Biopyrrin Test Service Pack

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Biopyrrines are end products of bilirubin oxidation reactions with reactive oxygen, and urinary Bioperine (UBP) levels may increase under oxidative stress. The authors examined the reference UBP level for healthy adults and its physiological variation in 40 healthy volunteers recruited from our university students (20 juniors and seniors) and compared the results with data on 8-hydroxy-2-deoxyguanosine. (8OHdG).

Methods

UBP and 8OHdG levels could be considered as markers of oxidative stress. UBP levels were measured with a competitive ELISA kit using the 24G7 biopyrrin antibody, according to the manufacturer’s protocol. 8OHdG levels were measured with a highly sensitive 8-OHdG verification kit. UBP and 8OHdG measurements 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.

• Subjects

A total of 40 healthy young adult volunteers who gave their consent to participate in the study (35 women and 5 men aged 20 to 23 years; body mass index <25.0 kg/m2) were recruited among third and fourth-year students non-smokers. 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 their third year (2017 urine samples) and collected when they were in their fourth year (2018 urine samples).

• UBP and 8OHdG measurements

UBP and 8-hydroxy-2-deoxyguanosine (8OHdG) levels could be considered as markers of oxidative stress. UBP levels were measured with a competitive ELISA kit using the 24G7 biopyrrin antibody (Metallogenics, Chiba, Japan), according to the manufacturer’s protocol. 8OHdG levels were measured with a highly sensitive 8-OHdG verification kit (JaICA, Fukuroi, Japan).

This kit can measure extremely low levels of 8OHdG and the specificity of the monoclonal antibody has been established. UBP and 8OHdG measurements 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, while morning urine samples were used to investigate intra-monthly variation and the effect of menstrual stress. All urine samples were stored at -80 °C protected from light; they were used for measurement immediately after thawing with protection from light.

• 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 are not normally distributed and have quantitative variables. StatFlex ver. 6 (Artec, Osaka, Japan) was used for all statistical analyses, and a P-value of <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 courses tended to have a higher UBP level than fourth-year students. Therefore, we compared current UBP levels in fourth-year students (samples collected in 2018) with their UBP level when they were in third grade (samples collected in 2017) to examine yearly 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 level of 8OHdG. 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, Biological sciences, Analytical chemistry, Oxidative stress, Quality of life, Occupational health, Educational psychology, Health education, Urinary biopyrrines, 8OHdG, Oxidative stress, Reference value, School stress