Association of Alkaline Phosphatase with Cardiovascular Disease in Patients with Dyslipidemia: A 6-Year Retrospective Study

  1. Petros Spyridonas Adamidis
  2. Matilda Florentin
  3. Evangelos Liberopoulos
  4. Amalia Despoina Koutsogianni
  5. Georgia Anastasiou
  6. George Liamis
  7. Haralampos Milionis
  8. Fotios Barkas

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Abstract

Background and Aim: Serum alkaline phosphatase (ALP) activity has been associated with atherosclerotic cardiovascular disease (ASCVD). We aimed to investigate the association of ALP with ASCVD in patients with dyslipidemia. Methods: We conducted a retrospective cohort study including consecutive adults with dyslipidemia followed-up for ≥3 years (from 1999 to 2022) in the outpatient Lipid Clinic of Ioannina University General Hospital, Greece. The primary endpoint was the association between baseline ALP and incident ASCVD after adjusting for traditional risk factors (i.e., sex, age, hypertension, diabetes, smoking, and dyslipidemia), baseline ASCVD, and lipid-lowering treatment. ALP levels were stratified by tertiles as follows: low: <67 U/L, middle: 67–79 U/L, high: ≥79 U/L. Results: Overall, 1178 subjects were included; 44% were males, and their median age was 57 years (range: 49–65). During a 6-year median follow-up (interquartile range: IQR: 4–9), 78 new ASCVD events (6.6%) occurred. A statistically significant association between baseline ALP levels and incident ASCVD was demonstrated (Odds Ratio, OR: 6.99; 95% Confidence Interval, CI: 2.29–21.03, p = 0.001). Subjects in the highest ALP tertile had the highest odds for ASCVD when compared with those in the lowest tertile (OR: 2.35; 95% CI: 1.24–4.41, p = 0.008). Conclusions: The present study indicates an association between ALP and the development of ASCVD in patients with dyslipidemia, which underscores the potential of ALP as a predictive tool or a therapeutic target in the realm of ASCVD prevention within this population.

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    RESEARCH QUESTIONS

    This study attempts to answer the following research questions:

    • Is there an association between baseline serum alkaline phosphatase (ALP) levels and the development of atherosclerotic cardiovascular disease (ASCVD) in patients suffering from dyslipidemia?

    • How does the risk of developing ASCVD vary across different tertiles of baseline ALP levels after adjusting for traditional risk factors (such as sex, age, hypertension, diabetes, smoking status, dyslipidemia), baseline ASCVD, and lipid-lowering treatment?

    • Can baseline ALP levels serve as a predictive biomarker for the development of ASCVD in patients with dyslipidemia?

    • Is there any potential for ALP to serve as a therapeutic target and reduce or prevent the development of ASCVD in patients with dyslipidemia receiving lipid-lowering treatment?

    RESEARCH MAIN GOAL AND ITS IMPORTANCE

    The main goal of this study is to examine whether baseline serum alkaline phosphatase (ALP) levels can be used as a predictive biomarker in the development of atherosclerotic cardiovascular disease (ASCVD), while adjusting for traditional risk factors in patients with dyslipidemia, and to explore the potential of ALP as a therapeutic target. This is important because, despite recent advances in lipid-lowering treatments using drugs like statins, many patients still face a significant risk of cardiovascular complications with high rate of mortality. Identifying new biomarkers such as ALP levels, could help in better risk management of the disease in patients. Also, understanding the connection between ALP levels and ASCVD could provide more insights into other risk factors of cardiovascular diseases beyond the traditional risk factors. This may lead to improved predictions and prevention strategies for this disease. If ALP levels is shown to be a significant factor in the pathogenesis of ASCVD, then it could open up new avenues, aimed at reducing the levels of ALP, in order to mitigate cardiovascular risks, as suggested by recent studies involving ALP-lowering treatments (1,2).

    RESEARCH MAIN APPROACH AND WHAT THE AUTHORS DID TO ADDRESS RESEARCH QUESTIONS

    The authors conducted a retrospective cohort analysis of 1178 Caucasian patients of Hellenic origin diagnosed with dyslipidemia. They were enrolled from the Outpatient Lipid Clinic of the University Hospital of Loannina, Greece, with a minimum follow-up duration of 3 years (1999-2002). Ethical approval and informed consent were obtained for all participants.

    The authors collected from patients detailed cardiovascular histories, medication profiles, demographic and clinical characteristics, with specific emphasis on the risk factors of cardiovascular diseases such as sex, age, smoking status, diabetes, hypertension and dyslipidemia. Blood pressure was measured following European Society of Hypertension (ESH) / European Society of Cardiology (ESC) guidelines. Fasting Plasma Glucose (FPG) levels and a comprehensive lipid profile (Total Cholesterol TC, Triglycerides TG, High-density Lipoprotein Cholesterol HDL-C, and Low-density Lipoprotein Cholesterol LDL-C) were analysed, with LDL-C calculated using the Friedewald formula for TG levels below 400 mg/DL.

    Other laboratory tests included complete blood count, creatinine, urea, electrolytes, liver enzymes, creatine kinase (CK), alkaline phosphatase (ALP), thyroid function, and urine analysis. Renal function was estimated using eGFR calculated with the CKD-EPI formula. Cardiovascular and metabolic conditions were defined based on established guidelines for ASCVD, diabetes, hypertension, impaired fasting glucose, and chronic kidney disease (CKD).

    Concomitant lipid-lowering treatments such as statins, ezetimibe, PCSK9 inhibitors, fibrates, colesevelam, and omega-3 fatty acids, with the intensity of statin therapy classified as "high", "moderate", or "low", based on expected LDL-C lowering effects. ALP levels were categorised into tertiles (low: <67 U/L, middle: 67-79 U/L, high: >79 U/L).

    Statistical analyses involved normality testing using the Kolmogorov-Smirnof test and logarithmic transformations where necessary. Parametric data were presented as mean ± standard deviation (SD), non-parametric data as mean inter quartile range (IQR), and categorical values as frequency counts and percentages. Paired sample t-test examined changes in numerical variables, and Pearson's and Spearman's correlation co-explored the relationship between ALP changes and other variables. Multivariate logistic regression analyzed the impact of ALP on ASCVD development, adjusting for confounders, and reported associations as odds ratios (OR) and 95% confidence intervals (CI). Significance level was set at P<0.05, and SPSS v21.0 was used for all analyses.

    RESEARCH MAIN FINDINGS

    According to the results presented in Table 3 by the authors, baseline alkaline phosphatase (ALP) levels were significantly associated with higher incidence of atherosclerotic cardiovascular disease (ASCVD) in the fully adjusted model 3 that adjusted for several risk factors of ASCVD, including lipid profile test, etc. Subjects in the high ALP tertiles have a significantly higher risk of developing ASCVD than those in low or middle ALP tertiles. Also, ALP levels as a continuous variable were significantly associated with the occurrence of ASCVD in both crude model 1 (no adjustment) and model 2 (adjusted for sex and age). In addition, in Table 2, it was recorded that significantly higher mean values of calcium and direct bilirubin (P<0.05) were found in the middle and low tertiles of ALP, respectively, to demonstrate some laboratory differences among the ALP tertiles, while Table 1 revealed significant differences in ALP tertiles among participant groups especially smoking status (P<0.001), lipid-lowering therapy (P<0.05), and alkaline phosphatase levels (P<0.001)

    WHAT IS MOST INTERESTING ABOUT THE RESEARCH

    What I find most interesting about this research is the strong association between baseline ALP levels and the incidence of ASCVD in patients with dyslipidemia. This association continued to be significant even after adjusting the models for various cardiovascular risk factors. The authors' findings are relevant in medicine and suggest that ALP could potentially serve as a therapeutic target or an independent predictive biomarker of ASCVD.

    RELATIONSHIP OF THE MANUSCRIPT TO PUBLISHED LITERATURE AND FUTURE RESEARCH

    This study relates to published literature by confirming the connection between elevated alkaline phosphatase (ALP) levels and increased risks of developing atherosclerotic cardiovascular disease (ASCVD) in patients with dyslipidemia (3-6). Findings from this study are also consistent with research involving older men and general population cohorts that identified ALP as a prognostic marker and therapeutic target for coronary heart disease and cardiovascular mortality (7,8). Another research was referenced in this study that explored the potential of reducing ALP levels through the use of pharmacological agents, such apabetalone, which has shown therapeutic promise in lowering major adverse cardiovascular events (MACEs) (9,10).

    Future research can focus their attention on understanding the precise biological mechanisms that link elevated ALP levels to ASCVD. This may include issues raised in this study such as exploring how ALP contributes to vascular calcification, inflammation and endothelial dysfunction. In addition, based on this study's reference to apabetalone (9, 25), future research could also involve clinical trials to test the efficacy of ALP-lowering treatments in reducing cardiovascular complications. Therapies could explore the use of existing drugs or new ones to target ALP. Future research may build on the current findings of this study, to further shed light on the role of ALP in cardiovascular diseases, and this could lead to improved prevention, diagnosis and treatment opportunities for patients.

    RESEARCH MAIN STRENGTH AND WEAKNESS

    A major strength of this study is the use of robust analytical models (Models 1, 2, 3) and comprehensive statistical analyses (Pearson's and Spearman's correlation coefficients, Multivariate logistic regression, and SPSS). This thorough data collection and analyses would enhance the accuracy of the authors' findings.

    The main weakness of this research is its retrospective cohort design. This retrospective nature limits the study's ability to establish a direct cause-and-effect relationship between elevated ALP levels and ASCVD. Although their association can be identified, nevertheless, it could not be firmly established. Due to the challenges encountered by the authors in data collection, there might have been potential biases in their selection and information gathering which will impact their findings.

    MAJOR ISSUES

    • The topic of this research mentioned a 6-year retrospective study, while the abstract mentioned 3 and 6. Can the authors clarify? Also the year specified (1999-2022) is not correct.

    Experimental design

    • No information was provided regarding the methodology, principles, and references involved in the analyses of various biochemical and laboratory parameters analysed in this study. For example, how was the lipid profile test conducted? What methods, principles and references were adopted? How do I reproduce this study?

    Table 1

    • The data of female participants is not indicated in the table, despite the fact that there are strong evidences suggesting females generally have a higher risk of cardiovascular diseases than males. Including this data may have shed more light on these findings.

    • Can the authors provide more explanations relevant to the interpretation of data presented in the tables? For example, I would like to know how they determined the number of follow-up years and the median of ALP tertiles, etc.

    • The significant difference at P<0.001 of ALP levels among ALP tertiles was not discussed.

    • No significant difference between low-density lipoprotein-cholesterol (LDL-C) and different tertiles of ALP seems a little bit odd. Since elevated levels of LDL-C are a major contributor to the development of ASCVD, and are associated with increased risk of inflammation and activities of ALP, one would expect a direct association between LDL-C levels and ALP activities. Whereas the results of this study show a significant difference in ALP levels at different tertiles of ALP at P<0.001, so this is conflicting.

    • If the follow-up year for total patient samples is 6 years, and since it's a 6-year retrospective study, then why is there a variation as large as 8 years in the follow-up *years* per ALP tertiles? Shouldn't the follow-up year be more evenly distributed? I need more clarification.

    Table 2

    • The assertion in Table 2 that "Laboratory variables also demonstrated differences with higher mean values of calcium and direct bilirubin found at the middle and lower tertiles of ALP, respectively (p<0.05)," appears to be untrue by mere looking at the data presented in the table. There's no significant difference (p<0.05) in the mean values of calcium and direct bilirubin at any ALP tertile, therefore there's no major laboratory differences among them. Besides, the correlation of these two variables (calcium, bilirubin) with ALP levels and incidence of ASCVD was not explained throughout this study.

    Table 3

    • Can the authors explain the meaning of "ALP was logarithmically transformed" under Table 3?

    • What is the contextual meaning of ALP as a continuous variable?

    • What's the value of "reference" for low ALP tertiles across all models in Table 3?

    MINOR ISSUES

    • In abstract, the 3 year cohort study specified should have been 1999-2002, not 1999-2022.

    • The percentage of female participants, their age, and patients placed on statin treatment at the beginning of this experiment was not stated.

    • What is the meaning of baseline characteristics in the context of this study?

    • Table 1: Cardiovascular disease not "cardiovascular diseaser"

    • "n-3 fatty acid" should be omega-3 polyunsaturated fatty acids (n-3 PUFAs).

    RESEARCH LIMITATIONS

    • This study exclusively consists of patients of Hellenic origin. Genetic and ethnic differences can affect both ALP levels and cardiovascular risk, this can limit the applicability of these findings when compared to other studies of heterogeneous ethnicity.

    • The study population is primarily composed of dyslipidemic patients. This may reduce the generalisability of the findings compared to more diverse demographic groups.

    • Lack of comprehensive data on inflammatory markers like C-reactive protein (CRP) also limits the understanding of the role of inflammation in the observed association between elevated ALP levels and ASCVD in dyslipidemic patients.

    • This study did not account for lifestyle factors such as dietary habits, and physical activities. These are recognised risk factors of ASCVD.

    • This study did not also perform detailed analysis of ALP isozymes due to lack of electrophoresis data, thus leading to an incomplete understanding of their roles or contributions to ASCVD.

    RECOMMENDATION

    I would recommend this interesting manuscript for publication and for others to read, provided that the issues raised are addressed.

    REFERENCES

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    2. A. Kizu, A. Shioi, S. Jono, H. Koyama, Y. Okuno, and Y. Nishizawa, "Statins inhibit in vitro calcification of human vascular smooth muscle cells induced by inflammatory mediators," Journal of Cellular Biochemistry, vol. 93, no. 5. pp. 1011–1019, 2004, doi: 10.1002/jcb.20207.

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    6. A. J. G. Hanley, K. Williams, A. Festa, L. E. Wagenknecht, R. B. D'Agostino, and S. M. Haffner, "Liver markers and development of the metabolic syndrome: the insulin resistance atherosclerosis study," Diabetes, vol. 54, no. 11, pp. 3140–3147, Nov. 2005, doi: 10.2337/DIABETES.54.11.3140.

    7. S. G. Wannamethee, N. Sattar, O. Papcosta, L. Lennon, and P. H. Whincup, "Alkaline phosphatase, serum phosphate, and incident cardiovascular disease and total mortality in older men," Arterioscler. Thromb. Vasc. Biol., vol. 33, no. 5, pp. 1070–1076, May 2013, doi: 10.1161/ATVBAHA.112.300826.

    8. M. Haarhaus, V. Brandenburg, K. Kalantar-Zadeh, P. Stenvinkel, and P. Magnusson, "Alkaline phosphatase: A novel treatment target for cardiovascular disease in CKD," Nat. Rev. Nephrol., vol. 13, no. 7, pp. 429–442, Jul. 2017, doi: 10.1038/nrneph.2017.60.

    9. M. Haarhaus et al., "Apabetalone lowers serum alkaline phosphatase and improves cardiovascular risk in patients with cardiovascular disease," Atherosclerosis, vol. 290, no. September, pp. 59–65, 2019, doi: 10.1016/j.atherosclerosis.2019.09.002.

    10. S. Picaud et al., "RVX-208, an inhibitor of BET transcriptional regulators with selectivity for the second bromodomain," Proc. Natl. Acad. Sci. U. S. A., vol. 110, no. 49, pp. 19754–19759, Dec. 2013, doi:10.1073/PNAS.1310658110/SUPPL_FILE/PNAS.201310658SI.PDF.

    Competing interests

    The author declares that they have no competing interests.

  2. Version published to 10.3390/jcdd11020060
  3. Version published to 10.20944/preprints202401.0093.v1