Species differences in bile acids II. Bile acid metabolism

One of the mechanisms of drug‐induced liver injury (DILI) involves alterations in bile acid (BA) homeostasis and elimination, which encompass several metabolic pathways including hydroxylation, amidation, sulfation, glucuronidation and glutathione conjugation. Species differences in BA metabolism may play a major role in the failure of currently used in vitro and in vivo models to predict reliably the DILI during the early stages of drug discovery and development. We developed an in vitro cofactor‐fortified liver S9 fraction model to compare the metabolic profiles of the four major BAs (cholic acid, chenodeoxycholic acid, lithocholic acid and ursodeoxycholic acid) between humans and several animal species. High‐ and low‐resolution liquid chromatography–tandem mass spectrometry and nuclear magnetic resonance imaging were used for the qualitative and quantitative analysis of BAs and their metabolites. Major species differences were found in the metabolism of BAs. Sulfation into 3‐O‐sulfates was a major pathway in human and chimpanzee (4.8%–52%) and it was a minor pathway in all other species (0.02%–14%). Amidation was primarily with glycine (62%–95%) in minipig and rabbit and it was primarily with taurine (43%–81%) in human, chimpanzee, dog, hamster, rat and mice. Hydroxylation was highest (13%–80%) in rat and mice followed by hamster, while it was lowest (1.6%–22%) in human, chimpanzee and minipig. C6‐β hydroxylation was predominant (65%–95%) in rat and mice, while it was at C6‐α position in minipig (36%–97%). Glucuronidation was highest in dog (10%–56%), while it was a minor pathway in all other species (<12%). The relative contribution of the various pathways involved in BA metabolism in vitro were in agreement with the observed plasma and urinary BA profiles in vivo and were able to predict and quantify the species differences in BA metabolism. In general, overall, BA metabolism in chimpanzee is most similar to human, while BA metabolism in rats and mice is most dissimilar from human.     

Mandibular Hader bar attachment overdenture wearers: A five-year longitudinal clinical evaluation by electromyographic analysis

IntroductionElectromyography is used to evaluate the muscle activity of temporalis and masseter muscles in a comparative clinical research on effectiveness of masticatory cycle by conventional overdenture and use of overdenture with Hader bar attachment for five years established on a scientific level and is first of its kind.AimThe purpose of this study is to determine the response of co-ordination of stomatognathic system and the functional status of long-term overdenture use, which can be recorded in EMG.Material and MethodsTen patients were treated with maxillary conventional complete denture and mandibular overdenture. Electrical activity of masseter and temporal muscles were obtained in 3 groups. (Gp. I) After insertion of conventional overdenture with copings; (Gp. II) Overdenture with Hader bar attachment and (Gp.III) five years of use of overdenture with Hader bar attachment.Results and conclusionThe mean and standard deviation for all the patients showed an increase in muscular activity of temporalis and masseter muscles after long-term rehabilitation (x = 0.405; 0.407 and s = 0.0668; 0.1344 respectively, p = 0.0042 and 0.0074 which is <0.05). This study concludes that overdenture with Hader bar system, after five years of function provide a sound justification for a viable alternative treatment modality to provide overdenture with attachment service to patients.     

Longitudinal studies in South Indian villages on Japanese encephalitis virus infection in mosquitoes and seroconversion in goats

Japanese encephalitis (JE) is endemic in Cuddalore district, Tamil Nadu, where Culex tritaeniorhynchus Giles was the major vector. We screened 45 100 adult female Cx. tritaeniorhynchus (902 pools) by enzyme-linked immunosorbent assay and isolated and confirmed JE virus (JEV) by using an insect bioassay system. We had 69 isolates of which 62 (90%) were identified as JEV. The average vector abundance per man hour for Cx. tritaeniorhynchus was 324.5 per month for the period June 1998-May 2000. The average minimum infection rate (MIR) per month in Cx. tritaeniorhynchus was 1.4 (range 0.0-5.6). Every year, a new batch of goats, 20 in the first year and 31 in the second year, born during the non-JE transmission period (January-June), aged <6 months and negative for haemagglutination inhibition (HI) antibodies were procured and placed in the villages as sentinels. Fortnightly, blood specimens were collected from these goats and tested for JE antibodies by HI test. Seroconversions (SCs) were recorded in 14 goats (70%) in the first year and 23 goats (74%) in the second year. JE HI antibody titres in goats were low (1:10-1:80) and these levels declined to undetectable levels in about 4 weeks following SCs. The time sequence of events indicated that four of five peaks of MIR in mosquitoes were followed 1-3 months later by peaks in the proportion of seroconverted goats. We suggest the screening of goats and cattle as a more feasible tool to stratify areas according to JE infection risk to the human population through the regular health system rather than screening mosquitoes using monoclonal antibodies, which is possible only in specialized laboratories.     

Pregnancy and rheumatic disease: “by the book” or “by the doc”

Pregnancy is an important condition that can affect and be affected by rheumatic disease. Overall, pregnancy is viewed as a Th2-predominant state, but several Th1-related cytokines are vital to early pregnancy. In rheumatoid arthritis for example, the majority of women improve by the beginning of the second trimester, but the majority (90%) will flare in the first 3 to 4 months postpartum. In contrast, systemic lupus erythematosus has an unpredictable course in pregnancy, leaving most rheumatologists to recommend a disease-quiescent state prior to conception. Other diseases such as scleroderma are less clear because the disease less commonly presents in the childbearing period. Many immunosuppressive medications for the rheumatic diseases are contraindicated in pregnancy because of their mechanisms of action leaving only a select few “safe” medications. Significant heterogeneity between the Food and Drug Administration (FDA) category for a medication and what a rheumatologist does in clinic leads to confusion on how a patient should be treated for active rheumatic disease both peripartum and postpartum, particularly if the patient is breastfeeding. We review the general state of pregnancy and how it is affected by prototypical rheumatic diseases including rheumatoid arthritis and systemic lupus erythematosus. In addition, we present the most commonly used disease-modifying antirheumatic drugs and immunosuppressants and explain the difference between the FDA category and clinical practice among rheumatologists. Finally, we provide some general recommendations on how to manage a rheumatic disease during pregnancy including: (a) preconception planning to ensure no teratogenic medications on board, (b) early disclosure of pregnancy to all caregivers including the rheumatologist, family physician, obstetrician, and maternal–fetal medicine specialist, and (c) planning of safe medication use for acute flare-ups and disease suppression peripartum and postpartum. Presented as Medical Grand Rounds at the University of Alberta.     

Sacrococcygeal neurofibroma: rare cause for chronic pelvic pain

Pelvic pain is a common gynecologic complaint. Retroperitoneal pelvic tumors are rarely a cause of pelvic pain. Neurofibroma is an uncommon pelvic retroperitoneal tumor, and only 17 cases are reported to date. A 38-year-old woman with chronic pelvic pain had a soft fixed mass that was the size of an orange in the right posterolateral fornix, with a normal uterus on pelvic examination, and a mass of 6.3 × 5.2 cm with mixed echotexture on the right side separate from both ovaries on transvaginal ultrasonography. A provisional diagnosis of retroperitoneal mass probably a retroperitoneal teratoma was made. Laparoscopy was performed; an ill-defined retroperitoneal soft tissue mass of about 6 cm was seen on the right pararectal and presacral area, displacing the rectum toward the left side. The mass was soft and jellylike without a cyst wall. Histopathologic study and immunohistochemistry results were consistent with neurofibroma of the sacrococcygeal regions. To our knowledge this is the third case of sacrococcygeal neurofibroma treated by complete laparoscopic excision. Gynecologists should keep sacrococcygeal neurofibroma as a differential diagnosis of pelvic pain with atypical location of a pelvic mass. A high index of suspicion and an appropriate imaging technique are needed for accurate diagnosis. Laparoscopy seems to be a safe and effective method of managing retroperitoneal presacral neurofibromas.     

Investigation of chloroform-methanol soluble wheat proteins and sphingolipids as potential dietary triggers of diabetes in BBdp rats

The present study is a follow-up to the study by Coleman (1990), which found that the incidence of diabetes reduced in non-obese diabetic (NOD) mice when chloroform-methanol soluble substances were removed from a cereal-based diet. We used a 2:1 chloroform-methanol (C-M) mixture to extract C-M soluble sphingolipids and proteins from wheat gluten. The C-M extract was subjected to silica gel chromatography and saponification to remove triglycerides, glycolipids and phospholipids. This sphingolipid-enriched fraction from wheat gluten was subsequently incorporated into a BioBreeding diabetes-prone (BBdp) rat diet. Five diets were fed over a 120-day period to BBdp rats: a negative control hydrolysed casein (HC) diet, a positive control National Toxicology Program 2000 (NTP 2000) diet, a wheat gluten (WG)-based diet, a WGGSLF diet containing the WG residue remaining after chloroform-methanol extraction, and a hydrolysed casein plus sphingolipid-enriched fraction (HCGSL) diet. There was a significant increase in diabetes incidence in rats fed the HC + wheat sphingolipid diet versus the HC diet at days 70 and 80 of the feeding study, but not at the end of the study (120 days). There were no significant differences in diabetes incidence in rats fed the WG diet and WG diet with sphingolipids removed. There were no significant differences in pancreatic insulitis scores or in the numbers of jejunal CD4+ and γδTCR + T cells in BBdp rats due to the dietary addition or absence of CM soluble substances from wheat gluten. Sera IFN-γ levels were significantly higher in the sphingolipid-removed WG diet group compared to the BBdp rats in the other four diet groups. Overall, the results of this study provide some evidence for a slight promotional effect of C-M soluble sphingolipids and proteins in wheat gluten on the incidence of diabetes and up-regulation of immune responses in the BBdp rat.     

Toxicity of multiwalled carbon nanotubes with end defects critically depends on their functionalization density

Carboxylated carbon nanotubes stand as the most promising nanovectors for biomedical and pharmaceutical applications due to their ease of covalent conjugation with eclectic functional molecules including therapeutic drugs, proteins, and oligonucleotides. In the present study, we attempt to investigate how the toxicity of acid-oxidized multiwalled carbon nanotubes (MWCNTs) can be tweaked by altering their degree of functionalization and correlate the toxicity trend with their biodistribution profile. In line with that rationale, mice were exposed to 10 mg/kg of pristine (p) and acid-oxidized (f) MWCNTs with varying degrees of carboxylation through a single dose of intravenous injection. Thereafter, extensive toxicity studies were carried out to comprehend the short-term (7 day) and long-term (28 day) impact of p- and various f-MWCNT preparations on the physiology of healthy mice. Pristine MWCNTs with a high aspect ratio, surface hydrophobicity, and metallic impurities were found to induce significant hepatotoxicity and oxidative damage in mice, albeit the damage was recovered after 28 days of treatment. Conversely, acid-oxidized carboxylated CNTs with shorter lengths, hydrophilic surfaces, and high aqueous dispersibility proved to be less toxic and more biocompatible than their pristine counterparts. A thorough scrutiny of various biochemical parameters, inflammation indexes, and histopathological examination of liver indicated that toxicity of MWCNTs systematically decreased with the increased functionalization density. The degree of shortening and functionalization achieved by refluxing p-MWCNTs with strong mineral acids for 4 h were sufficient to render the CNTs completely hydrophilic and biocompatible, while inducing minimal hepatic accumulation and inflammation. Quantitative biodistribution studies in mice, intravenously injected with Tc-99m labeled MWCNTs, clearly designated that clearance of CNTs from reticuloendothelial system (RES) organs such as liver, spleen, and lungs was critically functionalization density dependent. Well-individualized MWCNTs with shorter lengths (<500 nm) and higher degrees of oxidation (surface carboxyl density >3 μmol/mg) were not retained in any of the RES organs and rapidly cleared out from the systematic circulation through renal excretion route without inducing any obvious nephrotoxicity. As both p- and f-MWCNT-treated groups were devoid of any obvious nephrotoxicity, CNTs with larger dimensions and lower degrees of functionalization, which fail to clear out from the body via renal excretion route, were thought to be excreted via biliary pathway in faeces.     

Basal level insulin delivery: in vitro release, stability, biocompatibility, and in vivo absorption from thermosensitive triblock copolymers

The major goal of this study was to develop the biodegradable and biocompatible thermosensitive polylactic acid-polyethylene glycol-polylactic acid triblock copolymer-based delivery systems for controlled release of basal level insulin for a longer duration after single subcutaneous injection. Insulin was dispersed into aqueous copolymer solutions to prepare the delivery system. The in vitro release profile of insulin from delivery systems was studied at 37°C in phosphate-buffered saline. Stability of released insulin was investigated using circular dichroism, differential scanning calorimetry, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and skin histology were used to determine the in vitro and in vivo biocompatibility of the delivery systems, respectively. Streptozotocin-induced diabetic rat model was used to study the in vivo absorption and bioactivity of insulin. In vitro release studies indicated that the delivery systems released insulin over 3 months in structurally stable form. The delivery systems were biocompatible in vitro and in vivo. In vivo absorption and bioactivity studies demonstrated elevated insulin level and corresponding decreased blood glucose level in diabetic rats. Thus, the delivery systems released insulin at a controlled rate in vitro in conformationally and chemically stable form and in vivo in biologically active form up to 3 months.