Associations Of Fluorescent Oxidation Products(FlOPs)with Osteoporosis,bone Microstructure And Bone Turnover Markers

Osteoporosis(OP)is a common bone metabolic disease characterized by decreased bone strength and increased risk of fracture;it has become a global public health problem.Oxidative stress is defined as an imbalance between excessive reactive oxygen species(ROS)and the endogenous antioxidative defense in the body,in favor of a pro-oxidative status,which can cause oxidative damage to cells.Oxidative stress can disrupt the bone remodeling balance by promoting bone resorption and inhibiting bone formation,which in turn leads to the development of osteoporosis.Among traditional oxidative stress markers,malondialdehyde(MDA),8-hydroxy-2’-deoxyguanosine(8-OHd G),pentosidine(PTD),and nitrotyrosine(NT)reflect a single aspect of oxidative damage from lipids,DNA,sugars,and proteins,respectively.However,their assays suffer from large coefficients of variation and low sensitivity and specificity.Fluorescent Oxidation Products(Fl OPs),a global biomarker of oxidative stress,comprehensively reflect the oxidative damage of lipids,proteins,DNA,and carbohydrates.The existing methods for Fl OP measurement have low reliability,sensitivity and specificity,and laboratory factors that influence the stability of Fl OP assay are poorly understood.Fl OPs have been widely used in epidemiological studies of chronic diseases in the last two decades.Fl OPs were closely associated with oxidative stress-related diseases,such as cardiovascular disease,kidney disease,and cancer.The associations of Fl OPs with osteoporosis,bone microstructure,and bone turnover markers are still unclear.Objective:(1)To improve the reliability of existing methods for Fl OP measurement using fluorescent microplate technology and to explore their sensitivity,specificity,and stability in laboratory assays.(2)To clarify the association between Fl OPs and osteoporosis through population-based studies.(3)To explore the associations of Fl OPs with bone microstructure and bone turnover markers using an animal model of oxidative stress.Methods:1.Studies on the reliability,sensitivity,and specificity of Fl OP assay(1)Human study:Peripheral blood was collected from 16 healthy volunteers and8 patients with cardiovascular disease.Using fluorescent microplate technology,we detected plasma Fl OP levels at three wavelengths(including Fl OP＿320:excitation/emission wavelengths of 320/420 nm,Fl OP＿360:excitation/emission wavelengths of 360/420 nm,and Fl OP＿400:excitation/emission wavelengths of400/475 nm),because Fl OPs detected at different wavelengths reflect different oxidation products.We examined the coefficients of variation(CV)of the Fl OP assay at different plasma/extractant ratios to determine the optimal plasma/extractant ratio.A series of experiments were performed to determine whether the Fl OP assay was influenced by laboratory factors such as hemolysis,transportation,sample storage,and freeze-thawing.(2)Animal study:We established an animal model of D-galactose-induced oxidative stress,and measured the blood levels of C-reactive protein(CRP)and traditional markers of oxidative stress(i.e.,MDA,8-OHd G,PTD,and NT).We examined the sensitivity of Fl OPs in the measurement of global oxidative damage.We also tested the relationship between Fl OPs and inflammatory factors(CRP)to determine whether Fl OPs are specific indicators of oxidative stress.2.Studies on the association between Fl OPs and osteoporosisWe conducted three human studies to examine the association between Fl OPs and osteoporosis,as follows:(1)Using a cross-sectional study design,subjects were recruited from the Department of Medical Examination,a tertiary hospital in Jilin Province in 2019.Hip bone mineral density(BMD)was measured using dual-energy X-ray absorptiometry(DXA)instrument.The association between Fl OPs and hip BMD was analyzed using multivariable linear regression models.To compare whether Fl OPs are more sensitive than traditional biomarkers of oxidative stress for measuring bone health,we also examined the association between MDA and BMD.(2)We conducted a cross-sectional study.The calcaneal BMD of participants was measured using an ultrasound bone densitometer in middle-aged and older adults in the community.The relationship between Fl OPs and ultrasound BMD was tested by multivariable linear regression models.The speed of sound(SOS)and broadband ultrasound attenuation(BUA)are two important parameters that reflect ultrasound BMD.(3)We conducted a 1:2 age,sex,hospital,and specimen-matched case-control study.Participants were recruited from two tertiary-level hospitals in Jilin Province.All osteoporosis cases and controls were determined by BMD T-scores detected by DXA.We used conditional logistic regression models to estimate the odds ratios(OR)and 95%confidence intervals(CIs)for the associations between Fl OPs and osteoporosis3.Associations of Fl OPs with bone microstructure and bone turnover markersEight-week-old male Wistar rats were selected and randomly divided into D-galactose group and control group(injected with 0.9%saline),with 8 animals in each group.Blood was collected from the tail vein on days 0,30,and 60 of the experiment.On day 90,the animals were anesthetized and sacrificed,and femur and blood samples were collected.Plasma levels of Fl OPs,NT,MDA,8-OHd G,PTD,and serum levels of bone turnover markers[N-terminal lengthening peptide of type 1collagen(P1NP)and C-terminal telopeptide of type I collagen(CTX)]were measured.The femurs of rats were scanned using micro-CT to analyze the bone microstructure.Student’s t-test was used to compare the differences in trabecular bone parameters between D-galactose group and control group at 90 days.We used the two-way repeated measures ANOVA to assess whether there were trend differences between groups and within-groups for Fl OPs,NT,MDA,8-OHd G,PTD,P1NP,and CTX over time.Results:1.The reliability,sensitivity,and specificity of Fl OP assay(1)Human studyThe coefficient of variation of the Fl OPs assay was lowest when the plasma/extractant ratio was 1:20.The inter-batch CV of Fl OPs assay in the healthy population group was<3.6%and the intra-batch CV was<2.7%;the inter-batch CV of Fl OPs assay in the coronary heart disease group was<4.4%and the intra-batch CV was<2.0%.(2)Animal studyThe results of animal experiments showed that the correlation coefficients between Fl OP＿320 and Fl OP＿360 and D-galactose dose at 30 days were 0.816 and0.801,respectively,and these correlation coefficients were 3-12 times higher than those of MDA(r=0.183),8-OHd G(r=0.157),PTD(r=0.254)and NT(r=0.068).There was a significant relationship between Fl OP＿360 and CRP(r=0.225,P=0.045),whereas neither Fl OP＿320 nor Fl OP＿400 had a statistically significant correlation with CRP.Short-term and severe hemolysis affected Fl OP values,whereas short-term specimen delivery time<12 h,storage at-80°C for one year,and freeze-thawing≤5times did not significantly affect Fl OP values.2.The association between Fl OPs and osteoporosis(1)Association analysis between Fl OPs and hip BMDA total of 164 males were enrolled in the study,their average age was 56.6±2.0years.After adjusting for confounders,for each standard deviation increase in Fl OP＿320,femoral neck and total hip BMD decreased by 0.014 g/cm~2and 0.016g/cm~2,respectively.For each standard deviation increase in Fl OP＿360 and Fl OP＿400,total hip BMD decreased by 0.017g/cm~2and 0.019 g/cm~2,respectively.There was no significant association of MDA with femoral neck or total hip BMD(all P>0.05).(2)Association analysis between Fl OPs and ultrasound BMDA total of 491 study participants were included,with an average age of 65.2±9.7years.Among them,290(59.1%)were females.After adjusting for confounders,Fl OP＿320 was negatively associated with SOS(βfor per 1-SD increase=-2.562;P=0.018),and the associations of Fl OP＿360 and Fl OP＿400 with SOS were not statistically significant.No statistically significant association was observed between Fl OPs and BUA.(3)Association analysis between Fl OPs and osteoporosisWe identified 87 osteoporosis cases and 174 controls.After adjusting for confounders,higher levels of Fl OP＿320 were associated with increased odds of osteoporosis(OR for per 1 SD increase=1.49,95%confidence interval[CI]:1.01-2.20).Significant results were noted for serum Fl OP＿360(OR for per 1 SD increase=1.59,95%CI:1.07-2.37).We did not observe a significant association between Fl OP＿400 and osteoporosis.(3).Associations of Fl OPs with bone microstructure and bone turnover markersD-galactose treated rats,as compared with control rats,showed higher levels of Fl OP＿320 and MDA,and lower P1NP levels at 90 days(all P<0.05).Micro-CT scans showed that the treatment group had lower trabecular bone volume fraction(BV/TV)(0.07±0.03 vs.0.13±0.05;P=0.008)and volumetric BMD(225.4±13.8 vs.279.1±33.2 mg HA/cm~3;P=0.001)than the control group.The microstructure of the bone trabeculae was also damaged.Conclusions:1.Fl OP＿320,Fl OP＿360,and Fl OP＿400 measured by fluorescent microtiter plate technology have high reliability and stability;Fl OP＿320 have higher sensitivity and specificity in assessing oxidative stress status in vivo.2.Fl OPs,but not MDA,were negatively associated with total hip BMD,and Fl OP＿320 was negatively associated with femoral neck BMD and SOS.These results suggest that Fl OPs may be more sensitive than MDA in assessing the association between oxidative stress and bone health.3.Fl OP＿320 and Fl OP＿360 levels of osteoporosis.4.Higher levels of Fl OP＿320 were associated with bone microstructural disruption and decreased bone formation.