Immigrants from South Asia to Western countries have a high prevalence of type 2 diabetes mellitus (T2DM). We explored pathogenic factors that might contribute to the high risk of T2DM in Pakistani immigrants to Norway.t test or Mann–Whitney U test as appropriate. Spearman’s correlation coefficient and multiple linear regression analyses were used.A cross-sectional study was performed in 18 Pakistani and 21 Norwegian men and women with T2DM (age 29 – 45 years), recruited from two hospital out-patient clinics. Anthropometrics and a two-step euglycemic, hyperinsulinemic clamp with measurements of non-esterified fatty acids (NEFA) during clamp, was performed in all patients. Insulin sensitivity, given as the Glucose Infusion Rate (GIR) and Insulin Sensitivity Index (ISI), was calculated from the two euglycemic clamp steps. Fasting adipokines and inflammatory mediators were measured. Continuous variables between groups were compared using Student’s (low insulin): 339.8(468.0) vs 468.4(587.3) μmol/m2/min (p = 0.060), ISI(low insulin): 57.1(74.1) vs 79.7(137.9) μmol/m2/min (p = 0.012), GIR(high insulin): 1661.1(672.3) vs 2055.6(907.0) μmol/m2/min (p = 0.042), ISI(high insulin): 14.2(7.3) vs 20.7(17.2) μmol/m2/min (p = 0.014). Pakistani patients had lower percentage NEFA suppression 30 minutes into clamp hyperinsulinemia than Norwegians: 41.9(90.6)% vs 71.2(42.1)%, (p = 0.042). The relationship of ISI to BMI, leptin and interleukin-1 receptor antagonist also differed between Norwegians and Pakistanis.Despite having a lower BMI, Pakistani patients were more insulin resistant than Norwegian patients, during both low and high insulin infusion rates, after adjustment for sex and % body fat: median (interquartile range) GIRCompared with Norwegian patients, Pakistani patients with T2DM had lower insulin sensitivity, affecting both glucose and lipid metabolism. The relation of insulin sensitivity to BMI and some adipokines also differed between the groups. Immigrants from South Asia to Western countries have a high prevalence of Type 2 diabetes mellitus (T2DM)-4. The pThere is a well known association between obesity and T2DM, but South Asians develop T2DM at lower levels of body mass index (BMI) than Westerners, and are more insulin resistant for any given BMI,13. SeveAdipokines and inflammatory mediators derived from adipose tissue may be important in the pathogenesis of insulin resistance and T2DM. PossiblIn the present study, we investigated a group of young adult immigrants from Pakistan with T2DM, and compared them to a group of Norwegian patients of similar age. We chose to study young adult subjects, where disease duration was relatively short, to avoid comorbidities that might confound the interpretation of our data. Our aim was to explore differences between the groups with regards to pathogenic factors such as insulin resistance, obesity and inflammation, which might contribute to the high prevalence of T2DM in Pakistani immigrants to Norway.This was a cross-sectional study comparing young T2DM patients from two different ethnic groups.1c and anthropometrics were available. All Pakistani participants were first generation immigrants. One Pakistani woman was excluded on the first day of testing, because of difficulties in obtaining intravenous access. The remaining 39 patients were examined.Norwegian and Pakistani patients with T2DM, aged 45 years or younger, attending the out-patient clinics at Lovisenberg Deaconess Hospital between 1999 and 2005, and Aker University Hospital between 2003 and 2009,were eligible for inclusion. After searches in the two hospitals’ patient registries, 195 patients were randomly selected to receive an invitation to participate in the study. Exclusion criteria were: ethnicities other than Norwegian or Pakistani, positive GAD or IA2 auto-antibodies, age > 45 years, person unwilling or unable to give informed consent. Patients were contacted by letter with information about the study in Norwegian and Urdu, and patients whose telephone number could be obtained were also informed by phone. Nineteen Pakistani and 21 Norwegian sex-matched patients  with confirmed T2DM, agreed to participate, and were included in the study. Participant characteristics were similar to a subgroup of approximately 80 of the 155 patients not included, where data on HbAHeight and weight were measured with participants wearing light clothing and without any shoes on. Waist and hip circumferences were assessed with a tape measure at mid point between the lowest rib margin and the iliac crest, and at the level of the major trochanter, respectively. Bioelectrical impedance analysis (BIA) was performed on a Tanita Body Composition Analyzer BC-418 . All subjects were fasting and voided urine before measurement.2/min for a minimum of 100 minutes, until at least 30 minutes of stable euglycemia was obtained. This was directly followed by a 400 mU/m2/min insulin infusion, also for a minimum of 100 minutes, with at least 30 minutes of stable euglycemia at the end. The body surface area was calculated using Mostellers equation[To enhance comparability of examinations, all patients stopped taking oral antidiabetic drugs for two days, and insulin for at least 12 hours prior to examination. Patients were asked to refrain from strenuous physical exercise and alcohol intake during these two days, and to fast from midnight during the night before the examination. We performed a euglycemic, hyperinsulinemic clamp, using a modification of the method originally described by De Fronzo et al.. The claequation.2/min was established, and denoted GIR40 and GIR400 respectively. Because of varying metabolic clearance rates of insulin, the insulin levels measured at the end of each step of the clamp differed between patients. The insulin sensitivity index (ISI) was therefore also calculated, and expressed as the ratio of the GIR to the prevailing mean serum insulin levels ((GIR/I) × 100), denoted ISI40 and ISI400. Every 30 minutes during the 2 step euglycemic clamp, EDTA plasma for non-esterified fatty acid (NEFA) measurements was extracted and immediately frozen at -70°C.Human insulin  300 mU/mL and Glucose 200 mg/mL were infused through a teflon catheter in a vein at the left elbow of the patient. Insulin was diluted in NaCl 0.9%, after having first added 2 ml of the patients own blood, to avoid insulin sticking to the walls of the bag. All blood samples were drawn from a teflon catheter in a vein at the right elbow, kept open by a slow infusion of NaCl 0.9%. The right arm was kept at 37°C by a heating sleeve connected to a thermal control unit , to arterialize blood samples. Plasma glucose was measured every five minutes using a OneTouch Ultra glucose measuring device , with control measurements every 30 minutes by the glucose oxidase method, on a Glucose Analyzer II . At the end of each step of the clamp, three measurements of serum insulin were taken at ten-minute intervals. The glucose infusion rate in μmol/mTwo Norwegian and two Pakistani patients did not attain euglycemia during the first step of the clamp, and were excluded from this part of the clamp analyses. Two Norwegian and four Pakistani patients were not examined with the high-step clamp, due to contraindication of hyperosmolar glucose at high infusion rates (for the most insulin sensitive patients) and long duration of the low-step part of the clamp to reach euglycemia (for the most insulin resistant patients). After excluding these patients from clamp analyses, there were 46% men in the first step and 48% men in the second step of the clamp.1c was measured by high performance liquid chromatography on a Variant analyzer . Fasting serum total cholesterol, serum HDL cholesterol, and serum triglycerides were measured using a routine enzymatic method . Serum LDL cholesterol was calculated using the Friedewald equation[Fasting plasma glucose was measured by the glucose oxidase method on a Glucose Analyzer II (Beckman Instruments). Serum insulin was analyzed using the radioimmunoassay (RIA) kit, formerly from Linco Research Inc. , presently available from Millipore Corp. . Plasma HbAequation. NEFA wet test or Mann Whitney U tests were used for comparison of continuous variables between groups. For correlations, Spearman’s correlation coefficient (rs) was used. Multiple linear regression analyses were performed with log-transformation of parameters when needed, to obtain normally distributed residuals. A two-sided p-value <0.05 was considered significant, but owing to the large number of comparisons, particular attention should be directed towards analyses where p-values are <0.01. Statistical analyses were performed with SPSS 18.0 for Windows .Data are presented as mean ± SD, or median (interquartile range) as appropriate. We analysed non-normally distributed data log-transformed, or with the use of non-parametric methods. Student’s The study was carried out in accordance with the Helsinki Declaration, and approved by the Eastern Norway Regional Committee for Medical and Health Research Ethics. Informed, written consent was obtained from each participant before enrolment.1c, longer median duration of diabetes, and a tendency towards earlier onset of diabetes . Further clinical characteristics of the participating patients are presented in Additional fileThe two ethnic groups did not differ significantly in age, sex and fasting plasma glucose. In contrast, BMI and weight, including both total body fat mass and lean body mass were significantly higher in the Norwegian, compared with the Pakistani group Table . Despite40 showed a clear tendency towards lower values in the Pakistani group, and LogISI40 was significantly lower in the Pakistani compared to the Norwegian group  in a multiple regression analysis, suggesting a difference between the ethnic groups. , LogBMI , as well as ethnicity x LogBMI). We were, however, not able to confirm an ethnic difference in the relationship between insulin sensitivity and waist circumference by regression analysis (data not shown).When exploring the relationships between anthropometric characteristics and insulin sensitivity, we found a strong, significant correlation between ISI Figure . There wThere were no significant differences in fasting NEFA values between Norwegian and Pakistani patients, but initial NEFA suppression by hyperinsulinemia, expressed as percentage suppression of NEFA after 30 minutes of the clamp, was significantly more pronounced in the Norwegian group compared to the Pakistani group Figure .40 and leptin, as well as IL-1RA . In the Pakistani group, IL-1RA neither correlated to ISI, nor to leptin , but correlated positively to waist circumference.Table 1c nor diabetes duration were significantly associated with the ethnic differences found in insulin sensitivity in adjusted multiple regression analyses.In the present study we found significant differences in insulin sensitivity, measured by the euglycemic, hyperinsulinemic clamp, between young adult Norwegian and Pakistani patients with T2DM. There were also ethnic differences in diabetes duration and HbA1c, even in this young patient population, demonstrating longer duration of diabetes and poorer glycemic control in the Pakistani group. These differences, however, did not explain the lower insulin sensitivity in the Pakistani group per se, since neither HbAFurthermore, we demonstrated a different relationship between insulin sensitivity and BMI in the two groups. Although obesity is clearly an important factor in insulin resistance, BMI was a poor marker of metabolic disturbances in the Pakistani patients. Whereas Pakistani patients were all quite insulin resistant regardless of BMI, among the Norwegian patients there was a strong negative correlation between BMI and insulin sensitivity. To our knowledge, this is the first study, using the euglycemic clamp to measure insulin sensitivity, to show such ethnic differences in subjects with T2DM. Our results support and expand previously published results obtained in healthy South Asian immigrants,21.NEFA are normally suppressed by hyperinsulinemia. There is evidence that this suppression is impaired in T2DM, althougIt is noteworthy that leptin and IL-1RA were differently related to insulin sensitivity in the two ethnic groups. T2DM is now regarded as a disorder characterized by non-resolving inflammation. IL-1, released though activation of Nod-like receptor protein 3 (NLRP3) inflammasomes, seems to play an important role in the inflammatory processes. Il-1RA 1c and diabetes duration between the two patient groups may be regarded as a weakness. However, these differences were also present in patients who were not included in the study, and have been demonstrated in several other studies[The main strengths of our study include thorough patient examinations with gold standard methods for measurement of insulin sensitivity, and using two steps of hyperinsulinemia. The difference in HbA studies-12. ThusIn this study of Norwegian and Pakistani patients with T2DM we found significant differences in insulin sensitivity and the relationship between insulin sensitivity and obesity markers, which may impact on our understanding of the pathogenic mechanisms that place Pakistani subjects at higher risk of developing T2DM. Further studies are needed to elucidate a possible relationship between insulin sensitivity, various adipose tissue compartments and adipokines and related molecules in various ethnic groups.Anti-GAD: Anti-glutamic acid decarboxylate; Anti-IA2: Anti-protein tyrosine phosphatase; BIA: Bioelectrical impedance analysis; BMI: Body-mass index; ELISA: Enzyme linked immunosorbent assay; GIR40: Glucose infusion rate at the low insulin infusion clamp step; GIR400: Glucose infusion rate at the high insulin infusion clamp step; hsCRP: High sensitivity C-reactive protein; IL-1α: Interleukine-1 alpha; IL-1β: Interleukine-1 beta; IL-1RA: Interleukine-1 receptor antagonist; IL-6: Interleukine-6; ISI40: Insulin sensitivity index at the low insulin infusion clamp step; ISI400: Insulin sensitivity index at the high insulin infusion clamp step; NEFA: Non-esterified fatty acids; NLRP3: Nod-like receptor protein 3; RIA: Radio immuno assay; sTNF-R1: Soluble tumor necrosis factor-receptor 1; SD: Standard deviation; T2DM: Type 2 diabetes mellitus; %TBF: Percent total body fat.The authors declare that they have no competing interests.CW conceived of the study, participated in its design and coordination, researched data and wrote the manuscript. ETA researched data and critically revised the manuscript. TU performed analyses of inflammation markers and contributed to discussion. AEM performed analyses of inflammation markers. PMT participated in the design of the study and revised the manuscript. IFL conceived of the study and participated in patient recruitment. PAT provided the insulin and adipokine analyses and revised the manuscript. PA contributed to discussion and critically revised the manuscript. KIB conceived of the study, participated in its design, contributed to discussion and critically revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6823/13/49/prepubTable showing. Further clinical characteristics of patients according to ethnic group. Click here for fileTable showing. Correlations between parameters of insulin sensitivity, anthropometry, adipokines and inflammation. Click here for file


Colletotrichum species of fungi, is one of the most serious diseases affecting jute in China. The disease causes chlorotic regions with black brown sunken necrotic pits on the surfaces of stems. In late stages of disease, plants undergo defoliation, dieback and blight, which make anthracnose a major threat to jute fiber production and quality in China. In this study, 7 strains of Colletotrichum fungi were isolated from diseased jute stems from Zhejiang, Fujian, Guangxi, and Henan plantations in China. Multi-locus sequence  analysis coupled with morphological assessment revealed that C. fructicola, C. siamense and C. corchorum-capsularis sp. nov. were associated with jute anthracnose in southeastern China. C. fructicola and C. siamense were previously not associated with jute anthracnose. C. corchorum-capsularis is a new species formally described here. Pathogenicity tests confirmed that all species can infect jute, causing anthracnose, however the virulence of the 3 species differed. This report is the first associating these three species with jute disease worldwide and is the first description of the pathogens responsible for jute anthracnose in China.Anthracnose, caused by the  Corchorus capsularis L.) is an annual bast fiber crop. It is found predominantly in Southeast Asia After cotton, jute is the second cheapest and second most commercially available fiber crop, making it an abundant source of biodegradable and renewable lignocellulose fiberColletotrichum species, has recently become the most serious disease of jute in China. This disease results in sunken necrotic lesions on the surfaces of stems that limit fiber productivity and reduce fiber quality.Jute  of stem tissue from each plant were surface sterilized in 70% ethanol for 45 s followed by 1% NaClO for 1 min. Samples were then rinsed three times with sterilized water and dried on sterile tissue paper. Samples were placed on PDA and incubated at 25 °C for 2–4 days. Additionally, the leading edge of any fungal hyphae that grew from the tissues was transferred aseptically to PDA. Fungi were monitored for sporulation and spore masses were picked off with a sterilized wire loop and streaked on the surface of water agar. After incubation overnight at 25 °C, single germinated spores were picked up with a sterile needle and transferred to PDAet al., characterization of spore morphology and growth in culture were performed6Referred to the method described by Cai, 2 square of agar was inoculated on one side with conidia and then covered with a sterile cover slipAppressoria were obtained by use of a slide culture technique in which 1 cmIsolates were grown on PDA and incubated at 25 °C for 7 days. Mycelium was scraped from the colony surface using a sterile 10 μl pipette tip. Genomic DNA was extracted from the mycelium using the Biospin Fungus Genomic DNA Extraction Kit (BioFlux®) according to the manufacturer’s protocol. DNA concentrations were estimated visually on a 1% agarose gel by comparing band intensity with a 1 kb DNA ladder (Transgen Biotech®).Colletotrichum strains were amplified by PCR. Primer pairs for PCR amplifications were referred to the method described by Prihastuti, et al. The PCR products were examined by electrophoresis in 1% agarose gels, purified, and ligated into the pMD18-T vector . The vectors containing these gene fragments were transformed into Escherichia coli DH5α and DNA sequencing was performed by BGI Company, Shanghai, China. Sequences derived in this study are deposited in GenBank. The accession numbers of all sequences analyzed in this study are listed in Partial actin (ACT), calmodulin (CAL), β-tubulin (TUB2), glutamine synthetase (GS), glyceraldehyde-3-phosphate dehydrogenase (GPDH) genes and the complete rDNA-ITS (ITS) region from 7 Sequences from our isolates, together with reference sequences obtained from GenBank , were alIn addition, the Markov Chain Monte Carlo (MCMC) algorithm was used to regenerate the phylogenetic trees with Bayesian posterior probabilities in MrBayes v.3.2.1Colletotrichum boninense (MAFF 305972) was used as outgroup in this analysis. A second analysis was carried out to confirm the identity of five isolates with curved conidia based on a concatenated alignment of 6 genes: ACT, CAL, GAPDH, GS, ITS and TUB2. Colletotrichum lindemuthianum (CBS 151.28) was used as the outgroup in this second analysis. Phylogenetic trees were created in Figtree2 isolates were used in the initial MP analysis using a concatenated alignment for 4 genes: CAL, GAPDH, GS and TUB2. 6 conidia/ml with sterile water using a hemocytometer. Jute leaves and stems without symptoms of disease were washed with tap water, surface disinfected in 75% ethanol for 60 sec and 1% sodium hypochlorite for 5 min, and then washed 3 times with sterile distilled water and dried in a fume hood. Spore suspensions, or sterile water for the negative control, were sprayed on the jute leaves. Stems were inoculated by using a sterile scalpel to create superficial wounds in the stem epidermis. The wound was then inoculated with a 5-mm-diameter PDA disk selected from the edge of an actively growing culture. Stems inoculated with sterile PDA were used as a negative control. The inoculated plants were kept in plastic containers, covered with plastic wrap to maintain humidity, and incubated at 28 °C with 12/12 h fluorescent light and darkness. 10 jute seedlings were inoculated for each species; the experiment was performed in triplicate. The incidence of infection was calculated by the formula [Incidence (%) = (infected sites or leaves/inoculated sites or leaves) × 100%] at 12-days post inoculation. The incidence data was analyzed using SPSS software version 20.0 .The isolates that were characterized by morphology were also submitted to pathogenicity tests. Single spore isolates were incubated on PDA for 7 days at 28 °C. Conidial suspensions were prepared by adding 10 ml of sterile distilled water to the culture, swirling to isolate the conidia, and filtering through two layers of muslin cloth. Spore concentration was adjusted to 10Colletotrichum strains were isolated from diseased jute samples from the main jute growing regions  of China. Based on the morphological characterization on PDA, 2 strains produced conidia similar to C. gloeosporioides. 5 strains produced curved conidia, which is typical of fungi in the C. truncatun species complexIn total, 7 Colletotrichum strains isolated, including 2 strains from the C. gloeosporioides complex and 5 strains with curved conidia. C. gloeosporioides species complex. The strain, FAFU01, could be confidently identified as C. fructicola as it clustered together with the ex-epitype strain ICMP 18581 with 100% bootstrap support. Another strain, FAFU04, clustered with C. siamense strains with 100% bootstrap support, based on the combined datasets of partial CAL, GAPDH, GS and TUB2 sequence analysis. The other 5 strains did not cluster with any currently known species based on these 4 molecular markers. Therefore, a further 6 gene regions  of these five strains were sequenced and phylogenetic relationships were predicted using parsimony and Bayesian methods  that was black and withered.Host: FAFU02, FAFU03, FAFU05, FAFU06 and FAFU07 were isolated from the stems of jute  than that from C. corchorum (12.0–25.0 μm).Notes: All 5 strains form a distinct clade with 100% bootstrap support, indicating that they represent a distinct species. Referring to their colony characteristics, Prihastuti, H., Cai, L. & Hyde, K.D. Fungal Diversity 39:96 (2009).Corchorus capsularis, 20 June 2013, Xiaoping Niu and Hong Gao, culture FAFU01 = BPD-I18.Material examined: CHINA, Fujian Province, Putian, isolated from stems of Colletotrichum fructicola was originally reported as a pathogen of coffee berries in ThailandPyrus pyrifolia (Japan), Persea americana , Malus domestica (Brazil), Dioscorea (Nigeria), Theobroma and Tetragastris (Panama)Vitis (China)Mangifera indica (Brazil)C. fructicola based on morphology and multi-locus  phylogenetic analysis. In the phylogram, the strain clustered with C. fructicola (ICMP 18581) with 100% bootstrap support and posterior probability values of 1.00 .Coffea arabica berries, 12 December 2007, Prihastuti, H., culture CBS 130417 = ICMP 18642 = MFLUCC 090230 = BPD-I2.Material examined: CHINA, Guangxi Province, Nanning, cultured from stems of jute, 16 June 2013, Xiaoping Niu and Hong Gao, culture FAFU04 = BPD-I2. THAILAND, Chiang Mai, Mae Lod Village, on Colletotrichum siamense was provided by Prihastuti, et al. C. siamense was also reported as a pathogen of Hymenocallis sp. (China), Malus (USA), Jasminum (Vietnam), Dioscoria (Nigeria), Persea and Pistacia ProteaceaeC. siamense was isolated from stems of jute. The conidial shape and dimensions match the holotype of C. siamenseC. siamense (ICMP18642) and strain MFLUCC090230 with bootstrap support/posterior probability values of 100%/0.99 and 100%/1.00, respectively  but this was not significantly different from strain FAFU01. As for lesion size on stems, C. corchorum-capsularis strain FAFU02 produced the largest lesions .The pathogenicity of the Colletotrichum species on jute (C. capsularis) have been poorly studied, with reports focusing on C. gloeosporioides and C. corchorum18Colletotrichum species causing disease on jute used morphological and culture characterizations which restrains identification to species complexes rather than individual species. The current study represents the first identification of Colletotrichum species associated with anthracnose of jute in China using a muti-locus phylogenetic approach. In this study, we isolated 7 Colletotrichum strains representing 3 distinct taxa, 2 of which have not been previously associated with jute disease. Although this investigation is limited in the sampling scale and isolations obtained, it appears that jute may harbor more Colletotrichum species than previously expected.C. gloeosporioides that was previously reported to be one of the main causal agents of jute anthracnose. However, 2 members of the C. gloeosporiodes species complex were newly associated with jute anthracnose, C. fructicola  and C. siamense . Although C. fructicola and C. siamense were isolated only from symptomatic stems, pathogenicity tests showed that both species can also cause anthracnose on jute leaves. This could indicate that C. fructicola and C. siamense begin their lifecycles as endophytes and grow into opportunistic pathogensThe most striking finding of this study was the absence of the Colletotrichum fructicola was previously found to be an important pathogen on a variety of hosts131413of C. fructicola causing jute anthracnose. Similarly, Colletotrichum siamense is another species that had not been thought to cause anthracnose in jute in southeastern China. This species was originally isolated from coffee berries in Thailand, and was biologically and geographically diverse15et al. of ApMat sequence data recognized several clades within C. siamense, suggesting C. siamense may be a species complex32C. siamense (ICMP18642) with bootstrap support/posterior probability values of 100%/0.99  produced curved conidia  longer (18.3–26.3 μm) than that from C. corchorum (12–25 μm)Colletotrichum corchorum-capsularis is introduced by this study to accommodate this species.00%/0.99 , further conidia , which hense ICMP642 with C. corchorum-capsularis was the most virulent species with a mean incidence of disease of 83% on leaves, while C. fructicola and C. siamense showed mild virulence  in the most important jute producing areas of China. The most important causal agent was C. corchorum-capsularis. C. corchorum-capsularis encompasses the most virulent strains and appears to be responsible for most jute anthracnose in China . C. corchorum-capsularis shows phylogenetic divergence and is probably a species complex; further work with more discerning genes is required to characterize the new species. This is the first report to link C. fructicola and C. siamense to jute anthracnose. Both caused disease in Fujian and Guangxi provinces. Pathogenicity tests showed that both species could cause disease at similar frequencies. C. gloeosporioides, which is reported to be the main pathogen for jute anthracnose, was not found in this study, possibly because we did not survey in the whole vegetative period, and the collected strains were only from the infected stems.In the present study, we have combined morphological and molecular data to identify the species of How to cite this article: Niu, X. et al. Colletotrichum species associated with jute (Corchorus capsularis L.) anthracnose in southeastern China. Sci. Rep.6, 25179; doi: 10.1038/srep25179 (2016).


Introduction. Hydroxychloroquine (HCQ) overdose is rare and potentially deadly when consumed in large doses. Management of severe HCQ toxicity is limited and infrequently reported. This report presents the case of a massive ingestion of HCQ. Case Report. A 23-year-old female presents following an intentional ingestion of approximately 40 g of HCQ. Within six hours after ingestion, she developed severe hemodynamic instability resulting from myocardial irritability with frequent ventricular ectopic activity leading to runs of polymorphic ventricular tachycardia (PMVT) and ventricular fibrillation (VF) requiring multiple defibrillations. Additional treatments included intravenous diazepam, epinephrine, norepinephrine, sodium bicarbonate, and magnesium sulfate. Despite the ongoing hemodynamic instability, the patient was also treated with Intralipid (ILE) and received hemodialysis. Improvements in her hemodynamics were observed after 18 hours. She survived her massive overdose of HCQ. Conclusion. HCQ poisoning is rare but serious because of its rapid progression to life-threatening symptoms. Hemodynamic support, gastric decontamination, electrolyte monitoring and replacement, and management of arrhythmias are the mainstays of treatment. The combined role of dialysis and ILE in the setting of massive HCQ overdose may improve outcomes. Hydroxychloroquine (HCQ) overdose is rare and often lethal when ingested in large doses. There exists a paucity of data on management of HCQ toxicity as it is infrequently reported. The majority of treatment recommendations are extrapolated from chloroquine (CQ) poising , 2. Curr2: 16, pO2: 70, and bicarbonate: 8. Electrocardiograph performed on arrival demonstrated sinus rhythm with a widened QRS (140 ms) and QT interval (QTc 576 ms). See μmol/L; albumin: 39 g/L; lipase: 113 units/L; gamma-glutamyl transpeptidase (GGT): 30 units/L; alkaline phosphatase: 55 units/L; total bilirubin: 3 μmol/L; direct bilirubin: 1 μmol/L; alanine aminotransferase (ALT): 23 units/L; aspartate aminotransferase (AST): 14 units/L.A 23-year-old female with a past medical history of depression, borderline personality disorder, obsessive compulsive disorder, psychosis NOS, and congenital hydrocephalus with VP shunt presents following ingestion of approximately 40 g of HCQ. She had been using HCQ for the treatment of pruritus. She was also being treated with risperidone, clonazepam, and zopiclone for her psychiatric disorder and fluconazole at the time of presentation for the treatment of a vaginal yeast infection. At presentation, she had a Glasgow Coma Score (GCS): 15/15, temperature: 37.1°C, blood pressure (BP): 92/60 mmHg, pulse rate: 65/min, respiratory rate: 16/min, blood glucose: 3.9 mmol/L, and serum potassium: 3.0 mmol/L. The initial blood gas demonstrated a metabolic acidosis and respiratory alkalosis: pH: 7.33, pCOWithin three hours of her overdose, the patient became obtunded requiring intubation and mechanical ventilation. Hypotension was treated with crystalloid and vasopressor support. Gastric lavage and activated charcoal for decontamination were provided. Within six hours of her overdose, she demonstrated severe hemodynamic instability resulting from myocardial irritability with frequent ventricular ectopic activity leading to runs of polymorphic ventricular tachycardia (PMVT) and ventricular fibrillation (VF) requiring 18 defibrillations with 200J with biphasic defibrillator over the first 18 hours after ingestion. Given her protracted cardiac instability, additional treatments including intravenous diazepam 60 mg followed by infusion of 6 mg/hr for calcium channel stabilization , epinephrine 20 mcg/min, norepinephrine 30 mcg/min, sodium bicarbonate 30 mmol/hr, and magnesium sulfate 1 g/hr infusions were also initiated. The serum potassium three hours after the overdose was 1.5 mmol/L. In total, 380 mmol of potassium chloride was given over the first 18 hours administered in 20 mmol boluses. She also received intravenous sodium bicarbonate for alkalization. To treat refractory ventricular ectopy, a bolus dose of 1.5 mL/kg of 20% Intralipid ILE was initially given followed by a high dose infusion over approximately 30 minutes. A total of 500 mL of ILE was given. No further infusion of ILE was given after this initial dose. Vascular access for dialysis was established immediately after the bolus dose and thus dialysis overlapped briefly with the infusion of ILE. The runs of PMVT and VF were felt to be secondary to a preexisting channelopathy likely potentiated by fluconazole and risperidone. Ventricular pacing was reviewed but not considered indicated. The patient was started on piperacillin/tazobactam for possible aspiration pneumonia. Echocardiographic imaging revealed normal global ventricular contractility with an ejection fraction of 60%.The patient was started on intermittent hemodialysis (IHD) in an attempt to reduce HCQ levels despite a paucity of literature regarding its use. The pharmacokinetics suggests that much of the drug is not accessible to the dialyzer, but given the severity of the overdose and the impact of hemodynamics even a small amount removed was considered favorable. Plasma HCQ level was 6425 mol/L 12 hours after ingestion. A five-hour run of hemodialysis was initiated with end points being either improved hemodynamics or of significant decrease in blood levels. A F1000 Dialyzer was used with a dialysate flow rate of 800 cc/hr. In total, 135 L of blood was processed. As noted above, concurrent with the dialysis run, an ILE infusion was administered in an effort to create a “lipid sink” to sequester lipophilic HCQ. At 31 hours after ingestion, the plasma HCQ level was 4328 mol/L.Improvements in her hemodynamics were observed after administration of dialysis and ILE. She was subsequently weaned from the ventilator and extubated on day 3 and discharged from ICU on postadmission day six.Hydroxychloroquine is sold under the trade name Plaquenil and is an aminoquinoline derivative used in the prophylaxis and treatment of malaria. It is also used as an anti-inflammatory agent in rheumatoid arthritis and complications of lupus and connective tissue disorders with an off label use in the treatment of urticaria. Hydroxychloroquine is highly toxic in overdose resulting in rapid onset of hypotension, ventricular dysrhythmias, and cardiac arrest resulting in death –3. SeizuDescriptions of HCQ overdose are limited to case reports in the literature. Given this paucity of data, the current emergency treatment is modeled on the experience of CQ overdose. The mortality rate of a CQ overdose for adults is between 10 and 30% [Hydroxychloroquine has a dose-related cardiac sodium and potassium channel blocking effect resulting in delayed repolarization and slow intraventricular conduction. This results in bradycardia, hypotension, ventricular dysrhythmias, widened QRS, and prolonged QT interval . HypokalHydroxychloroquine is rapidly absorbed following ingestion. Peak plasma levels of HCQ occurred 2–4.5 hours after ingestion. In overdose, onset of symptoms usually occurs within 30 minutes. It is highly tissue bound with a large volume of distribution. Distributional half-life is 15–30 hours . Common Patients presenting to health care facilities less than one-hour after ingestion should be considered for gastric decontamination with gastric lavage and activated charcoal. The use of multiple-dose activated charcoal should be considered in severe HCQ poisoning , 8.Profound hypokalemia is a known effect of HCQ poisoning and appears to correlate with toxicity. The mechanism is related to reduced potassium efflux from the blockade of membrane channels. It is unknown whether HCQ causes direct cardiotoxicity or if it is partly due to the hypokalemia. ECG findings include prolonged QT and QRS intervals leading to life-threatening ventricular arrhythmias and cardiac arrest , 6. AggrThe clinical value of hemodialysis and peritoneal dialysis has not been established for the overdose of HCQ. The apparent volume of distribution for HCQ is approximately 600 L/kg. This implies that the drug rapidly becomes unavailable for removal after ingestion . The extIntravenous lipid emulsion is commercially available and termed Intralipid for treatment of local anesthetic systemic toxicity (LAST). Early basic science research by Weinberg et al. established that lipid emulsion successfully resuscitated rats and dogs from bupivacaine induced cardiac arrest , 19. ThiIn conclusion, HCQ poisoning is rare but serious because of its rapid progression to life-threatening symptoms. Hemodynamic support, gastric decontamination, electrolyte monitoring and replacement, and management of arrhythmias are the mainstays of treatment. The combined role of dialysis and ILE in the setting of massive HCQ overdose may improve outcomes by extending the “lipid sink” effect of ILE and normalizing electrolyte concentrations.


Background. The aim of the study was to analyse the effect of caffeic acid phenethyl ester (CAPE) on fluoxetine-induced hepatotoxicity in rats. Materials and Methods. Group I served as control. Group II received CAPE intraperitoneally. Group III received fluoxetine per orally. Group IV received fluoxetine and CAPE. The total antioxidant capacity (TAC), total oxidant status (TOS), oxidative stress index (OSI), and liver enzymes including paraoxonase-1 (PON-1), aspartate transaminase, and alanine transaminase levels were measured. Liver tissues were processed histopathologically for evaluation of liver injury and to validate the serum enzyme levels. Results. An increase in TOS and OSI and a decrease in TAC and PON-1 levels in serum and liver tissues of Group III were observed compared to Groups I and II. After treatment with CAPE, the level of TOS and OSI decreased while TAC and PON-1 increased in serum and liver in Group IV. Histopathological examination of the liver revealed hepatic injury after fluoxetine treatment and reduction of injury with CAPE treatment. Conclusion. Our results suggested that CAPE treatment provided protection against fluoxetine toxicity. Following CAPE treatment with fluoxetine-induced hepatotoxicity, TOS and OSI levels decreased, whereas PON-1 and TAC increased in the serum and liver. Fluoxetine is a selective serotonin reuptake inhibitor (SSRI) as a first-line drug which is used for treatment of depression and many neuropsychiatric disorders . Use of The studies on caffeic acid phenethyl ester (CAPE) have demonstrated that propolis, which is the active component in CAPE and used in traditional medicine, has antiviral, anti-inflammatory, immunomodulatory, and antioxidant characteristics. Also, CAPE has inhibitor effects on lipid peroxidation, as well as lipoxygenase and cyclooxygenase enzymes –16. CAPETotal oxidant status (TOS), total antioxidant capacity (TAC), paraoxonase-1 (PON-1), and OSI (Oxidative Stress Index) are usually measured to determine the toxicity level in damaged tissues . Local hThe objective of the present study was to analyse the effects of CAPE as an antioxidant against the hepatotoxicity caused by fluoxetine through analysis of TOS, TAC, PON-1, OSI, AST, and ALT and histopathological examination.Fluoxetine hydrochloride with code number PHR1394 and CAPE with catalog number C8221 were obtained from Sigma-Aldrich Co., LLC, Germany. Fluoxetine was diluted by saline solution. Dimethyl sulfoxide (DMSO) was used as a solvent for CAPE and the solution was diluted with saline.The authors acknowledge that the aforesaid experiments on animals were carried out in accordance with applicable laws and legislations of the Republic of Turkey. The study protocols were investigated and approved by the Ethics Committee on Animals of the University . Four groups including 8 rats which were monitored in individual cages at a room temperature of 22 ± 2°C were designed. Male Sprague Dawley rats (with a weight ranging between 320 and 340 g) used in the present study were kept under standard laboratory conditions and fed by standard rat feed and water.The procedure started with narcotization of the rats through intramuscular injection of ketamine hydrochloride on a dose of 80 mg/kg . Skins of the rats were shaved and 10% povidone iodine solution (Betadine) was used for sterilization.The present study was conducted in rats divided into four groups of 8 animals in each group. Group I served as control and received no drug or agent. Group II received CAPE intraperitoneally at 10 mmol/kg/day for 7 days. Group III received fluoxetine 10 mg/kg/day for 7 days. Group IV received fluoxetine 10 mg/kg/day plus CAPE 10 mmol/kg/day for 7 days. The total antioxidant capacity (TAC), total oxidant status (TOS), Oxidative Stress Index (OSI), and liver enzymes including paraoxonase-1 (PON-1), aspartate transaminase (AST), and alanine transaminase (ALT) were measured. Liver tissues were processed histopathologically for evaluation of liver injury and to validate the serum enzyme levels.Following final application of the medication at day 8, blood samples were collected directly from the heart through cardiac puncture. The blood samples were centrifuged at 4000 rpm for 10 minutes. Measurement of ALT and AST activities in the serum was performed according to the Reitman-Frankel method for all groups , 21. EnzTissue samples were obtained from the liver for histopathological examination. The tissues were prepared for biochemical analysis by clearing the residues and blood with saline solution. Additionally, the tissue samples were stored in plastic containers with 10% formaldehyde solution for histopathological examination. The tissue specimens were stored at −80°C, and transfer of samples was performed in dry ice. The specimens with an approximate weight of 0.30 to 0.50 g were placed into a tube to which 2 mL of Tris-HCl buffer was added. The tubes were placed in an ice-filled plastic container, and they were treated with a 50 mM phosphate-buffered saline with a pH of 7.0 in a homogenizer at 14,000 rpm for 1 to 3 minutes . The homogenate was centrifuged for 30 min. at 15,000 rpm, at +4°C. Supernatant samples were used for analysis of TAC, TOS, and PON-1.μmol H2O2 equiv/L) for serum and hydrogen peroxide per gram (μmol H2O2 equiv/gram) of protein for tissue samples.TAC and TOS values were detected through automated biochemical measurement using a novel measurement method developed by Erel . Methods p-nitrophenol at 412 nm wavelength absorbency which was revealed by hydrolysis of paraoxon in the sample was accepted as the PON-1 activity [ p-nitrophenol is 18,290 nm and one unit of paraoxonase activity equals 1 mol of paraoxon hydrolyzed per liter per minute at 37°C. U/L for serum and U/gr protein for tissue samples were used as a unit of PON-1 activity.Paraoxonase activity of PON-1 was measured with a commercial kit of Rel Assay Diagnostics  using a spectrophotometric method. The kit contained both substrate solution and Tris buffer. The substrate solution consisted of paraoxon and buffer solution . Activitactivity . The molFollowing the TAC and TOS measurements, the OSI levels were calculated by means of the following formula: OSI = (TOS)/(TAC). Oxidative Stress Index (OSI) is an indicator parameter of the degree of oxidative stress .Total antioxidant capacity (TAC), total oxidant status (TOS), paraoxanase-1 (PON-1), Oxidative Stress Index (OSI), ALT, and AST were analysed in the blood sample. Histopathological analysis as well as biochemical parameters, TAC, TOS, and PON-1, and OSI levels to evaluate oxidative stress were performed on the liver tissue samples , 21.μm slices. Tissues were stained with hematoxylin and eosin using standard protocol. The sections were examined under a light microscope using 200x magnification for assessment of the degree of liver injury by a liver pathologist blinded to the animal grouping.Tissues were carried in 10% formalin, and the paraffin blocks were prepared in 4 Liver Injury Score. Liver injury scores were as follows. Grade 0: none or slight injury, Grade 1: slight injury; cytoplasmic vacuolization and nuclear pyknosis, Grade 2: moderately increased nuclear pyknosis, increased loss of eosinophil, and intracellular margin in the cytoplasm, Grade 3: severe injury, hemorrhage, neutrophil infiltration, and severe necrosis causing disintegration of liver cells.The tissue injury through the grading above following histopathological analysis [analysis  was deteU test was used for binary comparisons. A p value of <0.05 was accepted as statistically significant.Statistical analysis was performed using SPSS for Windows 11.5 . Data were presented with descriptive statistics including mean ± standard deviation (SD) for parametric data and median (range between 25% and 75%) for nonparametric data. The normality of the parameters in serum and tissue groups was analysed with the Shapiro-Wilk test. The parametric data were analysed with an Anova test and a Tukey post hoc test for binary comparisons. The nonparametric data were analysed with a Kruskal-Wallis test, and a Mann-Whitney p < 0.05). The decrease in TOS, OSI, ALT, and AST levels and the increase in TAC and PON-1 levels were observed in Group IV when compared with Group III (p < 0.05) (p < 0.05)  . The bio < 0.05) . After t < 0.05) . The his < 0.05)  and cont < 0.05) . Slices  < 0.05) . Althoug < 0.05)  were lesOur results demonstrate that CAPE treatment decreased liver injury and serum oxidant enzyme levels caused by fluoxetine treatment. CAPE treatment also increased antioxidant enzyme levels in the present study. Furthermore, positive contribution of CAPE to recovery of hepatic modifications caused by fluoxetine was observed at tissue level. This indicated the benefit of CAPE on liver injury induced by fluoxetine.Increase of transaminase enzyme activity  in the serum as a sensitive indicator for liver injury was reported in hepatic cell injury . SeveralFluoxetine includes fluorine; if a drug has fluorine in its compound, increase in pharmacokinetic and pharmacodynamic characteristics along with toxic effects appears . One of OSI measurements provide a sensitive, novel index of oxidative stress and can reflect both oxidative and antioxidative parameters . In the PON-1 is an antioxidant enzyme that prevents oxidation of low-density lipoprotein. Activity of PON-1 decreases with an increase of oxidative stress , 38. UzaStudies on CAPE, the active component of propolis used in traditional medicine, showed antiviral, anti-inflammatory, immunomodulatory, and antioxidant characteristics of CAPE due to inhibitor effects on lipid peroxidation as well as lipoxygenase, cyclooxygenase enzymes –16. ÇakiJohnston and Wheeler reported a case of serious chronic hepatitis due to fluoxetine exposure , whereasThe present study indicates an increase in serum TOS, OSI, PON-1, and transaminase activity along with a decrease in TAC levels following hepatotoxicity caused by fluoxetine. Furthermore, moderate injury appeared histopathologically. CAPE was effective at reducing serum transaminase levels and oxidative stress parameters, at increasing antioxidant stress parameters, and at reversing the histopathological injury. The data presented above concludes that CAPE acts as an agent to protect the liver against oxidative alterations due to hepatotoxicity. We believe that CAPE may be used for fluoxetine-induced hepatotoxicity and that further clinical and laboratory surveys are required.


To investigate macular pigment optical density (MPOD) and serum concentration changes of lutein in Japanese subjects participating in a clinical trial in which two formulations of lutein and zeaxanthin supplements with different physiochemical properties are used.Thirty-six healthy volunteers were recruited into this prospective, randomized, parallel-group, double-masked comparative study at a single institute. Two products were used, FloraGLO® (Kemin Japan) and XanMax® (Katra Phytochem). The lutein particle size and zeaxanthin concentrations differed between the formulations. The subjects consumed one of the two supplements for a duration of up to 6 months. MPOD levels were measured by resonance Raman spectrometry at baseline and once a month until the end of the study. Serum lutein concentration was measured at baseline, month 3, and month 6. The subjects were also tested for contrast sensitivity, glare sensitivity, visual acuity, and in addition had a focal electroretinogram measured.p = 0.0133). Glare sensitivity improved in retinal responders in both supplement groups, while there were no remarkable changes in contrast sensitivity.The mean serum lutein concentrations increased significantly after the first three months, but the mean MPOD levels in either supplement group did not show any statistically significant increase. A detailed analysis, however, revealed three response patterns in both groups for the increase of MPOD levels and serum lutein concentration, i.e. “retinal responders”, who had an increase of both MPOD levels and serum lutein concentrations (n = 13), “retinal non-responders”, who had only increased serum concentrations and no change in MPOD levels (n = 20), and “retinal and serum non-responders”, who had neither MPOD level nor plasma concentration increases (n = 3). The subjects with low MPOD levels at baseline appeared to show increased MPOD levels at the 6 month time point upon lutein supplementation (r = -0.4090, No statistically significant differences could be detected for MPOD levels and serum lutein concentrations between the two investigated lutein supplement formulations. Responses to lutein supplementation regarding MPOD levels and serum lutein concentrations varied between subjects. Subjects with lower MPOD levels at baseline responded well to lutein supplementation. However, since the number of subjects was low, a further study with more subjects is needed to prove that subjects with low MPOD levels will benefit from lutein supplementation.UMIN000004593UMIN-CTR  After mixing for 1 minute, 5 mL of n-hexane was added to the sample and mixed for 2 minutes. The sample was centrifuged , and n-hexane extracts were evaporated under N2 gas. Finally, the residue was dissolved in 250μL of acetonitrile: methanol: chloroform = 60: 25: 15, and the solution was centrifuged . The supernatant was injected onto the HPLC system .Blood samples were taken from each subject at baseline, 3 months, and 6 months after the start of supplementation. Serum lutein analysis was conducted by the Diagnostic Division of Otsuka Pharmaceutical Co., Ltd. . For this purpose, 0.5 mL of serum was mixed with 25μL of ethyl 8’-apo-beta-caroten-8’-oate . This method has an advantage over a univariate approach with split-plot and repeated-measures analysis of variance, since it does not require a sphericity assumption.Statistical analyses were performed on a Macintosh personal computer using StatView software  or JMP software . All statistical tests were two-sided; When analyzing changes of MPOD levels and serum lutein concentrations, we found certain patterns between the change of MPOD levels and change of serum lutein concentrations. In order to differentiate these response patterns to lutein supplementation, we calculated the rates of change of MPOD levels and serum concentrations of lutein between baseline and six-month supplementation time point for each subject. Whenever the rate of MPOD change was 1.2 or higher (i.e. 20% increase), the MPOD levels were considered “increased” at the six month time point. The value of 1.2 is based on the relative standard deviation of 19.1% for RRS measurements. Whenever the rate of change in serum lutein concentration was 1.1 or higher (i.e. 10% increase), the serum lutein concentration was considered “increased” at the six month time point. The value of 1.1 was adopted from the relative standard deviation of 3.9% with the used measurement technique.The correlation between baseline MPOD levels and serum lutein concentrations, as well as the rate of MPOD and serum lutein concentration changes, were analyzed with Spearman's rank correlation coefficient test.The subject demographic data at baseline are shown in P = 0.6832, unpaired t-test). The changes of MPOD levels in either group are shown in P = 0.4957 for the FloraGLO group and P = 0.4878 for the XanMax group, MANOVA, P = 0.9788, MANOVA). P = 0.4830 for the FloraGLO group and P = 0.0997 for the XanMax group, MANOVA).Mean MPOD levels (in Raman counts) at baseline were 4358±1936 in the FloraGLO group and 4039±2546 in the XanMax group. There was no statistically significant difference between them . At 3 months after the start of supplementation, the serum concentration increased to 0.65±0.30 μg/ml in the FloraGLO group and to 0.60±0.20 μg/ml in the XanMax group, and these high levels continued until the end of supplementation. Changes in lutein serum concentration are shown in P = 0.0008 for the FloraGLO group and P = 0.0016 for the XanMax group, MANOVA, P = 0.3603, MANOVA). No remarkable difference was noted in the pattern of increasing serum lutein concentration in the three age groups (data not shown).The serum concentration of lutein at baseline was 0.37±0.12 μg/ml in the FloraGLO group and 0.42±0.13μg/ml in the XanMax group. There was no statistically significant difference between them , month 3 , and month 6 .There was no significant correlation between MPOD levels and serum lutein concentration in either group at baseline  showed increases in both MPOD levels and serum concentrations of lutein. These 13 subjects appeared to correspond to “retinal responders” according to the classification of Hammond et al . Twenty P = 0.0034, unpaired t-test). There was a significant difference in gender . Men were predominant in retinal-responders.There was no difference in serum lutein concentration between retinal responders and retinal non-responders, but the serum lutein concentrations in retinal and serum non-responders were higher than those in retinal responders and non-responders.P = 0.0010), the largest target size of the instrument. At month 6, glare sensitivities were significantly improved at the target size of 4.0, 2.5, 1.6 and 1.0 degree  and marginally improved at 6.3 degree (P = 0.0535). In retinal non-responders, contrast and glare sensitivity showed no significant improvements at six months at all size of targets with one each exception at 6.3 degree (P = 0.0010) in contrast sensitivity and 4.0 (P = 0.0207) in glare sensitivity.Contrast and glare sensitivities in retinal responders are shown in Macular ERG results are shown in The changes in MPOD levels and serum lutein concentrations with two kinds of supplementation containing same amounts of lutein but with different particle sizes and slightly different amounts of zeaxanthin have been investigated in this Asian population. The mean serum lutein concentrations increased, but the mean MPOD levels showed no increase in either supplement group. The mean MPOD levels also did not change when the three age groups were analyzed, although there was a tendency that MPOD levels in the older group were lower than in the younger group. No significant correlation between serum lutein concentration and MPOD levels were observed, as shown in previous studies ,17. ThusP = 0.001, t-test, Although the mean MPOD levels in the present subjects did not change, a more detailed analysis showed that thirteen subjects  showed increases in MPOD levels at one month after supplementation and continued gradual increases up to the end of supplementation . The serThe number of retinal and serum non-responders was too small for a statistical analysis, but these subjects had higher serum lutein concentrations and MPOD levels at baseline compared to the other two groups . This faConsidering the obtained results, the terms “retinal responder”, “retinal non-responder” and “retinal and serum non-responder” may not be appropriate to describe the character of the three response patterns. Retinal responders are subjects whose dietary consumption of lutein is insufficient to maintain high levels of MPOD. Therefore, they might be good candidates for lutein supplementation. Retinal non-responders are subjects who have a high ability for carotenoid uptake into the retina even with relatively small consumption of dietary lutein. Retinal and serum non-responders are subjects who have already high MPOD levels from sufficient dietary lutein consumption. These conclusions are supported by Tables The mean rate of MPOD level increase for retinal responders was 46% and 26% in the FloraGLO and XanMax groups, respectively. Our previous study measuring MPOD levels with the same instrument after 10 mg of FloraGLO lutein supplementation for three months showed a rate of MPOD level increase 〔 = (MPOD at the end of study − MPOD at baseline) / MPOD at baseline ×100%)〕of 24% for the average of 11 subjects . The difFunctional changes were assessed with contrast and glare sensitivity tests and macular ERGs. The relationship between macular pigment and visual functions have been previously investigated, with several reports suggesting an improvement of contrast sensitivity ,22, glarA shortcoming of the present study is a lack of information on genotype and dietary intakes of carotenoids for each subject. This is important because not only diet can influence ocular carotenoid status but there is also a well-known hereditability of macular pigment levels . GeneticS1 CONSORT Checklist(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S3 Data(XLSX)Click here for additional data file.S4 Data(XLSX)Click here for additional data file.S5 Data(XLSX)Click here for additional data file.S6 Data(XLSX)Click here for additional data file.S7 Data(XLSX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S1 Protocol(DOCX)Click here for additional data file.S2 Protocol(DOCX)Click here for additional data file.


The critical role of metabolic abnormality in hypertension is increasingly recognized, but its biomarkers are not clearly identified. In this study, 47 chemical compounds recorded by literature were employed as target metabolites of essential hypertension (EH). We detected their content in the plasma of EH patients and healthy subjects by using the Multiple Reaction Monitoring-Mass Spectrometry (MRM-MS). After screening the most altered compounds, acupuncture was used to treat patients for 3 months and these plasma metabolites were tested again. The results showed that oleic acid (OA) and myoinositol (MI) were the most important differential metabolites between the hypertensive plasma and the healthy plasma. They were also closely correlated with 24-hour blood pressure and nocturnal dipping. Moreover, plasma OA and MI could be restored to normal levels by acupuncture, accompanying with reduction of 24-hour systolic and diastolic blood pressure  and improvement of circadian blood pressure rhythm. This study demonstrated that plasma OA and MI were potential hypertension biomarkers and they could be used to preliminarily assess the treating effects such as acupuncture. Hypertension is a leading risk factor for cardiovascular, cerebral, and renal events. It accounts for at least 45% of deaths due to heart disease and 51% of deaths due to strokeTherefore, in this study, we employed Multiple Reaction Monitoring-Mass Spectrometry (MRM-MS), a new high-throughput method, to detect 47 kinds of low molecular weight plasma metabolites that have already been reported or considered as potential target molecules in the study of hypertension. We chose acupuncture, one of the signature treatment methods of traditional Chinese medicineA total of 113 eligible patients with mean age of 59.82 ± 8. 95  and 15 healthy subjects with mean age of 55.93 ± 6.30  were enrolled in the current study. The baseline characteristics and BP-related parameters such as gender, age, dietary habit, nationality, and other physiological parameters like blood sugar, total cholesterol, ALT, AST, BUN, sCr, HDL-C, and LDL-C demonstrated no significant difference in the two groups. However, BP was significantly different between EH patients and the healthy control, as shown in vs. healthy controls showed that the area under the curve (AUC) of OA is 0.859 (0.625–1), and 0.781 for MI(0.5–0.969) -galactose, Glycine, fructose, L-tyrosine, OA, MI, and urea were significantly changed in EH patients . Multivariate analysis by OPLS-DA analysis revealed that EH patients and healthy control subjects could be separated by the target metabolites . The cor5–0.969) .2 = 0.25), and MI was inversely correlated with 24-hour systolic (R2 = 0.22) and diastolic BP (R2 = 0.16). These two metabolites also showed correlation with systolic and diastolic BP nocturnal dipping. The OA showed a negative correlation with SBP nocturnal dipping (R = −0.44) and DBP (R = −0.37), while MI was positively correlated with SBP nocturnal dipping (R = 0.35)  . BesidesAfter finding OA and MI are the two potential biomarkers in EH patients. We used them to evaluate the effect of acupuncture treatment. Interestingly, the OPLS-DA results demonstrated that acupuncture could successfully restore the level of OA and MI to normal levels . VIP forAcupuncture simultaneously lowered 24-hour BP, improved circadian BP rhythm, along with reversing OA and MI abnormalities. All values of 24-hour, daytime and nighttime (systolic and diastolic) BP, and pulse are regulated by acupuncture  . The 95%The metabolic abnormality is getting increasingly concerned in the development and treatment of hypertension. A three population-based cohort study demonstrated that identification of metabolic biomarkers of cardiovascular diseases could be helpful for more accurate diagnosis and more precise treatment2-adrenergic receptor pathways. The other key metabolite, MI, is an insulin-sensitizing substance possessing insulin-mimetic properties3R). IP3R up-regulation in hypertension is associated with sensitization of Ca2+ release and vascular smooth muscle contractility. In hypertension, resistant arteries show elevated responsiveness to vasoconstrictor agonists, and this abnormality relies partly on enhanced Ca2+ signaling in vascular smooth muscle (VSM). Studies proved that levels of IP3R transcript and protein were significantly increased in mesenteric arteries of hypertensive animals, and pharmacological inhibition of the IP3R revealed a higher contribution of IP3-dependent Ca2+ release to vascular contraction in these arteries. IP3R up-regulation in VSM is associated with enhancement and sensitization of IP3-dependent Ca2+ release, resulting in increased VSM contraction in response to agonist stimulation3 changing, which is a crucial mechanism underlying BP elevation.OA is one of the most important free fatty acids (FFAs), accounting for 40% of FFAs in the healthy blood sample. Elevated plasma levels of FFA are considered to be associated with higher cardiovascular risk, induced by FFA-related oxidative stress in endothelial cells. In addition, OA can help lower the level of low-density lipoprotein (LDL) in the bloodstream, while unchanging the level of high-density lipoprotein (HDL). Although there is no evidence that dietary total OA is closely related to BP in individuals, OA from vegetable sources may contribute to preventing the elevated BP levels in general population11In addition, we found the levels of plasma OA and MI are independently related to blood pressure because there is no difference seen in other physiological parameters like blood sugar, total cholesterol, ALT, AST, BUN, sCr, HDL-C, and LDL-C between hypertension patients and health control subjects. As we know, high blood pressure is one of the components that results in metabolic syndrome. On the other hand, metabolites such as OA and MI are also related to some metabolic syndrome (such as high cholesterol and diabetes). Thus, whether the alteration of OA and MI is specifically resulted from hypertension is a great challenge. In this study, subjects with abnormal blood sugar, total cholesterol, ALT, AST, BUN, sCr, HDL-C, and LDL-C were excluded according to the inclusion/exclusion criteria. After applying the univariate analysis and the multivariate analysis, OA and MI were two compounds that significantly changed in disease condition as compared with the healthy subject. Moreover, these two contributed the most to separate disease plasma and health ones, as indicated in the PLS-DA analysis. Therefore, OA and MI are proved to be closely correlated with hypertension. However, this does not mean that the alteration of OA and MI in plasma has no relation with diabetes or high cholesterol. One obvious reason is that we have only studied borderline hypertension. Besides, other studies found that OA could induce a synergistic mitogenic response with angiotensin II in vascular smooth muscle cells in obese hypertensive patients, which consequently accelerated vascular disease. The increased OA level and enhanced activity of the renin-angiotensin axis in patients with the risk factor cluster could also interact with atherosclerosis1718Our study demonstrates the improvement in the plasma level of OA and MI can reflect the therapeutic effect of acupuncture to some extent. Acupuncture is a traditional therapeutic treatment for EH patients especially for those on early stage or with complications. Flachskampf’s large clinical trial3 level in rat brain and spinal cord, suggesting links to the PI system via its signal transduction pathways. In mild cerebral artery occlusion, acupuncture was shown to downregulate the significant increases in expression of Angiotensin II and influence its receptor-mediated phosphatidyl inositol signal pathway, consequently reducing vasoconstriction and improving blood supply to the ischemic region, and ultimately conferring beneficial effects on cerebral ischemia3 and diacylglycerol (DAG) in rat cerebral arteries, which may contribute to acupuncture’s effect in modulating vascular constriction and dilation to ameliorate cerebral microcirculationTill now, it is still not clear how those two metabolites are involved in acupuncture’s antihypertensive effect. Recent studies suggested that the hypoglycemic effect of acupuncture is probably attributable to the reduction of FFA24In conclusion, by a combination of laboratory tests with clinical effectiveness, which is much more comprehensive, OA and MI were found to be closely related to increased BP and possibly circadian BP rhythm disorder in this study. Besides, the restoration of OA and MI in the plasma can reflect the up-regulating BP and the improvement of circadian rhythm which was induced by acupuncture treatment. Future studies are welcome to examine our hypothesis by enlarging the sample size and by involving different drug-related studies.All procedures were designed according to the Declaration of Helsinki’srd teaching hospital of Chengdu University of Traditional Chinese Medicine. Essential hypertension (EH) patients and healthy subjects were included if they fulfilled all the following criteria. Inclusion criteria: (1) aged between 40 and 70 years; (2) diagnosed with stage I hypertension at the first visit, not with some type of secondary hypertension such as renal vascular disease, Cushing’s syndrome, hyperadrenocortism, or drug-induced hypertension; (3) free of other complicated cardiovascular, digestive, respiratory, urinary, blood, nervous, endocrine-system, or other severe primary diseases; (4) did not ingest any drugs or herbs during the 15 days before the start of the study; (5) were not participating in any other study; (6) agreed to cooperate with researchers in all research procedures after they were introduced into this study; and (7) provided written informed consent. Exclusion criteria included epilepsy, sleep apnea hypopnea syndrome, depression or anxiety, pregnancy or lactation, or women of reproductive age who intended to conceive within 1 year; any blood laboratory test abnormality; suffering from acute diseases in the past 2 weeks, such as high fever, or gastritis; or being prescribed any drug or being treated with acupuncture within the past three months. Healthy adults of similar age, body mass index (BMI), and gender were recruited as healthy controls. These healthy adults had no organic or functional disorders, were not infected with the common cold or other conditions, and had not taken medications or received acupuncture treatments within the past three months.All study participants were recruited from the 3Demographic information and regular physiological data were acquired at baseline. The 24-hour ambulatory BP was measured by an oscillometric device  within 24 hours before the commencement and the completion of acupuncture treatment. The basic acupuncture compulsory formula included two acupoints: Taichong (LR3) and Renying (ST9). Arbitrary acupoints included Taixi-KI3, Neiguan-PC6, Zusanli-ST36, and Quchi-LI11. No other acupoints or sham acupoints were used in this study. Licensed Chinese acupuncture therapists performing all treatment procedures have at least five years’ experience of acupuncture treatment. All acupoints were punctured with disposable stainless steel needles . Deqi sensation, which is essential to ensure the clinical efficacy of acupuncture, was subsequently induced by manipulations. The hypertensive patients received acupuncture therapy for 30 minutes in each session, three sessions a week, for six weeks. Every two weeks there was a two-day interval without treatment.Fasted venous blood (about 5 mL) of the patients was collected into vacutainer tubes with heparin sodium before and after acupuncture treatment at 8:00–9:00 a.m. respectively. Plasma was obtained by centrifugation at 1500 rpm at 4 °C for 15 min.Metabolites dramatically changed in Hypertension patients or closely related with the pathophysiological mechanisms of the cardiovascular system were identified31323334353637The analyses were performed on a QTRAP5500 mass spectrometer  equipped with a Shimadzu UFLC system, which consisted of Shimadzu LC-20AD XR pumps and a SIL-HTC autosampler . Liquid chromatography-tandem mass spectrometry data were acquired using a 5500 QTRAP triple-quadrupole mass spectrometer . LC−MS/MS analysis was performed on a 5500 QTRAP triplequadrupolelinear ion trap mass spectrometer  equipped with electrospray ionization (ESI) source interface operating in positive ion mode. Chromatography separation was achieved on an Eksigentekspert™ microLC 200 System . Two microliters of each sample was injected on a Luna 5 μm, 150 mm × 4.6 mm HPLC column . The mobile phases were: (A) 0.01% heptafluorobutyric acid, 0.1% formic acid in water, and (B) 0.01%heptafluorobutyric acid, 0.1% formic acid in methanol. The needle-rinse solvent was methanol. The HPLC flow rate was set at 0.8 mL/min and eluted with a gradient of 2–40% solvent B for 6 min, 40% solvent B for 4 min, 40–90% solvent B for 0.5 min, 90% solvent B for 0.5 min, 90–2% solvent B for 1 min, and followed by 2% solvent B for 3 min. The column was reequilibrated with 2% B for 1 min prior to the next injection. In QTRAP5500, the parameters were set to positive ionization mode, with curtain gas set at 40 psi, nebulizer gas at 60 psi, IonSprayTM voltage at 5500 V, ion source temperature at 600 °C, and CAD gas at medium and to negative ionization mode, with curtain gas set at 40 psi, nebulizer gas at 25 psi, IonSprayTM voltage at −4500 V, ion source temperature at 600 °C, and CAD gas at medium. The MRM transitions for the analyses, declustering potential (DP), entrance potential (EP), collision energy (CE), and the collision cell exit potential (CXP) are listed in http://www.metaboanalyst.ca/MetaboAnalyst/) to find differential metabolites between groups. Score scatter plot and loading plot visualized the separation of samples and the metabolites which were responsible for the separation. Data were also preprocessed using orthogonal signal correction (OSC) which was generally used in metabonomic analysis to filter the unrelated variations. Variable Importance Plot (VIP) that indicated the metabolites changed dramatically in different groups were identified and those whose VIP > = 1.0 were considered to be the key biomarker in this stage. The paired t test (P < 0.05) and fold-change   were also performed to strength the above results.Metabolic biomarker identification-For metabolomics data, the multivariate analysis and univariate analysis were performed as an integral to determine the most important plasma metabolites for EH and its treatment, or namely, the biomarkers. Pareto-scaled data were Partial least square discriminant analyzed (PLS-DA) by SIMCA-P  and MetaboAnalyst (www.graphpad.com).Clinical effect evaluation- clinical data and the effect of acupuncture were analyzed by paired t test  was used to analyze the correlation between the level of key metabolites and the clinical outcomes of EH.The statistical analysis framework included EH biomarker identification, clinical effect evaluation and biomarker validation according to clinical outcomes.How to cite this article: Yang, M. et al. A Targeted Metabolomics MRM-MS Study on Identifying Potential Hypertension Biomarkers in Human Plasma and Evaluating Acupuncture Effects. Sci. Rep.6, 25871; doi: 10.1038/srep25871 (2016).


Streptococcus pneumoniae, an important human pathogen responsible for more than 1.5 million child deaths worldwide. For that, cultured live bacterial cells were “shaved” with trypsin, and the recovered peptides were analyzed by LC/MS/MS. We selected 95 proteins to be produced as recombinant polypeptides, and printed them on an array. We probed the protein array with a collection of patient sera to define serodiagnostic antigens. The mass spectrometry proteomics data correspond to those published in Surface proteins play key roles in the interaction between cells and their environment, and in pathogenic microorganisms they are the best targets for drug or vaccine discovery and/or development. In addition, surface proteins can be the basis for serodiagnostic tools aiming at developing more affordable techniques for early diagnosis of infection in patients. We carried out a proteomic analysis of a collection of pediatric clinical isolates of  Sera were drawn either from patients with a diagnosis of pneumococcal pneumonia (the “patient” group), or from healthy children or patients affected by other pathologies different from pneumococcal pneumonia (the “control” group). Written informed consent was obtained from parents or legal guardians of participating children, for sera to be used within the project in which this work was designed, according to the principles expressed in the Declaration of Helsinki.3.2Twenty-four pneumococcal isolates from human patients with pleural empyema were kept, grown and “shaved” for surface protein identification as already described 3.3m/z range. MS/MS data were acquired in dependent scan mode, selecting automatically the five most intense ions for fragmentation, with dynamic exclusion set to on.All analyses were performed as described 3.4S. pneumoniae strains available at the UniProtKB site at the moment of the database construction .Tandem mass spectra were extracted using Thermo Proteome-Discoverer 1.0 (Thermo Fisher Scientific). Charge state deconvolution and deisotoping were not performed. All MS/MS samples were analyzed using Sequest , as described 3.5http://www.cmbi.ru.nl/locatep-db/cgi-bin/locatepdb.py). They were contrasted by several feature-based algorithms: TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0) for searching transmembrane helices; SignalP 3.0  for type-I signal peptides: those proteins containing only a cleavable type-I signal peptide as featured sequence were classed as secreted; LipoP (http://www.cbs.dtu.dk/services/LipoP) for identifying type-II signal peptides, which are characteristic of lipoproteins. GO annotations were retrieved from the UniProt Knowledgebase (http://www.uniprot.org/) and annoted using a webserver from the University of Adelaide (http://genomes.ersa.edu.au/BacteriaGO/submissions.php).Primary predictions of subcellular localization were assigned by using the web-based algorithm LocateP , and expressed in Escherichia coli BL21, as described 2+–agarose affinity chromatography from the E. coli intracellular fraction, dialyzed against PBS and used for protein array printing.Recombinant proteins were produced as double fusion fragments containing an 3.7Affinity-purified recombinant proteins were printed on glass slides in quintuplicate (6 ng/spot) as detailed in 3.8For analysis of antibody binding to recombinant fragments on the microarray, local background subtraction from 10 surrounding spots was performed and corrected median fluorescence intensity was used for initial data processing. Then, the mean background signal of negative controls was substracted from each raw spot value after sera hybridization. Negative controls represented hybridizations of non-pneumococcal proteins and buffer spots with sera and secondary antibodies. Both in non-pneumococcal proteins and buffer positions, no reaction with human sera was observed. After background substraction, negative or zero values were assigned a net value of 0. Then, outlier values for each spot were removed. The two different hybridizations for each serum were averaged to report the signal mean intensity (SMI) values, and the mean and standard deviation (SD) were obtained from the 5 printed spots per protein in each patient and control groups. Finally, data normalization by background was carried out using Microsoft Excel as described


During the last few years, scientific investigations have proposed diet based regimens to prevent several health ailments including obesity, hypercholesterolemia and diabetes. In this regard, a promising tool is the use of functional foods/nutraceuticals. Present research project was an attempt to explore nutraceutical worth of locally grown green tea variety (Qi-Men) against lifestyle related disorders.2 and T3) were prepared by adding catechins and epigallocatechin gallate (EGCG) @ 550 mg/500 mL and compared with control (T1). These functional drinks were tested in experimental rats modeling (Sprague Dawley). Based on diets, four studies were conducted i.e. trial-I , trial-II (high cholesterol diet), trial-III (high sucrose diet), trial-IV (high cholesterol + high sucrose diet). Rats were monitored daily for their feed and drink intake while body weight was measured on weekly basis. After period of 56 days rats were sacrificed and evaluated their serum lipid , glucose and insulin levels.Functional drinks (TResults for feed consumption by rats revealed that highest feed intake was recorded in group provided control drink than other groups. However, non significant differences were noted among all groups for drink consumption. Functional drinks resulted in significant reduction in body weight with maximum lowering noted in trial-II and III i.e. 10.73 to 8.49% and 10.12 to 10.49%, respectively. Likewise, cholesterol and LDL were substantially reduced with 14.42% decrease observed in trial-IV and 30.43% in trial-II, respectively. Furthermore, serum glucose and insulin levels were also lowered significantly in the trial-III and IV while in trial-I and II differences were non-significant. In contrast to lipid profile, experimental drink containing EGCG reduced the trait better than catechins based functional drink.The drinks supplemented with catechins and EGCG are effective against obesity, hypercholesterolemia and hyperglycemia. Changing lifestyle and poor dietary habits of people often lead to development of various maladies like obesity, diabetes, dyslipidemia and immune dysfunction. Obesity is now well-known as one of the global concerns because it is not only prevalent in the developed nations but also becoming common in the developing world with associated physiological threats. Dyslipidemia is more frequent in obese people with raised levels of LDL, triglycerides and cholesterol. Among contributory factors of obesity, dietary habits are considered one of reasons for its expansion. During the last few years, amplified consumption of carbohydrate and animal fat has contributed to increased incidence of atherosclerosis induced cardiovascular complications and diabetes mellitus. High cholesterol diet causes significant increase in serum cholesterol level along with sensitivity to oxidation. Sucrose rich diet induces hyper-triglyceridemia and hypercholesterolemia .Camellia sinensis) belonging to family Theaceae is one such example of functional drinks containing bioactive molecules holding cure against various diseases. Green tea contains appreciable amounts of phytochemicals especially catechins [Phytochemicals are endemic in the human diet from the ancient times to fight against diseases as most of the medicines have been derived from plants. In the recent era, diet based therapy has been revitalized globally and people are adopting the approach of using natural materials as an intervention against various ailments. During the last few years, scientific investigations have proposed several modules like diet based regimen to prevent life threatening disorders including obesity, hypercholesterolemia and diabetes. Among these strategies, a promising tool is the use of functional/nutraceuticals foods that not only improve consumer health and wellness but also reduce disease risk with minimal cost. Green tea  were prepared by addition of catechins and epigallocatechin gallate (EGCG) respectively. A control drink, T1 (without active ingredient) was also prepared for comparison purpose. Other ingredients included aspartame, citric acid, sodium benzoate, carboxy methyl cellulose (CMC), food grade color and flavor. After processing of drinks, active ingredients including catechins and EGCG were added individually @ 550 mg/500 mL in respective drinks. Briefly, catechins rich fraction from green tea was separated using water at constant temperature of 40°C and concentrated through a rotary evaporator  and dried through Vacuum Oven. Later, chloroform and ethyl acetate were used used to extract catechins from dried extracts. For extracting EGCG, water filtration followed by addition of caffeine and subsequent centrifugation at 16,600×g for 20 min was used. Later, pellet was decaffeinated and solvents (chloroform and ethyl hexanoate) fractionation was used for further separation of gallate rich moieties. The resultant aqueous fraction thus obtained was subjected to further partition with propyl acetate to get EGCG rich fraction that was further concentrated using a Rotary Evaporator and dried using Freeze Drier [Functional drinks  of National Institute of Food Science and Technology (NIFSAT). Later, National Institute of Health (NIH) provided the researchers with 120 infectious free Sprague dawley rats for the research purpose. The rats were housed in the Animal Room of National Institute of Food Science and Technology. Four different trials depending on different types of diets i.e. trial-I , trial-II (high cholesterol diet), trial-III (high sucrose diet) and trial-IV (high cholesterol + high sucrose diet) were conducted. Therfore, 120 rats were divided in four groups of thirty each. In each trial, rats were further divided into three subgroups comprising of ten rats in each subgroup. Rats were placed in standard polypropylene cages with stainless steel top grill. Rats were acclimatized by feeding basal diet (AIN-76A) for a period of one week. At the initiation of study some rats were sacrificed to get baseline values. The temperature (23 ± 2°C) and relative humidity (55 ± 5%) were maintained throughout the experiment with 12 hours light–dark period. Feed & drink intake was recorded daily whilst body weight on weekly basis throughout the experiment. Spilled diet and feces were also collected.1, T2, T3) were provided in graduated bottles for a period of 8 weeks , casein protein (10%), corn starch (66%), casein (10%) and cellulose (10%). The mineral and vitamin mixtures (AIN-76) were added @ 3% and 1%, respectively. The functional drinks , corn starch (26%), casein (10%), cellulose (10%) and salt & vitamin mixture (3 & 1%).Rats of group IV were fed on diet including corn oil (10%), corn starch (25%), casein (10%), cellulose (10%), salt & vitamin mixture (3 & 1%), cholesterol and sucrose in amount of 1% and 40% respectively.The following parameters were recorded separately in all rat modeling studies.Net feed intake of each group was measured daily by excluding spilled diet from the total diet during the entire study period . The funGain in body weight of experimental groups was measured weekly throughout the study period to monitor suppressing effect of functional drinks on body weight gain.et al. [Serum cholesterol level was determined using CHOD–PAP method . High deet al. . Total tet al. .In each study, rats serum samples were evaluated for glucose concentration by GOD-PAP method as described by Thomas and Labor  whereas Completely Randomized Design (CRD) was applied and resultant data was subjected to statistical analysis using Cohort version 6.1. Analysis of variance technique (ANOVA) was used to determine the level of significance and means were further compared through Duncan Multiple Range test.1 group (control drink). Whereas, comparatively, lower feed intakes were noted in T2  and T3  groups. In all studies, there was a progressive increase in feed intake with passage of time however; relatively lower feed intake was recorded in groups using experimental functional drinks with green tea active ingredients.Means regarding feed intake Figure  in all tMeans belonging to drink intake of rats Figure  in four 1) than functional drinks (T2 and T3). The trend for body weights showed differences in each trial e.g. higher body weights were recorded in high cholesterol fed diets. Moreover, body weights in all groups increased steadily in the first 28 days (4 weeks) but last four weeks provided insight about the differences in body weights. In all the trials, body weight of rats in experimental groups was less than placebo or control. The effects of catechins based functional drinks were more pronounced in high cholesterol diets, while EGCG based functional drink performed better in high sucrose diet trial and high cholesterol + high sucrose diet trial.Body weights of different groups of rats in all studies are shown in Figure 2 (drink containing catechins) was more effective in reducing cholesterol than T3 (drink containing EGCG) though the differences were non-significant. It is obvious from Table Table Table et al. [et al. [et al. [In present exploration, less feed consumption due to functional drinks is supported by the work of Babu et al. . Results [et al. . However [et al.  elucidatet al. [et al. [The current results showing reduced body weight gain in groups consuming drinks containing catechins or EGCG are in consistent with the study of Basu et al.  and Byun [et al.  Weight r [et al. ,17. Inhi [et al. .et al. [Results for decreased cholesterol and LDL levels are in consistent with the work of Kim et al. . Hypochoet al. . Tea catet al. .et al. [Results for non-significant effect of drinks on HDL are in agreement with previous work of Gomikawa et al. . The effet al. . Roghaniet al.  noted LDet al. . Crude cet al. . Moreoveet al. ,27.et al. [Low concentration of triglycerides by green tea might result from suppressed expression of stearoyl-CoA desaturase (SCD 1) gene. Because in liver triglyceride synthesis depends on the expression of the SCD 1 gene, that is involved in biosynthesis of oleate and palmitoleate, the major monounsaturated fatty acids of triglycerides. However, Watanabe et al.  is of thet al. [et al. [Reduced glucose level by green tea is supported by the work of Polychronopoulos et al.  that staet al. . EGCG exet al.  and causet al. . Additioet al. . Attenua [et al. . It is p [et al. . It can Our study demonstrates that drinks supplemented with catechins and EGCG are effective against obesity, hypercholesterolemia and hyperglycemia. Consumption of functional drink reduced the body weight. Functional drinks resulted in significant reduction in body weight that could be caused by a decreased feed intake. Moreover, improvement in lipid profile was observed in all trials but more pronounced in semi-diseased states i.e. trial-III and trial-II. Furthermore, serum glucose and insulin levels were also lowered significantly in the trial-III and IV. It was interesting to observe that catechins showed higher potency against hypercholesterolemia but EGCG reduced the hyperglycemia more effectively.