The rising prevalence of obesity and metabolic syndrome (MetS) has received increased attention since both place individuals at risk for Type II diabetes and cardiovascular disease. Insulin resistance (IR) has been implicated in the pathogenesis of obesity and MetS in both children and adults and is a known independent cardiovascular risk factor. However measures of IR are not routinely performed in children while MetS or severe obesity when present, are considered as clinical markers for IR.The study was undertaken to assess the utility of ATPIII defined metabolic syndrome (MetS) and severe obesity as predictors of insulin resistance (IR) in a group of 576 overweight children and adolescents attending a pediatric obesity clinic in Brooklyn.Inclusion criteria were children ages 3–19, and body mass index > 95th percentile for age. MetS was defined using ATP III criteria, modified for age. IR was defined as upper tertile of homeostasis model assessment (HOMA) within 3 age groups . Sensitivity, specificity, positive predictive values and odds ratios (OR) with 95% confidence intervals (CI) were calculated within age groups for predicting IR using MetS and severe obesity respectively.MetS was present in 45%, 48% and 42% of the respective age groups and significantly predicted IR only in the oldest group . Sensitivities were <55%; specificities <63% and positive predictive values ≤ 42% in all groups. Severe obesity was significantly associated with IR in both the 9–11 (p = .002) and 12–18 (p = .01) groups but positive predictive values were nonetheless ≤ 51% for all groups.The expression of IR in overweight children and adolescents is heterogeneous and MetS or severe obesity may not be sufficiently sensitive and specific indicators of insulin resistance. In addition to screening for MetS in overweight children markers for IR should be routinely performed. Further research is needed to establish threshold values of insulin measures in overweight children who may be at greater associated risk of adverse outcomes whether or not MetS is present. Insulin resistance (IR) and/or compensatory hyperinsulinemia are usually associated with obesity and are risk factors for cardiovascular disease (CVD) and type 2 diabetes in both adults and children -8. FurthData were collected on all overweight children and adolescents(3–19 years) with BMI > 95th percentile for age,  who attended a pediatric obesity program at an inner city University Hospital, in Brooklyn, New York from January1, 2002 through December 31, 2005. Five hundred and seventy six (576) patients with complete data for metabolic risk factors constituted the study sample. The study was approved by the Internal Review Board and informed consent to participate in the community based obesity program and to be included in the database was obtained from the parent or guardian and assent from the children when first enrolled. Each individual was assessed for the presence of MetS. Fasting plasma concentrations of glucose, insulin, triglycerides, and HDL cholesterol were assessed using standard laboratory methods. Weight and height were measured to the nearest 0.1 kg and 0.5 cm respectively. BMI was calculated as weight in kilograms divided by height in meters squared and converted to BMI-Z score standard units according to CDC age-sex tables . Waist cSince IR is affected by age and pubertal status, and tanner stages were not recorded on all patients in the database, the population was further divided into 3 age groups  reflecting prepubertal, pubertal and post pubertal stages and also to assess the age related associations of MetS and IR. Currently there are no accepted values to define insulin resistance in normal and overweight children, so we used the upper tertile of homeostasis model assessment (HOMA: fasting serum insulin (μunits/ml) × fasting plasma glucose (mmol/l)/22.5) within age group to denote higher IR consistent with previous work in both adults  and chilSensitivity, specificity, and positive predictive value of using MetS to identify higher IR were computed. In addition, similar to Weiss et al we dividOf the 576 participants, 122 were ages 3–8, 164 were ages 9–11 and 290 were 12–19. Almost all were African American (81.4 %) or Hispanic (16.0%). Table Defining higher IR by upper age-specific tertiles of HOMA, threshold values of 2.31 for ages 3–8, 4.29 for those 9–11 and 4.26 for the 12–19 year olds were obtained. Upper tertiles of fasting insulin for these age groups were 11.8, 21.1 and 20.5 (units) respectively. Adolescents with MetS , there was a prevalence of higher IR in 31% of the 3–8 yr old, 29% in the 9–11 yr old and 20% in those 12–19 yr old.Table In binary logistic regression models adjusting for sex, age and race, we found MetS and BMIZ were not significant predictors of higher IR (p = .55 and p = .24 respectively) for 3–8 year olds. For 9–11 year olds, MetS showed a non-significant trend (p = .13) while BMIZ was a significant predictor (p < .01). Both MetS and BMIZ were significantly associated with higher IR for 12–19 year olds (p < .01 for each). In sensitivity analyses, using upper quartile of HOMA instead of upper tertile to indicate higher insulin resistance gave similar results.The principle findings in our study were that the ATP III definition of MetS, as modified for the pediatric populations, and severe obesity are but modest predictors of IR, defined here as upper tertile of HOMA in this sample of overweight, children and adolescents. We found that although the prevalence of MetS was high and relatively similar in the three age groups, a statistically significant association with IR was evident only in adolescence. As described in table Although MetS is believed to be closely related to IR, and both are independently associated with serious adverse outcomes among adults, recent Currently there are no widely accepted values to define IR in either normal or overweight children. Hence, markers of IR such as HOMA as described by Matthews and coworkers  have beeSeveral reasons may account for the relatively low MetS sensitivity and specificity for identifying IR in this pediatric population. One is that in the ATP III definition all criteria are given equal importance in defining the syndrome such that impaired fasting glucose(IFG), the factor most closely associated with IR, need not be one of the ≥ 3 criteria for an individual to have MetS. If on the other hand IFG was a required criterion, the specificity for such a definition of MetS would be greater, while the sensitivity and positive predictive value would be far lower since very few (<4%) of the overweight children and adolescents in this sample had (IFG) ≥ 100 mg/dL. One reason for the relatively low proportion with elevated glucose may be that insulin resistance is generally associated with increased levels of insulin production. The ability of an insulin insensitive individual's beta cells to compensate for IR by producing extra quantities of insulin in response to a glucose load may decrease with age and elevSecond unlike the WHO definition for MetS , the cluOther reasons for the low sensitivity of Mets criteria to identify IR are that, the deleterious effects of the prolonged presence of elevated insulin levels may take time to manifest themselves with regard to increase in triglycerides, blood pressure as well as circulating glucose levels as shown in this study where the associations of IR and metabolic risk factors were evident in the adolescents but not in the younger age groups. Alternately, as shown in a recent study by Lambert et al for the Our study has several limitations. The sample was drawn from participants in an obesity clinic situated with a predominantly African American and Hispanic catchments area. Thus our findings can only be generalized to overweight non-white youth. Our findings are nonetheless relevant given the higher prevalence of MetS found in this population compared to other studies  in patieIn this study, we followed the example of others ,47, in uIt is also possible that the modified ATP III criteria for MetS that we used in this study may not be the most meaningful with regard to long-term clinical outcomes. The individual components and the specific cutoffs for children are not based on any prospective studies but have been defined by a consensus statement of an expert panel for adults. Since there is no universally accepted definition of MetS for children and the thresholds for adult ATP III criteria cannot be applied to a pediatric population given there are changes in the metabolic parameters as a function of age, we used modified ATP III adult criteria for children similar to that used by Cook et all which has been widely quoted and applied in different populations. It is pIn conclusion, the growing epidemic of childhood obesity, early onset of type II diabetes and clustering of cardiovascular risk factors has raised great concern. To date, this concern has led to increasing attention to the presence of MetS in children and adolescents. Even if MetS is an important marker or risk factor for CVD and type II diabetes in itself in a pediatric population, IR unaccompanied by MetS is also important.The major benefit of defining MetS in children is to draw attention to risk factor clustering associated with obesity and insulin resistance. While the convergence of IR and MetS becomes evident over time these data suggest that in addition to screening for Mets, surrogate markers of insulin resistance such as fasting insulin, HOMA or if resources allow, glucose tolerance test, could be a useful addition to routine evaluations of overweight children in order to alert clinicians to potential increased risk in those with IR even without MetS. Further studies are needed to assess clinically relevant threshold values of insulin measures in overweight children and adolescents of different ethnicities that can predict the development of CVD, diabetes and other clinical syndromes in patients both with and without the metabolic syndrome.National Cholesterol Education Program (NCEP)Cardiovascular Disease(CVD)Adult Treatment Panel (ATP)Metabolic Syndrome (MetS)Insulin Resistance (IR)Homeostasis model assessment (HOMA)Center for disease control (CDC)Impaired Fasting glucose (IFG)World Health Organization(WHO)The author(s) declare that they have no competing interests.All authors made a significant contribution in the conception, design and manuscript preparation.


One of the major controversies surrounding the metabolic syndrome (MetS) in type 2 diabetes is whether its single components act synergistically as risk factors for atherosclerotic vascular disease (AVD). We aimed to answer this by evaluating the relationship, and its various combinations to AVD in comparison to single traits in a population-based study with type 2 diabetes in Germany.4020 unselected patients with type 2 diabetes aged 35 – 80 years. MetS was: diabetes plus ≥ 2 traits of the MetS by AHA/NHBLI definition.AVD was: history of myocardial infarction and/or coronary revascularization and/or stroke. The occurrence of AVD in relation to overall MetS/single traits/combinations was presented as OR (95% CI). Multiple logistic regression, including established cardiovascular risk factors, modeled their associations.The prevalence of overall MetS was 74.4% and the OR for AVD was 1.41 (1.12–1.78), which however was higher for hypertension as single trait (OR 4.76). Different combinations of MetS presented a wide range of ORs (0.47 to 10.90) and strong sex differences. Some clusters of MetS including hypertension and low HDL-cholesterol presented a higher risk factor than single traits or their sum, whereas the others out of 11 possible carried no increased AVD risk. Multiple logistic regression showed independent association between AVD and overall MetS.The overall MetS in type 2 diabetes comprises 11 heterogenous clusters of traits. Overall MetS increases the risk of AVD in type 2 diabetes and individual traits in some clusters with hypertension and low HDL-cholesterol may act synergistically as risk factors particularly in women. Type 2 diabetes is associated with a high prevalence of the metabolic syndrome (MetS) -3. SinceIt is commonly recognized that T2DM is associated with multiple AVD risk factors. However data on the epidemiology of MetS in unselected patients with T2DM are scarce. The AHA/NHBLI criteria  provide The Diabetes in Germany (DIG) study is a population-based, prospective, observational study in patients with T2DM. This report will analyse the following questions in a large cross-sectional sample:1) What is the prevalence of the MetS by AHA/NHBLI criteria, its single traits and their combinations in a representative German population with T2DM?2) Is the MetS an independent risk factor for AVD compared to its single components in men and women?3) What is the AVD risk for different clusters of the MetS?1C, total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides, and creatinine. The traits of the MetS were defined based on the AHA/NHBLI criteria with one modification. Obesity was defined as BMI ≥ 30 kg/m2 since waist circumference was not measured routinely. Hypertension was diagnosed if blood pressure at the time of inclusion was ≥130/85 mmHg and/or the patients were treated with antihypertensive drugs. The same applies for dyslipidemia with respect to fibrates and nicotinic acid. Statines, however were not considered as equivalent of dyslipidemia.Two hundred and thirty eight practices , that represent a cross section of daily practice diabetes care in Germany, took part in the study. With respect to the distribution of sites in the federal states of Germany was found out an average frequency of 1.7 to 3.5 per 10,000 inhabitants, with exception of Saxony where the ratio was 16.9 to 10,000 resp. Inclusion criteria were clinical T2DM and age between 35 and 80 years. Exclusion criteria were: major cardiovascular event <3 months before entry , stroke, amputation), heart failure NYHAIV, macro-proteinuria >300 mg/24 h or creatinine >2 mg/dl, cancer disease <5 years before entry. The participating physicians  were asked to include ≥ 10 consecutive patients with T2DM per site. A total of 4,331 patients were recruited of whom 311 had to be excluded due to protocol violations; thus 4,020 were included in the final analysis. Medical history and drug intake were registered by a standardized questionnaire. Body mass index (BMI) and blood pressure were measured according to a standard protocol and all laboratory measurements were done by local laboratories that held a quality control certification. The following parameters were registered as baseline characteristics: sex, age, duration of diabetes, current smoking, BMI, waist circumference, systolic and diastolic blood pressure, fasting and postprandial blood glucose, HbAWe diagnosed triads (6) of the MetS (diabetes plus two other traits), quartets (4) (diabetes plus 3 other traits) and a quintet presenting all the 5 traits. Overall MetS was diagnosed when T2DM coexisted with ≥ 2 other traits of the MetS. Cardiovascular events were identified based on the medical history obtained by the physicians at the participating sites. The diagnosis of AVD was established if the following aggregated events were present: MI and/or coronary revascularization and/or stroke. Since the database did not allow differentiating amputations by cause we did not include peripheral vascular disease in the calculations for AVD. The Saxon Ethics Committee approved the study and informed consent was obtained from all patients.2 tests. The probability in the occurrence of AVD in relation to the MetS, its single traits and their combinations were estimated as odds ratio (OR) (95% CI). Binary logistic regression was used to model the associations of the overall MetS, its single traits, their combinations and of major established risk factors with AVD and the test of interaction by collinearity diagnostics.All data were analyzed using statistical software SPSS Version 11.5. Baseline characteristics were presented as means (SD) for continuous variables and frequency for all categorical variables. The statistical significance of the differences by sex was assessed by t-tests or χ1C of 7 (1.2)% for both sexes. Males had significantly higher postprandial glucose levels than females. Average LDL-cholesterol was 3.2 (1.0) mmol/l with significantly higher levels in women. Triglycerides (2.2 (1.7) mmol/l) were significantly higher in men and the same applied to creatinine. HDL-cholesterol was significantly higher in women with an overall level of 1.3 (0.4) mmol/l.Baseline characteristics of the patients by sex are given in table With respect to the prevalence of major cardiovascular diseases, 7.6% of the patients reported a previous MI  and 4.3% a stroke . The frequency of coronary revascularization was high  in relation to the frequency of MI. Coronary revascularization and MI were 2–3 times more frequent in males, but there was no significant sex difference for stroke. According to our definition 15.2% of our patients had AVD. Males had a significantly higher frequency than women: 20.0% and 9.8% resp. (p < 0.001).2and more than half (55.4%) had hypertriglyceridemia. In contrast, the percentage of people with low HDL-cholesterol was less than 10%. The only significant sex difference for single traits found was in the percentage of obesity with 44.4% in men and 55.9% in women (p < 0.05). Only 2.4% of the patients had no additional trait of the MetS with no sex difference. Duration of diabetes had no significant effect on prevalence of MetS.Elevated blood pressure was by far the most frequent single trait of the MetS found in 91.3%. Approximately every second patient had a BMI of ≥30 kg/mThe prevalence of different combinations which meet the diagnosis MetS is presented in table The ORs for AVD risk are presented in table We conducted logistic stepwise regression analysis to determine the relevance of the MetS as an independent risk factor for AVD in T2DM in the context of other major cardiovascular risk factors. We used 2 models; in the first we included the well-established risk factors: age, sex, smoking habit and LDL-cholesterol levels along with the single traits of the MetS. In the second model we considered the MetS as a whole, independently of the combination used, along with the above-mentioned established risk factors. In both models age, male sex, LDL-cholesterol levels and smoking were independently associated to AVD. In the first model T2DM with hypertension remained also independently associated to AVD as a trait of the MetS, while in the second model the MetS as a whole showed an independent association with AVD. There was no significant influence of interaction between the single traits of MetS on AVD. The calculated models predicted 85.1% in model 1 and 85.0% in model 2 resp of AVD or non AVD correctly.We found in this large population based study of patients with T2DM in Germany a prevalence of the overall MetS according to AHA/NHBLI criteria of 74.4% with no sex difference. However, the analysis of the 11 possible combinations which meet the diagnosis MetS reveals a striking heterogeneity in the prevalence of phenotypes and their ORs as AVD risk factors resp. Furthermore, we observed a distinct sex difference with respect to ORs for single traits as well as for all phenotypes of the MetS.III [The prevalence of overall MetS in DIG is comparable to other studies with T2DM in Europe, also they use different definitions -11 and s1C level of 7% and that 44.8% of them were receiving insulin treatment, which is known to increase HDL-cholesterol [As already described in other epidemiological investigations with T2DM and MetS hypertension was the most frequent co-morbidity with not less than 91.3%, with no sex difference. In a recently published study triglycerides were the strongest predictor to indicate the presence of MetS among patients with type 2 diabetes of a outpatient clinic . In contlesterol . ObesityFor the first time we present data on the distribution and prevalence of the 11 possible clusters hidden in the unspecified diagnosis overall MetS. In the DIG sample 62.5% of the people had two or three additional traits  and only 4.4% had a complete set of components of MetS. As expected from the high prevalence of hypertension, hypertriglyceridemia and obesity the prevalence of triads with these components was high: 55.9% for combination with hypertension plus hypertriglyceridemia, 50.7% for combination with hypertension plus obesity and 33.7% for hypertriglyceridemia and obesity as companions. Among quartets only the combination with these three traits was of relevance (31.9%). Of notice was the low frequency of remaining phenotypes with prevalences between 4.7 and 9.7%.Our data on OR for AVD suggest on the first look that the overall MetS is associated with an increased risk: OR for men 1.38 and for women 1.67 resp., but the corresponding ORs for hypertension as single trait were 4.22 and 7.69 resp. In the Botnia study the MetS by WHO definition in patients with T2DM did not increase the AVD mortality (RR 1.15 CI 0.68–1.94), while some of its individual components such as hypertension and albuminuria were highly significant predictors of cardiovascular events. Therefore a critical appraisal put a question to the clinical relevance of MetS  dyslipidIn the DIG study overall MetS could be confirmed as independent AVD risk factor in multiple regression analysis including established risk factors such as LDL-cholesterol, age, sex and smoking. The predictive power of this model was as good as for multivariate analysis taking the single traits into the model. We did not find a statistical interdependence of the single traits as risk factors. In a prospective, community-based study, using modified AHA/NHBLI definition, in patients with T2DM the increase in the number of traits was associated with higher CVD rates . AnotherThere are some limitations to our analysis. We made a cross-sectional evaluation and our results are subject, therefore, to survival bias. Some of the combinations of traits were present in a very low number so that the ORs could not be calculated or resulted no significant. In addition, the diagnosis of AVD was based on medical history and information obtained by home physicians without the participation of an adjudicator committee. On the other hand, this population sample represents the real-world scenario of T2DM in Germany.The metabolic syndrome is not more or less than a concept for an integrated approach to a cluster of metabolic diseases with hypertension . This stIn conclusion the MetS was found to be an independent risk factor for AVD in the DIG study together with established risk factors such as age, sex, LDL-cholesterol and smoking. The prognostic power was equal to models with the single trait as categorical variable. Hypertension as single variable had a higher AVD risk than overall MetS. However, triads of the MetS including hypertension and/or low HDL-cholesterol had twice as high a risk as hypertension alone. There are however other phenotypes of the MetS with no increased risk. Thus the metabolic syndrome comprises heterogenous clusters with respect to AVD risk. The data of the DIG study support the concept to focus on multiple risk factor strategy to prevent AVD in diabetes.The author(s) declare that they have no competing interests.


The binding of ring-labelled and side-chain labelled misonidazole to hypoxic cells in monolayer and spheroid cultures of mammalian cells has been compared. The kinetics and patterns of binding for the two labelled compounds are indistinguishable. This finding has implications for the mechanism of binding and for the design of misonidazole analogues which might be used to identify hypoxic zones in tumours.


We report longitudinal changes in the metabolic syndrome (MetS) in 2,458 participants from 480 families in the Family Heart Study. Participants were examined between 1994–96 (FHS-T1) and 2002–03 (FHS-T2), about 7.4 years apart. Additionally, the impact of medication on estimates of MetS prevalence, and associations of MetS with prevalent coronary heart disease (CHD) and type 2 diabetes (T2D) were studied.o) National Cholesterol Education Program (NCEP) MetS criteria. Two others considered the confounding of medications effects, respectively (m) lipid medications constituted a categorical diagnostic criterion for lipids variables, and (c) lipids and blood pressure variables were corrected with average clinical trials medications effects. Logistic regression of MetS on CHD and T2D, as well as the trend analysis of MetS by age, were performed.Three definitions for MetS prevalence were considered. One represented the original (o) to 28.8% in FHS-T2(o); from 19.7% in FHS-T1(m) to 42.5% in FHS-T2(m); and from 18.4% in FHS-T1(c) to 33.6% in FHS-T2(c). While we observed adverse changes in all risk factors, the greatest increase was for waist circumference (25%). The percentages of MetS were about 2 to almost 3 times higher in ages 50 years and older than in younger ages. The odds of having prevalent CHD were about 2.5 times higher in the subjects classified with MetS than without.MetS increased from 17.1% in FHS-T1(MetS percentages increased noticeably longitudinally and cross-sectionally with older age. These conclusions were reached with and without considering medication use, but correcting risk factors for medications use affects the MetS prevalence estimates. As found in other studies, MetS was associated with increased odds for prevalent CHD. The metabolic syndrome (MetS) is a combination of interconnected risk factors for obesity, insulin resistance and glucose intolerance, dyslipidemia, and hypertension. The Adult Treatment Panel III of the National Cholesterol Education Program (NCEP) defined a group of five clinical criteria for effective classification of MetS (see Methods) . An indiBased on the NCEP classification, it is reported, that a rapidly growing epidemic of metabolic syndrome is taking place in the United States -4. For eMetS is of special interest because it represents a complex disorder expressed by interrelated risk factors with unknown genetic and not well known environmental influences. Previous familial studies have shown that MetS has an important heritable component -8. WhileFHS) – Time 1 (FHS-T1) and FHS – Time 2 (FHS-T2), which represent two clinical visits with a mean age interval of about 7.4 years. The main purpose of our study was to assess the longitudinal trends of MetS, its association with age, prevalent CHD and T2D, and the impact of medication on the prevalence estimates of MetS.One of the main features of MetS is an increased risk of CHD and Type 2 diabetes (TD2) -12. AlexThe sampled population is part of the multi-center FHS supported by the National Heart, Lung, and Blood Institute. It is important to mention that the original FHS white familial sample was separated into two large groups, one random and one non-random, based on the participants' recruitment criteria for CHD and their familial risk for CHD. Details are provided elsewhere . Toriginal MetS definition as (o). The second method, labeled (m), considered medication use for TG and HDLC as a categorical effect and anyone on lipid lowering drugs was classified as positive for the NCEP lipid threshold (Table m) is similar to the recent criteria proposed by the Scientific Statement of AHA/NHLBI for diagnosing and managing MetS, with a difference that they defined the threshold of fasting glucose to be 100 mg/dl , but larger at the follow-up visit [FHS-T2(m) and FHS-T2(o) (7.9%). This emphasizes the fact that the expansion of the prevalence of lipid lowering medication use can raise the MetS prevalence estimation, and should be considered.The precision of defining MetS for epidemiological studies is made difficult by the fact that medication treatment can potentially change the level of each risk factor ,17. MediA third approach was performed in an attempt to impute the original levels of treated BP/lipids MetS risk factors (see Methods). This third method resulted in MetS percentages of 18.4% at baseline and 33.6% at follow-up , (m), and (c) methods.For the three methods applied, similar trends were noticed when familial samples were selected random/for CHD  and FHS-T2(c). Higher presence of prevalent CHD and T2D when participants were classified with MetS may imply that MetS and/or its contributing risk factors are associated with the development of CHD and T2D. Similar findings were reported in other publications [It was apparent, when comparing results of FHS-T1 vs. FHS-T2, that the presence of MetS in the sample was associated with higher odds for prevalent CHD. The odds of having prevalent CHD in the presence of MetS were about 2.5 times higher than in its absence in FHS-T1, in this study we assumed that there are correlated medication effects on LDL, HDL, and TG traits. Also for each medication prescribed there are over and under responders. By adjusting everyone on a medication, , by an average medication efficacy or a percentage there is a potential to over-/under estimate the original imputed HDL, TG, BP, and concurrently the MetS prevalence. Therefore to overcome this limitation, (where data are available), we recommend in similar studies for treated risk factors, their correction per participant with average effects of specific classes of medications matched to specific medication labels .We conclude that when estimating the MetS prevalence it is important to account for the medications use that confounds MetS risk factors. To account categorically for anti-hyperlipidemic medication use is a difficult task that can overestimate MetS prevalence. Our study demonstrated also that MetS is associated with a higher risk for prevalent CHD and T2D. This finding is supported by the scientific literature, which in addition shows that CHD mortality is higher in individuals with MetS than without ,28. MetSThe author(s) declare that they have no competing interests.All authors contributed equally.


The Family Blood Pressure Program is an ongoing, NHLBI-sponsored, multi-center program to study the genetic determinants of high blood pressure. The goal of this particular study was to study patterns of metabolic syndrome (MetS) in four ethnic groups: African Americans, Caucasians, Hispanics, and Asians.A major part of participants in three networks GENOA, HyperGEN and SAPPHIRe were recruited mainly through hypertensive probands. MetS was defined as a categorical trait following the National Cholesterol Education Program definition (c-MetS). MetS was also characterized quantitatively through multivariate factor analyses (FA) of 10 risk variables (q-MetS). Logistic regression and frequency tables were used for studying associations among traits.Using the NCEP definition, the Hispanic sample, which by design was enriched for type 2 diabetes (T2D), had a very high prevalence of MetS (73%). In contrast, its prevalence in Chinese was the lowest (17%). In African Americans and Hispanics, c-MetS was more prevalent in women than in men. Association of c-MetS with type 2 diabetes (T2D) was prominent in the Hispanics and African Americans, less pronounced in the Whites and Japanese, , and weakest in the Chinese sample.Using FA without rotation, we found that the main factor loaded obesity (OBS) and blood pressure (BP) in African Americans; OBS and insulin (INS) in Hispanics, in Japanese, and in Whites; and OBS alone in Chinese. In Hispanics, Whites, and Japanese, BP loaded as a separate factor. Lipids in combination with INS also loaded in a separate factor. Using FA with Varimax rotation, 4 independent factors were identified: "Obesity-INS," "Blood pressure," "Lipids-INS," and "Central obesity." They explained about 60% of the variance present in the original risk variables.MetS ethnic differences were identified. Ascertaining for hypertension or T2D increased the MetS prevalence in networks compared with the one in the US general population. Obesity was the most prominent risk factor contributing to both c-MetS and q-MetS. INS contributed in two important factors (obesity and lipids). The information imbedded into c-MetS trait /q-MetS factors scores can contribute in future research of the MetS, especially its utilization in the genetic analysis. Metabolic syndrome (MetS) is defined as a clustering of cardiovascular and type 2 diabetes risk factors including obesity, insulin resistance, dyslipidemia, and hypertension (HT). The genetic control of MetS is expected to be complex since it represents a syndrome of multifaceted abnormalities. Categorical and clinically applicable criteria were developed by the National Cholesterol Education Program (NCEP), which defined MetS as the presence in an individual of at least 3 out of 5 risk factors (increased waist circumference (WAIST), increased level of triglycerides (TG), low levels of high density lipoprotein cholesterol (HDL), HT, and fasting glucose (GLUC) ≥ 110 mg/dl) [We designated this dichotomous definition of MetS (presence or absence) as c-MetS . Quantitative factor analytic treatment of MetS was designated as q-MetS . Several earlier studies have employed multivariate techniques such as factor analysis (FA) to investigate MetS. This method transforms a set of MetS risk variables to a smaller set of latent factors. Most studies have reported 2 to 4 underlying factors, depending on the number of risk factors included, whether or not the Varimax rotation was used, and statistical decisions made .This study is an investigation of MetS in the Family Blood Pressure Program (FBPP) . The FBPThe goal of this particular study was to evaluate MetS in the rich FBPP database using both the c-MetS and q-MetS definitions. Common features and differences among the major ethnic groups were explored. Finally, the relationships of c-MetS with T2D and vascular heterogeneous atherosclerotic (VHA) events were also investigated.FBPP pooled database (version 3) of 13,592 participants from 4 different networks represents one of the largest compilations of ethnically diverse data. GenNet had only partial data for defining c-MetS and therefore, data from GenNet were excluded from analysis. GENOA (Genetic Epidemiology Network of Atherosclerosis) includes 3 field centers: the Jackson, MS center recruited African Americans; the one in Starr County, TX recruited Hispanics; the one in Rochester, MN, recruited Whites. HyperGEN (Hypertension Genetic Epidemiology Network) included field centers in Birmingham AL, which recruited African Americans; the rest of centers in Forsyth County, NC, in Framingham, MA, in Minneapolis, MN, and in Salt Lake City, UT recruited Whites. SAPPHIRe (Stanford Asian Pacific Program in Hypertension and Insulin Resistance), with 3 major field centers, recruited Asian Pacific populations of Chinese origin residing in Taiwan and of Japanese origin residing in Hawaii, and California. GENOA recruited African American, Hispanic, and White sibships with at least 2 hypertensive sibs (with HT onset before the age of 60). The Hispanic sibships were recruited with at least 2 sibs who were each diagnosed as type 2 diabetic (T2D). HyperGEN recruited African American and White hypertensive sibships with 2 or more hypertensive sibs, at least 1 of them having severe HT. In addition, HyperGEN recruited random samples of African American and White participants, and parents. SAPPHIRe recruited sib-pairs concordant and/or discordant for hypertension. For all participants the diet was uncontrolled and reflective of the "free-living" dietary habits of these populations.The We analyzed data from the FBPP where participants with data for defining c-MetS included: 1857 African Americans, 1799 Hispanics, and 1578 Whites in GENOA; 2010 African Americans, and 1888 Whites in HyperGEN; 1630 Chinese, and 581 Japanese participants in SAPPHIRe). In all networks, subjects with unknown ethnicity were excluded. As a result, information on a total of 11,343 participants was considered in the c-MetS study  > 35 kg/m2), WAIST (cm), waist-to-hip ratio (WHR), fasting insulin , fasting GLUC (mg/dl), SBP and DBP, mm Hg), low density lipoprotein cholesterol , HDL (mg/dl) and fasting triglycerides . To maintain consistency among the three networks, the minimum fasting time required was set at 8 hours.The categorical trait c-MetS was created employing the NCEP definition. c-MetS was defined by the presence of 3 or more of the following abnormalities in an individual: WAIST > 102 cm in men or > 88 cm in women, TG ≥ 150 mg/dl, HDL < 40 mg/dl in men or < 50 mg/dl in women, systolic blood pressure (SBP) ≥ 130 mm Hg and/or diastolic blood pressure (DBP) ≥ 85 mm Hg, or on treatment for HT, and GLUC ≥ 110 mg/dl or on treatment for diabetes [Type 2 diabetes was defined by a fasting GLUC ≥ 126 mg/dl, or current use of hypoglycemic medication or insulin that was documented at examination in the clinic, or diabetes reported on questionnaires. An age of onset ≥ 40 years was also required to diagnose T2D .In the FBPP pooled database, three important VHA variables were available from questionnaires: stroke or transient ischemic attacks, heart attack, and bypass or angioplasty. If any of these were reported in an individual, it was used to define the VHA status.2, age3, and field center within each gender-by-race-by-Network group. INS, TG, and HDL were transformed by natural logarithm to render them approximately normal. Likewise, GLUC was transformed as the inverse of the squared value (1/GLUC2). These transformations, together with standardization to zero mean and unit variance, prepared the data for FA. FA was performed by employing the FACTANAL function in S-plus version 6.1, Insightful Corp., Seattle. We applied an exploratory factor analysis where the extraction of the latent factors was performed based on the maximum likelihood estimation [The 10 risk variables of MetS were checked for normality and outliers. Each variable was adjusted for age, agetimation ,12. In stimation . The twotimation . Factor FREQ and LOGISTIC regression procedures of SAS. Means and standard deviations were estimated with SAS software v. 9.0., SAS Institute, NC [Odds ratios, prevalence rates, and their confidence intervals were estimated by utilizing the tute, NC .Mean age of participants ranged from 48 years in the HyperGEN African Americans to 58 years in the GENOA African Americans Tables , 2, 3. MAmong African Americans, twice as many were hypertriglyceridemic in GENOA than in HyperGEN, even though they were very comparable for all other MetS risk factors. This difference contributed to a relatively higher percentage of c-MetS in GENOA African Americans 41%) than in HyperGEN African Americans (34%). Whites in both networks were similar with respect to all NCEP thresholds  in African Americans, Hispanics, and Whites; TG had smaller weight in African Americans, but higher in Hispanics and Whites; in the Japanese, TG, HDL, BP, and to a smaller degree WAIST; and in the Chinese, HDL, BP, and less of TG. Hispanics also had a higher contribution of GLUC. Hispanics had a total of 73% of participants with at least 3 risk factors beyond the thresholds . Levels lower than the NCEP thresholds were more frequent in the Chinese and in the Japanese samples than in Hispanics, African Americans, or Whites. As expected, all networks and ethnicities selected had a high percentage of participants above the NCEP threshold for SBP/DBP or using hypertensive medications. For a better comparison, similar ethnicities were combined across networks . Looking at the q-MetS patterns, Asians had more a contribution of hypertension and dyslipidemia. Whites and African Americans showed a classical MetS as a combination of obesity, dyslipidemia, and hypertension, much like the Hispanics  using data from the National Health and Nutrition Examination Survey (NHANES), it is evident that our US samples have a higher prevalence of MetS than the general US population . In our The prevalence of MetS was comparable across Networks within the same ethnicity. However, there are ethnic differences in the prevalence of MetS. Prevalence of c-MetS is high in GENOA Hispanics Figure . They alConversely, Asians  are leaner than others. We recognize that SAPPHIRe exclusion criteria biased the obesity findings. They also had lower T2D prevalence . Other authors have concluded that African-American women and Hispanic men and women have the highest prevalence of MetS. They attributed this to higher BP, obesity, and diabetes in African Americans, and the high prevalence of obesity and diabetes in Hispanics . In the In our study, each of the ethnicities considered showed significant MetS and T2D associations. Young et al. (2003), in a longitudinal cohort study of 429,918 veterans with diabetes, found that African Americans and Native Americans had a higher odds ratio (1.3 and 1.5 respectively) for having early diabetic nephropathy than Whites . In the Four independent factors were identified when factor analysis was performed with Varimax rotation. Their pattern was very similar in African Americans, Hispanics, Whites, and Japanese, but not entirely so in Chinese. BMI, WAIST, and INS contributed together mainly in a factor labeled by us as "Obesity-INS." SBP and DBP contributed in a separate "BP" factor. A "Lipids-INS" factor was constructed mainly from contributions of LDL, HDL, TG, and INS. The last, "Central obesity" factor, was mostly an involvement of WAIST and WHR. These 4 factors were persistent also by gender in the HyperGEN data . When noIn general, our results about the structure of the factors, which reflect multivariate correlations of the variables studied, are supported by the literature. However, there are also differences that could be the result of variations in recruitment. In a study of Japanese Americans, it was found that visceral fat was a significant correlate of hypertension and independent of fasting INS . In contIn conclusion, patterns of the MetS were relatively similar across networks within ethnicity, but were statistically different among ethnicities. Overall, obesity was the most prominent compound risk factor expressed in both c-MetS and q-MetS. However, the degree of consistency in factor structures observed across ethnicities and networks is remarkable given that there are considerable differences in the Network-specific study designs. The notable exception of Hispanics in GENOA is quite understandable since the sample was also enriched for T2D. Thus, some of the differences especially in the prevalence of MetS are, at least in part, attributable to the study design differences. Nevertheless, the increase of MetS prevalence in our U.S. samples compared to the U.S. general population confirmed that there is an important link between HT and MetS. Together, our results underline that MetS is a compound phenotype, where obesity, dyslipidemia, and hypertension enable MetS. If we assume that obesity and dyslipidemia have separate biochemical pathways for their expression, it appears that the presence of INS in both latent obesity and lipids factors may be an indication that INS is an important contributor and possibly a connector of pathways in the development of MetS.The reported findings will be useful if they lead to innovations. One application of these results can be the genetic analysis of the new MetS created data. It is well known that a categorical trait has less power in detecting genetic linkage as compared to a quantitative trait for a complex phenotype. Two types of q-MetS factor scores (with and without Varimax rotation) provide ample opportunity to discover quantitative trait loci for MetS. Parallel with this work, we have undertaken a detailed genetic analysis of the MetS factors that will be reflected in another publication (unpublished observations). Qualitative and quantitative characterization of MetS in the rich Family Blood Pressure Program pooled data will help in getting a better understanding of the genetic inheritance underlying MetS and its interaction with the environmental causes.FBPP, Family Blood Pressure Program; NCEP, National Cholesterol Education Program; MetS, metabolic syndrome; c-MetS, qualitative MetS; q-MetS, quantitative MetS; OBS, obesity; T2D, type 2 diabetes; FA, factor analysis; VHA, vascular heterogeneous atherosclerotic events; HT, hypertension; BMI, body mass index; WAIST, waist circumference; WHR, waist to hip ratio; INS, insulin; GLUC, glucose; TG, triglycerides; LDL, low density lipoprotein cholesterol; HDL, high density lipoprotein cholesterol; SBP, systolic blood pressure; DBP, diastolic blood pressure; BP, blood pressure, CI-confidence interval.The author(s) declare that they have no competing interests.


Panax quinquefolius  on diabetes is yet to be elucidated. Recent studies show that Panax quinquefolius increases insulin production and reduces the death of pancreatic beta cells. Mechanism studies indicate that Panax quinquefolius improves cell's immuno-reactivity and mitochondrial function through various factors. Clinical studies show that Panax quinquefolius improves postprandial glycemia in type 2 diabetic patients. Further studies to identify the component(s) of Panax quinquefolius linked with pancreatic islets/beta cells in vitro and in vivo are warranted for better understanding of the full effects of Panax quinquefolius.The mechanism of the beneficial effects of  Increasing steadily in case numbers, diabetes mellitus has become a devastating illness with significant morbidity and mortality around the globe ,2. TherePanax quinquefolius L.  and Panax ginseng CA Meyer  [Panax ginseng increases blood flow and decreases fatigue. Experimental studies indicate that Panax ginseng alleviates oxidative stress generated by diabetes through the inhibition of lipid peroxidation [Panax quinquefolius has anti-aging effects, aids digestion [Panax quinquefolius may improve psychological conditions, immune function and conditions associated with diabetes  ,8,9. Accxidation . On the es Table . Overallenelzine . HistoriPanax ginseng can be distinguished from Panax quinquefolius by using ginsenoside profiles because Panax ginseng contains ginsenoside Rf which is absent in Panax quinquefolius [Panax ginseng were studied in diabetic C57BL/6J ob/ob mice. The results indicate that the total ginsenoside extract has significant anti-hyperglycemic and anti-obesity properties [-diabetic rats in a dosage dependent manner [The main active components in ginseng responsible for ginseng's medical value have been identified as glycosides. Glycoside is a naturally occurring substance consisting of a sugar and non-sugar moiety. Some glycosides belong to a family of compounds named saponins which produce froth under agitation by reducing water surface tension. A group of saponins in ginseng have been named ginsenosides and classified into subclasses as Ro, Ra, Rb, Rc, Rd, Re, Rf, Rg and Rh. These ginsenosides can be differentiated in thin layer chromatography (TLC) based on their retention factor value, which is the distance that ginsenosides travel up the TLC column . Panax guefolius . The antoperties . In the operties . Rh2, ant manner . Recent t manner .Panax quinquefolius lowers blood glucose in diabetic patients [Panax quinquefolius in stimulating insulin production/secretion and anti-apoptosis are dosage dependent, suggesting a biological specificity of ginseng water extracts on beta cells. A high dosage of Panax quinquefolius is probably required to reverse IL-1 beta induced apoptosis. According to a clinical study by Vuksan et al., Panax quinquefolius attenuated postprandial glycemia in both type 2 diabetic and non-diabetic patients. While no difference was found in postprandial glycemia between placebo and ginseng groups in non-diabetic subjects when administered together with glucose challenge, in subjects with type 2 diabetes significant reductions were observed when ginseng was taken 40 minutes before the glucose challenge [patients , increaspatients . The reshallenge .Panax quinquefolius has not been fully elucidated. According to our findings, Panax quinquefolius water extracts reversed an IL-1 beta induced increase of uncoupling protein-2 , decreased pro-apoptotic protein caspase-9 and increased the level of anti-apoptotic protein Bcl-2. This indicates that the anti-apoptotic effects of Panax quinquefolius may be achieved through regulating mitochondrial UCP-2 and ATP levels, thereby influencing insulin synthesis and apoptotic cascades in beta cells [Panax quinquefolius, there are other reports on additional factors targeted by ginseng such as immuno-reactivity [et al. reported that Panax quinquefolius exerted an anti-lipolytic effect on type 2 diabetic conditions through a signaling pathway different from that of insulin, suggesting that Panax quinquefolius not only affects pancreatic cells through increasing insulin production and cell viability, but also through creating an anti-lipolytic effect and targeting glucose receptors to counter hyperglycemia [Panax quinquefolius to improve hyperglycemia and treat diabetes.The mechanism for the beneficial effects of ta cells   significantly hinders insulin production and inhibits cell viability. During apoptosis, cells shrink; chromatin condenses; DNA is cleaved into pieces at internucleosomal regions . The celpoptosis . Previoucapase-9 . In a stpression . IL-1 bepression . Panax qof Bcl-2 . FurtherPanax quinquefolius products. It is also not certain whether different ginseng species would have the same effects on hyperglycemia. Sievenpiper et al. discovered that Panax ginseng showed both null and opposing effects on acute postprandial plasma glucose and insulin, which did not coincide with the findings with Panax quinquefolius [Panax ginseng has the same effects on humans as it does in animal models.Due to a lack of standardization in the herbal medicine industry, it is not certain whether the beneficial effects on hyperglycemia hold true for all uefolius . Reproduuefolius . FurtherPanax quinquefolius increases cell insulin production and cell viability through mediating mitochondrial proteins and apoptosis factors. These findings confirm that Panax quinquefolius improves pancreatic islet functions. The components of Panax quinquefolius that improve pancreatic beta cell function have not been identified. The precise actions of Panax quinquefolius components on other human cells such as muscle cells and lipocytes under diabetes therapy are yet to be investigated.The author(s) declare that they have no competing interests.ZW proposed to cooperate in this project and provided information for the manuscript. JL conceived this project, collected information and wrote the first draft of the manuscript. LL worked with JL to conceive this project, collected references andinformation, wrote and approved the manuscript. All authors approved the final manuscript.


The metabolic syndrome (MetS) concept is widely used in public health and clinical settings without an agreed pathophysiology. We have re-examined the MetS in terms of body fuels, so as to provide a coherent cross-cultural pathogenesis.National Health and Nutrition Examination Survey (NHANES 2001-2) with n = 2254 and Taiwanese National Health Interview Survey (NHIS) sub-set for hypertension, hyperglycemia and hyperlipidemia assessment (TwSHHH 2002), n = 5786, were used to compare different ethnicities according to NCEP-ATPIII (NCEP-tw) criteria for METS. Exploratory factor analysis (EFA) using principal components (PC) was employed to differentiate and unify MetS components across four ethnicities, gender, age-strata, and urban-rural settings.The first two factors from the PC analysis (PCA) accounted for from 55.2% (non-Hispanic white) to 63.7% (Taiwanese) of the variance. Rotated factor loadings showed that the six MetS components provided three clusters: the impaired energy regulation (IER) components , systolic and diastolic blood pressures (BPs), and HDL-cholesterol, where the IER components accounted for 25-26% of total variance of MetS components. For the three US ethnic subgroups, factor 1 was mainly determined by IER and HDL-cholesterol, and factor 2 was related to the BP components. For Taiwanese, IER was determinant for both factors, and BPs and HDL-cholesterol were related to factors 1 and 2 respectively.There is a MetS core which unifies populations. It comprises WC, TG and FPG as a core, IER, which may be expressed and modulated in various second order ways. The metabolic syndrome (MetS) was conceptualized on the basis that a cluster of metabolic phenomena could be observed in those prone to cardiovascular disease ahead of frank diabetes. It combined features of possible pathogenesis with risk evaluation. Its definition has been in evolution, but those currently used include that of the International Diabetes Federation (IDF) in 2005 ,2 and thThere have been efforts to create a unified explanatory model for this syndrome. The best known of these is that the MetS is a disorder of insulin sensitivity i.e. basically 'insulin resistance' and its sequelae, however it may be caused, as suggested by the IDF .It could be argued that the anatomico-physiological basis of the syndrome is in the splanchnic region  with disThe MetS and energy dysmetabolism are modulated by a number of factors including the psycho-social ,17 and eThere are often striking differences in the presentations of the MetS in Western and Eastern populations. We have taken advantage of such differences between the USA and Taiwan to test for hypothesis generation. In the case of the USA, we have used the National Health and Examination Survey (NHANES) of 2001-2002 data  and in tExploratory factor analysis (EFA) has been used for hypothesis generation. It is a multivariate correlation method to reduce a number of inter-correlated variables into smaller sets of factors with less or no association; it has been applied to study the clustering of metabolic abnormalities that contribute to the metabolic syndrome ,20,24,25The study was approved by the Institutional Review Board (ethics committee) of the NHRI  of Taiwan.lins  were selected with PPS. Four households were selected randomly from each selected lin, with each member of these households to be interviewed. The response rate for households was 91.1%, and it was 94.2% for individuals [Two datasets, one from each of Taiwan and the US were used for this study. The Taiwan data came from the Taiwanese Survey on Hypertension, Hyperglycemia, and Hyperlipidemia (TwSHHH) in year 2002 , a subseividuals . The deshttp://www.cdc.gov/nchs/nhanes.htm[The other dataset was the NHANES which comes from interviews, examinations, and laboratory tests based on blood and urine samples hanes.htm. The 200hanes.htm.As with the Taiwanese sample, when data were missing, or biologically implausible, the subject was not included in the analyses.When examining the relationships between measurements, sampling weights were not necessaryvarimax criterion for the clustering patterns of the six components from the first two factors for each of the ethnic groups. To assess the sensitivity of the estimated proportions, for each of the four ethnic groups, the EFA procedure was applied repeatedly for their communalities in each of the one thousand resampled datasets by bootstrapping [th and 975th millile of the sorted added-up communalities of the corresponding clustered components among one thousand bootstrapped datasets.Descriptive statistics, such as mean and standard deviation, were used to present the characteristics of the study populations. An EFA using the method of principal components  was appltrapping . The 95%The average ages and the metabolic components for the several ethnic groups are presented in Table In regard to urbanization, the analysis was only possible for Taiwanese Table . There wWe have not shown the findings for physical activity or alcohol consumption because of the high frequencies (>20%) of missing values in NHANES. However, in Taiwan, this was not the case .Pearson correlations are shown in Table The correlation between BMI and WC is less in Taiwanese (r = 0.75) than in US ethnic groups . With the exception of blood pressure, the remaining correlations between the metabolic syndrome components are similar for the various ethnic groups.For both the TW3HHH and NHANES datasets, all the distributions of the six components were skewed to the left. Therefore, a log-transformation was applied to each of the six components before analysis, so that the component variables were approximately normally distributed. Figure In the case of Taiwan, the IER cluster was closer to the BP cluster than in the US ethnic groups.Figure In contrast, females tended to have lower communalities in BPs than males except for Mexican-Americans, for whom the females had substantially higher communality. The 95% CIs obtained from bootstrap resamplings were reasonably narrow and homogeneous for all the clustered components across the four ethnic groups as well as the males and females separately, with Taiwanese having the narrowest bounds possibly due to the relatively large sample size Figure , Table 4Table In Figure The factor analysis for elderly Taiwanese (≥ 65 y) is similar to a factor analysis of the MetS in elderly Swedes (≥ 75 y)  where a This study utilized factor analysis to discover a consistent component core as a likely core of the metabolic syndrome. We found a similar pattern in different ethnic groups. It confirms our hypothesis about IER. Wijndaele et al.  also useÖhrvik et al.  have expAt present we can say that ethnicity, and urbanisation and, with the exception of elderly women in urban settings, display a common core to the MetS which could reflect IER. Where this does not apply may provide clues to the pathogenesis and consequences of the MetS. An example of this could be the report of Malan et al (2008)  that theVarious attempts have been made to provide a mechanistic construct on the many observations of the MetS cluster of 6 components with insulin resistance being the earliest and most preferred. For example, Alberti and colleagues recommended one single set of cut points for all components except waist circumference . NeverthA somewhat similar conceptualization of the patho-physiological basis of the MetS has been advanced by others ,37. DespIf additional components, like insulin, are included in the MetS definition, they will direct the unifying concept accordingly, in this case towards insulin resistance . In thisWhile we have not formally assessed insulin resistance, fasting glucose represents a partial measure of it, at least insofar as nocturnal gluconeogenesis and its determination by FFA flux in the splanchnic circulation is concerned. Seen this way, insulin resistance may be embraced by the broader and unifying phenomenon of IER.manipulate the datasets in accordance with a hypothesis and then test for it. Hence, we used an exploratory rather than a confirmatory approach.We propose that IER components  rather than the usual six components of MetS are the core metabolic disorder in the MetS, and have employed an exploratory factor analysis (EFA) to differentiate the clustering and inter-relationships among the 6 components of MetS. As the EFA has showed, this hypothesis is "confirmed" by the invariant (or homogeneous) pattern across four different ethnic groups. It might be asked why we did not employ a confirmatory factor analysis (CFA) to confirm our hypothesis at the beginning? It is the starting point for those authors who adopt the CFA approach ,40,41 inAs indicated, the conceptual IER segregates from the rest of the components of the MetS and provides a unifying capacity with which to consider inputs and consequences in more systematic ways. Greater sophistication in our hypothesis would involve reference to insinuated, but unobserved measurements (such as free fatty acid) or latent or partially captured factors (such as insulin resistance), at which point the CFA approach would be appropriate. Our findings provide a near-fundamental basis with which to take IER further through more structured hypotheses about phenotype, cellular biology and genomics. Such an enquiry would have public health and clinical relevance.The extent to which the non-IER MetS components and their health outcome significance differ between the ethnic groups studied is of interest We have noted in the results that, for Taiwan, the BP cluster is closer to the IER cluster than in the US ethnic populations. Furthermore, in the case of diastolic BP, where Taiwan has the highest values and prevalence, it is against a background of preference for a high Na/K molar ratio diet, and relatively high incidence of hemorrhagic stroke as featured in the annual Public Health reports. On the other hand, relatively greater North American lipoprotein cholesterol abnormalities Tables  and 2 apA more analytical study of the representative Taiwanese subjects by age, gender and locality provides further insights into the core and second order of the MetS components Table . With reOur segregation of the MetS into an energy-dependent core (IER) and other phenomena is consistent with emerging evidence on the regulation of cellular fuel metabolism and about mitochondrial function and disorder. In particular, the AMP-activated protein kinase cascade has been identified as a unifying system for energy control by Carling and others . It alsoIn both the US and Taiwan, the study populations were community-based and representative, not selected for health status and not institutionalised in this cross-sectional study. Their health characteristics are shown in part, by reported behaviours (eg tobacco smoking) and various risk factors, by the medication history, by anthropometry to detect body fatness and its distribution and by virtue of the components of the MetS itself. Although physical activity and alcohol intake were recorded, the frequency of missing values in the US ethnic groups precluded their reliable evaluation.The interpretation of the medication use is difficult. First, it is some confirmation of a risk or disease state eg hypertension, hyperlipidaemia or diabetes. Second, it means the observed MetS components will have been somewhat corrected depending on compliance, affordability, access to health care and public health education. In these respects the US and Taiwan are strikingly different, since Taiwan has a universal health insurance scheme , with 99% of the population covered, and the US does not. Nevertheless, in both places, remoteness, culture, education, socio-economic circumstances and other equity issues are likely to have influenced MetS component profiles. For example, our data showed age and gender might have effects on MetS, especially on urban older women. Notwithstanding these considerations, the MetS core is robust by setting, health status and where other differences will be inevitable.If the MetS can be understood in clearer etiologic and pathogenetic terms, it will pave the way for more rational and supportable public health preventive strategies and clinical interventions.It remains unclear as to how important the MetS is in the prediction of morbidity and mortality in different clinical settings and the understanding of IER may clarify this situation. From the public health point of view, the "physical activity transition" and its role in the genesis of MRFs as proposed by Katzmarzyk and Mason may operate principally through the IER pathway .Energy dysnutrition is a fundamental problem at all stages of economic development. It may operate at different levels from the environmental-human biology interface to every human cell, in turn to determine the likelihood of health and well-being. However, in public health and clinical practice, its measurement is available, not only by way of intake and expenditure with difficulty, but also by way of waist  circumference for abdominal fat stores and by fasting plasma glucose and triglycerides as fuels generated in the splanchnic circulation and region. These 3 components (IER) of the MetS cluster consistently across several ethnic groups in different localities; nevertheless, the phenomenon is not as pronounced in Taiwan as the US and its persistence into later life is less evident in urban Taiwanese women The IER cluster allows a step-wise first (core) and then second order approach to the understanding and application of the present form of the MetS, in relation to cardiovascular disease. This also allows for other variants of the MetS, more relevant, for example, to inflammatory or neoplastic disease, to be developed as second order phenomena. We are encouraged to take advantage of these findings in cohort and intervention studies, especially those which include genetic markers for energy metabolism, fat distribution, and various clinical outcomes.AHA: American Heart Association criteria for metabolic syndrome in 2004; BMI: Body Mass Indices; BPs: systolic and diastolic blood pressures; CFA: confirmatory factor analysis; CI: confidence interval; DBP: diastolic blood pressure; EFA: Exploratory factor analysis; FFA: free fatty acids; FPG: fasting plasma glucose; HDL: high-density lipoprotein; IDF: International Diabetes Federation criteria for metabolic syndrome; IER: Impaired Energy Regulation; LDL: low-density lipoprotein; MRF: metabolic risk factors; MetS: metabolic syndrome; NCEP-ATP III: National Cholesterol Education Program-Adult Treatment Panel III criteria for metabolic syndrome; NHANES: National Health and Nutrition E1xamination Survey; NHIS: National Health Interview Survey; NHLB: National Heart Lung and Blood Institute; PC: principal components; PCA: PC analysis; PPS: probability proportional to their size; SBP: systolic blood pressure; TG: fasting triglycerides; TwSHHH: Taiwan Three High Prevalence Survey on Hypertension, Hyperglycemia, and Hyperlipidemia; VLDL: very low density lipoprotein; WC: waist circumference.The authors declare that they have no competing interests.MLW, HYC, CCC and CCH were involved in the conception and design. WCC WSW and CCC were involved in the analysis and interpretation of the data. MLW, HYC, CCC and CCH drafted the article. MLW, HYC and CAH were responsible for critical revision of the article for intellectual content. MLW, HYC, CCC, CCH, WCC, WSW and CAH gave final approval of the article. HYC, CCC WCC, and WSW provided statistical expertise. HYC, WCC and WSW were involved in the collection, where relevant, and assembly of the data. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6823/10/11/prepub


A 44-year-old male developed acute fulminant hepatic failure of unknown etiology and expired within four days. His serial electroencephalograms (EEGs) showed diffuse background slowing on day one, which evolved into “alpha coma” and later into “spindle coma” over the ensuing two days. Such EEG transition is hitherto undescribed in patients with hepatic encephalopathy and gives fresh insight into the etiopathogenesis of specific EEG patterns in diffuse encephalopathy. Electroencephalogram (EEG) changes in hepatic encephalopathy vary from low-frequency alpha rhythm (8 Hz) admixed with bilateral theta activity that may later evolve into theta-delta slowing over both hemispheres, with or without triphasic waves. Presence of alpha coma and spindle coma pattern in the same patient has hitherto not been highlighted in cases of coma due to fulminant hepatic failure. We report this unusual EEG evolution occurring within a span of three days and discuss its possible electrophysiological basis and prognostic implications.A 44-year-old male with headache and malaise of one-day duration was found unconscious at home. He was last seen by his relative 4-5 hours prior to the present state, ambulant, and verbalizing. He had a history of complex partial seizures of extra temporal origin of 20 years duration with infrequent secondary generalization and was on Phenytoin 350 mg/day. His neuroimaging (MRI brain-1.5T) was normal. On admission, he was febrile with a Glasgow Coma Score of 3/15 without other focal abnormalities on neurological examination and was put on mechanical ventilation. His liver function tests revealed elevated serum glutamic-oxaloacetic transaminase  and glutamic-pyruvic transaminase . During serial monitoring they further rose to 50-100 times respectively in the next 48 hours. His total bilirubin was 4.5 mg% [N = 0.2-1.0 mg], with a direct fraction of 3.2 mg% [N = 0.4-0.6 mg%], serum ammonia 65 µmol/L [N = 11-35 µmol/L], alkaline phosphatase 156U/L [N = 50-136 U/L], and albumin 4.1 gm% [N = 4-5 g]. He had progressively worsening coagulation parameters [INR 2.1 to 3.4]. CSF study was normal and blood culture, urine toxic screen  and viral markers  were negative. Ultrasound scan of abdomen and CT scan of brain were normal.Serial EEGs were acquired on daily basis, ranging from forty- five minutes to four hours, but no continuous monitoring was done. On day one, it showed diffuse back ground slowing, which evolved into frontally dominant alpha frequency waves without reactivity, suggestive of “alpha coma” on day 2 . On the EEG changes in hepatic encephalopathy depend on the stage of coma. From low-frequency alpha rhythm (8 Hz) admixed with bilateral theta slowing (early stages), it may later evolve into theta-delta slowing over both hemispheres with or without triphasic waves. With increasing stupor sleep activity disintegrates. In severe coma, arrhythmic delta activity decreases, both in frequency and amplitude, evolving into electrocerebral silence.de novo abnormal pattern. Animal studies have shown that after hypoxic brain insult, alpha-like activity appears first with maximum amplitude in the amygdala. This activity could be blocked by bilateral ablation of the amygdala, but not by destruction of cerebral hemispheres.[Our patient's EEG done on day 1 showed nonreactive, generalized delta slowing, suggestive of a diffuse electrophysiological disturbance. This evolved into “alpha coma pattern” on second day. On the third day, his EEG revealed “spindle coma pattern”. Alpha coma is a term used when a predominant alpha frequency activity is noted in a comatose patient which is not posterior dominant, continuous and nonreactive. Three maispheres.5 Howeverispheres. In the aSpindle coma is an electroclinical entity in which physiologic sleep patterns, such as sleep spindles in the 12 to 14 Hz range, vertex sharp waves, and K complexes, superimposed on a background of delta and theta activity, occur synchronously in patients with altered consciousness. Spindle coma pattern is distinguished from physiologic slow wave sleep by the inability to rouse the patient to a normal level of consciousness. Head injCycling slow wave sleep patterns in EEG have been reported rarely during prolonged nocturnal recordings in patients with alpha coma. AlthoughPrevious reports denoted better outcomes with spindle coma rather than alpha coma. However,


FTO gene has previously been found to be associated with obesity in European Caucasian samples. The objective of this study is to examine whether this association extends to metabolic syndrome (MetS) and applies in non-Caucasian samples.The rs9939609 T>A single-nucleotide polymorphism (SNP) in the FTO rs9939609 SNP was genotyped in 2121 subjects from four different non-Caucasian geographical ancestries. Subjects were classified for the presence or absence of MetS according to the International Diabetes Federation (IDF) and National Cholesterol Education Program Adult Treatment Panel (NCEP ATP) III definitions.The Carriers of ≥ 1 copy of the rs9939609 A allele were significantly more likely to have IDF-defined MetS (35.8%) than non-carriers (31.2%), corresponding to a carrier odds ratio (OR) of 1.23 , with a similar trend for the NCEP ATP III-defined MetS. Subgroup analysis showed that the association was particularly strong in men. The association was related to a higher proportion of rs9939609 A allele carriers meeting the waist circumference criterion; a higher proportion also met the HDL cholesterol criterion compared with wild-type homozygotes.FTO rs9939609 SNP was associated with an increased risk for MetS in this multi-ethnic sample, confirming that the association extends to non-Caucasian population samples.Thus, the  The metabolic syndrome (MetS) is a clinical entity characterized by abdominal obesity, hypertension, hypertriglyceridemia, depressed plasma high-density lipoprotein (HDL) cholesterol and elevated glucose ,2. MetS FTO (fat mass and obesity associated) gene, encoding the human analogue of fused toes in mice. The FTO gene encodes a 2-oxoglutarate-dependent nucleic acid demethylase that is present in many tissues, but most abundant in the hypothalamus, the control center of energy balance ). Subgroup analysis of males showed that the carrier OR was 1.46 . By individual ethnic populations, a significant increased risk for MetS was observed for FTO rs9939609 A allele carriers only in the South Asian group (P = 0.027). Analysis of the NCEP ATP III definition for MetS showed a similar trend towards increased risk for FTO rs9939609 A allele carriers compared to TT homozygotes  .Analysis across the 4 study populations indicated that FTO rs9939609 A allele. The non-significant trends suggested that sample numbers were insufficient for detecting an association with this particular trait.No significant increased risk of MetS was observed overall for AA homozygotes, for either the IDF or NCEP ATP III definitions, upon repeating the analyses using a recessive model for the FTO rs9939609 A allele carriers met the MetS criteria for depressed HDL cholesterol (P = 0.0089), and there was a nonsignificant trend towards increased waist circumference and increased BMI for FTO rs9939609 A allele carriers   Table .FTO as a candidate gene for MetS in a sample from multiple non-Caucasian geographical ancestries showed 1) significant association with the FTO gene, with rs9939609 A allele carriers, particularly males, having an increased risk of MetS   a direct function of FTO [FTO locus.This is the first report of an association between the mitation . Despitefindings -11. Howey sample . Aside fFTO rs9939609 A allele carriers met the MetS criteria for depressed HDL cholesterol (P = 0.0089) than non-carriers. In addition, values were close to significance also for increased waist circumference (P = 0.080) and increased BMI (P = 0.060) among A allele carriers  and Greenland Inuit (NCEP ATP III definition). Thus, the overall association is primarily due to these two populations, with little influence from the Chinese and Oji-Cree. Again, replication of the FTO association in other larger study samples is essential for clarification. Further evidence against an association for the Chinese can be found in a recent study of 3,210 unrelated Chinese Han subjects from Shanghai and Beijing where no association found between the rs9939609 SNP and obesity [Although the meta-analysis across the four non-Caucasian study populations showed an association between MetS and the common rs9939609 SNP in  obesity .FTO. The observed consistent association between MetS and FTO in a non-Caucasian multi-ethnic study group, including populations which differ considerably in MetS prevalence, strengthens the likelihood that the FTO locus is related to obesity and MetS. Future replication of this association in larger sized samples and devising functional molecular studies will further enhance the validity of association and the causative relationship between the FTO variant and MetS.In summary, we report an association between MetS and the common rs9939609 SNP in The author(s) declare that they have no competing interests.SAA and RLP participated in the experimental design, data acquisition and analysis, interpretation of results, and manuscript writing. MRB participated in the analysis of the data. TKY, PB, SSA, SY, BZ, SBH, AJGH, PWC, and MWH were involved in the provision of patient samples and/or clinical data. RAH participated in the experimental design, data analysis and interpretation of results and manuscript writing. All authors approved the final manuscript.


The metabolic syndrome (MetS) may contribute to the excess cardiovascular burden observed in rheumatoid arthritis (RA). The prevalence and associations of the MetS in RA remain uncertain: systemic inflammation and anti-rheumatic therapy may contribute. Methotrexate (MTX) use has recently been linked to a reduced presence of MetS, via an assumed generic anti-inflammatory mechanism. We aimed to: assess the prevalence of the MetS in RA; identify factors that associate with its presence; and assess their interaction with the potential influence of MTX.MetS prevalence was assessed cross-sectionally in 400 RA patients, using five MetS definitions . Logistic regression was used to identify independent predictors of the MetS. Further analysis established the nature of the association between MTX and the MetS.P = 0.004).MetS prevalence rates varied from 12.1% to 45.3% in RA according to the definition used. Older age and higher HAQ scores associated with the presence of the MetS. MTX use, but not other disease modifying anti-rheumatic drugs (DMARDs) or glucocorticoids, associated with significantly reduced chance of having the MetS in RA (OR = 0.517, CI 0.33–0.81, The prevalence of the MetS in RA varies according to the definition used. MTX therapy, unlike other DMARDs or glucocorticoids, independently associates with a reduced propensity to MetS, suggesting a drug-specific mechanism, and makes MTX a good first-line DMARD in RA patients at high risk of developing the MetS, particularly those aged over 60 years. The MetTo date, five definitions for the MetS have been developed: The National Cholesterol Education Programme (NCEP) 2004  and NCEPSeveral of the individual components of the MetS have been shown to be influenced by demographic, anthropometric and RA-specific factors ,8,29, buIn this study we aimed to: (1) assess the prevalence of the MetS in a large RA population according to all definitions currently used, in order to develop a bench-mark allowing comparisons between other relevant studies in the future; (2) to identify demographic, anthropometric and RA-disease specific factors that may be associated with the presence of the MetS in RA patients; (3) to establish if anti-rheumatic drug use (in particular methotrexate), is associated with the presence of the MetS, and whether this occurs in a drug-specific manner or as a result of an overall anti-inflammatory effect.Four hundred RA patients fulfilling the 1987 revised American College of Rheumatology classification criteria , were rePatient data was obtained via case note analysis and a face-to-face interview performed by a rheumatologist. The dual approach facilitated the documentation of a detailed history to include: disease course/characteristics (including disease duration), drug use , co-morbid conditions, and family history of rheumatic and cardiovascular diseases. Details of current medication prescriptions were recorded at baseline (no prospective data was collected), and previous anti-rheumatic drug use were recorded via retrospective case note analysis and patient interview. Baseline demographics were recorded and anthropometric characteristics were measured as previously described . Current® 5,1FS chemistry system , with multilayered slides used to measure TC, HDL, and TGs, whereas a dual chamber package was used to assess LDL, apolipoprotein (Apo) A and ApoB. Insulin resistance was evaluated from fasting glucose and insulin using the Homeostasis Model Assessment of Insulin Resistance (HOMA IR) [Baseline blood samples were obtained from each patient and were analysed in a single laboratory. Blood tests included: C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), fasting lipid profile , HDL, low density lipoproteins (LDL), TG), rheumatoid factor, anti-cyclic citrullinated peptide antibodies, thyroid function tests), liver function tests, renal function, insulin and fasting glucose. All lipid components were analysed using the Vitros HOMA IR)  and the HOMA IR) , and wasHOMA IR) .For the purposes of this study, the prevalence of the MetS was analysed according to all existing definitions , or percentages, as appropriate. For the univariate analysis, chi-squared, t-test and Mann-Whitney U tests were used to test categorical, normally and not normally distributed data, respectively. The independence of the predictors of the MetS was tested in the multivariate models using binary logistic regression.This was carried out using SPSS 15.0 . The distribution of each variable was examined using Kolmogorov-Smirnov function. Results are expressed as mean ± standard deviation, median  and had a median age of 63.1 years. Patients had a median disease duration of 10 years, and had moderate disease activity (mean DAS28 score 4.2).Disease-modifying anti-rheumatic drugs (DMARDs) were widely prescribed among this cohort (340/387), either as monotherapy (218/387) or combination therapy (122/387). The breakdown of DMARD usage was: 218 (56%) patients were taking methotrexate, 114 (29.5%) sulphasalazine, 77 (19.9%) hydroxychloroquine and 16 (4.1%) leflunomide. Biologic therapy and glucocorticoids were prescribed in 45 (11.6%) and 56 (14.5%) patients, respectively. The use of other drugs known to influence components of the MetS included: statins in 83 (21.4%), anti-hypertensives in 171 (44.2%) and NSAIDs/cyclo-oxygenase-II inhibitors in 108 (27.9%) patients.P = 0.429; Table There was great diversity in the reported prevalence rates according to the definition used Table . The preResults presented are only for the MetS as defined by NCEP 2004, but were very similar using any of the other definitions, despite the difference in prevalence.P = 0.001), had shorter disease duration (P = 0.008), higher ESR (P = 0.006), higher HAQ scores (P = 0.036) and significantly less of them were treated with methotrexate (P = 0.001), compared with those who did not have the MetS , higher HAQ scores  and less methotrexate use  values remained significant independent predictors of the presence of the MetS in RA patients. Patients on methotrexate had half the odds of having the metabolic syndrome compared with those not taking methotrexate (odds ratio (OR) = 0.525, 95% confidence interval (CI) = 0.96 to 1.56, P = 0.003). The odds were not significantly altered when other DMARD, anti-TNF therapies, glucocorticoid use and NSAID medications were added to the model  were tested in a multivariate logistic regression model. Older age .The multivariate model was repeated, replacing ESR with DAS28 score and subsequently with CRP, to check for any differences among these potential confounders. The results were not found to differ significantly by using DAS28 or CRP instead of ESR , higher HDL levels (P ≤ 0.001), and lower fasting plasma glucose levels  but its prevalence depends on the definition used, in a very similar manner to that seen in the general population, with the IDF criteria reporting the highest and the EGIR criteria the lowest rates. We also demonstrate for the first time that, irrespective of the definition used, factors including older age and disease severity (HAQ) are associated with the presence of the MetS in patients with RA. More importantly, methotrexate therapy appears to significantly decrease the odds of having the MetS, independently of any of these factors, suggesting the possibility of a drug-specific protective mechanism.To date, four other studies have commented on the prevalence of the MetS in patients with RA, reporting prevalence rates ranging from 14% to 44% ,17,28,30P = 0.429). This was consistent across all definitions of the metabolic syndrome, apart from the EGIR classification, which diagnosed significantly more males than females (P < 0.001). These findings differ from those observed in the general population, where age-matched females have been reported to have significantly higher rates of the MetS [In this study we observed similar prevalence rates of the MetS among males and females . This demonstrated that the 'protective' effects of methotrexate on the presence of the MetS are only present in patients over the age of 60 years. These findings are unsurprising given that patients over the age of 60 years have a higher prevalence of the metabolic syndrome, upon which methotrexate can act.In order to gain further understanding of potential mechanisms that may underlie this phenomenon, we analysed the impact of methotrexate on the individual components of the MetS. Methotrexate use is associated with lower TG, higher HDL and lower fasting glucose levels, but did not appear to be associated with either blood pressure or obesity (as assessed by waist circumference). Although the inflammatory process has been shown to directly modify many of these parameters ,45,46, t2A, thus promoting reverse cholesterol transport [per se, but as a consequence of concurrent folic acid supplementation. Folic acid has been shown to suppress plasma homocysteine levels [P < 0.001), this did not result in significantly lower levels of homocysteine (P = 0.406). We also failed to demonstrate any significant impact of folate levels on the development of the MetS in a binary logistic model . Thus, although this mechanism is still plausible it is not supported by the findings of this study. All of the possible underlying mechanisms of action require further investigation in studies designed specifically for the purpose. However, it remains that the observation described in this study may be important in the clinical context. Methotrexate may be the most appropriate first-line DMARD therapy for RA patients at particular risk of developing the metabolic syndrome, such as the elderly and obese with severe, active RA of relatively short duration.These observations provide interesting insights into the potential mode of action of MTX. One possible mode of action may be through alterations in adenosine concentrations. Extracellular adenosine levels are increased by methotrexate and are known to mediate its anti-inflammatory effect ,48. To aransport . Anothere levels . This mae levels . Insuline levels . Thus, sThe association between methotrexate use and the MetS carries further complexities. A strongly significant negative association is apparent with all but the WHO definition. This phenomenon may be explained by differences in the components and cut-off values used in the definitions. The WHO is the only definition to include albumin/creatinine ratio as a criterion, a factor that was not found to be influenced by methotrexate use. Conversely, it could be explained in differences in the sensitivity and specificity of each definition. The use of the NCEP criteria in RA has been questioned over recent years, because it has been found to confer a low sensitivity for predicting insulin resistance  and may P < 0.001). However, apart from diabetes mellitus , all other components of the MetS had a non-significant association (data not shown).Over recent years, scepticism has arisen over whether the MetS is independently associated with CVD . This isIn addition to the originality of most of the findings, this study has several other strengths. These include the use of all of the existing MetS criteria for the first time in RA, in the largest RA population studied thus far: these data can be used for benchmarking purposes to compare past or future studies, irrespective of the MetS criteria they use. Also, the detailed, prospective data collection minimised selection and recall bias as well as missing data and allowed meaningful sub-analysis with corrections for multiple potential confounders. Despite this, the cross-sectional design is a major limitation and precludes the ability to prove the causality or directionality of the associations found. Our study was also limited to secondary care RA patients from a single geographical location in the UK and did not assess the MetS in local general population controls, although another study of patients with diabetes from the geographically neighbouring (6 miles) area of Wolverhampton suggest that the local population is demographically representative of the total UK population . We cannThe MetS is common among RA patients, and may contribute significantly to their excess cardiovascular morbidity and mortality. In order to aggressively address this issue and minimise the associated risk we suggest that the NCEP 2004 criteria should be used as an annual screening tool in RA patients over the age of 60 years to identify RA patients with the MetS. Consideration should be given to using methotrexate with folate supplementation as first-line DMARD therapy in RA patients deemed to be at the highest risk, such as the elderly with early severe active disease.Apo: apolipoprotein; CI: confidence interval; CRP: C-reactive protein; CVD: cardiovascular disease; DAS28: 28-joint disease assessment score; DMARD: disease-modifying anti-rheumatic drugs; EGIR: European Group for Study of Insulin Resistance; ESR: erythrocyte sedimentation rate; HAQ: health assessment questionnaire; HDL: high-density lipoprotein; HOMA IR: homeostasis model assessment of insulin resistance; IDF: International Diabetes Federation; LDL: low-density lipoprotein; MetS: metabolic syndrome; NCEP: National Cholesterol Education Programme; NSAIDs: non-steroidal anti-inflammatory drugs; OR: odds ratio; QUICKI: quantitative insulin sensitivity check index; RA: rheumatoid arthritis; TC: total cholesterol; TG: triglycerides; TNF: tumour necrosis factor; WHO: World Health Organization.The authors declare that they have no competing interests.TET analysed and interpreted the data and drafted the manuscript. VFP acquired, analysed and interpreted the data. HJ drafted the manuscript. KMD acquired the data. GDK made substantial contributions to the conception and design of the study and revised the draft manuscript. All authors read and approved the final manuscript.