Age- and Sex-Specific Reference Intervals for Renin and Aldosterone in Healthy Individuals in Yunnan Province, China

doi:10.21203/rs.3.rs-4325558/v1

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Age- and Sex-Specific Reference Intervals for Renin and Aldosterone in Healthy Individuals in Yunnan Province, China

https://doi.org/10.21203/rs.3.rs-4325558/v1

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Background

The Renin-Angiotensin-Aldosterone System (RAAS) is crucial involvement in both diagnostic and therapeutic strategies for hypertension. However, the thresholds for detection vary depending on the characteristics of the examined population. Numerous factors can influence the quantification of the PRC and PAC, including instrumentation, methodology, geographical location, ethnicity, body posture, dietary habits, sex, and age. This study aimed to establish age- and sex-specific reference intervals for renin and aldosterone in Yunnan Province, China.

Methods

A cross-sectional survey was conducted using a comprehensive dataset including age, sex, medical and medication history, family history, anthropometric measurements (height, weight, blood pressure, and heart rate), blood biochemical indices, plasma renin concentrations (PRC) and plasma aldosterone concentrations (PAC). A rigorous statistical analysis was conducted to investigate variations in renin and aldosterone levels by sex and age, facilitating the redefinition of groups. Subsequently, reference intervals for renin and aldosterone were established in the defined groups.

Results

This study involved 5200 ostensibly healthy individuals. Renin and aldosterone levels differed significantly across sex and age groups. Specifically, PRC was significantly lower in women than men, while PAC was significantly higher in men than women. PRC decreased with age, while PAC levels were lowest in the18–24 age group and peaked in the 25–64 age group.

Conclusions

Our findings underscore the crucial role of sex and age when precisely evaluating renin and aldosterone levels. This groundbreaking study established for the first time age- and sex-specific reference intervals for renin and aldosterone in healthy individuals in Yunnan Province, China.

Health sciences/Diseases/Endocrine system and metabolic diseases/Adrenal gland diseases

Health sciences/Health care/Diagnosis

renin

aldosterone

reference intervals

1.RAAS plays a significant role in the diagnosis and treatment of hypertension.

2.Thresholds for detection of renin and aldosterone vary based on population characteristics.

3.PRC and PAC can be influenced by factors such as instrumentation, methodology, geography, ethnicity, body posture, diet, sex, and age.

What this study adds:

1.Methodology: Conducted a cross-sectional survey with a comprehensive dataset to study variations in renin and aldosterone levels by sex and age in Yunnan Province, China.

2.Key Findings: Renin and aldosterone levels differ significantly between sexes and age groups.

3.Reference Intervals: For the first time, established age- and sex-specific reference intervals for renin and aldosterone in healthy individuals in Yunnan Province, China.

4.Implications: Highlights the importance of considering sex and age in accurately assessing renin and aldosterone levels, which can help improve diagnostic and therapeutic strategies for hypertension.

Hypertension is a prevalent chronic condition and a significant risk factor for cardiovascular disease (CVD). There is a continuous, independent, and positive correlation between blood pressure levels and cardiovascular risk [1]. Notably, a significant proportion of patients with uncontrolled blood pressure suffer from secondary hypertension that remains undiagnosed [2]. Primary aldosteronism (PA) is a common cause of secondary hypertension, increasing risks for myocardial infarction, stroke, and arrhythmias [3].

Screening for PA is the starting point for diagnosis. The aldosterone to renin ratio (ARR) is currently the most reliable screening method for PA [4], and laboratories must quantify both aldosterone and renin levels to determine the ARR. In addition, aldosterone has been shown to independently exacerbate target organ damage in patients with essential hypertension, independent of blood pressure levels [5, 6].

In recent years, radioimmunoassay has been gradually replaced by chemiluminescence for detecting renin and aldosterone. The radioimmunoassay is invasive, time-consuming, and environmentally contaminating, while chemiluminescence offers a more environmentally friendly and clinically valuable alternative [7]. Currently, reference intervals for renin and aldosterone in China are primarily derived from manufacturers’ instructions, often provided by authoritative laboratories abroad. Therefore, establishing reference intervals for the regional population is crucial for accurate CVD diagnosis and treatment.

However, there is a dearth of research on renin and aldosterone levels in healthy individuals from different regions of China. Solanki et al. showed that the ARR requires age- and sex-specific reference intervals [8]. Therefore, this study uses the fully automatic chemiluminescence method to quantify the PRC and PAC concentrations, aiming to establish age- and sex-specific reference intervals for renin and aldosterone in healthy individuals in Yunnan Province, China, to provide a basis for diagnosing and treating hypertension and CVD risk prediction.

Participants

Study Population: This study was derived from a cross-sectional survey of the status of CVD and risk factors among permanent residents of Yunnan Province, China. The study participants consisted only of ostensibly healthy permanent residents aged ≥ 18 years. A rigorous multi-stage stratified random sampling method was used to ensure a representative sample, which involved selecting individuals from four distinct districts/counties and both urban and rural settings. After a rigorous investigation, we collected data on 9996 individuals, which constituted the primary dataset of our study. This study was approved by the Ethics Committee of Fu Wai Hospital (ethics approval no. : 2020 − 1360), and all participating subjects signed an informed consent form.

Excludion Criteria: (1) A hypertension diagnosis and currently undergoing treatment with antihypertensive medication or an average of three blood pressure readings with a systolic blood pressure of ≥ 140 mmHg or a diastolic blood pressure of ≥ 90 mmHg. (2) Pregnancy and lactating. (3) A history of acute myocardial infarction, unstable angina, coronary balloon dilatation/stenting, coronary bypass surgery, stroke, valvular heart disease, heart failure, or acute infectious disease within the last two weeks. (4) A blood potassium level of < 3.5 mmol/L. (5) Abnormal renal function: an estimated glomerular filtration rate of < 60 mL//min/1.73 m². (6) Obesity: a body mass index of ≥ 28 kg/m2.

Biochemical and Physiological Measurements

We conducted an extensive data collection process, gathering participants’ basic information such as age, sex, medical history, medication history, and family history through carefully designed questionnaires. We also conducted thorough physiological measurements, including height, weight, blood pressure, and heart rate. Fasting (> 12 hours) venous blood samples were also obtained to assess various biochemical indices, including blood creatinine, blood potassium, PRC (µIU/mL), and PAC (ng/dL). All measuring instruments underwent meticulous calibration to ensure highly accurate data.

Blood pressure was measured using an HBP-1300 sphygmomanometer obtained from OMRON (Dalian) Company. We adhered to a strict protocol, taking three consecutive measurements separated by at least one minute. The acceptability criterion was set such that the difference between any two of the three systolic and diastolic blood pressure measurements should not exceed 10 mmHg. The measurements were repeated for cases where the data did not meet these criteria. The final blood pressure was determined by calculating the average of the three acceptable measurements.

All blood samples intended for biochemical analysis were sent to the Beijing Zhongtong Lambo Medical Laboratory for testing. However, the PRC and PAC were measured at the Laboratory Department of Fuwai Yunnan Hospital using the state-of-the-art Italian Soling Automatic Chemiluminescence Immunoanalyzer.

Statistical Analysis

Statistical analyses were conducted using the SPSS 26.0 statistical software. First, normality tests were performed on the PRC and PAC data, and data that do not follow a normal distribution are presented as median(P25-P75). Two groups (i.e., sex) were compared using the non-parametric Mann–Whitney U rank-sum test. Multiple groups (i.e., age) were compared using the Kruskal–Wallis H rank-sum test. When multiple comparisons were made, the significance level (P-value) of pairwise comparisons was adjusted using the Bonferroni correction method, ensuring rigorous statistical inferences. Groups were defined based on the comparison results, and based on the Clinical and Laboratory Standards Institute’s (CLSI) C28-A3 document [9], the percentile method (P2.5-P97.5) was used to establish reference intervals for non-normally distributed data. A significance level of P < 0.05 was considered statistically significant for all comparisons.

The Normality of PRC and PAC Levels

This cross-sectional study initially included 9996 ostensibly healthy adults. However, 4796 were excluded based on the exclusion criteria, leaving 5200 aged 18–93 years for analysis. PRC and PAC levels had a skewed distribution in healthy individuals in Yunnan Province, China (Fig. 1).

Comparisons of PRC and PAC Levels

Sex-Specific Differences in PRC and PAC Levels

PRC and PAC levels differed significantly by sex among healthy individuals in Yunnan Province, China (all P < 0.05; Table 1).

Table 1

Sex-specific differences in PRC and PAC levels.
Sex	PRC, µIU/mL	PAC, ng/dL
Males (n = 2297)	27.32 (16.15–44.49)	7.72 (5.51–11.10)
Females (n = 2903)	20.50 (11.57–35.18)	10.20 (6.93–15.10)
Z	−12.042	17.136
P	< 0.001^*	< 0.001^*

^* P < 0.05.

Age-Specific Differences in PRC and PAC Levels

PRC levels differed significantly across age groups, including 18–24, 25–34, 35–44, 45–54, 55–64, and ≥ 65 years (all P < 0.05). In contrast, PAC levels did not differ significantly between the 25–34, 35–44, 45–54, and 55–64 age groups (P > 0.05). Nevertheless, PAC levels in the 25–34, 35–44, 45–54 and 55–64 age groups differed significantly from those in the 18–24 and ≥ 65 age group (P < 0.05; Table 2).

Table 2

Age-specific differences in PRC and PAC levels.
Age, years	PRC, µIU/mL	PAC, ng/dL
18–24 (n = 1281)	30.06(18.71, 46.51)	7.64(5.10, 12.30)
25–34 (n = 1283)	27.40(16.51, 43.08) ^∗	9.65(6.56, 14.30)^∗
35–44 (n = 1175)	21.87 (12.38–36.02) ^∗°	9.52 (6.67–14.00)^∗
45–54 (n = 779)	18.18 (9.92–32.67) ^∗°^#	9.30 (6.61–12.60)^∗
55–64 (n = 362)	15.09 (8.38–27.61) ^∗°^#×	9.18 (6.58–13.00)^∗
≥ 65 (n = 320)	13.49 (7.10–23.86) ^∗°^#×∇	8.28 (5.60–12.20) °^#×∇
F	414.365	31.822
P	< 0.001	< 0.001

^∗ P < 0.05 compared to 18–24 years; ^° P < 0.05 compared to 25–34 year; ^# P < 0.05 compared to 35–44 years; ^× P < 0.05 compared to 45–54 years; ^∇ P < 0.05 compared to 55–64 years.

Establishing Reference Intervals for PRC and PAC Levels

Based on the CLSI C28-A3 document and using the percentile method M (P2.5-P97.5), we determined the reference intervals for PRC and PAC in ostensibly healthy individuals in Yunnan Province, China, to be 3.05–102.50 µIU/mL and 2.86–29.70 ng/dL, respectively. These reference intervals differed from those provided by the manufacturers (PRC: 4.4–46.1 µIU/mL; PAC: 3.0–35.3 ng/dL) and were broader. We also further established age- and sex-specific reference intervals for PRC and PAC in healthy individuals in Yunnan Province, China, to provide a more comprehensive understanding of hormone levels in this region.

Reference Intervals for PRC Levels

We observed PRC levels to differ significantly across various age groups (18–24, 25–34, 35–44, 45–54, 55–64, and ≥ 65 years). Therefore, we segmented the population into these five age-specific groups and revealed distinct reference intervals for PRC levels among healthy individuals in Yunnan Province, China, based on age and sex. For males aged 18–24, 25–34, 35–44, 45–54, 55–64, and ≥ 65 years, the reference intervals were 6.51–93.40, 5.52–106.23, 3.97–120.58, 3.37–120.43, 2.70–96.47, and 1.61–136.39 µIU/mL, respectively. For females, the corresponding reference intervals were 4.97–106.90, 3.67–92.55, 2.64–82.55, 1.68–87.23, 1.07–65.94, and 1.31–89.74 µIU/mL, respectively. Notably, the PRC intervals were generally lower in women than in men and decreased with age (Table 3 and Fig. 2).

Table 3

Reference intervals for PRC (µIU/mL).
Age, years	Males				Females
	n	median	P2.5	P97.5	n	median	P2.5	P97.5
18–24	618	32.48	6.51	93.40	663	28.76	4.97	106.90
25–34	587	30.68	5.52	106.23	696	24.36	3.67	92.55
35–44	489	27.74	3.97	120.58	686	18.13	2.64	82.55
45–54	324	23.05	3.37	120.43	455	14.82	1.68	87.23
55–64	146	20.01	2.70	96.47	216	12.78	1.07	65.94
≥ 65	133	14.36	1.61	136.39	187	12.18	1.31	89.74
All	2297	27.32	4.01	109.82	2903	20.5	2.41	90.63

Reference Intervals for PAC Levels: We observed that differences in PAC levels between those aged 25–34, 35–44, 45–54, and 55–64 years were nonsignificant. Therefore, the population was categorized into three age-specific groups: 18–24, 25–64, and ≥ 65 years. The analysis yielded reference intervals for PAC levels in healthy individuals in Yunnan Province, China. For males, the reference intervals were 2.09–19.82, 2.87–23.53, and 2.73–21.15 ng/dL, respectively. For females, the reference intervals were 2.46–37.76, 3.77–35.03, and 3.23–23.66 ng/dL. Our results indicate that PAC levels are higher in females than in males. In addition, PAC levels were lowest in the18–24 age group, whereas peaked in the 25–64 age group and lowest(Table 4 and Fig. 3).

Table 4

Reference intervals for PAC levels (ng/dL).
Age, years	Males				Females
	n	median	P2.5	P97.5	n	median	P2.5	P97.5
18–24	618	7.06	2.09	19.82	663	8.91	2.46	37.76
25–64	1546	8.18	2.87	23.53	2053	10.60	3.77	35.03
≥ 65	133	7.00	2.73	21.15	187	10.20	3.23	23.66
All	2297	7.72	2.41	22.76	2903	10.20	3.36	35.14

Extensive research in the field of medicine has shown that the RAAS plays a pivotal role in maintaining blood volume, electrolyte balance, and systemic vascular resistance, comprising three key components: renin, angiotensin II (AngII), and aldosterone [10]. This system orchestrates a cascade of hormone responses that are vital for homeostatic regulation [11]. Renin, a proteolytic enzyme, initiates this cascade by cleaving its substrate, angiotensinogen, to generate angiotensin I (AngI). Next, AngI is converted to AngII by the angiotensin-converting enzyme, marking a critical step in RAAS activation. Then, AngII stimulates the adrenal cortex to secrete aldosterone, ultimately culminating in blood pressure regulation. However, excessive AngII and aldosterone levels in the circulation and tissues can lead to deleterious effects, including fibrosis, inflammation, and hypertrophy. These pathological processes contribute to tissue remodeling and dysfunction, particularly affecting the cardiovascular and renal systems [12], emphasizing the need for tight RAAS regulation.

PA is the most prevalent form of secondary hypertension. It is distinguished by an overproduction of aldosterone that occurs independent of the usual regulator AngII, accompanied by suppressed renin secretion. The excess of aldosterone has emerged as a substantial contributor to CVD progression, potentially leading to detrimental cardiovascular outcomes such as myocardial fibrosis, left ventricular hypertrophy, congestive heart failure, and cardiac arrhythmias, independent of its effects on blood pressure or volume [13–15]. Fortunately, PA can be effectively managed through targeted aldosterone antagonists such as spironolactone and eplerenone or even adrenalectomy, making it a potentially treatable form of hypertension [13, 16].

The guidelines of the Endocrine Society [4] recommend the ARR as the first test to screen for the presence of PA, and its reliability hinges on the precise quantification of renin and aldosterone levels. However, the thresholds for detection vary depending on the characteristics of the examined population. Numerous factors can influence the quantification of the PRC and PAC, including instrumentation, methodology, geographical location, ethnicity, body posture, dietary habits, sex, and age [17, 18]. Therefore, it is imperative to establish renin and aldosterone reference intervals that accurately reflect the unique characteristics of specific populations for diagnosing and treating hypertension and cardiovascular risk assessment.

In recent years, high-throughput automated chemiluminescence assays have superseded radioimmunoassays in clinical laboratories due to their speed, convenience, and reproducibility. Our study used chemiluminescence to determine the PRC and PAC, allowing us to establish age- and sex-specific reference intervals for PRC and PAC in healthy individuals in Yunnan Province, China, for the first time. These findings could potentially contribute to improved hypertension diagnosis and treatment in this specific population.

Numerous studies have firmly established that sex and age influence the PRC and PAC, emphasizing the importance of determining age- and sex-specific reference intervals [8, 18, 19]. Studies reported that estrogen elevates the angiotensinogen level while simultaneously decreasing the renin and aldosterone levels [20, 21]. However, progesterone competes with aldosterone for the salt corticosteroid receptor and might trigger a compensatory mechanism leading to partial aldosterone elevation. Our study observed that PRC levels are lower in women than in men, while PAC levels are higher in women than in men. These findings are consistent with the above studies. However, our study had certain limitations. Specifically, we did not consider the impact of sex hormone levels, and we did not collect data about the menstrual cycle from female participants. These omissions could potentially introduce bias in the PAC measurements.

Age-dependent declines in RAAS activity have been demonstrated in normal humans in numerous studies involving many factors [22], renin and aldosterone levels decrease significantly with age, with renin decreasing more significantly in individuals aged > 60 years [4]. This age-related decline in renin and aldosterone levels highlights the need for age-specific reference intervals and underscores the importance of considering age as a factor when interpreting PAC and PRC results. Our findings uncovered a tendency for the PRC to decrease with age, while PAC levels were lowest in the18–24 age group and peaked in the 25–64 age group.

The intricate hormonal fluctuations that occur within the body and their impact on aldosterone and renin could potentially explain the observed variations between sexes across diverse age groups. The inconsistent age-related patterns in the PRC and PAC between sexes underscore the complexity and controversy surrounding these relationships. Therefore, further studies are warranted to explore the intricate effects of age and hormonal changes on renin and aldosterone levels, aiming to enhance our understanding of their physiological functions and potential clinical implications.

In conclusion, establishing reference intervals for renin and aldosterone is vital, especially given age- and sex-related disparities. Our study, which followed the CLSI C28-A3 guidelines, analyzed 5200 samples to derive age- and sex-specific reference intervals for renin and aldosterone in healthy individuals in Yunnan Province, China. However, these reference intervals may be specific to Yunnan’s unique geographical and demographic context. Therefore, it is imperative to collect comprehensive data on the populations of other provinces in China and establish their specific reference intervals. Furthermore, it must be noted that different assay methods may lead to different results. Therefore, clinical laboratories must establish their own reference intervals based on the assays they use to ensure the highest level of accuracy and reliability in clinical decision-making.

Competing interests

The authors declare no potential conflicts of interests.

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There is NO conflict of interest to disclose.

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You are reading this latest preprint version

Age- and Sex-Specific Reference Intervals for Renin and Aldosterone in Healthy Individuals in Yunnan Province, China

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Version 1

Abstract

Figures

What is known about the topic

Introduction

Materials and Methods

Participants

Biochemical and Physiological Measurements

Statistical Analysis

Results

The Normality of PRC and PAC Levels

Comparisons of PRC and PAC Levels

Establishing Reference Intervals for PRC and PAC Levels

Discussion

Conclusions

Declarations

References

Additional Declarations

Status:

Version 1