensive analysis of biomarkers using metabolomics techniques to identify novel urine biomarkers.

A high urinary urobilinogen/serum total bilirubin ratio indicates acute hepatic porphyria in patients with abdominal pain.

Acute hepatic porphyria (AHP) has always been a diagnostic dilemma for physicians due to its variable symptoms. Correct diagnosis mainly depends on the detection of an elevated urinary porphobilinogen (PBG), which is not a routine test and highly relies on the physician's awareness of AHP. In the present study, we identified a more convenient indicator during routine examinations to improve the diagnosis of AHP. We found that AHP patients showed a significant higher "FALSE" urinary urobilinogen level caused by urinary PBG during the urinalysis when detected by strips impregnated with Ehrlich reagent (P < 0.05). And a remarkable increase in the urinary urobilinogen/serum total bilirubin ratio was observed in AHP patients. The area under the ROC curve of this ratio for AHP was 1.000 (95% confidence interval 1.000-1.000, P < 0.01). A cutoff value of 3.22 for this ratio yielded a sensitivity of 100% and a specificity of 100% to distinguish AHP patients from the controls. Thus, we proved that a "falsely" high urinary urobilinogen level that was adjusted by the serum total bilirubin level (urinary urobilinogen/serum total bilirubin ratio) could be used as a sensitive and specific screening marker for AHP in patients with abdominal pain.

Annotation of the human adult urinary metabolome and metabolite identification using ultra high performance liquid chromatography coupled to a linear quadrupole ion trap-Orbitrap mass spectrometer.

Metabolic profiles of biofluids obtained by atmospheric pressure ionization mass spectrometry-based technologies contain hundreds to thousands of features, most of them remaining unknown or at least not characterized in analytical systems. We report here on the annotation of the human adult urinary metabolome and metabolite identification from electrospray ionization mass spectrometry (ESI-MS)-based metabolomics data sets. Features of biological interest were first of all annotated using the ESI-MS database of the laboratory. They were also grouped, thanks to software tools, and annotated using public databases. Metabolite identification was achieved using two complementary approaches: (i) formal identification by matching chromatographic retention times, mass spectra, and also product ion spectra (if required) of metabolites to be characterized in biological data sets to those of reference compounds and (ii) putative identification from biological data thanks to MS/MS experiments for metabolites not available in our chemical library. By these means, 384 metabolites corresponding to 1484 annotated features (659 in negative ion mode and 825 in positive ion mode) were characterized in human urine samples. Of these metabolites, 192 and 66 were formally and putatively identified, respectively, and 54 are reported in human urine for the first time. These lists of features could be used by other laboratories to annotate their ESI-MS metabolomics data sets.

The Combined Escherichia coli Nissle 1917 and Tryptophan Treatment Modulates Immune and Metabolome Responses to Human Rotavirus Infection in a Human Infant Fecal Microbiota-Transplanted Malnourished Gnotobiotic Pig Model.

Human rotavirus (HRV) is a major cause of childhood diarrhea in developing countries where widespread malnutrition contributes to the decreased oral vaccine efficacy and increased prevalence of other enteric infections, which are major concerns for global health. Neonatal gnotobiotic (Gn) piglets closely resemble human infants in their anatomy, physiology, and outbred status, providing a unique model to investigate malnutrition, supplementations, and HRV infection. To understand the molecular signatures associated with immune enhancement and reduced diarrheal severity by Escherichia coli Nissle 1917 (EcN) and tryptophan (TRP), immunological responses and global nontargeted metabolomics and lipidomics approaches were investigated on the plasma and fecal contents of malnourished pigs transplanted with human infant fecal microbiota and infected with virulent (Vir) HRV. Overall, EcN + TRP combined (rather than individual supplement action) promoted greater and balanced immunoregulatory/immunostimulatory responses associated with greater protection against HRV infection and disease in malnourished humanized piglets. Moreover, EcN + TRP treatment upregulated the production of several metabolites with immunoregulatory/immunostimulatory properties: amino acids (N-acetylserotonin, methylacetoacetyl-CoA), lipids (gamma-butyrobetaine, eicosanoids, cholesterol-sulfate, sphinganine/phytosphingosine, leukotriene), organic compound (biliverdin), benzenoids (gentisic acid, aminobenzoic acid), and nucleotides (hypoxathine/inosine/xanthine, cytidine-5'-monophosphate). Additionally, the levels of several proinflammatory metabolites of organic compounds (adenosylhomocysteine, phenylacetylglycine, urobilinogen/coproporphyrinogen) and amino acid (phenylalanine) were reduced following EcN + TRP treatment. These results suggest that the EcN + TRP effects on reducing HRV diarrhea in neonatal Gn pigs were at least in part due to altered metabolites, those involved in lipid, amino acid, benzenoids, organic compounds, and nucleotide metabolism. Identification of these important mechanisms of EcN/TRP prevention of HRV diarrhea provides novel targets for therapeutics development. IMPORTANCE Human rotavirus (HRV) is the most common cause of viral gastroenteritis in children, especially in developing countries, where the efficacy of oral HRV vaccines is reduced. Escherichia coli Nissle 1917 (EcN) is used to treat enteric infections and ulcerative colitis while tryptophan (TRP) is a biomarker of malnutrition, and its supplementation can alleviate intestinal inflammation and normalize intestinal microbiota in malnourished hosts. Supplementation of EcN + TRP to malnourished humanized gnotobiotic piglets enhanced immune responses and resulted in greater protection against HRV infection and diarrhea. Moreover, EcN + TRP supplementation increased the levels of immunoregulatory/immunostimulatory metabolites while decreasing the production of proinflammatory metabolites in plasma and fecal samples. Profiling of immunoregulatory and proinflammatory biomarkers associated with HRV perturbations will aid in the identification of treatments against HRV and other enteric diseases in malnourished children.

Evaluation of the iChem® Velocity™ urine chemistry analyzer in a hospital routine laboratory.

BACKGROUND: The novel urine chemistry analyzer iChem Velocity (IRIS Diagnostics) offers improved urinalysis automation options through integration with the well-established iQ200 urine microscopy analyzer. In the course of optimizing the workflow in our hospital routine laboratory, we evaluated the performance of the iChem Velocity. METHODS: A total of 257 random urine samples were analyzed with the iChem Velocity, iQ200, Clinitek Atlas (Siemens Healthcare Diagnostics) and by manual microscopy. RESULTS: Depending on the parameter, 93% (hemoglobin) to 100% (urobilinogen), the iChem Velocity and Clinitek Atlas results agreed within the same rank or within one level of difference. The Clinitek Atlas featured a higher sensitivity for hemoglobin (area under the curve 0.86) and leukocyte esterase (area under the curve 0.85) compared with the iChem Velocity (area under the curve for hemoglobin 0.73, leukocytes 0.78). Imprecision was highest for hemoglobin and leukocytes in a pathological sample pool. While the precision of the Clinitek Atlas for hemoglobin measurements was superior, the iChem Velocity was more precise in analyzing protein and pH. CONCLUSIONS: Through urinalysis automation with the iChem Velocity and iQ200, we achieved a reduction of hands-on time by 89%. The sensitivity of this new system should be further improved through ongoing development.

Acute generalized exanthematous pustulosis and Coombs-positive hemolytic anemia in a child following Loxosceles reclusa envenomation.

Previously reported cases of acute generalized exanthematous pustulosis secondary to brown recluse spider bite have been questioned due to lack of identification of the spider or because of the concomitant administration of antibiotics. We report a 9-year-old boy who arrived at the emergency department with a confirmed Loxosceles reclusa bite to the neck. On the third day of hospitalization, he developed hundreds of monomorphous, sterile pustules, initially in intertriginous areas. The eruption disseminated and was followed by pinpoint desquamation typical for acute generalized exanthematous pustulosis. During this he also developed late onset Coombs-positive hemolytic anemia and systemic loxoscelism. Sphingomyelinase in Loxosceles venom induces the production of interleukin-8 and granulocyte-macrophage colony-stimulating factor, cytokines involved in the pathogenesis of acute generalized exanthematous pustulosis, providing a mechanism by which Loxosceles reclusa bite may trigger acute generalized exanthematous pustulosis. We suggest that this case adds Loxosceles envenomation to the spectrum of agents that can trigger acute generalized exanthematous pustulosis.

Association of UGT1A1 Gly71Arg with urine urobilinogen.

Bilirubin is glucoronized by uridine diphosphate-glucuronosyl transferase 1A1 (UGT1A1) mainly in the liver, and excreted into bile. The conjugated form is metabolized into the unconjugated form, and then into urobilinogen by bacteria in the intestine. Unconjugated bilirubin and urobilinogen are absorbed into the blood stream. The kidney filtrates conjugated bilurubin and urobilinogen into urine. Accordingly, the reduced enzyme activity of UGTIAI may decrease serum conjugated bilirubin levels, resulting in a lower frequency of positive results of urine bilirubin and urobilinogen. This study examined the associations of UGTIAI Gly71Arg (UGTIAI *6) with urine bilirubin and urobilinogen, as well as serum AST, ALT and GGT. Subjects were 5,172 inhabitants 35 to 69 years old who participated in a cohort study in Nagoya from June 2008 to May 2010. Among them, data from 5,151 participants (1,465 males and 3,686 females) were available for analysis. The age-sex-adjusted odds ratio (OR) of ArgArg relative to GlyGly was 1.37 (95% confidence interval (95% CI), 0.55-1.23) for bilirubin, and 1.67 (95% CI, 0.86-3.26) for urobilinogen. Those of ArgArg+ArgGly were 0.87 (95% CI, 0.59-1.27) and 1.50 (95% CI, 1.17-1.94), respectively. AST, ALT and GGT levels had no associations with the genotype. Although the significant association for urobilinogen was contrary to the biological expectation, this study indicated that UGTIA1 Gly71Arg may be a genetic factor of urine urobilinogen.

Abnormal findings on dipstick urinalysis of out-patients with malaria in Abakaliki, Nigeria.

BACKGROUND & OBJECTIVES: Malaria, one of the major health challenges of the tropics affecting about 500 million people, particularly the children and pregnant women have been associated with changes in urine compositions. The present study was undertaken to document the urinary abnormalities in malaria patients based on malaria species and the level of malaria parasitaemia. METHODS: Febrile patients (n = 365) with positive Giemsa - stained blood films for malaria recruited from Outpatient Department of Ebonyi State University Teaching Hospital, Abakaliki participated in the study. Patients were classified into two categories (+ and ++) based on parasite density. Apparently healthy individuals (n = 81), without malaria parasite on both thick and thin films of comparable age and gender acted as control group. Urine sample (10 ml) was collected from each participant and analysed using standard laboratory methods and techniques. RESULTS: Seventy - four (20.3%) of the patients had Plasmodium falciparum malaria. Although all the urine parameters were higher in the malarial patients in comparison to the control, only bilirubinuria and urobilinogenuria were statistically significant (p <0.05). Also, bilirubinuria, urobilinogenuria, haematuria and proteinuria were significantly (p < 0.05) higher in P. falciparum infection than in infections with other malaria species, but only in P. falciparum infection, bilirubinuria and urobilinogenuria were significantly (p < 0.05) higher at higher parasitaemia. CONCLUSION: Even though positive blood film for malaria parasite remains the gold standard for the diagnosis of malaria, urinary abnormalities, such as bilirubinuria, urobilinogenuria, proteinuria and haematuria may aid in identifying patients with severe malaria parasitaemia, especially the falciparum malaria.

Urine markers of kidney disorders and their risk associations in HIV-infected patients attending Nyanza Provincial General Hospital in Kisumu, Western Kenya.

OBJECTIVE: To identify abnormal levels of urine metabolites and cells that serve as markers of existing kidney disorders in ambulatory HIV-infected patients. DESIGN: A cross sectional study. SETTING: Nyanza Provincial General Hospital's patient support centre. SUBJECTS: A total of 593 HIV infected patients were studied. INTERVENTION: Dipstick urinalysis test was used to screen mid stream urine to detect constituents with altered levels. RESULTS: Out of the 593 participants, the urine of 214 (36.1%) had abnormally altered levels of urine constituents, with more females afflicted than males [41.5% vs. 27.8%; OR 1.84 (1.28-2.63), chi2 = 11.08, p = 0.0009]. Urobilinogen was the most common urine metabolite while ketones were least commonly present. More participants had altered levels of leucocytes than erythrocytes in urine. Patients with pyuria were three times more likely to have elevated erythrocytes in their urine as well (chi2 = 34.37, p < 0.0001). Similarly, the risk of having proteinuria was three times higher in patients with pyuria (p < 0.0003, Fisher's test). Patients with erythrocytes in urine also had a threefold likelihood of having proteinuria (P < 0.0003, Fisher's test). Fewer ARV users had abnormal urine markers [15.7% vs 24.3% OR 0.62 (0.41-0.94), chi2 = 5.2, p < 0.05]. CONCLUSION: Metabolites and cellular markers of kidney disorders were prevalent in the urine of HIV patients especially females and those with pronounced immune depletion (CD4 counts equal to or below 500). ARVs use was associated with reduced manifestation of these markers.

Carboxyhemoglobin elevation after exposure to dichloromethane.

Inhalation of dichloromethane vapor in concentrations of 500 to 1000 parts per million for 1 to 2 hours promptly initiated the formation of significant quantities of carbon monoxide in human subjects. The evidence suggests that carbon monoxide may be a metabolite of dichloromethane and, that exposure to concentrations of dichloromethane below the industrial threshold limit values may result in the formation of carbon monoxide in amounts that exceed the allowable limits.

Bovine protoporphyria: the first nonhuman model of this hereditary photosensitizing disease.

Protoporphyria, a photosensitizing disease documented only in humans, was transmitted as a recessive trait to seven female calves. Cutaneous lesions were extensive, and erythrocyte and fecal protoporphyrin concentrations exceeded by far those of human protoporphyria. Average ferrochelatase activity was decreased to one-half of normal in the liver of carriers, and to about one-tenth of normal in liver, kidney, heart, spleen, lung, and marrow of protoporphyrics.

Renal excretion of urobilinogen in the dog.

The renal excretion of urobilinogen was studied in dogs by standard clearance techniques. The use of radiochemically pure tritiated mesobilirubinogen as a representative urobilinogen afforded much greater analytical precision than can be obtained with the usual colorimetric and fluorimetric techniques which are only semiquantitative. With constant plasma levels of urobilinogen, raising urinary pH from 5 to 8 increased urobilinogen excretion from about 30% to up to 200% of the filtered load. When urinary pH was kept constant, changes in blood pH had no effect on urobilinogen excretion. Increases in urinary flow had no effect on urobilinogen excretion when the urine was alkaline but increased excretion markedly during aciduria. Probenecid did not influence urobilinogen excretion by the kidney. It is concluded that urobilinogen is excreted by a three-component system of glomerular filtration, active secretion, and pH-dependent nonionic diffusion in the distal nephron. Urobilinogen is a weak acid, and this mode of excretion is similar to that of other weak, organic acids, such as salicylates. These results indicate that urinary pH and flow must be considered in the clinical interpretation of measurements of urinary urobilinogen.

Absorption of bile pigments by the gall bladder.

A technique is described for preparation in the guinea pig of an in situ, isolated, vascularized gall bladder that exhibits normal absorptive functions. Absorption of labeled bile pigments from the gall bladder was determined by the subsequent excretion of radioactivity in hepatic bile. Over a wide range of concentrations, unconjugated bilirubin-(14)C was well absorbed, whereas transfer of conjugated bilirubin proceeded slowly. Mesobilirubinogen-(3)H was absorbed poorly from whole bile, but was absorbed as rapidly as unconjugated bilirubin from a solution of pure conjugated bile salt. Bilirubin absorption was not impaired by iodoacetamide, 1.5 mM, or dinitrophenol, 1.0 mM, even though water transport was affected. This indicated that absorption of bilirubin was not dependent upon water transport, nor upon energy-dependent processes. The linear relationship between absorption and concentration of pigment at low concentrations in bile salt solutions suggested that pigment was transferred by passive diffusion. At higher pigment concentrations or in whole bile, this simple relationship was modified by interactions of pigment with bile salts and other constituents of bile. These interactions did not necessarily involve binding of bilirubin in micelles. The slow absorption of the more polar conjugates and photo-oxidative derivatives of bilirubin suggested that bilirubin was absorbed principally by nonionic, and partially, by ionic diffusion. Concentrations of pure conjugated bile salts above 3.5 mM were found to be injurious to the gall bladder mucosa. This mucosal injury did not affect the kinetics of bilirubin absorption. During in vitro incubation of bile at 37 degrees C, decay of bilirubin and hydrolysis of the conjugate proceeded as first-order reactions. The effects of these processes on the kinetics of bilirubin absorption, and their possible role in the formation of "white bile" and in the demonstrated appearance of unconjugated bilirubin in hepatic bile, are discussed.

Hemolysis during and after 21 days of head-down-tilt bed rest.

Hemoconcentration is observed in bed rest studies, descent from altitude, and exposure to microgravity. Hemoconcentration triggers erythrocyte losses to subsequently normalize erythrocyte concentration. The mechanisms of erythrocyte loss may involve enhanced hemolysis, but has never been measured directly in bed rest studies. Steady-state hemolysis was evaluated by measuring two heme degradation products, endogenous carbon monoxide concentration [CO] and urobilinogen in feces, in 10 healthy men, before, during, and after two campaigns of 21 days of 6° head-down-tilt (HDT) bed rest. The subjects were hemoconcentrated at 10 and 21 days of bed rest: mean concentrations of hemoglobin (15.0 ± 0.2 g/L and 14.6 ± 0.1 g/L, respectively) and erythrocytes (5.18 ± 0.06E6/µL and 5.02 ± 0.06E6/µL, respectively) were increased compared to baseline (all Ps < 0.05). In contrast, mean hemoglobin mass (743 ± 19 g) and number of erythrocytes (2.56 ± 0.07E13) were decreased at 21 days of bed rest (both Ps < 0.05). Indicators of hemolysis mean [CO] (1660 ± 49 ppb and 1624 ± 48 ppb, respectively) and fecal urobilinogen concentration (180 ± 23 mg/day and 199 ± 22 mg/day, respectively) were unchanged at 10 and 21 days of bed rest compared to baseline (both Ps > 0.05). A significant decrease in [CO] (-505 ppb) was measured at day 28 after bed rest. HDT bed rest caused hemoconcentration in parallel with lower hemoglobin mass. Circulating indicators of hemolysis remained unchanged throughout bed rest supporting that enhanced hemolysis did not contribute significantly to erythrocyte loss during the hemoconcentration of bed rest. At day 28 after bed rest, decreased hemolysis accompanied the recovery of erythrocytes, a novel finding.