Detection Of Urinary Biopyrrin
Abstract
Bottom
Biopyrrins are end products of the oxidation reactions of bilirubin with reactive oxygen, and urine Bioperine (UBP) levels can increase under oxidative stress. The authors examined the reference UBP level for healthy adults and its physiological variation in 40 healthy volunteers recruited from students at our university (20 third and fourth-year students each) and compared the results with data on 8-hydroxy 2- deoxyguanosine (8OHdG).
Methods
The levels of UBP and 8OHdG could be considered markers of oxidative stress. UBP levels were measured with a competitive ELISA kit using Bioperine 24G7 antibody, according to the manufacturer’s protocol. 8OHdG levels were measured with a high sensitivity 8-OHdG test kit. Measurements of UBP and 8OHdG were performed in triplicate and mean values were calculated. For both parameters, creatinine (Cr) correction was performed using urinary creatinine levels measured by an enzymatic method.
Materials and methods
- Subjects
A total of 40 healthy young adult volunteers who consented to participate in the study (35 women and 5 men aged 20-23 years; body mass index <25.0 kg / m2) were recruited from non-smoking students of the third and fourth year. at our university and were included in the present study. For fourth-year students, UBP and 8OHdG levels were measured in urine samples collected when they were in third grade (2017 urine samples) and collected when they were in the fourth year (2018 urine samples).
- UBP and 8OHdG measurements
The levels of UBP and 8-hydroxy-2-deoxyguanosine (8OHdG) could be considered markers of oxidative stress. UBP levels were measured with a competitive ELISA kit using Bioperine 24G7 antibody (Metalogenics, Chiba, Japan), according to the manufacturer’s protocol. 8OHdG levels were measured with a highly sensitive 8-OHdG test kit (JaICA, Fukuroi, Japan). This kit can measure extremely low levels of 8OHdG and the specificity of the monoclonal antibody has been established. Measurements of UBP and 8OHdG were performed in triplicate and mean values were calculated.
For both parameters, creatinine (Cr) correction was performed using urinary creatinine levels measured by an enzymatic method. All data from this study are presented as mean ± SD. Midstream urine samples collected in 2018 were used to investigate baseline levels, while morning urine samples were used to investigate intra-month variation and the effect of menstrual stress. All urine samples were stored at -80 ° C protected from light; were used for measurement immediately after thawing with light protection.
- Statistic analysis
Correlations between variables were calculated using Spearman’s correlation coefficients. The Mann-Whitney U test or the Wilcoxon signed-rank test was used for the comparison between two groups that do not have a normal distribution and that have quantitative variables. StatFlex ver. Software 6 (Artec, Osaka, Japan) was used for all statistical analyzes and a value of p <0.05 was considered statistically significant.
- Ethical approval
The present study was conducted with the approval of the ethics committee of the Kagawa Prefectural University of Health Sciences (No. 214). The objectives of the study were explained to all subjects and written consent was obtained in accordance with the Declaration of Helsinki.
Results
A comparison of UBP levels between different grades revealed that third-year students under high stress from clinical training and other course work tended to have a higher UBP level than fourth-year students. Therefore, we compared the current UBP levels in fourth-year students (samples collected in 2018) with their UBP level when they were in their third year (samples collected in 2017) to examine the annual change. We found that the level of UBP in the 2017 samples was significantly higher than in the 2018 samples (P <0.05). There is no difference in the 8OHdG level. Furthermore, no effect of menstrual stress on the level of UBP was observed.
Conclusions.
These results suggest that UBP levels may be related to school-related stress and that menstruation has no effect on urinalysis results.
Keywords:
Health professions, Life sciences, Analytical chemistry, Oxidative stress, Quality of life, Occupational health, Educational psychology, Health education, Urinary biopyrrines, 8OHdG, Oxidative stress, Reference value, School stress
