Reply to the ‘Comment on “The association between carotenoids and subjects with overweight or obesity: a systematic review and meta-analysis”’ by N. Shokri-mashhadi and S. Saadat, Food Funct., 2021, 12, DOI: 10.1039/D1FO01617B

Abstract

Here, we re-emphasize the purpose of our meta-analysis of the associations between carotenoids and subjects with excess body weight (Yao et al., 2021) and address some of the points raised in the commentary by Nafiseh Shokri-mashhadi and Saeed Saadat. The commentary focused on the methodological faults of our published meta-analysis research—but the research was rigorously conducted and scientifically sound, following strictly the relevant requirements of the Cochrane guidelines. Therefore, in the current reply, we included an elaboration and thorough discussion of the search strategy, inclusion and exclusion criteria, and tests of heterogeneity for the discussed meta-analysis in light of the comments made in the commentary. In conclusion, we believe that our study has made an essential contribution to the investigation of the significance of carotenoids in people with excess body weight.

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1. Review of the debated research

Obesity and being overweight in general are global epidemics and are due to excessive accumulation of body fat, which is associated with a variety of health-threatening diseases.¹ Some studies have shown that being overweight or obese has a variety of effects on the development of diseases such as arterial hypertension, type 2 diabetes mellitus, dyslipidemia, coronary heart disease, stroke, asthma, and arthritis.² In addition, excess body weight causes severe economic losses, increasing the burdens of the resulting diseases and leading to decreased productivity, disability, and premature death.³

Carotenoids constitute a family of more than 600 fat-soluble plant pigments,⁴ and are believed to be at least in part responsible for the abilities of fruits and vegetables to decrease the occurrence of human diseases such as cancer, coronary vascular disease, eye diseases, and osteoporosis.⁵ Currently, the most studied carotenoids are β-carotene, lycopene, astaxanthin, lutein, and zeaxanthin.⁴ There are substantial studies demonstrating that carotenoids can prevent cancer by affecting cell proliferation and differentiation,⁶ being involved in redox-dependent mechanisms,⁷ enhancing immune surveillance during tumorigenesis,⁴ and limiting clonal expansion of starting cells.⁴ The hypothesis that carotenoids prevent coronary vascular disease is supported by observational epidemiological and intervention trials, with studies reporting that the consumption of carotenoid-rich foods is associated with a reduced risk of cardiovascular disease.⁸ Lutein, zeaxanthin and meso-zeaxanthin, the only carotenoids found in the retina^9,10 and lens,¹¹ are responsible for absorbing blue light while inhibiting oxidative stress in the eye; therefore, they both may play a unique role in preventing eye diseases such as cataracts from developing.⁴ In addition, the antioxidant effect of carotenoids makes them useful in preventing the development of osteoporosis, which is associated with oxidative stress.¹²

A new idea has emerged in recent years that links carotenoids and the control of body fat accumulation, suggesting that carotenoids may influence the development and management of obesity. The latest animal experiments have shown that lycopene reduces body weight and abdominal fat in obese rats.¹³ In addition, a meta-analysis examining the effects of astaxanthin in animal models of diet-induced obesity-related diseases showed that astaxanthin is associated with a reduction in body weight and adipose tissue weight.¹⁴ The impact of carotenoids on obesity is the result of a combination of several factors, including the control of adipogenesis, adipocyte metabolism (the relative capacity of fat storage and oxidation), regulation of signaling and inflammatory mediators, and oxidative stress.¹⁵ Uncoupling protein 1 (UCP1) is a key factor involved in preventing excess fat accumulation and has also been evaluated to determine the mechanism of action of the seaweed extract astaxanthin against obesity.¹⁶ Expression of the UCP1 protein and its mRNA was observed in white adipose tissue (WAT) in mice enriched with astaxanthin or astaxanthin fractions, while only a very low expression of UCP1 was observed in control mice.¹⁷ Simultaneously, there is growing evidence that carotenoids and carotenoid metabolites are associated with inhibition of adipocyte differentiation (adipogenesis) and reduction of fat storage in mature adipocytes through inhibition of peroxisome proliferator-activated receptor γ (PPARγ), a major regulator of adipogenesis and mature adipocyte phenotype.¹⁵ In addition, considering that excessive reactive oxygen species (ROS) production in obese adipose tissue is a pro-inflammatory, pathogenic mechanism of obesity-related metabolic syndrome, the anti-inflammatory effects of carotenoids in adipose tissue may be related to the ability of carotenoids to reduce oxidative stress.¹⁸ These antioxidant properties are thought to mainly be derived from their ability to activate the NF-E2-related factor 2 (NRF2) pathway and cellular antioxidant defenses and to inhibit the nuclear factor kappa-B (NF-κB) pathway, thereby inhibiting downstream production of inflammatory cytokines and hence suppressing inflammation.^1,9–22

Considering the adverse impacts of obesity and being overweight on human health, our study focused on groups of overweight and obese subjects to determine the effect of serum carotenoid levels on them, and to investigate further the influences of carotenoid intervention on demographic characteristics of these subjects. Therefore, we included observational studies and randomized controlled trials (RCTs), primarily concentrating on information such as odds ratios (ORs), weight change, and body mass index (BMI) change.

2. Addressing criticisms and adhering to facts

Reading the commentary, one can extract three main criticisms:

2.1 Search strategy

In contrast to the statement in the commentary, we formulated standard and correct search terms and reviewed the results thoroughly. The search terms were based on the requirements of the Cochrane Handbook, using the methods of “combining subject terms with free words”, “combining different expressions of the same concept with OR”, “using as few operators as possible NOT” and “no time limit”. Meanwhile, to ensure the accuracy of the search, we added all types of compounds included in the family of carotenoids to the search terms, including carotenoids[Mesh] OR carotene OR beta-carotene[Mesh] OR alpha-carotene OR beta-carotene OR cryptoxanthin OR retinoids[Mesh] OR canthaxanthin[Mesh] OR lutein[Mesh] OR lycopene OR zeaxanthin OR zeaxanthin[Mesh]. In accordance with the requirements of the Cochrane Handbook, our researchers viewed the search results broadly and carefully to ensure that all retractions (e.g., as a result of fraudulent publications) and errata were identified. Furthermore, two researchers independently evaluated all studies and extracted the final eligible literature. For disagreements between the works of these two researchers, Dr. Cui, who is one of the co-authors and an expert in the field of nutrition, discussed these matters and helped resolve the disagreements.

2.2. Inclusion and exclusion criteria

In their commentary, the authors repeatedly cited the results of their own search^23–35 to claim that the included literature for this study was inadequate and to suggest that their search results needed to be incorporated. However, these papers were excluded because they did not meet the inclusion criteria of this study.

According to the study objectives, inclusion and exclusion criteria were developed after deliberation. The inclusion criteria of the RCTs in our meta-analysis were as follows: (1) RCTs compared carotenoid intervention and noncarotenoid intervention groups; (2) obesity or being overweight was defined based on a local criterion; and (3) the outcomes were quantitative data that could be extracted or calculated. Exclusion criteria were as follows: (1) single-arm studies or those without a placebo or a mixture of carotenoid and antioxidant intervention or unquantifiable doses of carotenoid intervention; (2) patients with cancer, who were pregnant, or were taking any medication that could influence carotenoid concentrations; and (3) nonhuman studies, reviews and conference literature.

Note that, first, we used rigorous criteria to ensure that the included RCTs were targeted to overweight or obese populations. In our research, in addition to the international standards to define overweight (BMI ≥ 25) and obese (BMI > 30) individuals, two definitions involving local standards were used: (1) an overweight individual was defined as one having a BMI for AGE > Z-score + 1 and ≤ + 2, and an obese individual was defined as one having a BMI for AGE > Z-score + 2; and (2) those with BMI-for-age between the 85^th and 95th percentiles were considered to be overweight, and those with BMI-for-age ≥ 95th percentiles were considered to be obese. A study was excluded if any member of the study population was neither overweight nor obese, or if the study population was not described as overweight or obese. These standards were included mainly to ensure the consistency of the study population and to improve the credibility and scientific validity of the study.

Second, we excluded literature that described an inclusion of a mixture of carotenoid and antioxidant interventions. We did so because certain nutrients, such as vitamin E, have antioxidant^36,37 functions similar to those of carotenoids and have been shown to have anti-obesity abilities³⁸—and an inclusion of intervention experiments with mixtures of nutrients and carotenoids could have led to incorrect interpretations of the results and affect the credibility of the study.

Finally, we excluded studies combining obesity with metabolic diseases such as diabetes, meaning that we only included patients with metabolically healthy obesity. Metabolically healthy obesity is conceptualised in various ways and usually defined as the absence of any metabolic disorders and cardiovascular disease, including type 2 diabetes, dyslipidaemia, hypertension and atherosclerotic cardiovascular disease (ASCVD) in obese people.³⁹ The physical status of metabolically healthy overweight and obese patients differed from that of overweight and obese patients with metabolic diseases. For example, obese diabetic patients have abnormal states including β-cell dysfunction,⁴⁰ and obese patients with metabolic syndrome are in pathological states of endoplasmic reticulum (ER) stress.^41,42 Furthermore, metabolic diseases may have an impact on the digestion and absorption of carotenoids, which could be detrimental to the analysis of the results of this study. One investigation showed that the concentrations of α-, β-carotenoids and the sum of the five most-studied carotenoids introduced above decreased significantly in patients with metabolic syndrome.⁴³ A cross-sectional study showed diabetic patients having low carotenoid levels, with statistically significant differences between them and healthy patients in the concentration of β-carotene, which is a risk factor for type 2 diabetes.⁴⁴ The basic information and reasons for exclusion of some literature^23–35 are shown in Table S1.†

2.3. Heterogeneity

To analyze the sources of heterogeneity in the observational studies, we performed subgroup analysis based on age (n = 7), sex (n = 8), region (n = 8), population type (n = 8) and carotenoid type (n = 8). Additionally, we used subgroup analyses based on the intervention time, region, population type and population sex to evaluate the source of heterogeneity for the RCT. Despite our having considered that different types of carotenoids might have varied effects on the obese population, we were limited in our ability to implement subgroup analysis by the number of articles available. It is anticipated that this area can be investigated thoroughly in the future to remedy this shortcoming.

Meta-regression, although a common method to address heterogeneity of effects between studies, was not carried out in this research. According to Yusuf S, the number of studies or sample sizes in a given subgroup of covariates may be too sparse for reliable inferences to be made using meta-regression.⁴⁵ Therefore, most experts recommend that not more than 1 covariate be evaluated in a single model/analysis for every 10 studies in the meta-analysis.⁴⁶ This would mean that at least 20 studies would be required to run a ‘multivariable’ (i.e., two or more covariates being addressed at the same time) meta-regression.⁴⁷ Obviously, our study did not satisfy these requirements for carrying out such a meta-regression.

3. Conclusions

We conducted a meta-analysis of all related studies to evaluate the association between carotenoids and overweight or obese subjects by developing a standardized search format, rigorously screening the literature, and performing all feasible analyses on the included literature. Consequently, we considered that our study has made a contribution to revealing the significance of carotenoids in excess bodyweight populations.

Author contributions

NY and SMY designed the research; NY, XTL, LW and WYH conducted the research; NY and SMY wrote paper; and NY had primary responsibility for the final content. All authors read and agreed with the final manuscript.

Conflicts of interest

The authors have no conflicts to declare.

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Footnote

† Electronic supplementary information (ESI) available: Table S1: The basic information and reasons for exclusion of some literature. See DOI: 10.1039/d1fo02621f

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