There has been no previous population-based study reporting the prevalence and risk factors of male lower urinary tract symptoms (LUTS) among men in mainland China. This cross-sectional study was conducted from 2013 to 2014 in three representative provinces of China: Guangdong, Hubei and Jiangsu. 3250 individuals participated in the interviews, which involved a questionnaire covering sociodemographic characteristics, lifestyle, dietary patterns and the International Prostate Symptom Score (IPSS). Blood was collected for lipids, glucose, insulin and reproductive hormone tests. The incidences of LUTS and its obstructive and irritative symptoms were calculated. Risk factors for LUTS were identified using multivariable logistic regression analysis. The prevalence of moderate to severe LUTS and its obstructive and irritative symptoms was 14.3%, 13.1% and 16.1%, respectively, and increased with age. The prevalence in Guangdong was much lower than that in Hubei and Jiangsu in different ages. Increased fasting plasma glucose and decreased HDL-C levels were associated with an increased risk of moderate to severe LUTS . Free testosterone < 0.22 ng/ml decreased the risk of moderate to severe LUTS and obstructive and irritative symptoms by about 20–30%. An inadequate daily intake of vegetables, fruit and water significantly increased the risk of LUTS by 1.3–to 2.0 times. In conclusion, the prevalence of LUTS in Chinese men is high and increases with age. Dietary patterns may be critical for the development of LUTS. Thus, dietary modifications could be a useful strategy for preventing the development of LUTS. Lower urinary tract symptoms (LUTS) is one of the most common clinical complaints in elderly men and has a significant effect on their quality of life . LUTS isWhile the identification of risk factors is important for the prevention of LUTS, apart from advancing age, there have been inconsistent conclusions about what may constitute a risk. Some studies have reported alcohol and cigarette consumption as an important risk factor for LUTS , 13. OthThe objective of this population-based study was thus to investigate the prevalence of LUTS in three representative provinces of China. We also aimed to evaluate the risk factors involved in the development of LUTS and its obstructive and irritative symptoms.Out of the 3358 volunteers who came to the clinics, 3283 were eligible for the study based on the inclusion and exclusion criteria outlined above. All of these volunteers were asked to complete the questionnaires, but only 3250 were used in the analysis because the other 33 questionnaires had unintelligible or missing information. Of these, 1087 participants came from Guangdong, 1037 from Hubei and 1126 from Jiangsu . For serum lipid levels, the HDL-C from subjects with moderate LUTS (1.7 mmol/l) was lower than subjects with mild (1.8 mmol/l) and severe LUTS (1.8 mmol/l) (p = 0.005). Nevertheless, the TC levels in subjects with severe LUTS (5.4 mmol/l) were significantly lower than for subjects with mild (5.7 mmol/l) and severe (5.5 mmol/l) LUTS (p = 0.008). As for testosterone levels, TT, but not FT, was higher in severe LUTS subjects (18.2 nmol/l) than in mild (16.4 nmol/l) and moderate (17.1 nmol/l) LUTS subjects (p = 0.03) , 366 subjects were diagnosed with moderate LUTS (IPSS 8–19) and 99 with severe LUTS (IPSS > 19). Age and BMI were significantly different among the groups , whereas HDL-C and TC were negatively related to IPSS . The results also showed a positive correlation between age, BMI, FBG and QoL , and a negative correlation between SBP, HDL-C, TC and QoL . Age was positively associated with obstructive and irritative symptoms , whereas HDL-C and TC were negatively correlated with both obstructive and irritative symptoms . Additionally, there was a positive relationship between HOMA-IR and irritative symptoms , and between FT levels and obstructive symptoms  . However, it remains a controversial question whether testosterone replacement therapy for the treatment of LOH effects LUTS , 38. FurThis study has several strengths. First, as a multicentre study, a large sample was selected from three representative areas. Second, the same group of well-trained urologists collected the data with good consistency. Additionally, we included a series of confounders, including demography, life habits, dietary patterns and some important serum parameters to draw a reliable conclusion. This study also has several limitations. On one hand, as a cross-sectional study, it was not possible to ascertain risk factors, and longitudinal studies are needed to better understand the development of LUTS. On the other hand, the diet questionnaire did not collect information on the detailed nutrients, which would be useful to further investigate the effect of dietary patterns on LUTS.In conclusion, Chinese adult men also have a high prevalence of LUTS and the prevalence varies significantly in different areas, although the prevalence is much lower than that in most other countries. Our results underline age as the strongest risk factor for LUTS. Increased FBG and decreased HDL-C levels were associated with an increased risk of LUTS. We also found evidence that dietary patterns, including inadequate daily intake of vegetables, fruit and water, increased the risk of LUTS. Although further studies are needed to confirm the results, it is possible that dietary modification could be useful for preventing the development of LUTS.This project was supported by the 12th Five-Year Plan of National Science and Technology of China (2012BAI32B03) and is part of the project “Reproductive health survey of Chinese middle-aged and elderly men.” The study proposal was approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (No. 20130311), and conducted according to their guidelines for research. All participants provided their full written informed consent.The study involved a cross-sectional multicentre survey carried out from March 2013 to May 2014, as described in our previous study . Three pThe subjects who participated in the study were required to meet the following inclusion criteria: 1) aged over 18 years old, 2) normally developed and 3) could communicate with the researchers and complete the questionnaires. The exclusion criteria included: 1) severe brain, cardiovascular, or reproductive endocrine disorders, kidney disease or prostatitis, 2) previous diagnosis of prostate cancer, 3) hormone replacement therapy in the past 3 months or 4) recent rectal examination, prostate biopsy or cystoscopy. All the subjects were required to complete a routine examination, questionnaire and blood collection.The routine examination involved height, weight and blood pressure measurements. Body Mass Index (BMI) was calculated by dividing weight (kg) by the square of height (m). Blood pressure, including systolic blood pressure (SBP) and diastolic blood pressure (DBP), were measured in the sitting position after a 5-min rest by using a mercury sphygmomanometer. SBP ≥ 140 mmHg and/or DBP ≥ 90 mmHg indicated hypertension. Hypertension was also diagnosed in the participants based on any previous diagnosis by doctors. The questionnaire involved a series of detailed questions, covering age, residence , education, history of hypertension, diabetes mellitus, dyslipidemia and treatment, history of cigarette and alcohol consumption, dietary habits and the the International Prostate Symptom Score (IPSS). Smoking status was classified into three groups: former (at least 100 cigarettes smoked in a lifetime), current and never smoking. Current smoking was further categorised into ≤ 20 and > 20 cigarettes per day based on the number of cigarettes consumed daily during the past year. Drinking status was similarly defined as former, current (at least 12 drinks in the previous year) or never drinking. For current drinkers, the estimation of usual weekly alcohol consumption was calculated by combining the intake of beer and wine together. Approximately, 12 bottles of beer was equivalent to 0.5 kg of alcohol. Current drinking was further categorised into ≤ 1 and > 1 kg/week based on the alcohol intake amount in the past year. For dietary habits, we mainly focused on the daily intake of vegetables, fruit and water. To obtain the information conveniently and reliably, we used standard containers as references to reflect the daily intake of vegetables, fruit and water. The participants were classified into different groups according to their estimated average daily intake of vegetables, fruit and water in the past year: vegetables: < 100 g, 100–200 g, 200–300 g and > 300 g per day; fruit :almost none, < 100 g, 100–200 g and > 200 g per day; and water: < 500 ml, 500–1000 ml, 1000–1500 ml and > 1500 ml per day.The IPSS is an internationally recognised valid method for the evaluation of LUTS and based on the answers to seven questions, concerned with the feeling of incomplete bladder emptying, frequency, intermittency, urgency, weak stream, straining and nocturia. The answers for each question are assigned points from 0 to 5. The total score can therefore range from 0 to 35 . It also contains a question about the quality of life (Qol), which is assigned a score from 1 to 6. The QoL scores were defined as mild (0–2) or moderate to severe (2–6). The questions 1, 3, 5 and 6 could also be categorised as the assessment of obstructive symptoms (≥ 5 out of 20 points was considered to indicate the presence of moderate to severe obstructive symptoms). The questions 2, 4 and 7 were categorised as the evaluation of irritative symptoms (≥ 4 out of 15 points was considered to indicate the presence of moderate to severe irritative symptoms) .After completing the questionnaire, venous blood samples were collected from the participants between 7:00–10:00 am after overnight fasting. The samples were stored at each centre temporarily and were all tested in the reproductive centre of Tongji Medical College. Serum total testosterone (TT) and sex hormone-binding globulin (SHBG) were tested using a chemiluminescent immunoassay method on a UniCel DxI 800 analyser . Free testosterone (FT) concentration was calculated by the Vermeulen formula . The serp value < 0.05 was considered for statistical significance. Data analysis was performed using SPSS 17.0 .Sociodemographic characteristics, lifestyle, dietary patterns and the prevalence of moderate to severe LUTS, QoL and obstructive and irritative symptoms in each centre were compared using Chi-square tests. The average age, BMI and serum parameters in each centre were compared with the analysis of one-way ANOVA. The participants were categorised into three groups of mild, moderate and severe LUTS according to their IPSS scores. A one-way ANOVA was performed to evaluate the difference of age, BMI, blood pressure and serum parameters across the ordered categories. The correlation of the IPSS, QoL and obstructive and irritative components with selected parameters was assessed using Pearson's partial correlation (controlling for the age of the subjects). Additionally, two multi-variable logistic regression models were used to assess the association between sociodemographic characteristics, lifestyle, dietary patterns and the risk of moderate to severe LUTS, QoL and the obstructive and irritative symptoms. First, we only adjusted for age because it was the main factor affecting the development of LUTS. Then, we adjusted all of the parameters except for research centre and TT, because the research centre had a collinearity with age and TT was not as important as FT. A two-sided


A high body mass index (BMI) has been proposed as an important risk factor for pancreatic cancer. However, this association of BMI with pancreatic cancer risk has not been confirmed in Asian populations.We evaluated the association between BMI (either at baseline or during early adulthood) and pancreatic cancer risk by conducting a pooled analysis of nine population-based prospective cohort studies in Japan with more than 340,000 subjects. Summary hazard ratios (HRs) were estimated by pooling study-specific HRs for unified BMI categories with a random-effects model.2 compared with 23 to <25 kg/m2, adjusted HR 1.71; 95% confidence interval [CI], 1.03–2.86). A J-shaped association between BMI during early adulthood and pancreatic cancer incidence was seen in men. In contrast, we observed no clear association among women, although there may be a positive linear association between BMI at baseline and the risk of pancreatic cancer .Among Japanese men, being obese at baseline was associated with a higher risk of pancreatic cancer incidence (≥30 kg/mPooling of data from cohort studies with a considerable number of Japanese subjects revealed a significant positive association between obesity and pancreatic cancer risk among men. This information indicates that strategies that effectively prevent obesity among men might lead to a reduced burden of pancreatic cancer, especially in Asian populations. Moreover, an early detection method for pancreatic cancer has yet to be established. Efforts to reduce the burden of pancreatic cancer should therefore be strongly focused on primary prevention.Pancreatic cancer is a major cause of cancer mortality in developed countries.5 Most of the findings on which this conclusion was came from Western populations, however, and it remains unclear whether it is also applicable to Asian populations, which substantially differ from Western populations in the prevalence of overweight and also in dietary factors, lifestyle characteristics, and genetic background. In fact, findings from prospective studies in Japan have been inconsistent,6–9 while two pooled analyses of pancreatic cancer mortality from Asian countries reported that there was no clear association.10,11The World Cancer Research Fund (WCRF) and American Institute of Cancer Research (AICR) concluded that the positive association between body-mass index (BMI) and pancreatic cancer risk is convincing.6,8,12–15 analyzing prospective data have examined the association between BMI during early adulthood (ages 18–21 years) and the risk of pancreatic cancer, with generally consistent evidence: all studies except one8 suggested a positive association between the two. The WCRF/AICR stated that greater childhood growth (marked as BMI at age ∼20 years) is a probable cause of pancreatic cancer.5 With respect to BMI change from younger age to older age, however, the results were not consistent.5,12–15 These findings are also mostly from Western populations, and evidence from Asian populations is again limited.Several studiesEvidence to date on the association between BMI and pancreatic cancer incidence from pooled analyses with substantial population sizes in Asia is scarce. In the present study, we evaluated the association between BMI and pancreatic cancer risk in Japan by conducting a pooled analysis of nine population-based prospective cohort studies comprising >340,000 subjects with unified BMI categories.16–18 In brief, the present study was conducted using data from nine representative ongoing large-scale population-based cohort studies in Japan, namely (i) the Japan Public Health Center-based Prospective Study (JPHC-I),19 (ii) the Japan Public Health Center-based Prospective Study (JPHC-II),19 (iii) the Japan Collaborative Cohort Study (JACC),20 (iv) the Miyagi Cohort Study (MIYAGI-I),21 (v) the Three-Prefecture Cohort Study in Miyagi (MIYAGI-II),22 (vi) the Three-Prefecture Cohort Study in Aichi (AICHI),22 (vii) the Takayama Study (TAKAYAMA),8 (viii) the Ohsaki Cohort Study (OHSAKI),23 and (ix) the Three-Prefecture Cohort Study in Osaka (OSAKA)22  have already published results on the association between BMI and the risk of pancreatic cancer in the respective cohort.24 0.97 and 0.85 in MIYAGI-I,25 and 0.93 and 0.97 in OHSAKI.26 Correlation coefficients for comparisons of BMI estimated from the questionnaire with BMI from the actually measured weight and height were 0.89 in men and 0.90 in women in JPHC-I and -II,27 0.91 for both sexes in MIYAGI-I,25 and 0.88 for both sexes in OHSAKI.26 JACC, which did not have information on the validation of BMI, utilized the same questions on weight and height as MIYAGI-I. MIYAGI-II, AICHI, and OSAKA also had no information on the validation of BMI, but used a similar question to that in JPHC-I and -II. Important covariates for pancreatic cancer, including smoking history, alcohol consumption, and diabetes, were also collected via a self-administered questionnaire.All studies collected information on height and weight at baseline using self-report. Five studies  collected self-reported, recalled weight during early adulthood (age 20 or 21). Body mass index (BMI) was calculated as weight in kilograms divided by the square of the height in meters in their questionnaires. Several studies have reported the validity of self-reported height and weight; respective correlation coefficients for height and weight in both sexes were 0.93 and 0.97 in TAKAYAMA,28 In OSAKA, sites of any cancers were coded using the International Classification of Diseases, Ninth Revision (ICD-9),29 except for one city in Osaka where the ICD 10th Revision (ICD-10)30 was used. Study outcome was defined as the incidence of pancreatic cancer  during the follow-up period of each study. Participants were followed from the date of completion of the baseline questionnaire  until the last date of follow-up in each study , date of death, date of loss to follow-up, or date of diagnosis of pancreatic cancer, whichever occurred first.We identified pancreatic cancer cases via local cancer registries or direct access to major local hospitals . In all 2) or extreme values of BMI change (more than 3 standard deviations from the mean). Patients with cancer at baseline were also excluded from all analyses.Participants were excluded from all analyses if they had missing or extreme values of BMI (BMI <14 or >40 kg/m2) at baseline was divided by sex-specific categories using identical cut points for men at <19, 19 to <21, 21 to <23, 23 to <25, 25 to <27, 27 to <30, and ≥30; and for women at <19, 19 to <21, 21 to <23, 23 to <25, 25 to <27, and ≥27. We defined 23 to <25 kg/m2 as the reference category. BMI (kg/m2) during early adulthood was divided using the same categories as BMI at baseline. BMI change (BMI at baseline–BMI during early adulthood) was classified using the following cut points for men: <−4, −4 to <−2, −2 to <2, 2 to <5, ≥5; and for women: <−2, −2 to <2, 2 to <5, 5 to <8, and ≥8. We defined −2 to <2 kg/m2 as the reference category. Smoking history was evaluated as pack-years, calculated by multiplying the number of packs consumed per day by the number of years of smoking, and then classified into three categories: 0, 0< and ≤20, and >20. Alcohol consumption for men was categorized into nondrinker, occasional drinker (less than once a week), and current drinker , and that for women as nondrinker, occasional drinker and current drinker (<23 and ≥23 ethanol g/day). Current drinkers were further categorized by the amount of alcohol consumed using cut points per 23 g ethanol, on the basis that some questionnaires collected data on the amount consumed in terms of one “go” (180 mL) of Japanese sake equivalent, which contains 23 g ethanol. A history of diabetes was categorized as “Yes” or “No”. Missing data of smoking history, alcohol consumption, and diabetes were coded using dummy variables.For categorical analyses, BMI  and their two-sided 95% confidence intervals (CI) of pancreatic cancer by each BMI category. We assessed the assumption of proportional hazards graphically using log-negative-log plot, and detected no major violations of the proportional hazards assumption. All studies analyzed two models in the estimation of HRs: model 1, age (and area within each study for JPHC and JACC)-adjusted HRs; and model 2, smoking history, alcohol consumption, and diabetes in addition to model 1. Model 2 of BMI change was additionally adjusted for BMI during early adulthood . We also evaluated HRs by excluding cases diagnosed early within three years after enrollment in each study. All analyses were performed by SAS version 9.3  or STATA version 14.1 .To be pooled in the estimation of summary statistics, we conducted study-specific analyses by sex.31 to obtain a single pooled estimate of the HR from the individual studies for each category.16 The extent of heterogeneity for each category was indicated by Cochran’s Q-statistic, which was considered statistically significant when P < 0.10. The I2-statistic was also reported to describe the percentage of total variation in the study-specific HRs which was due to heterogeneity.32 The dose-response relationship was examined using models in which actual BMI values were included as an explanatory variable, which would provide HRs by a 1-kg/m2 increase in BMI. Meta-analyses were done using the Stata’s ‘metan’ command (http://www.stata.com/stb/stb44).We applied a random-effects model2; accordingly, nine combinations were evaluated by setting BMI at baseline and BMI during early adulthood, with 21 to 25 as the reference category for both. This combined analysis was done using model 2 to estimate HRs. An interaction between BMI at baseline and BMI during early adulthood was assessed by adding a cross-product term  in the model.We also evaluated the joint effect of a combination of BMI at baseline and BMI during early adulthood. For this analysis, BMI at baseline and BMI during early adulthood were coded as <21, 21 to <25, and ≥25 kg/m2 accounted for 22.9% of males and 24.9% of females. These values are comparable to those reported in national surveys of the same age group.33The present pooled analysis included nine cohort studies comprising 345,799 subjects  with 1,593 incident pancreatic cancer cases  during 4,361,312 person-years of follow-up (average follow-up: 13.0 years) . Although HRs with the exclusion of early cases were not significant for those with a BMI at baseline ≥30, trends of association were consistent . Those with a BMI at baseline <21 kg/m2 appeared to be at slightly increased risk of pancreatic cancer, although without statistical significance. For BMI in early adulthood, we observed a J-shaped association with pancreatic cancer risk  , and estimated HR of pancreatic cancer by each category of BMI at baseline. After adjusting for age, area (within each study for JPHC and JACC), alcohol consumption, and history of diabetes, we observed similar tendency as the analysis without stratification Table  in the t2: aHR 0.55; 95% CI, 0.32–0.96) (P for interaction = 0.230). To assess the association between pancreatic cancer and BMI gain, we further evaluated the distribution of BMI during early adulthood in each category of BMI change  . Results32–0.96) , we obseI change . As show2, aHR 1.02; 95% CI, 1.00–1.05), although the P value was only marginally statistically significant (Table P for interaction = 0.333)  .This large study, the first pooled analysis to examine an association between BMI and pancreatic cancer incidence in the Japanese population, was conducted as a pooled analysis of data from nine population-based cohort studies comprising >340,000 subjects. Obesity at baseline was associated with a statistically significantly higher risk of pancreatic cancer incidence among Japanese men. For BMI during early adulthood, we observed a J-shaped association with pancreatic cancer incidence in men. Among Japanese women, in contrast, results suggested a positive linear association between BMI at baseline and pancreatic cancer incidence.13–15 This may be partly because of the small number of women in the higher BMI categories and the fact that we did not define the category that had the smallest risk of pancreatic cancer as the reference category. With respect to men, in contrast, a statistically significant increase in risk of pancreatic cancer was observed for the highest category of BMI at baseline (≥30 kg/m2), consistent with previous large studies from Western countries.12–15 In two pooled analyses of Asian populations that reported no clear association between BMI and pancreatic cancer risk,10,11 mortality was used as an end point. Using mortality instead of incidence is reasonable because pancreatic cancer has high case fatality and short survival time. On the other hand, early detection of pancreatic cancer is difficult, and diagnosis of pancreatic cancer requires advanced level of medical standards. Therefore, the two studies might include pancreatic cancer death without being diagnosed, which resulted in the possibility of misclassification for pancreatic cancer mortality. We consider that the difference of diagnostic accuracy may contribute to the discrepant finding. To date, only a few prospective studies have evaluated the association between pancreatic cancer and BMI at baseline in Japan.6–9 Three studies6,8,9 showed no significant association, while the fourth7 showed an inverse association among men and no association among women. The reason for this heterogeneity might be partly due to the limited statistical power of each study to detect the association. Our present pooled approach has likely resolved the statistical limitations of each of the individual studies, and the stability of the resulting estimation is therefore of marked importance to this field.Our study did not support the internationally reported association between obesity and pancreatic cancer among women.34 and three pooled analyses from Western countries13,14,35 showed a significant positive linear association, whereas a pooled analysis from Asian countries10 showed a non-significant positive linear association . In the present study, the aHR of BMI at baseline (per 1 kg/m2) was 0.97  in men and 1.02  in women  or obese (BMI ≥30 kg/m2) during early adulthood appeared to be associated with a higher risk of pancreatic cancer in men. In addition, men with a BMI <25 kg/m2 during early adulthood appeared to be at lower risk, even if they gained weight after early adulthood  in this large pooled analysis.Among other findings, a BMI gain ≥5 kg/m33,36 In addition, the most recent survey in Japan37 showed an increasing trend of overweight and obesity for male adults in all age groups, although the prevalence of obesity still remains low compared with Western populations. Our finding of a concrete association between pancreatic cancer risk and BMI suggests that the increased prevalence of overweight and obesity among Japanese men over decades might partly explain the recent increases in pancreatic cancer incidence in Japan.4Over the last several decades, average BMI in the Japanese population has increased constantly.38,39 which might contribute to the increase in pancreatic cancer incidence.40 In the present study, the significant positive association persisted when adjusted by history of diabetes, which indicated that obesity at baseline was associated with a higher risk of pancreatic cancer incidence that is independent of diabetes. However, since BMI partly could increase the risk of pancreatic cancer due to its effect on diabetes, diabetes may also be regarded as an intermediate variable.34 Indeed, it is difficult to exclude the effect of diabetes completely from the analysis. Therefore, this point should be investigated in future studies.On the other hand, the prevalence of diabetes has rapidly increased in recent decades,18 BMI was measured before pancreatic cancer incidence in all studies included in this pooled analysis, which excludes the possibility of selection bias and recall bias. In addition, the categories for BMI and the covariates used were identical across studies, which removed a potential source of heterogeneity that can occur when conducting a meta-analysis of published studies.17 Lastly, we observed non-significant heterogeneity of association across the studies.The present study has several strengths. It included most of the ongoing, large-scale prospective cohorts in Japan, with overlapping birth generations of the study subjects. The total number of subjects in this analysis is very large, warranting sufficient statistical power to detect an association between BMI and pancreatic cancer risk. Therefore, pooling of these cohorts allows for stable summary quantitative estimates of the effect of BMI among Japanese. Using incidence instead of mortality as an end point is advantageous because it enables the direct assessment of the risk contribution of BMI. In addition, because this study was not based on a meta-analysis of published studies, the possibility of publication bias is small.41 has shown that self-reported past body weight is highly correlated with measured weight, but that the accuracy of recall is influenced by age or elapsed time, current body size, and body weight variability. Therefore, although errors in self-reported information are unlikely to be strongly associated with the risk of pancreatic cancer incidence, the possibility remains of reporting bias, recall bias, and differential misclassification of BMI during early adulthood. With regard to BMI at baseline, most of the studies used validated questionnaires24–27 or equivalent questionnaires for current BMI measurement, and any impact of error in this analysis appears limited, if present at all. Second, we were unable to consider changes in BMI and potential confounders over time after enrollment because our analyses were conducted using only a baseline questionnaire. Third, although we considered potential confounders in the analysis, the possibility of residual confounding can never be fully excluded. Fourth, there is a possibility that the healthy participants with adequate BMI tend to move to the other regions for some reasons and are more likely lost to follow-up compared with the obese participants. This potential selection bias might have caused the underestimation of HRs for the participants with adequate BMI. Therefore, the result of the present study should be interpreted carefully. Lastly, as this is the aggregation of cohort studies, other anthropometric measurements, such as waist circumference or waist-to-hip ratio, were not evaluated because not all the studies collected such data. Epidemiological findings, mostly from Western populations, showed that alternative measures of adiposity might also be important indicators of pancreatic cancer risk.13,34,42 To promote understanding of the association between anthropometric factors and pancreatic cancer risk in Asian population, evaluation of comprehensive anthropometric measurements are needed in future studies.Several potential limitations of the present study warrant mention. First, all studies collected information on height and weight using self-report. Previous researchIn conclusion, this study revealed a possible association between BMI (either at baseline or during early adulthood) and pancreatic cancer incidence in an Asian population. Pooling of data from cohort studies with a considerable number of Japanese subjects revealed a significant positive association between obesity and pancreatic cancer risk among men. In contrast, we observed no clear association among women, although we did see a tendency toward a positive linear association between BMI at baseline and the risk of pancreatic cancer. This information indicates that reducing the burden of pancreatic cancer will require effective strategies to prevent obesity, particularly in Asian males.


Octopus vulgaris) is promising since the species has a relatively short lifecycle, rapid growth, and high food conversion ratios. However, recent attempts at successful paralarvae culture have failed due to slow growth and high mortality rates. Establishing an optimal nutritional regime for the paralarvae seems to be the impeding step in successful culture methods. Gaining a thorough knowledge of food regulation and assimilation is essential for paralarvae survival and longevity under culture conditions. The aim of this study, then, was to elucidate the characteristic metabolic organization of octopus paralarvae throughout an ontogenic period of 12 days post-hatching, as well as assess the effect of diet enrichment with live prey containing abundant marine phospholipids. Our results showed that throughout the ontogenic period studied, an increase in anaerobic metabolism took place largely due to an increased dependence of paralarvae on exogenous food. Our studies showed that this activity was supported by octopine dehydrogenase activity, with a less significant contribution of lactate dehydrogenase activity. Regarding aerobic metabolism, the use of amino acids was maintained for the duration of the experiment. Our studies also showed a significant increase in the rate of oxidation of fatty acids from 6 days after-hatching. A low, although sustained, capacity for de novo synthesis of glucose from amino acids and glycerol was also observed. Regardless of the composition of the food, glycerol kinase activity significantly increased a few days prior to a massive mortality event. This could be related to a metabolic imbalance in the redox state responsible for the high mortality. Thus, glycerol kinase might be used as an effective nutritional and welfare biomarker. The studies in this report also revealed the important finding that feeding larvae with phospholipid-enriched Artemia improved animal viability and welfare, significantly increasing the rate of survival and growth of paralarvae.The culture of the common octopus ( Octopus vulgaris in captivity. These practices remain promising since the species has a relatively short lifecycle, fast growth, and high food conversion ratios. However, under culture conditions, paralarvae of this cephalopod species show slow growth and high mortality rates, reflecting the main obstacle to successful culture. Nutritional deficiencies and food source imbalances are considered the primary causes for low paralarvae survival and growth, although other factors related to zootechnical conditions  cannot be ruled out  than LDH during anoxia, where the glycolytic pathway is prevalent.After the larval yolk reserves are exhausted, paralarvae depend solely on exogenous food. Octopuses in this stage survive on live prey, which are actively captured using bursts of anaerobic swimming Baldwin, . In vertAlthough metabolic capacities in adult cephalopods have been assessed in several studies  based on the European Union's directive on animal welfare for the protection of animals used for scientific purposes (Directive 2010/63/EU). Guidelines for the care and welfare of cephalopods proposed by Fiorito et al.  were folTwo experiments were carried out to characterize the metabolic profile in common octopus paralarvae. The first experiment analyzed the time course of metabolic capacities during the first 12 days of life. The second experiment analyzed the influence of food composition (with a diet rich in highly unsaturated fatty acids and phospholipids) on metabolic capacities and survival.Octopus vulgaris were captured by local fishermen using artisanal octopus traps in Tenerife coastal waters  and maintained in the facilities of the Oceanographic Centre of the Canary Islands (Spanish Institute of Oceanography). Adult specimens were kept in 1,000 L tanks (with a maximum density of 10 kg/tank) with water renovation (5 L/min), under oxygen saturation conditions and low light intensity. Two batches of paralarvae obtained from this broodstock were used in the experiment described below.To carry out both experiments, a total of 20 adult Artemia to go through a 500 μm outflow mesh located in the middle of the tank. Moderated flux aeration stones were placed on the edge of the tanks. Green water  was added at 8:00. The natural photoperiod was attained at a maximum intensity (around mid-day) of 300 lx. Temperature and oxygen were measured daily, and nitrite, ammonium, and salinity once a week  enriched for 20 h after hatching with freeze dried Isochrysis galbana . Artemia was supplied at 0.3 Artemia/mL divided in three times a day . In order to avoid enrichment lost, Artemia cultures were kept in the dark at 4°C with soft aeration until the moment of feeding.A total of 15,000 paralarvae, , were reared in triplicate during 12 days in 1,000 L black fiberglass cylinder-conical tanks with a flow-through seawater system at 60 mL/s from 18:00 to 8:00 . The renovation flow allowed the unfed Artemia to go through a 500 μm outflow mesh located in the middle of the tanks. Two moderated flux aeration stones were placed in front each other in the edges of the tanks. Green-water system using 5 × 105 cell/mL of Nannochloropsis sp  was added to the tanks before illumination. Temperature and oxygen were measured daily, and nitrite, ammonium and salinity once a week  were reared over 28 days in 500 L black fiberglass cylinder-conical tanks. Two fluorescent lights (OSRAM Dulux superstar 36W/840) were placed above each tank to attain 700 lx focused in the middle of the tank surface with a 12L:12D photoperiod (8:00–20:00). A flow-through seawater system equipped with 20, 5, and 1 μm filter cartridges and UV lamps was used. A water flow per tank of 1 L/min  was applied from 18:00 to 8:00. In similar protocol to the first experiment, the renovation flow allowed the unfed Artemia used in the first experiment, but in this case, the control group (C) was enriched with microalgae  or with an experimental diet of Marine Lecithin LC 60® . Each treatment was carried out in triplicate. To quantitate Artemia size over the duration of the experimental period, three prey sizes were used: first, nauplii from day 0 to 3; second, metanauplii from day 4 to 11; and third, 8 day old metanauplii from day 12 to 27. Enrichments and on-growing of Artemia were made according to Garrido et al. . In order to avoid enrichment loss, Artemia cultures were kept in the dark at 4°C with soft aeration until the moment of feeding.Paralarvae were fed with the same o et al. . ArtemiaNo dry weight was determined in Experiment 1. In the second experiment, 15 individuals' dry weight (DW) was determined for each treatment at day 0, 12, and 28. Paralarvae were euthanized in chilled seawater (−2°C), washed in distilled water, oven dried  and weighed. Specific growth rate  was calculated as (Ln DWf-Ln DWi) 100/(tf-ti), where DWf and DWi are the dry weight at final time (tf) and initial time (ti) respectively. Survival was assessed in both experiments at the termination of the experiment. Survival  was calculated as S = 100 Xf/(Xi-Xs), where Xf is the number of live individuals at the end of experiment, Xi is the initial number of individuals and Xs is the number of individuals sacrificed during the experiment.Due to the usual mortality in this type of culture and the large size of samples that require analyses, sampling could only be extended up to 12 days. Thus, sample collections in experiment 1 were carried out at 0, 3, 6, 9, and 12 days.Likewise in the second experiment, samples were taken at 12 days from C and LC groups, and also at 28 days in LC groups since this treatment showed greater survival allowing for a suitable sample size.Pools of 300 paralarvae per tank were taken in each sampling. Paralarvae were euthanized using ice seawater (−2°C), frozen in liquid nitrogen and stored at −80°C until further analysis.Pooled paralarvae of each sample were homogenized in four volumes of ice-cold 100 mM Tris-HCl buffer containing 0.1 mM EDTA and 0.1% (v/v) Triton X-100, pH 7.8. All procedures were performed on ice. Homogenates were centrifuged at 30,000 × g for 30 min at 4°C and the resultant supernatants were kept in aliquots and stored at −80°C for further enzyme assays.−1 cm−1, respectively.All enzyme assays were performed at 25°C using a PowerWaveX microplate scanning spectrophotometer  and run in duplicate in 96-well microplates . The optimal substrate and protein concentrations for the measurement of maximal activity for each enzyme in each tissue were established by preliminary assays. The millimolar extinction coefficients used for NADH/NADPH and DTNB, were 6.22 and 13.6 mMActivities of fructose 1,6-bisphosphatase , glycerol kinase , pyruvate kinase , glucose-6-phosphate dehydrogenase , citrate synthase , β-hydroxyacyl CoA dehydrogenase , glutamate pyruvate transaminase , glutamate oxaloacetate transaminase , and glutamate dehydrogenase  were determined as previously described by Hidalgo et al. . OctopinSoluble protein concentration in homogenates was analyzed using the method of Bradford , with bot-test. Multiple comparisons in experiment 1 were performed by mean of one-way ANOVA test and Tukey's HSD post hoc test. When normal distribution and/or homoscedasticity were not achieved, data were subjected to Kruskall–Wallis non-parametric test, followed by Games-Howell non-parametric multiple comparison test .Data were checked for normal distribution with one-sample Kolmogorov-Smirnoff test, as well as for homogeneity of variances with the Levene's test Zar,  and tranest Zar, . StatistTable The PK (glycolysis), G6PDH (NADPH provision), CS (oxidative metabolism), and GPT (amino acid catabolism) activities did not show significant changes during the period analyzed. Nevertheless, FBPase activity decrease at day 3, to subsequently increase and remain unchanged until the end of the experimental period.Activity of enzymes involved in anaerobic metabolism revealed that LDH reached maximum activity on day 6, after decreasing to values similar to the 0 day. ODH activity increased progressively from day 3 of life, reaching a significant maximum at 12 days old.Activity of β-oxidation of fatty acids (HOAD) showed a significant increase from day 6 onwards. Enzymes involved in protein metabolism, specifically, the activity of GOT, showed significant increase from day 3 post-hatching, remaining significantly higher until day 9. However, at day 12 the activity was similar to that of day 0. In turn, GDH activity increased gradually, reaching statistical significance at day 9 post-hatching. Again on day 12, activity significantly decreased.The HOAD/CS ratio increased since the sixth day forward, and the GPT/CS ratio was higher in newly hatched paralarvae and decreased from the third day onwards Figure .Artemia enriched with marine phospholipids on metabolic enzyme activity of hatchlings, 12 day and 28 day old paralarvae was studied. Table Artemia enriched with phytoplankton (Control Diet) or Artemia enriched with Marine Lecithin (LC diet). The results showed that DW was significantly higher in paralarvae from the LC group compared to the control group at 12 days old (P < 0.05). Also, survival in 28 day old paralarvae was significantly higher in the LC group compared to the control group (P < 0.05).In experiment 2, the effect of feeding with Artemia, and in 28 day old paralarvae fed on LC60-enriched Artemia (28LC). Of these activities, ODH, CS, HOAD, and GOT increased significantly in 12 day old paralarvae. The only difference among 12C and 12LC paralarvae was the significantly higher GyK activity in 12C. The 28 day old paralarvae fed on LC60-enriched Artemia (28LC) showed significant increases in GyK, PK, G6PDH, LDH, GOT, and GDH activities with respect to 12LC.Table Common octopus paralarvae have a planktonic lifestyle that lasts until the first 30–60 days of life. Remaining yolk reserves provide energy substrates during the first few days post-hatching, however paralarvae immediately begin to actively capture live prey . This would ensure the provision of oxaloacetate necessary for the CS reaction. The data indicate that aspartate (a substrate of GOT) and glutamate, representing almost half of the non-essential amino acids in the body of cephalopods, are relevant (Villanueva et al., In addition to the energy demands imposed by the capture of prey supported by anaerobic metabolism, paralarvae need a supply of energy for growth, food processing, and maintenance of routine activity. These demands are typically supported by aerobic metabolism. In the present study, CS activity did not show significant changes during the ontogenic period analyzed, although the slight tendency to increase from third day could be related to growth and development processes that impose greater energy needs. GOT activity increased significantly in parallel to the observed changes in CS, indicating a positive correlation between both activities (Artemia showed a 12% survival rate after 28 days, whereas less than 2% of the C group survived before that day. The results for SGR in the present study were similar to those reported previously for O. vulgaris paralarvae by Seixas et al. (Artemia as live prey. Conversely, SGR results were lower than those previously found when LC60-enriched Artemia was provided (Garrido et al., The main result in this nutritional assay was that the paralarvae that were fed on LC60-enriched s et al.  and Vills et al.  using HUArtemia, oxidation rate of fatty acids did not increase in LC group, either at 12 or 28 days. This result may indicate that rather than being used as fuel, the higher amount of lipids could be contributing to the development of the nervous system, essential in such early stages (Nixon and Mangold, O. vulgaris paralarvae hatchlings represents approximately one quarter of the animal's fresh weight (Packard and Albergoni, Regarding the possible beneficial effect of food enrichment, in spite of the increased lipid content of LC60-enriched In searching for a possible metabolic biomarker, GyK might be considered, since a significant increased activity was observed in both 12C and 28LC groups and a few days later an event of massive mortality took place. Although the role of G3P in cephalopods is controversial (Storey and Hochachka, de novo synthesis of glucose from amino acids and glycerol was also observed. Feeding of larvae with phospholipid-enriched Artemia improved animal welfare, since there was a significant increase in the rate of survival and growth. Regardless of the composition of the food, GyK activity significantly increased a few days prior to a massive mortality event. This data may, in fact, indicate an imbalance in the redox state that could be responsible for the observed high mortality. Thus, GyK might be used as a nutritional and welfare biomarker.In summary, throughout the ontogenic period studied, an increase in anaerobic metabolism takes place, probably related to the increased dependence of paralarvae on exogenous food. Such activity is mainly supported by ODH activity, although LDH activity also contributes to the process. Regarding fuels that support aerobic metabolism, the use of amino acids was maintained and a significant increase in the rate of fatty acid oxidation was observed from the sixth day post-hatching. A low, albeit sustained, capacity for AM Analytical procedures, interpretation of the findings, and design, writing, and revision of the manuscript. GC Analysis, interpretation of the findings, and design, writing, and revision of the manuscript. MH Analysis and interpretation of the findings, and revision of the manuscript. DG and MM octopus paralarvae cultures and execution of the experiments, revision of the manuscript. EA design and execution of experiments, interpretation of results and revision of the manuscript. All authors read and approved the submitted manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AT and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


A greenhouse pot test was conducted to study the impacts of replacing mineral fertilizer with organic fertilizers for one full growing period on soil fertility, tomato yield and quality using soils with different tomato planting history. Four types of fertilization regimes were compared: (1) conventional fertilizer with urea, (2) chicken manure compost, (3) vermicompost, and (4) no fertilizer. The effects on plant growth, yield and fruit quality and soil properties (including microbial biomass carbon and nitrogen,  Intensive agricultural production using inorganic fertilizers has led to increased yield, albeit at the expense of poor product quality, particularly under protected cultivation.Lycopersicon esculentum) is one of the most widely grown vegetables in Liaoning Province, where the tomato-growing area encompassed more than 85,000 hectares in 2014 , soil nutrient enrichment , 5, and 20 years.Eisenia fetida) to semi-decomposed cow manure; before use, the vermicompost was passed through a 2 mm sieve to remove the earthworms. Chicken manure compost was purchased from Ruiyuande Biotechnology Co., Ltd. . The nutrient content of the fertilizer is shown in Table A pot experiment was conducted from March to June of 2016 at the Greenhouse Base  of Shenyang Agricultural University in Shenyang City, Liaoning Province. Polyethylene plastic pots with a diameter of 30 cm and a height of 28 cm were used. Each pot was filled with 15 kg of air-dried soil that had been passed through 1 cm sieve. The basic physical and chemical properties of the soil are shown in Table 2O5, and 0.4 g K2O per kg soil that is equal to 900 kg N, 560 kg P2O5, and 900 kg K2O per hectare), compost (chicken manure compost), and vermicompost. A total of 8.30 g of chicken manure compost per kg soil (about 19 t chicken manure compost per hectare) and 13.10 g of vermicompost per kg soil (about 30 t vermicompost per hectare) was added to the compost and vermicompost treatments, respectively . Each treatment was repeated five times with a complete random arrangement within each block. The four fertilization treatments were CK (control: no fertilizer), urea , when the tomato seedlings had grown to the 3- to 4-leaf stage, seedlings of a consistent size were transplanted into each pot. The plants were watered to 100% field water-holding capacity using tap water. After this watering, all the treatments received the same amount of irrigation. Soil moisture was maintained at 18–20%  during the experiment, as determined gravimetrically, with the addition of tap water when necessary.The pots within a block were arranged randomly in the greenhouse, with the positions re-randomized every week. The greenhouse temperature ranged from 15 to 35°C. Natural light was supplied with no supplementary light.During the entire growth period of the tomato plants, soil samples were collected in duplicate from each pot on March 27 (seedling stage), April 26 (flowering stage), May 26 (fruiting stage), and June 25 . Part of the fresh soil was passed through a 2 mm sieve and stored at 4°C for other measurements, and the remainder of the soil was air-dried and passed through a 0.9 and a 0.15 mm sieve for the determination of physical and chemical properties.Soil physical and chemical properties were determined as previously described Bao, . The soi2SO4 (1/4 w/v). The MBC and MBN contents in the extract were measured with a Multi N/C®; 3100 analyzer  and were calculated using calibration factors of 0.45 , and the The soil microbial biomass carbon (MBC) and microbial biomass nitrogen (MBN) were determined according to Brookes et al.  and VancEach tomato fruit was weighed, and the total yield per plant was calculated. The plant height, stem diameter and leaf chlorophyll content (SPAD) were measured at 15 days (March 27), 45 days (April 26), 75 days (May 26), and 105 days (June 25) after transplanting. The chlorophyll content of the leaves at the same part of the second branch of the plant was measured using the CCM-200 Plus Chlorophyll Analyzer , and the average of three measurements was calculated. The chlorophyll content measurement was based on the difference in the light absorption rate at 653 and 931 nm.Fruit quality was determined in tomatoes of similar color when the second ear fruit reached at 75–85% maturity Li, . The vitThe data was presented as the arithmetic mean values with standard errors. One-way ANOVA was performed to compare the effects of different fertilization treatments on tomato yield and fruit quality. Two-ways ANOVA was done on stem diameter, plant height, SPAD, P < 0.001). There was no interaction for these three parameters between sampling days and fertilizer treatments . Overall, compared with CK treatment, the other fertilizer treatments (particularly compost and urea) largely promoted soil EC at the beginning of the experiment. Over time, soil EC decreased to a much lower level than at the beginning . At the final sampling period, after the entire growth stage, soil EC exhibited the following order: compost > vermicompost > urea > CK, regardless of years of continuous cropping.In all three types of soil, both soil EC and pH were influenced by sampling time, fertilizer treatment, and the interaction between them . In soil with 0 cropping years, treatment with urea, compost and vermicompost produced average (from four sampling times) reductions in soil pH of −0.45, −0.27, and −0.26, respectively, compared with CK treatment; after 5 cropping years, the reduction was −0.46, −0.42, and −0.42, respectively, compared with CK treatment; and after 20 cropping years, the reduction was −0.59, −0.23, and −0.14, respectively, compared with CK. At the final growth stage, soil pH exhibited the following orders: urea ≤ compost < vermicompost < CK after 0 cropping years; compost ≤ vermicompost < urea < CK after 5 cropping years; and urea < compost < CK < vermicompost after 20 cropping years.Soil pH was highest in the CK group at almost every stage and cropping span, whereas urea treatment produced the lowest soil pH at almost every stage in soil with 0 and 20 years cropping history  in the yield, sugar/acid ratio, vitamin C and soluble sugar of fruit were the highest in soil with 0 cropping years. These differences were significantly higher than those after 20 cropping years while the differences after 5 cropping years were between 0 and 20 years. Furthermore, the decrease (%) in nitrate and organic acids in soil with 0 cropping years were the highest among the three soil treatments.r = 0.459, P < 0.01; r = 0.447, P < 0.01). Vitamin C, soluble sugar, acid content and sugar/acid ratio were significantly correlated with the water-soluble organic carbon, A correlation matrix among the different yields, quality parameters, and soil parameters found in the present study is presented in Table Cucumis melo L.). Singh and Chauhan (Phaseolus vulgaris), finding that the vermicompost promoted the germination of seeds, the height of the plant, the number of leaves of each plant, length of leaves and width of leaves of each plant and the yield. Thus, applying organic fertilizer produced an improvement in plants even at the beginning of the growth cycle. The data analysis confirmed that the final tomato yield was correlated with soil N status , resulting in higher electrical conductivity even at the end of the growing period. Hashemimajd et al. (The results of the present study show that vermicompost and compost had a significant impact on soil microbial C and N values Figure , which ad et al.  comparedd et al.  even foud et al. , but didIn conclusion, our results suggest that vermicompost can improve the biochemical properties of soil under different years of continuous growth, thereby increasing tomato growth, yield, and fruit quality compared with urea. Moreover, considering the higher electrical conductivity and lower soil pH achieved by applying compost, vermicompost could be a better recommendation for soils. Especially for the new soil without tomato planting history, vermicompost can produce better improvements in fruit yield and quality compared with old tomato-planting soils Table . HoweverX-XW analyzed the data, wrote the first manuscript and modified it. FZ performed the experiment, collected the samples and collected the data. GZ and YZ assisted to manage the experiment and collect the data. LY designed the experiments, revised the manuscript and applied funding to support the study. All the authors discussed the results and commented on the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Since low counts trigger extensive investigations, determining prognostic cutoffs especially for different ethnicities and races is critical.Although absolute neutrophil counts (ANC) below 1.5x10A multiethnic cohort of 27,760 subjects, 65 years old and above, was utilized to evaluate the association of neutropenia with overall survival in different ethnicities and races.3/uL in non-Hispanic whites, 3.6±1.57x103/uL in non-Hispanic blacks and 4.3±1.54x103/uL in Hispanics (p<0.001). An ANC below 1.5x103/uL was associated with significantly shorter overall survival among whites , but not in blacks  or Hispanics , after adjustment for age, sex, comorbidities, anemia and thrombocytopenia. Using Cox regression multivariable models, an ANC below 1.1x103/uL in blacks was found to be associated with increased mortality . We found no association between neutropenia and mortality at any ANC cutoff in elderly Hispanics. In conclusion, neutropenia was found to be an independent prognostic variable in the elderly, when determined in race-specific manner. Most importantly, a cutoff of 1.1x103 neutrophils/uL may be a more prognostically relevant marker in elderly blacks and could serve as a novel threshold for further evaluation and intervention in this population.The mean ANC was 4.6±1.51x10 An absolute neutrophil count (ANC) of less than 1.5 x 10tropenia , has not3/uL has been conventionally considered the threshold below which increased susceptibility to infections is incurred, this may not be accurate across the different ethnic and racial groups. Moreover, the association between neutrophil count and morbidity or mortality risk may not be mediated by infection alone [Racial and ethnic differences in the distribution of circulating neutrophils were first observed as early as 1941 by Forbes . This obon alone –18.There are no large studies that have examined the relationship of low neutrophil counts with overall survival. It was, therefore, the aim of this study to 1) look at the association of racial/ethnic variations in neutrophil counts with mortality risk and 2) determine a race/ethnicity-specific lower limit for circulating neutrophils.With a primary hypothesis that the association of neutropenia, as currently defined, with overall survival differs between Caucasians and individuals of African ancestry, we wanted to further explore for a new race-specific neutrophil cutoff that would better identify the clinically important cases of neutropenia. We focused on subjects above the age of 65 years, an age population frequently seen with cytopenias, and at the same time, particularly vulnerable to their negative effects , 20.3/uL in non-Hispanic whites, 3.6 ± 1.57 x 103/uL in non-Hispanic blacks and 4.3 ± 1.54 x 103/uL in Hispanics (p<0.001). Neutropenia was prevalent in 3.0% of blacks, 0.7% of whites and 1.4% of Hispanics. Notable proportion of blacks (134/276) and Hispanics (72/136) with ANC<1.5 x 103/uL had isolated neutropenia, which was less frequent in whites (16/61). The unadjusted survival experience of neutropenic and non-neutropenic subjects in the different ethnic subgroups is shown in Figure The baseline characteristics of elderly individuals with and without neutropenia within each ethnic group are summarized in Table In multivariable analysis, neutropenia continued to be a significant predictor of higher mortality risk for whites , independent of age, sex, comorbidities, anemia and thrombocytopenia. On the contrary, it was associated with a lower, albeit statistically non-significant risk in blacks  and was not significantly associated with decreased overall survival in Hispanics , adjusted for the same covariates. In all three groups, age, male sex, Charlson Comorbidity Index (CCI), anemia and thrombocytopenia were independent predictors of mortality , compared to those with ANC at or above that threshold in the African American cohort. Using this cut-point, black patients with neutropenia were more likely to be anemic and thrombocytopenic too compared to those without neutropenia, similar to what was seen in white subjects with neutrophils below 1.5 x 103/uL , but relatively frequent in non-Hispanic black subjects (3.0%). These prevalence estimates are not far from what is reported in the literature by the largest to date study of neutropenia prevalence in the US .[3 cells/uL, as the established threshold in clinical and research practice, neutropenia has prognostic value for white, but not for black elderly subjects, and this was independent of other cytopenias, age, sex and comorbidity burden. By using this ANC cutoff, 41% of white elderly neutropenic subjects were noted to be pancytopenic, whereas pancytopenia was noted in only 11.6% of their black counterparts. Almost half of black neutropenic subjects had isolated neutropenia. Interestingly, an ANC below 1.5 x 103/uL fell more than 2 standard deviations (SD) away from the mean neutrophil count in the white subcohort, hence appropriately outside a reference range determined by mean ANC ± 2SD, as the one currently in effect. However, it was only 1.6 SD below the mean ANC in the African American subgroup, as blacks were shown to have, on average, significantly lower neutrophil counts than the whites. This finding supports the notion that, had a specific reference range for neutrophil counts in African Americans, been determined, it would include ANC of 1.5 x 103/uL, as a value within the ‘normal’ range.Using a large urban multiethnic, multiracial cohort of elderly subjects, we confirmed that neutropenia, defined as ANC below 1.5 x 10ctively). We found3 cells/uL that have a survival disadvantage, compared to those with higher counts. In our cohort, the majority of blacks labeled neutropenic were noted to have ANC of 1.1 x 103 cells/uL or above, an observation that is in line with the findings of Hsieh et al.[3 neutrophils/uL seems to discriminate better those neutropenic blacks with other concurrent cytopenias from those without and hence is more likely to uncover underlying bone marrow pathology. All together, these findings suggest that the “normal” range for ANC currently in use probably overestimates the prevalence of neutropenia in elderly individuals of African decent and that blacks with neutrophil counts above 1.1 x 103 cells/ul could, in reality, be functionally normal.We, then and most importantly, showed for the first time that it is those neutropenic black elderly individuals with an ANC below 1.1 x 10eh et al.. Finally3/ul (1.4%) was intermediate between that of non-Hispanic whites and blacks. However, Hispanic neutropenics were not identified as a subgroup at significant risk for shorter survival compared to those with higher counts, yet with a relative hazard in-between of what was shown in whites and African Americans. These findings could be interpreted on the basis of the racial heterogeneity of the hispanic ethnicity. This could also explain the different neutropenia prevalence estimate found in our study and that reported by Hsieh et al. for their hispanic group, consisted exclusively of Mexican-Americans with presumably different racial distribution [In our hispanic subcohort, consisted mainly by Puerto Ricans and Dominicans, the prevalence of neutrophil counts below 1.5 x 10ribution .We found no neutrophil cut-off in Hispanics at which neutropenia was significantly associated with increased mortality risk. It is possible that death events of undocumented subjects were not captured in this dataset in which date of death was obtained from the Social Security Death Index. Given the demographics of this cohort, this more likely affected the hispanic subgroup. Missing death events could have hindered our ability to more accurately determine the relationship of neutrophil counts with mortality risk in this ethnic group. Finally, since ethnic variations in neutrophil distribution are recently shown to be likely of racial origin , 13, futThere are several limitations in the present study. Blood counts are known to fluctuate during an acute illness or secondary to medications, yet we believe that the exclusion of subjects with recent hospitalizations and primarily the large sample size should have been adequately protective against such effects. The retrospective design prevented the accurate determination of the etiology of neutropenia. However, we took care in our analysis to control for clinical entities, incorporated in the Charlson comorbidity index, likely to influence neutrophil counts. This study, nevertheless, has certain strengths, as well. An ethnically and racially diverse urban outpatient elderly population provided a very large pool of data that facilitated a stratified and multivariable analysis. Although this cohort represents the population of a single institution, it consists of cases encountered in outpatient settings within the various facilities that comprise the Montefiore Medical system, improving the generalizability of the results.3 cells/uL could more accurately identify those cases of neutropenia with an underlying pathology, at risk for adverse outcomes. Consequently, this cutoff could serve as a novel threshold for evaluation and intervention in elderly subjects of this racial population. It should be the subject of future studies to confirm our findings in other multiethnic, multiracial cohorts and ultimately extend our understanding of the prognostic implications of neutropenia by ethnicity and race to other age groups.In conclusion, the present study of a large inner city cohort demonstrated that neutropenia could be an independent predictor of mortality in the elderly. However, its prognostic value is likely to vary among different ethnicities and races according to the definition used. In non-Hispanic blacks, setting the ‘lower limit of normal’ for neutrophils at 1.1 x 10st 1997 to May 1st 2008 and had a complete blood count drawn within 3 months from the index clinical encounter. To exclude acutely ill patients, those who had been hospitalized within a month from the date of the blood test were removed from the initial sample. Data on demographic and clinical parameters including blood counts and comorbidities were retrieved.The study cohort included all individuals at 65 years of age and above that had visited an ambulatory clinic within the Montefiore Medical system in the Bronx, NY, from January 1The cohort was generated with the use of Clinical Looking Glass (CLG®) from a multiethnic, multiracial inner-city population of patients. CLG is a software tool developed at Montefiore Medical Center that utilizes integrated clinical, demographic and administrative information to generate datasets accessible to statistical processing. The study was approved by the Einstein Institutional Review Board.Demographic data  are based on information obtained at patients' registration. Ethnicity and race were self-reported. Ethnicity was coded as Not Hispanic and Hispanic, while race was reported according to the Office of Management and Budget (OMB) 1997 classification . The Cha3/ul, hemoglobin >16.5 mg/dl in women and >18.5 mg/dl in men, and platelet count > 450x103/ul, respectively. There were limited observations for race other than the white and black among the Not Hispanic ethnic group; as such, along with those of unknown ethnicity and race, 4,017 additional individuals were excluded from the analysis. Because of missing data on race for Hispanics, the remaining subjects were categorized into three groups based on composite information on race and ethnicity, referred hereto as ethnic groups: non-Hispanic whites, non-Hispanic blacks (blacks or African Americans) and Hispanics. Due to lack of follow-up time, 545 observations were ultimately excluded from the survival analyses. The final sample was consisted of 27,760 individuals.The initial sample included 36,174 subjects. 3,852 subjects with leukocytosis, erythrocytosis and thrombocytosis, almost exclusively represented in the non-neutropenic subgroup, were excluded. Leukocytosis, erythrocytosis and thrombocytosis were defined, per established criteria, as WBC>11x103 cells/uL, based on the neutrophil count seen in the complete blood count at the index date for study inclusion. Continuous variables were compared with the Mann Whitney (U) test, due to violations of the normality assumption. Comparison of proportions was done with the Pearson's 2χ test.The distribution of absolute neutrophil count (ANC) was examined graphically and the mean difference by ethnic groups was compared with one-way analysis of variance (ANOVA). We proceeded to stratified analyses by the three categories. Stratification was based on the a priori assumption of neutropenia effect modification by race. Descriptive statistics compared age, sex, CCI, hemoglobin and platelet counts between neutropenic and non-neutropenic subjects within each separate stratum. Neutropenia was defined as ANC below 1.5 x 103/ul [The Kaplan Meier survival function was used to depict the unadjusted survival experience of neutropenic and non-neutropenic subjects within each cohort and differences were tested with the log-rank test. Cox Proportional Hazard (PH) multivariate regression models were subsequently constructed to look at the mortality risk associated with neutropenia, adjusted for age, sex, CCI, hemoglobin and platelet count. Those covariates were selected based on a priori determination of their clinically meaningful association with overall survival. The assumption of linearity for continuous and ordinal variables was evaluated with use of fractional polynomials and assessment of post-estimation partial residual plots, as well as with the dummy variable method. Age and CCI were, thus, treated as continuous variables, while hemoglobin and platelet count variables were dichotomized to denote anemia and thrombocytopenia. Anemia was defined, according to the World Health Organization (WHO) criteria of hemoglobin below 12g/dl and 13g/dl in female and male subjects, respectively, while thrombocytopenia was defined as a platelet count below 150 x 103/ul . The PH In order to explore for cohort-specific cutoffs for neutropenia, we initially assessed the relationship of mortality risk with absolute neutrophil count graphically, with partial residual plots, adjusted for age, sex, CCI, anemia and thrombocytopenia, in each ethnic group. Subsequently, Cox PH multivariable models, adjusting for the same covariates, were built using serially lower neutrophil thresholds, by decrements of 100cells/ul, till a statistically significant risk ratio was observed. The aforementioned methods were used to evaluate the linearity and PH assumptions.All tests were two-sided and an alpha 0.05 level was used to determine statistical significance. Statistical analyses were performed in Stata software, version 12.0.


Measures of compliance including serum β-carotene, lycopene and erythrocyte monounsaturated fatty acids were significantly higher in the MedDiet group at three and six months (p < 0.05). Our results indicate that a population of older Australians can adopt a Mediterranean diet over a six month period.Adherence to a Mediterranean diet (MedDiet) is thought to be achievable in non-Mediterranean regions, but this has yet to be investigated. We aimed to determine if an older Australian population could adhere to a MedDiet for six months. We conducted a randomised, parallel dietary intervention trial with two dietary arms: the Mediterranean diet (MedDiet) group and the habitual diet (HabDiet) control group. A 15-point Mediterranean diet adherence score and food and nutrient intakes were estimated from three-day weighed food records collected at baseline, two and four months. Erythrocyte fatty acids, serum carotenoids and urinary metabolites were assessed at baseline, three and six months. We enrolled 166 participants; 152 commenced and 137 completed the study . Adherence scores were significantly higher in the MedDiet group at two months  and four months . Consumption of vegetables, fruits, fish, legumes, nuts and olive oil significantly increased in the MedDiet group compared to the control, and discretionary food intake decreased ( In the mid-20th century, evidence emerged that populations living in the Mediterranean region had lower incidence of cardiovascular disease (CVD) than those living in northern Europe and North America . It was The MedDiet has received much attention from researchers with the majority of studies reporting positive health outcomes when close adherence is achieved ,6. LargeThis evidence led the US Department of Health and Human Services and US Department of Agriculture to recognise and include the MedDiet as one of two alternative healthy dietary patterns in the 2015–2020 US Dietary Guidelines . The US Epidemiological evidence shows that non-Mediterranean populations with a priori measured high adherence to a MedDiet have lower risk of CVD ,18. Inden = 85 MedDiet group, n = 81 HabDiet group), and 14 withdrew before commencement leaving 152  who commenced the study. Ten withdrew from the MedDiet group and 5 from the HabDiet group, thus 137 completed their 6 months visit (11% attrition from commencement). The study was conducted in 2 cohorts, the first (n = 69) conducted between August 2013 and April 2014, the second (n = 83) between May 2014 and February 2015. Those in the MedDiet group were prescribed a MedDiet for 6 months , while those in the HabDiet group followed their regular diets for 6 months in a parallel design. We aimed to assess dietary adherence through self-reported 3-day weighed food records (WFRs), daily checklists and also objective measures: fasting serum carotenoid levels, erythrocyte fatty acids and 24-h urinary metabolites. Dietary intake was assessed at baseline, 2 and 4 months. Urinary metabolites, erythrocyte fatty acids and serum carotenoids were assessed at baseline, 3 and 6 months. All clinic visits occurred at the Sansom Institute for Health Research, University of South Australia. Checklists were completed daily by those randomised to the MedDiet group only. Further to this, in an exit survey participants were asked to report on their experiences in relation to following the MedDiet. The study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures involving human subjects/patients were approved by the University of South Australia Ethics Committee (#31163). Written informed consent was obtained from all subjects/patients with an investigator present to answer and clarify any queries. Participants received $100 pro rata as an honorarium. The trial was registered with the Australia New Zealand Clinical Trials Register (ACTRN12613000602729), on the 27 May 2013 [The Mediterranean diet for cardiovascular and cognitive health in the elderly study (MedLey) protocol has been published ,24, and May 2013 .The diet was based on a literature review to determine approximate food and nutrient content of the MedDiet  and was Participants were educated at baseline by a study dietitian. They received written resources detailing the basic principles of the MedDiet, a list of Mediterranean fruits and vegetables, a list of serving sizes for key foods, recommendations for number of servings to consume daily and weekly, suggestions for recipe modification and eating out, a recipe book with appropriate Mediterranean recipes and checklists to record their daily intake of servings. They were encouraged to eat a variety of foods, have cooked and raw vegetables, use olive oil in baking, cooking and as a dressing/sauce and consume 50% of their cereals from whole-grain sources. This education session lasted between 45 min to 1 h. Participants were provided with foods to aid adherence, including extra virgin olive oil (750 mL per fortnight), plain and flavoured low fat Greek yoghurt (1 kg per week), unsalted peanuts, almonds and walnuts (~350 g per week), canned legumes and canned tuna. Combined, this was 30–35% of estimated energy requirements. These foods were chosen because they were reasonably shelf-stable, somewhat expensive but important components of the diet, and were offered every 2 weeks. After the initial baseline session, participants attended a 30 min session with the dietitian biweekly to discuss the diet, return checklists, have body mass assessed and collect food. At these sessions, advice tailored to each individual was given to help participants adhere closely to the diet. We aimed to maintain body mass to avoid the possible confounding effects of weight loss on outcomes, such as lipids and blood pressure. If weight loss or gain was occurring, personalised advice was given to counteract this, by either increasing key foods such as olive oil, whole-grain cereals, fruits and vegetables, or decreasing certain foods such as discretionary foods. In addition, participants could call or email the dietitian at any time during the week with questions or concerns. Other than these supports, participants were required to adapt to the MedDiet in their own capacity. Weighed food records were undertaken by participants in the week preceding commencement of the intervention to assess baseline dietary intake. They were then repeated between weeks 9–11 (2 months) and weeks 19–21 (4 months) during the intervention phase. WFRs were administered at the biweekly appointments rather than 3 and 6 month clinic visits to reduce participant burden. Participants were asked to record all food and beverage consumption on consecutive week days and one weekend day, i.e., either Thursday, Friday and Saturday, or Sunday, Monday and Tuesday. All participants received detailed verbal and written instructions from trained staff, digital kitchen scales and a paper diary in which to write their food intake. Upon return these were checked for completeness with the participant. Recorded foods were analysed using FoodWorks Professional Version 7.0.3016 , by a study dietitian and exported to Microsoft Access™  for analysis. Total quantity of flavonoids and carotenoids was calculated for all available foods from the following databases: United States Department of Agriculture (USDA) Database for the Flavonoid Content of Selected Foods (Release 3.1 and 2.1), USDA-NCC Carotenoid Database for U.S. Foods, and the USDA Database for the Isoflavone Content of Selected Foods (Release 2.0). Where necessary, data were supplemented with information from the online phenol explorer. To assess adherence to the MedDiet, we used a 15-point scoring system modified from the 9-point MedDiet score (MDS) created by Trichopoulou et al. . Total fTo monitor adherence throughout the duration of the trial, all participants randomised to the MedDiet group were asked to complete a semi-quantitative checklist. On a daily basis, participants were asked to record when foods were consumed, as half or whole servings using a checklist. Discretionary foods, small goods, red meat, eggs, red wine, skim milk and white potatoes were optional foods and had no minimum requirement. Total weekly servings for each food group were summed, and a percentage adherence for each food group was calculated by comparing actual consumption with recommended consumption. For foods where there was a minimum requirement, the equation was as follows: Participants gave 8-h fasting blood samples for the analysis of erythrocyte fatty acids and serum carotenoids. Samples were taken via venepuncture, centrifuged and stored at −80 °C until analysis. Erythrocyte fatty acid composition was determined as an indicator of longer term fatty acid status using direct transesterification as described elsewhere . CarotenBox 1Did you…Yes all of the timeYes most of the timeYes some of the timeNone of the time(1) Enjoy the yoghurt?(2) Enjoy the legumes?(3) Enjoy the tuna?(4) Enjoy the olive oil?(5) Enjoy the nuts?(6) Manage the red meat restrictions?(7) Manage the “extras” restrictions?(8) Manage with the milk restrictions?Question 9. The table below asks you about the foods you were asked to eat and restrict. Please tick the appropriate box:Question 10. What was/were the most difficult thing(s) about following the Australianised MedDiet?Question 13/15. Which, if any, components of the MedDiet are you likely to continue with now that the study is finished?The MedLey exit survey questioned volunteers on their experiences within the trial, including aspects of following the intervention diet. n = 64) would provide 80% power to detect a significant (p < 0.05) 0.5 standard deviation change in cognitive outcomes. For this study, adherence score was the primary outcome. With a sample size of n = 128, based on results from our pilot study [p < 0.05) 1 unit difference in the 15-point adherence score between groups (standard deviation = 2). We expected an effect size of at least a 2-unit change. Raw data is available as online t-tests were used for normally distributed variables, and Mann-Whitney U test for non-normally distributed variables. To assess differences in adherence scores, nutrient and food intake, urinary metabolites, erythrocyte fatty acids and serum carotenoids, analyses were performed using intention-to-treat linear mixed effects models with a group × time interaction term to determine overall differences in effects across time and at each time point. Significant was set at 0.05 (two sided). For food intakes, gender was controlled for in the model. To control for energy intakes, all food and nutrients intakes were analysed per MJ of energy intake. In a sensitivity analysis, the 10-point MDS was applied to our data, to enable comparison to previous studies. Gender-specific median intakes at baseline were used as cut-offs for baseline, 2 month and 4 month intakes. One point was awarded if intakes of vegetables, legumes, fruits and nuts, cereals, fish and monounsaturated fatty acid:saturated fatty acid (MUFA:SFA) was above the median, and 1 point awarded if intakes of meat/poultry and dairy were below the median. Additionally, 1 point was awarded for intakes between 10–50 g ethanol for males, and 5–25 g for females, so that minimum score was 0 and maximum was 9. To calculate adherence to the a priori MedDiet, we also calculated the MDS using cut-offs established from the Greek cohort enrolled in the EPIC study [Sample size for this study was based on the primary outcome measure of cognitive function and is explained in detail elsewhere . We calcot study  we estimIC study . Linear 2 in the MedDiet group, 27.1 ± 4.1 kg/m2 in the HabDiet group) at baseline. The first participants commenced in August 2013 and all had completed the 6-month period by February 2015. Of 152 participants who commenced, baseline data were complete for 150 participants for dietary intake and urinary metabolites, and 152 participants for erythrocyte fatty acids and carotenoids. All participants with baseline data were included in the analyses. p < 0.01), while they remained unchanged in the HabDiet group (p < 0.001). The sensitivity analysis revealed similar changes when the 10-point MDS was applied using sex-specific baseline medians, although when the sex-specific medians from the Greek cohort of the EPIC study were used, adherence scores were lower (Adherence scores increased in the MedDiet group significantly (et group . Scores re lower . Accordip = 0.05). Between groups, intakes of extra virgin olive oil, fruits, nuts, legumes, dairy, fish, meats and miscellaneous foods were significantly different at four months (diet*visit p < 0.05). Intakes of extra virgin olive oil, fruits, nuts, legumes, dairy, and fish were higher in the MedDiet group, while meat and miscellaneous food intakes were lower relative to the control group. Originally, 17 participants were allocated to energy levels 3 and 4, while 19 were allocated to energy level 2 and 27 to energy level 1. However, this was adjusted during the two and four week visits based on participant reports of satiety, and adherence levels. After the first month, 12 volunteers were allocated to energy level 1, 25 to energy level 2, 12 to energy level 3 and 31 to energy level 4. Mean energy intakes for energy levels 1, 2, 3 and 4 were 10 MJ, 9.6 MJ, 8.4 MJ and 7.8 MJ at four months, respectively. Of 70 MedDiet group participants who completed the study, 68 completed the exit survey. The responses to question 9 are shown in According to a 15-point adherence score, self-reported food intakes and objective biological markers, older Australians were able to increase adherence to the MedDiet over a six-month intervention period. There was a significant increase in MUFA:SFA ratio measured from both dietary intake and erythrocyte fatty acids, and a significant increase in olive oil consumption. Serum differences in β-carotene and lycopene between groups indicated those in the MedDiet were consuming more carotenoids after four months. Carotenoid intakes and serum levels of α-carotene both increased significantly in the MedDiet group, suggesting increased intake of fruits and vegetables, which was reflected by results from the WFRs. Previous studies including the EPIC and Women’s Health Study have shown serum carotenoids to correlate with fruit and vegetable intakes ,34,35,36The average 15-point adherence score moved from a medium to high level in the MedDiet group. In a sensitivity analysis, we also calculated the 10-point MDS, using both our sex-specific median intakes, and those from the Greek cohort of the EPIC study. All three scoring methods showed a significant increase in score amongst the MedDiet group over time compared to the control group. Scores increased by 23% according to our score, 22% according to the MDS using our medians, and 21% using the Greek EPIC medians, thus increases were similar across the three systems. Adherence was lower at all three time points when using cut-offs from the Greek data, suggesting that, although adherence did increase, the dietary intake achieved by our participants is still lower than a Mediterranean population. This could have consequences for health outcomes, however in their meta-analysis, Sofi et al.  showed tIn general, objective data supported the self-reported nutrient intake data, with some exceptions: changes in dietary intakes and urinary excretion were similar except for magnesium, where urinary excretion increased while dietary intake reportedly decreased, although this did not reach significance. Although 24-h urinary excretion of sodium and potassium has been linked to disease outcomes , many faIntake of Mediterranean foods including fruits, nuts, vegetables, legumes and fish increased, and non-core/non-Mediterranean foods decreased. As energy intakes did not change, core foods completely replaced the energy lost from discretionary foods. Across all four energy levels, energy intake was higher than the prescribed level, suggesting participants underestimated energy intake in their baseline WFRs. This is a common phenomenon with dietary recording . In a liExit survey results indicate that while not all components of the diet were perceived as easy to adopt, the majority of volunteers intended to continue most aspects of the diet after finishing the study. The most difficult things about following the MedDiet were the influences of friends/family, eating out and while holidaying, and food restrictions such as red meat, beer and discretionary foods. Considering the MedDiet restricts intakes of these foods below the national average intake for men and women, this is not surprising . Eating n = 23) found that money spent on foods before and after a three-week MedDiet intervention was not significantly different [n = 20) of participants with major depressive disorders. The estimated cost of food per week from the WFRs was $138 per person, compared to an estimated cost of a modified MedDiet at $112 per week per person [Limitations to this study must be acknowledged. Use of a priori adherence scores in non-Mediterranean countries has been criticised, as they do not necessarily compare intakes to a traditional MedDiet, and often exclude important food groups such as nuts . We deveifferent . In Austifferent . The invr person . Thus, cIn this study, a population of older, free-living Australians made substantial changes to their food and nutrient intake, resulting in changes in erythrocyte MUFA:SFA and serum carotenoids and an increased adherence to the MedDiet. From a feasibility perspective, our results suggest the MedDiet may be an option for dieticians and other health professionals to recommend to patients/clients with health issues. Importantly, data are still needed to confirm the health benefits of the MedDiet in Australia.