Second-trimester abortion attitudes and practices among maternal-fetal medicine and family planning subspecialists.

BACKGROUND: Patients deciding to undergo dilation and evacuation (D&E) or induction abortion for fetal anomalies or complications may be greatly influenced by the counseling they receive. We sought to compare maternal-fetal medicine (MFM) and family planning (FP) physicians' attitudes and practice patterns around second-trimester abortion for abnormal pregnancies. METHODS: We surveyed members of the Society for Maternal-Fetal Medicine and Family Planning subspecialists in 2010-2011 regarding provider recommendations between D&E or induction termination for various case scenarios. We assessed provider beliefs about patient preferences and method safety regarding D&E or induction for various indications. We compared responses by specialty using descriptive statistics and conducted unadjusted and adjusted analyses of factors associated with recommending a D&E. RESULTS: Seven hundred ninety-four (35%) physicians completed the survey (689 MFMs, 105 FPs). We found that FPs had 3.9 to 5.5 times higher odds of recommending D&E for all case scenarios (e.g. 80% of FPs and 41% of MFMs recommended D&E for trisomy 21). MFMs with exposure to family planning had greater odds of recommending D&E for all case scenarios (p < 0.01 for all). MFMs were less likely than FPs to believe that patients prefer D&E and less likely to feel that D&E was a safer method for different indications. CONCLUSION: Recommendations for D&E or induction vary significantly depending on the type of physician providing the counseling. The decision to undergo D&E or induction is one of clinical equipoise, and physicians should provide unbiased counseling. Further work is needed to understand optimal approaches to shared decision making for this clinical decision.

Outpatient Management of Delayed Graft Function Is Associated With Reduced Length of Stay Without an Increase in Adverse Events.

Delayed graft function (DGF) is a common and costly complication of kidney transplantation. In July 2011, we established a multidisciplinary DGF clinic managed by nurse practitioners to facilitate early discharge and intensive management of DGF in the outpatient setting. We compared length of stay, 30-day readmission, acute rejection, and patient/graft survival in 697 consecutive deceased donor kidney transplantations performed between July 2009 and July 2014. Patients were divided into three groups: no DGF (n = 487), DGF before implementation of the DGF clinic (n = 118), and DGF clinic (n = 92). Baseline characteristics including age, gender, panel reactive antibody, retransplantation rates, HLA mismatches, induction, and maintenance immunosuppression were not significantly different between pre- and post-DGF clinic groups. Length of stay was significantly longer in pre-DGF clinic (10.9 ± 6.2 vs. 6.1 ± 2.1 days, p < 0.001). Thirty-day readmission (21% vs. 16%), graft loss (7% vs. 20%), and patient death (2% vs. 11%) did not differ significantly between pre- and post-DGF clinic. Patients in the DGF clinic were less likely to develop acute rejection (21% vs. 40%, p = 0.006). Outpatient management of DGF in a specialized clinic is associated with substantially shorter hospitalization and lower incidence of acute rejection without significant difference in 30-day readmission or patient and graft survival.

Mental health needs in adolescents with intellectual disabilities: cross-sectional survey of a service sample.

BACKGROUND: Little research has been conducted on the mental health needs of adolescents with intellectual disability, despite the severity and rates of such needs being high throughout childhood and in adulthood. We have investigated the prevalence and predictors of mental health needs and service use in adolescents with intellectual disabilities. MATERIALS AND METHODS: Service-based sample (n = 75) in one catchment area. Individual assessments were carried out. The main outcome was the presence of mental health needs measured by the Developmental Behaviour Checklist. RESULTS: Prevalence of mental health needs increased from 51% as reported by parents to 67% as judged by clinical interviews. Caseness was associated with low adaptive functioning, diagnosis of autism and family history of mental illness. High scores on parent reports of participant mental ill-health showed negative correlations with adaptive functioning scores. Most individuals were in receipt of social and health care. Half of the participants had sought help for mental health needs. Almost half of those receiving medication were on psychiatric medication. CONCLUSIONS: Adolescents with intellectual disabilities may have considerable mental health problems which are functionally impairing yet frequently unidentified and hence untreated. Identification of those at risk and undertaking of a comprehensive needs assessment are essential to maximize potential and quality of life and to reduce further deficits and social exclusion.

Upregulation of cyclooxygenase-2 expression in porcine macula densa with chronic nitric oxide synthase inhibition.

The objective of this study was to investigate the effects of chronic inhibition of nitric oxide synthase (NOS) on cyclooxygenase-2 (COX-2) expression in the macula densa (MD) of swine, as well as the effects on expression of related proteins. Adult female Yucatan swine were given either tap water (control, n = 6) or water with N (G)-nitro-L-arginine methyl ester (L-NAME, 100 mg/liter, n = 5) for a minimum of 30 days. Duplicate samples of kidney were fixed or snap frozen. There was a significant (P = .0082) upregulation of COX-2 mRNA expression in the MD of L-NAME, as well as an apparent increase in COX-2 protein. Plasma renin activity also increased with L-NAME treatment (control, 0.34 ± 0.08 ng/ml; L-NAME, 1.26 ± 0.03 ng/ml; P = .00000003). There were no differences between groups in expression of either inducible NOS or renin protein or in serum electrolyte concentrations. In conclusion, with chronic inhibition of NOS, COX-2 in MD is upregulated, perhaps to compensate for loss of nitric oxide. Increases in COX-2 products may counteract renal arteriolar constriction and sustain renin release.

The integration of abortion into obstetrician-gynecologists' practice after comprehensive family planning resident training.

OBJECTIVES: To explore the impacts of routine family planning and abortion training during residency on abortion practice between three and ten years after residency. STUDY DESIGN: In 2018, we surveyed 771 graduated obstetrician-gynecologists at least three years after residency about their current abortion practice. Respondents consented to join a prospective cohort as part of routine, post-rotation evaluation of the Kenneth J. Ryan Residency Training Program in Abortion and Family Planning. We matched and then de-identified post-rotation and post-residency surveys, and conducted bivariate and multivariable analyses. RESULTS: Of 463 respondents (60% response rate), 188 (41%) reported that they provide abortions (median of eight abortions per month) in their current practice. Eighty-eight (19%) do not provide abortions but would if not restricted by their practice. One hundred-fifty respondents (32%) reported abortions are out of their practice scope or that someone else in their practice provides abortions, and 38 (8%) do not desire to provide abortion care. Two hundred twenty-six (54%) reported practice or hospital group restrictions to abortion care. In multivariable analyses controlling for demographics, training, attitude and practice factors; geographic location, practice restrictions and logistical barriers, among other variables, correlated with abortion practice (practice in the West: odds ratio (OR) 2.3; 95% confidence interval [CI], 1.3-4.2; p = 0.01; logistical barriers: OR 0.3, CI 0.1 to 0.7, p = 0.01; and practice restrictions OR 0.5, CI 0.3 to 0.8, p = 0.01). CONCLUSIONS: Nearly half of Ryan Program-trained obstetrician-gynecologists provide abortions. However, many barriers prevent the integration of abortion into practice. Healthcare providers and leaders should work to eliminate barriers to the provision of abortion care. IMPLICATIONS: Regardless of their intentions at the time of training, nearly half of Ryan Program-trained obstetrician-gynecologists provide abortions in practice, and another 19% would if not restricted by their practice. Integrated training is critical to abortion care, and efforts to overcome practice barriers could improve access to comprehensive health care.

Adaptive behaviour in Angelman syndrome: its profile and relationship to age.

BACKGROUND: Angelman syndrome (AS) is a neurodevelopmental disorder usually caused by an anomaly in the maternally inherited chromosome 15. The main features are severe intellectual disability, speech impairment, ataxia, epilepsy, sleep disorder and a behavioural phenotype that reportedly includes happy disposition, attraction to/fascination with water and hypermotoric behaviour. METHOD: We studied the level of adaptive behaviour and the adaptive behavioural profile in the areas of 'motor skills', 'language and communication', 'personal life skills' and 'community life skills' in a group of 25 individuals with genetically confirmed AS, to determine whether there is a specific adaptive behaviour profile. RESULTS AND CONCLUSIONS: None of the individuals, whatever their chronological age, had reached a developmental age of 3 years. A specific adaptive behaviour profile was found, with 'personal life skills' emerging as relative strengths and 'social and communication skills' as weaknesses.

Reduced incidence and severity of experimental autoimmune arthritis in mice expressing catalytically inactive A disintegrin and metalloproteinase 8 (ADAM8).

A disintegrin and metalloproteinase 8 (ADAM8), a catalytically active member of the ADAMs family of enzymes, is expressed primarily on immune cells and thus probably involved in inflammatory responses. ADAM8 is also produced by chondrocytes, and recombinant ADAM8 can induce cartilage catabolism. We therefore decided to test the role of ADAM8 in autoimmune inflammatory arthritis using transgenic mice expressing catalytically inactive ADAM8. Transgenic DBA/1J mice expressing an inactivating point mutation in the ADAM8 gene to change Glu330 to Gln330 (ADAM8(EQ)) were generated to evaluate the proteolytic function of ADAM8 in an lipopolysaccharide-synchronized collagen-induced arthritis (LPS-CIA) model of autoimmune arthritis. The systemic inflammatory reaction to LPS was also evaluated in these mice. Expression profiling of paw joints from wild-type mice revealed that ADAM8 mRNA levels increased at the onset of clinical arthritis and correlated well with cellular macrophage markers. When subjected to LPS-CIA, ADAM8(EQ) mice demonstrated decreased incidence and severity of clinical arthritis compared to wild-type mice. Histological examination of paw joints from ADAM8(EQ) mice confirmed marked attenuation of synovial inflammation, cartilage degradation and bone resorption when compared to wild-type mice. However, transgenic mice and wild-type mice responded similarly to LPS-induced systemic inflammation with regard to mortality, organ weights, neutrophil sequestration and serum cytokine/chemokine production. We conclude that ADAM8 proteolytic activity plays a key role in the development of experimental arthritis and may thus be an attractive target for the treatment of arthritic disorders while minimizing risk of immunocompromise.

The fragile X continuum: new advances and perspectives.

Fragile X syndrome is the world's most common hereditary cause of intellectual disability in men and to a lesser extent in women. The disorder is caused by the silencing of a single gene on the X chromosome, the Fragile X Mental Retardation Gene-1. A substantial body of research across the disciplines of molecular genetics, child psychiatry and developmental neuroscience bears testament to a decade of exciting and innovative science that has advanced our knowledge about the fragile X 'signature' or influence across cognitive and social development. The core aims of this review are to first discuss fragile X syndrome and premutation involvement in the context of current advances that demonstrate the dynamic nature of the genotype on phenotypic outcomes. Second, to discuss the implications of these recent advances for the development of clinical and educational interventions and resource tools that target specific phenotypic 'signatures' within the fragile X continuum.

A regiospecific monooxygenase with novel stereopreference is the major pathway for arachidonic acid oxygenation in isolated epidermal cells.

We investigated the enzymatic mechanisms responsible for AA oxygenation in homogenous cell suspensions obtained by trypsinization of epidermis from healthy subjects. Cell incubation with AA (0.3-150 microM) invariably resulted in the predominant generation of a compound identified as 12-hydroxyeicosatetraenoic acid (12-HETE) by HPLC and by both negative-ion chemical ionization and electron-impact mass spectrometry. Maximal amounts of 12-HETE were 126 +/- 21 pmol/10(6) cells (+/- SE), and concentration-response curves yielded half-maximal levels for 12-HETE similar to PGE2 at 2 microM AA. Two epoxyeicosatrienoic acids derived from AA were also identified. Stereochemical analysis by chiral-phase chromatography demonstrated that the epidermal cell 12-HETE was a mixture of the 12S- and 12R-hydroxy isomers in a molar ratio varying from 2:1 to 8:1 among subjects. Subcellular fractionation into 12,000 g pellet (containing mitochondria) and 100,000 g supernatant (cytosol) and pellet (microsome) demonstrated that greater than 99% of the 12-HETE was generated by enzymatic activity distributed equally in the two pellets. Both mitochondrial and microsomal activities were increased upon addition of NADPH and were inhibited by carbon monoxide, but the molar ratio of 12S/12R-HETE was threefold greater in microsomal than in mitochondrial fractions. The results demonstrate that human epidermis contains active membrane-bound monooxygenase(s) which preferentially generates 12-HETE from AA, exhibits a 12S stereopreference of hydroxylation, and suggests the presence of distinct mitochondrial and microsomal enzyme systems in epidermal cells.

Diacylglycerol synthesis de novo from glucose by pancreatic islets isolated from rats and humans.

Recent evidence has suggested that pancreatic islets isolated from rats synthesize 1,2-diacyl-sn-glycerol (DAG) de novo from glucose and that this process may constitute the long-sought link between the metabolism of glucose and the induction of insulin secretion. The cell-permeant diacylglycerol 1-oleoyl-2-acetyl-sn-glycerol (200 microM) has been found here to amplify both the first and second phases of insulin secretion from perifused human islets. Measurements of the mass of endogenous DAG in human pancreatic islets by enzymatic and by mass spectrometric methods indicate that levels of 200 microM may be achieved under physiologic conditions. Conversion of [14C]glucose to [14C]DAG has been demonstrated here to occur within 60 s of exposure of rat and human islets to stimulatory concentrations of glucose. This process has been found to be a quantitatively minor contributor to the total islet DAG mass after acute stimulation with glucose, however, and glucose has been found not to induce a rise in total islet DAG content within 20 min of induction of insulin secretion. In contrast to the case with rodent islets, two pharmacologic inhibitors of DAG-induced activation of protein kinase C (staurosporine and sphingosine) have been found not to influence glucose-induced insulin secretion from isolated human islets. These findings indicate that de novo synthesis of DAG from glucose does not participate in acute signal-response coupling in islets.

Interleukin 1 inhibits insulin secretion from isolated rat pancreatic islets by a process that requires gene transcription and mRNA translation.

Recombinant human IL 1 beta inhibits glucose-induced insulin secretion from isolated pancreatic islets and from purified beta-cells obtained by fluorescence-activated cell sorting (FACS) of dispersed islet cells. Brief (1 h) exposure of isolated islets to IL 1 produces sustained inhibition of insulin secretion for at least 17 h after the IL 1 has been removed from the culture medium. An inhibitory effect of IL 1 on insulin secretion is not observed when islets are coincubated with an inhibitor of DNA transcription (actinomycin D). This finding indicates that the inhibitory effect of IL 1 on insulin secretion requires transcription of one or more genes during the first hour of exposure of islets to IL 1. The inhibitory effect of IL 1 on insulin secretion also requires mRNA translation, because three structurally distinct inhibitors of protein synthesis (cycloheximide, anisomycin, and puromycin) prevent IL 1-induced inhibition of insulin secretion when added to islets after the 1-h exposure to IL 1. Two-dimensional gel electrophoresis of islet proteins metabolically labeled with [35S]methionine demonstrates that IL 1 augments the expression of a 65-kD (pl approximately 6.5) protein by greater than 2.5-fold. These findings indicate that biochemical events occurring within 1 h of exposure of islets to IL 1 lead to an inhibition of insulin secretion that persists for at least 17 h after the removal of IL 1. One of the early biochemical effects of IL 1 on islets is gene transcription (0-1 h), which is followed by mRNA translation (after 1 h). Our results suggest that the inhibitory effect of IL 1 on insulin secretion is mediated by protein(s) whose synthesis is induced by IL 1.

Diacylglycerol accumulation and microvascular abnormalities induced by elevated glucose levels.

The present experiments were undertaken to examine the hypothesis that glucose-induced increased de novo synthesis of 1,2-diacyl-sn-glycerol (which has been observed in a number of different tissues, including retinal capillary endothelial cells exposed to elevated glucose levels in vitro) and associated activation of protein kinase C may play a role in mediating glucose-induced vascular functional changes. We report here that twice daily instillation of 30 mM glucose over 10 d in a rat skin chamber granulation tissue model induces approximately a 2.7-fold increase in diacylglycerol (DAG) levels (versus tissues exposed to 5 mM glucose) in association with marked increases in vascular clearance of albumin and blood flow. The glucose-induced increase in DAG levels as well as the vascular functional changes are prevented by addition of 3 mM pyruvate. Pharmacological activation of protein kinase C with the phorbol ester TPA in the presence of 5 mM glucose increases microvascular albumin clearance and blood flow, and similar effects are observed with 1-monoolein (MOG), a pharmacological inhibitor of the catabolism of endogenous DAG. A pharmacological inhibitor of protein kinase C (staurosporine) greatly attenuates the rise in microvascular albumin clearance (but not the rise in blood flow) induced by glucose or by MOG. These findings are compatible with the hypothesis that elevated concentrations of glucose increase tissue DAG content via de novo synthesis, resulting in protein kinase C activation, and that these biochemical events are among the factors that generate the increased microvascular albumin clearance.

Xenotransplantation of cryopreserved equine squamous cell carcinoma to athymic nude and SCID mice.

Cryopreserved equine ocular squamous cell carcinoma (SCC) was inoculated subcutaneously into 15 athymic nude and 15 SCID mice. Xenotransplantation resulted in tumor growth in two athymic nude mice and 1 SCID mouse. Histological appearance and immunohistochemical characterization using cytokeratin 5/6 markers and p53 markers of the tumor grown in mice was in full accord with the original equine tumors. No evidence of metastasis was noted in any mouse. This model may serve as a relevant in vivo model for studying the biology of equine ocular SCC and for the testing of new therapeutic modalities.

The molecular biology of the group VIA Ca2+-independent phospholipase A2.

The group VIA PLA2 is a member of the PLA2 superfamily. This enzyme, which is cytosolic and Ca2+-independent, has been designated iPLA2beta to distinguish it from another recently cloned Ca2+-independent PLA2. Features of iPLA2beta molecular structure offer some insight into possible cellular functions of the enzyme. At least two catalytically active iPLA2beta isoforms and additionalsplicing variants are derived from a single gene that consists of at least 17 exons located on human chromosome 22q13.1. Potential tumor suppressor genes also reside at or near this locus. Structural analyses reveal that iPLA2beta contains unique structural features that include a serine lipase consensus motif (GXSXG), a putative ATP-binding domain, an ankyrin-repeat domain, a caspase-3 cleavage motif DVTD138Y/N, a bipartite nuclear localization signal sequence, and a proline-rich region in the human long isoform. iPLA2beta is widely expressed among mammalian tissues, with highest expression in testis and brain. iPLA2beta prefers to hydrolyze fatty acid at the sn-2 fatty acid substituent but also exhibits phospholipase A1, lysophospholipase, PAF acetylhydrolase, and transacylase activities. iPLA2beta may participate in signaling, apoptosis, membrane phospholipid remodeling, membrane homeostasis, arachidonate release, and exocytotic membrane fusion. Structural features and the existence of multiple splicing variants of iPLA2beta suggest that iPLA2beta may be subject to complex regulatory mechanisms that differ among cell types. Further study of its regulation and interaction with other proteins may yield insight into how its structural features are related to its function.

Mechanism of arachidonic acid-induced Ca2+ mobilization from rat liver microsomes.

Recent evidence indicates that unesterified arachidonic acid functions as a mediator of intracellular Ca2+ mobilization by inducing Ca2+ release from the endoplasmic reticulum of pancreatic islet beta cells in a manner closely similar to that of inositol 1,4,5-trisphosphate. To test the generality and explore the mechanism of this phenomenon we have examined the effects of arachidonic acid on calcium accumulation and release by hepatocyte subcellular fractions enriched in endoplasmic reticulum (microsomes). At concentrations above 0.017 mumol/mg microsomal protein, arachidonate induced rapid (under 2 min) 45Ca2+ release from microsomes that had been preloaded with 45Ca2+. Arachidonate also suppressed microsomal 45Ca2+ accumulation when present during the loading period, as reflected by reduction both of 45Ca2+ accumulation at steady state and of the rate of uptake. Neither the cyclooxygenase inhibitor indomethacin nor the lipoxygenase/cyclooxygenase inhibitor BW755C suppressed arachidonate-induced 45Ca2+ release, indicating that this effect was not dependent upon oxygenation of the fatty acid to metabolites. The long-chain unsaturated fatty acids oleate and linoleate were less potent than arachidonate in inducing 45Ca2+ release, and the saturated fatty acid stearate did not exert this effect. Albumin prevented 45Ca2+ release by arachidonate, presumably by binding the fatty acid. As is the case for inositol 1,4,5-trisphosphate, the ability of arachidonate to induce 45Ca2+ release was dependent on the ambient free Ca2+ concentration. Arachidonate did not influence microsomal membrane permeability or Ca2+-ATPase activity and may exert its effects on microsomal Ca2+ handling by activation of a Ca2+ extrusion mechanism or by dissociating Ca2+ uptake from Ca2+-ATPase activity.

Arachidonic acid metabolism in isolated pancreatic islets. VI. Carbohydrate insulin secretagogues must be metabolized to induce eicosanoid release.

Pancreatic islets stimulated with D-glucose are known to liberate arachidonic acid from membrane phospholipids and release prostaglandin E2 (PGE2). A component of the eicosanoid release induced by D-glucose has been demonstrated to occur without calcium influx and must be triggered by other coupling mechanisms. In this study, we have attempted to identify mechanisms other than calcium influx which might couple D-glucose stimulation to hydrolysis of arachidonate from membrane phospholipids in islet cells. We have found that occupancy of the beta cell plasma membrane D-glucose transporter is insufficient and that D-glucose metabolism is required to induce islet PGE2 release because 3-O-methylglucose fails to induce and mannoheptulose prevents PGE2 release otherwise induced by 17 mM D-glucose. The carbohydrate insulin secretagogues mannose and D-glyceraldehyde have also been found to induce islet PGE2 release, but the non-secretagogue carbohydrates L-glucose and lactate do not. Carbohydrate secretagogues are known to be metabolized to yield ATP and induce depolarization of the beta cell plasma membrane. We have found that depolarization by 40 mM KCl induces PGE2 release only in the presence and not in the absence of extracellular calcium, but exogenous ATP induces islet PGE2 release with or without extracellular calcium. Carbachol is demonstrated here to interact synergistically with increasing concentrations of glucose to amplify PGE2 release and insulin secretion. Pertussis toxin treatment is shown here not to prevent PGE2 release induced by glucose or carbachol but to increase the basal rate of PGE2 release and the islet cyclic AMP content. Theophylline (10 mM) exerts similar effects. Eicosanoid release in pancreatic islets can thus be activated by multiple pathways including muscarinic receptor occupancy, calcium influx, increasing cAMP content, and a metabolic signal derived from nutrient secretagogues, such as ATP.

Cloning and expression of a group IV cytosolic Ca2+-dependent phospholipase A2 from rat pancreatic islets. Comparison of the expressed activity with that of an islet group VI cytosolic Ca2+-independent phospholipase A2.

Stimulation of pancreatic islets with glucose induces phospholipid hydrolysis and accumulation of nonesterified arachidonic acid, which may play signaling or effector roles in insulin secretion. Of enzymes that catalyze phospholipid hydrolysis, islet beta-cells express low molecular weight secretory phospholipases A2 (PLA2) and a Group VI, Ca2+-independent PLA2 (iPLA2). Previous studies indicate that islets also express a protein recognized by antibodies against a Group IV, cytosolic, Ca2+-dependent PLA2 (cPLA2). To further examine the possible expression of cPLA2 by islets, we screened a rat islet cDNA library with a probe that recognizes cPLA2 sequence, and isolated a full-length cPLA2 cDNA. The rat islet cPLA2-deduced amino acid sequence is 96% identical to those of human and mouse cPLA2. Transfection of COS-7 cells with cPLA2 cDNA in an expression vector induced expression of Ca2+-dependent PLA2 activity and of a protein recognized by anti-cPLA2 antibody. Comparison of recombinant islet cPLA2 and iPLA2 activities expressed in transfected COS-7 cells indicated that iPLA2 but not cPLA2 is stimulated by ATP. Both activities are similarly sensitive to inhibition by arachidonyltrifluoromethyl ketone, but iPLA2 is more effectively inhibited by a haloenol lactone suicide substrate than cPLA2. RT-PCR experiments with RNA from purified islet beta-cells and from an alpha-cell-enriched population prepared by fluorescence-activated cell-sorting indicated that cPLA2 mRNA is more abundant in the beta-cell population. Immunoblotting analyses indicate that islets express cPLA2-immunoreactive protein, and that interleukin-1 does not affect its expression. The cPLA2 is thus one of at least three classes of PLA2 enzymes with distinct properties expressed in beta-cells.

Electrospray ionization tandem mass spectrometric analysis of sulfatide. Determination of fragmentation patterns and characterization of molecular species expressed in brain and in pancreatic islets.

The sphingolipid sulfatide is a component of myelin and some non-neuronal cells. Antibodies to sulfatide occur in some patients with autoimmune neuropathies and in patients with insulin-dependent diabetes mellitus (IDDM) caused by immunologic destruction of insulin-secreting pancreatic islet beta-cells. Distinct sulfatide molecular species may differ in immunogenicity, and facile means to identify sulfatide species in islets and other tissues obtainable in only small amounts could be useful. Electrospray ionization mass spectrometry (ESI/MS) permits structural determination of small quantities of phospholipids and is applied here to sulfatide analysis. We find that sulfatide standards are readily analyzed by negative ion ESI/MS, and tandem mass spectra of individual species exhibit some ions common to all species and other ions that reflect distinct fatty acid substituents in different sulfatide molecules. A signature ion cluster resulting from cleavage directed by the alpha-hydroxy group of sulfatide species with a hydroxylated fatty acid substituent identifies such species. Sulfatide profiles in tissue lipid extracts can be obtained by ESI/MS/MS scanning for common sulfatide ions and for ions reflecting fatty acid substituents. Islets are demonstrated to contain sulfatide and to exhibit a profile of species different from that of brain.

Arachidonic acid metabolism in isolated pancreatic islets. III. Effects of exogenous lipoxygenase products and inhibitors on insulin secretion.

Isolated pancreatic islets from the rat have been demonstrated by stable isotope dilution-mass spectrometric methods to synthesize the 12-lipoxygenase product 12-hydroxyeicosatetraenoic acid (12-HETE) in amounts of 1.7 to 2.8 ng per 10(3) islets. No detectable amounts of 5-HETE and only trace amounts of 15-HETE could be demonstrated by these methods. Nordihydroguaiaretic acid (NDGA) and BW755C have been demonstrated to inhibit islet 12-HETE synthesis and also to inhibit glucose-induced insulin secretion. Inhibition of insulin secretion and of 12-HETE synthesis exhibited similar dependence on the concentration of these compounds. Eicosa-5,8,11,14-tetrynoic acid (ETYA) also inhibited glucose-induced insulin secretion, as previously reported, at concentrations which inhibit islet 12-HETE synthesis. Exogenous 12-HETE partially reversed the suppression of glucose-induced insulin secretion by lipoxygenase inhibitors, but exogenous 12-hydroperoxyeicosatetraenoic acid (12-HPETE), 15-HPETE, 5-HPETE, 15-HETE, or 5-HETE did not reverse this suppression. These observations argue against the recently suggested hypothesis that islet synthesis of 5-HETE modulates insulin secretion. Suppression of glucose-induced insulin secretion by ETYA, BW755C and NDGA may be due to inhibition of the islet 12-lipoxygenase by these compounds. The possibility that other processes involved in glucose-induced insulin secretion are inhibited by ETYA, BW755C and NDGA cannot yet be excluded.

Arachidonic acid metabolism in isolated pancreatic islets. II. The effects of glucose and of inhibitors of arachidonate metabolism on insulin secretion and metabolite synthesis.

Isolated pancreatic islets from the rat incubated with 28 mM glucose have been found to secrete more insulin and to synthesize greater amounts of arachidonate lipoxygenase and cyclooxygenase products than islets incubated with 3 mM glucose. This effect was not apparent in studies examining metabolism of radiolabeled arachidonate and was revealed only when the metabolites were quantitated with mass spectrometric measurements. That the glucose-induced synthesis of arachidonate metabolites may participate in insulin secretion was suggested by studies with inhibitors of arachidonate metabolism. Eicosa 5,8,11,14 tetrynoic acid (ETYA) suppressed glucose-induced insulin secretion by 63-74% at a concentration (20 microM) which inhibited the synthesis of arachidonate lipoxygenase and cyclooxygenase products by 90%. Indomethacin (10 microM) completely prevented islet synthesis of cyclooxygenase products but did not influence glucose-induced insulin secretion. Although indomethacin did not inhibit the conversion of exogenous, 3H-labeled arachidonate to [3H]12-HETE, it did significantly inhibit (41-72%) the synthesis of 12-HETE from endogenous precursor. This is presumed to reflect indirect effects of indomethacin on hydrolysis of arachidonate from phospholipids, as recently reported in platelets. These studies constitute the first demonstration that glucose stimulates the synthesis of a lipoxygenase product (12-HETE) from endogenous arachidonate by isolated islets, and that suppression of 12-HETE synthesis with ETYA reduces glucose-induced insulin secretion from isolated islets.