The Relationship Between Whole-grain Intake And Type 2 Diabetes Mellitus And Impaired Glucose Regulation

Diabetes mellitus(DM)is a group of endocrine diseases that are marked by hyperglycemia,caused by insulin-secreting and/or insulin-action disorder.According to the etiology,DM is divided into type 1 diabetes mellitus(T1DM)and type 2 diabetes mellitus(T2DM),and T2DM is more common,accounting for 90%.Imparied glucose regulation(IGR),also called prediabetes,is a high-risk condition between normal blood glucose and diabetes hyperglycemia,which is more prone to developing T2DM.IGR consists of two states:imparied fasting glucose(IFG)and imparied glucose tolerance(IGT).The prevalence of T2DM has dramatically increased over the past few decades worldwide,leading to considerable increases in related mortality and economic cost.How to prevent and control the epidemic of T2DM has become major national public health problems.Due to their important role in diets,interest in the health effects of grain intake,and especially whole grains,is increasing worldwide.Several prospective studies have provided consistent evidence suggesting that higher intake of whole grains is associated with a lower risk of T2DM,cardiovascular disorders,obesity and some cancers.All these epidemiological studies on whole grains,however,generally assessed individual whole grain consumption through self-administered food frequency questionnaires(FFQ).Given the diversity of whole grain products in diets,it is always challenging in free-living populations to assess intake accurately,and some degree of random and measurement errors are inevitable due to the inaccurate identification of different whole grain constituents.A more objective estimation of whole-grain intake through use of a biomarker could strengthen such epidemiologic research.Alkylresorcinols(ARs)are phenolic lipids present almost exclusively in the bran layers of rye and wheat grain,but absent in highly refined white flour and most other cereal products.About half(45%-71%)of ARs are absorbed,and intact ARs and their main metabolites(3,5-dihydroxybenozoic acid,DHBA;3-(3,5-dihydroxyphenyl)-1-propanoic acid,DHPPA)have been detected in human plasma.Plasma ARs and their metabolites correlate significantly with whole-grain wheat and rye intake both in intervention studies and in observational studies.Recently,they have been suggested as biomarkers of whole-grain intake in epidemiologic studies.Considering that urine samples are less frequently available in epidemiologic studies,and the estimated half-life is significantly longer for DHPPA(16.3 h)than DHBA(10.1 h)and alkylresorcinols(5 h),DHPPA in plasma appears to be a better and specific biomarker of whole-grain wheat and rye intake.In view of the importance of dietary factors in the prevention and control of T2DM,several studies have already reported the relationship between whole grain consumption and the risk of T2DM,which assessed whole-grain intake through FFQ.To the best of our knowledge,no research has focused on the association between plasma DHPPA,a biomarker of whole grain intake,and the risk of T2DM and IGR.In this study,we aimed to examine whether plasma DHPPA as a biomarker of whole grain intake,is associated with risk of T2DM and IGR in a case-control study conducted among a Chinese middle-aged and older population.The main contents in our study are as follows:Part One Quantification of Alkylresorcinol Metabolite in Plasma by High Performance Liquid Chromatography-mass SpectrometryObjective:To establish a high performance liquid chromatography-mass spectrometry(LC-MS/MS)method with high sensitivity,high throughput and simple pretreatment,to quantificate AR metabolite concentrations in plasma.Method:Plasma samples were analyzed on a 20A high performance liquid chromatography system(Shimadzu,Japan)coupled with an AB Sciex Q-Trap 4500mass spectrometer(Applied Biosystems,Foster City,CA,USA).Mass spectrometry parameters,chromatographic parameters,and sample preparation were first optimized to achieve optimum sensitivity and separation.Finally,the current method validation was conducted according to European Medicines Agency Guideline on Bioanalytical Method Validation with respect to selectivity,lower limit of quantification(LLOQ),calibration curve,accuracy,precision,recovery,matrix effect,and stability.Results:DHPPA and syringic acid(internal standard,IS)were quantitated by multiple reaction monitoring(MRM)mode with negative electrospray ionization.The optimized mass transition ion-pairs(m/z)for quantitation were 181.1/95 for DHPPA,and 196.9/121 for IS,respectively.The chromatographic separation was performed on a BETASIL Phenyl Column(2.1mm×150mm,3?m,Thermo Scientific,USA),and analysis was completed with a gradient elution of acetonitrile(A)-0.1%aqueous acetic acid(B).The injection volume was 5?L.A protein precipitation method with acetonitrile as precipitant was used in the sample preparation.The method validation indicated that no significant endogenous interference was observed at the retention times of the analyte and IS.The LLOQ of DHPPA which was calculated on the basis of a signal-to-noise ratio of 10:1 was set to be 0.4 ng/mL.The calibration curve exhibited good linearity over the concentration range of 0.4-50 ng/mL for DHPPA(r~2=0.9997).The accuracy and precision were determined for LLOQ,low,medium and high QC levels,and all the values(<10%)were within the acceptable criterion,demonstrating that the developed method was accurate and reliable.The mean extraction recoveries were between 87.95%and 94.19%for DHPPA at three concentrations,and the IS-normalized matrix effects were between 102.88%and 108.84%for DHPPA.Furthermore,DHPPA were stable in plasma stored at room temperature for 24 h and-80°C for 1 year,and no obvious reduction was observed after three freeze-thaw cycles.Conclusion:The LC-MS/MS method established in this study was characterized by high sensitivity,accuracy,short running time and simple pretreatment,which is more suitable to be employed in epidemiologic studies.Part Two The Correlation Between Plasma Alkylresorcinol Metabolite and Intake of Cereal Fiber and Reliability of Fasting Plasma Alkylresorcinol Metabolite Concentrations Measured 4 Weeks ApartObjective:To explore the correlation between plasma DHPPA concentrations and intake of cereal fiber,and evaluate the reliability of fasting plasma AR metabolite concentrations measured 4 weeks apart.Method:According to the inclusion and exclusion Criteria,a total of 65 healthy volunteers were recruited in this study.The volunteers were given two dietary surveys conducted 4 weeks apart,and two fasting blood samples were separately collected in the next morning after dietary survey.All the volunteers agreed to maintain their previous dietary habits during the study period.Information of cereal fiber intake was obtained from 3-day food record which was conducted on three consecutive days:Thursday,Friday and Saturday.Basic characteristics were obtained by a standardized questionnaire,and anthropometric measurements were measured by trained project staff.Plasma AR metabolite(DHPPA)was measured by LC-MS/MS as previously described.Results:Two volunteers dropped out in this study,and three other volunteers were deleted because of great difference in daily energy intake(SD>600).Our study at last included 60 volunteers:42 males and 18 females.The mean age and BMI were24.72 years and 21.80 kg/m~2.The mean intake of cereal intake and total fiber was 3.43g/d and 9.50 g/d,respectively.Cereal fiber intake in both the first and the second dietary survey correlated significantly with plasma DHPPA during the same period even after adjustment for sex,age and BMI(First,r=0.432,P<0.001;Second,r=0.413,P<0.001).We further observed that plasma DHPPA was the independent predictor of cereal fiber intake,explaining 17%of the variance(adjusted r~2=0.173;P=0.001).Geometric mean concentrations of DHPPA in the two measurements were 13.44 nmol/L(95%CI=11.52-15.68)and 13.20 nmol/L(95%CI=11.11-15.69),respectively.No significant differences in levels of DHPPA were observed between the two measurements,and the intraclass correlation coefficient(ICC)was 0.486(95%CI=0.142-0.692)for plasma DHPPA.Conclusion:In this study,we observed a significant correlation between cereal fiber intake and plasma DHPPA,and moderate reliability in concentration of plasma DHPPA over time.Part Three Plasma Alkylresorcinol Metabolite and Risk of Type 2 Diabetes and Impaired Glucose RegulationObjective:To examine the association between plasma alkylresorcinol metabolite3-(3,5-dihydroxyphenyl)-1-propanoic acid(DHPPA),a biomarker of whole grain intake,and risk of T2DM and IGR in a Chinese population.Method:According to diagnostic criteria of the world health organization(WHO)in 1999,the study subjects were divided into T2DM patients,IGR patients and controls subjects with normal glucose tolerance(NGT).All cases were consecutively recruited from individuals who,for the first time,received a diagnosis of T2DM in the Department of Endocrinology,Tongji Hospital,Tongji Medical College,Wuhan,from February 2011 to December 2015.Concomitantly,we recruited control subjects from the general population undergoing a routine health checkup in the same hospital.All cases were frequency-matched with controls based on the following criteria:sex,and age(±5 years).Basic information was obtained by a standardized questionnaire or anthropometric measurements.Plasma AR metabolite(DHPPA)was measured by LC-MS/MS as previously described.Result:This study population consisted of 3,239 participants:1,060 newly diagnosed T2DM patients,736 newly diagnosed IGR patients,and 1,443 control subjects with NGT.Plasma DHPPA concentrations were significantly lower in T2DM and IGR patients compared with the control subjects(median:9.06(5.30-15.78)nmol/L,9.66(5.82-15.62)nmol/L,and 10.21(6.56-17.98)nmol/L,respectively,P<0.005).In multivariable logistic regression models,after adjustment for age,sex,BMI,and family history of diabetes,the ORs(95%CI)of T2DM and IGR were 0.57(0.45,0.73)and0.66(0.50,0.85)respectively,comparing the highest with the lowest quartile of plasma DHPPA concentrations.Further adjustment for other potential confounding factors did not affect the above results materially.The inverse association was also observed in all subgroups of participants grouped by age,sex,BMI,current smoking status,current alcohol consumption,physical activity,family history of diabetes,and history of hypertension.Conclusion:In this study,we observed that higher plasma DHPPA concentrations were associated with lower odds of T2DM and IGR.