Retigabine N-glucuronidation and its potential role in enterohepatic circulation.

The metabolism of retigabine in humans and dogs is dominated by N-glucuronidation (), whereas in rats, a multitude of metabolites of this new anticonvulsant is observed (). The comparison of the in vivo and in vitro kinetics of retigabine N-glucuronidation in these species identified a constant ratio between retigabine and retigabine N-glucuronide in vivo in humans and dog. An enterohepatic circulation of retigabine in these species is likely to be the result of reversible glucuronidation-deglucuronidation reactions. Rats did not show such a phenomenon, indicating that enterohepatic circulation of retigabine via retigabine N-glucuronide does not occur in this species. In the rat, 90% of retigabine N-glucuronidation is catalyzed by UDP-glucuronosyltransferase (UGT)1A1 and UGT1A2, whereas family 2 UGT enzymes contribute also. Of ten recombinant human UGTs, only UGTs 1A1, 1A3, 1A4, and 1A9 catalyzed the N-glucuronidation of retigabine. From the known substrate specificities of UGT1A4 toward lamotrigine and bilirubin and our activity and inhibition data, we conclude that UGT1A4 is a major retigabine N-glucuronosyl transferase in vivo and significantly contributes to the enterohepatic cycling of the drug.     

Measurement of albumin and low molecular weight proteins in the urine of newborn infants using a cotton wool ball collection method

Objective : The aim of this study was to investigate the inter-relationship between urinary excretion of alpha-1-microglobulin (AIM), retinol-binding protein (RBP) and albumin in term and premature neonates, with urine collected into cotton wool balls and extracted by a novel method. Subjects and methods : Sixty-four infants were studied on the first day of life; 26 had been born at term (37–42 weeks gestation) and 38 prematurely (24–28 weeks n = 16, 29–36 weeks n = 22). Urine collected into cotton wool balls was analysed following a new detergent extraction method, which resulted in a recovery rate of 94–107% for albumin, AIM, RBP and creatinine. Results : Urinary protein excretion, expressed as a ratio to urinary creatinine, decreased significantly with increasing gestational age (24–28 weeks, 29–36 weeks, 37–42 weeks: albuminxreatinine ratio mg/mmol mean 96.9, 31.7, 19.3; AIM: creatinine ratio mg/mmol mean 99.3, 37.0, 7.8; RBP: creatinine ratio mg/mmol mean 16.2, 3.8, and <0.01, below the limit of detection, respectively). When results were corrected for birthweight, this gestation-associated effect was still present for A1M and RBP, but not for albumin. In premature infants there was a significant positive correlation between AIM: creatinine ratio and RBP: creatinine ratio ( r = 0.85), and also between albumin and both AIM and RBP ( r = 0.82 and 0.77). Conclusion : Increased excretion of AIM, RBP and albumin at earlier gestational ages is probably due to proximal tubular immaturity, although tubular damage and also glomerular dysfunction cannot be excluded as possible explanations.     

Prophylactic total gastrectomy in the management of hereditary tumor syndromes

Purpose

Germline mutations in several genes confer a relevant lifetime risk of gastric cancer. In this context, an increasing involvement of a surgeon can be seen, mainly with the question of performing a prophylactic operation.

Methods

Patients with hereditary tumor syndromes predisposing for gastric cancer who received care leading to prophylactic total gastrectomy in our Center for Hereditary Tumor Syndromes were analyzed. For each patient, the multidisciplinary decision-making process, the perioperative course, and the histopathologic findings were assessed. Short-term morbidity was evaluated based on the medical reports.

Results

The analysis includes nine patients (six female, three male) with a median age of 41.6 (range 23–60) years. Indication for prophylactic total gastrectomy was based on family history and genetic analysis (eight patients with a germline mutation of the CDH1 gene and one patient with a SMAD4 mutation). Removal of the entire gastric mucosa was documented intraoperatively by fresh frozen section examination. Extended (DII) lymphadenectomy was performed in four patients. Histopathologic examination of gastrectomy specimens revealed six patients (6/9, 67 %) with multifocal signet ring cell carcinomas. In our series, prophylactic total gastrectomy was a safe procedure without mortality and low morbidity.

Conclusions

Patients with hereditary syndromes predisposing for gastric cancer should be evaluated for this curative procedure in a specialized center. Further research is necessary, and the implementation of nationwide registers including patients with prophylactic gastrointestinal operations due to hereditary tumor syndrome is advisable.

     

Consensus-recommendations for sirolimus in liver transplantation

Sirolimus is an m-TOR inhibitor without renal side effects and potentially protects against the development of malignancy. Due to a higher incidence of complications in two trials and an official warning in the drug information, the use of Sirolimus in liver transplantation is limited. The participants of this consensus meeting had to analyse and evaluate the literature with respect to the potential role of Sirolimus in liver transplantation. This consensus statement follows the scheme normally employed for the presentation of guidelines including the grading of evidence (1a-5) and the extent of recommendation (A-C). Moreover, the consensus included the experience of the authors with respect to the handling of Sirolimus after liver transplantation.     

Autoantibodies and autoantigens in autoimmune hepatitis

The diagnosis of autoimmune hepatitis (AIH) relies on the exclusion of viral, metabolic, genetic, and toxic etiologies of chronic hepatitis or hepatic injury. There are few parameters that positively predict the presence of AIH. Autoantibodies have been intensively evaluated in this respect and have led to the classification of AIH into three serological subgroups: antinuclear and smooth muscle antibody-positive (ANA/SMA, type 1), liver-kidney microsomal antibody-positive (LKM-1, type 2), and soluble liver antigen/liver-pancreas antigen antibody-positive (SLA/LP, type 3) AIH. Although there are few clinical implications resulting from this classification, autoantibody profiles indicate that AIH is a heterogenous group of entities. The molecular characterization of B cell autoimmunity has led to the identification of major phase I and phase II metabolic enzymes as well as structural and functional components of the cell nucleus as immunologic targets. Autoantibodies and their corresponding autoantigens are intensively studied to provide clues to the understanding of disease initiation, tissue specificity, and propagation of hepatic autoimmune diseases.     

Role of kidney in the catabolic clearance of human platelet antiheparin proteins from rat circulation

Stimulated platelets release at least two antiheparin proteins: platelet factor 4 (PF4) and low affinity platelet factor 4 (LA-PF4) from which beta-thromboglobulin (beta TG) is derived. We have found previously marked elevation of LA-PF4/beta TG antigen in platelet poor plasma of patients with chronic renal failure, whereas levels of PF4 remained normal. Therefore, we examined the role of the kidneys in the metabolic clearance of LA-PF4/beta TG and PF4. The supernates of aggregates of thrombin-stimulated human platelets were injected into sham operated control rats, nephrectomized rats, and into rats with acute ureteral ligation. The disappearance of human LA-PF4/beta TG antigen and PF4 in rat plasma determined by specific radioimmunoassays followed biphasic exponential curves. The half-lives (t1/2) for the fast and slow components of LA-PF4 in control rats were 6.4 and 68.4 min. Nephrectomy significantly increased these times to 9.7 and 144 min, while ureteral ligation resulted in no significant change. Comparison of the level of LA-PF4/beta TG antigen and of creatinine in aorta and in renal vein showed 25%-30% extraction of these compounds by the kidney. Less than 0.1% of the total LA-PF4 antigen injected was recovered in the urine of control rats. In contrast to these results, the clearance of PF4 was not affected by nephrectomy. In conclusion: (1) functional renal tissue is necessary for normal clearance of LA- PF4/beta TG, but renal excretion does not play a major role in its elimination suggesting that the protein is catabolized by the kidney; and (2) catabolic clearance of PF4 does not depend on functioning kidney tissue.     

Immunoglobulin G from patients with heparin-induced thrombocytopenia binds to a complex of heparin and platelet factor 4

Heparin-induced thrombocytopenia (HIT) is an important complication of heparin therapy. Although there is general agreement that platelet activation in vitro by the HIT IgG is mediated by the platelet Fc receptor, the interaction among the antibody, heparin, and platelet membrane components is uncertain and debated. In this report, we describe studies designed to address these interactions. We found, as others have noted, that a variety of other sulfated polysaccharides could substitute for heparin in the reaction. Using polysaccharides selected for both size and charge, we found that reactivity depended on two independent factors: a certain minimum degree of sulfation per saccharide unit and a certain minimum size. Hence, highly sulfated but small (< 1,000 daltons) polysaccharides were not reactive nor were large but poorly sulfated polysaccharides. The ability of HIT IgG to recognize heparin by itself was tested by Ouchterlony gel diffusion, ammonium sulfate and polyethylene glycol precipitation, and equilibrium dialysis. No technique demonstrated reactivity. However, when platelet releasate was added to heparin and HIT IgG, a 50-fold increase in binding of radio-labeled heparin to HIT IgG was observed. The releasate was then depleted of proteins capable of binding to heparin by immunoaffinity chromatography. Only platelet factor 4-immunodepleted releasate lost its reactivity with HIT IgG and heparin. Finally, to determine whether the reaction occurred on the surface of platelets or in the fluid phase, washed platelets were incubated with HIT IgG or heparin and after a wash step, heparin or HIT IgG was added, respectively. Reactivity was only noted when platelets were preincubated with heparin. Consistent with these observations was the demonstration of the presence of PF4 on platelets using flow cytometry. These studies indicate that heparin and other large, highly sulfated polysaccharides bind to PF4 to form a reactive antigen on the platelet surface. HIT IgG then binds to this complex with activation of platelets through the platelet Fc receptors.