|Year : 2019 | Volume
| Issue : 3 | Page : 69-75
Modulation of plasma triglycerides concentration by sterol-based treatment in subjects carrying specific genes
Ismael San Mauro Martin1, Javier Andrés Blumenfeld Olivares2, Elena Garicano Vilar1, María José Ciudad Cabañas3, Luis Collado Yurrita3
1 Research Centers in Nutrition and Health, Madrid, Spain
2 Pediatric Unit, El Escorial Hospital, San Lorenzo de El Escorial, Madrid, Spain
3 Department of Medicine, Faculty of Medicine, Complutense University of Madrid, Madrid, Spain
|Date of Submission||15-Apr-2019|
|Date of Acceptance||12-Jun-2019|
|Date of Web Publication||08-Nov-2019|
Dr. Ismael San Mauro Martin
Research Centers in Nutrition and Health, Paseo De La Habana, 43, Madrid 28036
Source of Support: None, Conflict of Interest: None
Introduction: Genetic load may indirectly influence on cardiovascular risk. This work aimed to analyze the influence of polymorphisms (APOA5 C56G-Ser19Trp, Prothrombin-G20210A, F5 Arg506Gln, MTHFR-C677T, LIPC-C-514T, LPA-I4300M, PPAR_ALPHA-L162V, APOA5-1131T > C, APOE-APOE2/3/4, and APOE-APOE2,3,4) in plasma triglyceride (TG) levels of patients ingesting plant sterols. Materials and Methods: Double-blind, crossover, controlled clinical trial was performed in 45 individuals (25 women). About 2.24 g sterols in milk and placebo milk were ingested daily during 3 weeks each, separated by a 2-week washout period. Blood draws and saliva genomic DNA was extracted. Results: APOA5-C56G-Ser19Trp, MTHFR-C677T, and PPAR_ALPHA-L162V greatly benefit from sterols intake. APOA5-C56G-Ser19Trp GG homozygous carriers lowered their TGs more than CG heterozygote carriers (P = 0.003). TT homozygous carriers of gene LIPC C-514T experienced an increase of TGs. Conclusions: Further studies are needed to establish which genotype combinations is the most protective against hypertriglyceridemia.
Keywords: Cardiovascular disease, genetic, nutrigenomic, plant sterol, triglycerides
|How to cite this article:|
San Mauro Martin I, Blumenfeld Olivares JA, Vilar EG, Ciudad Cabañas MJ, Collado Yurrita L. Modulation of plasma triglycerides concentration by sterol-based treatment in subjects carrying specific genes. Res Cardiovasc Med 2019;8:69-75
|How to cite this URL:|
San Mauro Martin I, Blumenfeld Olivares JA, Vilar EG, Ciudad Cabañas MJ, Collado Yurrita L. Modulation of plasma triglycerides concentration by sterol-based treatment in subjects carrying specific genes. Res Cardiovasc Med [serial online] 2019 [cited 2019 Nov 14];8:69-75. Available from: http://www.rcvmonline.com/text.asp?2019/8/3/69/270584
| Introduction|| |
Plasma triglyceride (TG) concentration is a complex polygenic trait that follows a skewed distribution in the population. It is a strong independent risk factor for cardiovascular disease (CVD), according to epidemiological evidence. There is frequent coexistence of elevated TGs with other conditions that affect CVD risk, such as depressed high-density lipoprotein (HDL) cholesterol, obesity, metabolic syndrome, pro-inflammatory and prothrombotic biomarkers, and type 2 diabetes.
Levels of TG above 100 mg/dl significantly increase the risk for heart attack. For each mmol/L increase in TGs (that is 88.5 mg/dL), the risk of coronary artery disease increases by 37% in women and 14% in men. Every percentage the TG level drops, so can the chance of heart disease or stroke. A man with a TG level of 300 mg/dL would have a risk of cardiovascular events roughly 28% higher than that of an otherwise comparable man who has a level of 100 mg/dL.
Common and rare variants in multiple genes, together with environmental influences, may collectively determine plasma TG concentration. The identification of patients with disordered metabolism susceptibility is enabled by the identification of genes and genetic variants associated with plasma-TG concentration. This identification allows for ameliorating CVD risk, thanks to the development of therapeutic interventions to improve plasma-TG concentration.
Global Lipids Genetics Consortium has provided in vitro and in vivo evidence supporting the involvement of specific genes in plasma TG metabolism. Naturally-occurring variants of the apolipoprotein A5 gene have been associated with increased TG levels and have been found to confer risk for CVDs. Functional analyses in vitro suggest that –1131T > C variant has minimal effect on APOA5 expression. The –1131T > C variant is also strong predictors of hypertriglyceridemia  and increased CVD risk. On the contrary, some studies did not observe differences between polymorphism 113G>T or 56C>G and plasmatic TGs concentrations. Some of the MTHFR gene polymorphisms are also associated with a congenital heart failure risk  and CVD, especially C677T genetic polymorphism. The hepatic lipase gene (LIPC) is responsible for the hydrolysis of TGs. Genetic studies and numerous epidemiologic studies have identified Lp (a) as a risk factor for atherosclerotic diseases such as coronary heart disease and stroke, as it is related to low-density lipoprotein cholesterol (LDL-c). In addition, genetic load may indirectly influence on cardiovascular risk. The APOE play an essential role in the catabolism of lipoproteins.
Complex interactions between genetic predisposition and the environment, in which genes manifest arise human health. The association of dietary recommendations with health will be influenced by individual differences in genetic variation, and this has yet to be reflected in dietary guidelines. Identifying the interplay between genes and dietary patterns holds promise for a new era of personalized medicine, whereby the recommended diet for best health is tailored toward how an individual's metabolism is genetically predisposed to respond to dietary intake. It would also serve as guide to intervene more effectively with dietary fiber, sterols, omega 3, and/or drugs.
Lipid metabolism is a well-defined responder to dietary intakes and a classic biomarker of cardiovascular health. For this reason, circulating lipid levels have become the key in shaping nutritional recommendations for better management of CVD.
A number of cholesterol-related gene-diet interactions are confirmed. Plant sterols/stanols are bioactive components with similar functions as that of cholesterol in mammals. They have been postulated as beneficial regulator agents for the control of CVD.,, Daily consumption of phytosterol-enriched foods is widely used as a therapeutic option to improve lipid levels in plasma. This has already been observed in studies that studied other lipids, such as LDL cholesterol.
Some early reports found moderately elevated plant sterol levels to be positively associated with vascular disease, although others suggested an inverse or lack of relationship between circulating plant sterols and cardiovascular risk.,
Plasma TG can be reduced by an average of 6%–9% by 2 g of plant sterols a day in hypertriglyceridemic patients, although this evidence warrants further evaluation. Future studies should be carried out with the aim of learning the increasing difference of dietary treatment data available in concordance with individual genomic profiles.
The aim of this study was to analyze the influence of polymorphisms APOA5 C56G Ser19Trp, Prothrombin G20210A, F5 Arg506Gln, MTHFR C677T, LIPC C-514T, LPA I4300M, PPAR_ALPHA L162V, APOA5 1131T>C, APOE APOE2/3/4, and APOE APOE2, 3, 4 in plasma TG levels of patients following a dietary treatment with plant sterols.
| Materials and Methods|| |
The study was designed as a randomized, double-blind, crossover, controlled clinical trial. Volunteers from primary care and endocrinology consultations at Hospital Clínico San Carlos and Hospital El Escorial, in Madrid, were recruited to participate. The purpose of the study was explained to participants prior to the trial, and those who voluntarily decided to participate signed a written informed consent form. The study was approved by the Bioethics Committees of Hospital Universitario Puerta de Hierro Majadahonda, in Madrid, and have followed the principles outlined in the Declaration of Helsinki for all human experimental investigations.
During a 3-week period, patients ingested daily two glasses (350 ml each) of either Naturcol milk with 2.24 g of plant sterols (supplied by Corporación Alimentaria Peñasanta, S. A., Asturias, Spain) or commercial skimmed milk, both available at the market during the study. The minimum consumption threshold was 80%. Treatment milk was composed by skimmed cow milk, plant sterols, and tea extract. Placebo skimmed milk was identical in sensorial and nutrient composition. Milk was presented in bottles with different color lids and no label to ensure neither the patients or the researchers were aware of the intervention assigned to each participant.
Groups were randomly distributed using random number tables. The intervention consisted of two phases of 3 weeks each separated by a 2-week washout period [Figure 1]. All patients participated in both groups. A 2-week wash system was implanted taking into account the metabolism and excretion process and that the functionality of the sterol in the human is not >2 days. Blood samples were taken at the beginning and at the end of each phase.
This study followed the ethical principles enshrined in the Helsinki Declaration, the recommendations for good clinical practice, current Spanish legislation regulating clinical research in humans, and protection of personal and bioethical data (Royal Decree 561/1993 on clinical trials and 14/2007, 3 July for biomedical research).
Forty-five patients completed the study – 25 women and 20 men with a mean age of 37.9 ± 7.5 years. Nine patients failed to complete the trial because of different genotyping and failure on completing plant sterols treatment (ingested <80% of consumption threshold).
The sample size calculation is performed by comparison of means, taking into account an estimated 10% drop:
A 95% confidence interval (CI) and a statistical power of 90% were used to calculate the sample size.
Inclusion and exclusion criteria
Men and women, aged 18–50 years, at cardiovascular risk according to lipid profile.
Cardiac pathology (myocardial infarction, angina); lactose intolerance, allergy to cow's milk proteins or plant sterols; obesity (body mass index [BMI] – >30); or pharmacological treatment (for cholesterol or fibrate TGs, statins) were excluded from the study. Patients already supplemented with plant sterols. Participants presenting active thyroid disease, liver disease, alcoholism, or any other conditions that dynamically alter lipid levels and/or diets.
Medical staff extracted analytical tests samples after a 12-h fast at the Clinical Analysis Unit in Hospital Clínico San Carlos and Hospital El Escorial, and in the San Carlos Specialty Unit in San Lorenzo de El Escorial.
The blood extraction was performed using the “blood collection with s-Monovette in aspiration” technique ,, with gel serum tubes. The samples were kept at 5°C ± 3°C after extraction, until the arrival at the laboratory. The centrifugation conditions were at T 20 20°C ± 5°C, for 10 min, at 1200 g. Stability was 1 week at 5°C ± 3°C. The ultraviolet-visible spectrophotometry technique was used.
The laboratory that performed the tests sits in Madrid and has the legal accreditations, certificates and standards (ISO 9001:2008 and accreditation 511/LE2114 according to criteria included in the UNE-EN ISO 15189 Standard).
Polymorphisms APOA5 C56G Ser19Trp, Prothrombin G20210A, F5 Arg506Gln, MTHFR C677T, LIPC C-514T, LPA I4300M, PPAR_ALPHA L162V, APOA5 1131T>C, APOE APOE2/3/4, and APOE APOE2, 3, 4 were studied.
Genomic DNA was extracted from saliva samples for genotyping the single-nucleotide polymorphisms. The genotyping was conducted using the Biobank Axiom1 96-Array from Affymetrix. Genotype calling was performed with respect to Affymetrix's best practice guideline including analysis with SNPolisher assuming a quality control rate of >0.97.,
The extraction and purification of DNA from saliva samples was carried out following the next procedure: to ensure high-quality and high molecular weight, each DNA extraction was run on agarose gel. To ensure maximum purity of the extracted DNA (ratio >1.7), the OD260/280 ratio was analyzed, that is, the optical density of the extracted DNA at 260 and 280 nm wavelength. All analyses were performed in duplicate, ensuring maximum precision and reliability in its results.
All laboratory analytical processes are carried in laboratories with Clinical Laboratory Improvement Amendment certification.
Additional variables and study factors
Anthropometric measurements were taken by a single trained researcher, ensuring the homogeneity and standardization of uniformity criteria and the methodology to follow. Weight, height, waist perimeter, BMI, fat percentage, subcutaneous fat percentage, and lean body mass (kg) were measured. Weight, BMI, and body composition were determined by means of tetrapolar multifrequency (20 and 100 kHz) electrical bioimpedance, InBody Model 230, following the usual standard protocol and the manufacturer's recommendations. Waist perimeter was measured with a flexible nonelastic metal measuring tape with a range of 0.1 mm–150 cm.
The TGs level was examined. Confounding factors were also taken into account with an affinity table after ingestion (>95%) and a record of food consumption frequencies in order to control the ingestion of foods that may influence the metabolism of lipids upward or downward; and by monitoring the nonmodification of baseline habits during the trial.
To see the effect of sterols on plasma TGs, only differences in the sterol group (not placebo) were observed and analyzed.
A descriptive analysis was first made of the sociodemographic and anthropometric data and the baseline and final lipid values under the ingestion of Naturcol and placebo. The normality of the lipid values was determined using the Shapiro–Wilk test. The efficacy of the intervention was verified by comparing the differences (final baseline) in the ingestion of milk with sterols and the placebo by applying Student's t-test for related samples, or the Wilcoxon rank sum test depending on the compliance with the assumption of normality of the lipid increases. The effect size and the proportion of the mean differences (MDs) were calculated with regard to the standard deviation of the baseline or milk with sterols as the case may be. The level of significance applied was 5%. Data were analyzed using the SPSS 21.0 (IBM Corp., Armonk, NY, U.S) statistical package.
| Results|| |
A total of 45 patients (25 women and 20 men) completed the trial. They had an average age of 37.9 ± 7.5 years and weighed 69 kg (BMI 23.7 kg/m2) [Table 1]. Baseline TGs were 115 mg/dl. There were no demographics differences between volunteer in both centers. [Table 2] shows descriptive statistic of genes and haplotypes.
|Table 1: Descriptive statistic of anthropometric measurements and lipid profile|
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APOA5 C56G Ser19Trp, MTHFR C677T, and PPAR_ALPHA L162V genes benefited the most from sterols intake in the diet [Figure 2]. In APOA5 C56G Ser19Trp carriers, GG homozygous carriers lowered their TGs more than CG heterozygote carriers, after ingestion of plant sterols (P = 0.003). A large difference can also be observed, in the case of LPA I4300M and MTHFR C677T genes, between the carriers of the homozygous (TT, CC, and TT, respectively) and heterozygous (TC and CT, respectively) variants, although the difference is no statistically significant (P = 0.121; P = 0.180). On the contrary to the previous cases, TT homozygous carriers of gene LIPC C-514T experienced an increase of TGs instead of lowering like CT heterozygote carriers.
|Figure 2: Percentage difference in triglyceride values with respect to before treatment, according to genes and haplotypes|
Click here to view
No statistically significant differences between the decrease percentage of TGs and the genotype were observed for the following genes: MTHFR C677T (P = 0.135), LIPC C-514T (P = 0.358), LPA I4300M (P = 0.132), APOA5 1131T > C (P = 0.703), APOE APOE2/3/4 (P = 0.599), and APOE APOE2, 3, 4 (P = 0.929). Only APOA5 C56G Ser19Trp and PPAR_ALPHA L162V genes incurred statistically significant changes on the levels of TG (P = 0.003; P = 0.001, respectively) after the treatment with sterols. Prothrombin G20210A and F5 Arg506Gln genes could not be subject to analytical study, because each gene had only one haplotype.
| Discussion|| |
Randomized clinical trials investigating a potential effect of sterol supplementation on hard clinical endpoints of atherosclerosis are still missing. However, important studies  evaluating the benefit of plant sterols consumption on cardiovascular risk biomarkers can still be found, as a meta-analysis of more than 40 clinical trials. A widely used therapeutic option to lower atherosclerotic disease risk and plasma cholesterol is the daily consumption of phytosterols-enriched foods. The matrix in which better effect is obtained is milk according to Clifton et al., who analyzed the differences of using plant sterols in different matrices.
The available data suggest that TG levels are reduced by 6%–20% at intakes of 1.5–2 g/day of plant sterol/stanol, with essentially no effect on HDL-cholesterol. Pooled analyses showed a modest reduction in plasma TGs of 6% and 4% for recommended intakes of plant sterols (1.6–2.5 g/day) or plant stanols (2 g/day), respectively. Indeed, evidence suggests a relationship between baseline TG levels and the magnitude of this effect, with 9% reduction when baseline TGs were 1.9 mmol/L (170 mg/dL), but no effect at baseline levels of 1.0 mmol/L (90 mg/dL).
On the contrary, four other trials reported that the TG levels were not significantly lower with sterol treatment MD-0.03 (95% CI-0.15–0.09) mmol/L, although there was considerable heterogeneity.,,,
Despite the clinical trial data demonstrating the cholesterol-lowering action of plant sterol supplementation, substantial variability in efficacy exists in responsiveness across individuals. Genetic explanations explaining this phenomenon appear to be gaining momentum. Lipid metabolism, of great importance on cardiovascular risk, is regulated by many genes whose variants may influence this risk. Genomic analysis was performed due to a variation in the effect of dietary treatment with plant sterols in milk on lipoprotein metabolism. With this analysis, we sought to understand which genetic polymorphisms could be associated with a higher or lower response effect to plant sterols treatment for hypertriglyceridemia.
Particularly, single-nucleotide polymorphisms within the genes coding for CYP7A1 and APOE, as well as possibly other genes including ABCG5 and ABCG8, exist as predictors of whether cholesterol levels will decrease or even increase subsequent to plant sterol administration.
Regarding the APOE2, its polymorphisms do not seem significantly related to coronary risk. On the contrary, it has been observed that the ε4 allele of the APOE4 is a major risk factor for coronary heart disease.
In mice, overexpression of the human APOA5 gene markedly decreases plasma TG concentration, whereas mice lacking the APOA5 gene become severely hypertriglyceridemic., The APOA5 C56G Ser19Trp allele can confer risk for development of large-vessel associated with stroke, exclusively. The APOA5 T-1131C allelic variant has been identified previously as a risk factor for all the stroke disease subgroups. The C allele variant increases the levels of TGs, and therefore, it is a risk factor for heart disease and ischemic stroke., This polymorphism has significant association with coronary heart disease risk. Thereby, the 56G allele differs from the latter APOA5 T-1131C allelic variant.
The relationship between the hepatic lipase gene and the influence on TGs seems to be an inverse relationship type, although it has not been clearly established. The influence that this gene has on the metabolism of glycerophospholipids  may also alter plasma concentrations thereof. Cardiometabolic parameters and cardiovascular risk factors were associated to the LIPC C-154T polymorphism by Posadas-Sánchez et al. Under a dominant model, the LIPC C-514T polymorphism was associated with increased hypertriglyceridemia (odds ratio [OR] = 1.36, P = 0.006) and coronary artery calcification (OR = 1.44, P = 0.015). The TT genotype, under dominant model too, was associated with increased levels of TGs/HDL cholesterol index (P = 0.046), as well as with the presence of small LDLs (P = 0.003).
The heterozygosity CT of the MTHFR C677T polymorphism increases the risk of congenital heart disease (OR = 2.249, 95% CI 1.305–3.877, P = 0.003), and the homozygous mutant genotype TT is associated with the risk of congenital heart disease (OR = 3.121, 95% CI 1.612–6.043, P = 0.001), compared with wild CC genotype.
Results suggest that genetic and metabolic biomarkers together may predict interindividual lipid level responsiveness to plant sterols intervention, and thus could be useful in devising individualized cholesterol lowering strategies. The results of these differences should undoubtedly be refuted by more research, as no accurate and concise explanation beyond them could be found. Samples of major population would help elucidate this issue. However, the classic nutrigenomics studies of polyunsaturated fatty acids and Framingham ,, have already observed these differences for years, as the mutation in some genes seemed not to fulfill the role of the gene. It has not supposed a clinical transcendence as suggestive as its important results announced.
| Conclusions|| |
Only APOA5 C56G Ser19Trp and PPAR_ALPHA L162V genes experienced statistically significant changes on the levels of TGs after the treatment with sterols, although LPA I4300M and MTHFR C677T genes also showed big differences between homozygous carriers (TT, CC, and TT, respectively) and heterozygous (TC and CT, respectively) variants, but with no statistically significant differences.
Further research on the genes and variants that modulate plasma TG would help develop markers for risk prediction and response to therapies.
The sample size is the major limitation of this study. It limits the extrapolation of the results to the Spanish population. A bigger sample would incur in a more sophisticated design and greater budget, which limits the number of publications aligned in a deeper analysis.
We would like to thank Corporación Alimentaria Peñasanta, S.A, the clinical analysis unit at Hospital Clínico San Carlos and Hospital El Escorial, in Madrid, and the Medicine Department of Universidad Complutense de Madrid and Microcaya S.A. for their collaboration.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]