Faecal calprotectin (FC) has been proposed to be a biomarker of gastrointestinal (GI) disease in systemic sclerosis (SSc). The purpose of this study was to extend cross-sectional observations and prospectively assess the variability of FC over time in SSc patients. We also aimed to examine FC in relation to immunosuppressive therapy. Finally we wanted to analyse FC in other rheumatic diseases to evaluate the specificity of FC for SSc GI disease.FC was measured in consecutive patients with SSc, primary Sjögren’s syndrome (pSS), rheumatoid arthritis (RA) and in healthy hospital workers. The intraindividual variability of FC in SSc was assessed with intra class correlation (ICC) and κ statistics. Associations between FC and objective markers of GI disease and immunosuppressive medication were investigated.FC was associated with micronutrient deficiency and GI pathology as assessed by cineradiography confirming our previous results. FC showed only a limited intra-individual variation in SSc, ICC = 0.69  and κ = 0.64 (95% CI: 0.56-0.73). Generalised immunosuppression did not have any significant impact on FC. FC was significantly higher in SSc patients compared to patients with pSS or RA as well as compared to healthy subjects.FC is a promising non-invasive biomarker for GI disease in SSc. In view of stable levels over time, FC could be a useful marker when novel, more specific drugs targeting the GI tract in SSc will be introduced. Faecal calprotectin (FC) is a validated non-invasive biomarker in inflammatory bowel disease (IBD),2. RecenGI manifestations of SSc are common and a major concern in this disease. Recent Symptomatic treatment of several GI manifestations of SSc is available, but current treatment regimens are insufficient,11. One Calprotectin, also known as s100A8/A9, MRP8/14 or calgranulin A/B, is a heterodimer composed of two small proteins. It constitutes a major (40% to 60%) part of the cytosolic proteins in neutrophils and is also present in other myeloid cells. Calprotectin is released from leukocytes both upon activation and cell death and levels of FC correlate to leukocyte influx in the GI lumen,14. The The primary aim of the present study was to extend our cross-sectional study and prospectively assess the variability over time of FC in SSc patients. A second aim was to monitor FC in relation to conventional immunosuppressive therapy in SSc. A third aim was to examine FC in other rheumatic diseases, that is, rheumatoid arthritis (RA) and primary Sjögren’s syndrome (pSS) in order to evaluate the specificity of pathological FC testing for SSc GI disease in a rheumatological setting. For comparison, FC was also assessed in healthy hospital workers.This study was conducted at the Rheumatology Clinic, Skåne University Hospital, Sweden, which is a referral centre for SSc. All patConsecutive patients with pSS according to the American-European Consensus Classification Criteria, visitingConsecutive patients with RA, positive for rheumatoid factor or anti-cyclic citrullinated peptide antibodies, awaiting either initiation of anti-TNF-α-therapy or orthopaedic surgery at the Rheumatology Clinic, Skåne University Hospital Lund, were invited to participate in this study as a second reference group to the SSc cohort.Patients with a history of IBD, GI malignancy, diverticulitis or alcohol abuse were excluded.Finally, healthy hospital workers, not currently treated with non-steroidal anti-inflammatory drugs (NSAIDs) or proton pump inhibitors (PPI) were asked to participate in this study.Sex and age at time of stool delivery were recorded for all subjects.From all SSc patients, the following data were collected: disease duration (defined as years since debut of first non-Raynaud’s manifestation), autoantibody profile and results from cineradiography. This examination has been proposed to be a core set variable in the clinical evaluation of the GI tract in SSc. CineradAs we have previously studied FC in relation to the modified Rodnan skin score, autoantibody status and disease subtype without any associations identified, these issues were not further investigated.All pSS patients were categorised according to the European League against Rheumatism (EULAR) Sjögren’s Disease Activity Index (ESSDAI) and the Disease Activity Score 28 based on C-reactive protein (CRP) and consequently, and three variables (DAS28-CRP (3)) was calculated for all RA patients,23.FC was measured in stool samples with a commercially available ELISA using a monoclonal antibody . The lower detection limit was 30 μg/g. Analyses were done at the Department of Immunology, Skåne University Hospital, Lund.Different cut-off levels of FC indicating organic disease have been suggested. Two cut-off levels were used in this study. Based on the majority of reports where the current ELISA has been used, FC levels above 50 μg/g were considered pathological,16,24. B12, zinc and folic acid. Subjects with pathological testing according to local laboratory standards were regarded as micronutrient deficient. Subjects with an iron/total iron-binding capacity ratio <0.16 were considered to be iron deficient[All SSc patients were subject to analysis of the following micronutrient deficiencies by laboratory examination of serum or plasma: iron, vitamin Beficient. PatientThe study was approved by the Lund University Ethics Committee, Lund, Sweden. All patients and healthy volunteers gave their written permission according to the Declaration of Helsinki to be included in the study.P-value of 0.05 was considered significant and 95% confidence intervals (CI) given when possible. Because of the interdependence between different variables analysed in this study, we chose not to use Bonferroni correction. All analyses were made using IBM SPSS Statistics version 20 .As previously described, the distribution of the log-transformed FC values allowed parametric statistics. StudentIn total, 93 SSc patients had FC measured at least twice during the study period. Of these patients, FC had been measured three times in 57 patients and at least four times in 27 patients. The main characteristics of the patients involved in this study are presented in Tables P = 0.010), Figure P = 0.001), Figure Eighty-three subjects had conclusive cineradiographies. Of these, patients with a pathological investigation had higher levels of FC  time span between the first (FC1) and the second (FC2) stool delivery was 369 (318 to 725) days. We saw only limited variation in FC over time . This was also true for patients with disease duration less than 400 days (n = 15) at time point 1, where no increase in FC could be seen over a median (IQR) time period of 358 (229 to 385) days . Notably, however, 11/15 of these early patients had pathological FC levels already at baseline.The consistency between FC1 and FC2 was good both when all patients were included, ICC = 0.69 (95% CI: 0.57 to 0.78), and when cases <30 μg/g were excluded, ICC = 0.62 (95% CI: 0.46 to 0.74). We saw The consistency and reliability of FC was not superior in patients with a short timespan compared to patients with a long timespan between FC1 and FC2 (data not shown).A separate analysis of patients who had FC measured at least three times further indicated that patients with normal, pathological or substantially elevated FC levels were also likely to show a similar FC result upon repeated testing Table .During the study, eight patients had FC measured both before and after initiation of immunosuppressive therapy. Median (IQR) disease duration at FC1 was 295 (156 to 575) days and the second FC was delivered 339 (190 to 434) days later. Immunosuppression was initiated because of interstitial lung disease, extensive skin fibrosis, myositis, arthritis, serositis or any combination of these manifestations, but not because of gastrointestinal disease. Four patients started immunosuppression with azathioprine, three with mycophenolate mofetil and one with cyclophosphamide.P = 0.243).FC was not different before compared to after initiation of immunosuppressive therapy .During the study, 15 patients delivered FC both before and after cessation of immunosuppressive therapy. The majority of the patients had been prescribed immunosuppressants because of interstitial lung disease and no one because of gastrointestinal disease. A majority of the patients (9/15) had been prescribed mycophenolate mofetil, four had been prescribed azathioprine and two cyclophosphamide. FC was not different before compared to after cessation of immunosuppression .Twenty-six SSc subjects were taking glucocorticoids at FC1; no relationship between FC and glucocorticoid intake was noted .FC was measured in patients with pSS n = 44) and RA (n = 24), and healthy controls n = 21) (Table  (Table 4 and RA P = 0.019). Furthermore, FC correlated with ESSDAI in these patients . Subgroup analysis of the different ESSDAI-components did not indicate any specific domain score of the ESSDAI to be solely responsible for this correlation. There was a weak correlation between FC and erythrocyte sedimentation rate , but no correlation between FC and immunoglobulin G (IgG) . There was no significant difference in FC between users and non-users of NSAIDs or PPI . Finally, among the pSS patients, FC did not correlate with glucocorticoid intake .Of note, patients with pSS had significantly higher FC values than healthy controls  and did not correlate with disease activity measured as DAS28-CRP(3) . There was a significant correlation between FC and glucocorticoid intake , in contrast to patients with pSS or SSc. RA subjects taking NSAIDs or PPI did not have significantly higher FC levels compared to non-users .In RA, FC was not significantly higher than in healthy controls : Disease Activity Score 28 based on CRP and 3 variables; ELISA: enzyme-linked immunosorbent assay; ESSDAI: EULAR Disease Activity Index; FC: faecal calprotectin; GI: gastrointestinal; IBD: inflammatory bowel disease; ICC: intra class correlation; IQR: interquartile range; PPI: proton pump inhibitor; pSS: primary Sjögren’s syndrome; RA: rheumatoid arthritis; SSc: systemic sclerosis; TNF-α: tumor necrosis factor-alpha.The authors declare that they have no competing interests.Conception and design: KA, TS, TM, RH. Acquisition of data: all authors. Analysis and interpretation of data: all authors. Drafting of article: KA Reviewing for intellectual content: TS, TM, IB, AS, RH. All authors read and approved the final manuscript.


In vitro and rodent studies have shown that arsenic (As) exposure can deplete glutathione (GSH) and induce oxidative stress. GSH is the primary intracellular antioxidant; it donates an electron to reactive oxygen species, thus producing glutathione disulfide (GSSG). Cysteine (Cys) and cystine (CySS) are the predominant thiol/disulfide redox couple found in human plasma. Arsenic, GSH, and Cys are linked in several ways: a) GSH is synthesized via the transsulfuration pathway, and Cys is the rate-limiting substrate; b) intermediates of the methionine cycle regulate both the transsulfuration pathway and As methylation; c) GSH serves as the electron donor for reduction of arsenate to arsenite; and d) As has a high affinity for sulfhydryl groups and therefore binds to GSH and Cys.Background: Objectives: We tested the hypothesis that As exposure is associated with decreases in GSH and Cys and increases in GSSG and CySS .n = 76), 10–100 (n = 104), 101–200 (n = 86), 201–300 (n = 67), and > 300 µg/L (n = 45). Concentrations of GSH, GSSG, Cys, and CySS were measured using HPLC.Methods: For this cross-sectional study, the Folate and Oxidative Stress Study, we recruited a total of 378 participants from each of five water As concentration categories: < 10 (Results: An interquartile range (IQR) increase in water As was negatively associated with blood GSH  and plasma CySS . We observed similar associations with urine and blood As. There were no significant associations between As exposure and blood GSSG or plasma Cys.Conclusions: The observed associations are consistent with the hypothesis that As may influence concentrations of GSH and other nonprotein sulfhydryls through binding and irreversible loss in bile and/or possibly in urine.http://dx.doi.org/10.1289/ehp.1205727Citation: Hall MN, Niedzwiecki M, Liu X, Harper KN, Alam S, Slavkovich V, Ilievski V, Levy D, Siddique AB, Parvez F, Mey JL, van Geen A, Graziano J, Gamble MV. 2013. Chronic arsenic exposure and blood glutathione and glutathione disulfide concentrations in Bangladeshi adults. Environ Health Perspect 121:1068–1074;  Arsenic (As) is a class I human carcinogen that causes several types of cancer . ChronicV), and then to dimethylarsinic acid (DMAsV)  undergoes methylation via nutrient-dependent one-carbon metabolism, first to monomethylarsonic acid (MMAs (DMAsV) . Arsenicdiseases . The prediseases .V) to arsenite (AsIII) (Glutathione (GSH), a tripeptide consisting of cysteine (Cys), glutamic acid, and glycine, is a critical component of the body’s primary antioxidant defense mechanism . GSH als (AsIII) . GSH can (AsIII) . Detoxif (AsIII) . Cells c (AsIII) .h) of the GSH/GSSG and Cys/CySS thiol/disulfide pairs may influence redox-sensitive signaling pathways and enzymes . Alternatively, homocysteine can be directed to the transsulfuration pathway and used in the synthesis of GSH  inducing the formation of ROS, which are then detoxified by GSH; b) consuming GSH during the reduction of AsV to AsIII (c) forming As/GSH complexes, which are substrates for the ATP-binding cassette membrane transporters that mediate efflux from cells (d) inhibiting glutathione reductase, thereby limiting the regeneration of GSH from GSSG  formom cells ; and d) rom GSSG . In vitrlete GSH . Thus, tlete GSH , additioWe conducted a cross-sectional study of 378 Bangladeshi adults to test the primary hypothesis that chronic As exposure is associated with reductions in GSH and increases in GSSG. We also wished to test the secondary hypothesis that chronic As exposure is associated with reductions in plasma Cys and increases in CySS.Participants and procedures. Participants in the current study, the Folate and Oxidative Stress (FOX) Study, were recruited between February and July of 2008 in Araihazar, Bangladesh. Potential participants were identified on the basis of well water As (wAs) concentrations obtained from a well survey in the year 2000 (a) were between 30 and 65 years of age; b) were not pregnant; c) were not taking nutritional supplements; d) did not have known diabetes, cardiovascular or renal disease, chronic obstructive pulmonary disease, or cancer; and e) had been drinking water from their current well for at least 3 months. Trained recruiters identified eligible participants, explained the nature of the study, obtained informed consent, and scheduled a field clinic visit. Because GSH is unstable and therefore requires that blood samples be processed immediately, all visits were conducted in the laboratory at our field clinic in Araihazar. During the field clinic visit, a trained interviewer administered a detailed questionnaire to each participant, and a physician collected a venous blood sample. Urine samples were collected in 50-mL acid-washed polypropylene tubes and frozen at –20°C.ear 2000  in ordern = 76), 10–100 µg/L (n = 104), 101–200 µg/L (n = 86), 201–300 µg/L (n = 67), and > 300 µg/L (n = 45).The primary aim of this study was to examine the dose–response relationship between As exposure and measures of oxidative stress. We aimed to recruit 75 participants each from five wAs concentration categories: < 10, 10–100, 101–200, 201–300, and > 300 µg/L. However, the final sample included more particpants with lower exposures because many households switched to lower-As wells after wells in the region were surveyed for As in 1999–2000 . TherefoOral informed consent was obtained by our Bangladeshi field staff physicians, who read an approved assent form to the study participants. This study was approved by the Bangladesh Medical Research Council and the institutional review board of Columbia University Medical Center.Sample collection and handling. After the initial processing of blood samples in the field clinic, the blood and plasma aliquots were immediately frozen at –80°C. Samples on dry ice were transported in batches to Dhaka, Bangladesh, by car and again stored at –80°C (blood and plasma) or –20°C (urine). Samples were then packed on dry ice in coolers and transported by air to Columbia University.wAs. Field sample collection and laboratory analysis procedures have been previously described in detail  in the Columbia University Trace Metals Core Laboratory by graphite furnace atomic absorption spectrometry  concentration, as described previously . The samples for plasma measurements were centrifuged for 1 min, and 200 µL of supernatant was transferred into Eppendorf tubes containing an equal volume of 10% perchloric acid and 0.2 M boric acid. For derivatization, plasma samples were centrifuged at 13,000 rpm for 2 min, 300 µL of supernatant was transferred to a fresh tube, and the pH was adjusted to 9.0. After incubating for 20 min at room temperature, dansyl chloride was added, and samples were incubated at room temperature in the dark for 24 hr. The derivatized samples were then stored at –80°C until delivered to Columbia University for analysis. Free dansyl chloride was extracted from thawed samples with 500 µL chloroform, and then 20 µL of the sample was injected onto the HPLC. Separation was achieved using a Supelcosil LC-NH2 column . Initial solvent conditions were 60% A , 40% B  run at 1 mL/min for 10 min. A linear gradient to 20% A, 80% B was run during the 10- to 50-min period. From 50 to 52 min, the conditions were returned to 60% A, 40% B. Metabolites were detected using a Waters 474 scanning fluorescence detector , with 335 nm excitation and 515 nm emission. Within-assay CVs were all between 0.05 and 0.10, and interassay CVs were between 0.11 and 0.18.Plasma folate. Plasma folate was analyzed by radio protein-binding assay . To determine folate concentrations, we used folic acid as pteroylglutamic acid for calibration, and its 125I-labeled analog as the tracer. The intraassay and interassay CVs were 0.06 and 0.14, respectively.Calculation of the reduction potential. The reduction potential (Eh) of the thiol/disulfide GSH/GSSG and Cys/CySS redox pairs  were calculated using the Nernst equation:Eh = Eo + RT/nF ln(acceptor/(donor)2,Eo is the standard potential for the redox couple, R is the gas constant, T is the absolute temperature, n = 2 for the number of electrons transferred, and F is Faraday’s constant (where constant . For GSHEh (mV) = −264 + 30 log[(GSSG)/(GSH2)],Eo value assumes a physiologic pH of 7.4. A more positive Eh value indicates a more oxidized redox state.where (GSH) and (GSSG) are molar concentrations, and the Statistical methods. We calculated descriptive statistics for characteristics of the study sample, As exposure variables , and outcome variables , both for the total sample and by sex. Bivariate associations were examined using scatter plots and Spearman’s correlation coefficients. To examine the bivariate associations between dichotomous covariates and As exposure variables or continuous outcome variables we used t-tests or the nonparametric Wilcoxon rank sum test.R2 between models for each outcome that included covariates only and corresponding models that included both the covariates and As exposure.We used linear regression models to further examine the associations between As exposure variables, as continuous variables, and the outcome variables, with and without adjustment for potential confounders. Age and sex were included in all covariate-adjusted regression models. Other covariates considered for inclusion in the regression models were variables reported to be associated with the exposures or outcomes based on previous publications and/or variables associated with the exposure and outcome variables in the present study population. These variables included television ownership (as a surrogate for socioeconomic status), cigarette smoking, body mass index (BMI), urinary creatinine, and plasma folate. We adjusted for GSH laboratory batch  in order to reduce extraneous variation in the outcome variables. We also calculated the change in h, plasma CySS, and plasma Cys Eh associated with an interquartile range (IQR) increase in each exposure. For outcome variables that were natural log-transformed (blood GSSG and plasma Cys) we report the ratio of estimated geometric means for an IQR change in As exposure.To facilitate comparisons among the different measures of exposure , we report the estimated change in the mean value of blood GSH, blood GSH ETo examine possible nonlinear relationships, we also created quintiles of As exposure variables and computed covariate-adjusted mean values of the outcome variables for categories of As exposure; plots of quintile-specific adjusted mean values were examined to determine if the association was approximately linear.We ran separate linear regression models to examine the covariate-adjusted associations between As exposure and the outcome variables stratified by sex or by folate status. We then used a Wald test to detect differences in the covariate-adjusted associations between As exposure and outcome variables by sex or by folate status. All analyses were performed using SAS ; all statistical tests were two-sided with a significance level of 0.05.a) had more years of formal education ; b) were more likely to report being ever-smokers ; c) were more likely to have a BMI < 18.5 ; d) were more likely to have plasma folate concentrations < 9 nmol/L , which is indicative of marginal folate status; and e) were more likely to have total homocysteine concentrations above the cutoff for hyperhomocysteinemia of 13 µmol/L . We observed other differences between the sexes that were specific to the present study sample: The males were older than the females  and had higher bAs concentrations . Blood GSH Eh and plasma Cys Eh also differed significantly by sex; median blood GSH Eh values for females indicated a more oxidized intracellular redox state compared with males , whereas median plasma Cys Eh values for males indicated a more oxidized extracellular redox state compared with females .The general characteristics of the study sample are shown in h (indicating a less oxidized redox state) than did females. Ever-smokers also had significantly higher bAs than never-smokers. Contrary to expectation, ever-smokers had higher blood GSH concentrations and lower blood GSH Eh than never-smokers. However, most smokers were males, and after stratification by sex, there were no statistically significant difference in blood GSH and blood GSH Eh by smoking status (data not shown). Participants who owned a television had lower wAs and uAs concentrations and higher blood GSH, blood GSSG, and plasma CySS concentrations than participants who did not own a television.Several covariates were associated with As exposure variables and/or outcome variables. All three measures of As exposure were negatively associated with BMI . Plasma folate was positively associated with blood GSSG. Age, BMI, and plasma folate were all positively associated with plasma CySS; age was also negatively associated with plasma Cys. We also observed differences by sex, smoking, and television ownership . Males had higher blood GSH and lower plasma Cys and blood GSH Ep = 0.01 for wAs; –54.0 µmol/L , p = 0.004 for uAs; and –33.4 µmol/L , p = 0.03 for bAs] and positively—but not always significantly—associated with blood GSH Eh   for wAs; –3.56 µmol/L  for uAs, and –3.09 µmol/L  for bAs] and negatively associated with plasma Cys Eh , indicating that As exposure is associated with a more reduced extracellular environment. The change in R2 values for plasma CySS models with adjustment for As exposure  were 2.7% for wAs, 1.2% for uAs, and 1.4% for bAs. Given that males and females in this study population differed substantially on several characteristics, all regression models were also run stratified by sex; the directions of associations in males and females were the same as in the overall sample, but in some cases did not reach statistical significance because of the smaller sample sizes (data not shown). We also examined the possibility that associations might differ by folate status  given the influence of folate on As methylation . In models using continuous As exposure variables, all three markers of As exposure were negatively associated with blood GSH  . The chatrations . In unadhylation . We obsea priori hypothesis, As exposure was not positively associated with blood GSSG, the oxidized form of GSH. Also, contrary to our secondary hypotheses, we observed no association between As and plasma Cys, and we saw a negative—rather than positive—association with plasma CySS.In this cross-sectional study of Bangladeshi adults chronically exposed to a wide range of As concentrations in drinking water, we observed that three different measures of As exposure  were all negatively associated with blood GSH. Contrary to our primary To our knowledge, only one other population-based study has examined the association between As exposure and GSH concentrations. ad libitum had elevated hepatic GSH after 2 months and decreased hepatic GSH after 4 months compared with mice administered As-free water. In male Wistar rats, Our findings and those of in vitro and animal model studies have reported that As exposure led to an increase in the production of ROS and a decline in GSH  and plasma CySS (1.2–2.7%). Our findings, as well as the inconsistent findings from other population-based studies examining associations between As exposure and blood markers of oxidative damage , raise tS-adenosylhomocysteine and homocysteine. In addition, As exposure increased the expression of cystathionine β-synthase, the enzyme that catalyzes the first step in GSH production via the transsulfuration pathway, and increased cellular reduced GSH by 5-fold , and an individual’s response to As might depend on their background level of oxidative stress. A mathematical model of one-carbon metabolism developed by We observed that As exposure was negatively associated with blood GSH and plasma CySS in a population chronically exposed to a wide range of As concentrations in drinking water. These findings, and the lack of association between As exposure and blood GSSG or plasma Cys, suggest that As may exert its greatest influence on GSH through binding and irreversible loss in bile and/or possibly in urine. Our findings do not lend strong support to the hypothesis that As induces levels of oxidative stress that are measurable in blood using these biomarkers and at these environmentally relevant exposures. However, our findings cannot rule out the possibility of As-induced oxidative stress in tissue or cellular compartments that we were unable to measure in healthy human volunteers.In the manuscript originally published online, Abu B. Siddique’s name was listed incorrectly. It has been corrected here.(532 KB) PDFClick here for additional data file.


We present the diagnostic approach of a patient with adrenal incidentalomas. A 72-year-old African American male had a CT scan of the abdomen showing right and left adrenal masses measuring 5 × 3.5 cm and 3.7 × 2.9 cm, respectively. The patient had negative hormonal workup. The radiologist insisted that the CT findings are consistent with adrenal hyperplasia, and therefore he underwent ACTH stimulation to rule out late-onset congenital adrenal hyperplasia (CAH). The stimulation test revealed that 17-hydroxyprogesterone and 11-deoxycortisol increased to levels high enough to confirm CAH, but cortisol had exaggerated response as well, thus making the diagnosis of CAH unlikely where metabolism is shifted to precursors. Subsequently, the patient underwent screening for Cushing's syndrome (CS) with a dexamethasone suppression test. Patient failed the suppresion test, raising the issue for subclinical CS (SCS), likely due to ACTH-independent macronodular adrenal hyperplasia. Our patient had been diagnosed with MGUS and so far there are only 3 case reports of extramedullary plasmacytoma arising from the adrenals. One was bilateral and one had functional abnormalities. Our differential diagnosis includes subclinical CS with aberrant receptors versus a functioning extramedullary plasmacytoma.  Adrenal incidentalomas are very common nowadays, and subclinical autonomous cortisol hypersecretion is the most frequent hormonal abnormality and it may be intermittent. This is why there is a need for periodic hormonal and morphological evaluation for several years . Also, tThe patient is a 72-year-old African-American male. After complaining of chronic back pain in 9/2009, he had a spine MRI that revealed arachnoiditis and prominence of bilateral adrenals. CT scan of abdomen/pelvis was done to further evaluate the adrenals that revealed lipid-rich adrenal masses bilaterally, right adrenal mass 5 × 3.5 cm, and left adrenal 3.7 × 2.9 cm . Both th Besides his chronic back pain, the patient had no other complaints. He was very adherent to his medication although his blood pressure on occasion was ranging from 150/78 to 160/96. His medications include diltiazem 240 mg daily, lisinopril 10 mg daily, omeprazole 20 mg, aspirin 81 mg, plavix 75 mg, and tramadol 50 mg as needed for pain.It was hypertension, hyperlipidemia, spinal stenosis, monoclonal gammopathy of uncertain significance (MGUS), and coronary artery disease. Patient was never a smoker. He rarely drank a glass of wine at social events and denied any use of illicit drugs or over-the-counter medication. It was breast cancer and colon cancer in one sister and his father, respectively. His mother died of stroke and father of colon cancer. After the CT scan results, the patient was sent to our endocrine clinic for evaluation. On physical exam, the patient has no cushingoid features. He is slightly overweight, with BMI of 26 and no evidence of abnormal skin pigmentation, striae or evidence of proximal muscle weakness. He was screened for depression as part of the standard of care, and the patient health questionnaire (PHQ-9) was negative. He denied obstructive symptoms at night and admits waking up in the morning refreshed from the night sleep. Patient underwent hormonal workup to rule out hormone-producing adrenal tumors three times, including 24-hour urine cortisol level, 24-hour urine metanephrines, serum aldosterone, and plasma renin activity (at that time patient was not on lisinopril), all of which were normal. During followup, CT scans showed the adrenal masses remained stable in size, but the radiologist insisted that the CT findings are consistent with adrenal hyperplasia. In view of the patient also being hypertensive, he underwent an ACTH (cosyntropin) stimulation test to rule out late-onset CAH . In our case the patient's cortisol went from baseline of 419 mmol/L to 1,848.53, 17-hydroxyprogesterone from 0.33 nmol/L to 11.69, and 11-deoxycortisol from 0.5772 mg/L to 10.97. ACTH at baseline was 1.034 pmol/L. CAH is unlikely in this case, since one would expect insufficient production of cortisol and a shift towards the precursors. Our patient had exaggerated response of both cortisol and the precursors. The patient subsequently underwent overnight 1 mg dexamethasone suppression test as screening for CS and also low dose (4 mg) suppression test. Patient failed both suppresion tests with morning cortisol level of 215.2 mmol/L and 204.16 mmol/L, respectively. Normal response should be morning cortisol less than 49.662 mmol/L, according to the 2008 Endocrine Society Guidelines. The above results were consistent with SCS, most likely secondary to aberrant receptors. Thereafter, patient underwent workup to rule out the ectopic expression of glucose-dependent insulinotropic peptide (GIP) receptor in zona fasciculata cells.The postdexamethasone suppression test baseline fasting cortisol level was 215 mmol/L and 2-hours after a mixed meal was 201, essentially ruling out GIP-dependent CS. The full workup for aberrant receptors could not be performed because TRH and GnRH are not available in the USA and the patient started developing chest discomfort, had a positive stress test, and underwent coronary artery catheterization with stenting. The patient is currently recovering well.ACTH (adrenocorticotropic hormone) stimulation test is traditionally used in the diagnosis of adrenal insufficiency and also to diagnose cases of CAH. In CAH where 21-hydroxylase or 11-beta hydroxylase deficiency is common, the ACTH stimulation shifts the steroidogenesis to the precursors . So 17-hydroxyprogesterone concentrations range typically from 10 to 100 nmol/L. Cortisol in healthy individuals should double from baseline and usually ranges from 386.26 to 689.75 mmol/L.Abnormal response to ACTH stimulation test has been shown in patients with major depression . Also, dβ-adrenergic, vasopressin, LH, FSH, TSH, and GIP receptors in the adrenals.As supported by the literature search, supraphysiologic response of cortisol to ACTH stimulus occurs with ACTH-independent CS, also known as ACTH-independent macronodular adrenal hyperplasia (AIMAH). It is ACTH independent because endogenous ACTH levels are low or normal, and cortisol secretion is regulated by hormones other than ACTH via aberrant expression of hormone receptors, like AIMAH accounts for <1% of overt CS. It usually presents in the 5th-6th decade of life as subclinical CS. It can be sporadic or familial . Supraphysiologic response of cortisol to ACTH occurs with AIMAH because the ACTH receptor gene remains expressed. However, there is also relatively inefficient hormonal synthesis with decreased expression of steroidogenic enzymes. The hormone secretion in AIMAH results from an increase in the number of adrenocortical cells rather than an augmented synthesis per cell and this is why steroid precursors may be found elevated –13. Diagnosis of subclinical CS is very challenging and currently there are no guidelines. However, according to some authors, the presence of at least two of the following: abnormal 1 mg dexamethasone suppression test, elevated 24 hr urine cortisol, ACTH below 2.2 pmol/L, constitutes diagnosis . The adrUnfortunately, the workup for aberrant receptors, to confirm ACTH-independent macronodular adrenal hyperplasia, is very tedious and some of the suggested stimuli like TRH or GnRH are not commercially available in the USA. Thus, we were unable to perform the protocol on our patient, but the workup for subclinical Cushing's syndrome was positive and the abnormal response to ACTH stimulation test is suggestive of our diagnosis. The identification of aberrant receptors provides new opportunities in pharmacological therapy as an alternative to adrenalectomy. The patient had negative workup for GIP receptor, but in view of his CAD, we can give a trial of b-blocker in case his aberrant receptors are beta-adrenergic . There iAnother consideration arises because of the patient's previous diagnosis of MGUS. Thus far, in the literature there have been 3 cases of extramedullary plasmacytoma arising from the adrenals. One was actually bilateral and one was a functioning tumor secreting catecholamines –20. Of cSubclinical Cushing's syndrome can be a cause of increased morbidity and mortality and the physician should be vigilant to diagnose and treat when appropriate. Since there is no consensus currently it relies on the clinician's judgment how to manage these challenging cases.


Bariatric surgery is the most effective current treatment for severe obesity. Capacity to perform surgery within Canada’s public health system is limited and potential candidates face protracted wait times. A better understanding of the gaps between demand for surgery and the capacity to provide it is required. The purpose of this study was to quantify and characterize the bariatric surgery-eligible population in Canada in comparison to surgery-ineligible subjects and surgical recipients.Data from adult (age > 20) respondents of the 2007–09 nationally representative Canadian Health Measures Survey (CHMS) were analyzed to estimate the prevalence and characteristics of the surgery-eligible and ineligible populations. Federally mandated administrative healthcare data (2007–08) were used to characterize surgical recipients.2 (95% CI 39.3 to 40.9 kg/m2) and, compared to the surgery-ineligible obese population, were more likely to be female (62 vs. 44%), 40–59 years old (55 vs. 48%), less educated (43 vs. 35%), in the lowest socioeconomic tertile (41 vs. 34%), and inactive (73 vs. 59%). Self-rated mental health and quality of life were lower and comorbidity was higher in surgery-eligible respondents compared with the ineligible populations. The annual proportion of Canadians eligible for surgery that actually underwent a publicly funded bariatric surgery between 2007–09 was 0.1%. Surgical recipients (n = 847) had a mean age of 43.6 years (SD 11.1) and 82% were female. With the exception of type 2 diabetes, obesity-related comorbidity prevalence was much lower in surgical recipients compared to those eligible for surgery.In 2007–09, an estimated 1.5 million obese Canadian adults met eligibility criteria for bariatric surgery. 19.2 million were surgery-ineligible (3.4 million obese and 15.8 million non-obese). Surgery-eligible Canadians had a mean BMI of 40.1 kg/mThe proportion of bariatric surgery-eligible Canadians that undergo publicly funded bariatric surgery is very low. There are notable differences in sociodemographic profiles and prevalence of comorbidities between surgery-eligible subjects and surgical recipients. Furtheractually receive surgery. Furthermore, no prior study has characterized the surgery eligible Canadian population in comparison to the population considered surgery-ineligible and the population currently receiving bariatric surgery. These data would help inform the current state of bariatric care in Canada, facilitate comparisons with other countries, and assist future health care delivery planning. For example, those responsible for setting volume targets for bariatric surgery in Canada can use these data to understand the current surgical volumes and estimate remaining gaps. Comparing similar data from other countries would allow one to rank surgical volumes in Canada against other health care systems and determine if they require adjustment. To address these knowledge gaps, we linked several contemporary and nationally representative surveys and administrative databases to characterize the population eligible for bariatric surgery in Canada and compare them with the patients currently receiving bariatric procedures.High demand, driven primarily by the large number of potentially eligible surgical candidates, is thought to be a major contributor to these lengthy wait times . HoweverData from the Canadian Health Measures Survey  were analyzed to identify subjects meeting guideline-concordant eligibility criteria for bariatric surgery . The CHMA complex, stratified sampling design was used to obtain estimates representative of the Canadian population . The CanBlood pressure (BP) was measured electronically with calibrated BpTRU automated oscillometric devices  using previously described methods . CertifiThe diagnosis of asthma, osteoporosis, dyslipidemia, and mood disorders  was based upon self-report. Respondents were considered hypertensive if they self reported hypertension, were receiving treatment with antihypertensive medication or if the average of five their screening blood pressure measurements were ≥ 140 mm Hg systolic or ≥ 90 mm Hg diastolic. Respondents were considered diabetic if they self reported diabetes, were taking diabetes medications or if their fasting blood glucose level was ≥ 7.0 mmol/L. Blood pressure thresholds to diagnose hypertension in patients with diabetes were systolic BP levels ≥ 130 mm Hg or diastolic BP levels ≥ 80 mm Hg. Last, subjects were asked to rate their mental health and their quality of life as excellent, very good, good, fair or poor .2or a BMI of 35–39.9 kg/m2 and a major obesity-related comorbidity  and, therefore eligible for bariatric surgery (n = 2850) [2), overweight (BMI 25–29.9 kg/m2) and normal/underweight (BMI ≤ 24.9 kg/m2) categories.We examined patients aged 20–60 years who, in accordance with Canadian bariatric guidelines, had a BMI ≥ 40 kg/mThe Canadian Institute for Health Information Discharge Abstract Database (CIHI-DAD) and the National Ambulatory Care Reporting System (NACRS) were used to identify patients who received publicly funded bariatric surgery at a Canadian hospital in fiscal 2007–08 . The CIHI-DAD contains acute-care hospital discharge data for all Canadian provinces excluding Quebec, while NACRS contains information on day surgeries performed across Canada (excluding Quebec and Alberta) ,17. CIHIPatients undergoing bariatric surgery were identified using the CCI code for bariatric procedures (1.NF.78.^^). Patient age and sex, the type of bariatric procedure, obesity-related co-morbidities, length of stay, in-hospital mortality and in-hospital complications were obtained for 2007–08 fiscal year to coincide and overlap with administration of the CMHS to the Canadian population.Analyses were primarily descriptive in nature, consisting of estimates of means and proportions. For CHMS data, population-representative estimates were obtained by applying respondent-specific survey weights  and bootTo estimate the annual proportion of eligible patients actually receiving bariatric surgery between 2007–09, we divided 1695  by the surgery-eligible population estimate derived from CHMS data [2 (95% CI 39.3 to 40.9 kg/m2) and, compared to the surgery-ineligible obese population, were more likely to be female (62 vs. 44%), 40–59 years old (55 vs. 48%), less educated (43 vs. 35%), in the lowest socioeconomic tertile (41 vs. 34%), and inactive (73 vs. 59%). Mean BMI levels were 40.1 kg/m2 for surgery-eligible population (95% CI 39.3 to 40.9) and 32.7 (95% CI 25.7 to 26.5 kg/m2) for the obese surgery-ineligible population. Surgery-eligible Canadians were more likely than obese non-eligible subjects to be female (62 vs. 44%), aged 40–59 years old (55 vs. 48%), less educated (43 vs. 35%), in the lowest socioeconomic tertile (41 vs. 34%), and physically inactive (73 vs. 59%)  from April 1, 2007 to March 31, 2008  preferentially select healthier patients to undergo surgical intervention or related to known under-coding of chronic stable comorbidities that do not impact surgical risk or length of stay . The lacLimitations of our analysis are inherent to the data sources examined and the fact that we compared two different, albeit population-representative, data sources (survey data and administrative data). The CHMS was a voluntary survey and, as with all surveys of this type, the generalizability of the results depends upon the absence of systematic differences between responders and non-responders. Certain obesity-related comorbidities  considered to be indications for bariatric surgery were not captured in the CHMS and some comorbidities were self-reported. For these reasons, the number of surgery-eligible subjects may have been underestimated. Three potential limitations of the administrative CIHI data should be noted. First, BMI is not captured within administrative databases; therefore, we assumed but could not directly verify that surgical programs are following current guideline-concordant eligibility criteria when selecting surgical candidates. Published data from the largest publicly funded Canadian bariatric programs suggest that this assumption is justified -32. SecoIn conclusion, our results quantify and characterize the bariatric eligible population in Canada in relation those considered ineligible and those receiving surgery. These data are useful to depict the current state of bariatric surgery delivery in Canada, thus facilitating comparison to other jurisdictions and serving as a benchmark for ongoing and future initiatives related to bariatric health care delivery in this country. Our findings raise a number of questions regarding the adequacy and appropriateness of publicly funded bariatric surgery care delivery in Canada. In this regard, we note that four of the five founding principles of the Canada Health Act  are not currently being met. These principles are comprehensiveness , public administration (obviating the need for private service delivery), universality , accessibility (in a timely manner), and portability . We propose that future efforts should focus on fully characterizing these care gaps; clarifying the role of privately delivered care; examining the value of prioritization methods to streamline and optimize care; and ensuring accessibility for suitable candidates seeking bariatric surgery.RP, SWK, SM and AMS are supported by an alternative funding plan from the Government of Alberta and the University of Alberta. AMS is supported by an Alberta Health Services Chair in Obesity Research and Management. SWK and SM hold salary support awards from Alberta Heritage Foundation for Medical Research and Alberta Innovates-Health Solutions. The authors declare no competing interest with respect to this work.RP developed the initial study concept, which was refined after input from all of the other authors. HC and RP performed data analysis. RP wrote the initial draft, which was critically revised by all other authors. All authors read and approved the final manuscript.


Drosophila melanogaster as our model organism to address this question.Mitochondria are organelles found in nearly all eukaryotic cells that play a crucial role in cellular survival and function. Mitochondrial function is under the control of nuclear and mitochondrial genomes. While the latter has been the focus of most genetic research, we remain largely ignorant about the nuclear-encoded genomic control of inter-individual variability in mitochondrial function. Here, we used P ≤1 × 10-5). Gene-centered regression models showed that 2–3 SNPs can explain 31, 13, and 18% of the phenotypic variation in state 3, state 4, and P:O ratio, respectively. Most of the genes tagged by the SNPs are involved in organ development, second messenger-mediated signaling pathways, and cytoskeleton remodeling. One of these genes, sallimus (sls), encodes a component of the muscle sarcomere. We confirmed the direct effect of sls on mitochondrial respiration using two viable mutants and their coisogenic wild-type strain. Furthermore, correlation network analysis revealed that sls functions as a transcriptional hub in a co-regulated module associated with mitochondrial respiration and is connected to CG7834, which is predicted to encode a protein with mitochondrial electron transfer flavoprotein activity. This latter finding was also verified in the sls mutants.We quantified mitochondrial state 3 and state 4 respiration rates and P:O ratio in mitochondria isolated from the thoraces of 40 sequenced inbred lines of the Drosophila Genetic Reference Panel. We found significant within-population genetic variability for all mitochondrial traits. Hence, we performed genome-wide association mapping and identified 141 single nucleotide polymorphisms (SNPs) associated with differences in mitochondrial respiration and efficiency (D. melanogaster. The integrative genomic approach used in our study allowed us to identify sls as a novel hub gene responsible for the regulation of mitochondrial respiration in muscle sarcomere and to provide evidence that sls might act via the electron transfer flavoprotein/ubiquinone oxidoreductase complex.Our results provide novel insights into the genetic factors regulating natural variation in mitochondrial function in  A primary role of mitochondria is to utilize oxygen and nutrients to form adenosine triphosphate (ATP) via a process called oxidative phosphorylation (OxPhos) . In addies (ROS)  and physes (ROS) . Howeveres (ROS) . Thus, mes (ROS) .2), which are generated in the Krebs cycle, are fuelled into complexes I (NADH dehydrogenase) and II (succinate dehydrogenase) of the ETC. The electrons are then transferred through the complexes III (cytochrome bc1 oxidoreductase) and IV (cytochrome c oxidase) ultimately reducing oxygen to water, with protons concurrently pumped across the mitochondrial inner membrane in complexes I, III, and IV. This establishes an electrochemical potential difference across the inner membrane and a motive force for protons to re-enter through ATP synthase (complex V). ATP synthase captures the potential energy released upon protons re-entry by converting adenosine diphosphate (ADP) and inorganic phosphate to ATP. In this manner, electron transport is coupled to OxPhos [2 is consumed in the presence of saturating amounts of respiratory substrate and ADP). However, it has been known for several decades that under normal conditions protons leak back through the mitochondrial membrane into the matrix via a mechanism that does not involve ATP synthase [uncouples respiration from OxPhos. Proton leak increases exponentially with the membrane potential (“non-Ohmic” pattern) [2 is consumed in the presence of respiratory substrate and absence of ADP) in isolated mitochondria. Thus, mitochondria in the intact cell would normally respire at a rate somewhere between state 3 and state 4 respiration rates depending on the energy demand, substrate availability, oxygen, ADP availability, and proton leak back into the matrix. The mechanisms that account for proton leak are poorly understood, but phospholipids and fatty acid composition of the mitochondrial inner membrane and the expression and activation of uncoupling proteins (UCPs) are proposed contributors to the leak [The OxPhos system consists of five large multi-protein complexes, four of which (complexes I-IV) make up the ETC . During o OxPhos . In a pesynthase . This unpattern)  and is gthe leak ,10.Drosophila melanogaster as our model organism. We chose D. melanogaster for several reasons. First, this organism has emerged in recent years as a powerful model to elucidate the genomic basis that controls naturally occurring variation in quantitative traits, such as mitochondrial respiration traits [D. melanogaster possesses four genes coding for close relatives of the UCPs. One of the four fly genes (Bmcp) has been shown to be a Drosophila mitochondrial uncoupler of OxPhos [In all Metazoa, the large OxPhos complexes are encoded mainly by nuclear genes and, to a small extent, by mitochondrial genes. The only exception is represented by complex II subunits that are entirely encoded by nuclear genes . Given tn traits . Second,n traits -18, withn traits ,20. Thirf OxPhos . Finallyf OxPhos -25. ThusD. melanogaster genomics resource [D. simulans flies [D. melanogaster is also particularly amenable for studying the nuclear-encoded genomic control of naturally occurring variation in mitochondrial function since previous population studies have shown that the level of naturally occurring variation in the mtDNA of D. melanogaster is low compared to other Drosophila species [D. melanogaster have been reported to be the least diverse with a single dominant haplogroup [First, we investigated whether there is variability in mitochondrial respiration and coupling efficiency among 40 inbred, sequenced lines of the Drosophila Genetic Reference Panel (DGRP), a newly established resource . This waresource . Previouns flies ,29. Howens flies . Also, ins flies . Additions flies . Hence,  species ,34. Westsls as a transcription regulator of mitochondrial respiration in D. melanogaster. The product of sls is a protein with homology to the NH2-terminal half of vertebrate titin [The 40 DGRP lines were previously quantified for transcript abundance  and theite titin . As in mte titin . Our resWe found significant differences in the function of thoracic mitochondria isolated from the 40 DGRP lines Figure . Our resD. simulans flies under similar mitochondrial respiration conditions used in our study. Furthermore, studies in mammalian models reported that female rodents had higher mitochondrial oxidative capacity and efficiency for substrate oxidation across several tissues [X chromosome spend relatively more time under selection in females due to their asymmetric inheritance [The sex-related differences in mitochondrial function observed in our study are consistent with the well-recognized differences between males and females in the control of substrate metabolism and energy homeostasis that occur across different species . Also co tissues -43. The  tissues . Througheritance -46 and aeritance . Since feritance .Variation in mitochondrial respiration can be influenced by differences in mitochondrial density. To address this issue, we measured the activity of the marker enzyme citrate synthase (CS) in a panel of eight of the 40 inbred wild-type lines. The eight lines were selected to represent the range of variability in mitochondrial respiration rates and coupling efficiency seen in our sample. We measured CS activity in whole-fly homogenates and isolated mitochondria and calculated the mitochondrial protein density from the ratio between the CS activity of the whole-fly homogenates and that of the isolated mitochondria as described in . Only onP = 0.584) . Given that in our study phenotypic correlations are mainly estimates of their genetic component, the results suggest that different genes regulate adaptive variation in these two traits. However, the P:O ratio was negatively correlated with state 3 in the sex-pooled analysis (P = 0.005)   in the analysis averaged across sexes and in males (P = 0.01). State 4 was also negatively correlated with copulation latency in females (P = 0.017). Furthermore, we found that state 3 was positively associated with glycogen levels in females (P = 0.03) and P:O ratio with body weight in males (P = 0.009). Although the correlations were not significant after Bonferroni correction for multiple tests (significance threshold: P < 0.003), they are in agreement with the physiological relationship among mitochondrial efficiency, nutrient availability, cell proliferation and death, and ROS production reported in mammals [Next, we asked whether there was a phenotypic correlation between mitochondrial respiratory rates and coupling efficiency among the 40 lines. We did not observe any correlation between state 3 and state 4 in both the sex-pooled analysis  Figure D and the mammals , and hig mammals . Over 90 mammals . The mag mammals . A balan mammals . Additio mammals ,56, whic mammals . But in P ≤1 × 10-5  is associated with variation in state 3 and P:O ratio, respectively. For the GWA analyses, we used 1,312,183 SNPs with a minor allele frequency of at least 10%. The analysis averaged across sexes detected 40, 6, and 25 SNPs associated with variation in state 3, state 4, and P:O ratio, respectively, at a  insects . Consistelopment , suggestand cGMP . Althougand cGMP ,60) and and cGMP  suggestiand cGMP . The reg oxidase ,61. Simi oxidase . Our ana-proteins [dopamine receptor 2, tyramine β hydroxylase , which represent the largest group of integral membrane proteins involved in signal transduction and exert a wide variety of biological functions, including neurotransmission, photoreception, chemoreception, metabolism, and cell differentiation and migration ,64. GPCRproteins . Additioproteins . For exaproteins , the mosproteins . Interestopamine ), locomoproteins ), star , alphab pathway ), still activity ), and RhSNF4/AMP-activated protein kinase gamma subunit and variation in mitochondrial state 4 respiration rates  causes AMPK activation in flies [SNF4/AMP-activated protein kinase gamma subunit locus may be maintained as part of a mechanism controlling the production of ROS and therefore their signaling activities [Another intriguing finding is the association between an intronic variant in the in flies . Furtherin flies ,72. Overtivities . Additioper se, as well as fitness and several life-history traits within and across populations [Together, the above observations indicate that there is great utility in using the DGRP as a primary screen for follow-up testing of mutants of focal genes. Also, a notable result of our GWA study is that none of the nuclear genes encoding the subunits of the ETC enzyme complexes or the components required for the assembly and function of the respiratory chain was depicted by the SNPs that showed statistically significant association with the mitochondrial phenotypes. A growing body of evidence indicates that epistatic interactions between natural genetic variants in the mitochondrial and nuclear genomes affect mitochondrial function ulations ,29,74-77sls as a novel candidate gene influencing inter-individual variability in state 3 respiration rate  , one (synonymous coding), and two (intronic and splice site intronic) were independently associated with state 3, state 4, and P:O ratio, respectively, at a nominal P ≤1 × 10-4   insertion into the exon 6 and intron 2, respectively, of the sls gene in the w1118 strain [slsd00134 flies, on average, had 17% lower mitochondrial state 3 . Additionally, we did not find any difference in mitochondrial protein density between w1118 and slsd00134 (slsd00134: 0.58 ± (standard error) 0.04 and w1118: 0.62 ± 0.06; P = 0.555) or slsd07587  flies. Thus, these results are consistent with a regulation of mitochondrial respiration by sls. Our GWA analysis pinpointed asticity . The 75.40 lines . Of thes8 strain . We asseP-value of 0.01 as a threshold for our analysis and found 105, 59, and 89 QTTs associated with state 3, state 4, and P:O ratio, respectively, in the analysis averaged across sexes , which encodes a DNA binding transcription factor [salm plays a critical role in the development of Drosophila indirect flight muscles [salm induces the fate of fibrillar muscles  during Drosophila metamorphosis by regulating the gene expression profile of components of the sarcomere, including sls and CG7834[Electron-transfer-flavoprotein, beta polypeptide gene [sls and other QTTs in the co-regulated state 3 module 4  in homozygous mutant and control flies. Given that slsd07587 is a mutant generated by the insertion of a P-element in the intron 2 of the sls gene . We found that the expression of both sls isoforms was significantly reduced in the slsd07587 flies  activity. Across different species, ETFs accept electrons from the FADH2 produced in the first step of the fatty acid β-oxidation and transfer them to the mitochondrial membrane-bound ETF ubiquinone oxidoreductase complex [sls might control mitochondrial respiration rates by affecting the respiratory chain [Based on our results from the correlation network analysis, we hypothesized that the ry chain .D. melanogaster exhibit genetic variation in mitochondrial respiration and efficiency. The GWA data also suggest that natural variation regulating long-term adaptation of D. melanogaster flight muscle mitochondrial function occurs in nuclear-encoded genes involved in a network of interconnected signaling pathways induced by extracellular stimuli, such as neurotransmitters, to maintain cellular homeostasis. Finally, the integrative genomic approach used in our study allowed us to identify sls, with homology to the human Titin (TTN) gene, as a novel hub gene responsible for the regulation of mitochondrial respiration in muscle sarcomere and to provide evidence that sls might act via the ETF ubiquinone oxidoreductase complex.The present study demonstrates that natural populations of D. melanogaster inbred lines used in this study are a subset of the sequenced DGRP lines, which were established from a sample of isofemale lines collected in the Raleigh Farmer’s Market (North Carolina) and inbred to near-homozygosity by 20 generations of full-sib mating [slsd00134 and slsd07587 stocks were obtained from the Harvard Exelixis Stock collection (https://drosophila.med.harvard.edu/) and the w1118 (stock no: 6326) co-isogenic control line from the Bloomington Stock Center (http://flystocks.bio.indiana.edu). Each stock was maintained at constant parental density for at least two generations to minimize environmental effects. To control for larval density, we allowed the parents of the experimental flies to mate for 3 hours to generate egg collections on apple juice/agar medium in laying plates. After 24 hours, groups of 100 first-instar larvae were picked from the surface of the medium and put into replicate vials. To minimize the influence of genetic variation in reproduction on energy metabolism, all the phenotypic assays were performed on virgin flies that were randomly collected from the replicate vials for each line under brief CO2 exposure. For mitochondrial function assays, seven replicate vials per line were used, with each vial containing 20 single-sexed individuals aged 3–5 days. All lines were tested over a 2-year period and the lines and replicate vials were assayed in random order. Flies were reared in vials containing 10 ml of standard cornmeal, agar, molasses, and yeast medium, at a constant temperature of 25°C, relative humidity, and 12hr/12hr light/dark cycle.The 40 unrelated wild-derived b mating . The slsMitochondria were isolated from the thorax of the flies as described previously , with mi, Innsbruck, Austria) with 0.2 mg/ml of freshly isolated mitochondria incubated in respiration medium . Oxygen pH 7.2; . As impl reached . P:O ratDatLab Version 4.1.0.8.All assays were performed within three hours of mitochondrial isolation. Data was analyzed using the software y = Âµ + L + S + LxS + ε, where μ is the overall mean, L and S are the main effects of Line (Random) and Sex (Fixed), LxS is the random effect of sex-by-line interaction, and ε is the within-vial error variance. Pearson phenotypic correlations among traits were calculated by SAS PROC CORR using pooled data and data stratified by sex.All statistical analyses were performed using SAS version 9.1. We used a mixed model ANOVA to partition variation in each trait among the inbred lines according to the model, y = Âµ + M + S + M × S + L(M) + ε to evaluate each segregating site, where M is the effect of SNP (marker) genotype, L is line, and S is sex. Genotype-phenotype associations were also performed for males and females separately using the reduced model y = Âµ + M + ε. We calculated the standardized effect size (a/σG) as one-half the difference between marker classes divided by the overall genotypic standard deviation [r2 measure to compute linkage disequilibrium among significant markers [The line mean of each mitochondrial parameter was associated with all segregating sites in the DGRP present in four or more DGRP lines, and having sequence coverage levels greater than two and less than thirty . We usedeviation . We used markers .P < 10-8 for r2 between SNPs). We imputed SNP genotypes for markers with missing data. We fitted SNPs to the model, beginning with the most significant marker, until the r2 for variance was maximal. We identified haplotypes among replicate line means and analyzed the data for sexes combined and sexes separate using the model y = Âµ + H + L(H) + ε, where H is haplotype and L is line. We estimated the phenotypic variance explained as σH2/(σH2 + σL2 + σE2), where σH2 is the among-haplotype variance component, σL2 is the among-line variance component, and σE2 is the error component.To estimate the amount of genetic variance explained by the SNPs, we applied multiple regression models using gene-centered forward selection. We chose SNPs for the model that were highly significant and not in strong LD with each other  and extracting the residuals to compute pair-wise transcript correlations for module construction [The genetic correlations between all QTTs associated with each trait at truction . Modulestruction .t test.We isolated total RNA using the TriPure RNA isolation kit (Roche). Isolated RNA was then used to make cDNA, using the First Strand Synthesis kit (Invitrogen). We performed RT-qPCR using a SYBR Green Master mix and 50 ng total of cDNA per reaction and run in a Stratagene Mx3000P® qPCR machine. The primers used for qRT-PCR are listed in Additional file 2: Flavin Adenine Dinucleotide; ADP: Adenosine Diiphosphate; UCP: Uncoupling Protein; mtDNA: Mitochondrial DNA; CS: Citrate Synthase; PKA: CAMP-Dependent Protein Kinase; GPCR: G-Protein Coupled Receptors; EGF: Epidermal Growth Factor; AMPK: AMP-Activated Protein Kinase; ETF: Electron Transfer Flavoprotein.DGRP: Drosophila Genetic Reference Panel; GWA: Genome-Wide Association; QTT: Quantitative Trait Transcript; ATP: Adenosine Triphosphate; OxPhos: Oxidative Phosphorylation; ETC: Electron Transport Chain; ROS: Reactive Oxygen Species; NADH: Nicotinamide Adenine Dinucleotide; FADHThe authors declare that they have non- financial competing interests.DRM, PJL, and MD conceived the study and participated in its design and coordination. PJL, SB, DRM, and JCS performed research. STH, TFCM and MD analyzed the data. MD wrote the first draft of the manuscript. All authors read, critically revised the manuscript, and approved the final manuscript.Variation in mitochondrial protein density among eight of the 40D. melanogasterDGRP wild-derived inbred lines. This file includes the distribution of line means for mitochondrial protein density in eight of the 40 DGRP lines and scatter plots of correlations between mitochondrial traits.Click here for fileList of SNPs significantly correlated with mitochondrial respiration and coupling efficiency in the analyses averaged across sexes (Panel A) and stratified by sex (Panel B). This file includes SNP position, FlyBase accession number and name of the genes tagged by the SNPs and near intergenic SNPs, P-value from the ANOVA of the difference in trait means between the two SNP classes, minor allele frequency of the SNPs.Click here for fileList of transcripts significantly correlated with mitochondrial respiration and coupling efficiency. This file includes Affymetrix Probe identification number, FlyBase accession number and name of the gene transcript, P-value from the regression analyses, and gene ontology information. Click here for fileModules of correlated transcripts associated with mitochondrial function. This file shows the average correlation of a transcript with all other transcripts in its module (degree) and the average correlation of all transcripts in the module (average degree). (XLSX 31 kb)Click here for fileNucleotide sequence of primers used for qRT-PCRs. This file reports the gene symbol, direction of the primer, and nucleotide sequence. (DOCX 11 kb)Click here for file


Multiple epiphyseal dysplasia (MED) is a relatively common skeletal dysplasia mainly involving the epiphyses of the long bones. However, it is a genetically heterogeneous group of diseases sharing certain aspects of the radiologic phenotype. In surveys conducted in East Asia, MATN3 was the most common causative gene, followed by COMP. In this study, the authors compared clinical manifestation of MED patients caused by MATN3 and COMP gene mutations, as well as subsequent orthopaedic interventions.Fifty nine molecularly-confirmed MED patients were subjects of this study. The MATN3 gene mutation group comprised of 37 patients . The COMP gene mutation consisted of 22 cases . Medical records and radiographs were reviewed, and questionnaire surveys or telephone interviews were conducted.At the first presentation, the mean age was 8.8 ± 2.8 years (mean ± standard deviation) in the MATN3 group, and 8.5 ± 3.5 years in the COMP group (p = 0.670). The height in the COMP group was significantly shorter than those in the MATN3 group (p < 0.001). Gait abnormality at the first visit (p = 0.041) and the lastest follow-up (p = 0.037) were statistically significant difference. Hip pain (p = 0.084), limitation of daily activity (p = 0.075) at the latest follow-up tended to be more frequent in the COMP group. Hip dysplasia was more common in the COMP group, having significantly larger acetabular angle (p = 0.037), smaller center-edge angle (p = 0.002), severe Stulberg classification (p < 0.001), and smaller femoral head coverage (p < 0.001).Clinical manifestations of MED caused by MATN3 were milder than manifestations of the COMP mutation group. These differences in clinical manifestation and prognosis justify molecular differentiation between the two genotypes. Multiple epiphyseal dysplasia (MED) is a relatively common skeletal dysplasia mainly involving the epiphyses of long bones. MED clinically manifests as mild short stature, intermittent joint pain, angular or rotational deformity of the extremities, short hands and feet, and/or precocious osteoarthritis. Radiologic characteristics include delayed ossification, hypoplasia and irregularity of long bone epiphyses, and varying degrees of flattening of vertebral bodies . It is cStudies of Caucasian and East Asian populations showed ethnic differences in the distribution of causative genes for MED. In an early study on Caucasian population reported the proportions of DTDST as 14%, COMP as 10%, MATN3 as 10%, and unknown as 66% in 29 MED patients . SubsequIndividual clinical and radiographic characteristics of MEDs caused by COMP, MATN3, type IX collagen genes or DTDST have been described -14. ConsThis retrospective study was approved by the institutional review board at Seoul National University Hospital. Fifty-nine MED patients, who were molecularly confirmed to have either MATN3 or COMP mutations, were subjects in this study.The MATN3 group was comprised of 37 subjects . Thirty patients were probands, and the remaining 7 subjects were affected family members of 7 probands. They harbored 7 different mutations in the MATN3 gene as previously reported [The COMP group was comprised of 22 subjects . Twenty patients were probands, and the remaining 2 subjects were affected family members of 2 probands. They harbored 16 different mutations in the COMP gene as previously reported . The meaMedical records were reviewed. Data at the first presentation were recorded, including age, gender, chief complaint, presence or absence of joint pain, and gait abnormalities . The most concerning symptom at first presentation was selected as the chief complaint. Data at latest follow-up were recorded, including height, presence or absence of joint pain, and gait abnormality. Height was converted into z-score using age-matched reference data of the same ethnic group. The patients were asked as to whether the current symptom(s) interfered with their daily life.Sequential radiographic studies of hip joints were evaluated for occurrences of avascular necrosis (AVN) of the femoral head and of aberrant development of the hip joint. AVN was defined as 1) sequential radiographic change of all or part of stages including sclerosis, subchondral crescent sign, resorption of the preexisting bone tissue, and re-ossification Figure , and 2) In order to evaluate acetabular development and femoral head coverage, acetabular angle (AA),  center-eType and purpose of the surgical intervention were categorized and recorded.t-test. Categorical parameters were analyzed using Pearson’s Chi-square test when all the cells had frequency more than 5, or using Fisher’s exact test when any cell had frequency of 5 or less. Distribution of head deformity according to the Stulberg classification was compared using linear by linear association test. The z-score for height, AA, CEA, femoral head coverage, and number of surgical procedures were compared by the Mann–Whitney test. Variance of height and radiographic parameters were compared between the two groups using Levene’s test. A p-value less than 0.05 was considered as statistically significant.Age at the first visit of probands was compared between the two groups using student’s The mean age at first presentation was 8.8 ± 2.8 years (mean ± SD) in the MATN3 group and 8.5 ± 3.5 years in the COMP group (p = 0.670). Chief complaints included gait abnormality, knee angular deformities, pain at the hip, knee, or ankle joints, and short stature. In the MATN3 group, pain at the knee joint was the most common chief complaint, followed by pain at the hip joint, genu valgum deformity, and gait abnormality in decreasing order of frequency. In the COMP group, gait abnormality was the most common chief complaint, followed by hip pain and knee pain. There were no statistically significant differences in proportions of chief complaints between the two groups. Of the clinical manifestations detected by interview or physical examination at the first visit, gait abnormality was significantly more common in the COMP group than in the MATN3 group p = 0.041), but incidences of various types of pain were not significantly different between the two groups  Figure . All theAccording to the criteria described above, AVN of the femoral head had occurred in 4 of 37 (10.8%) MATN3-MED patients, and 1 of 22 4.5%) COMP-MED patients (p = 0.641). On the other hand, aberrant development of the hip was commonly observed in the COMP group, but most MATN3-MED patients developed with normal-appearing hip joints unless complicated by the AVN. Among patients older than 10 years, the COMP-MED patients exhibited more dysplastic hip joints. AA was significantly larger in the COMP group  than in the MATN3 group  (p = 0.037), and CEA was significantly smaller in the COMP group  than in the MATN3 group  (p = 0.002)  were performed for growing children, while acute correction by osteotomy or gradual correction by distraction osteogenesis were reserved for the skeletally mature patients Table . Hip proThis study compared clinical and radiological characteristics of MED caused by MATN3 mutations versus COMP mutations, from an orthopaedic point of view. Characteristics of various MED genotypes have been described in a small number of patients or in families -14,20-22The clinical characteristic of MATN3-MED has only been reported in studies of a single family ,21,22 orOrthopaedic intervention is frequently indicated in patients with MED. In this series, correction of angular deformity at the knee joint was the most common surgical procedure for MED, without significant differences between groups Table . During Surgical intervention at the hip joints was less common than at the knee joints, but the prognosis for hip joints seems to be more guarded, especially in COMP-MED patients. The knee joint surgeries were angular deformity correction procedures with straightforward outcome. On the other hand, the hip surgeries were to improve hip joint function in AVN or hip dysplasia secondary to aberrant hip development, in order to increase the femoral head coverage or containment, however the efficacy has yet to be determined. In our series, AVN occurred in both groups. MATN3-MED patients, who showed abnormal radiographic findings at the hip joint during early childhood, usually developed normal-appearing hip joints unless complicated by AVN. On the other hand, many COMP-MED patients showed persistent aberrant hip development, and ended up with deformed and sometimes subluxated hip joints, which may keep the hip joints symptomatic, even at adolescence. This difference is reflected by the significant difference in the frequency of hip surgery between the two groups Table . HoweverAbnormal appearance of the femoral head in MED is sometimes confused with AVN, and genuine AVN also develops in MED patients, which should be strictly differentiated from the MED itself. Mackenzie et al. reported a series of AVN associated MED . From thThis study has several limitations. It was designed as a retrospective multicenter study, and clinical manifestations could only be categorized on an all-or-none basis, instead of more a sophisticated standardized scoring system. Most patients have not reached skeletal maturity at the latest follow-up; hence, this study represents interim follow-up results. Nevertheless, the study was able to demonstrate significant differences in clinical characteristics between MATN3-MED and COMP-MED.Although MATN3-MED and COMP-MED are currently categorized under the same clinical diagnosis of MED, the long term prognosis of the joints, especially hip joint, should be different. Unless complicated by AVN, MATN3-MED patients are expected to develop normal appearing hips at skeletal maturity. In contrast, COMP-MED patients seem to experience more clinical symptoms in childhood, and are more likely to develop early onset osteoarthritis of the hip due to aberrant development.In comparison between MATN-3 and COMP-MED, the COMP-MED patients were shorter in height, and had more gait abnormality and hip dysplasia, while the MATN3-MED patients had a relatively milder phenotype from an orthopaedic point of view. These differences in clinical manifestation and prognosis justify distinguishing between the COMP and MATN3 molecular subtypes of MED.Written informed consent was obtained from the patient’s guardian/parent/next of kin for publication of this report and any accompanying images.The authors declare that they have no competing interests.SGS collected and analyzed data and wrote the manuscript. HRS, HWK, JSS, CYC, MSP, CWO, CHJ, and KSS collected and analyzed data. WJY and IHC discussed the results and revised the manuscript. OHK analyzed data and gave radiologic support. SSP analyzed data and supported genetic analysis. TJC planned the study, collected data and wrote the manuscript. All authors edited the drafted version of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/15/84/prepub


Methodology. Perioperative records of 1167 patients who underwent ERCP were divided on the basis of anesthesiologist assisting these procedures either on regular basis (Group R) or on ad hoc basis (Group N). Comparisons were made for anesthesia times, complication rates, and airway interventions. Results. Across all American Society of Anesthesiologists (ASA) Classes, regular anesthesiologists were more efficient . Within Group R, anesthesia times across all ASA classes were comparable. In Group N, anesthesia times for higher ASA status patients were significantly longer . Intubation rates (0.76% versus 12.8%) and median minimal oxygen saturation (100% versus 97.01%) were significantly higher in Group R. Had Group R anesthesiologists performed all procedures, the hospital could have saved US $ 758536 (based upon operating room time costs). Conclusion. Experience in providing deep sedation improved patient safety and decreased the operating room turnaround time, thereby lowering operating room costs associated with these procedures.The present study evaluates the effect of anesthesiologist's experience in providing deep sedation for endoscopic retrograde cholangiopancreatography (ERCP) on cost and safety.  Achieving “efficiency without compromising safety” is the new mantra in medicine. Specialization and training in a chosen area have already been implemented successfully in many fields of anesthesiology. Many areas like cardiac, obstetrics, pediatric, and neuroanaesthesia have already received recognition with dedicated fellowships programs. However, anesthesia for many procedures that are done by anesthesiologists in remote diagnostic/therapeutic locations (out of operating room (OR)) is poorly studied. Most of these procedures are conducted under deep sedation (previously termed “Monitored Anesthesia Care (MAC)”). The risk of complications in out of OR is similar to OR anesthesia . Althoug The effect of anaesthesiologist's experience (in the setting of ERCP) on the safety and cost of providing deep sedation has not been studied. In the current analysis, we have tried to address this aspect of anesthesia care in patients undergoing ERCP with anesthesia assistance. After institutional review board approval, perioperative records of patients who underwent ERCP at the Hospital of the University of Pennsylvania from 2010 to 2011 were analyzed. In this retrospective exploratory study, the following patient-specific and procedure-specific data were extracted: demographic profile and comorbidities (ASA class), airway evaluation,  MMP, indication for ERCP, method of airway management, minimal oxygen saturation during the procedure, persistent cardiovascular complications if any, duration of ERCP, total duration of procedure, and any procedure cancellation after the start of the procedure due to anesthesia issues. Total duration of procedure was defined as the time from the patient entering the gastroenterology (GE) suite to leaving the suite. Duration of ERCP was defined as the time interval between endoscopy probe insertion to its removal. For all patients anesthesia time (for this study) was calculated as the time difference between “total duration” and “duration of ERCP.” Hospital of the University of Pennsylvania has two gastroenterology suites, one attached to the main hospital block and the other attached to outpatient block . The procedures done in surgicenter are supervised by a small group of anesthesiologists with interest and extensive experience in providing deep sedation. This group of anesthesiologists involved in Surgicenter primarily involved with GE procedures was labeled as “regular” (R) for the study purpose. The GE suite attached to the main hospital is supervised by anesthesiologists primarily involved in general anesthesia in the main operating room complex and are assigned to GE suite only on an ad hoc basis. This group of anesthesiologists was labeled as “nonregular” (N). GE suites at both of these locations are equivalent in terms of available monitoring, resuscitation equipment, and anesthesia delivery systems. The use of the term “surgicenter” in our setting might be misleading; hence, an explanation is necessary. Unlike most surgicenters in the country, we do not have any restrictions on the ASA class of the patients or procedural difficulty. It is not uncommon to provide anesthesia care for patients with severe aortic stenosis, pulmonary hypertension, severe (chronic obstructive airway disease COPD), pacemaker/defibrillator, cardiomyopathy evaluated for cardiac transplant, and so forth. Because the backup help available (including ICU admission) is similar in both facilities, we do not make any distinction, and this is the hospital's stated policy. However, the surgicenter patients are cared by a small group of anesthesiologists. This allowed us to make comparison of naturally segregated groups, yet comparable populations. Comparisons were made for complications, airway methods, and anesthesia times and estimated operating room costs attributable to anesthesia times. P value of 0.05). Descriptive statistics were used to define distribution of parameters among the groups. Baseline nonparametric, ordinal data among the groups were compared using Mann-Whitney's test. Equality of variances of parametric variables was tested using Levene's test for equality. One-way ANOVA was used to compare the mean anesthesia times of ASA subgroups within both Group R and Group N. Post hoc analysis was done using Tukey's HSD test. Student's unpaired t-test was used to compare means of parametric data between the groups. Pearson's Chi-square test was used to relate airway method frequency data to specifically ASA grade (IV) and also with MMP class IV patients. Automatic linear regression modeling of SPSS was used to evaluate the relative effect of combined factors: anaesthesiologist group, ASA status, airway intervention, and MMP on variations in anesthesia time. The statistical analysis was done using SPSS Version 20  for Macintosh. The level of statistical significance was set to allow an alpha error of 5% (2) was 27.56 ± 7.01 . Mean BMI values in both groups were found to be statistically equivalent (P = 0.07) using unpaired student's t-test. In Groups N and R 79.57% (376 +33) and 56.35% (361 + 7) were of ASA class III and IV (combined), respectively. The MMP distribution between both groups was compared using Wilcoxon rank-sum test and was found to be similar in both groups (P = 0.714).A total of 1167 ERCP procedures were performed, of which 653 (56%) were assisted by regular (Group R) and 514 (44%) were assisted by nonregular (Group N) anesthesiologists. Group R had 233 (35.68%) females and 420 (64.31%) males, whereas Group N had 207 (40.27%) females and 307 (59.72%) males. The mean age of patients in both groups was statistically comparable . The overall observed mean BMI (kg/mt-test (P < 0.001). Anesthesia time in Group N (48.63 ± 21.53) was almost twice that of Group R (24.83 ± 12.96). Mean anesthesia times of individual ASA grades within each group were compared using one-way ANOVA. Within Group N, highly significant difference was found between anesthesia times of ASA subgroups (P < 0.001), whereas the same was not significant in Group R (P = 0.518). On post hoc analysis, all ASA groups had statistically similar anesthesia times in Group R, whereas in Group N ASA III/IV had significantly higher time than ASA I/II. (P < 0.05)   Unpaired Group N .P < 0.001). The median minimal saturation in Group R was 100% (60–100%) and in Group N was 97.01% (40–100%) (P < 0.001), with higher number of ASA IV and III patients in Group N. Distribution of MMP score was similar between both groups (P = 0.8). The airway interventions in both the groups are represented in (P = 0.992).Minimum documented pulse oximeter saturation values were compared using Man-Whitney's test and were significantly lower in Group N (40–100%) . All theented in ,  and MMP 1% (P > 0.05). SPSS derived automated linear regression modeling was used to evaluate the predictability of anesthesia time for regular versus nonregular anesthesiologists. Factors like airway assessment (MMP), ASA status, and type of airway device used could predict anesthesia duration with only 41.6% accuracy. Among these, anesthesiologist's skill (regular versus nonregular) accounted for 50% of predictability, type of airway intervention accounted for 47%, ASA status 3% (P < 0.001) and 1475.75 ± 71.346$ (P < 0.001), respectively. The mean ERCP procedural duration was higher in Group N (29.77 ± 1.44 minutes) by 10.61 ± 1.55 minutes than in Group R (19.08 ± 0.80 minutes), this accounted for $ 657.82 ± 96.10 (29.76%) of the mean total cost difference. However, anesthesia time alone amounted for the remaining 70.24% of mean total cost differences. The anesthesia time showed poor correlation with ERCP procedure time. . We calculated the cost based on an average operating room cost in USA  of $ 62 In the current analysis, we have compared the performance of two groups of anesthesiologists providing deep sedation to similar patient groups in two different areas. Although it is a retrospective review, the strength of results lies in the fact that comparisons could be made between similar groups. The results highlight the time-based efficiency of the anesthesiologists providing deep sedation on a regular basis along with increased safety and a possible economic advantage. This is supported by significantly lower anesthesia times and higher values of minimal oxygen saturation noted during the procedures supervised by “regular anesthesiologists.” Our analysis shows that had the procedures in Group N been performed by Group R anesthesiologists, it could have saved $ “758536 ± 36668” in a single center, along with increased patient safety. Extrapolating these figures to an estimated half a million ERCPs done yearly in the USA,  it would The Group R anesthesiologists in this analysis had been involved in procedures performed under deep sedation on a regular basis. Most had been anesthesia consultants for around 10 years and had spent an average of 20–40% of their clinical time in GI endoscopy suite. Increased experience lead to comparable anesthesia times irrespective of associated co-morbidities/ASA status as suggested by statistically similar anesthesia times for patients cared by Group R . Higher number of ASA III/IV patients in group N was expected to increase anesthesia times; however, linear regression model evaluating the predictability of ASA status on anesthesia time (whether sicker patients required more time) showed poor regression coefficient and thus negated the same. On an average, anesthesiologists infrequently involved with procedures conducted with deep sedation (Group N) took almost 1.8 times more anesthesia time across all patients. This increased time was not only higher in sicker patients (ASA IV) but also proportionately larger in relatively healthier patients (ASA I) as well. Within Group N, increasing level of patient's sickness (ASA I to ASA IV) increased the anesthesia time by a factor of 1.6, while such a significant increase was not noted for ASA IV patients of Group R . In spit The debate “to intubate or not to intubate” patients presenting for ERCP under deep sedation continues. In the current study, the indications for endotracheal intubation were not clear in Group N. Most of these indications (as documented in patient's anesthesia charts) were well-known risk factors for airway difficulties under general anesthesia . Due to semiprone positioning, the possibility of upper airway obstruction due to tongue fall with sedation is low . AnestheUnlike procedures conducted under general anesthesia, where perioperative complications strongly relate to preoperative ASA status, ERCP in multiple other trials have shown no significant relation among the two , 12. OurCommonest complications of deep sedation are respiratory depression (leading to desaturation) and hypotension. ERCP-based studies show that these are primarily transient, reversible and resolve with simple airway interventions or vasopressor boluses, leaving no sequelae . None ofThe present analysis has limitations of being a retrospective review. The documentation is generally done with no plans of analysis at a later date. Details for few parameters in some patient's anesthesia charts were missing, thus bringing down the actual sample size pertinent to that parameter. It would have been interesting to divide anesthesia times into “induction” and “recovery” phases to further analyze where the level of experience helps, but due to limitations in recording methodology, the same was not possible. We could not compare long-term complications rates as anesthesia charts used for analysis had records of immediate periprocedural period only. Hemodynamic variations like hypotensive episodes were also not compared as no significant persistent episode requiring specific attention (other than boluses of vasopressors) was recorded. The number of ASA IV patients in Group R was much smaller than that in Group N. Although this was incidental, it decreased the statistical significance of results.