| Researchs on healthy issues caused by environmental cadmium polluted havebeen always a hot spot among public health in recent decades. Many researchs haveshowed that cadmium is one of the most accumulable toxins in the body, chroniccadmium exposure was associated with kidney injury, skeleton damage,cardiovascular disease, etc, positively. Cadmium exposure is a hazardous factor formultiple lesions, leading to increased mortality, especially in women, and renal wouldbe the earliest attacked organ.Internationally, the level of urinary cadmium was used as the internal index toindicate cadmium exposure, and effect of biomarkers such as urinary β2-MG andNAG were widely served as indicators to reflect early renal damage, played animportant role in clinical diagnosis and treatment for cadmium poisoning populations.As we know little about physiological metabolism which caused by cadmium, aswell as the injury mechanism. The present study intends to use the metabonomicsmethods to explore the typical metabolites which associated with environmentalcadmium exposure in long-term, to seek evidence of damage casused byenvironmental cadmium exposure from the perspective of metabolic pathways, and toprovide clues for searching biomarkers related to environmental cadmium exposure.ObjectiveTo explore the significantly changed metabolites in urine from those whoexposed to cadmium, environmentally, in long-term, and wish to provide clues forsearching biomarkers related to environmental cadmium exposure. Methods1. Epidemiological study1.1RespondentsA mining region with high environmental cadmium was chosen as the exposedarea, and an area with no certain history of heavy metal pollution was adopted as thecontrol area, where the economy level and the living habits of villagers were similarto the exposed area. The inhabitants whose residence time of local was more than15years in both areas, and all of them eat the locally produced rice and vegetables as themain food, who aged from45to70were adopted in the present study. The exclusioncriteria of the study were those who had an occupational exposure histoty of cadmium,smoking habit, liver disease, kidney disease and chronic metabolic disease, etc.1.2Collection, transportation and storage of samplesCollection, transportation and storage of rices and vegetablesA rice and a leafy vegetable produced by each respondent were collected,numbered and recorded; All the fresh food samples was sent to the laboratory as quickas possible, and stored at-4℃until analysis.Collection, transportation and storage of urine samplesThe morning urine from each patricipant was gathered to send to the laboratorywith a cold-chain transpotation, and made into several tubes for relevant analysis;Urinary cadmium, creatinine, β2-MG and NAG testing should be done in8hours aftersample collection; The samples for HPLC-Q/TOF testing were preserved under-80℃refrigerator.1.3Indices and methods for sample testingIndices of external exposure from environmental cadmiumThe content of cadmium in rices and vegetables were used as the external indicesof cadmimu exposure, and the ICP-MS was employed for detecting.Index of internal exposure from envrionmental cadmiumThe content of urinary cadmiu was used as the internal index of cadmiumexposure, and the ICP-MS was employed for detecting. Kidney injury biomarkersThe indices of urinary creatinine, NAG and β2-MG were used as the kidneyinjury biomarkers associated with cadmium exposure. The urinary creatinine wastested by use of Jaffe’s assay, the urinary NAG and β2-MG were detected by use ofimmunoturbidimetry.2. Urinary metabonomics analysis2.1The platform for metabonomics chemical analysisPretreatmentAfter thawing at room temperature, the urine samples had a2min vortex, and100μL were added in internal standard solution, centrifuged for10min aftersufficient mixing, and the supernatant fluid was centrifuged a second time beforeanalysis.Chromatographic conditionsC18reversed phase chromatographic column was used, the column temperaturewas kept at50℃, the injection volume was5μL; Mobile phase of A: pure water+0.1%formic acid, mobile phase of B: methanol+0.1%formic acid, the flow rate was0.4mL/min, and use gradient elution program.MS conditionsESI source, the voltage of capillary was3500V, the temperature of drying gaswas350℃, the flow rate was10.0L/min, the atomization pressure was30psi, and themass scan range was50to1000.2.2The platform for metabonomics data processingSPSS13.0was used for rank sum test and drawing ROC curve, etc; MPP12.1was employed to recognize the chromatograms and pretreat them; SIMCA-P13.0wasapplied to fit PCA and OPLS-DA models, and screen the micromolecular metaboliteswith larger VIP values.3. Quality ControlBefore the investigation, all investigators were trained, and all the respondentswere selected accordance with the inclusion criteria and exclusive criteria, strictly; Urine, rice and vegetable samples were transported with cold-chain; Thedetermination of each index was borne by the professional, and make sure of thestability of instrument before sample detection, the order of each vial was randomizedwith bland samples and quality control samples, alternately, and the RSD of qualitycontrol should be less than10%.Results1. Epidemiological data1.1Demographic informationAccording to the objective and exclusion criteria, the present study includes60women, and there were30individuals in both exposed group and control group, theage of them in each group were55.3±6.99years old and59.7±7.52years old.1.2External exposure levels30rices and vegetables from each group were determined, compare with controlgroup, the content of cadmium in rice and vegetables from exposed group were higher,and the differences were statistically significant.(P <0.001)1.3Internal exposure levelsThe median content of urinary cadmium in exposed group and control groupwere1.95and8.33, respectively, the content of urinary cadmium in exposed groupwas higher than control group, and the difference was statistically significant.(P <0.001)1.4The levels of biomarkers related to kidney injuryThe median concentration of urinary creatinine in exposed group and controlgroup were85.25mg/dL and84.38mg/dL, respectively, the difference has nostatistically significant (P>0.05); The median concentration of urinary NAG inexposed group and control group were6.18U/g.Cr and5.50U/g.Cr, respectively, thedifference has no statistically significant (P>0.05); And the median concentration ofurinary β2-MG in exposed group and control group were0.07mg/g.Cr and0.04mg/g.Cr, respectively, the difference has no statistically significant (P>0.05). The results indicate that compare with the control group, we haven’t found any renaldamage in the individuals from cadmium exposed area as yet.2. Metabonomics analysis2.1Stability and precision of the detection systemThe RSD of abundance and retention time of internal standard substance were5.13%and0.06%in ESI+, and were2.00%and0.06%in ESI-. The results indicatedthat the stability and precision were sufficient for metabonomics analysis.2.2Pattern recognitionBy using HPLC-Q/TOF,2351valid metabolites were detected in urine from bothgroups; after non-parametric test, compare with control group, there were139micromolecular metabolites had changed in exposed group (P <0.05); the139metabolites were applied to conduct pattern recognition to fit PCA and OPLS-DAmodels, the models showed different urine metabolic profiles from two group, theestablished OPLS-DA model can explain96.7%of the urinary total variation from thetwo groups, and the accuracy for discriminating unknown samples were95.3%, whichmeans a good result for the established model. And the results illustrated there mustbe some micromolecular metabolites in the urine from exposed group showed adifferent characteristic compared with the control group.2.3Analysis of changed metabolites caused by cadmium exposure9micromolecular metabolites which were significantly different in both groupswere screened by use of VIP and z value, all the9metabolites were tend to rise incadmium exposed group, the AUC value of them were ranged from0.658to0.714,showed that we could well distinguish environmental cadmium exposed group fromcontrol group by observing these changed metabolites. They would provide someclues for further searching for characteristic metabolites which were associated withenvironmental cadmium exposure. The9significantly changed metabolites could beclassified as species of aromatic compounds, nucleoside compounds, lipid compoundsand amino acids. Conclusions1.9micromolecular metabolites were identified by using HPLC-Q/TOF, and all thesesignificantly changed metabolites were tend to rise in cadmium exposed groupcompared with control group. These metabolites are mainly involved in amino acidmetabolism, lipid metabolism and nucleotide metabolism;2. By comparing with the classical biomarker of early renal injury caused byenvironmental cadmium exposure, metabonomics analysis tend to find the subtlechanges and physiological/biochemical process earlier, the result may provide a cluefor exploring cadmium damage mechanism and finding relevant biomarkers. |
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