Long chain fatty acids and related pro-inflammatory, specialized pro-resolving lipid mediators and their intermediates in preterm human milk during the first month of lactation.

This study aimed to measure longitudinal quantities of the long chain fatty acids, their biologically active terminal metabolites and related intermediates (also called oxylipins) in preterm human milk expressed during the first month of lactation. In a prospective cohort, breast milk was collected throughout the first month of lactation in 30 women who delivered preterm infants. Eighteen bioactive lipids and their intermediates were quantified via solid phase extraction and LC-MS/MS. Analysis by GC-FID quantified the fatty acid precursors. Arachidonic acid (ARA) and docosahexaenoic acid (DHA) milk concentrations significantly declined throughout the first month. Oxylipin concentrations did not change during lactation. Positive associations existed between ARA and thromboxane B2, eicosapentaenoic acid and 18-hydroxyeicosapentaenoic acid, and between DHA and PDX and 14- and 17-hydroxydocosahexaenoic acids. DHA concentrations were 1.5 times higher and 14-HDHA was 1.7 times higher in milk from women taking DHA supplements. This investigation showed conditionally essential fatty acids, ARA and DHA, decreased in preterm milk, suggesting a need to supplement their intake for the breast milk-fed preterm infant. Positive associations between parent fatty acids, bioactive lipids and intermediates, as well as sensitivity of milk to maternal fatty acid intake, support consideration of a comprehensive approach to providing fatty acids for preterm infants through both maternal and infant supplementation.

From actors to agents in socio-ecological systems models.

The ecosystem service concept has emphasized the role of people within socio-ecological systems (SESs). In this paper, we review and discuss alternative ways of representing people, their behaviour and decision-making processes in SES models using an agent-based modelling (ABM) approach. We also explore how ABM can be empirically grounded using information from social survey. The capacity for ABM to be generalized beyond case studies represents a crucial next step in modelling SESs, although this comes with considerable intellectual challenges. We propose the notion of human functional types, as an analogy of plant functional types, to support the expansion (scaling) of ABM to larger areas. The expansion of scope also implies the need to represent institutional agents in SES models in order to account for alternative governance structures and policy feedbacks. Further development in the coupling of human-environment systems would contribute considerably to better application and use of the ecosystem service concept.

Interlaboratory comparison of the assessment of P450 activities in human hepatic microsomal samples.

1. Although the importance of in vitro technology in supporting drug development is widely accepted, there is no real consensus about which approaches should be taken, which substrates should be used, or on the reliability and application of in vitro data. Consequently, as part of a collaborative project to characterize human liver with respect to the major forms of cytochrome P450, an interlaboratory comparison of the analysis of samples for form-specific activities was undertaken. 2. Microsomal fractions were isolated from five different human liver samples taken from the liver bank maintained at the Royal Postgraduate Medical School (RPMS). Aliquots from the five samples were sent to the 11 collaborating laboratories for characterization using their in-house, form-specific assays for cytochrome P450 activities. Although each laboratory assayed protein concentration, total cytochrome P450 content and enzyme activities were calculated using the protein estimation generated by RPMS to eliminate this possible source of variability. 3. With the exception of one laboratory, all estimates of protein concentration were similar (coefficient of variation, CoV, 9-13%) and the rank-order of the five samples was consistent across the laboratories. There was greater variability in the estimates of total cytochrome P450 content (CoV 28-43%), although again rank order of the samples across laboratories was fairly consistent. 4. The various laboratories used a number of different probe substrates, together with a range of conditions (substrate concentration, time of incubation, amount of protein), to assay for activity of CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4. However, apart from the occasional outlier, the five samples were ranked for activity of all these forms of cytochrome P450 with a high degree of consistency by the various laboratories and the choice of substrate had no appreciable effect on the ranking of the samples. 5. While this interlaboratory comparison has shown that greater consistency in the approach to in vivo determination of drug-metabolizing activity is desirable, there was little indication that any particular approach or substrate was superior to the others.

Adjustment of iron intake for dietary enhancers and inhibitors in population studies: bioavailable iron in rural and urban residing Russian women and children.

Although determining iron intakes is essential in assessing adequacy of iron in the diet, estimating iron availability may be more useful for evaluating whether iron requirements are met. Our objectives were to describe the dietary information, analytical steps, and computer algorithms needed for iron bioavailability adjustments and to demonstrate the effects of various dietary factors on calculated iron absorption. Our study was based on 9890 women and children participating in the Russian Longitudinal Monitoring Survey. Between August 1992 and February 1993, two 24-h recalls were collected from each participant, and total, heme and nonheme iron intakes were calculated. Nonheme iron availability was adjusted for meat, fish and poultry and vitamin C consumed in the same meal and then further adjusted for tea and phytates. We found mean total iron intakes to be comparable to those of women of reproductive age in the United States and lower than those of United States children. When these intakes were adjusted for enhancers and inhibitors of absorption, the iron bioavailability in these vulnerable Russian groups was extremely low. Mean bioavailable iron as well as the 25th-75th percentile ranges of intake were below the bottom of the range of requirements, indicating that iron adequacy in this population may be considerably less than expected based on total iron intakes alone. Furthermore, rural and urban food availability had a significant effect on iron bioavailability. Future research on dietary iron adequacy should be based on estimates of available iron by collecting meal-level dietary data and using detailed information on mixed dishes and phytates.

A novel class of cyclic beta-dicarbonyl leaving groups and their use in the design of benzisothiazolone human leukocyte elastase inhibitors.

Human leukocyte elastase (HLE) has been proposed to be a primary mediator of pulmonary emphysema, and inhibitors of this enzyme should be effective in the treatment of emphysema and other pulmonary diseases. We have discovered a novel class of alicyclic and heterocyclic leaving groups which share one common structural feature, a cyclic beta-dicarbonyl. This design concept for leaving groups has not been previously reported. A structure-activity relationship has been developed and the concept extended to several types of alicyclic and heterocyclic beta-dicarbonyl systems. This work led to the identification of a potent (K*i of 0.066 nM) and tissue stable (in vitro: blood t1/2 = 160 min, liver t1/2 > 240 min) benzisothiazolone HLE inhibitor, WIN 65936 (13b).

Picornavirus inhibitors: trifluoromethyl substitution provides a global protective effect against hepatic metabolism.

Several modifications of the oxazoline ring of WIN 54954, a broad spectrum antipicornavirus compound, have been prepared in order to address the acid lability and metabolic instability of this compound. We have previously shown that the oxadiazole analogue 3 displayed comparable activity against a variety of rhinoviruses and appeared to be stable to acid. A monkey liver microsomal assay was developed to examine the metabolic stability in vitro of both compounds, and it was determined that WIN 54954 displayed 18 metabolic products while 3 was converted to 8 products. Two major products of 3 were determined by LC-MS/MS to be monohydroxylated at each of the terminal methyl groups. Replacement of the methyl on the isoxazole ring with a trifluoromethyl group, while preventing hydroxylation at this position, did not reduce the sensitivity of the molecule to microsomal metabolism at other sites. However, the (trifluoromethyl)oxadiazole 9 not only prevented hydroxylation at this position but also provided protection at the isoxazole end of the molecule, resulting in only two minor products to the extent of 4%. The major product was identified as the monohydroxylated compound 23. The global metabolic protective effect of trifluoromethyl group on the oxadiazole ring was further demonstrated by examining a variety of analogues including heterocyclic replacements of the isoxazole ring. In each case, the trifluoromethyl analogue displayed a protective effect when compared to the corresponding methyl analogue.

Detection of a species-specific antigen of Gardnerella vaginalis by Western blot analysis.

Western blot analysis was used to identify antigenic components of Gardnerella vaginalis. Polypeptides bound to nitrocellulose membranes were probed with murine antisera raised to two strains of G. vaginalis, and antibody-antigen complexes were detected with 125I-labelled antimouse immunoglobulin followed by autoradiography. Although there was inter-strain variation in immunogenic polypeptide profiles, all 23 strains of G. vaginalis examined contained a common antigen of molecular mass 41 kDa. This antigen was not found in any of six other bacterial genera.

The bioavailability and metabolism of trilostane in normal subjects, a comparative study using high pressure liquid chromatographic and quantitative cytochemical assays.

High pressure liquid chromatographic (HPLC) analysis of plasma taken over 8 h from ten normal male subjects medicated with 120 mg of trilostane revealed that the drug is rapidly metabolised into at least one metabolite, 17-keto trilostane. Both compounds were detected in the blood stream at concentrations greater than 2 X 10(-7) M within an hour and were cleared from the blood by 6-8 h. Approximately 3 times the concentration of metabolite was detected compared to the parent compound in most samples analysed. There were large subject to subject variations in the handling of drug. Standard curves of pure 17-keto trilostane and trilostane were parallel as assessed by cytochemical bioassay. This assay is based upon the inhibition of 3 beta-hydroxysteroid dehydrogenase activity in unfixed tissue sections of the dioestrous rat ovary. The relative potency of the metabolite compared to trilostane was 1.71 (95% confidence 1.5-2.0) over the dose range 0.15-1.5 microM. Thus, the metabolite may be the major active agent when trilostane is administered for clinical purposes. In a further 4 volunteers, who also received 120 mg trilostane and were sampled over an 8 h period, plasma was analysed independently by HPLC and cytochemical assays. In the majority of cases the bioactivity recorded (relative to a trilostane standard curve) was substantially higher than the molar sum of circulating trilostane and 17-keto-trilostane (as assessed by HPLC). However, if the relative potency of 17-keto-trilostane is taken into consideration, correlation between the two assays was excellent (r = 0.947, n = 18, P less than 0.001). This also suggests that no further active metabolites were present in the plasma samples. The drug profiles seen in the second study were essentially the same as described for the first 10 volunteers. The combination of a bioassay, which detects trilostane-like bioactivity, and HPLC, which reveals the type of metabolism, should aid our understanding of the clinical value of this potentially important drug.

Clinical and microbiological study of non-gonococcal urethritis with particular reference to non-chlamydial disease.

A double-blind placebo-controlled study of minocycline in 221 men with non-gonococcal urethritis (NGU) was undertaken. Techniques were used which enabled diagnoses of chlamydial and mycoplasmal infections to be made within 24 hours of a patient attending a clinic. All patients from whom Chlamydia trachomatis was isolated were treated with minocycline, while patients from whom Ureaplasma urealyticum or Mycoplasma hominis was isolated, or from whom no micro-organisms were isolated, were treated on a double-blind basis with either minocycline or placebo. Chlamydia were isolated from 77 (35%) patients and were eradicated by minocycline from 76 (99%). Ureaplasmas were isolated initially from 96 (43%) patients. Treatment with minocycline eradicated them from 43 of 52 (83%) patients, and they disappeared from six of 31 (19%) patients who were treated with placebo. After one week significantly more patients had responded clinically to minocycline than to placebo. The response to minocycline was not influenced by the microbiological status of the patients, which suggests that ureaplasmas are playing a similar role to chlamydia in the pathogenesis of the disease and that an antibiotic-sensitive micro-organism may be producing disease in the isolate-negative group. An immunological approach is required to resolve the problem of the persistent urethral inflammation which occurred despite eradication of the micro-organisms.

The effect of Mycoplasma pulmonis on fertilization and preimplantation development in vitro of mouse eggs.

The effect of Mycoplasma pulmonis suspension on mouse on mouse fertilization and preimplantation development in vitro was examined. When sperm were preincubated with M. pulmonis, fertilization of eggs occured less consistently than when untreated sperm were used. There was also a highly significant and consistent reduction in embryonic develoment in the treated group, with relatively few embryos reaching the blastocyst stage. The adverse effects were not seen with M. fermentans or with M. pulmonis organisms inactivated by heating or sonication before incubation with gametes. Likewise, preincubation of M. pulmonis in medium containing tetracycline, an antibiotic which interferes with protein synthesis, resulted in embryonic development similar to that seen in the untreated group. These results suggest that the deleterious effects obtained with viable mycoplasmas are due to some substance produced by their active metabolism. Other results indicate that the egg is susceptible to M. pulmonis for a limited time, since incubation of two-cell embryos with mycoplasmas had no effect on development to the blastocyst stage. The findings are discussed in relation to the possible association between human infertility and mycoplasma infection.