The effect of astaxanthin supplementation on inflammatory markers, lipid profile, and anthropometric indices in patients with heart failure: a randomized controlled trial.

The study initially enrolled 80 participants, with 10 withdrawals during the study period (5 from each group), as shown in Fig.  1 . Two participants in the intervention group reported gastrointestinal adverse effects, including nausea and vomiting. All data were analyzed according to the ITT principle. Fig. 1 Flow diagram of study. Flow diagram of study. Comprehensive data on participants’ baseline characteristics, dietary intake, and physical activity have been reported in our previously published study 21 . To avoid redundancy, only summarized data are presented in the current manuscript, while detailed datasets are provided in the supplementary materials. As summarized in Table  1 , baseline characteristics were comparable between the intervention and control groups, except for smoking frequency ( p  = 0.045). The full dataset is provided in Supplementary Table S1 . Table 1 Comparison of the demographic and basic clinical characteristics of the two groups at the beginning of the study. Variable Group P a ASX (n = 40) Placebo (n = 40) Age (year) 61.82 ± 11.75 60.20 ± 12.81 0.556 BMI (kg/m 2 ) 25.72 ± 4.42 26.37 ± 4.82 0.535 Waist circumference (cm) 100.14 ± 15.45 102.60 ± 15.10 0.472 Gender (male) 30 (75) 27 (67.5) 0.459 Family history (yes) 28 (70) 29 (72.5) 0.805 Smoking (yes) 11 (27.5) 4 (10) 0.045 Medications  Blood thinners 25 (62.5) 29 (72.5) 0.340  Beta-blocker 16 (40) 20 (50) 0.369  Diuretic 27 (67.5) 27 (67.5) > 0.99  Calcium channel blocker 2 (5) 1 (2.5) 0.556  Angiotensin II blocker 22 (55) 21 (52.5) 0.823  ACE inhibitor 2 (5) 0 (0) 0.152  Diabetes drugs 22 (55) 27 (67.5) 0.251  Lipid-reducing drugs 31 (77.5) 26 (65) 0.217  Antiarrhythmic 3 (7.5) 5 (12.5) 0.456  Levothyroxine 4 (10) 8 (20) 0.210 Data are expressed as mean ± SD for quantitative variables and numbers (%) for qualitative variables. a Performed by an independent samples t -test for quantitative variables and the Chi-square test for qualitative variables. P  < 0.05 was considered statistically significant. BMI: Body mass index; ACE: Angiotensin-converting enzyme. Comparison of the demographic and basic clinical characteristics of the two groups at the beginning of the study. Data are expressed as mean ± SD for quantitative variables and numbers (%) for qualitative variables. a Performed by an independent samples t -test for quantitative variables and the Chi-square test for qualitative variables. P  < 0.05 was considered statistically significant. BMI: Body mass index; ACE: Angiotensin-converting enzyme. As shown in Table  2 , dietary intake data indicated a statistically significant difference only in vitamin A intake between the groups, while no other macronutrient or micronutrient differences were observed. The complete dietary profile is available in Supplementary Table S2. Table 2 Dietary intakes of the participants. Nutrients Group P a ASX (n = 40) Control (n = 40) Energy (kcal/day) 0.182  Baseline 1830.84 ± 468.32 2000.13 ± 655.94  Midpoint 1705.74 ± 384.88 1922.17 ± 509.04  End 1810.35 ± 417.78 1863.21 ± 486.43 Carbohydrate (g/day) 0.155  Baseline 253.99 ± 66.37 280.76 ± 92.46  Midpoint 243.46 ± 55.77 275.85 ± 80.31  End 262.64 ± 65.65 272.40 ± 75.40 Protein (g/day) 0.398  Baseline 75.92 ± 23.76 79.54 ± 26.82  Midpoint 64.60 ± 17.99 70.33 ± 18.75  End 67.08 ± 18.03 68.85 ± 17.54 Fat (g/day) 0.394  Baseline 61.48 ± 27.02 67.08 ± 26.66  Midpoint 58.18 ± 24.62 65.44 ± 22.69  End 60.98 ± 23.15 61.58 ± 22.44 Cholesterol (mg/day) 0.060  Baseline 261.94 ± 108.63 295.63 ± 138.23  Midpoint 230.19 ± 94.44 269.06 ± 114.22  End 190.66 ± 78.62 226.92 ± 102.98 SFA (g/day) 0.137  Baseline 17.37 ± 6.33 19.24 ± 7.02  Midpoint 15.96 ± 5.57 17.79 ± 5.87  End 15.61 ± 4.28 17.54 ± 7.11 MUFA (g/day) 0.093  Baseline 18.78 ± 6.74 21.39 ± 9.28  Midpoint 17.09 ± 5.15 20.65 ± 6.48  End 18.61 ± 5.29 18.72 ± 6.72 PUFA (g/day) 0.961  Baseline 18.91 ± 17.68 18.87 ± 13.74 14.26 (4.60–86.99) 14.51 (5.52–71.71)  Midpoint 18.37 ± 16.85 19.61 ± 12.61 14.76 (5.49–102.22) 17.77 (4.57–76.65)  End 19.42 ± 16.07 17.74 ± 12.99 14.34 (5.64–86.25) 15.31 (3.85–86.02) Total dietary fiber (g/day) 0.409  Baseline 16.36 ± 7.10 17.65 ± 6.84  Midpoint 17.17 ± 5.06 19.48 ± 5.84  End 22.93 ± 8.11 22.18 ± 6.48 Vitamin A (RAE) 0.023  Baseline 825.12 ± 426.62 1070.98 ± 683.83 650.75 (320.75–1892.04) 909.22 (120.81–2976.12)  Midpoint 746.70 ± 312.10 892.80 ± 423.99 720.06 (274.99–1305.98) 791.77 (324.49–1923.69)  End 745.38 ± 387.52 889.13 ± 559.32 671.18 (199.31–1766.53) 756.70 (177.03–2712.85) Vitamin E (mg/day) 0.851  Baseline 4.18 ± 3.77 6.02 ± 5.62 2.63 (0.81–16.73) 4.19 (0.63–23.93)  Midpoint 6.17 ± 4.62 5.69 ± 4.65 4.24 (0.87–16.48) 4.01 (1.12–22.65)  End 6.83 ± 6.18 5.97 ± 3.76 5.70 (1.94–37.79) 4.66 (1.12–17.49) Vitamin C (mg/day) 0.363  Baseline 97.71 ± 41.65 109.85 ± 67.07  Midpoint 104.05 ± 50.58 102.97 ± 48.42  End 110.61 ± 51.48 121.10 ± 46.09 Selenium (mg/day) 0.813  Baseline 64.83 ± 55.40 60.19 ± 60.33 44.13 (3.37–240.75) 41.02 (3.59–277.00)  Midpoint 49.42 ± 38.28 46.45 ± 34.48 37.00 (5.52–165.60) 39.18 (3.66–151.55)  End 45.37 ± 39.73 48.31 ± 34.44 33.99 (2.66–170.74) 52.93 (2.98–102.67) Zinc (mg/day) 0.752  Baseline 11.08 ± 6.17 11.34 ± 4.93 8.49 (5.44–32.32)  Midpoint 9.32 ± 4.45 10.03 ± 3.47 8.44 (5.24–32.20)  End 9.92 ± 4.68 9.88 ± 3.96 8.56 (5.53–26.69) 9.16 (4.08–26.72) Data are presented as mean ± standard deviation for normally distributed variables and median (first quartile-third quartile) for non-normally distributed variables. a P value obtained from a repeated measures ANOVA test. SFA: Saturated fatty acids; MUFA: Monounsaturated fatty acids; PUFA: Polyunsaturated fatty acids; RAE: Retinol activity equivalent. Dietary intakes of the participants. Data are presented as mean ± standard deviation for normally distributed variables and median (first quartile-third quartile) for non-normally distributed variables. a P value obtained from a repeated measures ANOVA test. SFA: Saturated fatty acids; MUFA: Monounsaturated fatty acids; PUFA: Polyunsaturated fatty acids; RAE: Retinol activity equivalent. As shown in Supplementary Table S3, no significant differences were observed between groups in physical activity levels at baseline, mid-intervention, or study end ( p  = 0.646). Table  3 presents the effects of astaxanthin supplementation on inflammatory biomarkers. The mean TNF-α level significantly decreased in both groups relative to baseline. However, the between-group comparison of mean changes indicated a significantly greater reduction in the intervention group ( p  = 0.017), which remained significant after adjusting for potential confounders ( p  = 0.013). The mean hs-CRP also decreased significantly within both groups by the end of the study. However, the between-group difference in hs-CRP changes was not statistically significant ( p  = 0.250). After controlling for confounding variables, no statistically significant difference was observed between groups at the end of the 8-week intervention ( p  = 0.132). Although the mean MCP-1 level decreased in the intervention group, this reduction was not statistically significant. In contrast, the control group showed a significant increase in MCP-1 from baseline to the end of the study ( p  < 0.001). A significant between-group difference in mean changes of MCP-1 was observed ( p  = 0.001), which remained significant after adjusting for confounders ( p  = 0.004). Table 3 Comparison of inflammatory markers before and after intervention within and between groups. Variable Group P b P c Observed power* ASX (n = 40) Placebo (n = 40) TNF-α (pg/ml) > 0.99  Baseline 10.63 ± 2.98 9.98 ± 2.82  End 7.44 ± 4.10 8.73 ± 3.71  Mean changes − 3.19 ± 0.58 − 1.26 ± 0.46 0.017 0.013   P a < 0.001 0.009 hs-CRP (mg/L) –  Baseline 12.92 ± 21.87 15.64 ± 28.85 4.45 (4.00–8.97) 7.05 (4.05–12.12)  End 6.19 ± 17.32 6.91 ± 11.87 2.70 (1.42–4.27) 3.79 (2.52–5.00)  Mean changes − 6.72 ± 2.08 − 8.73 ± 3.08 0.250 0.132   P a < 0.001  0.99  Baseline 20.90 ± 11.57 16.70 ± 7.96 18.55 (12.02–24.90) 14.25 (11.20–20.45)  End 18.43 ± 9.06 23.18 ± 11.70 15.90 (11.42–22.62) 19.70 (13.80–30.25)  Mean changes − 2.47 ± 1.41 6.48 ± 1.55 0.001 0.004   P a 0.110 < 0.001 Values are presented as mean ± standard deviation (SD) for baseline and end-of-study values, and mean ± standard error (SE) for changes. Median (first quartile-third quartile) is presented for variables with non-normal distribution. a Paired t -test for compare baseline and end-of-study values in each group. b ANCOVA for comparing the end-of-study values of outcomes between the two groups by adjusting the baseline values. c ANCOVA for comparing the end-of-study values of outcomes between the two groups by adjusting the baseline values and confounding variables including gender, smoking, and mean vitamin A. *Observed power was calculated only for outcomes with between-group p -values < 0.10. TNF-α: tumor necrosis factor-alpha; MCP-1: monocyte chemoattractant protein-1; hs-CRP: high-sensitivity C-reactive protein. Comparison of inflammatory markers before and after intervention within and between groups. Values are presented as mean ± standard deviation (SD) for baseline and end-of-study values, and mean ± standard error (SE) for changes. Median (first quartile-third quartile) is presented for variables with non-normal distribution. a Paired t -test for compare baseline and end-of-study values in each group. b ANCOVA for comparing the end-of-study values of outcomes between the two groups by adjusting the baseline values. c ANCOVA for comparing the end-of-study values of outcomes between the two groups by adjusting the baseline values and confounding variables including gender, smoking, and mean vitamin A. *Observed power was calculated only for outcomes with between-group p -values < 0.10. TNF-α: tumor necrosis factor-alpha; MCP-1: monocyte chemoattractant protein-1; hs-CRP: high-sensitivity C-reactive protein. As shown in Table  4 , VLDL levels significantly decreased in both groups by the end of the study, with a more pronounced and statistically significant reduction observed in the astaxanthin group ( p  = 0.004). The between-group comparison of mean changes also favored the intervention group, remaining significant after adjusting for potential confounders ( p  = 0.024). Total cholesterol exhibited a non-significant reduction in the astaxanthin group ( p  = 0.097), whereas the control group showed a slight but statistically non-significant increase. Although the between-group difference did not reach statistical significance after adjustment ( p  = 0.061), lower total cholesterol levels were observed in the astaxanthin group. LDL levels did not change significantly within either group; however, the control group showed a greater numerical increase (~ 10 units). After adjustment, the between-group comparison did not reach statistical significance, although lower LDL levels were observed in the astaxanthin group ( p  = 0.085). Table 4 Comparison of lipid profile before and after intervention within and between groups. Variable Group P b P c Observed power* ASX (n = 40) Placebo (n = 40) HDL (mg/dl) –  Baseline 44.02 ± 11.24 40.12 ± 8.94  End 37.03 ± 8.52 36.72 ± 7.32  Mean changes − 7.00 ± 1.79 − 3.41 ± 1.46 0.636 0.905   P a < 0.001 0.025 LDL (mg/dl) (0.40)  Baseline 68.07 ± 39.07 65.32 ± 42.53 56.50 (45.00–83.5) 56.50 (41.25–76.00)  End 68.86 ± 24.32 73.90 ± 31.90  Mean changes 0.79 ± 5.90 9.58 ± 6.02 3.00 (− 7.75 to 18.00) 13.23 (− 1.75 to 31.00)   P a 0.894 0.120 LDL/HDL (0.4)  Baseline 1.56 ± 0.68 1.66 ± 0.91  End 1.83 ± 0.60 2.04 ± 0.85  Mean changes 0.26 ± 0.10 0.38 ± 0.12 0.232 0.090   P a 0.014 0.004 Cholesterol (mg/dl) (0.47)  Baseline 141.65 ± 49.94 135.25 ± 52.62  End 128.08 ± 32.26 136.79 ± 41.88  Mean changes − 13.57 ± 7.99 1.54 ± 7.75 0.173 0.061 − 8.44 (− 30.75 to 14.25) 4.00 (− 17.75 to 35.25)   P a 0.097 0.843 Chol/HDL –  Baseline 3.22 ± 0.92 3.50 ± 1.20  End 3.60 ± 0.95 3.87 ± 1.13  Mean changes 0.38 ± 0.15 0.37 ± 0.18 0.517 0.197   P a 0.017 0.044 VLDL (mg/dl) (0.62)  Baseline 28.35 ± 11.09 32.35 ± 22.90 25.00 (19.25–36.75) 26.00 (18.00–38.75)  End 24.16 ± 12.63 29.50 ± 12.59 20.50 (15.00–28.93) 25.50 (19.00–38.48)  Mean changes − 4.19 ± 1.79 − 2.85 ± 3.47 0.019 0.024 − 4.5 (− 9.00 to 0.00) 0.00 (− 8.75 to 5.75)   P a 0.004 0.409 TG (mg/dl) –  Baseline 141.92 ± 55.54 161.97 ± 114.20  End 124.58 ± 62.48 140.95 ± 65.46  Mean changes − 17.34 ± 9.34 − 21.02 ± 16.64 0.435 0.272 − 13.00 (− 45.50 to 14.00) 0.17 (− 43.75 to 22.50)   P a 0.071 0.214 AIP –  Baseline 0.49 ± 0.19 0.55 ± 0.24  End 0.49 ± 0.21 0.60 ± 0.29  Mean changes − 0.002 ± 0.03 0.05 ± 0.04 0.132 0.280 − 0.005 (− 0.12 to 0.1) 0.02 (− 0.09 to 0.14)   P a 0.949 0.215 Values are presented as mean ± standard deviation (SD) for baseline and end-of-study values, and mean ± standard error (SE) for changes. Median (first quartile-third quartile) is presented for variables with non-normal distribution. a Paired t-test to compare baseline and end-of-study values in each group. b ANCOVA for comparing the end-of-study values of outcomes between the two groups by adjusting the baseline values. c ANCOVA for comparing the end-of-study values of outcomes between the two groups by adjusting the baseline values and confounding variables including gender, smoking, and mean vitamin A. *Observed power was calculated only for outcomes with between-group p -values < 0.10. HDL: high-density lipoprotein; LDL: low-density lipoprotein; TG: triglycerides; VLDL: very-low-density lipoprotein; Chol: cholesterol; AIP: atherogenic index of plasma. Comparison of lipid profile before and after intervention within and between groups. Values are presented as mean ± standard deviation (SD) for baseline and end-of-study values, and mean ± standard error (SE) for changes. Median (first quartile-third quartile) is presented for variables with non-normal distribution. a Paired t-test to compare baseline and end-of-study values in each group. b ANCOVA for comparing the end-of-study values of outcomes between the two groups by adjusting the baseline values. c ANCOVA for comparing the end-of-study values of outcomes between the two groups by adjusting the baseline values and confounding variables including gender, smoking, and mean vitamin A. *Observed power was calculated only for outcomes with between-group p -values < 0.10. HDL: high-density lipoprotein; LDL: low-density lipoprotein; TG: triglycerides; VLDL: very-low-density lipoprotein; Chol: cholesterol; AIP: atherogenic index of plasma. The LDL/HDL ratio increased significantly in both groups from baseline. Although the adjusted increase was slightly higher in the control group, the between-group difference was not statistically significant ( p  = 0.090). HDL levels significantly declined in both groups, with no significant difference in changes between groups, even after adjustment. No statistically significant effects were observed on other lipid parameters or the atherogenic index of plasma (AIP) ( p  > 0.05). Table  5 presents the effects of astaxanthin supplementation on waist circumference, body weight, and BMI. According to the findings, astaxanthin supplementation did not result in statistically significant changes in any of the assessed anthropometric parameters compared to the control group ( p  > 0.05). Table 5 Comparison of anthropometric indices before and after intervention within and between groups. Variable Group P b P c ASX (n = 40) Placebo (n = 40) Weight (kg)  Baseline 73.95 ± 13.05 76.11 ± 13.74  End 73.97 ± 12.81 76.09 ± 13.70  Mean changes 0.01 ± 0.13 − 0.02 ± 0.12 0.972 0.819 0.10 (− 0.59 to 0.47) 0.20 (− 0.27 to 0.40)   P a 0.913 0.882 Waist circumstances (cm)  Baseline 100.14 ± 15.45 102.60 ± 15.10  End 100.08 ± 15.41 102.50 ± 15.06  Mean changes − 0.05 ± 0.07 − 0.11 ± 0.06 0.654 0.872 0.00 (− 0.15 to 0.10) 0.00 (− 0.20 to 0.10)   P a 0.435 0.095 BMI (kg/m 2 )  Baseline 25.72 ± 4.42 26.37 ± 4.82  End 25.72 ± 4.33 26.37 ± 4.81  Mean changes − 0/001 ± 0.04 0.004 ± 0.04 0.824 0.738 0.05 (− 0.20 to 0.20) 0.10 (− 0.10 to 0.20)   P a 0.984 0.900 Values are presented as mean ± standard deviation (SD) for baseline and end-of-study values, and mean ± standard error (SE) for changes. Median (first quartile-third quartile) is presented for variables with non-normal distribution. a Paired t -test to compare baseline and end-of-study values in each group. b ANCOVA for comparing the end-of-study values of outcomes between the two groups by adjusting the baseline values. c ANCOVA for comparing the end-of-study values of outcomes between the two groups by adjusting the baseline values and confounding variables including gender, smoking, and mean vitamin A. BMI: body mass index. Comparison of anthropometric indices before and after intervention within and between groups. Values are presented as mean ± standard deviation (SD) for baseline and end-of-study values, and mean ± standard error (SE) for changes. Median (first quartile-third quartile) is presented for variables with non-normal distribution. a Paired t -test to compare baseline and end-of-study values in each group. b ANCOVA for comparing the end-of-study values of outcomes between the two groups by adjusting the baseline values. c ANCOVA for comparing the end-of-study values of outcomes between the two groups by adjusting the baseline values and confounding variables including gender, smoking, and mean vitamin A. BMI: body mass index. Sensitivity Analysis. To assess the robustness of the findings, a per-protocol sensitivity analysis was conducted including only participants who completed the intervention and had complete outcome data. Overall, the results were generally consistent with those obtained from the ITT analysis. The primary findings regarding TNF-α, MCP-1, VLDL, hs-CRP, anthropometric indices, and other lipid parameters remained unchanged. However, in the per-protocol analysis, the reduction in total cholesterol became statistically significant in the astaxanthin group compared with the control group ( p  = 0.041). Detailed results of the per-protocol analyses are presented in Supplementary Tables S3–S5.

This randomized, double-blind, placebo-controlled clinical trial was conducted on 80 adult patients with HF. The study was designed and carried out in accordance with the CONSORT (Consolidated Standards of Reporting Trials) guidelines. A completed CONSORT checklist is provided in the supplementary file. This study has been officially registered in the Iranian Registry of Clinical Trials (ID: IRCT20200429047235N3). Additionally, the study protocol received approval from the Ethics Committee of Isfahan University of Medical Sciences (approval code: IR.MUI.MED.REC.1402.099). Patients were recruited from Chamran Heart Hospital in Isfahan. Eligibility required a diagnosis of stage C or D HF, as defined by the American Heart Association (AHA) criteria 22 , and with left ventricular ejection fraction (LVEF) < 50%. Participants were eligible if they were adults (≥ 18 years) with a confirmed diagnosis of heart failure and were willing to participate in the study. Key exclusion criteria included pregnancy or lactation, use of antioxidant supplements within the previous three months, significant comorbidities (e.g., hepatic, renal, or pulmonary diseases), inflammatory or malignant conditions, recent acute cardiovascular events or surgery, and adherence to specific diet or exercise programs. Participants were also excluded during the study if they experienced adverse reactions, had poor compliance (< 80% of supplement intake), or had changes in their medication regimen. Inclusion and exclusion criteria have been described in detail in the published study protocol in the Trials journal, which provides comprehensive information on participant selection and study procedures 23 . Written informed consent was obtained from all participants prior to their enrollment in the study. The sample size for this study was calculated a priori based on total antioxidant capacity (TAC), which was the primary outcome of the broader doctoral research project and has been reported separately 21 . The calculation was performed using a significance level of 5% (Z = 1.96), a statistical power of 80% (Z = 0.84), and a standardized effect size (Δ) of 0.7. Ultimately, a total of 80 participants were enrolled 24 . Detailed methodology and rationale for sample size estimation are described in the published protocol 23 . Participants were randomized into two groups. The intervention group received one oral capsule containing 20 mg of astaxanthin per day for 8 weeks. The supplement contained non-esterified astaxanthin in the cis-isomer form and was produced by Zist Fanavari Taravat Zendegi. The control group received placebo capsules containing 20 mg of maltodextrin, which were matched in appearance (including color, smell, and taste) to ensure blinding. The placebo was manufactured by Osina Shimi under the brand name Foodchem. The intervention duration of 8 weeks was selected based on prior human clinical studies indicating that this time frame is sufficient to detect changes in inflammatory and metabolic biomarkers following astaxanthin supplementation 25 – 27 . In addition, this duration is consistent with the pharmacokinetic properties of astaxanthin, a lipophilic carotenoid that is absorbed via lipoproteins and gradually distributed into lipid-rich tissues, with evidence suggesting progressive accumulation and attainment of a relatively stable steady-state level during chronic supplementation 28 . An 8-week period was therefore considered appropriate to allow sufficient systemic exposure and biological effect manifestation, while also being feasible in terms of patient adherence in individuals with HF. The dose of 20 mg/day astaxanthin was selected based on evidence from previous human clinical trials in cardiometabolic and inflammatory conditions, where doses ranging from 2 to 20 mg/day have been shown to be safe and well tolerated 24 , 29 . Higher doses within this range (12–20 mg/day) have been more consistently associated with improvements in oxidative stress and inflammatory biomarkers 30 , 31 . Therefore, the upper end of the tested safe range was chosen to maximize potential therapeutic effects while maintaining an acceptable safety profile. Compliance with the intervention was assessed by counting returned capsules at each follow up visit. In addition, participants were regularly contacted to encourage adherence. Subjects with less than 80% compliance were excluded from the study. Randomization and allocation procedures were conducted as described in our previously published study protocol 23 .Briefly, eligible participants were randomly assigned to intervention or control groups using block randomization with permuted blocks of size four. The randomization sequence was generated using an online randomization tool (Sealed Envelope™; https://www.sealedenvelope.com/simple-randomiser/v1/lists ). Allocation concealment and blinding were ensured by an independent staff member who was not involved in participant recruitment, enrollment, or outcome assessment. This individual prepared and coded the supplement containers as Code A and Code B. The allocation codes were kept fully concealed from participants, investigators, and laboratory personnel until completion of the study. Dietary intake was assessed using 3-day food records collected at baseline, midpoint, and at the end of the study. This method has been previously validated for accuracy and reliability 32 . Participants were instructed to record all foods and beverages consumed over two weekdays and one weekend day, including portion sizes, to estimate habitual dietary intake. Physical activity levels were evaluated at the same time points using the International Physical Activity Questionnaire (IPAQ), which has been validated in various populations, including Iranian adults 33 . This questionnaire assesses the frequency and duration of physical activities at different intensity levels. The results were expressed as metabolic equivalent task minutes per week (MET-min/week).Further details regarding these assessment methods and the overall study design have been described in the published study protocol 23 , which provides comprehensive information on participant selection, intervention procedures, and outcome measurements. The study protocol was prospectively registered, further supporting the transparency and reproducibility of the trial. Anthropometric indices, including BMI, waist circumference, and body weight, were assessed at baseline and after 8 weeks of intervention. Body weight was measured to the nearest 0.1 kg in the morning under fasting conditions, with participants barefoot and wearing minimal clothing, using a digital scale (model 707, range 0.1–150 kg; Seca, Germany). Height was measured to the nearest 0.1 cm in a standing position without shoes using a non-elastic stadiometer (model 208 Portable Body Meter Measuring Device; Seca, Germany). BMI was calculated as weight (kg) divided by the square of height (m²). Waist circumference was measured to the nearest 0.1 cm at the narrowest point between the lower rib margin and the iliac crest using a non-stretchable tape while the participant was standing upright. To determine the concentrations of blood biomarkers, participants were instructed to visit Chamran Hospital Laboratory at the beginning and end of the study following a 10–12 h overnight fast. At each time point, 10 mL of venous blood was drawn. The blood samples were then centrifuged at 3,500 rpm to separate the serum. The isolated serum was transferred into microtubes and stored at − 80 °C until analysis. Serum levels of total cholesterol, low-density lipoprotein cholesterol (LDL-C), HDL-C, and TG were measured using enzymatic colorimetric methods. hs-CRP was assessed via a turbidimetric method. Inflammatory biomarkers, including MCP-1 and TNF-α, were quantified using ELISA kits from Karmania Pars Gene Company. The intention-to-treat (ITT) principle was applied, including all randomized participants in their original assigned groups regardless of protocol adherence or loss to follow-up. Handling of missing data – Missing outcome data (primarily due to loss to follow-up) were handled using regression-based imputation via the Missing Value Analysis (MVA) procedure in SPSS. We assumed that missingness was missing at random (MAR); that is, the probability of a missing post-intervention value depended on the observed pre-intervention value of the same variable but not on the unobserved post-intervention value itself. This assumption is reasonable because baseline (pre-intervention) measurements were complete and are known to be strong predictors of post-intervention outcomes. For each outcome variable with missing post-intervention data, the imputation model included the corresponding pre-intervention value of the same variable as the sole predictor. Using participants with complete pre- and post-intervention data, SPSS estimated a linear regression equation considering Post-intervention as dependent variable should be estimated and Pre-intervention as predictor. For each case with a missing post-intervention value, the observed pre-intervention value was entered into regression equation, and the predicted value was used as the imputed value. To strengthen confidence in the findings, a sensitivity analysis was performed comparing the results obtained from the complete-case analysis (per-protocol analysis) with those from the imputed dataset after regression imputation. Continuous variables were described using mean ± standard deviation (SD) for normally distributed data and median with interquartile range (IQR) for skewed data, as assessed by the Shapiro–Wilk test and Q–Q plot visualizations. Categorical data were presented as counts and percentages. Non-normal positively skewed data were subjected to natural logarithmic transformation. Group differences at baseline were examined using independent samples t-tests for continuous variables and chi-square tests for categorical variables. Within-group changes over time were assessed using paired t-tests. Between-group comparisons of outcome changes were conducted using analysis of covariance (ANCOVA), with adjustments for relevant covariates. Because smoking status and dietary vitamin A intake differed between groups at baseline, these variables were additionally included as covariates in the adjusted analyses. Dietary intake and physical activity trends were evaluated through repeated-measures ANOVA. A two-tailed P-value of less than 0.05 was considered statistically significant. All statistical analyses were carried out using SPSS software, version 20 (IBM Corp., Armonk, NY, USA).

Astaxanthin supplementation at a dose of 20 mg/day for 8 weeks was associated with reductions in selected inflammatory markers, particularly TNF-α and MCP-1, in patients with HF. Limited effects were observed on lipid parameters, with only VLDL showing a significant reduction, while no significant changes were detected in hs-CRP, other lipid indices, or anthropometric measures. These findings suggest that astaxanthin may have modest anti-inflammatory effects in patients with HF; however, its broader metabolic effects remain uncertain. In addition, given the exploratory nature of several secondary outcomes, these findings should be interpreted with caution. Future studies with larger sample sizes, longer intervention durations, and varying dosages are needed to further clarify the clinical efficacy and potential therapeutic role of astaxanthin in HF management.

This study demonstrated that astaxanthin supplementation at 20 mg/day for 8 weeks significantly reduced TNF-α and MCP-1, whereas no significant effect was observed on hs-CRP in patients with HF. One possible explanation for this finding is that hs-CRP is a relatively stable inflammatory marker that may require longer periods to exhibit measurable changes, particularly in patients with chronic conditions such as HF 30 , 34 . Its effects on lipid metabolism were modest; only VLDL decreased significantly, while total cholesterol showed a non-significant reduction after adjustment. No significant improvements were observed in LDL, HDL, TG, or anthropometric indices. These findings may suggest that short-term supplementation, in the absence of dietary or physical activity modifications, may be insufficient to induce measurable changes in body composition and lipid profile parameters in patients with HF. Longer-duration interventions may produce more definitive results. It should also be noted that dietary vitamin A intake was significantly higher in the control group. Given the antioxidant properties of vitamin A, this imbalance could have influenced oxidative and inflammatory status. However, vitamin A intake was adjusted for in the statistical analysis, and therefore its potential confounding effect on the observed outcomes is likely minimized. Interestingly, Despite the higher vitamin A intake in the control group, greater reductions in inflammatory markers were still observed in the astaxanthin group. The effects of astaxanthin and other antioxidant supplements may vary depending on the administered dose and duration of intervention. Previous studies have reported beneficial effects of astaxanthin at doses ranging from 6 to 20 mg/day, with higher doses generally associated with more pronounced improvements in inflammatory and metabolic parameters. For example, studies using lower doses (e.g., 6–12 mg/day) have demonstrated modest or selective effects, whereas higher doses (e.g., 20 mg/day) and longer intervention periods (≥ 12 weeks) have been associated with more consistent improvements in certain biomarkers 24 , 35 . Evidence from cardiovascular related research suggests that astaxanthin may exert beneficial effects through modulation of oxidative stress and inflammatory pathways, which are central mechanisms in the pathophysiology of HF 12 , 28 . Although direct evidence in HF populations is still limited, previous studies in cardiovascular and cardiometabolic contexts have reported improvements in inflammatory biomarkers following astaxanthin supplementation, supporting its potential relevance in HF 36 – 38 . In line with these findings, studies in conditions characterized by chronic systemic inflammation have also investigated the effects of astaxanthin. Jabbarpour et al. found that administration of 12 mg of astaxanthin for 8 weeks in women with polycystic ovary syndrome (PCOS) significantly decreased TNF-α but had no effect on CRP levels 39 . Similarly, in patients with rheumatoid arthritis, daily supplementation with 20 mg of astaxanthin for 8 weeks failed to produce any significant change in CRP concentrations 30 . In another study, supplementation with 6 mg of astaxanthin for 12 weeks in women with endometriosis led to a significant decrease in TNF-α, aligning with the outcomes of our research 40 . In a systematic review and meta-analysis, Xia et al. reported that astaxanthin supplementation for at least 12 weeks and at daily doses exceeding 12 mg was associated with a reduction in CRP levels 31 . The beneficial effects of astaxanthin observed in the present study may be explained through several interconnected molecular mechanisms. Previous experimental and preclinical studies have suggested that astaxanthin may modulate key signaling pathways involved in inflammation and oxidative stress, particularly nuclear factor-kappa B (NF-κB) and nuclear factor erythroid 2–related factor 2 (Nrf2) 41 , 42 . Activation of NF-κB plays a central role in the transcription of pro-inflammatory cytokines such as TNF-α and MCP-1, whereas Nrf2 regulates the expression of antioxidant enzymes and other cytoprotective proteins. Potential mechanisms proposed in previous studies include inhibition of NF-κB activation and enhancement of Nrf2 signaling, which may contribute to reductions in pro-inflammatory mediators and improved antioxidant defense 41 . In addition, astaxanthin has been reported to reduce lipid peroxidation by scavenging reactive oxygen species and protecting membrane lipids from oxidative damage 11 . Evidence from clinical studies has also demonstrated that astaxanthin supplementation can improve oxidative stress markers and inflammatory profiles in populations with chronic inflammatory and cardiometabolic conditions 43 , 44 . Although the observed reductions in TNF-α and MCP-1 were relatively modest, these inflammatory mediators play important roles in the progression and severity of HF. Elevated circulating levels of TNF-α and MCP-1 have been associated with adverse cardiovascular outcomes, endothelial dysfunction, and chronic inflammatory activation in patients with HF 6 , 8 , 14 . Therefore, even moderate reductions in these biomarkers may be clinically relevant. However, the long-term clinical implications of these changes remain uncertain and require confirmation in larger clinical trials. The magnitude of reduction observed in the present study appears generally comparable to the modest anti-inflammatory effects reported in previous astaxanthin supplementation studies conducted in chronic inflammatory and metabolic disorders 19 , 26 , 44 . In addition to these pathways, previous studies have proposed potential regulatory effects of astaxanthin on inflammatory microRNAs such as miR-146a 45 . NF-κB is a central mediator of the inflammatory response, activated by pro-inflammatory cytokines 46 . Under inflammatory conditions, macrophages produce large quantities of pro-inflammatory mediators such as cytokines, chemokines, cyclooxygenase-2 (COX-2), and matrix metalloproteinases (MMPs) 47 . Previous experimental evidence also suggests that astaxanthin may attenuate MCP-1 upregulation through modulation of inflammatory signaling pathways, particularly NF-κB-related pathways 48 . Previous studies have demonstrated that astaxanthin reduces MCP-1 and pro-inflammatory cytokines such as interleukin-6 (IL-6) and TNF-α 36 , 49 . Previous studies have also reported that astaxanthin supplementation may downregulate the gene expression of several pro-inflammatory mediators, including IL-6, TNF-α, and COX-2. The reduction in MMP levels and pro-inflammatory cytokines may be attributed to astaxanthin-mediated inhibition of the NF-κB transcription factor 36 , 42 , 47 . However, these mechanisms were not directly assessed in the present study and should be interpreted with caution. Another finding of this study was a significant reduction in VLDL levels following 8 weeks of astaxanthin supplementation in patients with HF. In addition, a non-significant reduction in total cholesterol levels was observed after adjustment. A slight increase in LDL and the LDL/HDL ratio was noted in the control group compared to the intervention group. No significant changes were detected in other lipid profile parameters or the AIP. The lipid profile plays a main role in lipid homeostasis and cardiovascular health. In one study, supplementation with 8 mg of astaxanthin for 3 months in healthy men reduced fatty acid oxidation 50 . In a high-fat diet mouse model, astaxanthin increased hepatic LDL receptor expression, reduced expression of 3-Hydroxy-3-Methylglutaryl-Coenzyme A (HMG-CoA) reductase, and upregulated sterol regulatory element-binding protein 2 (SREBP-2), which may collectively contribute to its hypocholesterolemic effects. Enhanced β-oxidation of fatty acids may also reduce triglyceride synthesis and subsequently limit hepatic VLDL secretion 51 . Furthermore, astaxanthin has been shown to potentially suppress the expression of SREBP1c , cause downregulation of lipogenic gene expression 52 . However, findings across studies remain inconsistent. A meta-analysis concluded that astaxanthin supplementation had no significant effect on lipid profile parameters 53 . In the study by Urakaze et al., only apo-E levels were significantly reduced, while no notable changes were observed in total cholesterol, TG, or HDL levels 54 . In contrast, another trial reported that 12 mg of astaxanthin significantly increased HDL in individuals with hyperlipidemia 55 . Similar to our findings, Shokri et al. showed that 8 weeks of astaxanthin supplementation reduced VLDL levels in patients with diabetes 25 . In the study by Rustaie Rad, no significant changes in lipid profile were observed after 12 weeks of supplementation in patients with type 2 diabetes 56 . Conversely, Saeidi et al. found that 20 mg of astaxanthin for 12 weeks significantly decreased total cholesterol, triglycerides, and LDL, while increasing HDL in obese men 35 . Evidence suggests that astaxanthin may support a favorable lipid profile by reducing lipid peroxidation, suppressing inflammation, and enhancing antioxidant defense mechanisms 11 , 12 . The third finding of our study indicated that 8 weeks of astaxanthin supplementation did not result in statistically significant changes in any anthropometric indices compared to the control group. These results are consistent with previous studies conducted in various populations. Two recent meta-analyses also concluded that astaxanthin supplementation had no significant effect on body weight, BMI, or waist circumference 20 , 31 . Similarly, a 12-week supplementation with 6 or 12 mg of astaxanthin in overweight and older adults did not result in significant changes in body weight or BMI 57 . Additionally, supplementation with 20 mg of astaxanthin for 12 weeks in overweight individuals yielded similar results, with no significant changes observed in weight, waist and hip circumference, BMI, or body composition (including body fat percentage, lean mass, or visceral fat) 58 . Contrary to these findings, Saeidi et al. reported that 12-week supplementation with 20 mg of astaxanthin significantly reduced body weight, BMI, and body fat percentage in obese men 35 . Another study found that 8 mg of astaxanthin for 8 weeks reduced visceral fat in patients with type 2 diabetes 25 . Some studies suggest that astaxanthin may enhance insulin sensitivity, leading to improved glycemic control and reduced fat storage. Furthermore, it may inhibit lipid peroxidation and prevent the accumulation of fat in visceral tissues 25 , 50 , 59 . It is important to note that patients with HF frequently experience fluid retention, muscle wasting, and metabolic alterations, which may complicate the measurement of weight, waist circumference, and BMI. These factors could potentially obscure any true effects of astaxanthin on body composition. Additionally, the relatively short duration of the intervention may not have been sufficient to elicit measurable changes in anthropometric or body composition parameters. The absence of statistically significant effects for hs-CRP, anthropometric indices, and several lipid parameters should be interpreted cautiously, as the present study may have been underpowered to detect modest changes in some secondary outcomes. Furthermore, differences in baseline inflammatory status, disease severity, medication use, and individual metabolic responsiveness may have influenced the magnitude of response to astaxanthin supplementation among participants. A key strength of this study lies in its randomized, double-blind design, the application of intention-to-treat analysis, and the use of objective biochemical and clinical endpoints. However, several limitations should be acknowledged. Plasma astaxanthin concentrations were not measured, limiting confirmation of bioavailability and pharmacokinetic interpretation. Additionally, variability in astaxanthin absorption and metabolism among participants, particularly in patients with HF who may have altered metabolic and gastrointestinal function, could not be evaluated and may have contributed to differences in individual responses to the intervention. Although oxidative stress biomarkers were measured in this trial, they were not included in the present manuscript as they have been reported elsewhere 21 . The present manuscript represents part of a broader randomized clinical trial evaluating the effects of astaxanthin supplementation on inflammatory, oxidative stress, metabolic, and clinical outcomes in patients with HF. Although oxidative stress biomarkers and disease symptoms from this trial have been reported previously 21 , the present manuscript focuses specifically on inflammatory markers, lipid profile parameters, and anthropometric indices, which were not included in the previous report. As a result, the ability to directly link the observed findings to antioxidant mechanisms within the scope of the present manuscript may be limited. Furthermore, dietary vitamin A intake was significantly higher in the control group, which may have influenced oxidative and inflammatory status. Although this variable was adjusted for in the statistical analysis, residual confounding cannot be entirely excluded. Similarly, smoking status differed between groups at baseline, and although statistical adjustment was performed, residual confounding related to smoking cannot be completely excluded. The relatively short duration of the intervention (8 weeks) may have been insufficient to observe changes in certain outcomes. Because the sample size calculation was based on the primary outcome of the broader trial, the present study may have been underpowered to detect smaller but potentially clinically meaningful differences in several secondary outcomes. Therefore, the absence of statistically significant effects for some secondary endpoints should not necessarily be interpreted as evidence of no effect. In addition, because multiple secondary outcomes were evaluated, the possibility of type I error due to multiple comparisons cannot be completely excluded. Therefore, findings related to secondary outcomes with limited statistical power should be interpreted with caution. Additionally, no imaging or echocardiographic assessments were performed to evaluate structural or functional cardiac changes. Future research should incorporate pharmacokinetic assessments, longer follow-up periods, and comprehensive clinical evaluations, including imaging-based outcomes, to better elucidate the effects of astaxanthin in patients with HF.

Heart failure (HF) is a progressive clinical syndrome resulting from structural or functional cardiac disorders that impair the ventricle’s ability to fill with or eject blood 1 . Globally, the burden of HF is increasing, with an estimated 26 million people affected 2 . In Iran, its prevalence reaches approximately 8%, which is notably higher than in other Asian countries 3 . HF is associated with high morbidity, reduced quality of life, frequent hospitalizations, and elevated healthcare costs 4 , 5 . One of the key pathophysiological mechanisms contributing to HF is chronic inflammation and oxidative stress 6 . Elevated serum levels of inflammatory biomarkers such as tumor necrosis factor-alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and high-sensitivity C-reactive protein (hs-CRP) are often observed in patients with HF 7 , 8 . These biomarkers promote myocardial remodeling, endothelial dysfunction, and progression of HF 9 , 10 . Dyslipidemia represents another major concern in patients with HF, typically marked by elevated triglycerides (TG) and very-low-density lipoprotein (VLDL) levels, alongside reduced high-density lipoprotein (HDL). These lipid abnormalities contribute to the development of atherosclerosis and exacerbate cardiovascular outcomes 11 , 12 . Additionally, patients with HF often exhibit metabolic alterations, including abnormal anthropometric indices such as increased waist circumference and body mass index (BMI), which further exacerbate systemic inflammation and insulin resistance 13 , 14 . Astaxanthin, a red-pigmented carotenoid found in marine organisms such as salmon, shrimp, and microalgae (Haematococcus pluvialis), has demonstrated potent antioxidant and anti-inflammatory properties 15 , 16 . Unlike other carotenoids, astaxanthin can cross the blood-brain barrier and cell membranes, making it biologically effective 17 , 18 . Previous research has demonstrated that astaxanthin may reduce serum CRP and TNF-α levels while also improving lipid profiles in metabolic syndrome and polycystic ovary syndrome 19 , 20 . Despite the growing body of evidence supporting the anti-inflammatory and lipid-modulating effects of astaxanthin, most existing studies have been conducted in populations with metabolic disorders or other chronic inflammatory conditions. To date, evidence regarding the effects of astaxanthin supplementation in patients with HF remains limited. In a previous report from our randomized controlled trial, astaxanthin supplementation was shown to improve oxidative stress biomarkers and selected clinical outcomes in patients with HF 21 . However, the effects of astaxanthin on inflammatory biomarkers, lipid profile parameters, and anthropometric indices have not been reported previously. Given the important role of inflammation and metabolic disturbances in the progression of HF, further evaluation of these outcomes may provide additional insight into the potential therapeutic role of astaxanthin in this population. Therefore, the primary objective of this study was to evaluate the effects of 8-week supplementation with 20 mg/day astaxanthin on inflammatory markers in patients with HF. Secondary outcomes included lipid profile parameters and anthropometric indices. We hypothesized that astaxanthin supplementation would reduce systemic inflammation and improve lipid profile in patients with HF.

Below is the link to the electronic supplementary material. Supplementary Material 1 Supplementary Material 1