Carotenoids in familial hypobetalipoproteinemia disorders: Malabsorption in Caco2 cell models and severe deficiency in patients.

BACKGROUND: Familial hypobetalipoproteinemias (FHBL) are rare genetic diseases characterized by lipid malabsorption. We focused on abetalipoproteinemia (FHBL-SD1) and chylomicron retention disease (FHBL-SD3), caused by mutations in microsomal triglyceride transfer protein (MTTP) and SAR1B genes, respectively. Treatments include a low-fat diet and high-dose fat-soluble vitamin supplementations. However, patients are not supplemented in carotenoids, a group of lipid-soluble pigments essential for eye health. OBJECTIVE: Our aim was to evaluate carotenoid absorption and status in the context of hypobetalipoproteinemia. METHODS: We first used knock-out Caco-2/TC7 cell models of FHBL-SD1 and FHBL-SD3 to evaluate carotenoid absorption. We then characterized FHBL-SD1 and FHBL-SD3 patient status in the main dietary carotenoids and compared it to that of control subjects. RESULTS: In vitro results showed a significant decrease in basolateral secretion of α- and ß-carotene, lutein, and zeaxanthin (-88.8 ± 2.2 % to -95.3 ± 5.8 %, -79.2 ± 4.4 % to -96.1 ± 2.6 %, -91.0 ± 4.5 % to -96.7 ± 0.3 % and -65.4 ± 3.6 % to -96.6 ± 1.9 %, respectively). Carotenoids plasma levels in patients confirmed significant deficiencies, with decreases ranging from -89 % for zeaxanthin to -98 % for α-carotene, compared to control subjects. CONCLUSION: Given the continuous loss in visual function despite fat-soluble vitamin treatment in some patients, carotenoid supplementation may be of clinical utility. Future studies should assess the correlation between carotenoid status and visual function in aging patients and investigate whether carotenoid supplementation could prevent their visual impairment.

Validation of Knock-Out Caco-2 TC7 Cells as Models of Enterocytes of Patients with Familial Genetic Hypobetalipoproteinemias.

Abetalipoproteinemia (FHBL-SD1) and chylomicron retention disease (FHBL-SD3) are rare recessive disorders of lipoprotein metabolism due to mutations in MTTP and SAR1B genes, respectively, which lead to defective chylomicron formation and secretion. This results in lipid and fat-soluble vitamin malabsorption, which induces severe neuro-ophthalmic complications. Currently, treatment combines a low-fat diet with high-dose vitamin A and E supplementation but still fails in normalizing serum vitamin E levels and providing complete ophthalmic protection. To explore these persistent complications, we developed two knock-out cell models of FHBL-SD1 and FHBL-SD3 using the CRISPR/Cas9 technique in Caco-2/TC7 cells. DNA sequencing, RNA quantification and Western blotting confirmed the introduction of mutations with protein knock-out in four clones associated with i) impaired lipid droplet formation and ii) defective triglyceride (-57.0 ± 2.6% to -83.9 ± 1.6%) and cholesterol (-35.3 ± 4.4% to -60.6 ± 3.5%) secretion. A significant decrease in α-tocopherol secretion was also observed in these clones (-41.5 ± 3.7% to -97.2 ± 2.8%), even with the pharmaceutical forms of vitamin E: tocopherol-acetate and tocofersolan (α-tocopheryl polyethylene glycol succinate 1000). MTTP silencing led to a more severe phenotype than SAR1B silencing, which is consistent with clinical observations. Our cellular models thus provide an efficient tool to experiment with therapeutic strategies and will allow progress in understanding the mechanisms involved in lipid metabolism.

Nutri-Score: Its Benefits and Limitations in Children's Feeding.

Reducing the burden of noncommunicable diseases (NCDs) is one of the top priorities of public health policies worldwide. One of the recognized means of achieving this objective is to improve the diet quality. The Nutri-Score (N-S) is a [five-color-A, B, C, D, E letters] front-of-pack labeling logo intended to help consumers quickly identify the healthier prepackaged foods within a food category. Available studies have shown that the N-S is an efficient tool to achieve this aim in terms of consumers' awareness, perception, understanding, and purchasing and that its use may help to reduce the prevalence of NCDs. The N-S is currently implemented on a voluntary basis in 7 European countries and a discussion is underway within the European Commission to achieve a harmonized mandatory label. However, no study on the putative impact of the N-S on children's dietary patterns and health is available. The N-S is not applicable to infants' and young children's formulas and to specific baby foods, the compositions of which are already laid down in European Union regulations. The N-S does not replace age-appropriate dietary guidelines. As children consume an increasing number of adult type and processed foods, the relevance of the N-S for children should be evaluated considering the children's high specific requirements, especially in younger children. This is especially necessary for fitting fat and iron requirements, whereas protein-rich foods should be better framed. Moreover, efforts should be made to inform on how to use the N-S and in education on healthy diets.

Guidance for the diagnosis and treatment of hypolipidemia disorders.

The Abetalipoproteinemia and Related Disorders Foundation was established in 2019 to provide guidance and support for the life-long management of inherited hypocholesterolemia disorders. Our mission is "to improve the lives of individuals and families affected by abetalipoproteinemia and related disorders". This review explains the molecular mechanisms behind the monogenic hypobetalipoproteinemia disorders and details their specific pathophysiology, clinical presentation and management throughout the lifespan. In this review, we focus on abetalipoproteinemia, homozygous hypobetalipoproteinemia and chylomicron retention disease; rare genetic conditions that manifest early in life and cause severe complications without appropriate treatment. Absent to low plasma lipid levels, in particular cholesterol and triglyceride, along with malabsorption of fat and fat-soluble vitamins are characteristic features of these diseases. We summarize the genetic basis of these disorders, provide guidance in their diagnosis and suggest treatment regimens including high dose fat-soluble vitamins as therapeutics. A section on preconception counseling and other special considerations pertaining to pregnancy is included. This information may be useful for patients, caregivers, physicians and insurance agencies involved in the management and support of affected individuals.

Lipids Responsible for Intestinal or Hepatic Disorder: When to Suspect a Familial Intestinal Hypocholesterolemia?

ABSTRACT: Familial intestinal hypocholesterolemias, such as abetalipoproteinemia, hypobetalipoproteinemia, and chylomicron retention disease, are rare genetic diseases that result in a defect in the synthesis or secretion of lipoproteins containing apolipoprotein B.In children, these conditions present with diarrhoea and growth failure, whereas adults present with neuromuscular, ophthalmological, and hepatic symptoms. Simple laboratory investigations have shown that diagnosis can be made from findings of dramatically decreased cholesterol levels, deficiencies in fat-soluble vitamins (mostly vitamin E), endoscopic findings of the characteristic white intestinal mucosa, and fat-loaded enterocytes in biopsy samples. Genetic analysis is used to confirm the diagnosis. Treatment is based on a low-fat diet with essential fatty acid supplementation, high doses of fat-soluble vitamins, and regular and life-long follow-up.The present study examines cases and literature findings of these conditions, and emphasises the need to explore severe hypocholesterolemia and deficiencies in fat-soluble vitamins to not miss these rare, but easy to diagnose and treat, disorders.

Religious dietary rules and their potential nutritional and health consequences.

BACKGROUND: The vast majority of the world population declares affiliation to a religion, predominantly Christianity and Islam. Many religions have special dietary rules, which may be more or less strictly adhered to. METHODS: Religious food rules were collected from holy books and religious websites as well as their translation into dietary practices. The literature was searched for potential associations between these rules and potential nutritional consequences. RESULTS: Jewish, Islamic and Indian religions support prolonged breastfeeding. Religious avoidance of alcohol is probably beneficial to health. When strictly applied, a few rules may lead to nutritional inadequacies, mainly in populations living in unfavourable socio-economic or environmental conditions. In Jewish and Muslim observants, animal slaughtering procedures may increase the risk of iron deficiency. Jews may be at risk of excess sodium intake related to home-prepared foods. A vegan diet, as observed by some believers, often by drifting from original precepts, or by some Hindus or Buddhists, may result in vitamin B12, calcium, iron, zinc, selenium and n-3 fatty acids deficiencies. CONCLUSION: When implemented in accordance with the rules, most religious food precepts are not detrimental to health, as suggested by the fact that they have more or less been followed for millennia. Nevertheless, some practices may lead to nutritional inadequacies, such as iron, calcium, vitamin D and vitamin B12 deficiencies. Patients with low socio-economic status, children and women of childbearing age are of particular risk of such deficiencies. Being aware of them should help health professionals to take an individualized approach to decide whether to supplement or not.

Comparison of α-Tocopherol, α-Tocopherol Acetate, and α-Tocopheryl Polyethylene Glycol Succinate 1000 Absorption by Caco-2 TC7 Intestinal Cells.

(1) Background: vitamin E is often supplemented in the form of tocopherol acetate, but it has poor bioavailability and can fail to correct blood tocopherol concentrations in some patients with severe cholestasis. In this context, α-tocopheryl polyethylene glycol succinate 1000 (TPGS) has been of value, but very little is known about the mechanisms of its absorption. The aim of our work was to evaluate the mechanisms of absorption/secretion of TPGS compared to tocopherol acetate (TAC) and α-tocopherol by human enterocyte-like Caco-2 TC7 cells. (2) Methods: two weeks post-confluence Caco-2 cells were incubated with tocopherol- or TAC- or TPGS-rich mixed micelles up to 24 h and, following lipid extraction, TAC and tocopherol amounts were measured by high performance liquid chromatography (HPLC) in apical, cellular, and basolateral compartments. (3) Results: at equivalent concentrations of tocopherol in the apical side, the amounts of tocopherol secreted at the basolateral pole of Caco-2 cells are (i) significantly greater when the tocopherol is in the free form in the micelles; (ii) intermediate when it is in the TAC form in the micelles (p < 0.001); and (iii) significantly lower with the TPGS form (p < 0.0001). Interestingly, our results show, for the first time, that Caco-2 cells secrete one or more esterified forms of the vitamin contained in TPGS at the basolateral side.

Normal serum ApoB48 and red cells vitamin E concentrations after supplementation in a novel compound heterozygous case of abetalipoproteinemia.

BACKGROUND AND AIMS: Abetalipoproteinemia (ABL) is a rare recessive monogenic disease due to MTTP (microsomal triglyceride transfer protein) mutations leading to the absence of plasma apoB-containing lipoproteins. Here we characterize a new ABL case with usual clinical phenotype, hypocholesterolemia, hypotriglyceridemia but normal serum apolipoprotein B48 (apoB48) and red blood cell vitamin E concentrations. METHODS: Histology and MTP activity measurements were performed on intestinal biopsies. Mutations in MTTP were identified by Sanger sequencing, quantitative digital droplet and long-range PCR. Functional consequences of the variants were studied in vitro using a minigene splicing assay, measurement of MTP activity and apoB48 secretion. RESULTS: Intestinal steatosis and the absence of measurable lipid transfer activity in intestinal protein extract supported the diagnosis of ABL. A novel MTTP c.1868G>T variant inherited from the patient's father was identified. This variant gives rise to three mRNA transcripts: one normally spliced, found at a low frequency in intestinal biopsy, carrying the p.(Arg623Leu) missense variant, producing in vitro 65% of normal MTP activity and apoB48 secretion, and two abnormally spliced transcripts resulting in a non-functional MTP protein. Digital droplet PCR and long-range sequencing revealed a previously described c.1067+1217_1141del allele inherited from the mother, removing exon 10. Thus, the patient is compound heterozygous for two dysfunctional MTTP alleles. The p.(Arg623Leu) variant may maintain residual secretion of apoB48. CONCLUSIONS: Complex cases of primary dyslipidemia require the use of a cascade of different methodologies to establish the diagnosis in patients with non-classical biological phenotypes and provide better knowledge on the regulation of lipid metabolism.

Efficacy of two vitamin E formulations in patients with abetalipoproteinemia and chylomicron retention disease.

Abetalipoproteinemia (ABL) and chylomicron retention disease (CMRD) are extremely rare recessive forms of hypobetalipoproteinemia characterized by intestinal lipid malabsorption and severe vitamin E deficiency. Vitamin E is often supplemented in the form of fat-soluble vitamin E acetate, but fat malabsorption considerably limits correction of the deficiency. In this crossover study, we administered two different forms of vitamin E, tocofersolan (a water-soluble derivative of RRR-α-tocopherol) and α-tocopherol acetate, to three patients with ABL and four patients with CMRD. The aims of this study were to evaluate the intestinal absorption characteristics of tocofersolan versus α-tocopherol acetate by measuring the plasma concentrations of α-tocopherol over time after a single oral load and to compare efficacy by evaluating the ability of each formulation to restore vitamin E storage after 4 months of treatment. In patients with ABL, tocofersolan and α-tocopherol acetate bioavailabilities were extremely low (2.8% and 3.1%, respectively). In contrast, bioavailabilities were higher in patients with CMRD (tocofersolan, 24.7%; α-tocopherol acetate, 11.4%). Plasma concentrations of α-tocopherol at 4 months were not significantly different by formulation type in ABL or CMRD. This study provides new insights about vitamin E status in ABL and CMRD and suggests the potential of different formulations as treatment options.

Non-alcoholic fatty liver disease severity and metabolic complications in obese children: impact of omega-3 fatty acids.

Although n-3 polyunsaturated fatty acids (PUFA) revealed promising therapeutic results in non-alcoholic fatty liver disease (NAFLD), which is considered as the most prevalent cause of chronic hepatic disease, inconsistencies are calling for further confirmatory trials to demonstrate therapeutic efficacy and safety. The study, registered as NCT02201160 on www.clinicaltrials.gov, was designed to compare two groups of NAFLD with a different severity, and to evaluate the efficacy of n-3 PUFA supplementation. Twenty young male participants of French Canadian origin with NAFLD were enrolled and classified into moderate (mNAFLD) and severe (sNAFLD) fatty liver groups, according to transaminase levels, ultrasonography, NAFLD Activity Score and Fatty Liver Index (FLI). The sNAFLD patients were assigned to consume 2 g of n-3 PUFA for 6 months. sNAFLD patients displayed higher insulinemia, insulin resistance (IR), oxidative stress (OxS), systolic blood pressure and the risk lipid indicators of cardiovascular diseases. Supplementation of n-3 PUFA for 6 months resulted in a significant increase in concentrations of eicosapentaenoic and docosahexaenoic acids in red blood cells along with an attenuation of hepatic steatosis as reflected by the reduction of the FLI, ALT and ALT/AST ratio. Moreover, the n-3 PUFA improved the lipid profile and carotid intima-media thickness, while reducing metabolic and OxS markers as well as raising adiponectin. In conclusion, NAFLD severity was essentially related to IR. Treatment with n-3 PUFA has an evidently beneficial effect on liver steatosis and related metabolic abnormalities. Furthermore, the cross association of omega-3 index with cardiometabolic markers may serve as a predictor for cardiovascular risk disorders in NAFLD.

Dietary oxidized n-3 PUFA induce oxidative stress and inflammation: role of intestinal absorption of 4-HHE and reactivity in intestinal cells.

Dietary intake of long-chain n-3 PUFA is now widely advised for public health and in medical practice. However, PUFA are highly prone to oxidation, producing potentially deleterious 4-hydroxy-2-alkenals. Even so, the impact of consuming oxidized n-3 PUFA on metabolic oxidative stress and inflammation is poorly described. We therefore studied such effects and hypothesized the involvement of the intestinal absorption of 4-hydroxy-2-hexenal (4-HHE), an oxidized n-3 PUFA end-product. In vivo, four groups of mice were fed for 8 weeks high-fat diets containing moderately oxidized or unoxidized n-3 PUFA. Other mice were orally administered 4-HHE and euthanized postprandially versus baseline mice. In vitro, human intestinal Caco-2/TC7 cells were incubated with 4-hydroxy-2-alkenals. Oxidized diets increased 4-HHE plasma levels in mice (up to 5-fold, P < 0.01) compared with unoxidized diets. Oxidized diets enhanced plasma inflammatory markers and activation of nuclear factor kappaB (NF-κB) in the small intestine along with decreasing Paneth cell number (up to -19% in the duodenum). Both in vivo and in vitro, intestinal absorption of 4-HHE was associated with formation of 4-HHE-protein adducts and increased expression of glutathione peroxidase 2 (GPx2) and glucose-regulated protein 78 (GRP78). Consumption of oxidized n-3 PUFA results in 4-HHE accumulation in blood after its intestinal absorption and triggers oxidative stress and inflammation in the upper intestine.

Oil composition of high-fat diet affects metabolic inflammation differently in connection with endotoxin receptors in mice.

Low-grade inflammation observed in obesity is a risk factor for cardiovascular disease. Recent studies revealed that this would be linked to gut-derived endotoxemia during fat digestion in high-fat diets, but nothing is known about the effect of lipid composition. The study was designed to test the impact of oil composition of high-fat diets on endotoxin metabolism and inflammation in mice. C57/Bl6 mice were fed for 8 wk with chow or isocaloric isolipidic diets enriched with oils differing in fatty acid composition: milk fat, palm oil, rapeseed oil, or sunflower oil. In vitro, adipocytes (3T3-L1) were stimulated or not with lipopolysaccharide (LPS; endotoxin) and incubated with different fatty acids. In mice, the palm group presented the highest level of IL-6 in plasma (P < 0.01) together with the highest expression in adipose tissue of IL-1ß and of LPS-sensing TLR4 and CD14 (P < 0.05). The higher inflammation in the palm group was correlated with a greater ratio of LPS-binding protein (LBP)/sCD14 in plasma (P < 0.05). The rapeseed group resulted in higher sCD14 than the palm group, which was associated with lower inflammation in both plasma and adipose tissue despite higher plasma endotoxemia. Taken together, our results reveal that the palm oil-based diet resulted in the most active transport of LPS toward tissues via high LBP and low sCD14 and the greatest inflammatory outcomes. In contrast, a rapeseed oil-based diet seemed to result in an endotoxin metabolism driven toward less inflammatory pathways. This shows that dietary fat composition can contribute to modulate the onset of low-grade inflammation through the quality of endotoxin receptors.

Guidelines for the diagnosis and management of chylomicron retention disease based on a review of the literature and the experience of two centers.

Familial hypocholesterolemia, namely abetalipoproteinemia, hypobetalipoproteinemia and chylomicron retention disease (CRD), are rare genetic diseases that cause malnutrition, failure to thrive, growth failure and vitamin E deficiency, as well as other complications. Recently, the gene implicated in CRD was identified. The diagnosis is often delayed because symptoms are nonspecific. Treatment and follow-up remain poorly defined.The aim of this paper is to provide guidelines for the diagnosis, treatment and follow-up of children with CRD based on a literature overview and two pediatric centers 'experience.The diagnosis is based on a history of chronic diarrhea with fat malabsorption and abnormal lipid profile. Upper endoscopy and histology reveal fat-laden enterocytes whereas vitamin E deficiency is invariably present. Creatine kinase (CK) is usually elevated and hepatic steatosis is common. Genotyping identifies the Sar1b gene mutation.Treatment should be aimed at preventing potential complications. Vomiting, diarrhea and abdominal distension improve on a low-long chain fat diet. Failure to thrive is one of the most common initial clinical findings. Neurological and ophthalmologic complications in CRD are less severe than in other types of familial hypocholesterolemia. However, the vitamin E deficiency status plays a pivotal role in preventing neurological complications. Essential fatty acid (EFA) deficiency is especially severe early in life. Recently, increased CK levels and cardiomyopathy have been described in addition to muscular manifestations. Poor mineralization and delayed bone maturation do occur. A moderate degree of macrovesicular steatosis is common, but no cases of steatohepatitis cirrhosis. Besides a low-long chain fat diet made up uniquely of polyunsaturated fatty acids, treatment includes fat-soluble vitamin supplements and large amounts of vitamin E. Despite fat malabsorption and the absence of postprandial chylomicrons, the oral route can prevent neurological complications even though serum levels of vitamin E remain chronically low. Dietary counseling is needed not only to monitor fat intake and improve symptoms, but also to maintain sufficient caloric and EFA intake. Despite a better understanding of the pathogenesis of CRD, the diagnosis and management of the disease remain a challenge for clinicians. The clinical guidelines proposed will helpfully lead to an earlier diagnosis and the prevention of complications.