Cluster analysis of angiotensin biomarkers to identify antihypertensive drug treatment in population studies
BMC Medical Research Methodology volume 23, Article number: 131 (2023)
The recent progress in molecular biology generates an increasing interest in investigating molecular biomarkers as markers of response to treatments. The present work is motivated by a study, where the objective was to explore the potential of the molecular biomarkers of renin-angiotensin-aldosterone system (RAAS) to identify the undertaken antihypertensive treatments in the general population. Population-based studies offer an opportunity to assess the effectiveness of treatments in real-world scenarios. However, lack of quality documentation, especially when electronic health record linkage is unavailable, leads to inaccurate reporting and classification bias.
We present a machine learning clustering technique to determine the potential of measured RAAS biomarkers for the identification of undertaken treatments in the general population. The biomarkers were simultaneously determined through a novel mass-spectrometry analysis in 800 participants of the Cooperative Health Research In South Tyrol (CHRIS) study with documented antihypertensive treatments. We assessed the agreement, sensitivity and specificity of the resulting clusters against known treatment types. Through the lasso penalized regression, we identified clinical characteristics associated with the biomarkers, accounting for the effects of cluster and treatment classifications.
We identified three well-separated clusters: cluster 1 (n = 444) preferentially including individuals not receiving RAAS-targeting drugs; cluster 2 (n = 235) identifying angiotensin type 1 receptor blockers (ARB) users (weighted kappa κw = 74%; sensitivity = 73%; specificity = 83%); and cluster 3 (n = 121) well discriminating angiotensin-converting enzyme inhibitors (ACEi) users (κw = 81%; sensitivity = 55%; specificity = 90%). Individuals in clusters 2 and 3 had higher frequency of diabetes as well as higher fasting glucose and BMI levels. Age, sex and kidney function were strong predictors of the RAAS biomarkers independently of the cluster structure.
Unsupervised clustering of angiotensin-based biomarkers is a viable technique to identify individuals on specific antihypertensive treatments, pointing to a potential application of the biomarkers as useful clinical diagnostic tools even outside of a controlled clinical setting.
The recent progress in molecular biology generates an increasing interest in investigating molecular biomarkers as markers of diagnosis, prognosis or response to treatment. For example, the present work is motivated by the molecular epidemiology of hypertension study, where the objective was to explore the potential of the molecular biomarkers of RAAS to identify the undertaken antihypertensive drug (AHD) treatments in the general population. Hypertension is a leading cause of death worldwide  and a primary risk factor for various comorbidities . RAAS targeting AHD treatments are central to the treatment of hypertension, which include angiotensin-converting enzyme inhibitors (ACEi) and angiotensin receptor blockers (ARB), either monotherapy or combined with a diuretic. Combinations of an ACEi or an ARB with a calcium channel blocker or a diuretic is the recommended first-line treatment for hypertension .
Population-based epidemiological studies provide an opportunity to assess AHD effectiveness in real non-clinical contexts. While these studies generally have a much larger scale than clinical controlled studies, they often lack high-quality documentation of the AHD treatment, especially when linkage to electronic health records is not available. In the absence of efficient drug information retrieval systems, treatment self-reporting is imprecise and subject to classification bias [4, 5]. On the other hand, sample biobanking guarantees the possibility to measure extensive sets of molecular biomarkers afterwards and, for example, to reconstruct the most likely AHD treatment a posteriori using specific statistical methods. This was possible thanks to the recent advance in modern techniques such as liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) to simultaneously measure the RAAS biomarkers in biobanked blood samples .
In the present study, we investigated whether unsupervised cluster analysis of biomarkers of the RAAS may help to identify the undertaken AHD treatment. To this end, we measured the three RAAS biomarkers angiotensin I, angiotensin II and aldosterone using LC-MS/MS  in biobanked serum samples from 800 participants from the Cooperative Health Research In South Tyrol (CHRIS) study , where AHD classification was constructed accurately through automatic drug package barcode scanning upon participation.
We evaluated the agreement between estimated clusters and the objective classification obtained via drug box barcode scanning. The resulting clusters were characterized based on available clinical information. Finally, to identify possible reasons of imperfect classification, we performed a lasso penalized regression and assessed which clinical characteristics, among those typically associated with different AHD treatments, were related to each biomarker while accounting for the effects of the clustering and the AHD treatments.
Study design and participants
This study was based on the CHRIS study, a single-center, population-based study designed to investigate the molecular, behavioral and environmental determinants of human health, whose baseline assessment was carried out between 2011 and 2018 [7, 8]. Blood was collected from CHRIS study participants following overnight fasting. After immediate pre-analytical sample processing, sample storage at − 80 °C was performed as described in [7, 8]. Health-related information was collected through either self- or interviewer-administered interviews based on standardized electronic questionnaires. Participants were requested to bring any boxes of medications taken in the preceding week to the study center. Drug information was retrieved via scanning the drug box barcodes, automatically classified according to the Farmadati database (https://www.farmadati.it/), and stored in the CHRIS database.
At the time when the present study was set up, the CHRIS study included N = 6075 participants. Budget limitations allowed to measure the RAAS biomarkers on a random sample of 800 samples. Taking into account that the smallest treatment group included 100 individuals, we sampled 8 age- and sex-matched groups based on the AHD treatment: (1) normotensive; (2) untreated hypertensive; (3) participants taking other drugs not prescribed as AHD (referred to as non-AHD); (4) participants on ACEi monotherapy; (5) participants on ACEi combined with diuretics (ACEi + diuretics); (6) participants on ARB monotherapy; (7) participants on ARB in combination with diuretics (ARB + diuretics); and (8) participants on beta blocker monotherapy treatment (Beta blockers). The diuretic used in the single-pill combinations was always hydrochlorothiazide. Additionally, 5 participants in ACEi + diuretics and 5 in the ARB + diuretics were taking furosemide.
Blood pressure (BP) was measured in supine position with the Omron digital automatic BP Monitor M10-IT at the end of a 20-minute resting electrocardiogram. The mean of three measurements taken at 2 minutes intervals was recorded. Hypertension was defined as: reporting the use of an AHD (ATC codes starting with C02, C03, C04, C07, C08, and C09) or having a diastolic BP (DBP) of ≥ 90 mmHg or a systolic BP (SBP) of ≥ 140 mmHg, according to established guidelines . Diabetes mellitus (DM) was defined as a positive answer to the question “Do you have diabetes mellitus?” or the reporting of glucose-lowering drugs (ATC codes: A10) or by measured levels of glycated haemoglobin (HbA1c) ≥ 6.5% (7.8 mmol/L) or glucose ≥ 126 mg/dl (7 mmol/L) . Estimated glomerular filtration rate (eGFR) was obtained from serum creatinine using the CKD-EPI formula . Serum levels of total cholesterol, cortisol, potassium and sodium were determined as previously described .
Quantification of RAAS biomarkers
Equilibrium Angiotensin I, angiotensin II and aldosterone levels were simultaneously determined using RAAS Triple A testing (Attoquant Diagnostics GmbH, Vienna, Austria) via liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) analysis as previously described . Briefly, equilibration of serum samples was performed at 37 °C for one hour, followed by stabilization through addition of an enzyme inhibitor cocktail. Samples were spiked with stable isotope-labeled internal standards for each analyte, and subjected to C-18-based solid-phase-extraction followed by LC-MS/MS analysis using a reversed-phase analytical column operating in line with a Xevo TQ-S triple quadruple mass spectrometer (Waters). Internal standards were used to correct for peptide recovery of the sample preparation procedure for each analyte in each individual sample.
The biomarkers were quantified from integrated chromatograms considering the corresponding response factors determined in appropriate calibration curves in serum matrix, on condition that integrated signals exceeded a signal-to-noise ratio of 10. The lower limits of quantification for angiotensin I, angiotensin II and aldosterone, defined as the lowest concentrations tested showing a coefficient of variation (CV) < 20% according to FDA criteria, are 5 pg/ml for each of the three biomarkers, corresponding to 3.9, 4.8 and 13.9 pmol/L, respectively. At 50 pmol/L, the inter-assay CVs for the three biomarkers are 10.2%, 6.1%, and 7.9%, respectively, while the corresponding intra-assay CVs are 8.6%, 4.4%, and 5.2%, respectively.
The distributions of angiotensin I, angiotensin II and aldosterone were skewed to the left, hence they were log-transformed to achieve normality (Fig. 1a). First, to determine whether the three RAAS biomarkers could identify participants in different AHD groups, we conducted K-means unsupervised cluster analysis [11, 12] by assigning each observation to one of k groups based on a similarity feature computed from the biomarkers’ covariance matrix. Cluster membership is computed as the sum of the squared distance between data points and the cluster center using the Euclidean distance [13, 14]. We inspected the identified clusters using principal components (PCs), which were obtained as the linear combinations of the normalized three RAAS biomarkers and their corresponding loadings or weights. The k-means method was chosen after comparison with the alternative unsupervised machine learning approaches such as hierarchical and fuzzy clustering . Selection of the best method as well as the optimal number of clusters was based on the Silhouette score , which was evaluated for a number of clusters between 2 and 6. We included a sensitivity analysis to determine whether the obtained optimal number of clusters remain the same if the candidate clusters were increased between 2 and 8.
We assessed the agreement between AHD treatment identified by the clusters and the eight groups using the weighted kappa (κw) inter-agreement coefficient . κw is a modification of the Cohen’s index  to deal with chance agreement between classifiers, and defined based on conditional probability that two classifiers will agree given that disagreement will occur by chance. Computational details are provided in . Sensitivity and specificity were estimated considering the objective AHD classification obtained by the drug box barcode scanning as the gold standard.
Next, we assessed differences of the clinical characteristics among the clusters using one-way analysis of variance (ANOVA) or chi‐squared test where appropriate. If the ANOVA test indicated evidence of significant difference between clusters, we performed pairwise comparisons using Tukey multiple test correction procedure. Finally, we fitted a lasso penalized regression model  to assess whether any clinical predictor could explain the residual variance of each RAAS biomarker, which was not explained by the clusters or the treatment. We fitted a model for each biomarker as the response variable, setting clinical characteristics as fixed-effect predictors and the identified clusters and the treatment group as random-effect terms. The rationale to introducing the identified clusters and treatment groups as random effect was to capture the residual variability not accounted for by the fixed effect predictors.
The lasso penalization was applied to obtain a parsimonious predictive model that should not suffer from the between-predictor pairwise correlation (Supplementary Fig. 1): coefficients are constrained by imposing a penalty to drop the less influential predictors from the model by shrinking their coefficients to zero [19, 20]. The penalty level was tuned by selecting a penalty parameter \(\lambda\) using k-fold cross-validation (CV), with the aim to minimize the mean squared error (MSE). We set k = 8 and the smallest MSE was observed at \(\lambda\)=0.03, 0.02 and 0.01, for angiotensin I, angiotensin II and aldosterone, respectively (Fig. 1b). The statistical significance level was set at 0.05 in all analyses. All analyses were performed with the R software v4.0.5, using the packages stats v4.2.0 and cluster v2.1.2  for cluster analysis and glmnet v4.1.3 and glmmLasso v1.5.1  for penalized regression analysis.
Characteristics of study participants
Clinical characteristics of each group are described in Table 1. Study participants were 43 to 90 years old, and 54% were females. Normotensive participants had the lowest SBP and DBP levels, while the hypertensive group had the highest BP levels. In the treatment groups, mean BP was within the control limits, with exception of ACEi users, whose mean SBP levels were > 140 mmHg. AHD treatment groups showed larger BMI levels compared to normotensive individuals, with maximal BMI levels in the ARB + diuretics group. Additional clinical characteristics are reported in Supplementary Table 1.
Figure 2 illustrates the distributions of the three RAAS biomarkers across AHD groups. Angiotensin I, angiotensin II and aldosterone showed a similar joint profile in all AHD groups that do not include an ACEi or ARB (normotensive, hypertensive, non-AHD, and Beta blockers). In contrast, more elevated angiotensin I and depleted aldosterone levels were observed in the ACEi, ACEi + diuretics, ARB, and ARB + diuretics groups. Angiotensin II was depleted in the ACEi groups, and elevated in the ARB groups.
The Silhouette index analysis identified the unsupervised k-means clustering with three clusters as the optimal clustering solution compared to the alternative hierarchical and fuzzy clustering methods (Fig. 3a). The obtained optimal clustering solution remains the same for increased number of candidate clusters (supplementary Fig. 2). Consequently, the k-means cluster analysis of angiotensin I, angiotensin II, and aldosterone identified three well-separated clusters (Fig. 3b). The three PCs explained, respectively, 62%, 28%, and 10% of the RAAS biomarkers total variability. Cluster 1, 2, and 3, included 55%, 30% and 15% of the study participants, respectively. The three RAAS biomarkers showed substantially different distributions (one-way ANOVA test P < 0.0001) and distinct patterns over the three clusters (Fig. 3c): angiotensin I was lowest in cluster 1, intermediate in cluster 2 and largest in cluster 3, with non-overlapping distributions between clusters 1 and 3. Angiotensin II peaked in cluster 2 and showed lowest levels in cluster 3, with nearly non-overlapping distribution between clusters 2 and 3. Aldosterone was relatively less variable across the clusters, yet the difference between the clusters was statistically significant.
Identification of AHD group by clusters
We evaluated to what extent the three clusters were able to identify different AHD treatment groups. Figure 4 depicts the study participants stratified by the eight AHD groups and by clusters. Cluster 1 comprised individuals from all groups but with strong preponderance of individuals from the normotensive, hypertensive, non-AHD, and beta blockers groups, that is, cluster 1 seems to represent individuals who are not on RAAS-targeting treatment (ATC = C09). Cluster 2 was enriched for individuals on ARB with or without diuretics. Cluster 3 included only individuals on ACEi with or without diuretics.
Results of agreement, sensitivity and specificity along with 95% confidence intervals (CI) from the analysis of the classification properties of the three clusters are shown in Table 2. We observed highest agreement between cluster 3 and ACEi (κw = 82%), with 55% sensitivity and 90% specificity, and between cluster 3 and ACEi + diuretics (κw = 78%, sensitivity = 67%, specificity = 92%). When joining ACEi and ACEi + diuretics groups together, cluster 3 showed κw = 82% (95%CI: 79-86%), 61% (95%CI: 54-68%) sensitivity, and 100% (95%CI: 99-100%) specificity. Cluster 2 had best agreement with ARB (κw = 74%, sensitivity = 73%, specificity = 83%) and ARB + diuretics (κw = 51%, sensitivity = 69%, specificity = 76%). When joining ARB and ARB + diuretics groups together, cluster 2 showed 71% (95%CI: 64-77%) sensitivity and 84% (95%CI: 81-87%) specificity. Cluster 1 showed a sensitivity between 77% and 91% to identify individuals from the normotensive, hypertensive, non-AHD and beta blocker groups (sensitivity = 85% when joining the four groups together). Specificity of cluster 1 to assess that an individual is either normotensive, hypertensive, non-AHD or beta blocker user but not a RAAS AHD user was 74% (95%CI: 69-78%).
Clinical features of the clusters and association with RAAS biomarkers
The clinical characteristics of each cluster are described in Table 3. Cluster 1 was characterized by higher average SBP and DBP than clusters 2 and 3. Individuals in clusters 2 and 3 displayed more cardiometabolic abnormalities. Cluster 2 participants had elevated fasting blood glucose and HbA1c levels. Consistently, this cluster had the largest proportion of individuals with DM. Cluster 3 participants had lower eGFR and higher BMI than the other clusters. Clusters didn’t differ in terms of cholesterol, cortisol, sodium and potassium levels. For BMI, HbA1c, Glucose, eGFR, SBP, DBP showing evidence of difference between clusters from the one-way ANOVA, post-hoc pairwise comparisons were performed adjusting for multiple testing. BMI is higher in participants taking ARB and ACEi enriched groups, compared with cluster 1, and eGFR is lower in participants taking ARB and ACEi enriched groups, compared with cluster 1. SBP is lower in ARB compared with cluster 1. The results were reported in supplementary Table 2.
If, after removing both the cluster and the treatment effects, the clinical characteristics considered above were still associated with angiotensin I, angiotensin II and aldosterone levels, that would indicate the presence of variability not captured by the two classifiers (clusters, treatment) and thus possible reasons for imperfect agreement between them. The lasso model fitting results are shown in Table 4. After removing the effect of the clusters and the treatment, angiotensin I was still associated with age, sex, eGFR, DBP, and DM. Angiotensin II was still associated with age, sex, eGFR, and SBP. Aldosterone was associated with sex, BMI, eGFR, and cortisol.
We investigated to what extent unsupervised cluster analysis applied to measured RAAS biomarkers may help identify individuals from the general population according to the most likely AHD treatment. Our results show that unsupervised clustering can reliably identify individuals on ACEi monotherapy or in combination with diuretics. To a lower extent, clustering can also identify ARB users, with or without diuretics. This is in line with several studies reporting changes in the biomarkers in response to RAAS targeting treatment [6, 23]. Furthermore, normotensive, untreated hypertensive and beta blockers groups were classified in the same cluster 1. This implies that the clustering based on the three biomarkers was able to separate those classified as agents acting on RAAS (ACEi and ARB), from the remaining groups, including beta blockers. Despite beta1-adrenergic receptor blockade suppresses renin release directly acting on juxtaglomerular cells , an impact of beta blockers on the analyzed biomarkers was not evident in our study. One explanation could be that the prescribed beta blockers were still not titrated to the optimal dosage, therefore not leading to a sufficient inhibition of renin release. However, renin secretion is a complex phenomenon and is regulated by multiple factors not limited to the sympathetic nervous system. Cluster 3, representing ACEi users, showed nearly perfect specificity, meaning that individuals who are not on ACEi would be very unlikely to be falsely classified as ACEi user. On the other hand, cluster 3 exhibited limited sensitivity, with nearly four out of ten ACEi users being missed by this classifier. Cluster 2, mainly representing ARB users, showed higher sensitivity, with seven out of ten ARB users that would be correctly identified by this classifier, but imperfect specificity, allowing some non-ARB user to enter this group.
Individuals in clusters 2 and 3 exhibited more cardiometabolic issues as compared to cluster 1. Clusters 2, encompassing ARB users, included a higher rate of individuals with DM and, consistently, individuals in this cluster aveh higher blood glucose and HbA1c levels. Individuals in cluster 3, encompassing ACEi users, had lower levels of eGFR and higher BMI. This is consistent with current clinical protocols, which prioritize assignment of ACEi and ARB for individuals with metabolic syndrome like diabetic hypertensive  and kidney disease  patients. The identified clusters were not different in terms of cortisol, potassium and sodium levels. The absence of an association with potassium, whose level can be depleted by thiazide diuretics reflects the inability of cluster analysis to discriminate those on diuretic treatment among those taking ACEi or ARB.
The penalized regression analysis of residual variability after removing the effect of the identified clusters and treatment groups, highlighted residual strong associations of sex with all three RAAS biomarkers, and age with angiotensin I and angiotensin II. Also higher eGFR, which indicates better kidney function, was associated with lower levels of all three RAAS biomarkers. A lower SBP was associated with higher angiotensin II levels, according to expectations since a drop in BP triggers RAAS to increase BP through increased release of angiotensin . The detection of these associations after removal of the treatment effect and of the cluster effect, indicates the presence of additional factors acting on angiotensin I, angiotensin II and aldosterone levels that might explain the imperfect agreement between clusters obtained through unsupervised statistical analysis and objective AHD classification up on participation. In particular, there is a known differential prescription of AHD by sex . However, given we adjusted the analyses for drug groups and groups were sex-matched, it is more likely that the residual association with sex is of purely biological origin.
The main feature of our study was the analysis of RAAS biomarkers typically analysed only in clinical context in a population-based scenario. This was possible thanks to a novel quantification method that allows RAAS biomarker quantification in frozen samples, thus allowing use in general population studies conducted outside of controlled clinical settings. On the other hand, limitations should be highlighted. This study relied on cross-sectional measurement which might not be reflective of the actual health status of an individual over an extended period, especially for what concerns BP. Imperfect discrimination by cluster analysis could be explained by heterogeneous counter-regulatory renin release mechanisms and by ACEi/ARB escape phenomenon, the latter dependent on individual drug response . Classification based on barcode scanning of drug boxes provides great precision but our study could not take into account adherence to treatment since drug levels were not measured. This unaccounted variability may have additionally contributed to the imperfect agreement between clusters and drug groups as noncompliance is known to affect sensitivity and specificity of treatment screening . While we identified a promising unsupervised procedure to identify underlying AHD treatment targeting the RAAS system, our limited sample size has prevented us an independent replication and calibration of the clustering algorithm in an independent setting is warranted. We considered enhancing the potential of the clustering by including biomarkers other than the RAAS, such as glucose, cholesterol, SBP and DBP. But the addition of these biomarkers did not improve drug identification compared to the identification achieved by using RAAS biomarkers only. Finally, our study was lacking by design the measure of urinary sodium excretion, representing dietary sodium intake.
In conclusion, our study has demonstrated that the unsupervised clustering of angiotensin-based biomarkers in previously biobanked samples is a viable technique to identify individuals on specific antihypertensive treatments from the general population, pointing to the potential application of the biomarkers as useful clinical diagnostic tools even outside of a controlled clinical setting.
Cooperative Health Research In South Tyrol
Angiotensin type 1 receptor blockers
Angiotensin-converting enzyme inhibitors
diastolic blood pressure
systolic blood pressure
estimated glomerular filtration rate
coefficient of variation
analysis of variance
mean squared error
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Authors thank CHRIS study participants from the middle and upper Vinschgau/Val Venosta, the general practitioners, the personnel of the Hospital of Schlanders/Silandro, the field study team. The CHRIS biobank was assigned the “Bioresource Research Impact Factor” (BRIF) code BRIF6107. The authors thank the Department of Innovation, Research and University of the Autonomous Province of Bozen/Bolzano for covering the Open Access publication costs.
The CHRIS study was funded by the Department of Innovation, Research and University of the Autonomous Province of Bolzano-South Tyrol and supported by the European Regional Development Fund (FESR1157). The present research was conducted within the project “Molecular profiling of uncontrolled and treatment resistant hypertension in the general population: the HyperProfile study” funded by Department of Innovation, Research and University of the Autonomous Province of Bolzano, grant “Legge Provinciale 14/2006”, unique project code D52F19000130003.
Ethics approval and consent to participate
The CHRIS study was approved by the Ethical Committee of the Healthcare System of the Autonomous Province of Bozen/Bolzano, protocol no. 21/2011 (19 Apr 2011). All participants gave written informed consent. All methods were carried out in accordance with relevant guidelines and regulations.
Consent for publication
Marko Poglitsch is the CEO of Attoquant diagnostics that performed the biomarker measurements.
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Arisido, M.W., Foco, L., Shoemaker, R. et al. Cluster analysis of angiotensin biomarkers to identify antihypertensive drug treatment in population studies. BMC Med Res Methodol 23, 131 (2023). https://doi.org/10.1186/s12874-023-01930-8
- Antihypertensive drugs
- Cluster analysis
- Lasso regression
- CHRIS study