Exploring Serum Bile Acid Biomarkers To Predict Gallstones With Excessive Heat Syndrome Based On Systematic Review And Metabolomics

1.BackgroundAs one of the most common hepatobiliary diseases,the incidence of gallstone disease is increasing annually,seriously affecting the quality of life of patients and consuming healthcare resources.Current treatment options,whether surgical or conservative,are limited.Based on an understanding of the mechanisms of stone formation,exploring early warning signs before stone formation and interrupting stone formation in a timely manner are undoubtedly the best strategies to intervene for this disease;however,there is not enough research to support this idea.Traditional Chinese Medicine(TCM)has a rich experience in the treatment of gallstone disease.Gallstone disease belongs to the TCM category of "hypochondriac pain" and"jaundice",which consider that the essential pathogenies are endogenous dampness,endogenous heat,qi stagnation,blood stasis,phlegm retention,and spleen asthenia.The common syndromes are excessive syndromes and heat syndromes.Gallstone disease can be effectively intervened at an early stage through the concept of TCM syndrome differentiation and treatment variation.However,the difference of recognition between different medical theories greatly limits the clinical application and promotion of Chinese medicine resources in the prevention of gallstone disease.Therefore,the precise identification of biomarkers in the early stages,based on differentiating the symptoms,not only facilitates the early detection of the disease,in compliance with the medical strategy of "early prevention",but also lays the necessary foundation for establishing TCM prevention and treatment plan.Bile acids are a group of similarly structured small molecules;as an important component of bile,they have been used in the treatment of gallstone disease since the early days.Modern research has revealed that bile acids,as a group of small molecules,have an even more important and rich physiological role in neuroendocrine regulation and are particularly important in the formation of gallstones,where small changes in the bile acid pool can have a significant impact on stone formation.Therefore,disorders in the bile acid profile play a key role in the formation of gallstones and can be a suitable candidate for early biomarkers of gallstone disease.Overall changes in bile acid profile can more accurately characterize disease states than single bile acid.Metabolomics can identify specific changes in metabolite profile with high sensitivity,enabling analysis of overall characteristic changes in bile acid profile and is a preferred strategy for early identification of biomarkers of pre-disease.2.Objectives2.1 Systematic review and meta-analysis were used to comprehensively evaluate the specific change characteristics of bile acids between different samples in the process of gallstone disease,and to explore bile acid biomarkers with predictive potential for gallstone disease.2.2 Based on metabolomics of serum bile acid profile in patients with gallstones with excessive heat syndrome,cross-section study and longitudinal study were combined to find out the specific characteristics of serum bile acid changes in patients with excessive heat syndrome at different periods,locate the warning signals of bile acid profile 6-12 months before the onset of the disease,and provide metabolic indications and time nodes for early intervention in the formation of gallstones.Lay the foundation for the research and development of early prevention and treatment drugs.3.Methods3.1 Systematic review and meta-analysis:Our study was completed under the guidance of the PRISMA 2020 statement.Eight electronic databases were searched including PubMed,the Cochrane Library,EMBASE,Web of Science,China Biology Medicine Disc,China National Knowledge Infrastructure,Wanfang databases and VIP Information Resource Integration Service Platform with key words and variants for "Gallstones" and "Metabolomics".Two authors independently screened eligibility papers which detected the bile acid profiles in various biological samples for both case group and control group by metabolomics.The risk of bias and methodological quality of the included studies were measured by the Newcastle-Ottawa Scale.The qualitative and quantitative reviews were performed to summarize the changes of bile acid profiles in the patients with gallstones compared with healthy subjects.Meta-analysis was performed by R software.3.2 Detection of serum bile acid profile in different stages of excessive heat syndrome gallstones and exploration of disease warning signals:Twenty patients with new onset excessive heat syndrome gallstone disease were included and their clinical information and serum samples were collected 6-12 months before and 12 months after the onset of the disease.Serum samples were collected from 30 patients with a one-year or more gallstone history and 50 healthy individuals matched according to age,sex,height,and weight.Gas chromatography time-of-flight mass spectrometer was applied to detect and analyze all serum samples,combined with multivariate statistical analysis such as principal component analysis and partial least squares,to locate characteristic changes in the bile acid profiles at different periods of the disease and to identify early warning signs.4.Results4.1 Systematic review and meta-analysis:A total of 2112 studies were identified from eight Chinese and English databases and 30 studies were included which contained 2313 participants and reported 39 bile acids or their ratios.Qualitative review showed that blood samples were the most used for detection which serum TCA,GCDCA,GCA,TCDCA,GDCA and DCA were increased;UDCA was decreased in gallstones patients.In bile samples,TCA,GCDC A,GDCA,DCA and TDC A raised obviously and GCA declined in control group.Fecal samples were seldom used for bile acid profiles determination,the existing studies only indicated that fecal TBA was elevated in patients with gallstone.Meta analysis was performed in 16 studies and showed that serum TBA(MD=1.36μmol/L,95%CI=0.33;2.4)was elevated however bile TBA(MD=-36.96mmol/L,95%CI=-52.32;-21.6)was declined in patient with gallstones.GCA(MD=0.83μmol/L,95%CI=0.06;1.6)and TCA(MD=0.51μmol/L,95%CI=0.18;0.85)were both increased in serum sample;TCDCA(MD=2.64mmol/L,95%CI=0.16;5.12)and the ratio of CA and CDCA(MD=0.25,95%CI=0.15;0.36)was rising,however GCDCA(MD=-13.82mmol/L,95%CI=-21.86;-5.78)was falling in bile sample of gallstone patients compared to healthy populations.Statistical differences were only detected for 3 species of bile acids,serum GCDCA(MD=0.87mmol/L,95%CI=0.23;1.520),bile GCA(MD=-4.66mmol/L,95%CI=-8.68;-0.64)and bile GDCA(MD=-4.18mmol/L,95%CI=-8.29;-0.08),after removing the high-risk studies.Subgroup analysis was feasible for 4 serum bile acids in 7 studies based on metabolomics technology.It demonstrated completely opposite results in DCA by using the two different technologies.In addition,GCDCA and TCDCA were rising with low heterogeneity yet TCA was growing slightly with high heterogeneity in patients with gallstones.4.2 Detection of serum bile acid profile in different stages of excessive heat syndrome gallstones and exploration of disease warning signals:Serum samples were collected in 120 cases,including 20 cases in the NGD,20 cases in the NGD-BL,30 cases in the RGD and 50 cases in the HC.There was no statistical difference between the 4 groups in terms of sex,age,total cholesterol,low density cholesterol,high density lipoprotein,aspartate aminotransferase,creatinine,blood uric acid,alpha-fetoprotein,carcinoembryonic antigen,triglycerides,alanine aminotransferase,body weight,and gallbladder polyps(P>0.05).In total,38 bile acids were identified and quantified,while 32 correlated bile acids total concentrations or concentration ratios were introduced into the analytical model together as variables,such as conjugated bile acids(the total concentration of GCA,GCDCA,GDCA,GLCA,GHDCA,GUDCA,GDHCA,G-λ-MCA,TCA,TCDCA,TDCA,TLCA,THDCA,TUDCA,T-α-MCA,T-λ-MCA and T-Ω-MCA),primary bile acids(the total concentration of CA,CDCA,GCA,GCDCA,TCA and TCDCA),primary conjugated bile acids(the total concentration of GCA,GCDCA,TCA and TCDCA),glycine bile acids(the total concentration of GCA,GCDCA,GDCA,GLCA,GHDCA,GUDCA,GDHCA and G-λ-MCA),CA Species(the total concentration of CA,GCA and TCA),CDCA Species(the total concentration of CDCA,GCDCA and TCDCA),UDCA Species(the total concentration of UDCA,GUDCA and TUDCA),GUDCA/TUDCA(the ratio of GUDCA and TUDCA).Series of comparisons for 70 bile acid variables between groups were performed and revealed significant changes in bile acid profiles across the periods of gallstone disease.Serum TBA was significantly different among the four groups(P=0.012),showing a trend of increasing first and then decreasing,which presented that the TBA in HC was the lowest,and TBA in NGD-BL was the highest.It further proved that changes in bile acid profile already began to be present in the 6-12 months prior to the development of gallstone disease.The results showed that:i 15 differential bile acids between the HC and NGD-BL,of which the up-regulated bile acids in the NGD-BL were GCA,GCDCA,GUDCA,THDCA,Ω-MCA,conjugated bile acids,primary bile acids,primary conjugated bile acids,glycocholic acid,CA species,CDCA species,GUDCA/TUDCA,and the down-regulated bile acids were 12-KCDCA,GLCA and GHDCA/THDCA;ii there were 9 differential bile acids between NGD-BL and NGD,with TLCA and 12-KCDCA being upregulated in the NGD and GCA,GCDCA,DCA,TDCA,GUDCA,primary bile acids,and primary conjugated bile acids being downregulated;iii there were 3 differential bile acids between the HC and NGD,of which TCA was upregulated and GHDCA and GHDCA/THDCA were downregulated in the NGD;iv 13 differential bile acids between the HC and RGD,CA,TCA,TCDCA,HDCA,α-MCA acid,Ω-MCA and GUDCA/TUDCA were upregulated in the RGD group and MCA,7,12-DKLCA,12-KCDCA,GLCA,TUDCA and CDCA/CA were downregulated;v there were 10 differential bile acids between NGD and RGD,with LCA,LCA/(GLCA+TLCA)and GHDCA/THDCA being upregulated,while CDCA/CA,CDCA/LCA,TLCA,UDCA,TUDCA,UDCA Species and UDCA/(GUDCA+TUDCA)being downregulated.Based on the above changes in the bile acid profile it was further found that a panel of 4 bile acids,containing GCA,GCDCA,GUDCA,and 12-KCDCA,was identified as an early potential warning sign for the development of gallstone disease.5.ConclusionOur study provided a systematic summary on the bile acid profiles in different samples of gallstone disease by metabolomics.The results demonstrated serum TBA was significantly increased,specifically TCA,GCDCA,GCA,TCDCA,GDCA and DCA were increased,while UDCA was decreased;Bile TBA decreased,specifically TCA,GCDCA,GDCA,DCA,and TDCA decreased,but GCA,TCDCA increased;Fecal TBA was elevated.Therefore,serum GCA and TCA showed potential to be the predictive biomarkers for gallstones.The bile acid profiles in different periods of excessive heat syndrome gallstone disease were detected based on metabolomics,which revealed significant changes in bile acid profiles across the periods of gallstone disease.Serum TBA showed a trend of increasing first and then decreasing,showing that the TBA in HC was the lowest,and TBA in NGD-BL was the highest,which further proved that changes in bile acid profile already began to be present in the 6-12 months prior to the development of gallstone disease.A panel of 4 bile acids,including GCA,GCDCA,GUDCA,and 12-KCDCA,was identified as an early potential warning sign that gallstone disease will occur in healthy individuals in 6-12 months.The results support the findings of the systematic review and meta-analysis.More significantly,compared with the single bile acid biomarker obtained by systematic review,the panel of bile acids biomarkers can better characterize the disease state and play a more accurate warning role.This study provided a metabolic indication and time point basis for early intervention in gallstone formation,laying the foundation for the development of early prevention and treatment drugs.