P < 0.05): LRP2 rs2544390, rs1800592 between UCP1 and TBC1D9, APOA5 rs662799, VWF rs7965413, and rs1411766 between MYO16 and IRS2. Furthermore, we performed multiple logistic regression analyses, including an SNP term, a CP term, and an SNP × CP interaction term for each CP and SNP that was significantly associated with MetS. We identified a novel SNP × CP interaction between rs7965413 and platelet count that was significantly associated with MetS . This association of the SNP × CP interaction with MetS remained nominally significant in multiple logistic regression analysis after adjustment for either the number of MetS components or MetS components excluding obesity. Our results reveal new insight into platelet count as a risk marker for MetS.Although many single nucleotide polymorphisms (SNPs) have been identified to be associated with metabolic syndrome (MetS), there was only a slight improvement in the ability to predict future MetS by the simply addition of SNPs to clinical risk markers. To improve the ability to predict future MetS, combinational effects, such as SNP—SNP interaction, SNP—environment interaction, and SNP—clinical parameter (SNP × CP) interaction should be also considered. We performed a case-control study to explore novel SNP × CP interactions as risk markers for MetS based on health check-up data of Japanese male employees. We selected 99 SNPs that were previously reported to be associated with MetS and components of MetS; subsequently, we genotyped these SNPs from 360 cases and 1983 control subjects. First, we performed logistic regression analyses to assess the association of each SNP with MetS. Of these SNPs, five SNPs were significantly associated with MetS ( Metabolic syndrome (MetS) is characterized by a clustering of metabolic abnormalities, including central obesity, insulin resistance, dyslipidemia, and hypertension; moreover, it has been identified as a common precursor to the development of cardiovascular disease (CVD) . The preTo explore risk markers for predicting MetS development, many studies have been performed utilizing health check-up data among different groups of people, such as company employees , people In some studies, several combinations of clinical risk markers have also been explored based on health check-up data. We have previously shown that the combination of the γ-glutamyl transpeptidase (γ-GTP) level and WBC count was the most significant combinatorial risk marker associated with MetS based on the health check-up data of company employees . We haveCOBLL1 and GRB14 is significantly associated with fasting insulin levels .We initially assessed the association between MetS diagnosed in 2009 and 98 genotyped SNPs. Five out of 98 SNPs were found to be significantly associated with MetS , with VWVWF rs7965413 and PLT in Based on the results of the screening analysis, we focused on the five SNPs listed in 4/μL and assessed an interaction effect between SNP rs7965413 and dichotomous PLT for MetS (P = 0.014). Among rs7965413 genotypes, OR was unchanged in subjects with PLT count ≥ 23.8×104/μL, which is the median value of PLT count in the study participants. On the other hand, in subjects with PLT count < 23.8×104/μL, OR decreased as the number of minor allele T increased.Furthermore, we transformed PLT count into a dichotomous value based on the median value of platelet count across all subjects, which was equal to 23.8×10for MetS . MultiplI2 = 90.2; P = 0.001).Finally, we assessed the association of rs7965413 with PLT count in each group of cases and controls. In the case group, the minor allele T of rs7965413 was significantly positively associated with PLT. In the control group, the minor allele T was significantly negatively associated with PLT . There wLRP2 rs2544390, rs1800592 between UCP1 and TBC1D9, APOA5 rs662799, VWF rs7965413, and rs1411766 between MYO16 and IRS2. Furthermore, we identified a novel SNP × CP interaction for MetS, which was the interaction between VWF rs7965413 and platelet count. These SNPs and associated interaction are expected to be useful as risk markers for MetS development.We performed a case-control study of MetS based on the health check-up data of Japanese male employees and found significant associations of five SNPs with MetS, including As shown in Tables VWF rs7965413 was significantly associated with MetS. VWF rs7965413 is located in the promoter region of the VWF gene. The VWF gene encodes von Willebrand factor (vWF). vWF promotes platelet adhesion and aggregation at sites of vascular injury, so it plays a prominent role in the formation of arterial thrombus [VWF gene cause von Willebrand disease because of deficiency of vWF. It was also reported that vWF was associated with insulin level and insulin resistance [vWF antigen (vWF:Ag) level in men significantly increased across insulin quintiles [vWF was reported to be associated with homeostasis model assessment—insulin resistance, which is an index of insulin resistance, and MetS [vWF plasma levels and CVD is expected and has been investigated. Several studies have reported that elevated vWF levels are positively associated with risk for coronary heart disease (CHD) [VWF promoter variant rs7965413 had significantly lower vWF:Ag levels than the C allele  [VWF promoter variant that was different from rs7965413 was also associated with risk for CHD [VWF promoter variants contribute significantly to CVD risk. We found that risk of MetS decreased as the number of T alleles of rs7965413 increased. This result may indicate a causal relationship between rs7965413 and CVD that is mediated via vWF:Ag and MetS.As shown in thrombus . Mutatiosistance . For exauintiles . Furtherand MetS . Thus, ase (CHD) ,28. Keigt study)  in a pop for CHD . However for CHD , it is uvWF:Ag levels, and vWF promotes platelet adhesion. The statistical interaction term between rs7965413 and platelet count is expected to reflect the biological interaction between vWF and PLT. This result reveals new information regarding platelet count as a risk marker for MetS. Moreover, for rs7965413, frequencies of the T allele across different ethnicities were as follows: African, 0.372; American, 0.445; Asian, 0.427; European, 0.621 in the 1000 Genomes Project Phase I version 3. This SNP was observed across different races, so the interaction may also be observed in other race/ethnic groups.As shown in VWF, rs7342306, was associated with platelet count [2 = 0.123, D’ = 0.401). Thus, the contribution of rs7965413 to platelet count is expected to be independent of the contribution of rs7342306 to platelet count. Our results indicate significant heterogeneity between case and control groups. The association of rs7965413 with platelet count might not be detected in the GWAS because of this heterogeneity.We found the association of rs7965413 with PLT count in both case and control groups. A GWAS recently showed that an SNP upstream of et count . AlthougVWF gene were associated with traits related to blood pressure, which was one of MetS components. Ruixing et al. reported that VWF rs1063856 (Thr789Ala) was significantly associated with hypertension in women [VWF rs2239153 was significantly associated with salt sensitivity [2 = 0.0, D’ = 0.013 for rs1063856; r2 = 0.066, D’ = 0.288 for rs2239153). Thus, the contribution of rs7965413 to MetS is expected to be independent of the contribution of these variants to blood pressure.It was also reported that variants in the in women . Defago sitivity . These vLRP2 rs2544390, rs1800592 between UCP1 and TBC1D9, APOA5 rs662799, and rs1411766 between MYO16 and IRS2. Of these SNPs, APOA5 rs662799 was frequently reported to be associated with MetS and dyslipidemia, which was one of MetS components, based on several populations, including Japanese [UCP1 and TBC1D9 is an A→G point mutation at the—3826 position in the 50 flanking region of the UCP1 gene. The UCP1 gene is a candidate gene for obesity and type 2 diabetes mellitus because the gene has been found to decrease mitochondrial membrane potential and increase thermogenesis [LRP2 rs2544390 is located in the LRP2 gene. It has been reported that the T allele of this SNP is associated with higher serum UA [MYO16 and 120 kb centromeric to IRS2. This SNP was reported increase susceptible to diabetic nephropathy, as determined by a GWAS in European-American subjects with type 1 diabetes [As shown in Japanese , ChineseJapanese , and CauJapanese  populatiogenesis . Our resdiabetes  and was Our study has some limitations. First, this is a case-control and exploratory study that does not establish a cause-and-effect relationship. Future studies are thus necessary to evaluate the predictive potential of SNP × CP interactions as risk markers in prospective cohorts. We assumed that the interaction is linear; that is, the per-allele effect of an SNP changes across the continuous spectrum of a CP. However, if the interaction effect is nonlinear or a threshold effect exists, in which case the association would only be present in one extreme of the CP distribution, this analysis is not suitable and other analytical methods should be applied.In conclusion, our data demonstrate associations of five SNPs with MetS and of an interaction between SNP rs7965413 and platelet count for MetS. Our results reveal new insight into PLT count as a risk marker for MetS.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


As a first approximation of the effect of global soil variability on plant growth response to CO2, we evaluated the effect of CO2 on tall fescue (Festuca arundinacea) grown in soils representing 10 of the 12 global soil orders plus a high-fertility control. Plants were grown in small pots in continuously stirred reactor tanks in a greenhouse. Elevated CO2 (800 ppm) increased plant biomass in the high-fertility control and in two of the more fertile soils. Elevated CO2 had variable effects on foliar mineral concentration—nitrogen was not altered by elevated CO2, and phosphorus and potassium were only affected by CO2 in a small number of soils. While leaf photosynthesis was stimulated by elevated CO2 in six soils, canopy photosynthesis was not stimulated. Four principle components were identified; the first was associated with foliar minerals and soil clay, and the second with soil acidity and foliar manganese concentration. The third principle component was associated with gas exchange, and the fourth with plant biomass and soil minerals. Soils in which tall fescue did not respond to elevated CO2 account for 83% of global land area. These results show that variation in soil physical and chemical properties have important implications for plant responses to global change, and highlight the need to consider soil variability in models of vegetation response to global change.Soils vary widely in mineral nutrient availability and physical characteristics, but the influence of this variability on plant responses to elevated CO Further analysis of topics for the 57 unique publications highlight the scarcity of consideration of the range of possible soil impacts on CO2 responses  and was reduced on a heavy clay soil (a Vertisol) with lower plant available water  include cereal crops that provide over half of the calories and protein consumed by humans Cordain,  and are 2 we grew tall fescue (Festuca arundinaceae Schreb.), a temperate C3 grass, in elevated and ambient CO2 on 13 different soils, representing ten of the twelve soil orders, and a high fertility control, and assessed plant growth, mineral acquisition, gas exchange, and non-structural carbohydrate accumulation.In order to begin to characterize the effects of the wide range of soils on plant responses to elevated CO−1 m−2 (This relatively low lighting intensity reflects both the attenuation of solar radiation through both greenhouse roof and the mylar covering of the CSTRs and the difficulty of controlling heat load from the HID lighting supplied to each CSTR).Tall fescue was grown in eight Continuous Stirred Tank Reactors  and the other receiving elevated , temperature, photosynthetically active radiation (PAR), and relative humidity for each CSTR were recorded every 16 min. CO2 concentrations were relatively stable, with mean values  were randomized within each of the eight CSTRs, for a total of 112 pots. Pots were irrigated manually with distilled water every day.Approximately 20 seeds of tall fescue  were broadcast on the surface of each pot on Jan 23, 2007. The pots were covered in clear plastic until germination, after which pots were thinned to 15 plants potNeotyphodium sp. was assessed from a sample of 100 seeds and two growing tillers per pot (from two replicates), using a commercial immunoblot detection kit .The presence of the endophytpe max, area basis) of a young, fully expanded leaf in one plant per pot was determined on weeks 8 and 10 at mid-day with a LI-6400 portable photosynthesis system  with leaf temperature set to 20°C. Measurements were made at 600, 800, and 1000 μmol m−2 s−1 PAR, after measurements at 200–1000 μmol m−2 s−1 PAR on a subset of plants showed that maximum photosynthetic rate occurred in the 600–1000 μmol m−2 s−1 range. Also at week 10 the net pot CO2 exchange (including soil) was measured with a Li-6200 Infrared Gas Analyzer system  using a 12 liter chamber in which the whole plant and pot were enclosed for 2 min. During these measurements temperature ranged from 25 to 28°C, and light (PAR) ranged from 320 to 380 μmol m−2 s−1, reflecting the ambient growing conditions.Leaf photosynthesis  was frozen at −80°C. These were later processed to quantify ethanol soluble sugars and starch from approximately 100 mg of ground tissue with the enzyme-coupled colorimetric method described by Hendrix .All remaining above-ground tissue was oven dried at 60°C for 48 h to determine total dry weight. A sample of about 0.2 g of dried and ground leaf tissue was digested in a microwave Miller, . The dil2 as the main plot factor and soil the subplot factor. Data were analyzed in R . Pair-wise Tukey comparisons for the effect of CO2 in each soil were obtained using multcomp  Principal component analysis (PCA) was used to characterize the principal sources of variability in the data. PCA was carried out for all observations with growth, photosynthesis, soil analysis and leaf mineral concentrations as variables using Neotyphodium endophyte, and Neotyphodium colonization did not differ among soils or CO2 treatments.About 40% of the seeds and 95% of all tillers tested positive for the 2, where biomass of CTR increased by about 60% , but did not increase biomass in the other soils  in biomass compared to CTR, and did not produce sufficient tissue for all analyses to be completed. They were therefore eliminated from most of the subsequent analyses. The remaining soils produced 50–80% less shoot biomass than CTR, indicating significant differences among soils , as would be expected. Furthermore, the C:N ratio was not altered by CO2 or the CO2 × soil interaction , though it did differ among the soils , of magnesium in CTR (p = 0.038) and OXI3 (p = 0.003), of manganese in OXI3 (p < 0.001); of sodium in SPO (p < 0.001); and of zinc in CTR (p < 0.001) and SPO (p = 0.033).The ANOVA analysis for leaf mineral concentrations found highly significant differences (p < 0.001) for the soil effect for all leaf nutrient concentrations measured. However, the results for CO2 increased leaf photosynthesis  in most of the soils, but not in CTR, ALF, MOL, and OXI3, reflecting a significant soil × CO2 interaction  did not differ significantly between elevated and ambient CO2 overall (p = 0.389), but varied between soils (p < 0.001), and there was no interaction between CO2 and soil (Table 2 exchange rate (nmol CO2 s−1) increased in CTR, ALF, ARD, and MOL, but not in the others  were excluded. Since % Sand, % Silt, and % Clay sum to 100%, we excluded % Sand from this analysis. Similarly, since CEC is reflective of soil Ca and Mg, we excluded CEC.Combining COPrincipal components analysis yielded four principal components (PC) which explained about 65% of the variability in the data Figure . PC1 29.2 (p = 0.029), but sucrose concentrations were not affected by CO2 (p = 0.181). Levels of NSC were influenced by the different soils  responded differently in this experiment Table . Starch max) did not show a strong correlation with foliar N  and marginally with CO2 (p = 0.079). A graphical analysis .Maximum photosynthetic rate  but no effect of CO2 (p = 0.410), or their interaction (p = 0.131). Three rough groupings of soils are differentiated here. CTR and MOL, with a low N:P ratio, INC1, AND, and OXI3, with a high N:P ratio, and ARD, ALF, OXI1, SPO, ULT, and VRT with intermediate values.Analysis of N:P ratios Figure , Table 22 on the growth and physiology of Festuca encountering different chemical and physical soil characteristics presented by soils from 9 of the 12 soil orders, spanning the global range of soil variability. The effect of elevated CO2 on growth, photosynthesis, and leaf chemistry depended on the soil in which the plants were grown. Since we provided adequate irrigation, we assume that the responses we observed mostly reflect the ability of Festuca to acquire nutrients from the different soils under contrasting CO2 regimes.In this experiment we evaluated the effect of elevated COFestuca, despite its wide range of adaptability . This suggests that other factors may also be important.We found large differences between soils in the effects of elevated CO2 under nutrient-limited conditions  also showed contrasting results  showed the expected increase in response to elevated CO2 in six of the soils, shoot CER was not altered by elevated CO2. The difference between leaf and canopy photosynthetic responses to elevated CO2 was not a simple product of changes in biomass as shoot CER was normalized by biomass. There was a significant negative relationship (log-linear) between shoot CER and shoot biomass . However, shoot CER was positively related (log-linear) with leaf N . There are reports of growth dilution of leaf N by elevated CO2 , the relationship of leaf N concentration and canopy CER suggests that there are fundamental differences in photosynthetic N use between plants grown in different soils.The carbon exchange rate for whole shoot and leaf photosynthesis . Without more detail on the soil the reported N:P ratios represent it is difficult to interpret these results—it is possible that the range of N:P ratios reported does not represent the range of soil variability we are testing here. Alternatively, the wide range of N:P ratios we report here may indicate that fescue is not well adapted to some of these soils. However, correlation between the divergence of observed N:P ratio from the “optimum” value of 13 and biomass was very low (r = −0.25) suggests that divergence of N:P ratio from some optimum is not strongly related to biomass.In a review of research on N:P ratios, Güsewell  reporteds Figure . A ranges Figure , with a 2 can immobilize sufficient N to lead to increasing N limitation with elevated CO2. We saw little support for this in this study. Foliar N had limited influence on Amax  as been reported in plants exposed to elevated CO Nguyen, . These c2, suggesting a trend of dilution of minerals as was observed clearly with CTR, ALF and ULT. The risk of mineral dilution and the consequent loss of food and forage quality has been mentioned by others , soil pH, foliar P, and foliar Mn (with the opposite sign). This is not surprising, as soil pH governs soil mineral availability, and it is well known that in acidic soils low foliar concentrations of P and high concentration of Mn can inhibit growth  and PC12 (2% of the variation), and CO2 was also loaded on PC5 (5.4% of variation). This suggests that variability associated with CO2 was relatively low in this data set compared with that associated with plant responses to diverse soils. This suggests that more work is needed with highly diverse soils to better map the potential responses of plants to global change variables.The fact that the first four PCs only captured 65% of the variation in the data indicates that the relationships between photosynthesis, leaf mineral content, and soil physical and chemical properties is complex and highly dimensional—a small number of variables will not adequately describe the range of differences seen. The strongest loadings for CO2  sites without considering how the FACE sites reflect the global diversity of soils. Such caution has been suggested by others who have noted that the distribution of soils limited by acidity . This highlights the urgent need to better understand the real effects of climate change on plant growth across a representative range of soils, and its implications for food production and ecosystem management.In this study elevated COld Table , account2 and the importance of soil variables in explaining these differences suggests that a more nuanced consideration of the consequences of soil variability on plant responses to global change may be warranted. The need for studies that address multiple climate change variables and their interactions, particularly with soil, has been noted by others  include multiple representatives for each soil order; (3) use intact soil monoliths to assess soils in a more natural context; (4) include more than one species of plant, to address the issue of plant adaptation to specific soils. Furthermore, the methodological challenge of accurately estimating root biomass and length for plants grown in widely differing soils needs to be solved.While we acknowledge the limitations in our study in the use of only one plant species, small pots, and the lack of water stress treatments, the differences between soils in the response to elevated COThe 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.


Almost 150 years after the first identification of Neandertal skeletal material, the cognitive and symbolic abilities of these populations remain a subject of intense debate. We present 99 new Neandertal remains from the Troisième caverne of Goyet (Belgium) dated to 40,500–45,500 calBP. The remains were identified through a multidisciplinary study that combines morphometrics, taphonomy, stable isotopes, radiocarbon dating and genetic analyses. The Goyet Neandertal bones show distinctive anthropogenic modifications, which provides clear evidence for butchery activities as well as four bones having been used for retouching stone tools. In addition to being the first site to have yielded multiple Neandertal bones used as retouchers, Goyet not only provides the first unambiguous evidence of Neandertal cannibalism in Northern Europe, but also highlights considerable diversity in mortuary behaviour among the region’s late Neandertal population in the period immediately preceding their disappearance. Neandertal funerary practices remain at the forefront of palaeoanthropological research, generating heated debates following the revision of old data and new excavations at key sites such as La Chapelle-aux-Saints19101315Here we provide new data on the diversity of Neandertal mortuary behaviour, focusing on a small area of their known range, Northern Europe, during Marine Isotope Stage (MIS) 3 (ca. 60–30 thousand years ago), in order to identify small-scale processes during this short period that witnessed the disappearance of the NeandertalsThe Troisième caverne (or “Third cave”) of Goyet, excavated in the latter half of the 19th and beginning of the 20th century, and again at the end of the 1990s202122Several human remains from different levels were published by Dupont14C) dated, 15 were sampled for stable isotope analyses, and 10 for DNA extraction  the revision of the human skeletal material, (ii) systematic sorting of the faunal collections to check for unidentified human remains , and ii. A good traction . Based otraction . Althougtraction , with lotraction .14C dating (see Methods). The ecology of the Goyet Neandertals was also investigated using δ13C and δ15N isotope composition of bone collagen14C dates obtained from the newly identified skeletal material place the Goyet Neandertals to ca. 40.5–45.5 ky calBP. However, when the youngest ages, which likely reflect undetected bone collagen contamination, are excluded  were identified. Observed only on a single radius alongside several femurs and tibias, notches are likely connected to the fracturing of fresh diaphyses and marrow extraction. Percussion pits are common and probably indicate failed attempts at fracturing bones. Both percussion notches and pits were also identified on eight bones  were refit to at least one other, with the number of specimens per refit set ranging from 2 to 8 tibia I; . Several37et al.While animal bone retouchers are common in European Middle Palaeolithic contexts , only a sample from Dupont’s excavation was examined  or the LRJ.Although the Goyet late Neandertals date to 40.5–45.5 ky calBP, the lack of reliable contextual information makes it impossible to associate them with any of the technocomplexes from the site. However, coeval Mousterian assemblages are known from sites in the Mosan Basin, as at unit 1A of Scladina23In terms of the region’s late Neandertal mortuary practices, four sites within an approximately 250 km radius around Goyet produced Neandertal remains reliably dated to between 50–40 ky calBP . Interes555860Considerable diversity is evident in the mortuary behaviour of the late Neandertal populations of Northern Europe, possibly involving both primary and secondary deposits, alongside other types of practices, including cannibalism. Despite low genetic diversity amongst late Neandertal populations, the presence of various late Middle Palaeolithic technocomplexes, as well as the LRJ, nevertheless suggests significant behavioural variability amongst these groups in Northern Europe.Grand Curtius Museum (Liège), the Cercle d’Histoire et d’Archéologie du Pays de Genappe (Genappe), and the Préhistosite de Ramioul (Ramioul), whose origin is less secure. The Neandertal remains presented here were found among the first two collections only. The numbering system of the specimens and their origin are discussed in The assessment of the Goyet collections included material housed at the RBINS and Royal Museums of Art and History (RMAH) in Brussels, which originate from the Troisième caverne, as well as collections from the et al.386768et al.http://www.minidome.be). Based on the polynomial texture mapping technique, the dome consists of 260 LEDs and a single fixed camera, which captures an image with each LED individually lit. The results allow to display an object interactively under varying lighting to reveal all of the details of its surface. Additionally, 3D models of the retouchers made on Neandertal bones obtained using a white light 3D measurement system (http://www.mechscan.co.uk/) are available at http://virtualcollections.naturalsciences.be/virtual-collections/anthropology-prehistory/human-remains/goyet.After determining the composition of the faunal assemblage sampled from Dupont’s collection , a total65All sampled specimens were untreated (glued or varnished), newly identified Neandertal bones, except for tooth 2878–2D see . Specimeet al.13C, 15N) used an elemental analyser NC 2500 connected to a Thermo Quest Delta+XL mass spectrometer. The degree of chemical preservation of collagen is expressed as the atomic ratio of Ccoll:Ncoll, whose acceptable range of variation is 2.9–3.6coll) should be above 5%coll:Ncoll atomic ratios span a narrow range (3.2–3.4), indicating exceptionally well-preserved collagen for all bone specimens .


IRF5 gene and some rheumatic disorders. In this study, we aimed to evaluate whether three different haplotype blocks within the IRF5 locus which have been shown to alter the protein function are involved in the risk of CV events occurring in Spanish RA patients.Rheumatoid arthritis (RA) is a complex polygenic inflammatory disease associated with accelerated atherosclerosis and increased cardiovascular (CV) disease risk. Interferon regulatory factor 5 (IRF5) is a regulator of type I interferon induction. Recently, researchers have described an association between multiple single-nucleotide polymorphisms of the IRF5 polymorphisms  representative of each haplotype group were genotyped by performing TaqMan assays using a 7900HT Fast Real-Time PCR System with tissue from a total of 2,137 Spanish patients diagnosed with RA. Among them, 390 (18.2%) had experienced CV events. The relationship of IRF5 genotypes and haplotypes to CV events was tested using Cox regression.Three IRF5 rs2004640 GG and IRF5 rs10954213 GG genotypes against the risk for CV events after adjusting the results for sex, age at RA diagnosis and traditional CV disease risk factors was observed (hazard ratio (HR) = 0.6, 95% confidence interval (CI) = 0.38 to 0.92, P = 0.02; and HR = 0.58, 95% CI = 0.36 to 0.95, P = 0.03, respectively). Moreover, we detected a protective effect of the GTG haplotype against the risk for CV events after adjusting the results for potential confounding factors .Male sex, age at RA diagnosis and most traditional risk factors  were associated with increased risk for CV events in the RA population. Interestingly, a protective effect of both IRF5 gene variants are associated with risk of CV events in patients with RA.Our results reveal that  Rheumatoid arthritis (RA) is a chronic inflammatory rheumatic disease associated with an increased risk for cardiovascular (CV) events and CV disease–related deaths compared to the general population . BecauseIFN genes has been observed in peripheral white blood cells of patients with several autoimmune diseases [IFN gene transcription [Type I interferons (IFNs) are signaling molecules involved in both innate and adaptive immunity. Misregulated expression of type I diseases . In thiscription ,11. Thiscription , such ascription .IRF5 gene, which is located in chromosome 7q32.1. Several genetic studies have described the relevance of multiple single-nucleotide polymorphisms (SNPs) of the IRF5 gene in different rheumatic disorders, such as RA and lupus erythematosus [IRF5 variants, designated as groups 1, 2 and 3, have been found to be independently associated with these inflammatory disorders and with different functional roles. Group 1 includes SNPs tagging a 30-bp in-frame insertion/deletion variant of exon 6 that alters protein stability. The association of group 2 SNPs seems to be explained by the T allele of IRF5 rs2004640 polymorphism that allows the expression of an alternative isoform and is associated with significantly higher levels of IRF5 expression. The association of group 3 SNPs is probably due to the rs10954213 A allele that creates an early polyadenylation site which leads to higher IRF5 expression [IRF5 protein is encoded by the ematosus ,14. In tTaking into account all of these considerations together in the present study, we analyzed the potential role of one tagging SNP of each group of independent signals  in the risk for CV disease in a large and well-characterized cohort of patients with RA.IRF5 rs2004640, rs2070197 and rs10954213 polymorphisms.A set of 2,137 Spanish patients with RA were included in the present study. Blood samples were obtained from patients recruited from University Hospital Lucus Augusti (Lugo), Marqués de Valdecilla University Hospital (Santander), Hospital de Bellvitge (Barcelona) Hospital Clínico San Carlos, La Paz University Hospital, Hospital de La Princesa, University Hospital Gregorio Marañón and 12 de Octubre University Hospital (Madrid). Informed written consent was obtained from all participants. The study was approved by the ethics committees of Galicia for University Hospital Lucus Augusti; of Cantabria for Marqués de Valdecilla University Hospital; of Cataluña for Hospital de Bellvitge; and of Madrid for Hospital Clínico San Carlos, La Paz University Hospital, Hospital de La Princesa, University Hospital Gregorio Marañón and 12 de Octubre University Hospital. All the patients fulfilled the RA classification criteria published by the American College of Rheumatology in 1987 and 2010 ,16. In aData on the main demographic data, clinical characteristics, CV disease risk factors and CV events of patients enrolled in the study are shown in Table IRF5 rs2004640, rs2070197 and rs10954213 polymorphisms were genotyped with predesigned TaqMan SNP genotyping assays in a 7900HT Fast Real-Time PCR System according to the conditions recommended by the manufacturer . Negative controls and duplicate samples were included to check the accuracy of genotyping.Patient DNA was obtained from peripheral blood using standard methods. The IRF5 locus among the RA patients was estimated as the Nelson-Aalen cumulative HR. Comparative values between the different genetic models of inheritance for IRF5 polymorphisms were estimated using the Akaike Information Criterion (AIC). Statistical significance was defined as P ≤ 0.05, and all analyses were performed using Stata SE/12 statistical software .The genotype data were checked for deviation from Hardy-Weinberg equilibrium (HWE) using a previously described method . The relIRF5 rs2004640, rs2070197 and rs10954213 polymorphism genotype distributions were in Hardy-Weinberg equilibrium. The genotyping success rate was greater than 98% in all cases. Genotype and allele frequencies of the IRF5 rs2004640, rs2070197 and rs10954213 polymorphisms were in agreement with the data in the HapMap database [The P < 0.05 in all cases)     (Table IRF5 rs2070197 polymorphism according to the presence or absence of CV events (Table Table s) Table . InteresP = 0.012).In a further step, we combined the three polymorphisms to create haplotypes Table . As showIRF5 locus. Additional file IRF5 rs2004640 and rs1095213 polymorphisms decreased CV disease risk in 0.7% and 1% of patients, respectively  [IRF5 rs2004640, rs2070197 and rs10954213) as representatives of three different IRF5 haplotype blocks [IRF5 polymorphisms in the risk of CV disease. Interestingly, when we studied each of the polymorphisms separately, our results revealed a protective effect of the IRF5 rs2004640 GG and IRF5 rs10954213 GG genotype variants against the risk of CV events in RA. Moreover, when we analyzed all the genetic variants together to create haplotypes, our results revealed a protective effect of the GTG haplotype  against the risk of CV disease. Because the protective effect of the mutant allele of the IRF5 rs2004640 and rs1095213 polymorphisms is seen only in homozygosis, we can conclude that our study is adjusted to a recessive model of inheritance. Additionally, the results derived from our study are in accordance with those obtained in other pathologies [IRF5 rs2004640 polymorphism and subclinical atherosclerosis [Outside the HLA region, IFN pathway genes, which encode cytokines with critical modulatory effects on innate and adaptive immunity, have been shown to represent a key component of the genetic network leading to autoimmune processes. In this context, several polymorphisms in the duction) ,11 are aduction) ,14. Becaclerosis .The results obtained in this study provide additional evidence on the potential role that genetic factors may play in the development of CV disease in RA. The search for genetic markers associated with CV disease in RA may be important to establishing a better characterization of RA patients at risk of CV disease. Improved understanding of these underlying genetic may be useful to establish future therapeutic targets to decrease the risk of CV disease in RA patients.IRF5 gene variants are associated with CV disease in RA patients.Our results reveal that ACR: American College of Rheumatology; Anti-CCP: Anti–cyclic citrullinated protein/peptide antibody; CI: Confidence interval; CV: Cardiovascular; HLA: Human leukocyte antigen; HR: Hazard ratio; IFN: Interferon; IL: Interleukin; IRF: Interferon regulatory factor; RA: Rheumatoid arthritis; SD: Standard deviation; SLE: Systemic lupus erythematosus; SNP: Single-nucleotide polymorphism; TNF: Tumor necrosis factor.The authors declare that they have no competing interests.MGB, RLM and FG carried out genotyping, participated in the study design and data analysis and helped to draft the manuscript. SC and BFG were involved in the acquisition and interpretation of data and in revising the manuscript critically for important intellectual content. JL carried out data analysis and interpretation. AC and CGJ performed the carotid ultrasound examination and were involved in the acquisition and interpretation of data as well as study coordination, and they helped to draft the manuscript. BU, JAMF, TP, CGV, LRR, AB, DPS FJLL, PC and RB participated in the acquisition and interpretation of data and helped to draft the manuscript. JM and MAGG made substantial contributions to the study’s conception and design, data acquisition and study coordination, and they helped to draft the manuscript. All authors read and approved the final manuscript.JM and MAGG share senior authorship of this article.IRF5 locus at 5 years.Proportion of the CV risk for RA patients due to age, smoking history, hypertension and the genetic variants at the Click here for fileIRF5 polymorphisms.Comparison between different genetic models of inheritance for Click here for file


Different protocols have been suggested to treat aluminum phosphide (ALP) poisoning. We aimed to evaluate the possible therapeutic effect of hyperinsulinemia/euglycemia (HIE) in treatment of ALP poisoning.In a prospective interventional study, a total of 88 ALP-poisoned patients were included and assigned into HIE group undergoing glucose/insulin/potassium (GIK) protocol and a control group that was managed by routine conventional treatments. The 2 groups were then compared regarding the signs and symptoms of toxicity and their progression, development of complications, and final outcome to detect the possible effect of GIK protocol on the patients’ course of toxicity and outcome.P < 0.001) and better outcomes . Regression analysis showed that GIK duration was an independent variable that could prognosticate mortality . The risk of mortality decreased by 4.5% each hour after initiation of GIK.The 2 groups were similar in terms of demographic characteristics and on-arrival vital signs and lab tests. Using GIK protocol resulted in significantly longer hospital stays (24 vs 60 hours; GIK protocol improves the outcome of ALP poisoning and increases the length of hospital stay. ALP poisoning was reported in only 59 cases between 1900 and 1958 with 26 of the poisoned patients dead.,4 This rate has dramatically increased within the recent 35 years due to its significant ingestion to commit suicide. It is one of the most common causes of death due to poisoning in Iran with an increased incidence of fatal ALP poisoning from 5.2 to 37 per million of population in 2006 and 2013 in Tehran.–7Annually, almost 300,000 deaths occur worldwide due to pesticide poisoning, probably some of whom die due to aluminum phosphide (ALP) intoxication. ALP was first introduced as a pesticide in India. Animal and human studies showed that high exogenous insulin had strong positive inotropic effects.–11 Hyperinsulinemia–euglycemia (HIE)—by administrating glucose, insulin, and potassium (GIK)—assists in myocardial uptake of carbohydrates which are the preferred fuel substrate of the heart under stressed conditions. The putative mechanism is correction of calcium antagonist or beta-blockers-induced hypoinsulinemia, leading to improved cell carbohydrate metabolism, increased myocardial contractility and peripheral vascular resistance, and correction of acidosis. The treatment is not expected to improve conduction block or bradycardia. HIE was first introduced as a possible treatment of ALP poisoning in 2008. Although this treatment was tested on only 5 patients at the time of publication of the preliminary results with it and is currently recommended as a treatment in ALP poisoning, no study has proved its efficacy even retrospectively.ALP poisoning has no specific antidote, and therefore, different modalities have been suggested for its treatment, to date. Patients generally die due to multi-organ failure. From the factors associating with poor prognosis, cardiocirculatory collapse and metabolic acidosis are probably the most important ones that do not respond to conventional therapies.1.1Although mortality of ALP poisoning has been reported to be as high as 90% in some studies, no antidote is available to treat the patients.1.2The aim of the present study is to evaluate the probable therapeutic effect of GIK protocol in treatment of ALP poisoning.22.1A prospective interventional study was conducted using a convenient sampling of all patients with ALP ingestion. Further confirmation of ALP exposure was done by silver nitrate test (SNT) or development of clinical signs and symptoms of toxicity. Participants entered into either the intervention group  or control group . Conventional standard therapy included using inotropes, fluids and electrolytes resuscitation, intubation, mechanical ventilation, and antiarrhythmic agents if indicated.2.2,5Loghman-Hakim Hospital Poison Center is the main referral hospital for poisoned patients in Tehran . Over 24,000 to 27,000 intoxicated patients refer to this center annually, of whom, almost 10,000 to 12,000 are hospitalized. There are almost 230 to 280 ALP suspected cases annually.2.3 the desired 95% level of confidence, the desired ± 10% precision of the estimate, a sample size of 81 was achieved to study the effect of new intervention in reducing mortality. Forty-four subjects per group were recruited after consideration of dropouts.Considering an average mortality of 70% in aluminum phosphide poisoning,2.4Between 2006 and 2012, sampling was conveniently performed. Whenever the authors were on shift and available at hospital and confirmed ALP poisoning in referred cases, the senior attending physician (first author) visited the patients and entered them into intervention group. There was no gender preference for the allocation. When other attending physicians of the department were on shift, patients were included in the conventional therapy group (control group). Written consent forms were taken from the patients or their next of kin if they were included in the intervention group. All relevant information pack was provided to next of kin and the patients. Patients with a positive history of ALP exposure with either a positive SNT result or a systolic blood pressure (SBP) of <80 mm Hg/a serum bicarbonate level of 15 meq/L or less/a pH of 7.2 or less were considered to be ALP-poisoned and included , antiarrhythmic medications, calcium gluconate, magnesium sulfate, bicarbonate, and inotropes. The intervention group was treated on the basis of the suggested protocol for study conduction Fig. , while t2.6Patients’ data including age, sex, on-arrival vital signs and lab tests, minimum systolic and diastolic blood pressures (SBP and DBPs) during the course of treatment until discharge or death, signs and symptoms on admission, treatments given, hospital stay, and the final outcome were recorded and entered into social package for statistical sciences software version 17.2.7Outcome measures were patients’ mortality and their length of stay.2.8t test was used. Mann–Whitney U test was performed to compare differences between 2 independent groups when the dependent variable was continuous, but not normally distributed. For the assessment of association between categorical variables, a chi-square test was applied. Pearson correlation coefficient was used for assessing the severity of association between continuous variables. Logistic model determined independent variables predicting death in these patients. Odds ratio (OR) and 95% confidence interval (CI) were used for expressing the severity of this association. A P value less than 0.05 was considered to be statistically significant.Kolmogorov–Smirnov test was used to determine normal and non-normal distributions of the variables, for whose description, mean (±SD) and median (interquartile range) were given. For qualitative variables, percent of frequency was provided. To compare normally distributed continuous variables between GIK and conventional standard therapy groups, 3From a total of 153 cases referred with possible ALP ingestion, 67 were excluded due to unavailable or negative SNT results as well as lack of development of clinical manifestations. These cases might have ingested nontoxic tablets or might actually be mild ALP-poisoned whose diagnoses could not be confirmed , hospital stay (24 vs 60 hours), and death (72.7% vs 50%) significantly differed between the 2 groups. Although the patients in the standard conventional group had higher blood pressures and seemed more stable regarding their vital signs, they survived less hours and died more . It should be mentioned that median [IQR] (range) of insulin dose/h, duration of GIK administration, and cumulative dose of insulin were 35  (6–94), 15  (2–134), and 525   in the GIK group, respectively. Figure P < 0.001).When comparing the survivors and nonsurvivors of the 2 groups Table , we founP < 0.02). Regression analysis showed that GIK duration was an independent variable that could prognosticate mortality ; model significance and Nagelkerke R square of 0.032, 0.610 showed that risk of mortality decreased by 4.5% each hour after initiation of GIK.All 8 patients with bradycardia died , they switch to glucose utilization. Hypoinsulinemia may prevent glucose uptake by myocytes ensuingloss of inotropy and decreased peripheral vascular resistance. This may explain the fact that hyperglycemia is a poor prognostic factor in ALP poisoning as confirmed by our results and shown by previous studies. As tissue perfusion falls, decreased delivery of glucose deprives myocytes of needed fuel. Continuation of this cycle leads to hemodynamic deterioration, shock, and death.,19Use of HIE and GIK protocol was first advocated in treatment of toxicity by beta-blockers and calcium channel blockers (CCB) in clinical toxicology. Engebretsen et al described that efficacy of HIE was due to increased inotropy and increased intracellular glucose transport. They mentioned different high-dose insulin treatment protocols. When first introduced, insulin doses were cautiously initiated at 0.5 IU/kg bolus dose followed by a 0.5 to 1 IU/kg/h continuous infusion. With increasing clinical experience and publication of animal studies, high-dose insulin was recommended. Doses up to 1 IU/kg of bolus insulin followed by doses as high as 1 to 10 IU/kg/h of continuous infusion were even advocated. Although the optimal regimen is still to be determined, bolus doses up to 10 IU/kg and continuous infusions as high as 22 IU/kg/h have been administered with good outcomes and minimal adverse events.The exact mechanism of action of HIE therapy is poorly defined. It improves inotropy and peripheral vascular resistance and reverses acidosis by improving myocyte carbohydrate uptake and utilization. In addition, this therapy may promote the metabolism of lactate and limit metabolic acidosis common in ALP poisoning. showed promising results with this protocol in 5 cases of ALP poisoning. Although this treatment is currently advocated as a routine treatment in ALP-poisoned patients in our center, no study has evaluated its efficacy in these patients.The possible therapeutic effect of GIK protocol on ALP-poisoned patients was first suggested in 2008 when Hassanian-Moghaddam,21 Even in the survivors of both groups, minimum SBP was less in the patients of the GIK group (P = 0.04). The other interesting findings were the probable protective effect of hyperventilation and tachycardia. The more the patients hyperventilated, the more they survived, and all patients with bradycardia died. These findings emphasize the possible role of hyperventilation in treatment of ALP-poisoned patients; meanwhile, higher PCO2 levels and bradycardia may be used as poor prognostic factors in this toxicity.As the results show, GIK protocol can significantly increase the hospital stay and improve the final outcome of the patient. Insulin—as a vasodilator—may also explain the difference of minimal SBP between the groups. Other clinically relevant indices such as frequency or duration of mechanical ventilation and time or total dose of vasoactive support were not evaluated, either. Prospective randomized controlled trials are warranted to evaluate the efficacy of this treatment, although it seems that depriving the patients from this treatment seems unethical.Type of patient selection (nonblinded randomization) is definitely a limitation of the present study. We only entered the patients who referred during our own shifts when we could be available for at least the first hours of initiation of GIK protocol. The nature of shock was not well established as the patients were not investigated by echocardiography or Swan Ganz catheter or even lactometer. Also, we did not document the possible complications of our treatments especially insulin. Exclusion of early deaths (those within the first 2 hours after admission) is definitely another limitation of our study which was inevitable because GIK sometimes takes several hours to affect.Other advanced novel techniques like extracorporeal life support may save the patients and GIK protocol may increase its success rate.5Insulin probably improves inotropy by altering cell metabolism from fatty acids to carbohydrates and restoring calcium flux resulting in improvement of cardiac contractility. In ALP poisoning, GIK protocol stabilizes cardiovascular system, increases hospital stay, and improves the survival rate.


Concerns have increasingly been raised about the medical economic burden in Japan, of which approximately 20% is attributable to cardiovascular disease, including coronary heart disease and stroke. Because the management of risk factors is essential for the prevention of cardiovascular disease, it is important to understand the relationship between cardiovascular risk factors and medical expenditure in the Japanese population. However, only a few Japanese epidemiological studies analysing data on health checkups and medical insurance have provided evidence on this topic. Patients with cardiovascular risk factors, including obesity, hypertension, and diabetes, may incur medical expenditures through treatment of the risk factors themselves and through procedures for associated diseases that usually require hospitalization and sometimes result in death. Untreated risk factors may cause medical expenditure surges, mainly due to long-term hospitalization, more often than risk factors preventively treated by medication. On an individual patient level, medical expenditures increase with the number of concomitant cardiovascular risk factors. For single risk factors, personal medical expenditure may increase with the severity of that factor. However, on a population level, the medical economic burden attributable to cardiovascular risk factors results largely from a single, particularly prevalent risk factor, especially from mildly-to-moderately abnormal levels of the factor. Therefore, cardiovascular risk factors require management on the basis of both a cost-effective strategy of treating high-risk patients and a population strategy for reducing both the ill health and medical economic burdens that result from cardiovascular disease. As this medical economic burden is increasing drastically each year,1 concerns regarding this issue have been raised.In Japan, the majority of outpatient medical services and hospitalizations are provided at approved clinics and hospitals within the medical insurance system, which regulates medical costs nationwide and is compulsory for all Japanese residents.1 Because the management of risk factors is essential for the prevention of cardiovascular disease, it is important to understand the relationship between cardiovascular risk factors and medical expenditure in the Japanese population. However, only a few Japanese epidemiological studies analysing data on health checkups and medical insurance have provided evidence on this topic.Cardiovascular disease, including coronary heart disease and stroke, accounts for 20.8% of the total medical expenditure in the Japanese population and is the leading cause of medical economic burden.4–12 It was conducted in 7 towns and 1 village in Shiga prefecture in the central part of Japan and included 4535 community dwellers between the ages of 40 and 69 years who were beneficiaries of NHI, an insurance group for self-employed individuals (eg farmers and fishermen) as well as retirees and their dependants. The participants were followed up for 10 years following their health checkup in 1989–1991 in order to calculate the monthly medical expenditure during the follow-up period and to identify deceased cases. If a participant withdrew or died, the follow-up period was terminated at that time. The study obtained information on medical expenditures per participant and information on cases that withdrew from the NHI system or died, using the monthly claim history from the Shiga NHI organizations. The medical expenditure recorded in this study was restricted to the fee schedule range used in the medical insurance system in Japan, calculated as the sum of the expenditures from the insurance organization and from the beneficiary.The Shiga National Health Insurance (NHI) cohort study was one of the earliest Japanese studies on this topic.The arithmetic mean medical expenditure was calculated for participants grouped according to baseline characteristics measured at the health checkup. When subtracting the arithmetic mean medical expenditure of participants without the risk factor of interest from the corresponding mean of those with the factor, this residual medical expenditure crudely represented the average medical expenditure attributable to the risk factor of interest. Data on medical expenditure was logarithmically transformed to normalize the positively skewed medical expenditure distribution. Analysis of covariance was then used to compare medical expenditure strictly among characteristic groups after adjustment for potential confounding factors, including age, sex, and other cardiovascular risk factors. Log-transformed values were ultimately expressed as geometric mean medical expenditure. The present study hypothesized that a patient with a cardiovascular risk factor incurred medical expenditures not only through treatment of the factor itself but also through very expensive procedures required for associated severe vascular diseases. To support this hypothesis, the hazard ratio for all-cause mortality was also calculated using a Cox proportional hazards model incorporating the same covariates.13 and the Japan Society for the Study of Obesity,14 the participants were categorized into one of the following 4 groups: body mass index (BMI) <18.5 kg/m2; BMI 18.5–24.9 kg/m2; BMI 25.0–29.9 kg/m2; and BMI ≥30.0 kg/m2. A J-shaped relationship existed between BMI and medical expenditure, with the nadir of the curve occurring at a BMI of 18.5–24.9 kg/m2, even after adjustment for potential confounders ; pre-hypertension (SBP 120–139 mm Hg and/or DBP 80–89 mm Hg); stage 1 hypertension (SBP 140–159 mm Hg and/or DBP 90–99 mm Hg); and stage 2 hypertension (SBP ≥160 mm Hg and/or DBP ≥100 mm Hg). Medical expenditure tended to increase with the severity of hypertension, especially in men .According to the 7th report of the Joint National Committee in the United States,P < 0.01).11 The risk of all-cause mortality tended to be higher in diabetic than in non-diabetic participants, with a multivariate-adjusted hazard ratio of 1.45 (95% CI 0.86–2.47).Medical expenditure was higher in diabetic participants, who were defined as having a history of diabetes as assessed by a self-reported questionnaire, than in non-diabetic participants .8 Proteinuria tended to increase the risk of all-cause mortality, with a multivariate-adjusted hazard ratio of 1.60 (95% CI 0.64–4.03).Medical expenditure was higher in participants who had proteinuria, identified using a spot urine dipstick test, than in those who did not have this condition , relative to those with neither hypertension nor diabetes.Hypertension and diabetes coexist in a single individual mainly due to insulin resistance accompanied by compensatory hyperinsulinemia,s Figure .7 These 10 Although this pattern was similar in participants both with and without obesity, defined as a BMI ≥25.0 kg/m2, obesity resulted in additional medical expenditures related to hypertension, diabetes, and hypercholesterolemia in each group.Medical expenditure broadly increased with the number of concomitant cardiovascular risk factors, including hypertension, diabetes, and hypercholesterolemia, which was defined as a serum total cholesterol ≥220 mg/dL.The Shiga NHI cohort study estimated medical expenditures attributable to cardiovascular risk factors from a population perspective using the population attributable fraction concept as follows:2) accounted for 3.1% of that incurred in the entire population.9The medical expenditure attributable to obesity  accounted for 23.7% of that incurred in the entire population Figure .5 The me10 The percentages were 13.1% for the presence of any 1 of the 3 metabolic disorder without obesity, 3.4% for the presence of any 2 or 3 of the 3 metabolic disorders without obesity, 2.4% for obesity alone, 1.8% for the presence of any 1 of the 3 metabolic disorders plus obesity, and 2.9% for the presence of any 2 or 3 of the 3 metabolic disorders plus obesity.The medical expenditure attributable to obesity and/or metabolic disorders, including hypertension, diabetes, and hypercholesterolemia, accounted for 23.6% of that incurred in the entire population.21The second Shiga NHI cohort study included NHI beneficiaries from all 26 local municipalities of Shiga prefecture and collected data from their health checkup in 2000 and medical expenditures for up to 6 years between the fiscal years of 2000 and 2005.n = 33 213) who were grouped by age and sex according to the number of cardiovascular risk factors each participant had at baseline: none of 4 cardiovascular risk factors ; any 1 of the 4 factors; any 2 of the 4 factors; and any 3 or 4 of the 4 factors. These 4 cardiovascular risk factors were defined as follows: hypertension, defined as SBP ≥140 mm Hg and/or DBP ≥90 mm Hg; hypercholesterolemia, defined as serum total cholesterol ≥240 mg/dL; high blood glucose, defined as casual blood glucose ≥200 mg/dL; and smoking, defined as currently smoking. The study estimated the adjusted mean medical expenditure on a real scale for the 4 age- and sex-specific groups using a gamma regression model, which is a type of generalised linear model.22 This model has recently been considered the best modelling approach to handle medical expenditure data that allows for potential confounders. The mean medical expenditure increased with the number of concomitant cardiovascular risk factors, irrespective of age or sex.21 The mean medical expenditure was 6-to-7-fold higher for participants at age 80 who had any 3 or 4 of the 4 risk factors  compared with those at age 50 who had none of the 4 risk factors .The mean medical expenditure per year during the follow-up period was calculated for participants 40 years of age or older  and the Employee’s Health Insurance scheme .24 In fact, male and female study participants who ranked in the top 1% for medical expenditures incurred costs of at least 1 807 710 and 1 437 650 Japanese yen/year, respectively (data edited for currency translation), while the respective medical expenditure sums in these top 1% male and female groups accounted for 25.6% and 21.2% of medical expenditures incurred in the entire male and female populations 40–69 years of age (n = 314 622).23 The corresponding male and female groups showed median cumulative hospitalization periods of 38 and 32 days, respectively. The study compared the risk of being in the top 1% medical expenditure group in the year following the baseline survey among age- and sex-specific participants grouped according to blood pressure and antihypertensive medication status at baseline. Participants who were not taking antihypertensive medication were categorized into 1 of the following 5 groups defined according to the 2007 criteria of the European Society of Hypertension and the European Society of Cardiology25: optimal blood pressure (SBP <120 mm Hg and DBP <80 mm Hg); normal-to-high-normal blood pressure (SBP 120–139 mm Hg and/or DBP 80–89 mm Hg); grade 1 hypertension (SBP 140–159 mm Hg and/or DBP 90–99 mm Hg); grade 2 hypertension (SBP 160–179 mm Hg and/or DBP 100–109 mm Hg); and grade 3 hypertension (SBP ≥180 mm Hg and/or DBP ≥110 mm Hg). The remaining participants, who were taking antihypertensive medication, were classified into one of the following 2 groups: well-controlled hypertension on treatment (SBP <140 mm Hg and DBP <90 mm Hg on medication); and poorly-controlled hypertension on treatment (SBP ≥140 mm Hg and/or DBP ≥90 mm Hg on medication). The risk of incurring such extremely high medical expenditures increased with more severe, untreated hypertension in men aged 40–54 or 55–69 years and in women aged 40–54 years (Figure Moturu et al suggested that a very small portion of patients accounted for a substantial percentage of the medical expenditure in the entire population.26 Therefore, cardiovascular risk factors require management on the basis of 2 strategies for reducing both the ill health and medical economic burdens resulting from cardiovascular disease. One is a cost-effective strategy of treating high-risk individuals based on the assumption that the treatment of cardiovascular risk factors themselves is essential for preventing cardiovascular disease in individuals with risk factors. The second is a population strategy which aims to shift the entire population to more desirable physical profiles from current ones. Risk factors that are directly linked to undesirable lifestyle habits, such as smoking and excessive alcohol intake, might be important targets for this purpose, as lifestyle modifications may lead directly to cost-effective elimination of the corresponding risk factors and subsequently to reduction of the overall medical economic burden.12,27Patients with cardiovascular risk factors may incur medical expenditures though both treatments of the risk factors themselves and procedures for associated diseases that usually require hospitalization and sometimes result in death. Untreated risk factors may cause medical expenditure surges, mainly due to long-term hospitalization, more often than risk factors preventatively treated by medication. On an individual patient level, medical expenditures increase with the number of concomitant risk factors. For single risk factors, personal medical expenditure may increase with the severity of that factor. However, on a population level, the medical economic burden attributable to cardiovascular risk factors results largely from a single, particularly prevalent risk factor, especially from mildly-to-moderately abnormal levels of the factor. This is explained by Rose’s theory that ‘a large number of people exposed to a small risk may generate many more cases than a small number exposed to high risk’.1,28,29 Individuals with cardiovascular risk factors, including mildly elevated blood pressure and mildly deregulated lipid and glucose metabolism, especially those with obesity, are considered candidates for incurring medical expenditure; consequently, various modes of intervention are used in these individuals, depending on the number and severity of risk factors. Therefore, medical staff and health care planners need to acknowledge the importance of this issue. Larger cohort studies are expected to provide more reliable and useful evidence on this subject. It is also important to contribute new statistical analysis methods and interesting ideas to this topic in future studies.To reduce both the ill health and medical economic burdens resulting from cardiovascular disease, the Japanese national government introduced a nationwide public health strategy in 2008.Abstract in Japanese.


Background: Cereal crops, particularly wheat, are a major dietary source of starch, and the bioaccessibility of starch has implications for postprandial glycemia. The structure and properties of plant foods have been identified as critical factors in influencing nutrient bioaccessibility; however, the physical and biochemical disassembly of cereal food during digestion has not been widely studied.Objectives: The aims of this study were to compare the effects of 2 porridge meals prepared from wheat endosperm with different degrees of starch bioaccessibility on postprandial metabolism  and to gain insight into the structural and biochemical breakdown of the test meals during gastroileal transit.Design: A randomized crossover trial in 9 healthy ileostomy participants was designed to compare the effects of 55 g starch, provided as coarse (2-mm particles) or smooth (<0.2-mm particles) wheat porridge, on postprandial changes in blood glucose, insulin, C-peptide, lipids, and gut hormones and on the resistant starch (RS) content of ileal effluent. Undigested food in the ileal output was examined microscopically to identify cell walls and encapsulated starch.Results: Blood glucose, insulin, C-peptide, and glucose-dependent insulinotropic polypeptide concentrations were significantly lower  after consumption of the coarse porridge than after the smooth porridge (P < 0.01). In vitro, starch digestion was slower in the coarse porridge than in the smooth porridge . In vivo, the structural integrity of coarse particles (∼2 mm) of wheat endosperm was retained during gastroileal transit. Microscopic examination revealed a progressive loss of starch from the periphery toward the particle core. The structure of the test meal had no effect on the amount or pattern of RS output.Conclusion: The structural integrity of wheat endosperm is largely retained during gastroileal digestion and has a primary role in influencing the rate of starch amylolysis and, consequently, postprandial metabolism. This trial was registered at isrctn.org as ISRCTN40517475. Diets containing a high proportion of foods with a low glycemic index (GI)Compelling evidence reveals that food structure plays a critical role in influencing the bioaccessibility of starch and other macronutrients and the subsequent metabolic responses –11. CurrA common misconception is that any food containing whole grains, even in flour form, is slowly digested. However, the high GI of white and whole-meal wheat bread is linked to structural characteristics of the constituent flour, which contains mostly ruptured cells with high-bioaccessibility starch , 19. PreThe aim of this postprandial ileostomy study was to provide insight into the structural and biochemical degradation of wheat endosperm  during gastroileal digestion and its effects on metabolic responses . We hypothesized that porridge prepared from coarsely milled wheat endosperm (containing intact cells) would be digested more slowly and therefore elicit a lower glycemic response than a nutritionally matched porridge prepared from finely milled endosperm (lacking intact cells). This unique study in healthy participants with an ileostomy has provided new insight into both the mechanistic aspects of starch digestion and its metabolic consequences.Healthy participants aged 20–76 with ileostomies were recruited between September 2012 and July 2013 via the Ileostomy Association in London. All participants had undergone a proctocolectomy for ulcerative colitis, pure colonic Crohn disease, or lower bowel cancer at least 1 y before taking part in the study. The study protocol was registered as ISRCTN40517475 and approved by the relevant ethics committee in the United Kingdom (REC 12/LO/1016). All volunteers gave their written informed consent after being provided with oral and written information about the aims and protocol of the study.2) <20 or >35, fasting plasma glucose >7 mmol/L, serum cholesterol >7.8 mmol/L, or serum triacylglycerols >3 mmol/L. Fasting plasma glucose concentration, BMI, blood pressure, liver function, and blood cell counts were confirmed to be within prescribed limits before enrollment in the study. All participants were otherwise healthy, with no history of diabetes or signs of diagnosed gastrointestinal conditions, as confirmed by means of a full medical history and examination. The usual dietary habits of individuals were assessed from a 3-d diet diary, and the nutrient intake was analyzed with the use of NetWisp v.3.0 (Tinuviel Software).All participants had healthy small bowels with well-functioning ileostomies with <10 cm of small intestine resected. Exclusion criteria included the following: BMI (in kg/mThis study was one part of a 2-branch investigation (study 1 was on lipid bioaccessibility and study 2 on starch bioaccessibility) with common participant recruitment and screening processes for both branches. The participant characteristics, study details, and results presented in this publication are for the starch study only.A randomized crossover study design was used in which each participant received 2 experimental meals (in random sequence) separated by at least 1 wk. The random allocation of the treatment sequence was based on a computer-generated random number sequence. The study investigators generated the random allocation sequence, enrolled participants, and assigned participants to interventions. Because of the different physical appearance and texture of the test meals, study participants were not blinded; however, all samples were coded so that those investigators assessing outcomes  were blinded. All screening and study visits took place at the Clinical Research Facilities at St. Thomas’ Hospital, London.On the day preceding the study, participants were provided with a standardized low-fiber evening meal and advised to drink plenty of water. Participants were also instructed to avoid food and drink high in sugar, caffeine, or alcohol and to refrain from strenuous exercise both on the day preceding the study and throughout the study period.An overview of the study day protocol is provided in Meals low in resistant starch (RS) were provided after blood and effluent collections at 4 h, and again at 8 h (dinner with dessert), to minimize interference with ileal RS measurements. Participants were required to eat an identical menu for both study visits. Foods for all meals were purchased from Sainsbury’s, London. Lunch  consisted of a leafy salad with cheese and either tofu, chicken breast, beef, or tuna, served with yogurt and juice (from concentrate), and contained <4 g starch. Dinner consisted of mashed potato with fish, chicken, lamb, or beef and cooked carrots, and was followed by a high-calorie  dessert. Dinner with dessert contained up to 50 g starch in total, but the majority (at least 85%) of the starch in this meal originated from mashed potato, which contained starch that previously was found to be readily digested and absorbed  of the specific test meals . This caTest meals consisted of 2 freshly prepared porridges made from 77 g durum wheat endosperm, cooked in water and flavored with 17 g low-sugar black currant jam (Sainsbury’s Reduced Sugar Blackcurrant Jam) and 61 g jelly . Proximate analysis of the durum wheat  was performed by Premier Analytic Services, and additional direct measurements of available carbohydrate (starch and sugar) contents were performed with the use of a modified Megazyme kit  method . On the Supplemental Figure 1) confirmed that the coarsely milled particles contained a high proportion of intact cells, whereas particles <0.21 mm consisted almost entirely of ruptured cells. Durum wheat (Triticum durum L. “Svevo”), donated by Millbo, was selected for this study because its fracture properties make it particularly well-suited to the milling techniques used.The durum wheat endosperm component of the meal was provided in the form of finely milled flour (<0.2 mm) to create a smooth porridge or coarsely milled macroparticles (∼2 mm) to create a coarse porridge. The smooth and coarse porridges were predicted to be high- and low-bioaccessibility starch test meals, respectively. The endosperm material was prepared from de-branned durum wheat grains with the use of a Satake TH050 roller-mill (Satake Europe), as described previously . MicrostThe porridges were cooked under standardized heating conditions by simmering smooth or coarse wheat endosperm in water (while stirring vigorously) to achieve total and uniform gelatinization of the starch. Next, the porridge was allowed to cool for 5 min, during which time the jam and jelly flavoring was stirred in. The liquid component of the meal was standardized at 449 mL by providing the required volume of drinking water to make up for evaporative losses. The meal was served immediately thereafter to prevent further starch retrogradation, which is known to decrease the susceptibility of starch to amylolysis . Both teC∞). Because the Prussian blue assay quantifies products as maltose (not glucose) and either underestimates or fails to account for other minor starch digestion products , it slightly underestimates the extent of overall starch digestion. For purposes of comparison, therefore, all digestibility data were expressed as a percentage of total hydrolyzable starch based on maltose equivalents, with the use of extracted durum wheat starch, which had been hydrothermally processed to fully gelatinize the starch  during duodenal digestion is an important determinant of the duration and magnitude of the glycemic response , 22, 23.e starch , as a ree starch .2 EDTA-coated tubes preloaded with either 100 μL aprotinin  for GIP and PYY analyses or 40 μL dipeptidyl-peptidase-IV inhibitor (Merck Millipore) for GLP-1 and cholecystokinin analyses, and kept on ice for 15 min followed by centrifugation and plasma storage. All blood samples were centrifuged at 1500 × g for 15 min at 4°C, and aliquots of the supernatant were collected and stored at either −40°C  or −80°C (for gut hormones) until analyzed.Venous blood samples for glucose, insulin, C-peptide, triacylglycerols, and NEFA analyses were collected and handled as described previously . Blood sIleal effluent was collected every 2 h for up to 10 h and overnight at the convenience of participants. Effluent samples were obtained from the participants, who emptied the contents of their ileostomy pouches into Whirl-Pak (Nasco) specimen bags at predetermined time points. Immediately after each effluent collection, the sample was weighed. A small subsample was taken directly into the fixative , and the remaining effluent was then homogenized . For starch and sugar analysis, ∼2 g of the homogenized effluent was weighed into 4 × 15 mL BD Falcon tubes (BD Biosciences) preloaded with 95% (vol:vol) ethanol to inactivate any residual enzyme activity. These samples were stored overnight at 4°C before starch and sugar analysis. Subsamples of the homogenized effluent were also taken for determination of dry matter, and these were frozen immediately (−80°C) to prevent further degradation. Overnight samples were collected into specimen bags by the participants ad libitum and subsequently placed in absorbent padding between eutectic freezer blocks  in a polystyrene box to freeze the samples at the time of collection. These frozen samples were stored for future analysis.Plasma glucose, serum triacylglycerol, and serum NEFA concentrations were determined on an iLab 650 auto-analyzer (Instrumentation Laboratories) with the use of Glucose Oxidase and Triglyceride IL test kits (Instrumentation Laboratories) and a Randox NEFA kit (Randox Laboratories). Serum insulin and C-peptide and plasma GLP-1, GIP, and total PYY (1–36 and 3–36 forms) concentrations were determined by standard methods as described previously . Cholecyg at 22°C, and the resulting supernatant was analyzed for total reducing sugar, while the pellet was analyzed for total starch content. Total reducing sugar was determined by 3,5-dinitrosalicylic acid assay in which 15 μL of the supernatant (diluted in water if necessary) was transferred into a 96-well plate  to which was added 5 μL of 3M NaOH and 20 μL 3,5-dinitrosalicylic acid reagent consisting of 1.0% (wt:vol) 3,5- dinitrosalicylic acid, 1.6% (wt:vol) NaOH and 30% (wt:vol) sodium potassium tartrate, prepared in deioinized water. Standard samples containing known concentrations of maltose were also included. The plate with samples was then sealed and incubated in a water bath at 90°C for 5 min, after which 200 μL deionized water was added. The absorbance of standards and samples were then measured at 544 nm with the use of a FluoStar Optima plate reader (BMG Labtech), thereby enabling total reducing sugar concentrations to be calculated as maltose equivalents. Starch determinations were carried out with the use of a scaled-down version  present in the ileal effluent, because the sugars are likely to be products of starch degradation , as described in previous studies , 29. To  version  of the M1, in which t is the time interval between ingestion and ileal recovery of RS for samples 0–N.Mean transit time (MTT) was calculated  as shown2 with 5% wt:vol potassium iodide] and viewed on a Zeiss Axioskop 2 mot plus light microscope. Images were captured with a Zeiss AxioCam HRc and AxioVision v3.1 microscope software (Carl-Zeiss).For examination by light microscopy, samples of ileal effluent were immersed in Karnovsky’s fixative , and left to fix at room temperature for at least 24 h. The samples were subsequently rinsed in sodium cacodylate buffer (0.1M), dehydrated through increasing concentrations of ethanol, and then infiltrated with freshly prepared LR white resin mixture (Sigma-Aldrich) prepared in 1.98% (wt:wt) benzoyl peroxide with the use of absolute ethanol as the transition solvent. Finally, resin-embedded samples were sealed in BEEM 00 capsules and polymerized at 60 ± 2°C for 24 h. The cured samples were trimmed and sectioned (0.5–1.0 μm) on an Ultracut E Reichert-Jung microtome mounted with a glass knife. Sections were stained with Lugol’s iodine [2.5% (wt:vol) It tests were used to identify time points with significantly different values between the 2 meals. The Bonferroni method was used to correct for multiple comparisons. All statistical analyses were performed on data obtained between 0 and 240 min, and additional subanalysis was performed for glucose, insulin, C-peptide, and GIP data obtained from AUC values calculated for the 0–120 min period, because this is the most dynamic part of the blood response curves to a carbohydrate-rich meal and is a common indicator of glycemia . Graphs and incremental AUCs were obtained with SigmaPlot 12.5 (Systat Software). Statistical analysis was performed with IBM SPSS Statistics 20.0. Data were checked for normality with the use of a Shapiro-Wilks normality test before analysis. A repeated-measures ANOVA was performed with time and meal as “within” factors to evaluate the effect of the test meals on postprandial responses. When significant meal × time effects were detected, Student’s paired glycemia . StatistThe cooked particles of wheat endosperm used to make the smooth and coarse porridge meals were found to have contrasting starch amylolysis characteristics  and one further participant withdrew during the second intervention because of illness . Because we could not rule out the possibility that the abdominal discomfort experienced by this participant was related to the test meal, no further interventions were attempted with this individual. A total of 9 participants (7 women and 2 men) completed the study and participant characteristics are shown in A consort flow-chart of participants through the study is shown in P = 0.038) and over 240 min for insulin (P = 0.046), C-peptide (P = 0.018), and GIP (P = 0.024), but not for glucose (P = 0.229). Also, significant meal × time interactions over 240 min were observed for glucose (P < 0.001), insulin (P = 0.035), C-peptide (P < 0.001), and GIP (P < 0.001). After 120 min, the incremental AUC for glucose, insulin, C-peptide and GIP were 33%, 43%, 40%, and 50% lower (P < 0.01), respectively, after consumption of coarse porridge than after smooth porridge. The smooth porridge, which consisted of highly bioaccessible starch, elicited a rapid and large glycemic response within the first 60 min, followed by a reduction in glucose with an overshoot to nearly 1 mmol/L below the fasting value. In contrast, the coarse porridge elicited a smaller rise in blood glucose and insulin concentrations, with a much slower rate of decline after 90 min; indeed, the glucose concentrations remained above fasting even after 4 h. The peak concentrations of glucose, insulin, C-peptide, and GIP were 12%, 32%, 37%, and 60% higher (P < 0. 01), respectively, after smooth than after coarse porridge and were reached within 90 min of ingesting the test meals.Plasma glucose (0–120 min), serum insulin, C-peptide, and plasma GIP concentrations were significantly lower after the coarse porridge  than after the smooth porridge containing high-bioaccessibility starch , significant meal × time effects were observed over 240 min for GLP-1 (P = 0.009) and PYY (P = 0.053). Plasma GLP-1 and PYY concentrations increased rapidly after test meal consumption and returned to baseline concentrations within 2 h of test meal ingestion. Peak concentrations after smooth and coarse porridge consumption were not significantly different . Cholecystokinin concentrations fluctuated throughout the 4 h period, and no clear pattern was evident (data not shown).Although there was no main effect of the meal on GLP-1 and PYY . Statistical analysis showed that the test meals had no effect on the pattern of the response curves ; however, there was a tendency toward a lower triacylglycerol response after the coarse porridge  are shown in porridge .Supplemental Figure 4). A total of ∼3 g of RS  was recovered at the terminal ileum during the 10 h after ingestion of both smooth and coarse porridge meals . For both test meals, the amount of RS recovered over the entire 24-h collection period, estimated from cumulative data, was higher than the 10-h values . However, when considering the microstructural observations (see the next section) it seems likely that the starch measured in the overnight samples may have originated from sources other than the test meals . Thus, the RS recovered during the first 10 h may be more representative of the RS content of the porridge test meals than the 24-h samples.The smooth and coarse porridges did not have any significant effects on the total amount of ileal output or meal transit, or on the amount of RS in ileal effluent  were freshly prepared, differing only in particle size (2 mm compared with <0.2 mm), and were swallowed with minimal mastication. This design ensured that any observed metabolic effects could be attributed to the structural properties of the test meals during gastroileal digestive transit.Our in vitro digestion experiments clearly demonstrated that starch entrapped within the cells of the coarse wheat endosperm particles was hydrolyzed (by α-amylase) at a considerably slower rate than the exposed starch in the fine flour particles. Hence, the structural integrity of dietary fiber, as intact cell walls, can strongly influence the rate at which entrapped starch is made bioaccessible and thereby affect the time course of glucose absorption into the portal blood.On the basis of the in vitro data showing marked differences in the rate of amylolysis between coarse endosperm and flour, we hypothesized that the same materials would also elicit differences in the in vivo glycemic responses. Indeed, the coarse porridge evoked significantly lower glycemic, insulinemic, and C-peptide responses compared with the smooth porridge. The magnitude of the reductions in glycemia and insulinemia achieved in the present study were similar to, if not greater than, the glycemic reductions achieved in some previous studies with the addition of soluble fiber or use of whole grains , 34. OurMicroscopic examination of ileal effluent revealed that coarse (2-mm) particles of wheat endosperm retained structural integrity during the first 10 h of gastroileal transit but that the majority of encapsulated starch was eventually digested from seemingly intact cells. To our knowledge, this is the first time that such microstructural observations have been reported for wheat endosperm digested in the upper gastrointestinal tract of humans. After 10 h, the majority of the test meal had reached the terminal ileum, and only negligible amounts of RS remained , irrespective of particle size. This is consistent with previous ileostomy studies of white wheat bread and cornflakes  and suggIt has been suggested that limiting the bioaccessibility of nutrients may reduce the magnitude of gut hormone responses , 38. TheAlthough the test meals were rich in starch and contained little fat (1.6 g/portion), postprandial reductions in triacylglycerol and NEFA concentrations were observed. These reductions probably reflect the net effect of the insulin-mediated uptake of fatty acids and the inhibition of lipolysis in adipose tissue , 43. No Triticum aestivum (bread wheat), are affected by processing and digestion, but the results of the current work on durum wheat, which is used in bread- and pasta-making, demonstrate proof of principle.Preserving the natural structural integrity (intact cells) of wheat endosperm, and indeed other edible plant tissues, during processing could provide a means of limiting the rate and/or extent of energy uptake from food postingestion and may have significant and important applications in the prevention and management of cardiometabolic diseases. Although wheat breads containing 50–80% whole grains have previously been shown to elicit lower glycemic responses than white bread , granaryThere is now convincing evidence that the tissue structures of some plant foods  can be retained to a greater or lesser extent during mastication  and gastIn conclusion, these results provide the first evidence that the structural integrity of wheat endosperm is a major factor in influencing starch bioaccessibility and that manipulating the structural integrity of the endosperm has the potential to greatly influence postprandial metabolism.


Also there was reduced epididymal sperm count, poor grade of sperm motility, and lower percentage of normal sperm morphology significantly. Coadministration with RPO, however, has a protective effect against lead toxicity by decreasing H2O2 production, increased GSH level, and increased sperm qualities especially. This shows that RPO has a potential to attenuate the toxic effect of lead on testicular cells preventing possible resultant male infertility.To study the protective effect of Red Palm Oil (RPO) on testicular damage induced by administration of lead acetate on male Sprague-Dawley rats, 28 rats divided into four groups of 7 animals each were used. They were administered orally with RPO (1 mL and 2 mL) and lead acetate (i.p.) 6 mg/kg body weight/day, respectively. Treatment was conducted for 8 weeks, and 24 hrs after the last treatment the rats were sacrificed using cervical dislocation. Sperms collected from epididymis were used for seminal fluid analyses; while the testes sample was used for ROS and oxidative enzyme activities assessment. Statistical analysis was carried out using GraphPad Prism 5.02 statistical analysis package. Administration of lead acetate increased generation of reactive oxygen species (ROS) significantly ( Elaeis guineensis). It originated from tropical Africa and now spread to most part of the world. RPO is an important produce from oil palm in Southeast Asia, West Africa, and South America [ in vitro and in vivo [Red palm oil (RPO) is produced from the fruit of the oil palm tree  can cause nephropathy and colic-like abdominal pains. Concentrations of blood lead >40 μg/dL seem to be associated with a decrease in sperm count, sperm volume, sperm motility, and morphological alterations [Lead is a toxic heavy metal that can damage nervous connections  and cause blood and brain disorders. Lead poisoning typically results from ingestion of food or water contaminated with lead but may also occur after accidental ingestion of contaminated soil, dust, or lead based paint . Long-teerations , 12. Chrerations . The efferations . Reproduerations , 16. In erations .Industrial lead exposure is rampant among people working in some industries such as paint and battery making companies. Leaded gasoline is still in use in many developing countries resulting in environmental lead pollution. The incidence of lead poison around the world remains unabated as recent cases are still reported in countries like Nigeria, China, and Japan –13, 16. Red palm oil from Linkjon commercial processing oil mill, certified after quality indices were determined for moisture content, free fatty acid (FFA), acid value, and saponification value using standard methods by National Agency for Food and Drug Administration and Control (NAFDAC), was purchased from Sabo oil market in Ilesa, Osun State. Nigeria and all other chemicals were purchased from Sigma Chemicals, USA. The administered dose of lead acetate was selected according to our previous study .Twenty-eight healthy adult male Sprague-Dawley rats  were housed in clean polypropylene cages and maintained in animal housing facility with constant 12 h/12 h dark and light cycle. All animal handling procedure was approved by the College Ethics Committee.n = 7), labeled as groups I, II, III, and IV. Group I represents control and received water as placebo. Group II was administered (i.p.) with 6 mg/kgbw lead acetate only. Groups III and IV intraperitoneally received 6 mg/kgbw of lead acetate and 1 and 2 mL of RPO, respectively. Feed and water were made available ad libitum. The rats were divided into four groups . This procedure including evaluation of sperm motility and morphology was done as reported in our previous study .2O2, LPO, and GSH levels were assayed as described by Adedara and Farombi, 2014 [Samples from testes were homogenized in 50 mM tris-HCL buffer (pH7.4) containing 1.15% potassium chloride and the homogenate cold (4°C) centrifuged at 10,000 ×g for 15 minutes. The supernatant was collected for the estimation of markers of oxidative stress; Hbi, 2014 , 19.Tissues were fixed in Bouin's fluid for routine H&E histological preparation and photomicrographs of the sections were taken at different magnifications.t-test was used to compare difference between means. The difference of means was considered significant at p < 0.05.The results were statistically analyzed using the Prism 5 for Windows . The Mean ± Standard Error of Means of the data was calculated. One-way ANOVA was used to check the significance differences within and between groups while Student's Mortality. No mortality was recorded during the course of the experiment.p < 0.05) with 15.2% and 11.05% decrease in body weight, respectively, while the final body weight of group IV animals increased by 14.28% , while it is seen in groups III and IV that were coadministered with RPO that there are no significant changes in the enzymes activities when compared to group I, meaning that the testes were protected from lead which induces oxidative stress  declined body weight (15.2%) in lead acetate only treated groups when compared to the control. Also weight loss of 11.05% was recorded in group treated with 1 mL of RPO against a weight gain of 14.28% in group treated with 2 mL RPO. RPO attenuated the weight loss in lead treated animals in doses dependent manner. The higher the doses of palm oil, the lesser the weight lost. Although the observed initial animal body weight range for the experiment is wide, this did not in any way significantly influenced the change observed. Whereas increased food consumption by the animals in RPO (higher dose group) is plausible, the overall effects of these changes are statistically factored in the design of the experiment.Heavy metals are widely distributed in the environment. Environmental discharge of lead due to the use of petroleum products , construction works, paint removal, demolition, vehicle batteries, and car repairs contribute to airborne lead pollution  and possThe seminal fluid assessments (sperm count and sperm motility) were significantly decreased in the lead treated groups  compared with group I; this shows that lead toxicity causes oligospermia. Similar observations were recorded in experimental animal experimental models exposed to heavy metals , 23, 24.The percentage of the sperms with normal morphology in lead treated groups  was decreased with high prevalence of sperm head abnormality compared with control; this concurs with the earlier report which indicated that lead altered testis histology resulting in structural defects in sperms . Also, t2−), hydrogen peroxide (H2O2), hydroxyl radical (OH), nitric oxide (NO), hypochlorous acid (ClOH), peroxide (ROOH), and peroxynitrite (ONOO) [2O2 and LPO in this study shows a significant increase in the amount of generation of these free radicals in the lead acetate only treated group and a similar decrease in the RPO administered groups in a dose dependent manner. GSH is an intercellular antioxidant which is usually in high concentration within cell. In mice treated with heavy metals there were significant decreases in the levels of GSH and SOD in kidney and testicular tissues [Reactive oxygen species (ROS) are highly reactive forms of oxygen, free radicals (FR), which are short-lived intermediate containing one or more unpaired electrons. They include an array that has superoxide anion (Oe (ONOO) . These he (ONOO) , 29. The tissues ; our res tissues . Prevent tissues  and RPO Our results suggest that RPO offers positive protection against lead acetate induced testicular injuries. However, further studies are warranted to elaborate on the definite mechanism of its antioxidative potential.