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Purpose: Although cyclooxygenase (COX)-2 inhibitors could represent the most effective chemopreventive tool against colorectal cancer (CRC), their use in clinical practice is hampered by cardiovascular side effects. Consumption of ω-3-polyunsaturated fatty acids (ω-3-PUFAs) is associated with a reduced risk of CRC. Therefore, in this study, we assessed the efficacy of a novel 99% pure preparation of ω-3-PUFA eicosapentaenoic acid as free fatty acids (EPA-FFA) on polyps in ApcMin/+ mice.Experimental design: ApcMin/+ and corresponding wild-type mice were fed control diet (Ctrl) or diets containing either EPA-FFA 2.5% or 5%, for 12 weeks while monitoring food intake and body weight.Results: We found that both EPA-FFA diets protected from the cachexia observed among ApcMin/+ animals fed Ctrl diet (P < 0.0054), without toxic effect, in conjunction with a significant decrease in lipid peroxidation in the treated arms. Moreover, both EPA-FFA diets dramatically suppressed polyp number (by 71.5% and 78.6%, respectively; P < 0.0001) and load (by 82.5% and 93.4%, respectively; P < 0.0001) in both small intestine and colon. In addition, polyps less than 1 mm in size were predominantly found in the EPA-FFA 5% arm whereas those 1 to 3 mm in size were more frequent in the Ctrl arm (P < 0.0001). Interestingly, in the EPA-FFA groups, mucosal arachidonic acid was replaced by EPA (P < 0.0001), leading to a significant reduction in COX-2 expression and β-catenin nuclear translocation. Moreover, in the EPA-FFA arms, we found a significant decrease in proliferation throughout the intestine together with an increase in apoptosis.Conclusions: Our data make 99% pure EPA-FFA an excellent candidate for CRC chemoprevention. Clin Cancer Res; 16(23); 5703–11. ©2010 AACR.

Supplementary Figures S1-S3 and Supplementary Tables S1-S3.

Background Arachidonic acid (AA) and docosahexaenoic acid (DHA) are crucial for neural and visual development after premature birth. Preterm infants usually require tube feeding (TF) until the achievement of adequate oral feeding skills; the impact of TF on DHA and AA delivery has not been investigated yet. This study aimed to evaluate the effect of different TF techniques on the delivery of AA and DHA contained in human milk (HM). Methods HM samples (65 mL each) were collected and divided into three 20-mL aliquots. The remaining 5 mL served as baseline. Three TF techniques were simulated (1 for each aliquot): gravity bolus feeding (BF), 3-hour continuous feeding using a horizontal feeding pump, and 3-hour continuous feeding with the feeding pump angled at 45°. For horizontal continuous feeding (HCF) and 45° angled continuous feeding (ACF), aliquots delivered between 0 and 90 minutes (T1) and 91 and 180 minutes (T2) were collected separately. AA and DHA concentration was analyzed by gas chromatography/mass spectrometry and compared among the TF methods. DHA and AA delivery at T1 and T2 was also evaluated. Results Fifty-one simulated feeds were performed. DHA and AA amounts after BF and ACF did not differ significantly compared with baseline, whereas HCF resulted in significantly lower DHA and AA concentration. During T2, ACF delivered almost twice the DHA and AA amounts compared with T1. Conclusion The delivery of HM AA and DHA is significantly affected by TF, with potential clinical implications. When BF is not tolerated, ACF might represent a feasible alternative to reduce TF-related DHA and AA loss.

Patients with long-standing ulcerative colitis (UC) have an increased colorectal cancer (CRC) risk. In this pilot study we evaluated the effect of Eicosapentaenoic acid as free fatty acid (EPA-FFA) supplementation on mucosal disease activity, colonic differentiation markers and microbiota composition in UC patients. Twenty long-standing UC patients in stable clinical remission and with fecal calprotectin (FC) > 150 µg/g were enrolled (T0) and supplemented with EPA-FFA 2 g/daily for 90 days (T3). Endoscopic and histologic disease activities were measured by Mayo and Geboes scores, respectively. HES1, KLF4, STAT3, IL-10 and SOCS3 levels were determined using western blotting and qRT-PCR, while phospho-STAT3 levels were assessed by western blotting. Goblet cells were stained by Alcian blue. Microbiota analyses were performed on both fecal and colonic samples. Nineteen patients completed the study; seventeen (89.5%) were compliant. EPA-FFA treatment reduced FC levels at T3. Patients with FC > 150 µg/g at T3 (n = 2) were assumed as non-responders. EPA-FFA improved endoscopic and histological inflammation and induced IL-10, SOCS3, HES1 and KLF4 in compliant and responder patients. Importantly, long-term UC-driven microbiota composition was partially redressed by EPA-FFA. In conclusion, EPA-FFA supplementation reduced mucosal inflammation, promoted goblet cells differentiation and modulated intestinal microbiota composition in long-standing UC patients.

ContextAbnormal cortisol metabolism in polycystic ovary syndrome (PCOS) has been invoked as a cause of secondary activation of the hypothalamic–pituitary–adrenal axis and hence androgen excess. However, this is based on urinary excretion of cortisol metabolites, which cannot detect tissue-specific changes in metabolism and may be confounded by obesity.ObjectiveTo assess cortisol clearance and whole-body and tissue-specific activities of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1 (HSD11B1)) in PCOS.DesignCase–control study.SettingMedical center.PatientsA total of 20 overweight–obese unmedicated Caucasian women with PCOS, aged 18–45 years, and 20 Caucasian controls matched for age, BMI, body fat distribution, andHSD11B1genotypes (rs846910 and rs12086634).Main outcome measuresCortisol metabolites were measured in 24 h urine. During steady-state 9,11,12,12-[2H]4-cortisol infusion, cortisol clearance was calculated and whole-body HSD11B1 activity was assessed as the rate of appearance of 9,12,12-2H3-cortisol (d3-cortisol). Hepatic HSD11B1 activity was quantified as the generation of plasma cortisol following an oral dose of cortisone. Subcutaneous adipose HSD11B1 activity andHSD11B1mRNA were measured,ex vivo, in biopsies.ResultsUrinary cortisol metabolite excretion, deuterated cortisol clearance, and the rate of appearance of d3-cortisol did not differ between patients with PCOS and controls. However, hepatic HSD11B1 conversion of oral cortisone to cortisol was impaired (PHSD11B1mRNA levels and activity were increased (PConclusionsTissue-specific dysregulation of HSD11B1 is a feature of PCOS, over and above obesity, whereas increased clearance of cortisol may result from obesity rather than PCOS.

Inflammatory bowel diseases are associated with increased risk of developing colitis-associated colorectal cancer (CAC). Epidemiological data show that the consumption of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) decreases the risk of sporadic colorectal cancer (CRC). Importantly, recent data have shown that eicosapentaenoic acid-free fatty acid (EPA-FFA) reduces polyp formation and growth in models of familial adenomatous polyposis. However, the effects of dietary EPA-FFA are unknown in CAC. We tested the effectiveness of substituting EPA-FFA, for other dietary fats, in preventing inflammation and cancer in the AOM-DSS model of CAC. The AOM-DSS protocols were designed to evaluate the effect of EPA-FFA on both initiation and promotion of carcinogenesis. We found that EPA-FFA diet strongly decreased tumor multiplicity, incidence and maximum tumor size in the promotion and initiation arms. Moreover EPA-FFA, in particular in the initiation arm, led to reduced cell proliferation and nuclear β-catenin expression, whilst it increased apoptosis. In both arms, EPA-FFA treatment led to increased membrane switch from ω-6 to ω-3 PUFAs and a concomitant reduction in PGE2 production. We observed no significant changes in intestinal inflammation between EPA-FFA treated arms and AOM-DSS controls. Importantly, we found that EPA-FFA treatment restored the loss of Notch signaling found in the AOM-DSS control and resulted in the enrichment of Lactobacillus species in the gut microbiota. Taken together, our data suggest that EPA-FFA is an excellent candidate for CRC chemoprevention in CAC.

Background Oral administration of the omega-3 fatty acid eicosapentaenoic acid (EPA), as the free fatty acid (FFA), leads to EPA incorporation into, and reduced growth of, experimental colorectal cancer liver metastases (CRCLM). Design: We performed a Phase II double-blind, randomised, placebo-controlled trial of EPA-FFA 2 g daily in patients undergoing liver resection surgery for CRCLM. The patients took EPA-FFA (n=43) or placebo (n=45) prior to surgery. The primary end-point was the CRCLM Ki67 proliferation index (PI). Secondary end-points included safety and tolerability of EPA-FFA, tumour fatty acid content and CD31-positive vascularity. We also analysed overall survival (OS) and disease-free survival (DFS). Results The median (range) duration of EPA-FFA treatment was 30 (12–65) days. Treatment groups were well matched with no significant difference in disease burden at surgery or preoperative chemotherapy. EPA-FFA treatment was well tolerated with no excess of postoperative complications. Tumour tissue from EPA-FFA-treated patients demonstrated a 40% increase in EPA content (p=0.0008), no difference in Ki67 PI, but reduced vascularity in ‘EPA-naive’ individuals (p=0.075). EPA-FFA also demonstrated antiangiogenic activity in vitro. In the first 18 months after CRCLM resection, EPA-FFA-treated individuals obtained OS benefit compared with placebo, although early CRC recurrence rates were similar. Conclusions EPA-FFA therapy is safe and well tolerated in patients with advanced CRC undergoing liver surgery. EPA-FFA may have antiangiogenic properties. Remarkably, limited preoperative treatment may provide postoperative OS benefit. Phase III clinical evaluation of prolonged EPA-FFA treatment in CRCLM patients is warranted. Trial Identifier: ClinicalTrials.gov NCT01070355.

As pre-clinical and clinical research interest in ω-3 polyunsaturated fatty acids (PUFA) increases, so does the need for a fast, accurate and reproducible analytical method to measure fatty acids (FA) in biological samples in order to validate potential prognostic and predictive biomarkers, as well as establishing compliance in ω-3 PUFA intervention trials. We developed a LC-ESI-MS/MS method suitable for high throughput development to measure FAs and validated it in the context of treatment with the ω-3 PUFA eicosapentaenoic acid (EPA). Uniquely we directly compared the LC-ESI-MS/MS method to a GC-MS protocol. We demonstrated the LC-ESI-MS/MS method is accurate and reproducible, with coefficients of variation consistently below 15% for each PUFA analysed. The relative FA content values correlated well with those obtained by GC-MS (r2 = 0.94, p < 0.001 for EPA) in vitro. The data obtained following analysis of FA content of liver tissues from mice fed an eicosapentaenoic acid enriched diet showed similar results to that of published studies in which GC-MS was used. The LC-ESI-MS/MS method allows concomitant analysis of unesterified (free, unbound) and esterified (bound) FAs in biological samples, allowing investigation of different PUFA pools in cells and tissues.

Aberrant NOTCH1 signalling is critically involved in multiple models of colorectal cancer (CRC) and a prominent role of NOTCH1 activity during inflammation has emerged. Epithelial to Mesenchymal Transition (EMT), a crucial event promoting malignant transformation, is regulated by inflammation and Metalloproteinase-9 (MMP9) plays an important role in this process. Eicosapentaenoic Acid (EPA), an omega-3 polyunsaturated fatty acid, was shown to prevent colonic tumors in different settings. We recently found that an extra-pure formulation of EPA as Free Fatty Acid (EPA-FFA) protects from colon cancer development in a mouse model of Colitis-Associated Cancer (CAC) through modulation of NOTCH1 signalling. In this study, we exposed colon cancer cells to an inflammatory stimulus represented by a cytokine-enriched Conditioned Medium (CM), obtained from THP1-differentiated macrophages. We found, for the first time, that CM strongly up-regulated NOTCH1 signalling and EMT markers, leading to increased invasiveness. Importantly, NOTCH1 signalling was dependent on MMP9 activity, upon CM exposure. We show that a non-cytotoxic pre-treatment with EPA-FFA antagonizes the effect of inflammation on NOTCH1 signalling, with reduction of MMP9 activity and invasiveness. In conclusion, our data suggest that, in CRC cells, inflammation induces NOTCH1 activity through MMP9 up-regulation and that this mechanism can be counteracted by EPA-FFA.

The ability to induce changes in cell membrane properties is nowadays considered an additional mechanism to explain the pharmacological effects of non-steroidal anti-inflammatory drugs (NSAIDs). We previously demonstrated that the NSAID Licofelone, a dual cyclooxygenase/5-lipoxygenase inhibitor, triggers apoptosis in HCA-7 colon cancer cells independently from the inhibition of these enzymes. Here, we provide evidence that, in HCA-7 cells, the pro-apoptotic effect of this drug relies, at least in part, on its ability to inhibit epidermal growth factor receptor (EGFR) signalling by a decrease of cell membrane fluidity. Indeed, Licofelone induced a relevant change in the relative proportions of some saturated, monounsaturated and polyunsaturated fatty acids constituting HCA-7 phospholipid fraction, and significantly increased the levels of cholesterol in HCA-7 cell membrane. All of these changes resulted in a remarkable decrease of membrane fluidity. Such phenomenon was associated with the block of EGFR kinase activity and of its downstream targets, the p44-42 mitogen-activated protein kinase (MAPK) and AKT cascades, whose inhibitions were found to induce apoptosis in HCA-7 cells. Overall, these findings provide a new additional mechanism by which NSAIDs are effective toward colon cancer cells.

BACKGROUND AND PURPOSE The omega-3 polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (EPA) has antineoplastic activity at early stages of colorectal carcinogenesis, relevant to chemoprevention of colorectal cancer (CRC). We tested the hypothesis that EPA also has anti-CRC activity at later stages of colorectal carcinogenesis, relevant to treatment of metastatic CRC, via modulation of E-type PG synthesis. EXPERIMENTAL APPROACH A BALB/c mouse model, in which intrasplenic injection of syngeneic MC-26 mouse CRC cells leads to development of liver metastases, was used. Dietary EPA was administered in the free fatty acid (FFA) form for 2 weeks before and after ultrasound-guided intrasplenic injection of 1 × 106 MC-26 cells (n= 16 each group). KEY RESULTS Treatment with 5% (w w-1) EPA-FFA was associated with a reduced MC-26 mouse CRC cell liver tumour burden compared with control animals (median liver weight 1.03 g vs. 1.62 g; P < 0.034). Administration of 5% EPA-FFA was also linked to a significant increase in tumour EPA incorporation and lower intratumoural PGE2 levels (with concomitant increased production of PGE3). Liver tumours from 5% EPA-FFA- treated mice demonstrated decreased 5-bromo-2-deoxyuridine-positive CRC cell proliferation and reduced phosphorylated ERK 1/2 expression at the invasive edge of tumours. A concentration-dependent reduction in MC-26 CRC cell Transwell® migration following EPA-FFA treatment (50–200 µM) in vitro was rescued by exogenous PGE2 (10 µM) and PGE1-alcohol (1 µM). CONCLUSIONS AND IMPLICATIONS EPA-FFA inhibits MC-26 CRC cell liver metastasis. EPA incorporation is associated with a ‘PGE2 to PGE3 switch’ in liver tumours. Inhibition of PGE2-EP4 receptor-dependent CRC cell motility probably contributes to the antineoplastic activity of EPA.

Inflammatory bowel diseases are associated with increased risk of developing colitis-associated colorectal cancer (CAC). Epidemiological data show that the consumption of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) decreases the risk of sporadic colorectal cancer (CRC). Importantly, recent data have shown that eicosapentaenoic acid-free fatty acid (EPA-FFA) reduces polyp formation and growth in models of familial adenomatous polyposis. However, the effects of dietary EPA-FFA are unknown in CAC. We tested the effectiveness of substituting EPA-FFA, for other dietary fats, in preventing inflammation and cancer in the AOM-DSS model of CAC. The AOM-DSS protocols were designed to evaluate the effect of EPA-FFA on both initiation and promotion of carcinogenesis. We found that EPA-FFA diet strongly decreased tumor multiplicity, incidence and maximum tumor size in the promotion and initiation arms. Moreover EPA-FFA, in particular in the initiation arm, led to reduced cell proliferation and nuclear β-catenin expression, whilst it increased apoptosis. In both arms, EPA-FFA treatment led to increased membrane switch from ω-6 to ω-3 PUFAs and a concomitant reduction in PGE2 production. We observed no significant changes in intestinal inflammation between EPA-FFA treated arms and AOM-DSS controls. Importantly, we found that EPA-FFA treatment restored the loss of Notch signaling found in the AOM-DSS control and resulted in the enrichment of Lactobacillus species in the gut microbiota. Taken together, our data suggest that EPA-FFA is an excellent candidate for CRC chemoprevention in CAC.

Supplementation with n-3 polyunsaturated fatty acids (n-3 PUFAs) may be beneficial for patients with inflammatory bowel diseases (IBD). In this study we analyzed the pharmacokinetic profile of eicosapentaenoic acid (EPA), as the free fatty acid (FFA), in an enteric-coated preparation, in 10 ulcerative colitis (UC) and 10 Crohn’s disease (CD) patients and 15 healthy volunteers (HV). Subjects received 2 g daily of EPA-FFA for 8 weeks. Plasma phospholipid and red blood cell (RBC) membrane fatty acid content were measured by gas chromatography-mass spectrometry. There was a rapid incorporation of EPA into plasma phospholipids by 2 weeks and a slower, but highly consistent, incorporation into RBC membranes (4% total fatty acid content; coefficient of variation 10–16%). There was a concomitant reduction in relative n-6 PUFA content. Elongation and desaturation of EPA into docosahexaenoic acid (DHA) via docosapentaenoic acid (DPA) were apparent and DHA content also increased in membranes. EPA-FFA is well tolerated and no difference in the pharmacokinetic profile of n-3 PUFA incorporation was detected between IBD patients and HV. Our data support the concept that EPA can be considered the “universal donor” with respect to key n-3 PUFAs and that this enteric-coated formulation allows long term treatment with a high level of compliance.

Purpose: Although cyclooxygenase (COX)-2 inhibitors could represent the most effective chemopreventive tool against colorectal cancer (CRC), their use in clinical practice is hampered by cardiovascular side effects. Consumption of ω-3-polyunsaturated fatty acids (ω-3-PUFAs) is associated with a reduced risk of CRC. Therefore, in this study, we assessed the efficacy of a novel 99% pure preparation of ω-3-PUFA eicosapentaenoic acid as free fatty acids (EPA-FFA) on polyps in ApcMin/+ mice. Experimental design: ApcMin/+ and corresponding wild-type mice were fed control diet (Ctrl) or diets containing either EPA-FFA 2.5% or 5%, for 12 weeks while monitoring food intake and body weight. Results: We found that both EPA-FFA diets protected from the cachexia observed among ApcMin/+ animals fed Ctrl diet (P < 0.0054), without toxic effect, in conjunction with a significant decrease in lipid peroxidation in the treated arms. Moreover, both EPA-FFA diets dramatically suppressed polyp number (by 71.5% and 78.6%, respectively; P < 0.0001) and load (by 82.5% and 93.4%, respectively; P < 0.0001) in both small intestine and colon. In addition, polyps less than 1 mm in size were predominantly found in the EPA-FFA 5% arm whereas those 1 to 3 mm in size were more frequent in the Ctrl arm (P < 0.0001). Interestingly, in the EPA-FFA groups, mucosal arachidonic acid was replaced by EPA (P < 0.0001), leading to a significant reduction in COX-2 expression and β-catenin nuclear translocation. Moreover, in the EPA-FFA arms, we found a significant decrease in proliferation throughout the intestine together with an increase in apoptosis. Conclusions: Our data make 99% pure EPA-FFA an excellent candidate for CRC chemoprevention. Clin Cancer Res; 16(23); 5703–11. ©2010 AACR.

Colorectal cancer (CRC) is one of the major causes of cancer death worldwide. The development of novel anti-CRC agents able to overcome drug resistance and/or off-target toxicity is of pivotal importance. The mammalian target of rapamycin (mTOR) plays a critical role in CRC, regulating protein translation and controlling cell growth, proliferation, metabolism and survival. The aim of this study was to explore the effect of a combination of three natural compounds, eicosapentaenoic acid-free fatty acid (EPA-FFA), epigallocatechin-3-gallate (EGCG) and proanthocyanidins (grape seed [GS] extract) at low cytotoxic concentrations on CRC cells and test their activity on mTOR and translational regulation. The CRC cell lines HCT116 and SW480 were treated for 24 h with combinations of EPA-FFA (0-150 mu M), EGCG (0-175 mu M) and GS extract (0-15 mu M) to evaluate the effect on cell viability. The low cytotoxic combination of EPA-FFA 150 mu M, EGCG 175 mu M and GS extract 15 mu M completely inhibited the mTOR signaling in HCT116 and SW480 cells, reaching an effect stronger than or comparable to that of the mTOR inhibitor Rapamycin in HCT116 or SW480 cells, respectively. Moreover, the treatment led to changes of protein translation of ribosomal proteins, c-Myc and cyclin D1. In addition, we found a reduction of clonal capability in both cell lines, with block of cell cycle in G(0)G(1) and induction of apoptosis. Our data suggest that the low cytotoxic combination of EPA-FFA, EGCG and GS extract, tested for the first time here, inhibits mTOR signaling and thus could be considered for CRC treatment.

Phytosterols, which are plant sterol, are significantly present in most intravenous lipid emulsions (ILEs) and are considered toxic substances leading to parenteral nutrition-associated cholestasis (PNAC). Glutamine on the contrary, is considered protective against the occurrence of PNAC. In this study we pursue several objectives. First we want to determine the plasma amount of phytosterols deriving from different oil sources in rats receiving parenteral nutrition, and compare it with those in rats on standard oral diet. Subsequently, we want to verify whether phytosterols have an impact on the occurrence of cholestasis. Finally, we intend to verify whether the addition of glutamine changes the outcomes. To this end, plasma phytosterols (β-sitosterol, campesterol, and stigmasterol) were quantified by gas chromatography-mass spectrometry after performing a lipid extraction procedure in 15 weaning rats randomly divided into 3 groups receiving parenteral nutrition with soy bean oil (IL), IL plus glutamine (ILG), and chow respectively for five days. Diets were isocaloric and isonitrogenous. After killing, blood and liver samples were taken to determine plasma phytosterols and check liver histology. Every sample was triple-tested. Among phytosterols, sitosterol had the highest plasma level in all the groups. Its highest concentrations were found in the ILG group, followed by the IL group. Plasma stigmasterol levels were significantly higher in both the IL and ILG groups. Plasma campesterol levels were similar across groups including the one receiving chow. None of the liver specimens showed PNAC. In conclusion, sitosterol and stigmasterol levels were highly present in the plasma, particularly in all rats receiving parenteral nutrition. No cases of PNAC appear to have occurred. Addition of glutamine to the parenteral formulation seemed not to matter, as PNAC did not occur.

ObjectiveRegeneration of cortisol by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) within liver and adipose tissue may be of pathophysiological importance in obesity and the metabolic syndrome. single nucleotide polymorphisms (SNPs) in HSD11B1, the gene encoding 11β-HSD1, have been associated with type 2 diabetes and hypertension in population-based cohort studies, and with hyperandrogenism in patients with the polycystic ovary syndrome (PCOS). However, the functional consequences of these SNPs for in vivo 11β-HSD1 expression and activity are unknown.MethodsWe explored associations of well-characterised hormonal and metabolic phenotypes with two common SNPs (rs846910 and rs12086634) in HSD11B1 in 600 women (300 with PCOS) and investigated 11β-HSD1 expression and activity in a nested study of 40 women from this cohort.ResultsHSD11B1 genotypes (as single SNPs and as the combination of the two minor allele SNPs) were not associated with PCOS. Women who were heterozygous for rs846910 A and homozygous for rs12086634 T (GA, TT genotype) had a higher risk of metabolic syndrome, regardless of the diagnosis of PCOS (odds ratio in the whole cohort=2.77 (95% confidence interval (CI) 1.16–6.67), P=0.023). In the nested cohort, women with the GA, TT genotype had higher HSD11B1 mRNA levels in adipose tissue, and higher rates of appearance of cortisol and d3-cortisol (16.1±0.7 nmol/min versus 12.1±1.1, P=0.044) during 9,11,12,12-2H4-cortisol (d4-cortisol) steady-state infusion.ConclusionsWe conclude that, in a population of Southern European Caucasian women with and without PCOS, alleles of HSD11B1 containing the two SNPs rs846910 A and rs12086634 T confer increased 11β-HSD1 expression and activity, which associates with the metabolic syndrome.

BACKGROUND AND AIM: Despite the increasing evidence of MAP/DNA isolation in Crohn's disease (CD), its potential pathogenetic role remains unclear. To further clarify the possible relationship between MAP and CD, we investigated the presence of IS900 DNA fragment in feces from Crohn's disease and ulcerative colitis (UC) patients and from healthy controls (HC). METHODS: Stool samples were collected from 31 CD, 20 UC, and 23 HC and stored at -20°C in 200-mg aliquots. DNA was extracted. MAP presence was detected with a specific PCR amplifying a 409-bp fragment from IS900. The specificity of PCR for IS900 was confirmed sequencing three positive products. Statistical analysis was performed using the Chi-square test. RESULTS: Twenty-one of 31 CD (68%), 13 of 20 UC (65%) and 11 of 23 HC (48%) were MAP-positive (CD vs. HC: p = ns; UC vs. HC: p = ns). With the limits of a small sample size, the IS900-positive percentage in CD and UC was higher than HC, although the difference was not statistically significant. CONCLUSIONS: The possibility to track the MAP presence in human feces represents a new approach to the "MAP hypothesis". Detection of MAP DNA in feces is very common, reaching very high prevalence both in CD and in UC and even in HC. Our findings seem consistent with a high prevalence of MAP asymptomatic infection among the general population and so the possible involvement of MAP in CD pathogenesis could be linked to a specific immune defective response.

Mitochondrial optic neuropathies, that is, Leber hereditary optic neuropathy and dominant optic atrophy, selectively affect retinal ganglion cells, causing visual loss with relatively preserved pupillary light reflex. The mammalian eye contains a light detection system based on a subset of retinal ganglion cells containing the photopigment melanopsin. These cells give origin to the retinohypothalamic tract and support the non-image-forming visual functions of the eye, which include the photoentrainment of circadian rhythms, light-induced suppression of melatonin secretion and pupillary light reflex. We studied the integrity of the retinohypothalamic tract in five patients with Leber hereditary optic neuropathy, in four with dominant optic atrophy and in nine controls by testing the light-induced suppression of nocturnal melatonin secretion. This response was maintained in optic neuropathy subjects as in controls, indicating that the retinohypothalamic tract is sufficiently preserved to drive light information detected by melanopsin retinal ganglion cells. We then investigated the histology of post-mortem eyes from two patients with Leber hereditary optic neuropathy and one case with dominant optic atrophy, compared with three age-matched controls. On these retinas, melanopsin retinal ganglion cells were characterized by immunohistochemistry and their number and distribution evaluated by a new protocol. In control retinas, we show that melanopsin retinal ganglion cells are lost with age and are more represented in the parafoveal region. In patients, we demonstrate a relative sparing of these cells compared with the massive loss of total retinal ganglion cells, even in the most affected areas of the retina. Our results demonstrate that melanopsin retinal ganglion cells resist neurodegeneration due to mitochondrial dysfunction and maintain non-image-forming functions of the eye in these visually impaired patients. We also show that in normal human retinas, these cells are more concentrated around the fovea and are lost with ageing. The current results provide a plausible explanation for the preservation of pupillary light reaction despite profound visual loss in patients with mitochondrial optic neuropathy, revealing the robustness of melanopsin retinal ganglion cells to a metabolic insult and opening the question of mechanisms that might protect these cells.

Increased peripheral metabolism of cortisol may explain compensatory ACTH-dependent adrenal steroidogenesis and hence hyperandrogenism in polycystic ovary syndrome (PCOS). Previous studies have described an increased 5alpha-reduction of cortisol or impaired regeneration of cortisol by 11beta-HSD1 in PCOS. However, these observations may be confounded by obesity. Moreover, the relationship between alterations in cortisol metabolism and the extent of adrenal androgen hyper-secretion in response to ACTH has not been established. This study aimed to examine the association between cortisol metabolism and ACTH-dependent adrenal hyperandrogenism in PCOS, independently of obesity.We compared 90 PCOS women (age 18-45 yr) stratified by adrenal androgen responses to ACTH1-24 and 45 controls matched for age and body weight.PCOS women were stratified as normal responders (NR), intermediate responders (IR), and high responders (HR) to 250 microg ACTH1-24: NR (no.=27) had androstenedione and DHEA responses within 2 SD of the mean in controls; IR (no.=43) had DHEA responses2 SD above controls; HR (no.=20) had both androstenedione and DHEA responses2 SD above controls.All groups were similar for age, body weight, and body fat distribution. Basal testosterone, androstenedione, and 5alpha-dihydrotestosterone plasma levels were similarly elevated among the 3 groups of PCOS compared with controls, whereas basal DHEA-S was higher in HR (2.8+/-1.2 microg/ml) and IR (2.4+/-1.1 microg/ml) than in NR (1.8+/-0.8 microg/ml) and controls (1.7+/-0.6 microg/ml). The HR group had the lowest basal plasma cortisol levels (101+/-36 ng/ml vs IR 135+/-42 ng/ml, NR 144+/-48 ng/ml, and controls 165+/-48 ng/ml; all p0.01), but the greatest cortisol response to ACTH1-24 (Delta(60-0)cortisol 173+/-60 ng/ml vs IR 136+/-51 ng/ml, NR 114+/-50 ng/ml, and controls 127+/-50 ng/ml; all p0.01), and the highest urinary excretion of total and 5beta-reduced cortisol metabolites (eg 5beta-tetrahydrocortisol/ cortisol ratio 25.2+/-15.3 vs IR 18.8+/-10.7, NR 19.7+/-11.4, and controls 17.2+/-13.7; all p0.05). There were no differences in urinary excretion of 5alpha-reduced cortisol metabolites or in 5alpha-dihydrotestosterone/testosterone ratio between groups.Adrenal androgen excess in PCOS is associated with increased inactivation of cortisol by 5beta-reductase that may lower cortisol blood levels and stimulate ACTH-dependent steroidogenesis.

The present study was conducted in 15 essential hypertensives to evaluate the modifications of plasma levels of an endogenous Na/K ATPase inhibitor, blood pressure, forearm hemodynamics and plasma renin activity (PRA) elicited by an intravenous saline infusion (0.9% NaCl at the mean rate of 0.22 mL/min/kg body weight for 2 h). The response to saline was determined in the whole hypertensive population as well as in two subgroups of patients classified according to their rate of PRA suppression in response to volume expansion by comparison with normotensive controls (Normal- and Low-suppressors: N-S, L-S). Over the whole group of hypertensive patients, NaCl load provoked an increase in Na/K ATPase inhibitory activity, measured by enzyme-coupled assay, which was linearly related to PRA decline (r = 0.73) and to the increase in mean blood pressure (r = 0.57). These effects were clearly enhanced by considering L-S patients alone. Urinary Na/K ratio after saline infusion was significantly higher in L-S as result of a lesser potassium excretion in this subgroup. Our results support the hypothesis that acute volume expansion with saline causes an increase in plasma levels of an endogenous sodium pump inhibitor with hemodynamic effects and whose release is related to the individual handling of infused fluids and to the degree of renin-angiotensin-aldosterone suppression.

Increasing experimental evidences suggest an involvement of an endogenous Na + K + ATPase inhibitor in regulating water and electrolytes balance as well as in the pathogenesis of hypertension. However, conflicting results on the nature and the chemical structure of this substance still make it difficult to understand exactly its physiological mechanism of action. In the present study an attempt was made to purify a Na + K + ATPase inhibitor from hypertensives' plasma by solid phase extraction followed by 2 HPLC steps using reverse and normal phase columns. The fractions, from both columns, were able to inhibit Na + K + ATPase, 3H-ouabain binding to enzyme, ouabain sensitive 86Rb uptake and pNPPase activity in a manner not affected by boiling. Ultrafiltration experiments demonstrate that inhibitory activity is largely due to a low-molecular weight substance. These findings seem to confirm the presence in hypertensives plasma of a Na + K + ATPase inhibitor with some similarities with ouabain.

In a search for the role of long-term hypocaloric feeding on the expression of the erythrocyte Na pump in obesity, we examined three groups of subjects. Group 1 consisted of 10 obese subjects who had been under treatment for a long period of time with a very-low-calorie diet (500 kcal/d) while group 2 consisted of 10 age-, sex-, and body mass index-matched obese subjects on their usual diet; in the third group, 12 normal-weight subjects on a free diet served as controls. There was no difference between the groups in the number of erythrocyte binding sites per cell. On the contrary, the Na-K-ATPase activity was significantly lower in the obese group 1 (0.35 +/- 0.09 mumol Pi x mg protein-1 x h-1) compared to that observed in the obese group 2 (0.42 +/- 0.07, P less than .05) and in control subjects (0.45 +/- 0.06, P less than .05). Sex, duration of hypocaloric feeding, and the amount of weight loss before the study in the obese group 1 seemed not to be related to the Na pump parameters. We conclude that long-term severe hypocaloric feeding may be a factor in altered erythrocyte Na-K-ATPase in obese individuals.

Cardiopulmonary receptors influence renin release in a variety of physiological situations and in a fashion related to the degree of peripheral venous distensibility. We studied two groups of borderline hypertensives (BHTs) with different capacities to suppress plasma renin activity in response to saline infusion (0.20 mL/kg/per minute for 2 hours). Those BHTs with low suppressive capacity (L-supp) showed an increased venous distensibility in comparison with those with high suppressive capacity (H-supp). Saline infusion led to a significant increase in blood pressure only in L-supp BHTs, which was associated with enhanced 24-hour postloading natriuresis and raised plasma levels of an Na/K ATPase inhibitor (+12.2%). This result underlines the importance of venous distensibility as a determinant of pressor and humoral response to acute volume expansion.

Background: Infusion of lipid emulsions rich in polyunsaturated fatty acids (PUFAs) may increase lipid peroxidation, which is counteracted mainly by superoxide dismutase (SOD) (a zinc-, copper-, and manganese-dependent enzyme), selenium-dependent glutathione peroxidase (Se-GSHPx), and α-tocopherol. Objective: We investigated lipid peroxidation and antioxidant status in patients receiving home parenteral nutrition (HPN) providing variable amounts of a lipid emulsion rich in PUFAs, and α-tocopherol, zinc, copper, and manganese as recommended by the American Medical Association, and no selenium. Design: Serum malondialdehyde, plasma α-tocopherol, selenium, Se-GSHPx, PUFAs, and red blood cell Se-GSHPx and SOD were evaluated in 12 patients and in 25 healthy control subjects. Malondialdehyde was also assessed in a group of 40 healthy control subjects. Results: Patients had significantly higher concentrations of malondialdehyde and SOD and lower α-tocopherol concentrations and selenium nutritional status. Linear regression analysis showed that malondialdehyde was associated with the daily PUFA load (r = 0.69, P< 0.03) and with plasma α-tocopherol (r = -0.59, P< 0.05), but stepwise multiple regression analysis confirmed only the association between malondialdehyde and α-tocopherol; plasma α-tocopherol was associated with the daily PUFA load (r = -0.65, P< 0.04) and with the duration of HPN (r = -0.74, P< 0.02). Conclusions: In HPN patients, the peroxidative stress due to lipid emulsions rich in PUFAs is counteracted primarily by α-tocopherol. The dosages of α-tocopherol, zinc, copper, and manganese recommended by the American Medical Association appear sufficient to sustain SOD activity but inadequate to maintain α-tocopherol nutritional status. HPN formulations should be supplemented with selenium.