Effects of Nutritional Supplementation on Apolipoprotein B– Related Biomarkers in Adults with Dyslipidaemia: A Protocol for a Systematic Review and Meta-Analysis

Technical Summary: Effects of Nutritional Supplementation on Apolipoprotein B–Related Biomarkers

Problem Statement
Cardiovascular disease (CVD) remains the leading cause of global mortality, with dyslipidaemia serving as a principal modifiable risk factor. While Low-Density Lipoprotein Cholesterol (LDL-C) has traditionally been the primary therapeutic target, it fails to capture the total burden of atherogenic particles, leaving a significant "residual risk" in many patients. Apolipoprotein B (ApoB) has emerged as a more accurate quantitative index of atherogenic particle number, as each circulating atherogenic lipoprotein (LDL, VLDL, IDL, and Lp(a)) carries exactly one ApoB molecule. Consequently, ApoB, the ApoB/Apolipoprotein A1 (ApoA1) ratio, and non-High-Density Lipoprotein Cholesterol (non-HDL-C) are increasingly recognized as superior prognostic markers and are being integrated into contemporary lipid management guidelines.

Despite the widespread use of nutritional supplements (e.g., omega-3 fatty acids, vitamins, minerals, CoQ10) to improve lipid profiles, existing evidence focuses predominantly on conventional lipid parameters (LDL-C, HDL-C, triglycerides). There is a critical evidence gap regarding the effects of these interventions on ApoB-related biomarkers. Nutritional interventions that produce modest changes in LDL-C may exert substantially different effects on ApoB and the overall atherogenic lipoprotein burden, yet no comprehensive systematic review has synthesized this specific evidence across multiple supplement categories.

Methodology
This document is a study protocol for a systematic review and meta-analysis, developed in accordance with the PRISMA-P 2015 statement and prospectively registered with PROSPERO (CRD420261430196). The review will adhere to the Cochrane Handbook for Systematic Reviews of Interventions (version 6.5).

• Search Strategy: Comprehensive electronic searches will be conducted in PubMed/MEDLINE, Scopus, and the Cochrane Central Register of Controlled Trials (CENTRAL) from inception to the final search date. Reference lists and forward citations will also be screened.
• Eligibility Criteria (PICOS):
  • Population: Adults (≥18 years) with dyslipidaemia (hypercholesterolaemia, hypertriglyceridaemia, mixed dyslipidaemia), with or without comorbidities (e.g., obesity, diabetes, CVD).
  • Intervention: Oral nutritional supplements administered for a minimum of eight weeks. Included categories are vitamin preparations, mineral supplements, omega-3 fatty acids, CoQ10, and multicomponent formulations. Herbal medicines, probiotics, fiber supplements, and lifestyle-only interventions are excluded.
  • Comparator: Placebo, usual care, standard care, or no intervention.
  • Outcomes: Primary outcomes are changes in ApoB concentration and the ApoB/ApoA1 ratio. Secondary outcomes include non-HDL-C, conventional lipid parameters, and adverse events.
  • Study Design: Parallel-group Randomized Controlled Trials (RCTs). Crossover trials, observational studies, and non-randomized designs are excluded.
• Data Management: Two reviewers will independently screen studies, extract data, and assess risk of bias using the Cochrane Risk of Bias 2 (RoB 2) tool. Disagreements will be resolved by consensus or a third reviewer.
• Statistical Analysis: Random-effects meta-analyses will be performed using restricted maximum likelihood (REML) estimation. Results will be expressed as Mean Differences (MD) or Standardized Mean Differences (SMD) with 95% Confidence Intervals (CIs). Analyses will be stratified by supplement category to address clinical heterogeneity. Subgroup and sensitivity analyses are planned to explore sources of heterogeneity and robustness.
• Evidence Certainty: The certainty of evidence will be evaluated using the GRADE framework.

Key Contributions
As a protocol, this paper does not present empirical results but outlines a rigorous methodological framework to address a specific gap in the literature. Its primary contributions include:

1. Defining a New Scope: It establishes the first comprehensive plan to synthesize evidence specifically on ApoB, ApoB/ApoA1 ratio, and non-HDL-C in response to nutritional supplementation, moving beyond the traditional focus on LDL-C.
2. Stratified Analysis: The protocol proposes stratifying meta-analyses by supplement category (e.g., omega-3s vs. vitamins vs. minerals) to provide nuanced insights into how different biological mechanisms affect atherogenic particle burden.
3. Methodological Rigor: It commits to high-standard reporting (PRISMA-P 2015, PRISMA 2020), duplicate independent review processes, and the use of GRADE for evidence certainty, ensuring the future review will be robust and reliable.

Results
No results are presented in this document, as it is a protocol for a future study. The paper details the planned outcomes (changes in ApoB, ApoB/ApoA1 ratio, and non-HDL-C) and the statistical methods that will be used to generate these results once the systematic review is completed.

Significance and Claims
The authors claim that this systematic review will address a significant evidence gap by evaluating nutritional interventions using biomarkers that are increasingly central to cardiovascular risk assessment. The significance of the work lies in its potential to:

• Inform Clinical Practice: Provide evidence-based guidance on whether nutritional supplements can effectively reduce atherogenic particle burden, potentially influencing lipid management strategies.
• Align with Contemporary Guidelines: Offer data aligned with modern guidelines (e.g., ESC/EAS, AHA/ACC) that endorse ApoB and related biomarkers as risk-enhancing factors or secondary targets.
• Guide Future Research: Highlight the need for future trials to report ApoB-related endpoints and support the development of targeted nutritional strategies for dyslipidaemia.

The authors acknowledge limitations, including anticipated clinical heterogeneity across trials and the restriction to English-language publications, which may introduce language bias. They maintain a modest stance, noting that the review aims to synthesize existing evidence rather than propose new interventions, and that the findings will depend on the availability and quality of the underlying RCTs.