Accuracy of immunoassay and mass spectrometry urinary free cortisol in the diagnosis of Cushing's syndrome.

PURPOSE: Urinary free cortisol (UFC) determination by highly specific methods as mass spectrometry instead of commercially available antibody-based immunoassays is increasingly recommended. However, clinical comparisons of both analytical approaches in the screening of Cushing's syndrome (CS) are not available. The aim of this study was to evaluate the diagnostic value of mass spectrometry versus immunoassay measurements of 24 h-UFC in the screening of CS. METHODS: Cross-sectional study of 33 histologically confirmed CS patients: 25 Cushing's disease, 5 adrenal CS and 3 ectopic CS; 92 non-CS patients; and 35 healthy controls. UFC by immunoassay (UFCxIA) and mass spectrometry (UFCxMS), urinary free cortisone (UFCo) and UFC:UFCo ratio were measured, together with creatinine-corrected values. Sensitivity, specificity, AUC and Landis and Koch concordance index were determined. RESULTS: AUC for UFCxIA and UFCxMS were 0.77 (CI 0.68-0.87) and 0.77 (CI 0.67-0.87) respectively, with a kappa coefficient 0.60 and strong Landis and Koch concordance index. The best calculated cutoff values were 359 nmol/24 h for UFCxIA (78 % sensitivity, 62 % specificity) and 258.1 nmol/24 h for UCFxMS (53 % sensitivity, 86 % specificity). The upper limit of UFCxIA and UCFxMS reference ranges were 344.7 and 169.5 nmol/24 h respectively. Sensitivity and specificity for CS diagnosis at these cutpoints were 84 and 56 % for UFCxIA and 81 and 54 % for UFCxMS. CONCLUSIONS: According to our data, both methods present a very similar diagnostic value. However, results suggest that lower cutoff points for mass spectrometry may be necessary in order to improve clinical sensitivity.

Clinical onset of celiac disease after duodenal switch: a case report.

To our knowledge, this is the first reported case of clinical onset of celiac disease (CD) following duodenal switch surgery. A 61-year-old obese woman developed severe diarrhea soon after bariatric surgery (BS), which was unresponsive to standard medical treatment. After the most common diarrhea etiologies in the immediate postoperative period have been excluded, serological tests were performed. Final diagnosis was determined by anti-tissue transglutaminase antibody positivity. In light of this case report, we propose that CD should be ruled out in any patient presenting with typical or atypical symptoms after BS, regardless of the latency of onset.

Long-term outcome of insulin pump therapy: reduction of hypoglycaemia and impact on glycaemic control.

AIMS: To determine the long-term outcome of continuous subcutaneous insulin infusion (CSII) in Type 1 diabetes according to Catalan National Health Service indications. METHODS: Retrospective observational study including 178 patients with Type 1 diabetes who started CSII treatment in our centre (2003-2008). All patients were followed in our CSII programme for outpatients for at least 5 years. Data on annual HbA1c levels were collected, and the main indication for starting CSII was analysed. RESULTS: Twenty-seven of 178 patients were excluded because of loss to follow-up or withdrawal from CSII, thus 151 patients (aged 37.4 ± 10.5 years, 64% women) were analysed. The main indications for starting CSII were suboptimal metabolic control (60.9%), severe hypoglycaemia/hypoglycaemia unawareness (25.5%) and others (13.6%). HbA1c was 64 ± 13 mmol/mol (8.0 ± 1.2%) at the start of CSII and 62 ± 13 mmol/mol (7.8 ± 1.2%) after 5 years in the total cohort (P = 0.1). The severe hypoglycaemia rates were 0.66 ± 1.61 and 0.17 ± 0.42 episodes/patient/year (P < 0.001). In patients with suboptimal metabolic control, HbA1c decreased from 68 ± 12 mmol/mol (8.4 ± 1.1%) to 64 ± 14 mmol/mol (8.0 ± 1.3%) (P = 0.016), with 37.4% of those in this group having an HbA1c ≤ 58 mmol/mol (7.5%) after 5 years. In patients starting CSII due to severe hypoglycaemia the problem was considered resolved in 93%, and in 64% of those starting CSII because of suboptimal glycaemic control, HbA1c improved significantly. CONCLUSIONS: CSII therapy achieves and maintains its efficacy mainly in terms of reducing severe hypoglycaemia. In the whole group of patients, the reduction in HbA1c is transient and disappears after 5 years.

Allergic fetal priming leads to developmental, behavioral and neurobiological changes in mice.

The state of the mother's immune system during pregnancy has an important role in fetal development and disruptions in the balance of this system are associated with a range of neurologic, neuropsychiatric and neurodevelopmental disorders. Epidemiological and clinical reports reveal various clues that suggest a possible association between developmental neuropsychiatric disorders and family history of immune system dysfunction. Over the past three decades, analogous increases have been reported in both the incidence of neurodevelopmental disorders and immune-related disorders, particularly allergy and asthma, raising the question of whether allergic asthma and characteristics of various neurodevelopmental disorders share common causal links. We used a mouse model of maternal allergic asthma to test this novel hypothesis that early fetal priming with an allergenic exposure during gestation produces behavioral deficits in offspring. Mothers were primed with an exposure to ovalbumin (OVA) before pregnancy, then exposed to either aerosolized OVA or vehicle during gestation. Both male and female mice born to mothers exposed to aerosolized OVA during gestation exhibited altered developmental trajectories in weight and length, decreased sociability and increased marble-burying behavior. Moreover, offspring of OVA-exposed mothers were observed to have increased serotonin transporter protein levels in the cortex. These data demonstrate that behavioral and neurobiological effects can be elicited following early fetal priming with maternal allergic asthma and provide support that maternal allergic asthma may, in some cases, be a contributing factor to neurodevelopmental disorders.

Autophagy in the brain of neonates following hypoxia-ischemia shows sex- and region-specific effects.

Autophagy is responsible for the bulk degradation of cytoplasmic contents including organelles through the lysosomal machinery. Neonatal hypoxia-ischemia (HI) causes cell death in the brain by caspase-dependent and independent pathways. Ischemic insults also increase the formation of autophagosomes and activate autophagy. This study assessed the possible sex- and region-specific differences of autophagy activity in neonates subjected to HI brain injury. HI males had a modest decrease in lysosome numbers with no effect on LC3B-II protein in the cortex. In contrast, HI females had decreased lysosome numbers and their LC3B-II protein expression was significantly increased in the cortex following HI. In the hippocampus, both HI males and all females had increased numbers of autolysosomes suggesting activation of autophagy but with no effect on lysosome numbers, or Beclin-1 or LC3B protein levels. Males and females had increases in caspase 3/7 activity in their cortices and hippocampi following HI, though the increases were three to sixfold greater in females. The present data: (a) confirm greater caspase activation in the brains of females compared to males following HI; (b) suggest a partial failure to degrade LC3B-II protein in cortical but not hippocampal lysosomes of females as compared to males following neonatal HI; (c) all females have greater basal autophagy activity than males which may protect cells against injury by increasing cell turnover and (d) demonstrate that autophagy pathways are disturbed in regional- and sex-specific patterns in the rat brain following neonatal HI.

Maternal immune activation and strain specific interactions in the development of autism-like behaviors in mice.

It is becoming increasingly apparent that the causes of autism spectrum disorders (ASD) are due to both genetic and environmental factors. Animal studies provide important translational models for elucidating specific genetic or environmental factors that contribute to ASD-related behavioral deficits. For example, mouse research has demonstrated a link between maternal immune activation and the expression of ASD-like behaviors. Although these studies have provided insights into the potential causes of ASD, they are limited in their ability to model the important interactions between genetic variability and environmental insults. This is of particular concern given the broad spectrum of severity observed in the human population, suggesting that subpopulations may be more susceptible to the adverse effects of particular environmental insults. It is hypothesized that the severity of effects of maternal immune activation on ASD-like phenotypes is influenced by the genetic background in mice. To test this, pregnant dams of two inbred strains (that is, C57BL/6J and BTBR T(+)tf/J) were exposed to the viral mimic polyinosinic-polycytidylic acid (polyI:C), and their offspring were tested for the presence and severity of ASD-like behaviors. To identify differences in immune system regulation, spleens were processed and measured for alterations in induced cytokine responses. Strain-treatment interactions were observed in social approach, ultrasonic vocalization, repetitive grooming and marble burying behaviors. Interestingly, persistent dysregulation of adaptive immune system function was only observed in BTBR mice. Data suggest that behavioral and immunological effects of maternal immune activation are strain-dependent in mice.

[Assessment of ophthalmologic control of the diabetic patient in primary care]. / Evaluación del control oftalmológico del paciente diabético en atención primaria.

OBJECTIVE: To evaluate ophthalmic control in patients with diabetes mellitus (DM) in primary care (PC). DESIGN: Crossover observation study. SETTING: Hellín Health Centre (Albacete). PATIENTS: 137 diabetics selected at random, excluding pregnant women and children under 14. MEASUREMENTS AND MAIN RESULTS: The variables of age, sex, type of diabetes (DM1/DM2), years of evolution, ophthalmic control and appearance of diabetic retinopathy (DR) were studied. Mean age was 68.5 +/- 10.9 years. 52% were women (n = 72). 88% had DM2 (n = 121). Time of evolution from diagnosis was < 5 years in 46% (n = 63), 6-10 years in 23% (n = 32), 11-15 years in 14% (n = 19), > 15 years in 5% (n = 7) and nothing was recorded for 12% (n = 16). 41% (n = 56) of the patients had not had an ophthalmic evaluation. Of the 59% (n = 81) referred to the ophthalmology out-clinic (OOC), 65 patients followed recommendations of the Spanish Society of Retina and Vitreous Humour, and 16 were referred for other reasons, of which cataracts was the most common eye pathology. Of these 81 patients referred to the OOC, 29% did not have DR (n = 23), and 44% did (n = 36). Of this 44%, 14 were light, 15 moderate, 3 severe and 4 proliferating cases. The reports were not sent back to PC in 27% of cases (n = 22). CONCLUSIONS: Ophthalmic referral in PC is still deficient for a large number of DM patients. DR of varying degrees was detected in a high number of cases who did have an ophthalmic check-up. Communication between PC and ophthalmic care concerning diabetic patients must improve.

Metabolism of 8,11,14,17-eicosatetraenoic acid by human platelet lipoxygenase and cyclooxygenase.

Human platelets metabolize 8,11,14,17-eicosatetraenoic acid primarily into 12-hydroxy-8,10,14,17-eicosatetraenoic acid. Several other hydroxy acids were also produced in small amounts via an indomethacin insensitive pathway. Platelet cyclooxygenase metabolized this acid only into 12-hydroxy-8,10,14-heptadecatrienoic acid. It was not possible to detect any cyclic products even though vesicular gland cyclooxygenase metabolizes this (n-3) acid to 17,18-dehydroprostaglandin E1 (Oliw, E.H., Sprecher, H. and Hamberg, M. (1986) J. Biol. Chem. 261, 2675-2683).

Metabolism of (n-6) and (n-3) polyunsaturated fatty acids by human platelets.

Human platelets metabolize 7,10,13,16-docosatetraenoic acid and 4,7,10,13,16-docosapentaenoic acid into 22-carbon thromboxanes 19-carbon HHT analogs and 14-hydroxy fatty acids via the lipoxygenase pathway. Conversely the two analogous (n-3) acids, 7,10,13,16,19-docosapentaenoic acid and 4,7,10,13,16,19-docosahexaenoic acid, are metabolized only into a pair of isomeric 11- and 14-hydroxy fatty acids. These findings suggest that platelets may contain more than one lipoxygenase.

Decreased utilization of [2-3H]glycerol in phospholipid and neutral glyceride biosynthesis in the retina of streptozotocin-diabetic rats.

The effect of streptozotocin (STZ)-induced diabetes on lipid uptake was studied in the rat retina. The intravitreal incorporation of [2-3H]glycerol was followed in retinal lipids from the first day of diabetes (acute state) up to 20 wk (chronic state). Total lipid incorporation decreased 50% 2 d after injection of STZ; the labeling remained lower than the control values throughout the 20 wk period studied. In the chronic state of diabetes, the time-course of the incorporation of [2-3H]glycerol during the first 2 h after its injection displayed a pattern of incorporation similar to that of the controls. The possibility that the decreased utilization of [2-3H]glycerol in the lipid pathway results from a competition between the injected [3H]glycerol and the glycolytic pathway during diabetes in rat retina in vivo is discussed.

Synthesis of two hydroxy fatty acids from 7,10,13,16,19-docosapentaenoic acid by human platelets.

Platelets metabolize 7,10,13,16,19-docosapentaenoic acid (22:5(n-3] into 11-hydroxy-7,9,13,16,19- and 14-hydroxy-7,10,12,16,19-docosapentaenoic acid via an indomethacin-insensitive pathway. Time-dependent studies with 20 microM substrate show a lag in the synthesis of both the 11- and 14-isomers which was not observed for the synthesis of thromboxane B2 (TXB2), 5,8,10-heptadecatrienoic acid, and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) from arachidonic acid. When platelets were incubated with increasing concentrations of 22:5(n-3), the 11- and 14-isomers were not produced until the substrate concentration exceeded 5 microM unless arachidonic acid was also added to the incubations. The stimulatory effect of arachidonic acid was not blocked by indomethacin thus suggesting that 12-hydroperoxyeicosatetraenoic acid or 12-HETE derived from arachidonic acid may activate the platelet lipoxygenase(s) which metabolize 22:5(n-3). Incubations containing 20 microM 22:5(n-3) and increasing levels of [1-14C]arachidonic acid show that the (n-3) acid inhibits the synthesis of both 5,8,10-heptadecatrienoic acid and TXB2 from arachidonic acid. At the same time, 12-HETE synthesis increased due to substrate shunting to the lipoxygenase pathway.

High content of 22:6 (docosahexaenoate) and active [2-3H]glycerol metabolism of phosphatidic acid from photoreceptor membranes.

This study describes the content, fatty acid composition and [2-3H]glycerol metabolism of phosphatidic acid of rod outer segment membranes from vertebrate retinas. A relatively high content of phosphatidic acid was observed in rod outer segment membranes isolated from rat, toad and bovine retinas. In bovine retinas, about 65% of the acyl groups of phosphatidic acid were composed of docosahexaenoate. Arachidonate and docosapentaenoate represented about 4 and 5%, respectively, of the total, whereas stearate was the most common saturated acyl chain. An active [2-3H]glycerol metabolism in the phosphatidic acid of these membranes was found when whole retinas were incubated with the precursor for short periods prior to subcellular fractionation. Our results suggested that the pool of phosphatidic acid enriched in docosahexaenoate may arise from de novo biosynthesis or from phospholipid degradation by a phospholipase D enzyme, and that it is not metabolically related, in any major fashion, to the diacylglycerols of rod outer segment membranes.

Chain elongation and desaturation of eicosapentaenoate to docosahexaenoate and phospholipid labeling in the rat retina in vivo.

The metabolism of [1-14C]eicosapentaenoic acid in the retina after intravitreal injections in the adult rat eye was studied. The acylation of eicosapentaenoic acid and the appearance of labeled docosapentaenoate and docosahexaenoate in individual phospholipids was observed at 3, 5 and 30 min after injection. The elongation and desaturation products represented about 8 and 4%, respectively, of the total radioactivity of phospholipids 3 min after injection. The highest labeling was found in phosphatidylcholine, phosphatidylinositol and phosphatidic acid. The uneven labeling profiles and the specific activities in individual phospholipids suggested that, in addition to the deacylation-acylation route for the introduction of polyenoic acyl groups into phospholipids, acylation may also take place during the synthesis of phosphatidic acid, followed by channeling to phospholipids.

The rat retina is a useful in vivo model to study membrane lipid synthesis: rates of biosynthesis of neutral glycerides and phospholipids.

The phospholipid composition was studied in the whole rat retina, as well as in its subcellular fractions. A relative enrichment of phosphatidic acid, phosphatidylethanolamine, and phosphatidylserine was observed in rod outer segments (ROS) in comparison with entire retina: nuclear-photoreceptor inner segments-synaptic bodies (P1) and synaptosomal-mitochondrial (P2) fractions. Phosphatidylcholine was the predominant phospholipid class found in all subcellular fractions analyzed. The microsomal fraction was relatively enriched in phosphatidic acid and in phosphatidylinositol. In addition, the rat eye has been used as an in vivo system to study membrane lipid synthesis. After intravitreal injections of [2-3H]glycerol a rapid labeling of retinal glycerolipids took place. Up to 120 min after injection only the glycerol backbone of lipids was labeled. Phosphatidic acid and diacylglycerol displayed rapid rates of synthesis and breakdown. Fastest rates of labeling were attained by phosphatidylcholine followed by phosphatidylinositol. Differences were found when in vitro labeling by [2-3H]glycerol was compared with intravitreal injections. Labeling of phospholipids of subcellular fractions by intravitreally injected [2-3H]glycol showed that most of the label accumulated in microsomal phosphatidylcholine and phosphatidylinositol. Diacylglycerols and phosphatidylethanolamine also took up 10 and 20% respectively of the precursor. It is concluded that the rat eye is a useful experimental model to study synthesis and metabolism of membrane lipids in the retina.

Propranolol increases the biosynthesis of phosphatidic acid, phosphatidylinositol and phosphatidylserine in the toad retina. Studies in the entire and subcellular fractions.

1. Intact toad retinas incubated for short periods of time with [2-3H]glycerol were subject to subcellular fractionation. 2. The composition and labeling of glycerolipids were studied in the following subcellular fractions: rod outer segments (ROS), nuclear-photoreceptor inner segment synaptic body (P1), synaptosomal-mitochondrial (P2), microsomal and cytosolic. 3. It was concluded that the biosynthetic de novo route was followed by [2-3H]glycerol in the toad retina since radioactive was located solely in the glycerol backbone of lipids and phosphatidic acid specific activity was the highest. 4. Propranolol produces an increase in the biosynthesis of acidic phospholipids and inhibition in the biosynthesis of zwitterionic lipids in the entire toad retina. The effect was mainly located in microsomes and in the soluble fraction during the first minutes of incubation, being spread afterwards to other fractions. 5. These data are consistent with the view that enzymes of the biosynthesis of glycerolipids are modified in the retinal endoplasmic reticulum by propranolol, providing a useful tool to evaluate the regulation of the pathway.