High-molecular-weight phthalates, such as diisononyl phthalate (DINP) and diisodecyl phthalate (DIDP), are used primarily as polyvinyl chloride plasticizers.We assessed exposure to DINP and DIDP in a representative sample of persons ≥ 6 years of age in the U.S. general population from the 2005–2006 National Health and Nutrition Examination Survey (NHANES).We analyzed 2,548 urine samples by using online solid-phase extraction coupled to isotope dilution high-performance liquid chromatography–tandem mass spectrometry.p < 0.01) among children than among adolescents and adults.We detected monocarboxyisooctyl phthalate (MCOP), a metabolite of DINP, and monocarboxyisononyl phthalate (MCNP), a metabolite of DIDP, in 95.2% and 89.9% of the samples, respectively. We detected monoisononyl phthalate (MNP), a minor metabolite of DINP, much less frequently (12.9%) and at concentration ranges (> 0.8 μg/L–148.1 μg/L) much lower than MCOP . Adjusted geometric mean concentrations of MCOP and MCNP were significantly higher (The general U.S. population, including children, was exposed to DINP and DIDP. In previous NHANES cycles, the occurrence of human exposure to DINP by using MNP as the sole urinary biomarker has been underestimated, thus illustrating the importance of selecting the most adequate biomarkers for exposure assessment. Phthalates are high-production-volume chemicals. Some phthalates are used as solvents and additives and are often found in personal care products, such as cosmetics, lotions, and perfumes, and in the coatings of some medications . Others,Historically, toxicologic studies of phthalates have focused on liver effects in rodents and the toxicologic relevance of these effects to humans , 2003b. After exposure, phthalates are rapidly metabolized to their corresponding hydrolytic monoesters, which can be further transformed to hydrophilic oxidative products, conjugated, and eliminated . For theBiomonitoring data have consistently shown widespread human exposure to multiple phthalates . By contAssessing human exposure to phthalates is of interest because of their potential adverse health effects, particularly among the young. Data for several phthalates are available, but exposure information for high-molecular-weight phthalates, including DINP and DIDP, is inadequate  or rather limited . The aims of this study are 3-fold. First, we present for the first time nationally representative data on the concentrations of oxidative metabolites of DINP and DIDP in urine among those people in the U.S. general population ≥ 6 years of age, stratified by age group, sex, and race/ethnicity. Second, we evaluate the exposure to DINP and DIDP, based on the urinary concentrations of their metabolites MCOP and monocarboxyisononyl phthalate (MCNP), respectively, according to select sociodemographic factors. Finally, we also discuss the validity of MNP as a biomarker of exposure to DINP and compare its sensitivity with that of MCOP.Since 1999, the CDC has conducted the National Health and Nutrition Examination Survey (NHANES) annually. NHANES provides data, released in 2-year intervals, to evaluate the health and nutritional status of the civilian, noninstitutionalized U.S. population of all ages. NHANES includes household interviews, standardized physical examinations, and collection of medical histories and biological specimens. Some of these specimens are used to assess exposure to environmental chemicals.For this study, we analyzed 2,548 spot urine specimens collected during one of three daily examination sessions from a one-third subset of 2005–2006 NHANES participants ≥ 6 years of age. The representative design of the survey was maintained, because the subset was a random sample of the total NHANES population. The National Center for Health Statistics (NCHS) Institutional Review Board reviewed and approved the study protocol. All participants gave informed, written consent; parents or guardians provided consent for participants < 18 years of age.The urine samples were shipped on dry ice to the National Center for Environmental Health at the CDC and stored at −20°C or below until analyzed. The analytical method for measuring 15 phthalate monoesters, including MNP and oxidized metabolites of DINPs (MCOP) and DIDPs (MCNP), in 100 μL urine has been described in detail elsewhere . The ana0, where S0 is the standard deviation as the concentration approaches zero  and SUDAAN  to perform statistical analyses. SUDAAN calculates variance estimates that account for the complex, clustered design of NHANES. As recommended by NCHS, we used sample population weights to produce estimates that are representative of the U.S. population. We used the logoot of 2  to the cn = 195) were included only in the total population estimate. For each age, sex, and race/ethnic group, we calculated geometric means (GMs)  and distribution percentiles for both volume-based (micrograms per liter) and creatinine-corrected concentrations (micrograms per gram creatinine). We also determined weighted Pearson correlations among the creatinine-corrected concentrations (log10 transformed) of MCOP, MCNP, and MNP in the 334 samples with detectable concentrations of all three compounds. Statistical significance was set at p < 0.05.We stratified age, reported in years at the last birthday, in four groups . On the basis of self-reported data, we categorized race/ethnicity as non-Hispanic black, non-Hispanic white, and Mexican American. Participants not defined by these racial/ethnic categories . We categorized income as < $20,000, $20,000–$45,000, and > $45,000 to obtain a comparable number of participants per group. For the multiple regression models, we used the variables described previously and all their possible two-way interactions to calculate the adjusted GM concentrations (in micrograms per liter) of MCOP and MCNP. These variables were log10 transformed, because the distributions of concentrations of these phthalate metabolites and creatinine were skewed.We used multiple regression to examine whether several variables  were associated with the logTo arrive at the final model for each analyte, we used backward elimination with SUDAAN to remove the nonsignificant interactions one at a time. Covariates with nonsignificant main effects were then removed one at a time, and the model was rerun to determine whether the beta coefficients for covariates with significant main effects or interactions changed by > 10%. If any did, we retained the relevant nonsignificant covariate in the model. Once the backward procedure was completed, covariates and interactions between covariates were added back into the model one at a time to determine whether any were significant, in which case they were retained in the final model.We also constructed a 2 × 2 table to examine the suitability of the urinary concentrations of MNP and MCOP as DINP exposure biomarkers.In most of the samples analyzed, we detected MCNP (89.9%). Similarly, we detected MCOP in most samples (95.2%), but we detected MNP much less frequently (12.9%). More important, in 82.4% of participants with detectable concentrations of MCOP, which is a sensitive biomarker of exposure to DINP, MNP, the hydrolytic metabolite of DINP, was undetectable . The GM p < 0.0001) good to moderate correlations between the creatinine-corrected concentrations of MCOP and both MNP [Pearson correlation coefficient (R) = 0.63] (R = 0.46). We also observed a significant (p < 0.0001) but rather weak correlation (R = 0.25) between the concentrations of MCNP and MNP  than the R2 for the model unadjusted for these two variables . For both phthalate metabolites, adjusted GM concentrations for children were significantly higher (p < 0.01) than for all other age groups; differences between adolescents and adults and between younger and older adults did not reach statistical significance . Persons in the medium income level had significantly higher adjusted GM concentrations of MCNP than those in the low-income level (p = 0.03); other differences between household income groups did not reach statistical significance , the results were very similar to those presented in The final MCOP and MCNP models included household income, age group, and logvariates . The logificance . Adjusteificance . If instWe also conducted weighted univariate and multiple logistic regressions to examine the association of the concentrations of MCOP and MCNP above the 95th percentile  with sex, age group, race/ethnicity, household income, and examination session. We found no covariates significantly associated with the likelihood of MCNP or MCOP exceeding the 95th percentile (data not shown).We detected MCOP at concentrations ranging from > 0.7 μg/L to 4,961 μg/L in 95.2% of persons examined. By contrast, MNP, the hydrolytic DINP metabolite, was detected in only 12.9% of people and at lower concentration ranges (> 0.8–148.1 μg/L). The only DIDP metabolite evaluated, MCNP, was detected in 89.9% of persons at concentrations of > 0.6–672.6 μg/L. These data suggest that at least 90% of the general U.S. population is exposed to DINP and DIDP. These data are in agreement with previous evaluations of the urinary concentrations of MCOP  and MCNPConsistent with expectations, urinary concentrations of MCOP and MNP, which are metabolites of the same parent compound, correlated well with each other. We also found a fair correlation between the urinary concentrations of MCNP and MCOP. Commercial DINP and DIDP formulations are complex mixtures of C8–C10 or C9–C11 phthalates, respectively, enriched in C9 (DINP) or C10 (DIDP) isomeric phthalates , 2002b. Of interest, the person with the highest urinary concentration of MCOP in this study also had the highest urinary concentration of the hydrolytic metabolite of DINP, MNP. These data are in agreement with a previous report on the metabolism and elimination of four DINP metabolites, including MCOP and MNP, in a male adult volunteer after administration of a single oral dose of deuterium-labeled DINP of 1.27 mg/kg body weight . In thatWe observed that adjusted GM concentrations of MCOP and MCNP were dependent upon age and income. The differences in urinary concentrations of MCOP and MCNP among the various demographic groups examined may reflect differences in the consumption of food or use of consumer products containing DINP and DIDP, respectively. For the first time, we report here the urinary concentrations of some DINP and DIDP metabolites among school-age children and adolescents in the United States. Although we do not have data for toddlers and preschool-age children, our data confirm that exposure to these two phthalates occurs at young ages.Variability in a person’s exposure to phthalates can result from changes in use of personal care products, diet, or other activities. Although the urinary concentrations of phthalate metabolites can be used to assess a person’s exposure at a single point in time, the predictive ability of one spot sample to categorize exposure over longer time periods will differ among phthalate metabolites; for MCOP and MCNP this temporal variability is unknown. However, several reports suggest that, although some metabolites display more temporal variability than others, the concentrations of several phthalate metabolites in a single urine sample can provide a reliable ranking to classify exposure of an individual to phthalates for up to several months . FurtherGenerating high-quality biomonitoring data requires state-of-the-art analytical chemistry methods as well as controlled sampling protocols and quality control/quality assurance procedures . In addiAlthough the frequency of detection and concentration ranges of MNP in NHANES 2005–2006 and NHANES 1999–2000 are quite similar , NHANES We measured the urinary concentrations of one oxidative metabolite (MCOP) and the hydrolytic metabolite (MNP) of DINP, as well as MCNP, an oxidative metabolite of DIDP, in the general U.S. population. MCOP and MCNP were detectable in most persons examined, whereas MNP was detected in only about 12%. These NHANES data suggest that the U.S. general population is often exposed to DIDP and DINP and highlights the need for additional studies to identify potential sources of DINP and DIDP. Of interest, the significantly higher frequency of detection and urinary concentrations of MCOP than of MNP confirm the validity of MCOP as a biomarker for DINP exposure assessment and suggests widespread exposure to DINP. More important, these NHANES data suggest that the occurrence of exposure to DINP has been underestimated by using MNP as the sole DINP urinary biomarker.


The role of the process of apoptosis is investigated in the pathogenesis of many autoimmune diseases; however at present, there is not much information about its role in dermatitis herpetiformis. Skin biopsies were taken from 18 DH patients and from 10 healthy subjects. The localization and expression of Bax, Fas, FasL, TRAIL, TRAIL-R in skin lesions, and perilesional skin were studied by immunohistochemistry. Expression of Bax, Fas, and Fas ligand was detected in the keratinocytes in skin biopsies from DH patients. Expression of TRAIL and TRAIL receptor was confirmed in epidermis, infiltration cells, and some fibroblasts. The expression of examined molecules in biopsies from healthy people was observed only in single cells. There were statistically significant differences between lesional, perilesional, and healthy skin of control group in Bax expression analysis and between lesional skin and control group in Fas, FasL, and TRAIL expression. There were statistically significant differences between control group and perilesional skin in Bax and FasL expression. Our results show that selected proapoptotic molecules may take part in pathogenesis of dermatitis herpetiformis, but the role of apoptosis in this process is not clear. Dermatitis herpetiformis (DH) is a chronic subepidermal autoimmune bullous diseases characterized by skin and intestinal lesions. Skin lesions include polymorphic eruption , mainly distributed over the shoulders, elbows, backs, buttocks, and knees. They are usually symmetric and accompanied by severe pruritus. These symptoms are usually associated with asymptomatic and gluten-sensitive enteropathy. Diagnosis of DH is established on the results of direct immunofluorescence test (DIF) revealing granular deposits of IgA in the top of the papillae and the presence of circulating IgA antibodies directed against endomysium and/or tissue, and epidermal transglutaminase . Skin lesions in DH are histologically characterized by neutrophilic infiltrate leading to destruction of basement membrane zone (BMZ) proteins. Impairment of type IV collagen, laminin, and entactin results in degradation of anchoring fibres and blister formation. , 2 To daThe role of the process of apoptosis is investigated in the pathogenesis of many autoimmune diseases; however at present, there is not much information about its role in subepidermal blistering diseases.Apoptosis, also known as programmed death of a cell, is an outcome of the intracellular cell “suicide,” which is regulated by cellular pathways of passing the signal. It prevents many pathological processes, for example, autoimmunization and neoplasm , 4.Up to this day, two pathways of apoptosis have been described, that is, intrinsic and extrinsic and the basis for its distinction is a way of activation of the procaspases which initiate them , 6.B-cell leukemia/lymphoma-2) as a result of, for example, medicine administered or the destruction of DNA structure [2+, thermal shock, active toxins, the disturbance of the electron transport, or DNA damage [The intrinsic one, called mitochondrial, is connected with the activation of cytochrome c by the proapoptotic genes belonging to Bcl-2 family (tructure . The feaA damage .death domain (DD), by means of which the activation of the procaspases inside the cell occurs [The extrinsic pathway is associated with attaching the ligands to the receptor belonging to the superfamily of TNF receptors, which possess the so-called l occurs .The intrinsic and extrinsic pathways stimulating apoptosis are linked to each other by, for example, Bid protein, belonging to Bcl-2 family .TNF-related apoptosis-mediating protein), and DR-6 [The proteins composing the superfamily of TNF receptor also take an active part in the process of apoptosis. These are Fas, TRAIL-R1 (DR4), TRAIL-R2 , TNF-R1, TNF-R2, TRAMP  was performed on 18 patients  with dermatitis herpetiformis, who were treated in Department of Dermatology and Venereology of Medical University of Lodz. The patients were before treatment, at an active stage of the disease, that is, with skin lesions  developed. The lesions were accompanied by itch of different intensification. DH was diagnosed based on clinical picture, histological, and immunological findings.10 healthy volunteers, selected according to their sex and age, made up the control group.All the participants of the experiment gave an explicit consent in writing before entering the study and the study protocol was approved by The Local Ethical Committee of Medical University of Lodz .The biopsies from all patients were taken from lesional and from uninvolved skin (trunk) before administration of any treatment . In control group, biopsy specimens were taken from buttock or abdominal skin of healthy volunteers.μm) were used for routine H + E staining and for immunohistochemistry in DAKO EnVision detection system using immunoperoxidase method. The following primary monoclonal antibodies were used: Bax  (1 : 650), TRAIL  (1 : 200), TRAIL-R  (1 : 200), Fas, Fas ligand  (1 : 200).Paraffin-embedded sections . In order to retrieve the antigenicity of tissues and allow them to react with antibodies, sections were prepared in bath (98°C-1 h) or microwave oven. Then the sections were washed with TRIS buffer (pH 7.6) for 5 min. Activity of endogenous peroxidase was inhibited with 0.3% hydrogen peroxide solution in methanol for 30 minutes. Primary antibody solution directed against human antigens was put on these sections. After incubation with diluted antibodies for 60 minutes at room temperature or for 12 hours at 4°C, they were washed with TRIS buffer twice. DAKO EnVision double-step visualization system was then used in order to visualize the antigen-antibody reaction. In cases of positive immunohistochemical reaction, cellular nuclei were stained with Meyer haematoxylin for 2 minutes. After dehydratation and processing through series of acetones and xylenes, the sections were fixed in DPX. For every antibody a negative control was performed using TRIS buffer instead of antibody.In each specimen staining intensity of TRAIL and TRAIL receptor, Fas as well as Fas ligand in the epithelium and inflammatory infiltrates were recorded by two independent observers in 4–7 adjacent high power fields and graded from 0 (staining not detectable), 1 , 2 (weak immunostaining intensity in most cells), and 3 (strong staining in most cells). The mean grade was calculated by averaging grades assigned by the two authors and approximating the arithmetical mean to the nearest unity.Morphometric analysis was used for Bax . The percentage of Bax immunopositive cells was estimated by counting in each slide 500 cells in 4–7 adjacent high power fields (semiautomatic function).t-test was applied where appropriate after evaluation of distribution. Mann-Whitney test was used where necessary. The difference was considered statistically significant when P < 0.05.All data are shown as mean ± SD. Student's Bax protein expression was discovered in the cytoplasm of keratinocytes in samples of lesional skin (mean immunoexpression 39.742 ± 7.295) . The expFas expression in lesional skin was detected in the cytoplasm of keratinocytes (0.75 ± 0.347) . In uninFas ligand expression was revealed in the basal layer of the epidermis and inflammatory infiltrates in lesional skin (0.923 ± 0.427) . In uninImmunostaining of TRAIL was detected in the cytoplasm of keratinocytes as well as in inflammatory cells, and some fibroblasts in lesional skin (1.555 ± 0.783) . In uninImmunostaining of TRAIL receptor was revealed in inflammatory infiltration, in the cytoplasm of keratinocytes as well as in some fibroblasts in lesional skin (0.735 ± 0.634) , in uninThere were statistically significant differences between lesional, perilesional, and healthy skin of control group in Bax expression analysis. The expression of Fas and TRAIL was higher in lesional skin than in perilesional, and in healthy group. FasL expression was significantly higher in skin lesions and perilesional skin than in control group. In TRAIL-R analysis, there was no statistically significant difference, although the expression of TRAIL-R was more intense in uninvolved than in lesional skin.Statistical analysis was presented in Apoptosis is claimed to be involved in a number of chronic inflammatory and neoplastic skin diseases such as contact dermatitis, toxic epidermal necrolysis, acantholytic dermatoses, and systemic lupus erythematosus , 12, 13,Caproni et al.  examinedProapoptotic Bcl-2 family consists of many proteins for example, Bax, that takes part in intracellular pathway of apoptosis. In nonapoptotic cells, Bax and Bcl-2 form heterodimers keeping the homeostasis . In our Caproni et al.  also exaThe results of our study showed significant difference of Bax expression between lesional, perilesional, and healthy skin; that is why the overexpression of Bax protein seems to take part in DH pathogenesis, but explanation of the precise mechanism of starting the intrinsic pathway of apoptosis needs more studies.Extrinsic pathway of apoptosis starts for example, after activation of Fas receptor  by its ligand FasL . In physAlso in other bullous diseases, Fas was examined, Morawej et al.  describeAlso our earlier research , consideSimilar data showed Caproni et al.  describe Fas has not only proapoptotic activity, but also proinflammatory one , 25. Infβ, TNF-α, IFN-γ, and IL-15), viral infections  can induce expression of Fas in epidermis [Many factors, such as UV radiation, cytokines  and TRAIL-R2 DR5), which transduces the death signal and induces apoptotic cell death , 31. Alt, which tadhesion , 34. In adhesion , 35 thusIn vitro TRAIL is a stronger proapoptotic factor than TNF. In spite of that, it seems that most of cells are resistant to TRAIL-induced apoptosis. TRAIL protein is constitutively present on many cells, but in T and NK cells and dendritic blood cells, it is synthesized after their stimulation [ The role of TRAIL in physiological condition is not completely known. mulation . Interfemulation .The role of TRAIL in autoimmune diseases is discussed. Some researchers suggest that high level of TRAIL can increase disease activity, whereas some others present opposite opinion. TRAIL can induce the process of apoptosis of dendritic cells and neutrophils and its high expression was seen in T cells infiltrating skin in atopic dermatitis . In our The recent study of Wu et al.  reportedHigher expression of TRAIL receptor in perilesional rather than lesional skin seems interesting, but these results were not statistically significant. The more intense in uninvolved than in lesional skin expression of TRAIL receptor DR4 on keratinocytes and cells of inflammatory infiltration may suggest its role in maintaining inflammatory process and damage to dermoepidermal junction.The proteins from intrinsic (Bax) and extrinsic (Fas and TRAIL) pathway of apoptosis activation were assessed in our study. However, some authors hold the opinion that the recognition of autonomy of these two apoptotic ways is a simplification, as there are proteins which connect these two ways, for example, Bid protein . Also Su Caproni et al.  state thin vitro research seems to be needed to clarify the role of apoptosis in DH.Our earlier study  considerOur results as well as studies conducted by other authors showed that the role of apoptosis in DH pathogenesis is not clear. The exact stimulus triggering apoptosis in DH skin lesion still remains unknown, but the present study provides strong evidence that there is a difference between expression proapoptotic proteins in skin lesions, perilesional skin, and healthy skin. Cell death by apoptosis is a physiological process that enables the elimination of cells without causing an inflammatory response, but inflammation can provoke apoptosis so this mechanism in DH should be more explained.


Colin Masters died on April 6 from cancer, after making outstanding and pioneering contributions to biochemistry and biotechnology in Australia and to the development of Griffith University in SE Queensland as a major research based university.Colin was born in Taihape, a small town on the north island of New Zealand. In his view, the rural setting was an ideal background for his early childhood, full of friendship and scope for youthful adventures, such as canoeing on the local rivers, and exploring the caves, cliffs and other features of the local farmland. Subsequently, he attended a number of different primary schools (as his parents moved from town to town), and then won a scholarship to King’s College (one of NZ’s leading private schools) for his secondary education. There, in his final year, he was the top science student and gained a scholarship to university. He was also a school prefect, head of a house, played in the first fifteen and the first eleven, and represented the school at athletics, gymnastics and swimming.Casting back to his early boyhood, Colin was fascinated by science from an early age, and when only 10 years old had set up quite a sophisticated chemistry laboratory in a shed at the rear of his parent’s property, where he spent many hours experimenting. He maintained this interest in science throughout secondary school and university, and graduated from Auckland University with a Master’s degree in science.His first employment after graduating was as a medical scientist in charge of a biochemical laboratory in Auckland. He was keen to further advance his interest in this subject, and moved to a teaching and research position at the University of Queensland in Brisbane in 1960. He stayed there for 15 years, eventually heading the Department of Biochemistry, before accepting a position as the foundation professor of biochemistry at Griffith University. In subsequent years at Griffith University, he served as Dean of Science, and then as Pro-Vice-Chancellor, and on several occasions Acting Vice Chancellor—all the time maintaining his interest in science, and eventually being the recipient of three doctoral degrees. Among his many other academic roles, he chaired key university wide committees, including the academic, education and research committees, and was responsible for introducing many major educational streams, which are now features of the university’s offerings. He was also a member of the State Government Working Party, which advised on the tertiary entrance system in Queensland, and served on the inter-university Advisory Council, which oversaw the physical and academic development of Bond University.Many of these aspects of his career were brought into focus at his retirement ceremony in 1992, when he was described by the University Chancellor as a gentleman of science, a man of outstanding scientific achievements, broad international reputation, high academic integrity, excellent administrative abilities, and a calm and efficient style of administration.In addition to his heavy teaching and administrative duties at the University of Queensland and Griffith University, he still managed to carve out an outstanding career in research. He was internationally recognized as a top researcher in his field; an original thinker, who championed several major paradigm shifts in biomedical science. As such, he was often invited to collaborate in research with leading scientists in other countries, and lauded for his seminal development of major innovatory concepts, which enabled important breakthroughs in areas of contemporary interest such as biotechnology, genetic disease, mammalian growth processes, and medication metabolism. In the pursuance of this research, he published more than 300 research papers in international journals, and was an invited lecturer at many overseas research institutes and universities. He was recognized internationally as a leader in several fields of biochemistry and biotechnology, including mammalian development, molecular biology, protein interactions, peroxisomal biogenesis and metabolic control mechanisms. In addition, Colin was renowned for his expert, calm and successful role as a research postgraduate student and postdoctoral supervisor of many scientists, some of whom hold Professorial and other senior positions within universities and other research organizations in Australia and internationally.During his career and in his years of retirement, Colin Masters also wrote several books as advanced texts on aspects of his research, a number of which were republished in several languages. His most recent book, ‘DNA and Your Body, What you should know about Biotechnology’, though, was directly aimed at the general public. DNA of course is well known as the molecule that programs our human potential . Aware of the momentous changes that are occurring in DNA biology and the dramatic effect that these advances have on human health and attitudes , he wrote this book in a form, which was readily comprehensible to a general reader to facilitate a broader understanding of these issues among the general public.Besides writing books on science in his retirement years, Colin also participated in several areas of community interest. As one example, he made many contributions to the game of bridge in Queensland, writing a definitive history, well known to adherents of the game (‘Mind Games’), and spent several years in administrative roles. Upon his retirement  from the central management committee of the Queensland Bridge Association, for example, he was commended for his years of valuable service to the game by the president of the Australian Bridge Federation, and described as a man of integrity, dignity, character and courage.In addition to his academic interests, Colin Masters maintained an active interest in sports during the whole of his career. Several years of representative rugby and athletics in New Zealand, were followed by an active interest in tennis and golf in his later years in Australia.Colin is survived by his wife Josephine, three sons, Greg, Alistair and Scott, and six grandchildren.Roger S HolmesJuly 2012M Hinks and CJ Masters. Developmental changes in ruminant lactate dehydrogenase. Biochemistry 3: 1789–1791 (1964).RS Holmes, CJ Masters and EC Webb. A comparative study of vertebrate esterase multiplicity. Comparative Biochemistry and Physiology 26: 837–852 (1968).N Kingsbury and CJ Masters. Molecular weight interrelationship in the vertebrate esterases. Biochimica Biophysica Acta 200: 58–69 (1970).C Gibson and CJ Masters. On the lactate dehydrogenase of preimplantation mouse ova. FEBS Letters 7: 277–279 (1970).CJ Masters and RS Holmes. Isoenzymes and ontogeny. Biological Reviews 47: 309–361 (1972).BJ Kitchen, CJ Masters and DJ Winzor. Effects of lipid removal on the molecular size and kinetic properties of bovine plasma arylesterase. Biochemical Journal 135: 93–99 (1973).CJ Masters and RS Holmes. Isoenzymes, multiple enzyme forms, and phylogeny. Advances in comparative Physiology and Biochemistry 5: 109–195 (1974).CJ Masters and RS Holmes. Haemoglobin, Isoenzymes and Tissue Differentiation. North Holland Publishers (1975).RI Brinkworth, CJ Masters and DJ Winzor. Evaluation of equilibrium constants for the interaction of lactate dehydrogenase isoenzymes with reduced NAD by affinity chromatography. Biochemical Journal 151: 631–636 (1975).M Don and CJ Masters. On the comparative turnover rates of the lactate dehydrogenase isozymes in rat tissues. International Journal of Biochemistry 7: 215–220 (1976).CJ Masters and RS Holmes. Peroxisomes: new aspects of the cell physiology and biochemistry of these oxidative organelles. Physiological Reviews 57: 816–882 (1977).RS Holmes and CJ Masters. Genetic control and ontogeny of microbody enzymes: a review. Biochemical Genetics 16: 171–190 (1978).TP Walsh, DJ Winzor, FM Clarke, CJ Masters and DJ Morton. Binding of aldolase to actin-containing filaments. Evidence of interaction with the regulatory proteins of skeletal muscle. Biochemical Journal 186: 89–98 (1980).D Crane, RS Holmes and CJ Masters. Proteolytic modification of the mouse liver catalase. Biochemical and Biophysical Research Communications 104: 1567–1572 (1982).E Klucis, D Crane and CJ Masters. Sequential alterations in the microlocalization of catalase in mouse liver after treatment with hypolipidemic drugs. Molecular and Cellular Biochemistry 65: 73–82 (1984).C Masters and D Crane. The role of peroxisomes in lipid metabolism trends in Biochemical Sciences 9: 314-317 (1984).DI Crane, AC Hemsley and CJ Masters. Purification of peroxisomes from livers of normal and clofibrate-treated mice. Analytical Biochemistry 148: 436–445 (1985).S Reid and CJ Masters. On the ontogeny and interactions of glyceraldehyde-3-phosphate dehydrogenase. Mechanisms of Ageing and Development 35: 209–219 (1986).L Humphreys, S Reid and CJ Masters. Evidence for the spatial separation of the binding sites for substrate and for cytoskeletal proteins on the enzyme aldolase. International Journal of Biochemistry 18: 7–13 (1986).N Chen, DI Crane and CJ Masters. Analysis of the major integral membrane proteins of peroxisomes from mouse liver. Biochim et Biophysica Acta-Biomembranes 945: 135–144 (1988).DI Crane, J Zamettia and CJ Masters. Alterations in the integrity of peroxisomal membranes in livers of mice treated with peroxisome proliferators. Molecular and Cellular Biochemistry 96: 153–161 (1990).CJ Masters. Cellular differentiation and the microcompartmentation of glycolysis. Mechanisms of Ageing and Development 61: 11–22 (1991).W Murrell, D Crane and C Masters. Ontogenic characteristics of cavian aldolase. Mechanisms of Ageing and Development 65: 35–50 (1992).CJ Masters and DI Crane. On the role of the peroxisome in ontogeny, ageing and degenerative disease. Mechanisms of Ageing and Development 80: 69–83 (1995).CJ Masters and DI Crane. The peroxisome: a vital organelle. Cambridge University Press (1995).CJ Masters. Cellular signaling: the role of the peroxisome. Cellular Signaling 8: 197–208 (1996).CJ Masters. Omega-3 fatty acids and the peroxisome. Molecular and Cellular Biochemistry 165: 83–93 (1996).CJ Masters. Gluconeogenesis and the peroxisome. Molecular and Cellular Biochemistry 166: 159–168 (1997).C Masters and D Crane. On the role of the peroxisome in cell differentiation and carcinogenesis. Molecular and Cellular Biochemistry 187: 85–97 (1998).C Masters. DNA and Your Body: What You Need to Know about Biotechnology. University of NSW Press (2005).


Evaluate and compare the utility of serum folate receptor alpha (FRA) and megakaryocyte potentiating factor (MPF) determinations relative to serum CA125, mesothelin (MSLN) and HE4 for the diagnosis of epithelial ovarian cancer (EOC).Electrochemiluminescent assays were developed for FRA, MSLN and MPF and used to assess the levels of these biomarkers in 258 serum samples from ovarian cancer patients. Commercial assays for CA125 and HE4 were run on a subset of 176 of these samples representing the serous histology. Data was analyzed by histotype, stage and grade of disease. A comparison of the levels of the FRA, MSLN and MPF biomarkers in serum, plasma and urine was also performed in a subset of 57 patients.Serum and plasma levels of FRA, MSLN and MPF were shown to be highly correlated between the two matrices. Correlations between all pairs of markers in 318 serum samples were calculated and demonstrated the highest correlation between HE4 and MPF, and the lowest between FRA and MPF. Serum levels of all markers showed a dependence on both stage and grade of disease. A multi-marker logistic regression model was developed resulting in an AUC=0.91 for diagnosis of serous ovarian cancer, a significant improvement over the AUC for any of the individual markers, including CA125 (AUC=0.84).FRA has significant potential as a biomarker for ovarian cancer, both as a stand-alone marker and in combination with other known markers for EOC. The lack of correlation between the various markers analyzed in the present study suggests that a panel of markers can aid in the detection and/or monitoring of this disease. In 2012, it is estimated that 22,280 women will be diagnosed with ovarian cancer and 15,500 will die of the disease (SEER fact sheet). Ovarian cancer is considered a “silent killer” because of the absence of specific symptoms until late in the disease when 75% of the cases are diagnosed, five year survival rates are less than 30%, and a 70% recurrence rate is expected. Early diagnosis, when the cancer is confined to the ovary, can increase the 5-year survival rate to 90%. Because of the high fatality rate and relatively low prevalence of the disease, a sensitive and specific screening tool for asymptomatic women is needed. As such, effective and reliable diagnostic assays need to be highly sensitive and specific for the screening and detection of early stage ovarian cancer, especially in asymptomatic women.CA125, a membrane-associated mucin found on the apical membrane of epithelial cells of the ocular surface, respiratory tract and female reproductive tract, is elevated in approximately 80% of women with late-stage ovarian cancer. It is the gold standard diagnostic marker to detect recurrent ovarian cancer and monitor response to treatment. However, the usefulness of CA125 as a marker cannot be extended to diagnosis as 20% of ovarian cancers do not express CA125 [Human epididymis protein 4 (HE4), normally expressed in the epididymis, endometrial glands and respiratory tract ,7, is upSoluble mesothelin (MSLN) has a history as a biomarker for mesothelioma diagnosis, prognosis and monitoring -19. It iAlthough the literature is sparser than that for MSLN, a few studies have looked at MPF as a biomarker for mesothelioma , ovarianPanels of biomarkers, able to cover the molecular heterogeneity of ovarian cancer ,38, or sOne such biomarker is folate receptor alpha (FRA), a glycosylphosphatidylinositol (GPI)-anchored protein involved in folate transport into cells that is expressed in breast, lung, clear cell renal, ovarian and endometrial carcinomas, and non-small cell lung adenocarcinoma -58. FRA In the present work, novel, sensitive electrochemiluminescent assays were developed for the soluble forms of FRA, MSLN and MPF and were evaluated in a large cohort of ovarian cancer patient samples. Further, the diagnostic utility of these markers was compared to CA125 and HE4 in a subset of serous ovarian cancer samples. Finally, a multi-marker logistic regression model was developed that demonstrates increased diagnostic performance relative to any single marker.Samples were obtained from various commercial vendors with Institutional Review Board approvals and patient consent and were collected between 2009 and 2011. This study included serum samples from 258 ovarian cancer cases, of which 215 were serous and 47 were non-serous carcinoma (five unknown), and 60 age-matched control samples from women without disease. All non-serous ovarian cancer samples , were from women with stage I or II tumors.In addition, serum, plasma and urine samples were collected from 57 women (37 with ovarian cancer and 20 without the disease as controls) to compare sample matrices. All serum, plasma and urine samples were collected by standard techniques and processed/frozen within 30 min of collection. All samples were stored at -80°C, and thawed and aliquotted prior to analysis. Patient demographics including date of diagnosis, histology, stage, and age Table  were obtThe generation and characterization of monoclonal antibodies (MAbs) to FRA have been described previously . MAbs MN6-construct and purified by metal-chelate chromatography. All antigens were demonstrated to be >98% pure by both SDS-PAGE and analytical SEC.Purified, recombinant human FRA and human MSLN were the kind gift of Dr. Earl Albone . MPF was prepared as a HTM NHS-Ester, Meso Scale Discovery (MSD®), Rockville, MD) and label:protein was determined according the manufacturer’s instructions. The conjugation ratio was set to 20 labels per MAb for all assays.The FRA, MSLN and MPF assays all utilized ECL technology from MSD . For theSamples , standards or controls were added to wells of a 96-well plate previously coated with capture antibody and incubated at RT for two-hours. The Ruthenium labeled detection MAbs were diluted in assay buffer, added to washed plates and incubated for an additional two hours at RT. Plates were washed, read buffer added and signals measured using an MSD DISCOVERY WORKBENCH®. Optimal sample dilutions were: FRA (80-fold dilution of urine and a 20-fold dilution of serum and plasma), MSLN (60-fold dilution of urine and an 80-fold dilution of serum and plasma) and MPF (4-fold dilution of urine and a 20-fold dilution of serum and plasma).CA125 and HE4 measurements were performed by Myriad RBM  on a Luminex 100 instrument on 176 serum samples from women with serous ovarian tumors.Pearson’s correlation coefficient was performed to determine the correlation among the various biomarkers. Pairwise comparisons of biomarker levels between normal controls and stages and grades of ovarian cancer were made using the Mann–Whitney U test. Receiver Operating Characteristic (ROC) analysis was employed to determine the performance of these markers by stage and grade of disease. ROC Area Under the Curve (AUC) calculations were based on 95% confidence intervals. Logistic regression models were developed and used to assess the performance of a panel of biomarkers relative to CA125. All comparisons were two-sided and a P-value ≤0.05 was considered statistically significant except where otherwise stated. Statistical analyses were performed in MedCalc version 12.30 , SPSS version 19 (IBM), GraphPad Prism version 6.00  and Microsoft Excel version 2010.The intraday reproducibility and sensitivity of the ECL assays for FRA, MSLN and MPF were assessed at levels between 0.01 and 5000 pg/mL  and grade of disease and is summarized in Table Relative to histotype, FRA and MSLN both showed the highest levels and most significant discrimination with normals in the serous sub-type with p-values <0.0001. Both endometrioid and mucinous sub-types could also be discerned from normal by both FRA and MSLN although to lower significance than the serous sub-type Figure . ROC curThe serum levels of FRA, MSLN and MPF also differed by stage Figure  and gradBased on the data presented above and the preponderance of samples in our cohort from patients with serous ovarian cancer, a more detailed analysis of marker distributions and, importantly, comparisons to levels of CA125 and HE4 was performed on a subset of 176 serous ovarian samples.r=0.53) of these two markers in serum is somewhat surprising and may reflect different mechanisms by which these two proteins enter the circulation. Further, MPF and MSLN, molecular entities derived from the same gene product, were only correlated to r=0.66. This is most likely a reflection of the fact that MPF is a soluble, proteolytic cleavage product of the gene product whereas MSLN is GPI-anchored and requires additional processing to be released into the circulation. Interestingly, the strongest correlation of markers was for MPF and HE4 at r=0.83. The relatively low correlations between these 5 serum markers suggests involvement in different and varied biological processes in ovarian cancer and suggests their usefulness in a multi-marker panel for improved discriminatory ability over CA125 alone.Pairwise comparisons between FRA, MSLN, MPF, CA125 and HE4 were made through use of Pearson’s correlation coefficient Table . FRA wasThe serum levels of FRA, MSLN, CA125 and HE4 readily discriminated between serous ovarian cancer and control serum samples Figure , with p-An assessment of each of the five biomarkers in the cohort of 176 serous ovarian cancer samples relative to stage of disease is presented in Figure Similarly, all five biomarkers were assessed relative to grade of disease Figure . As can Given the diagnostic performance of the individual markers, and in particular the relatively weak correlation between markers, we applied logistic regression modeling in an attempt to define a combination of markers that would increase the diagnostic potential relative to the best single performing marker, CA125. In the resulting model, CA125 and MSLN were shown to increase the probability of a diagnosis of ovarian cancer as their values increased, whereas MPF showed the opposite effect. HE4 added slightly to the performance of the model, but it was not significant and therefore not included. The best model included CA125, FRA, MSLN and MPF and as can be seen in the ROC analysis presented in Figure CA125, first introduced in the mid-1980s, remains the gold standard with respect to detection and monitoring of ovarian cancer. In recent years other markers, in particular HE4 have gained acceptance, with somewhat limited utility -67, and FRA has been the subject of intense research as a potential therapeutic target in the last several years primarily because of its highly restricted expression profile in normal tissues  and highThe described assays showed excellent limits of detection in the low pg/mL range and wide dynamic ranges up to at least 5000 pg/mL. From a practical point of view, such assays will require less in the way of repeat sample testing due to high marker levels. Importantly, FRA, MSLN and MPF were all shown to distribute equivalently between serum and plasma allowing flexibility in the choice of sample matrix. On the other hand, markers such as osteopontin are known to distribute more into the plasma fraction, restricting the sample type. FRA, MSLN and MPF were all shown to be detectable in urine samples from both healthy women and women with serous ovarian cancer. The clinical utility of MSLN measurement in urine for ovarian cancer has previously been described. In view of the ease of urine sample collection, the clinical utility of diagnostic assays assessing levels of FRA and MPF in urine is evident.The data presented here demonstrates that for each of the markers analyzed – CA125, HE4, MSLN, MPF and FRA – there was a preferential expression in the serous histotype. Recent work from our laboratory using immunohistochemical techniques for the detection of FRA, have shown a similar preferential expression in serous carcinomas . Taken tThe analyzed markers showed low to moderate correlations with each other. Surprisingly, CA125 and MSLN were not highly correlated even though CA125 has been described to be the ligand for MSLN and to be involved in the metastatic process in ovarian cancer. More surprisingly, perhaps, is the moderate correlation between MSLN and MPF since these molecules should be present in a 1:1 molar ratio given the fact that they derive from the same RNA and that MPF is simply a proteolytic product of the initial gene product. However, since MSLN is a GPI-anchored protein whereas MPF is soluble, these findings, as with the correlations of the other described markers, most likely reflect a combination of the route by which the markers enter the circulation and, importantly, the clearance from the circulation. For example, while MSLN, MPF and FRA are detectable in urine, CA125 is not. Further, FRA has been shown to bind to megalin, both in the kidney and liver and, as such, is removed from circulation . The gloHowever, as described herein, combining markers with CA125 does increase the diagnostic performance of the marker panel over CA125 alone, allowing for the development of a multi-marker panel that increases the sensitivity of detection of early stage disease while retaining specificity, the ultimate goal in ovarian cancer diagnosis.CA125 remains the best single biomarker for diagnosis and monitoring of ovarian cancer. However, additional markers are sought for use independently or in combination with CA125 to improve the sensitivity for ovarian cancer detection whilst retaining specificity. The current study presents data on the utility of a novel marker, folate receptor alpha, FRA; with respect to discrimination between ovarian cancer, for example, the serous histotype, and normal controls. Further, data was presented for additional markers including MSLN and MPF and the use of these markers in a multi-marker panel that outperforms CA125 alone.Development of additional markers for use either individually or in a panel for the diagnosis or detection of ovarian cancer, especially early stage disease, is critical. The novel ECL assays described herein provide a powerful tool for such development.AJCC: American joint committee on cancer; AUC: Area under the curve; BMI: Body mass index; CV: Coefficient of variance; ECL: Electrochemiluminescence; EOC: Epithelial ovarian cancer; FDA: Food and drug administration; FRA: Folate receptor alpha; GPI: Glycosylphosphatidylinositol; LLOD: Lower limit of detection; MAb: Monoclonal antibody; MPF: Megakaryocyte potentiating factor; MSLN: Mesothelin; ROC: Receiver operator characteristic.The authors declare no conflicting interests in this study.DJO and ES conceived and designed the experiments. PO, RH and LM performed the experiments. RPT, LMP and DJO analyzed the data. LMP and DJO wrote the paper. All authors read and approved the final manuscript.


Estrogen has been reported to accelerate cutaneous wound healing. This research studies the effect of young coconut juice (YCJ), presumably containing estrogen-like substances, on cutaneous wound healing in ovairectomized rats.Four groups of female rats (6 in each group) were included in this study. These included sham-operated, ovariectomized (ovx), ovx receiving estradiol benzoate (EB) injections intraperitoneally, and ovx receiving YCJ orally. Two equidistant 1-cm full-thickness skin incisional wounds were made two weeks after ovariectomy. The rats were sacrificed at the end of the third and the fourth week of the study, and their serum estradiol (E2) level was measured by chemiluminescent immunoassay. The skin was excised and examined in histological sections stained with H&E, and immunostained using anti-estrogen receptor (ER-α an ER-β) antibodies.Wound healing was accelerated in ovx rats receiving YCJ, as compared to controls. This was associated with significantly higher density of immunostaining for ER-α an ER-β in keratinocytes, fibroblasts, white blood cells, fat cells, sebaceous gland, skeletal muscles, and hair shafts and follicles. This was also associated with thicker epidermis and dermis, but with thinner hypodermis. In addition, the number and size of immunoreactive hair follicles for both ER-α and ER-β were the highest in the ovx+YCJ group, as compared to the ovx+EB group.This study demonstrates that YCJ has estrogen-like characteristics, which in turn seem to have beneficial effects on cutaneous wound healing. One of the challenges of the current and coming centuries is the aging population. Age-related diseases like vascular and metabolic diseases often result in poor peripheral circulation leading to delayed wound healing, which in turn could lead to serious conditions like amputation.In Thailand, and like the rest of the world, this has resulted in a significant burden on the government and other health care providers, which was estimated to cost millions of Baht per year. Considering the high prevalence and the extended time frame for treatment of established ulcers, there seems to be a distinct lack of effective management modalities for chronic wounds . To dateSex steroid hormones seem to have a profound influence on regulating skin maintenance and turnover. Estrogen maintains dermal thickness, and promotes both the maintenance of the extracellular matrix collagen levels and the structural integrity of the skin. In postmenopausal women, estrogen reduction causes deleterious skin changes, resulting in delayed cutaneous healing that is associated with increased inflammation, reduced matrix deposition, and unregulated protease activity . Exogenosome estrogenic properties, which were able to significantly reverse some pathologies associated with hormonal imbalance [Young coconut juice (YCJ) has been used in Thailand by menopausal women for decades to alleviate some symptoms associated with the decrease in the level of estrogen. We have recently shown that YCJ possesses mbalance ,9. In thCocos nucifera L., Arecaceae) was collected from Tungngai district, Hat Yai, Songkhla, Thailand. It was then authenticated by Associate Professor Dr. Sanan Subhadhirasakul at the Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat Yai, Thailand. YCJ was dried, and the powder form was kept at -30°C until used. It was freshly reconstituted and prepared for oral intake every day. A complete description of YCJ, including its preparation and administration, is reported in our previous publications [Young coconut juice  included in this study. The first group consisted of ovx rats, the second group included sham-operated rats, the third group consisted of ovx rats injected intraperitoneally with exogenous estrogen  once a day, and the fourth group included ovx rats that received YCJ (100 mL/kgBW/day). The third and fourth groups were treated for one week (7 days treatment) and two weeks (14 days treatment). The dose of EB and YCJ in this study was based on the one reported in our earlier study, and in which dose standardization and optimal administration were set -10. In tSome of the biopsies were processed into paraffin blocks, and ten 5μm sections were collected from each block and mounted on 3-aminopropyltriethoxysilane -coated slides. The first two sections were stained with H&E, and were used for light microscopy examination and anatomical orientation. Three of the remaining eight sections were immunostained with anti-ER-α monoclonal antibody . Another three sections were immunostained with anti- ER-β polyclonal antibody . The remaining two sections were used as negative controls. The immunostaining technique was described previously ,9. Negat2. Mean ± SEM was used to compare the four groups.Counting of all parameters was performed by two independent observers. An eye piece micrometer was mounted on a light microscope, and counting was made at x400 magnification. Readings from both observers were then added and the average was determined. Three areas of wound depth were determined and averaged from the perpendicular epidermis to the deepest part of the granulation tissue. Three areas of wound width: the top, the middle, and the bottom of wound were measured and averaged. The epidermal thickness was defined as the distance between the stratum granulosum and the stratum basale. The distance between the stratum basale and the upper part of hypodermis was measured for assessing the dermal thickness. Hypodermal thickness was measured as the distance between the bottom line of the dermis and the muscularis adiposa. Each reported thickness is an average of 25 measurements. Hair follicles were detected from the largest size of 10 hair follicle diameters in the dermis and hypodermis, averaged, and compared between each group. Image analysis and quantification of all parameters were performed using an Image Pro Plus program . Data was expressed as numbers per μmAll the rats were sacrificed on the first day of the fourth and the fifth week, and their serum was collected and measured for estradiol (E2) using the chemiluminescent immunoassay (CIA) technique .Statistical analysis was performed using the Kruskal-Wallis and the Mann-Whitney U-tests. Results were reported as mean ± SEM. P < 0.05 was considered significant. Correlation analysis between hair follicle diameter in the dermis and hypodermis and serum E2 level was performed using Spearman's rank correlation coefficient test.Our results showed that the circulating level of E2 was significantly (p < 0.01) less in the ovx+YCJ group following 14 days intake, as compared to the other groups. Such a trend was also seen following seven days intake, except when compared with the ovx group Figure . Using tGross examination of the skin clearly showed that all wounds (ovx wounds and excision ones) of the ovx +YCJ group had significantly p < 0.01) the fastest healing rate, which was associated with less redness, swelling, and exudates, as compared to the other groups . This was associated with higher number and thicker dermal collagen fibers, as well as with significantly (p < 0.01) thicker epidermis Figures . The amoFor centuries, YCJ has been used for its various therapeutic effects, but not for wound healing. We were the first research group to report back in 2006 that ovx rats receiving YCJ at 100 mL/kgBW had significantly better wound healing, including less scarring, brighter skin, and softer hair, as compared to controls [in vitro stimulatory effect of a SERM, Raloxifene, on collagen biosynthesis by human skin fibroblasts, as compared to traditional estrogen treatment [Our results demonstrated that the intake of YCJ over one week was not enough to produce significant microscopic healing changes in the wound area, as compared to controls. However, such changes were prominent when the intake continued for one more week. This was evident in the accelerated wound healing that was characterized by reduced wound depth and width, increased thickness of the epidermis and dermis, thicker and more abundant collagen fibers and hair follicles, and density of immunostaining against ER-α, and ER-β in the epidermis, dermis, and hypodermis. Such results seem to provide evidence to the observation that YCJ has phytoestrogenic properties, and that the latter could both act as SERM and play a role in the homeostasis of the epidermis, dermis, and hypodermis. This is supported by our recent experiment in which we used gas chromatography-mass spectrometry to confirm that the phytoestrogens of YCJ were sitosterol, stigmasterol, and campesterol . This isreatment . This isreatment . Pirilä reatment . They obreatment . Pirilä reatment . Shusterreatment . Azzi anreatment .Both estrogen and SERMs act through binding to homodimeric and heterodimeric ERs. SERMs, in addition, have the potential to selectively induce ER isoforms in a specific target tissue, thus amplifying further the effects on down-stream gene expression. Estrogen acts via ER, and there are currently two known estrogen receptors, denoted ER-α and ER-β. ER-α is expressed in the skin of both humans and rodents, whereas conflicting results have been reported regarding the expression of ER-β in the skin -20. TherOur results showed that the number of hair follicles and their diameter either in the dermis or in the hypodermis of the ovx+YCJ group was the highest and largest, respectively, while the dermis and hypodermis of the ovx+EB group contained the lowest number of follicles and their diameter was the smallest. In contrast, we observed a strong negative correlation between the serum E2 level and the number and size of the hair follicles in the ovx+YCJ and ovx+EB groups. These findings indicate that YCJ had an agonistic effect on hair follicles, while exogenous estrogen (EB) has an antagonist effect. In agreement with our findings, E2 was found to effectively block hair growth in female mice . In humaThe importance of hair follicles in skin biology does not rest solely with its ability to produce hair. Hair follicles are self-renewing, contain reservoirs of multipotent stem cells that are capable of regenerating the epidermis, and, accordingly, are thought to be actively involved in accelerating wound healing ,26. The In summary, we are reporting some novel findings in relation to the effects YCJ on cutaneous wound healing. YCJ does not only increase epidermal and dermal thicknesses, but also increases the number as well as the size of hair follicles and it restores collagen fiber size. Another beneficial effect of YCJ is the reduction of hypodermal thickness. One might argue that the reduction of hypodermal thickness may indicate reduced fat tissue leading to thinning of the skin and, thus, accelerating appearance of age wrinkles. However, the appearance of age wrinkles does not solely depend on the amount of adipose tissue present, but also, and in a more significant manner, on the amount and thickness of collagen fibers. It was clear in the photos presented in this study that the amount and size of collagen fibers of the ovx+YCJ group were similar to the control groups . Moreover, our lab has conducted a separate study, which focused on examining and quantifying collagen and elastic fibers using electron microscopy and image analysis. The data obtained confirm the results and argument presented in the current study, and will be presented in a separate manuscript due to the large amount of photos included in the current study.The encouraging findings presented in this study could have some exciting clinical implications, and could highlight the importance of conducting a clinical trial on the effects of the use of YCJ to treat chronically-delayed cutaneous ulcer healing in the elderly.The authors hereby declare that there is no conflict of interest in this manuscript.NR: Experimental design, conduction of animal study, and analysis of data, and drafting the initial manuscript. FS: Histological analysis, analysis and interpretation of data, presentation of photos and figures, re-writing the manuscript, and critically revising the manuscript for important intellectual content. IS: Participating in the experimental animal work. KS: Participating in the analysis of YCJ. PS: Immunohistochemistry. PB: Image analysis. WR: Participating in the analysis of YCJ. WM: Participating in the histological analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/12/252/prepubFigure S1. Wound following a seven day treatment period. Wound following a seven day treatment period. A = sham-operated group, B = Ovx group , C = Ovx+EB group, D = Ovx+YCJ group.Click here for fileFigure S2. Wound following 14 days treatment period. Wound following 14 days treatment period. A = sham-operated group, B = Ovx group , C = Ovx+EB group, D = Ovx+YCJ group.Click here for file


Autoimmune hepatitis (AIH) is a unique form of immune-mediated disease that attacks the liver through a variety of immune mechanisms. The outcomes of AIH are either acute liver disease, which can be fatal, or, more commonly, chronic progressive liver disease, which can lead to decompensated liver cirrhosis if left untreated. AIH has characteristic immunological, and pathological, features that are important for the establishment of the diagnosis. More importantly, most patients with AIH have a favorable response to treatment with prednisolone and azathioprine, although some patients with refractory AIH or more aggressive disease require more potent immune-suppressant agents, such as cyclosporine or Mycophenolate Mofetil. In this paper, we discuss the immunological, pathological and clinical features of AIH, as well as the standard and alternative treatments for AIH. Autoimmune hepatitis (AIH) is a chronic, immunologically mediated inflammatory liver disorder of unknown etiology –3. It isAIH is the cause of 11–20% of all cases of chronic hepatitis in Western countries , 13 and AIH is characterized by the loss of immune tolerance to antigens that are present on hepatocytes, as well as by impaired immune regulation , 35. No β1*1501 is associated with protection from AIH [The major histocompatibility complex (MHC), which is also known as human leukocyte antigen (HLA), is located on chromosome 6 and is the most commonly reported gene associated with AIH , 37, 38.from AIH , whereasfrom AIH . Among Mfrom AIH , whereasfrom AIH . α agents adalimumab and infliximab has been demonstrated [Antigens that are most likely triggered by autoantigen mimicry are thought to play a role in the initiation of the immune system-mediated damage that is associated with AIH , 26, 35.nstrated –54. It instrated . Howevernstrated . Furthernstrated . Moreovenstrated . Severalnstrated –61. Khatnstrated –65. In onstrated . We recenstrated . in vitro and in vivo [The immune response that targets the liver in AIH involves cytotoxic T lymphocytes, which damage the hepatocytes via the production of interleukins ). However, the molecular target of the T lymphocyte response has not yet been identified , 67. Sev in vivo .The presence of autoantibodies is one of the distinguishing features of AIH. Immunofluorescence (IFL) is the main technique that is currently used to screen for the autoantibodies that may be diagnostically relevant to liver disease , 76.ANAs exhibit nuclear staining in the kidney, stomach, and liver because ANAs are specific for nuclear antigens (first described by Miescher and Fauconnet in 1954) . AlthougSMAs were first described in patients with autoimmune liver disease in 1965 . Since tStudies have shown that a subset of patients who are positive for SMAs also demonstrate F-actin staining , 90. TheThe anti-soluble liver antigen auto-antibodies were first described by Manns et al. in 1987 . These aThe LKM-1 antibodies were first described in 1974 by Rizzetto et al.  The targThe presence of LKM-2 antibodies has been described in patients with drug-induced hepatitis that is caused by tienilic acid (which was subsequently withdrawn from the market) . LKM-3 antibodies have been described in patients with chronic hepatitis D , 110 andThe LC-1 antibodies, which target the enzyme formiminotransferase cyclodeaminase (FTCD), are present in 32% of the LKM-1-associated cases of AIH type 2 –122. HowThe ASGPR is a major liver-specific protein , 126 andP-ANCAs are detected by IFL using a neutrophil substrate , 132. A Antibodies to dsDNA have been detected in patients with AIH type 1. The presence of these antibodies has been associated with high immunoglobulin G (IgG) levels and a poor immediate response to corticosteroid treatment compared to anti-dsDNA-negative patients .Anti-chromatin staining is detected in 39% of the ANA-positive AIH patients and is more commonly detected in males with AIH. These antibodies are associated with high levels of IgG and active disease . The presence of AMA-M2 has been reported in 8–20% of typical AIH patients. Furthermore, AMA-M2-positive AIH is associated with the presence of DRB1*03, DRB1*04, and DRB1*13, the histological features of chronic active hepatitis and probable or improbable pretreatment scores –141. HowThe elevated levels of serum IgG and low C3- and C4-complement levels in AIH patients were first described by Thompson et al. in 1973 . High leAIH type 1 is the predominant form of AIH and affects all age groups, although it is more commonly diagnosed in individuals between 20 and 40 years of age , 21, 24.AIH type 2 is characterized by the detection of LKM-1 antibodies, which are directed predominantly against CYP2D6 . AIH typIn total, 10–15% of the AIH patients are negative for ANAs, SMAs, and LKM-1 antibodies at the time of clinical presentation. However, 25% of these patients will demonstrate detectable levels at a later time during the course of the disease. An additional 20% will be positive for other nonstandard AIH markers, such as the SLA, LC-1, or P-ANCA antibodies, which will prove helpful in establishing the diagnosis of AIH , 135. PaIn our experience, we have diagnosed all of the adult patients in our center to have AIH type 1 based on biochemical, immunological, and histological features , 62. UnpAIH type 1 is the most common type and represents approximately 80% of all cases. Most patients with AIH who are younger than 18 years of age are diagnosed to have AIH type 1. In addition, there is a female predominance of 75% in these cases , 26. The clinical presentation of AIH can be in the form of asymptomatic liver disease with abnormal liver test results , 29, 115There is no single diagnostic marker for AIH. Therefore, the diagnosis is based on a combination of clinical, biochemical, immunological, and histological findings . The diaInitial clinical data should be collected from the patient to evaluate the history of alcohol use and the use of any hepatotoxic medication , 155. LiThe previously described autoantibodies are often not sufficient for the diagnosis of AIH because these can be detected in many other conditions and their levels can sometimes be undetectable during the course of the disease. , 145 TheElevated levels of serum IgG are included in both the revised scoring system and the simplified criteria that are used for the diagnosis of AIH , 143. AcA liver biopsy is recommended for the definitive diagnosis of AIH. This test rules out features of alternative liver diseases and identifies features of AIH variants and overlapping syndromes , 11. MorPlasma cell infiltration involving both the portal and peripheral areas is typically observed in AIH , 98, 159The severity of the inflammatory activity can often predict the progression of the disease . In addiANAs and SMAs may also be present in patients with chronic hepatitis (CH) B and C, although these SMAs often lack specificity for F-actin , 166. SiSerum levels of ALT and AST that are 10-fold higher than the upper boundary of the normal range or the combination of elevated ALT and AST levels with elevated levels of serum IgG or with the histological evidence of interface hepatitis are absolute indicators for treatment , 35, 177The indication for treatment is uncertain in patients with mild laboratory and histological abnormalities , 177. IfTreatment is not indicated in patients who fail to present evidence of biochemically or histological, active disease, patients with decompensated cirrhosis that are listed for a liver transplant, and patients with established cirrhosis without inflammatory activity , 15, 16.The AIH treatment is divided into 2 phases, which consist of the induction of remission and the subsequent maintenance of the remission , 11, 15.The remission of previously symptomatic patients is defined as the recovery from symptoms, the normalization of biochemical markers of disease and inactive liver histology , 11, 15.The treatment of AIH with corticosteroids and AZA is accompanied by the risk of the many side effects of both drugs, as shown in The decision to withdraw patients from immune-suppressive therapy should not be attempted if there is a chance of relapse . BecauseA symptomatic, biochemical, and histological relapse of AIH may develop as the steroid dose is tapered or following withdrawal from immune-suppressive therapy , 186. ThA high dose of prednisolone together with 150 mg/day of AZA should be attempted prior to the use of an alternative agent that was not used with the initial therapy .Budesonide: budesonide is a synthetic steroid with a high first-pass metabolism in the liver [he liver  that hashe liver . Howeverhe liver . Multiplhe liver , 193, whhe liver .Deflazacort is another alternative corticosteroid that has been used in a small number of patients, although further studies examining its use for the treatment of AIH are needed .Mycophenolate Mofetil (MMF): MMF is a purine antagonist that has been used as an initial treatment for refractory or relapsed AIH patients [patients , 196. Nipatients ) have inpatients . Howeverpatients , 196. Bepatients . Two of patients .Cyclosporine and tacrolimus: These drugs are calcineurin inhibitors and potent immune-suppressing agents (tacrolimus is more potent than cyclosporine). Pilot trials on cyclosporine and tacrolimus have demonstrated that these drugs are effective for the induction of remission in refractory AIH patients [patients , 198–200Alternative treatments in cases of failure due to the initial immune-suppressive therapy and in cases of posttreatment relapse are the following.Other medications that have been evaluated alone or in combination with steroids in a small numbers of patients who failed to respond to standard AIH therapy include D-penicillamine, cyclophosphamide, and ursodeoxycholic acid , 16, 188α) has been shown in an animal model to induce AIH [α drug infliximab may be effective as an alternative treatment for AIH [Tumor necrosis factor alpha (TNFduce AIH . When usduce AIH , 53. Rec for AIH . However for AIH , 203.Tregs also play a major role in the pathogenesis of AIH, and Treg-mediated therapy is another future alternative that requires additional study , 11, 198Abatacept is an agent that blocks the immune system by acting on cytotoxic T cells. This drug has been approved for the treatment of rheumatoid arthritis and is another suggested future therapy for AIH .In total, 2.3% of AIH patients require liver transplants, and a diagnosis of AIH is the indication for liver transplantation in 4–6% of all liver transplant patients , 16, 204Liver transplantation may be urgently required in AIH patients with severe acute refractory presentation of fulminant liver failure , 206. LiAIH has been reported to develop after liver transplantation for other diseases , 210 andAcute presentation with fulminant hepatitis has also been reported in cases of AIH . CorticoAIH occasionally develops during pregnancy or shortly after delivery. This AIH presentation demonstrates a favorable outcome in most patients , 215. PrApproximately 20% of all cases of AIH develop in patients over 60 years of age , 32, 219AIH may coexist with the biochemical, radiological and histological features of other forms of autoimmune liver disease, namely, PBC and PSC , 221.An AIH-PBC overlap is the most commonly recognized form . A diagnThe AIH-PSC overlap is less recognized compared to the AIH-PBC overlap and is more common in children than adults . PatientHCC is much less commonly reported in AIH patients compared to other causes of liver cirrhosis –226. The


Breast cancer incidence is increasing. The survival rate varies and is longer in high-income countries. In Brazil, lower-income populations rely on the Unified Public Health System  for breast cancer care. The goal of our study is to evaluate the survival of patients with operable breast cancer stages I-III at a Brazilian public hospital that treats mostly patients from the SUS.A cohort study of patients who underwent surgery for breast cancer treatment at the Clinical Hospital of the Federal University of Minas Gerais from 2001 to 2008 was performed, with a population of 897 cases. Information on tumor pathology and staging, as well as patients’ age and type of health coverage (SUS or private system) was collected. A probabilistic record linkage was performed with the database of the Mortality Information System to identify patients who died by December 31th, 2011. The basic cause of death was retrieved, and breast cancer-specific survival rates were estimated with the Kaplan-Meier method. The Cox proportional hazards model was used for univariate and multivariate analysis of factors related to survival.A total of 282 deaths occurred during the study’s period, 228 of them due to breast cancer. Five-year breast cancer-specific survival rates were 95.5% for stage I, 85.1% for stage II and 62.1% for stage III disease. Patients from the SUS had higher stages at diagnosis , and in the univariate but not multivariate analysis, being treated by the SUS was associated with shorter survival . In the multivariate analysis, larger tumor size, higher histologic grade, higher number of positive nodes and age older than 70 years were associated with a shorter breast cancer-specific survival.Five-year breast cancer survival was comparable to other Brazilian cohorts. Patients treated by the SUS, rather than by the private system, had shorter survival times, mostly due to higher initial stage of the disease. Breast cancer is the most common malignant neoplasm among women in the world. The incidence is increasing, especially in low and middle-income countries . In 2012A patient’s survival is related to several prognostic factors, including number of positive lymph nodes, tumor size, hormone receptor status, histological type and grade, and patient’s age . SocioecIn Brazil, most of the population does not have private health insurance, and relies on the Unified Public Health System  for care, which provides patients with screening, diagnosis, and breast cancer treatment ,11. In 2Studies from Brazil and other countries were retrieved from the PubMed and LILACS databases in February 14, 2013, using the search terms breast cancer, survival, and Brazil. Seven hospital cohort studies that separated patients by stage and were not aiming to evaluate specific prognostic markers or new treatments were selected. For PubMed, English language was used, and for LILACS, both the English and Portuguese languages were used. Findings from these observational cohorts in different Brazilian hospitals suggested that 5-year breast cancer-specific survival rates have ranged from 90% to 97% for stage I, 87.8% to 89% for stage II and 51% to 73% for stage III breast cancer diagnosed since the 1990s ,12-16. IIn this article, we present new estimates of survival for Brazilian female patients with operable breast carcinoma (stages I-III). We provide estimates for both overall survival rates and breast cancer-specific survival rates, calculated as the probability of surviving breast cancer in the absence of other causes of death . We alsoWe designed a cohort study of patients with invasive operable breast carcinoma in stages I-III surgically treated at HC-UFMG from 2001 to 2008. The study protocol was approved by the UFMG Ethics Committee on March 7, 2012 (project CAAE number 0660.0.203.000-11).The cases were retrieved from files of the Breast Pathology Laboratory of the UFMG School of Medicine. We selected all specimens related to surgical treatment of breast cancer.in situ, as well as 2 patients with axillary metastasis only (unknown primary site), 1 patient with unknown tumor stage, 27 patients with unavailable primary tumor sample at our institution , 7 patients with metastatic breast cancer who underwent palliative surgery only, 14 patients who underwent surgery for recurrent breast cancer, 1 patient who moved to a different state while on treatment and 4 patients with missing date of birth and mother’s name. Eight hundred ninety-seven cases were available for the final analysis.Among the 1119 patients who underwent surgery for breast cancer treatment at HC-UFMG from 2001 to 2008, we excluded 166 cases of ductal and lobular carcinoma In addition to date of birth and type of health plan (private insurance or SUS), we recorded twelve variables related to breast cancer diagnosis and treatment: patient’s age, tumor size (T), regional lymph node status (N), age, laterality (right or left), having bilateral cancer, histopathological type , histologic tumor grade (according to the Nottingham grading system) , type ofhttp://www.iesc.ufrj.br/reclink/) [We retrieved information on survival status, and date and cause of death from the Mortality Information System (MIS) of the Ministry of Health in Brazil for the years 2001 through 2011. The MIS is a national, computerized index of death record information that was implemented in 1975. Over the years, the completeness of death registration in the MIS has improved substantially, reaching 93.5% as of 2007 in Minas Gerais [In the Mortality Information System, causes of death are classified according to the International Classification of Diseases, version 10 (ICD 10) , by a teAfter reading all causes of death described in each death certificate, we applied the coding by the Surveillance, Epidemiology, and End Results (SEER), of the U.S. National Cancer Institute to estimate breast cancer-specific survival. Cases with unknown death causes were not excluded . When thAn alternative analysis was performed, considering only the basic cause of death, as selected by technicians from the State’s Secretaries in Health, which is used for national mortality statistics. The methods reported by SEER were also used in this situation.We estimated Kaplan-Meier curves to describe the survival of this cohort over 5- and 10-year periods. We used the log-rank test to compare the survival distributions of different subgroups in our data. Since the date of the first biopsy was not available for all patients who had surgery as the primary treatment, survival interval was calculated in months from date of surgery in patients who did not undergo neoadjuvant chemo- or hormone therapy and from biopsy date in patients who underwent such therapies. Also, we tried to keep the staging as accurate as possible by using the clinical stage at the date of biopsy or the pathological stage at the date of surgery.Age was categorized in three subgroups: up to 35 years, 36–69 years, and 70 years and older.Mean age and standard deviation (SD) were calculated. The chi-square test was used to compare categorical variables. The chi-square test for a linear trend was used to compare the frequencies of tumor stage over the years of the study, as well as tumor stage in each age category. The significance level was defined as 0.05. The Cox proportional hazards model was used for hazard ratio (HR) and 95% confidence interval (CI) estimation in the univariate analysis and for multivariate survival analysis with a stepwise backward conditional strategy. Variables with statistical significance (p < 0.05) in the univariate analysis were initially used for the multivariate model, except for type of surgery, performing axillary node dissection, and use of neoadjuvant therapy, since we had incomplete data on treatment, to avoid biasing the results. For instance, patients diagnosed at higher stages probably underwent adjuvant systemic therapies later on. However, we did not have the data to confirm this information. Only variables with a p value bellow 0.05 were kept in the final multivariate model. All statistical analyses were performed with the SPSS software, version 17.0 .Five-year breast cancer-specific survival for the entire cohort was 78.5%, and 10-year survival was 64.5%. The cause-specific survival was 95.5% at 5 years for stage I, 85.1% for stage II, and 62.1% for stage III disease. Overall survival was 92.1% for stage I, 81.8% for stage II, and 58% for stage III disease. Only a small proportion of our patients were followed over a 10-year period ; among those in stage I, 10-year survival rate was 91.2%, 69.8% for stage II, and 43% for stage III patients.The median period of follow-up was 64 months (range 1–131 months). Among the 897 patients, 282 (31.44%) died during follow-up, out of whom 228 (80.9%) died from breast cancer and 54 (19.1%) from other causes. Cardiovascular diseases (ICD 10 chapter IX) was a frequent cause of death unrelated to breast cancer, with 16 cases . Four patients had unattended deaths , and 3 patients  had deaths from unknown causes.Table The stage at diagnosis was higher among patients from the SUS . The frequencies of stages did not change over the years .2 for a linear trend = 0.22) but was associated with a smaller proportion of patients undergoing neoadjuvant systemic therapies .In the univariate analysis, breast cancer in patients older than 70 years of age was associated with significantly lower chances of survival compared to patients 35 to 69 years old. Also, higher histologic tumor grade, larger tumor size, and higher number of involved lymph nodes were associated with lower survival Figure , Table 1Having bilateral breast cancer and having lobular or special-type carcinomas was not associated with a shorter survival time. In terms of therapy, undergoing neoadjuvant systemic therapy, undergoing mastectomy and undergoing axillary node dissection were associated with shorter survival time, but these variables are highly correlated to tumor stage Table .In the multivariate analysis, tumor size remained an important prognostic factor. Patients with tumors larger than 5 cm (T3) had an HR of dying due to breast cancer of 2.31 (CI 1.41-3.80) compared to patients with tumors measuring up to 2 cm  would have been censored and not considered to have died from breast cancer. In such cases, information contained in the death certificate suggested breast cancer as a contributing cause of death, and we decided to be conservative and, as done by other authors, consider the patient as having died from breast cancer . These pWhen patients with deaths that were correctly classified as due to breast cancer were excluded , higher stage (stage III versus stages I and II) remained associated with a higher HR of dying from other causes . After reading other death causes present in the death certificate and reassigning the basic death cause, this effect disappeared (p = 0.16).et al. [et al. [Five-year breast cancer-specific survival for the entire cohort was 78.5%. Our survival findings are in accordance with earlier studies that were based on different Brazilian cohorts. The study by Ayala  describeet al.  showed aet al.  showed s [et al.  showed aFor patients diagnosed in the United States in the years 2001 and 2002 , 5-year overall survival was 87.8% for stage I, ranged from 74% to 81.4% for stage II  and from 41% to 66.7% for stage III disease  .Studying breast cancer survival and prognostic factors gives us insight into the natural history of the disease. Many prognostic factors have been studied over the years. The factor with the highest impact on survival is lymph node invasion (N). Tumor size (T) and distant metastasis (M) also play an important role, as well as lymph vascular invasion, positivity for hormone receptors, and over-expression of the HER2 protein . Many otSocioeconomic status is also an intervening factor -8. Most in situ tumors, which has been shown in a previous publication [The Brazilian SUS also provides breast cancer screening with mammography according to national guidelines : since 2lication , but theet al. [et al. [In our study, patients 70 years old and older had shorter breast cancer-specific survival. Schonberg et al.  showed aet al. ,36. Comoet al. ,33. Thus [et al. ,37.Patients up to 35 years of age were not more likely to die from breast cancer than patients 36–69 years of age. This could be due to our small number of cases at this age (only 47 women were younger than 35 years of age). These patients are unlikely to die from other causes when diagnosed with breast cancer ,38. Womeet al.[Our study has some limitations. First, the possibility of having wrongly classified a woman as being dead or alive exists, due to possible errors in the Mortality Information System. Three variables  were used in the record linkage to minimize this bias. Also, fathers’ names and patients’ addresses were used to confirm the pair as a true one. The medical records for a small sample of patients  were checked. Only one patient was identified as having moved to another state, and since information on life status could be wrong, she was excluded from the study. Second, since high-quality data were only available in surgical treatment and neoadjuvant therapies, we chose not to include these variables in the multivariate Cox model, to avoid bias. The inclusion of patients who underwent neoadjuvant systemic therapies is unlikely to have affected our results; those patients had more advanced tumors at diagnosis and thus would very likely have undergone chemotherapy after surgery. Also, information on socioeconomic status, such as family income and educational level, were not available. Paim et al. reportedOn the other hand, our study also has strengths. Selecting patients from pathology reports has the advantage of providing good-quality data regarding stage, histologic tumor grade, and type. The information on histologic grade was missing in only 11 patients, either because the invasive component was too small (microinvasive tumor) or because the patient underwent neoadjuvant systemic therapy and the tumor sample prior to the systemic treatment was insufficient to assess histological grade. Even though we have limited information on treatment due to the origin of our data, this study brings insight into recent survival of women with operable breast cancer at a tertiary health facility that treats mostly low-income patients.Different methods are used for survival analysis. Overall mortality, cause-specific mortality, and relative survival have all been used as endpoints ,37,40. TCancer-specific survival depends on the data quality of death certificates, as well as in appropriate coding of reported causes of death. In Brazil, data quality has improved over the years , but stiDifferent populations are subject to innumerous differences in life expectancy, life styles, and access to health care that could affect their survival, both from breast cancer and from other causes ,9,23,27.In our study, 5-year breast cancer-specific survival was comparable to the one estimated for other Brazilian cohorts. Comparisons with estimates for high-income countries showed mixed results, which may be due to differences in the socioeconomic, demographic and health characteristics of the population subgroups analysed in each study. Also, patients treated by the SUS had a shorter survival rate than those treated through the private system, mostly due to higher initial stage of the disease. Patients older than 70 years had shorter survival time in comparison with patients 36–69 years of age. After reassigning the cause of death reported in the death certificate, more patients were considered to have died from breast cancer than when using only the basic cause of death, suggesting that one should be aware of the possible pitfalls of national cancer mortality statistics.The authors declare that they have no competing interests.DB planned the study, gathered the sample, performed the record linkage, statistical analysis and wrote the manuscript. CMT planned the study, aided in the record linkage and statistical analysis and critically revised the manuscript. HG planned the study, analyzed all pathology samples and critically revised the manuscript. All authors approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/434/prepub


This study aimed to investigate the associations among the levels of plasma atrial natriuretic peptide (ANP) and electrolytes and essential hypertension (EH) in Uygur, Han and Kazakh populations in Xinjiang. A total of 724 hypertensive participants of different ethnicities from Xinjiang  and 741 normal controls  were enrolled. The associations of ANP with serum potassium, serum sodium and blood pressure were assessed. In the normal control (NT) group, the concentration of ANP was higher in the Uygur population than in the Han population, and the concentration in the Han population was higher than that in the Kazakh population (P<0.05). In the EH group, the serum potassium levels of the Uygur and Han populations were higher than that of the Kazakh population (P<0.05). The ANP concentration in the Uygur ethnic group was higher than that in the Han population, which was in turn higher than that in the Kazakh participants (P<0.05). In the Kazakh population, the serum potassium level was significantly lower in the EH group compared with that in the NT group (P<0.05). The serum potassium level was significantly lower and the ANP concentration significantly higher in the EH group compared with those of NT groups in the Uygur and Han populations (P<0.05). Age and body mass index (BMI) were associated with hypertension in the Uygur, Han and Kazakh populations. Low serum potassium may be a risk factor of hypertension in individuals of Uygur and Kazakh ethnicity. Plasma ANP may be a regulatory factor involved in the development of hypertension in Uygur and Han populations. Essential hypertension (EH) is a common cardiovascular disease, affecting ∼25% of adults globally. It is expected that by 2025, its prevalence will have increased to 60%; a total population of 1.56 billion may suffer from hypertension . In viewet al has been demonstrated to reduce BP , and anoWe therefore aimed to evaluate the association between serum sodium, potassium and ANP in hypertensive patients of Uygur, Kazakh and Han ethnicity in Xinjiang.This study was conducted in accordance with the Declaration of Helsinki and with approval from the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University . Written informed consent was obtained from all participants. The study adopted a random cluster sampling method. The participants recruited were between 30 and 60 years old. They were divided into a hypertension group (EH) and a control group (NT) according to ‘the China high blood pressure control guide’ diagnosis standard . The UygIn the EH group, patients with secondary high BP, excessive drinking, secondary medication history or a history of diabetes or liver, kidney or thyroid disease were excluded. In the NT group, individuals were excluded due to liver, kidney or thyroid dysfunction, a history of diabetes or high BP.2 and obese if their BMI was ≥30 kg/m2.We adopted a random method for evaluating the study participants by a questionnaire survey and BP, height, weight and the abdominal circumference were measured. The questionnaire survey content included: general situation, history of hypertension, two weeks of taking BP medication, smoking and drinking, hypertension, family history, etc. When measuring height and weight, shoes and hats were removed. The smallest units of height and weight were 1 cm and 0.5 kg respectively. From the height and weight measurements, the body mass index (BMI) was calculated and the participants were identified as being overweight if their BMI was ≥25 kg/mNurse-derived conventional BP was measured after 10 min of rest in the seated position within 30 min of obtaining blood samples from the opposite arm to that subjected to venesection. Five consecutive BP readings were obtained using an appropriately sized cuff, 30–60 sec apart. The average of the five readings was taken as the BP.+-sparing diuretics and loop diuretics. After centrifuging, samples were stored at −20°C until the time of analysis. Serum potassium and sodium levels were measured using a Hitachi 7020 biochemical analyzer . A plasma ANP radioimmunoassay kit was provided by the North Biotechnology Research Institute , and procedures were carried out according to the manufacturer’s instructions, using a GC-1500 γ radiation counter.Blood samples were obtained from participants having an empty stomach after 10 min of rest between 10:00 and 12:00 in the morning. Participants were allowed to continue taking all routine medications when blood was taken, with the exception of KFor database management and statistical analysis, SPSS software, version 13.0  was employed. Data are shown as the mean ± SD unless otherwise specified. Comparison between two groups used a t-test. Comparison between three groups used one-factor analysis of variance. Correlations with BP were determined from multivariate regression models with age, BMI, serum potassium, serum sodium and the concentration of ANP included in the regression models.With the existence of hypertension as the dependent variable and age, BMI and serum potassium as covariates, the logistic regression equation was as follows: BP=−1.52 + 0.14age - 2.04serum potassium + 0.16BMI. It indicates that in the Kazakh population, hypertension, in addition to being affected by age and BMI, also has low serum potassium as a risk factor.With the existence of hypertension as the dependent variable and age, BMI, serum potassium and ANP as covariates, the logistic regression equation was as follows: BP=−4.65 +0.05age - 0.49serum potassium + 0.17BMI. It indicates that in the Uygur population, hypertension, in addition to being affected by age and BMI, also has low serum potassium as a risk factor.Using the existence of hypertension as the dependent variable and age, BMI and serum potassium as covariates, the logistic regression equation was as follows: BP=−10.27 + 0.15age + 0.14BMI. It indicates that hypertension in Han individuals is mainly affected by age and BMI.The etiology and pathogenesis of EH have not yet been fully elucidated; at present most of the theories accept that genetic factors and environmental factors play a role. Among the environmental factors, lifestyle , dietary factors , and aging may lead to high BP. In most areas of Xinjiang, high-salt diets are prevalent, and diets also tend to be low in potassium. A number of epidemiological studies suggest that there is close relationship between electrolyte and BP levels –17. ANP + intake promote the excretion of sodium and curb volume expansion, which may prevent salt-mediated elevations in BP (The results suggest that there is significant hypokalemia in the hypertensive Uygur, Kazakh and Han populations and there is an association between hypokalemia and hypertension in Uygur and Kazakh populations. However, no significant difference was observed among the serum potassium levels in the three ethnic groups of patients with normal BP. Considerable evidence has shown that potassium deficiency plays a key role in hypertension and its sequelae –20. The ns in BP .In the present study, no significant differences were observed in serum sodium among the different ethnicities, but the concentration of ANP was highest in the Uygur population, intermediate in the Han and lowest in the Kazakh. Atrial stretch is the main factor affecting ANP synthesis and secretion. In addition, salt intake, elevated extracellular fluid osmolarity, enhanced blood volume, rapid heart rate and neurohumoral factors directly affect the pressure on the atrial muscle and the release of ANP.The results suggest that in this case there was no difference in serum sodium levels among the different ethnic groups of patients with hypertension. However, the level of ANP in Uygur is higher than Han and Kazakh hypertensive, and the level of ANP in Han is higher than Kazakh hypertensive. Serum sodium levels are able to stimulate ANP effects on BP regulation, this is why the incidence of hypertension was the highest in the Xinjiang region.