Development of ß-sheet structure in Aß aggregation intermediates diminishes exposed hydrophobic surface area and enhances proinflammatory activity.

Three decades of research, both in vitro and in vivo, have demonstrated the conformational heterogeneity that is displayed by the amyloid ß peptide (Aß) in Alzheimer's disease (AD). Understanding the distinct properties between Aß conformations and how conformation may impact cellular activity remain open questions, yet still continue to provide new insights into protein misfolding and aggregation. In particular, there is interest in the group of soluble oligomeric prefibrillar Aß species comprising lower molecular weight oligomers up to larger protofibrils. In the current study, a number of strategies were utilized to separate Aß protofibrils and oligomers and show that the smaller Aß oligomers have a much different conformation than Aß protofibrils. The differences were consistent for both Aß40 and Aß42. Protofibrils bound thioflavin T to a greater extent than oligomers, and were highly enriched in ß-sheet secondary structure. Aß oligomers possessed a more open structure with significant solvent exposure of hydrophobic domains as determined by tryptophan fluorescence and bis-ANS binding, respectively. The protofibril-selective antibody AbSL readily discerned conformational differences between protofibrils and oligomers. The more developed structure for Aß protofibrils ultimately proved critical for provoking the release of tumor necrosis factor α from microglial cells. The findings demonstrated a dependency on ß-sheet structure for soluble Aß aggregates to cause a microglial inflammatory response. The Aß aggregation process yields many conformationally-varied species with different levels of ß-structure and exposed hydrophobicity. The conformation elements likely determine biological activity and pathogenicity.

Development of a Simple and Effective Lipid-A Antagonist Based on Computational Prediction.

Sepsis is a serious medical condition characterized by bacterial infection and a subsequent massive systemic inflammatory response. In an effort to identify compounds that block lipopolysaccharide (LPS)-induced inflammation reported herein is the development of simple Lipid-A analogues that lack a disaccharide core yet still possess potent antagonistic activity against LPS. The structure of the new lead compound was developed based on predictive computational experiments. LPS antagonism by the lead compound was not straightforward, and a biphasic effect was observed suggesting a possibility of more than one binding site. An IC50 value of 13 nM for the new compound was determined for the possible high affinity site. The combination of computational, synthetic, and biological studies revealed new structural determinants of these simplified analogues. It is expected that the acquired information will aid future design of LPS targeting glycopharmaceuticals.

Fetal exposure to polybrominated diphenyl ethers and the risk of hypospadias: focus on the congeners involved.

BACKGROUND: Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants, and their endocrine-disrupting properties have focused growing attention regarding their teratogenic potential. We have recently documented that mothers of children born with hypospadias had been exposed to statistically higher levels of PBDE during pregnancy than mothers of healthy controls. However, it is not known which congeners of PBDE are associated with this putative teratogenic effect. OBJECTIVES: To identify PBDE congeners associated with increased risk for hypospadias. STUDY METHODS: Hair samples from mothers were analyzed and compared between hypospadias cases and healthy controls for eight PBDE congeners using gas chromatography mass spectrometry (GC/MS). Polybrominated diphenyl ether levels were measured in the 0- to 3-cm segment closest to the skull of maternal hair as a proxy for in utero exposure of mothers who lived in the same environment for the duration of their pregnancy. RESULTS: Median maternal hair levels of five PBDE congeners (28, 47, 99, 153, and 154) and of total PBDE (∑PBDE) were significantly higher among mothers of infants with hypospadias (n = 152) than among controls (n = 64). Apparent greater differences in the lower brominated congeners, especially in BDE-47 and BDE-99, may be due to the fact that they had been used in larger amounts, and their persistence properties confer longer exposure. CONCLUSIONS: The majority of the lower brominated PBDE congeners measured in maternal hair exhibited higher PBDE body burden during pregnancy in mothers of infants who were born with hypospadias.

Adverse placental effect of formic acid on hCG secretion is mitigated by folic acid.

AIMS: Formic acid has recently been detected in maternal blood and umbilical cord blood of infants born to alcohol abusing mothers. This toxic metabolite of methanol requires folate for detoxification. We hypothesized that formic acid produced in the maternal circulation will transfer across the placenta and will be toxic to the placenta. Our objectives were, first, to determine whether formic acid transfers across the human placenta and whether it is toxic to the placenta and second, to determine whether folate can decrease transplacental transfer of formic acid and mitigate toxicity. METHODS: Dual perfusion of a single placental lobule ex vivo was used to characterize the transfer of formic acid across the placenta. After a 1-h control period, formic acid (2 mM) was introduced into the maternal circulation with (n = 4) or without folate (1 µM) (n = 4) and was allowed to equilibrate for 3 h. RESULTS: Formic acid transferred rapidly from the maternal to the fetal circulation, and transfer was not altered with the addition of folate. Compared with the control period, there was a significant decrease in hCG secretion (P = 0.03) after addition of formic acid. The addition of folic acid to the perfusate mitigated the decrease in hCG. CONCLUSIONS: Formic acid rapidly transfers across the placenta and thus has the potential to be toxic to the developing fetus. Formic acid decreases hCG secretion in the placenta, which may alter steroidogenesis and differentiation of the cytotrophoblasts, and this adverse effect can be mitigated by folate.

Bilateral hybrid oncocytoma and renal cell carcinoma.

A 26-year-old female presented with abdominal pain and distension in 2003. Clinical evaluation and imaging were suggestive of bilateral benign renal solid masses. Fine needle aspiration showed tubular cells only. Patient was kept under periodic follow up. She reported 4 years later with increase in pain and size of masses, and underwent bilateral staged nephron sparing surgery. The histopathology was reported as bilateral oncocytoma. Two years after surgery, she developed epidural spinal cord compression and liver metastasis. A decompression laminectomy and biopsy revealed conventional renal cell carcinoma (RCC). To our knowledge this is the first case report of sporadic bilateral synchronous hybrid RCC and oncocytoma in a young woman, with spinal epidural metastasis.

Free and bound enantiomers of methadone and its metabolite, EDDP in methadone maintenance treatment: relationship to dosage?

OBJECTIVES: To determine the effects of metabolism and protein binding on the relationship between administered dose, blood levels of R methadone and biological response by measuring the free and protein-bound forms of the R and S enantiomers of methadone and EDDP, its metabolite. DESIGN AND METHODS: To measure free and total drug, trough levels were collected from 45 methadone clients. To measure free methadone, samples were filtered using ultrafiltration with a MW weight cut-off of 10,000 and extracted using liquid-liquid extraction. The solvent was evaporated and samples reconstituted in mobile phase for analysis by LC/MS/MS. Total analyte was determined by extracting unfiltered samples. Enantiomeric separation of methadone and EDDP was by chiral chromatography. RESULTS: The presence of unmetabolized methadone suggested that none of the patients were very fast metabolizers. R and S forms were metabolized at the same rate at all administered doses. Free R methadone levels correlated both with methadone dose and with the total amount of R methadone. The free fraction of R methadone (%free R) was higher at lower doses than at high doses, varied from 5 to 25% and was inversely proportional to the total dose of administered drug in a relationship that was logarithmic and non-linear. CONCLUSIONS: By measuring the free, biologically active form of the drug, we were unable to account for the large variations in dose required between different patients to prevent the onset of withdrawal symptoms. The reason for the large range in dosage may be multifactorial.

Alcohol and breast feeding: calculation of time to zero level in milk.

OBJECTIVE: To create a nomogram that will guide lactating women who drink socially on how to avoid neonatal exposure to ethanol through breast milk. DESIGN: Pharmacokinetic modeling of ethanol elimination from milk based on reference values. Calculation of the time to zero alcohol in breast milk for a range of doses and body weights. RESULTS: The elimination of alcohol and time-to-zero levels in breast milk are described in a nomogram as a function of the amount of alcohol consumed and the body weight of the woman. CONCLUSIONS: Careful planning of a breast feeding schedule, by storing milk before drinking and/or waiting for complete alcohol elimination from the breast milk, can ensure women that their babies are not exposed to any alcohol.

Motherisk alert: Folic acid fortification of flour--three years later.

Fortification of flour with folic acid aims to prevent neural tube defects. In Canada, flour fortification began in 1997. The objective of this study was to quantify the changes in women's erythrocyte folic acid levels. Comparing these levels among normocytic women revealed an almost twofold increase (from 517+/-215 nmol/L in 1995 to 901+/-318 nmol/L in 1998) (P<0.00001). While the mean value increased substantially, even now, women at the low range of measured values (367 nmol/L) confer a relative risk of 3.2 for neural tube defects. For these women, higher intake of nutritional folic acid and perinatal supplementation of folic acid tablets continue to be crucial.

Is chronic poisoning with acetaminophen in children a frequent occurrence in Toronto?

BACKGROUND: Acetaminophen is a common cause of poisoning in children. Recent American studies suggest that acetaminophen poisonings pose serious risks in children, particularly in the case of chronic poisoning caused by therapeutic error. OBJECTIVE: To evaluate whether chronic acetaminophen poisoning in children is a frequent occurrence in a large, Canadian, urban population. PATIENTS AND METHODS: Retrospective study. Charts of all patients admitted to The Hospital for Sick Children, Toronto, Ontario from January 1, 1990 to June 31, 1996 with an acetaminophen overdose were reviewed. RESULTS: A total of 110 patients were admitted within the study period; only four of whom were preschool children (younger than five years of age). Among the preschool children, three had an acute overdose and one had possible chronic poisoning by therapeutic error. All preschool children were treated with N-acetylcysteine; one developed hepatotoxicity (aspartate aminotransferase or alanine aminotransferase greater than 1000 U/L) after presenting 24 h after acute ingestion. Of the remaining patients, all were adolescents; 102 had acute intentional overdose and four had staggered intentional overdoses. Fifty-three adolescents were treated with N-acetyl cysteine. Hepatotoxicity was present in 13 of 63 adolescents (21%). No patients required liver transplantation or died. CONCLUSIONS: Contrary to American experience, chronic acetaminophen poisoning, including therapeutic error in children in Toronto, is a rare occurrence--most cases of acetaminophen poisonings are acute intentional ingestion in adolescents.

Heavily smoking women who cannot quit in pregnancy: evidence of pharmacokinetic predisposition.

SUMMARY: Smoking in pregnancy is associated with a well-characterized increase in perinatal risks. Despite their wish to discontinue smoking, some pregnant women cannot stop. To characterize nicotine and cotinine levels in women who could not quit smoking after the first trimester, the authors recruited 19 white women (age 17-41 years) between 14-23 weeks of gestation who could not quit smoking. They started smoking at ages 11-22 years (mean 14.5) and smoked for 17 +/- 6 years. They had their first cigarettes 5-60 minutes after waking up (mean 12). Nicotine levels were compared with those expected in white patients in the general population, and the cotinine levels per cigarette smoked were compared with the population-based values. Sixteen of the 19 women had nicotine levels substantially lower than those expected. The mean level of serum cotinine produced by one cigarette per day was 19.1 +/- 15.8 ng/mL (range 6.1-67). The expected levels in white patients in the general population are 13 +/- 7.7 ng/mL. The data suggest that pregnant women who cannot quit heavy smoking in the second trimester form a selective group with pharmacokinetic predisposition to a high rate of nicotine metabolism.

Contamination of antibiotics resulting in severe pediatric methadone poisoning.

OBJECTIVE: To report an accidental contamination of antibiotic suspension by methadone that occurred in a retail Canadian pharmacy, leading to severe poisoning in a young child. CASE SUMMARY: A 4 1/2-year-old healthy Asian boy was prescribed amoxicillin suspension for cough and fever. Shortly after receiving the second dose of 5 mL he became drowsy and less responsive. On admission, he was arousable by deep pain, and pinpoint pupils were noted. A urine sample sent for a toxicology screen revealed the presence of methadone and its metabolite. Blood methadone concentrations were 0.23 and 0.14 mg/L, five and nine hours after the second dose of amoxicillin was given, respectively. The amoxicillin suspension was tested for methadone and was found to have a concentration of 2.4 g/L. The child gradually improved and was discharged on day 4 in good condition. The pharmacy in which the antibiotic was dispensed has been a dispensing center for a local methadone maintenance program, and methadone was accidentally mixed with the antibiotics. DISCUSSION: In this case, a near fatal outcome occurred when methadone was inadvertently mixed with antibiotics in a community pharmacy. A literature search revealed two previous reports of opiate toxicity in children following ingestion of oral antibiotic preparations. CONCLUSIONS: Prompt action is needed in Canadian pharmacies that dispense methadone in order to minimize such errors in the future. General practitioners, pediatricians, and emergency department physicians should recognize and suspect this rare cause of opiate toxicity in a child. In a patient presenting with a decreased level of consciousness and miosis, with or without respiratory depression, naloxone administration should be considered, whether or not a history of opioid ingestion is obtained.

Urine fingerprinting: detection of sample tampering in an opiate dependency program.

Methadone treatment programs commonly monitor patient compliance by screening urine samples for drugs of abuse. Our experience suggests that re-submission of urine samples (for example, providing a urine sample that is either not that of the patient or was previously submitted) is often used as a method of sample tampering. We have developed an algorithm that combines urine sodium, chloride, creatinine and pH values with urine drug screening results to effectively detect resubmitted samples. Given the widespread use of urine drug screening in drug and alcohol rehabilitation programs, we believe this technique has significant practical benefits. This technique may also have an application in forensic identification of duplicate samples.

Pancreaticogastrostomy for reconstruction of pancreatic stump after pancreaticoduodenectomy for ampullary carcinoma.

BACKGROUND: Management of the pancreatic stump after pancreaticoduodenectomy (PD) is still a matter of debate. Pancreaticojejunostomy (PJ) is used commonly but is associated with a significant incidence of pancreatic leaks. Pancreaticogastrostomy (PG) is an alternative that has been reported to be safer. METHODS: The study is a retrospective analysis of all patients having PD for ampullary carcinoma in one surgical unit at All India Institute of Medical Sciences over 18 years, with PG being the only drainage procedure for the pancreatic stump. RESULTS: Among 125 patients having PD for ampullary carcinoma, overall morbidity rate was 28%, mortality rate was 4.8%, with no cases of leakage from the pancreaticogastrostomy. CONCLUSIONS: In world literature (including the current series), the leakage rate of PG is 2.5% (14 of 553) with only 2 deaths (2 of 14) due to leakage from PG. Our large experience and these data conclusively prove the safety of pancreaticogastrostomy, which should be the drainage procedure of choice for the pancreatic stump following pancreaticoduodenectomy.