Age- and sex-specific reference intervals for blood urea nitrogen in Chinese general population

Blood urea nitrogen (BUN) is a nitrogenous end product of protein metabolism. This study aims to explore the age- and sex-specific distribution of BUN among healthy Chinese adults. A total of 24,006 BUN values from healthy adults (14,148 males and 9858 females) were included in the cross-sectional study. Males had a higher median BUN value compared to females (4.6 mmol/L vs. 4.1 mmol/L). BUN values showed a positive correlation with body mass index (BMI), cholesterol, and blood sugar (P < 0.0001). However, eGFR showed a negative correlation with the BUN reference value (P < 0.0001) in both sexes. Multiple linear regression analysis confirmed that the positive associations of BUN levels and age were statistically significant after adjusting confounding factors (P < 0.001). Thus, the serum BUN values increased by 0.21 mmol/L for males and 0.282 mmol/L for females per 10 years. The BUN values corresponding to the 1st, 2.5th, 50th, 97.5th, and 99th percentiles for any specific age in both sex were also calculated. These results indicate that the serum BUN reference value is significantly affected by age and gender, and thus, its interpretation is age- and sex-dependent.


Results
Population characteristics. The population under the study consisted of 14,148 males and 9858 females.
The baseline characteristics of the male and female groups are described in Table 1. Males have higher BMI, blood pressure, total cholesterol, uric acid, as well as BUN, and lower estimated glomerular filtration rate (eGFR), compared to females. There was no significant difference in age and glucose between the two groups. Figure 1A illustrates a histogram for the age and gender distribution of the population. The median BUN values  The relation between BUN values and age. As showed in Fig. 2A, the mean BUN level varied and increased steadily with age in males and females populations. In both genders, there were significant differences in BUN levels between different age groups (P < 0.0001). In brief, the estimated mean levels of BUN increased by 1.5 mmol/L from 3.7 mmol/L (95% CI 3.37 mmol/L-4.03 mmol/L) in the youngest female group to 5.2 mmol/L (95% CI 4.94 mmol/L-5.49 mmol/L) in the oldest. Similarly, ANOVA revealed up to 1.06 mmol/L higher estimated mean levels of BUN in the oldest age group in male populations, 4.56 mmol/L (4.31 mmol/L-4.81 mmol/L) compared to the youngest, 5.62 mmol/L (5.38 mmol/L-5.86 mmol/L). The rate of increase in the mean serum BUN value was 0.228 mmol/L/y, and 0.282 mmol/L/y in males and females, respectively. Linear regression analysis was carried out between age and BUN values for males and females, respectively, because there was a significant interaction between BUN values and age in both sex (P < 0.001) (Fig. 2B). Moreover, after adjusted eGFR, the relationship between BUN values and age remain unchanged in both males and females group (Fig. 2C,D). These results indicated BUN values increased stably with age in both sex.

Correlation analysis between BUN values with other clinical variables.
According to the correlation coefficient (r) in both sex groups, BUN values showed positive correlations with body mass index (BMI), www.nature.com/scientificreports/ cholesterol, and blood sugar (P < 0.0001) ( Table 2). However, eGFR showed a negative correlation with the BUN reference values (P < 0.0001). Interestingly, the triglycerides, total protein, and albumin showed significantly negative correlations with BUN values in the male group, while a positive correlation in the female group. However, the relationship between BUN and eGFR did not change with additional adjustment of age and BMI, in both males and females groups. BUN values were not significantly related to blood uric acid (UA) in males groups (P = 0.23) but showed a significant positive correlation in females group (P < 0.0001).

Multiple linear regression analysis of the relationship between BUN values and clinical variables.
Collinearity diagnostics among clinical variables were analyzed to choose the most suitable model. All the VIF values of clinical items were below 10 in both males and females, indicating that no collinearity existed in these clinical variables (Supplementary Table S1). Therefore, a linear model was used in this study. Multiple linear regression analysis confirmed that the positive associations of BUN levels and age were statistically significant after adjusting confounding factors in males (β = 0.21, 95% confidence interval, CI 0.187-0.233, P < 0.001), and females population (β = 0.282, 95% CI 0.255-0.309, P < 0.001). Thus, the increase in the rate of serum BUN values was 0.21 mmol/L and 0.282 mmol/L per 10 years, respectively. These results indicated that BUN levels of both sex groups increased steadily with age, although additionally adjusted for BMI and eGFR, albumin, and triglyceride. Conversely, a negative association between BUN levels and eGFR was observed in males (β = − 0.013, 95% CI − 0.015 to − 0.011, P < 0.001) and females (β = − 0.007, 95% CI − 0.009 to − 0.006, P < 0.001) population (Table 3).
Reference values for BUN in age-and sex-specific groups. The profiles and trends of reference intervals for BUN in different sex-and age-groups are tabulated ( Table 4). The BUN values in the lower 2.

Discussion
In this study, we formulated the first age-and sex-specific reference BUN values for healthy Chinese populations. These results suggest that BUN levels are stable and significantly higher in males compared to females irrespective of age. Moreover, BUN reference values increased with age in the healthy population up to around 70-86 years for both healthy adult males and females. The relationship between BUN and age remained unchanged in both males and females groups, even though adjusted for eGFR and BMI. The concentration of urea nitrogen in blood or serum is related to protein intake, endogenous protein catabolism, hydration state, hepatic urea synthesis, and renal urea excretion. BUN is widely used in daily clinical practice as one of the serum markers to estimate renal function and predict cardiovascular events caused by acute heart failure 12 . Matsue et al. found that BUN increased with age in males and females from the general population (4484 subjects) without cardiovascular comorbidities 12 , which was consistent with our results. Another study showed that 19 young patients had lower mean plasma urea 18 ± 8 mg/dL compared to 15 older subjects (27 ± 7 mg/dL) 13 . However, few studies have reported that normal reference levels vary with age and sex.
Although age-associated glomerular function impairment affects BUN excretion resulting in lower fractional urea excretion with age 13 , this present study excluded population with eGFR < 60 mL/min/1.73 m 2 . Moreover, the relationship between BUN and age remained unchanged in both male and female groups after being adjusted for BMI and eGFR. Thus, suggesting that the age-related increase of BUN was not affected by eGFR but related to decreasing fractional urea excretion with age. Further fundamental research is needed to elaborate on the exact mechanisms involved in the increase of plasma urea with age. The normal range of urea nitrogen in blood or serum is 5 to 20 mg/dL or 1.8 to 7.1 mmol urea per liter 14 . Thus, physicians must not misdiagnose renal dysfunction for observing plasma BUN > 8.0 mmol/L in healthy elderly.
Urea is the primary metabolite derived from dietary protein and tissue protein turnover. A significant interaction was observed between age and sex in BUN. Age-related BUN is higher in females than males, while sex-related differences for protein synthesis can partially explain sex differences in BUN levels. A study showed that females, irrespective of their muscle mass, BMI, and age, have higher fractional synthesis rates of muscle proteins and higher whole-body protein turnover. Protein metabolism does not appear to be related to the change of androgens with age 6,7 . These findings support our results of sex differences in age-related change in BUN.
BUN is a simple clinical variable and useful prognostic information for patients admitted for decompensated heart failure 15 . Fonarow et al. used the acute decompensated heart failure national registry database and identified the BUN level of 43 mg/dL as the best prognostic biomarker discriminator between hospital survivors and non-survivors 16 . The BUN level of every 10 mg/dL above a normal cutoff (17 mg/dL) increases the death risk of patients with heart failure by 21% 17 . Thus, suggesting that even mild elevation in BUN level could affect the Table 4. Blood urea nitrogen values (mmol/L) centiles of the age groups in males and females population. The 50th centile is shown as a point estimate only, as a reference point. www.nature.com/scientificreports/ assessment of heart failure in a population. Our study aimed to present serum BUN reference values to the Chinese population. The results showed an increase in BUN of 0.21 mmol/L per decade in males with a comparable rise in females (0.282 mmol/L per decade). Therefore, we recommend that the age-and sex-adjusted BUN reference range should be considered while diagnosing heart failure diseases in older adults to avoid over-evaluation or misevaluation of poor prognosis from slightly increased BUN values. This study has several significant limitations. Firstly, the study was retrospective and lacked data on dietary protein intake, nutritional status, and muscle mass from males and females of the same age group. Whether these additional factors can affect BUN values were not assessed in both sex. Women's menstrual cycle, oral contraceptives and HRT can regulate female hormone levels, and a recent study has shown that hormone levels can affect BUN levels. We lack relevant data to further analyze the BUN reference interval of women at different stages of the menstrual cycle and exclude the interference of hormone levels 18 . We lack data to further analyze the reference intervals of blood urea nitrogen in women at different stages of the menstrual cycle. Secondly, this is a single-center study, and the datasets primarily consisted of subjects of Han nationality. Thus, the results do not represent the general population and may not apply to other ethnicities or regions. Recently, a study reported that the BUN reference value is significantly affected by geographical environment and location, such as latitude, altitude, annual mean temperature, annual mean relative humidity, and annual precipitation. BUN reference values of healthy Chinese adults are lower in the east and higher in the west 5 . As the elderly over 70 years rarely undergo a routine health check, their number is relatively insufficient, which could be another limitation. Thus, our results on the normal BUN reference range of a population with over 70 years old may not have enough power to evaluate this age group.

Conclusions
In this study, we report that the reference values of serum BUN were age-and sex-related variable, and varied widely in the general population. This study is the first set of comprehensive and robust age-and sex-specific reference intervals determined for BUN in the general Chinese population. Our results clearly showed the importance of age and sex while assessing a patient's condition based on BUN values.