Metabolic signature of the pathogenic 22q11.2 deletion identifies carriers and provides insight into systemic dysregulation.

Large deletions at chromosome 22q11.2 are known to cause severe clinical conditions collectively known as 22q11.2 deletion syndrome. Notwithstanding the pathogenicity of these deletions, affected individuals are typically diagnosed in late childhood or early adolescence, and little is known of the molecular signaling cascades and biological consequences immediately downstream of the deleted genes. Here, we used targeted metabolomics to compare neonatal dried blood spot samples from 203 individuals clinically identified as carriers of a deletion at chromosome 22q11.2 with 203 unaffected individuals. A total of 173 metabolites were successfully identified and used to inform on systemic dysregulation caused by the genomic lesion and to discriminate carriers from non-carriers. We found 84 metabolites to be differentially abundant between carriers and non-carriers of the 22q11.2 deletion. A predictive model based on all 173 metabolites achieved high Accuracy (89%), Area Under the Curve (93%), F1 (88%), Positive Predictive Value (94%), and Negative Predictive Value (84%) with tyrosine and proline having the highest individual contributions to the model as well as the highest interaction strength. Targeted metabolomics provides insight into the molecular consequences possibly contributing to the pathology underlying the clinical manifestations of the 22q11 deletion and is an easily applicable approach to first-pass screening for carrier status of the 22q11 to prompt subsequent verification of the genomic diagnosis.

Effects of Long-Term Storage on the Biobanked Neonatal Dried Blood Spot Metabolome.

Over 2.5 million neonatal dried blood spots (DBS) are stored at the Danish National Biobank. These samples offer extraordinary possibilities for metabolomics research, including prediction of disease and understanding of underlying molecular mechanisms of disease development. Nevertheless, Danish neonatal DBS have been little explored in metabolomics studies. One question that remains underinvestigated is the long-term stability of the large number of metabolites typically assessed in untargeted metabolomics over long time periods of storage. Here, we investigate temporal trends of metabolites measured in 200 neonatal DBS collected over a time course of 10 years, using an untargeted liquid chromatography tandem mass spectrometry (LC-MS/MS) based metabolomics protocol. We found that a majority (71%) of the metabolome was stable during 10 years of storage at -20 °C. However, we found decreasing trends for lipid-related metabolites, such as glycerophosphocholines and acylcarnitines. A few metabolites, including glutathione and methionine, may be strongly influenced by storage, with changes in metabolite levels up to 0.1-0.2 standard deviation units per year. Our findings indicate that untargeted metabolomics of DBS samples, with long-term storage in biobanks, is suitable for retrospective epidemiological studies. We identify metabolites whose stability in DBS should be closely monitored in future studies of DBS samples with long-term storage.

Multi-omics to predict changes during cold pressor test.

BACKGROUND: The cold pressor test (CPT) is a widely used pain provocation test to investigate both pain tolerance and cardiovascular responses. We hypothesize, that performing multi-omic analyses during CPT gives the opportunity to home in on molecular mechanisms involved. Twenty-two females were phenotypically assessed before and after a CPT, and blood samples were taken. RNA-Sequencing, steroid profiling and untargeted metabolomics were performed. Each 'omic level was analyzed separately at both single-feature and systems-level (principal component [PCA] and partial least squares [PLS] regression analysis) and all 'omic levels were combined using an integrative multi-omics approach, all using the paired-sample design. RESULTS: We showed that PCA was not able to discriminate time points, while PLS did significantly distinguish time points using metabolomics and/or transcriptomic data, but not using conventional physiological measures. Transcriptomic and metabolomic data revealed at feature-, systems- and integrative- level biologically relevant processes involved during CPT, e.g. lipid metabolism and stress response. CONCLUSION: Multi-omics strategies have a great potential in pain research, both at feature- and systems- level. Therefore, they should be exploited in intervention studies, such as pain provocation tests, to gain knowledge on the biological mechanisms involved in complex traits.

Neonatal metabolome of caesarean section and risk of childhood asthma.

BACKGROUND: Birth by caesarean section is linked to an increased risk of developing asthma, but the underlying mechanisms are unclear. OBJECTIVE: To elucidate the link between birth by caesarean section and asthma using newborn metabolomic profiles and integrating early-life gut microbiome data and cord blood immunology. METHODS: We investigated the influence of caesarean section on liquid chromatography mass spectrometry metabolomic profiles of dried blood spots from newborns of the two independent Copenhagen Prospective Studies on Asthma in Childhood cohorts, i.e. COPSAC2010 (n=677) and COPSAC2000 (n=387). We assessed the associations between the caesarean section metabolic profile, gut microbiome data and frequency of cord blood regulatory T-cells (Tregs) at 1 week of age. RESULTS: In COPSAC2010, a partial least square discriminant analysis model showed that children born by caesarean section versus natural delivery had different metabolic profiles (area under the curve (AUC)=0.77, p=2.2×10-16), which was replicated in COPSAC2000 (AUC=0.66, p=1.2×10-5). The metabolic profile of caesarean section was significantly associated with an increased risk of asthma at school age in both COPSAC2010 (p=0.03) and COPSAC2000 (p=0.005). Caesarean section was associated with lower abundance of tryptophan, bile acid and phenylalanine metabolites, indicative of a perturbed gut microbiota. Furthermore, gut bacteria dominating after natural delivery, i.e. Bifidobacterium and Bacteroides were correlated with caesarean section-discriminative microbial metabolites, suggesting maternal microbial transmission during birth regulating the newborn's metabolism. Finally, the caesarean section metabolic profile was associated with frequency of cord blood Tregs. CONCLUSIONS: These findings propose that caesarean section programmes the risk of childhood asthma through perturbed immune responses and gut microbial colonisation patterns reflected in the blood metabolome at birth.

Genetic regulation of spermine oxidase activity and cancer risk: a Mendelian randomization study.

Spermine oxidase (SMOX) catalyzes the oxidation of spermine to spermidine. Observational studies have reported SMOX as a source of reactive oxygen species associated with cancer, implying that inhibition of SMOX could be a target for chemoprevention. Here we test causality of SMOX levels with cancer risk using a Mendelian randomization analysis. We performed a GWAS of spermidine/spermine ratio to identify genetic variants associated with regulation of SMOX activity. Replication analysis was performed in two datasets of SMOX gene expression. We then did a Mendelian randomization analysis by testing the association between the SMOX genetic instrument and neuroblastoma, gastric, lung, breast, prostate, and colorectal cancers using GWAS summary statistics. GWAS of spermidine/spermine ratio identified SMOX locus (P = 1.34 × 10-49) explaining 32% of the variance. The lead SNP rs1741315 was also associated with SMOX gene expression in newborns (P = 8.48 × 10-28) and adults (P = 2.748 × 10-8) explaining 37% and 6% of the variance, respectively. Genetically determined SMOX activity was not associated with neuroblastoma, gastric, lung, breast, prostate nor colorectal cancer (P > 0.05). A PheWAS of rs1741315 did not reveal any relevant associations. Common genetic variation in the SMOX gene was strongly associated with SMOX activity in newborns, and less strongly in adults. Genetic down-regulation of SMOX was not significantly associated with lower odds of neuroblastoma, gastric, lung, breast, prostate and colorectal cancer. These results may inform studies of SMOX inhibition as a target for chemoprevention.

Bioenergetic and Proteomic Profiling of Immune Cells in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients: An Exploratory Study.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a heterogeneous, debilitating, and complex disease. Along with disabling fatigue, ME/CFS presents an array of other core symptoms, including autonomic nervous system (ANS) dysfunction, sustained inflammation, altered energy metabolism, and mitochondrial dysfunction. Here, we evaluated patients' symptomatology and the mitochondrial metabolic parameters in peripheral blood mononuclear cells (PBMCs) and plasma from a clinically well-characterised cohort of six ME/CFS patients compared to age- and gender-matched controls. We performed a comprehensive cellular assessment using bioenergetics (extracellular flux analysis) and protein profiles (quantitative mass spectrometry-based proteomics) together with self-reported symptom measures of fatigue, ANS dysfunction, and overall physical and mental well-being. This ME/CFS cohort presented with severe fatigue, which correlated with the severity of ANS dysfunction and overall physical well-being. PBMCs from ME/CFS patients showed significantly lower mitochondrial coupling efficiency. They exhibited proteome alterations, including altered mitochondrial metabolism, centred on pyruvate dehydrogenase and coenzyme A metabolism, leading to a decreased capacity to provide adequate intracellular ATP levels. Overall, these results indicate that PBMCs from ME/CFS patients have a decreased ability to fulfill their cellular energy demands.

Changes in the gene expression profile during spontaneous migraine attacks.

Migraine attacks are delimited, allowing investigation of changes during and outside attack. Gene expression fluctuates according to environmental and endogenous events and therefore, we hypothesized that changes in RNA expression during and outside a spontaneous migraine attack exist which are specific to migraine. Twenty-seven migraine patients were assessed during a spontaneous migraine attack, including headache characteristics and treatment effect. Blood samples were taken during attack, two hours after treatment, on a headache-free day and after a cold pressor test. RNA-Sequencing, genotyping, and steroid profiling were performed. RNA-Sequences were analyzed at gene level (differential expression analysis) and at network level, and genomic and transcriptomic data were integrated. We found 29 differentially expressed genes between 'attack' and 'after treatment', after subtracting non-migraine specific genes, that were functioning in fatty acid oxidation, signaling pathways and immune-related pathways. Network analysis revealed mechanisms affected by changes in gene interactions, e.g. 'ion transmembrane transport'. Integration of genomic and transcriptomic data revealed pathways related to sumatriptan treatment, i.e. '5HT1 type receptor mediated signaling pathway'. In conclusion, we uniquely investigated intra-individual changes in gene expression during a migraine attack. We revealed both genes and pathways potentially involved in the pathophysiology of migraine and/or migraine treatment.

Integrating genetics with newborn metabolomics in infantile hypertrophic pyloric stenosis.

INTRODUCTION: Infantile hypertrophic pyloric stenosis (IHPS) is caused by hypertrophy of the pyloric sphincter muscle. OBJECTIVES: Since previous reports have implicated lipid metabolism, we aimed to (1) investigate associations between IHPS and a wide array of lipid-related metabolites in newborns, and (2) address whether detected differences in metabolite levels were likely to be driven by genetic differences between IHPS cases and controls or by differences in early life feeding patterns. METHODS: We used population-based random selection of IHPS cases and controls born in Denmark between 1997 and 2014. We randomly took dried blood spots of newborns from 267 pairs of IHPS cases and controls matched by sex and day of birth. We used a mixed-effects linear regression model to evaluate associations between 148 metabolites and IHPS in a matched case-control design. RESULTS: The phosphatidylcholine PC(38:4) showed significantly lower levels in IHPS cases (P = 4.68 × 10-8) as did six other correlated metabolites (four phosphatidylcholines, acylcarnitine AC(2:0), and histidine). Associations were driven by 98 case-control pairs born before 2009, when median age at sampling was 6 days. No association was seen in 169 pairs born in 2009 or later, when median age at sampling was 2 days. More IHPS cases than controls had a diagnosis for neonatal difficulty in feeding at breast (P = 6.15 × 10-3). Genetic variants known to be associated with PC(38:4) levels did not associate with IHPS. CONCLUSIONS: We detected lower levels of certain metabolites in IHPS, possibly reflecting different feeding patterns in the first days of life.

Studying Autism Using Untargeted Metabolomics in Newborn Screening Samples.

Main risk factors of autism spectrum disorder (ASD) include both genetic and non-genetic factors, especially prenatal and perinatal events. Newborn screening dried blood spot (DBS) samples have great potential for the study of early biochemical markers of disease. To study DBS strengths and limitations in the context of ASD research, we analyzed the metabolomic profiles of newborns later diagnosed with ASD. We performed LC-MS/MS-based untargeted metabolomics on DBS from 37 case-control pairs randomly selected from the iPSYCH sample. After preprocessing using MZmine 2.41, metabolites were putatively annotated using mzCloud, GNPS feature-based molecular networking, and MolNetEnhancer. A total of 4360 mass spectral features were detected, of which 150 (113 unique) could be putatively annotated at a high confidence level. Chemical structure information at a broad level could be retrieved for 1009 metabolites, covering 31 chemical classes. Although no clear distinction between cases and controls was revealed, our method covered many metabolites previously associated with ASD, suggesting that biochemical markers of ASD are present at birth and may be monitored during newborn screening. Additionally, we observed that gestational age, age at sampling, and month of birth influence the metabolomic profiles of newborn DBS, which informs us on the important confounders to address in future studies.

Gestational age-dependent development of the neonatal metabolome.

BACKGROUND: Prematurity is a severe pathophysiological condition, however, little is known about the gestational age-dependent development of the neonatal metabolome. METHODS: Using an untargeted liquid chromatography-tandem mass spectrometry metabolomics protocol, we measured over 9000 metabolites in 298 neonatal residual heel prick dried blood spots retrieved from the Danish Neonatal Screening Biobank. By combining multiple state-of-the-art metabolome mining tools, we retrieved chemical structural information at a broad level for over 5000 (60%) metabolites and assessed their relation to gestational age. RESULTS: A total of 1459 (~16%) metabolites were significantly correlated with gestational age (false discovery rate-adjusted P < 0.05), whereas 83 metabolites explained on average 48% of the variance in gestational age. Using a custom algorithm based on hypergeometric testing, we identified compound classes (617 metabolites) overrepresented with metabolites correlating with gestational age (P < 0.05). Metabolites significantly related to gestational age included bile acids, carnitines, polyamines, amino acid-derived compounds, nucleotides, phosphatidylcholines and dipeptides, as well as treatment-related metabolites, such as antibiotics and caffeine. CONCLUSIONS: Our findings elucidate the gestational age-dependent development of the neonatal blood metabolome and suggest that the application of metabolomics tools has great potential to reveal novel biochemical underpinnings of disease and improve our understanding of complex pathophysiological mechanisms underlying prematurity-associated disorders. IMPACT: A large variation in the neonatal dried blood spot metabolome from residual heel pricks stored at the Danish Neonatal Screening Biobank can be explained by gestational age. While previous studies have assessed the relation of selected metabolic markers to gestational age, this study assesses metabolome-wide changes related to prematurity. Using a combination of recently developed metabolome mining tools, we assess the relation of over 9000 metabolic features to gestational age. The ability to assess metabolome-wide changes related to prematurity in neonates could pave the way to finding novel biochemical underpinnings of health complications related to preterm birth.

[The role of the specialist nurse in parenteral nutrition : An observational study of complications with home parenteral nutrition in adult cancer patients] / Étude observationnelle prospective des complications chez l'adulte atteint de cancer et bénéficiant d'une nutrition parentérale à domicile.

Background : In France, home parenteral nutrition (HPN) is managed by two parallel healthcare systems : in approved specialist centers (HPN > 12 weeks), and outside of these approved specialist centers (HPN<12 weeks). Objective : To prospectively evaluate infectious and vascular complications in adult cancer patients undergoing HPN administered via a central venous line, outside of approved specialist HPN centers. Methods : Our observational prospective study included adult patients with cancer, hospitalized for 48 hours or more, and under HPN. They had a WHO performance status of ≤ 2 and had had a nutritional consultation before discharge. Results : 25 patients were included in the study, with a median age of 63 years [19­74]. Weight loss of ≥ 5% was reported in 79% of patients. The Ingesta score was < 7 in 96% of cases. 87% of patients presented chill or body temperature variation episodes, with a median of 2 episodes [1­6] per patient. The median delay between end of hospitalization and the first chill episode was 11 days [1­85]. A vascular complication (obstruction without thrombosis) was reported in one patient. Discussion : This high number of infectious episodes requires improvement of patient care when it comes to strictly adhering to the recommendations. Getting the approved specialist HPN centers to work together and share care protocols could be the first important step.

Étude observationnelle prospective des complications chez l'adulte atteint de cancer et bénéficiant d'une nutrition parentérale à domicile.

Background : In France, home parenteral nutrition (HPN) is managed by two parallel healthcare systems : in approved specialist centers (HPN > 12 weeks), and outside of these approved specialist centers (HPN<12 weeks).Objective : To prospectively evaluate infectious and vascular complications in adult cancer patients undergoing HPN administered via a central venous line, outside of approved specialist HPN centers.Methods : Our observational prospective study included adult patients with cancer, hospitalized for 48 hours or more, and under HPN. They had a WHO performance status of ≤ 2 and had had a nutritional consultation before discharge.Results : 25 patients were included in the study, with a median age of 63 years [19-74]. Weight loss of ≥ 5% was reported in 79% of patients. The Ingesta score was < 7 in 96% of cases. 87% of patients presented chill or body temperature variation episodes, with a median of 2 episodes [1-6] per patient. The median delay between end of hospitalization and the first chill episode was 11 days [1-85]. A vascular complication (obstruction without thrombosis) was reported in one patient.Discussion : This high number of infectious episodes requires improvement of patient care when it comes to strictly adhering to the recommendations. Getting the approved specialist HPN centers to work together and share care protocols could be the first important step.

Dietary habits, metabolic health and vitamin D status in Greenlandic children.

OBJECTIVE: To compare the dietary habits of children living in northern villages and in the capital of Greenland, given the reported transition from traditional to westernised diet in adults over recent decades, and to explore the association between consumption of marine mammals and fish (MMF) and the children's metabolic profile and vitamin D status. DESIGN: Children answered an FFQ encompassing sixty-four individual food types pooled into six food categories. Their pubertal stage, body fat, fitness level, metabolic profile (non-HDL-cholesterol, glycated Hb, insulin, glucose, high-sensitivity C-reactive protein) as well as serum 25-hydroxyvitamin D (25(OH)D) concentration were evaluated. SETTING: Siorapaluk and Qaanaaq (north of Greenland) and Nuuk (west). PARTICIPANTS: Children aged 6-18 years (n 177). RESULTS: MMF were most frequently eaten by children from Siorapaluk (mean (sd): 73·4 (14·1) times/month), followed by children from Qaanaaq (37·0 (25·0) times/month), and least often eaten by children from Nuuk (23·7 (24·6) times/month; P < 0·001). Children from Qaanaaq consumed 'junk food' more frequently (P < 0·001) and fruits and vegetables less frequently (P < 0·01) than children from Nuuk. MMF consumption was positively associated with serum 25(OH)D concentration (P < 0·05), but the overall prevalence of vitamin D deficiency was high (18 %). No association was found between MMF consumption and metabolic parameters. CONCLUSIONS: The dietary transition and influence of western diets have spread to the north of Greenland and only the most remote place consumed a traditional diet highly based on MMF. We found no strong associations of MMF consumption with metabolic health, but a positive association with vitamin D status.

Management of Cancer Cachexia and Guidelines Implementation in a Comprehensive Cancer Center: A Physician-Led Cancer Nutrition Program Adapted to the Practices of a Country.

CONTEXT: Cancer-associated cachexia is correlated with survival, side-effects, and alteration of the patients' well-being. OBJECTIVES: We implemented an institution-wide multidisciplinary supportive care team, a Cancer Nutrition Program (CNP), to screen and manage cachexia in accordance with the guidelines and evaluated the impact of this new organization on nutritional care and funding. METHODS: We estimated the workload associated with nutrition assessment and cachexia-related interventions and audited our clinical practice. We then planned, implemented, and evaluated the CNP, focusing on cachexia. RESULTS: The audit showed a 70% prevalence of unscreened cachexia. Parenteral nutrition was prescribed to patients who did not meet the guideline criteria in 65% cases. From January 2009 to December 2011, the CNP team screened 3078 inpatients. The screened/total inpatient visits ratio was 87%, 80%, and 77% in 2009, 2010, and 2011, respectively. Cachexia was reported in 74.5% (n = 2253) patients, of which 94.4% (n = 1891) required dietary counseling. Over three years, the number of patients with artificial nutrition significantly decreased by 57.3% (P < 0.001), and the qualitative inpatients enteral/parenteral ratio significantly increased: 0.41 in 2009, 0.74 in 2010, and 1.52 in 2011. Between 2009 and 2011, the CNP costs decreased significantly for inpatients nutritional care from 528,895€ to 242,272€, thus financing the nutritional team (182,520€ per year). CONCLUSION: Our results highlight the great benefits of implementing nutritional guidelines through a physician-led multidisciplinary team in charge of nutritional care in a comprehensive cancer center.

Meal context and food preferences in cancer patients: results from a French self-report survey.

PURPOSE: The present study examined patient self-reports of descriptions, experiences and consequences of meal disturbances and food preferences within a cultural context (i.e., French meal traditions) in various treated cancer patients along their disease trajectory. METHODS: Over 800 questionnaires were sent to 20 cancer treatment centres in France. During a 9-month period, 255 questionnaires were received from five centres. Inclusion criteria included those French patients over 18 years of age, could read and understand French, had an Eastern Cooperative Oncology Group score between 0 and 2, experienced treatment-induced nutrition changes and/or had decreased oral intake. Dietetic staff assessed clinical characteristics while patients completed a 17-item questionnaire. RESULTS: The majority of patients were diagnosed with breast, gastro-intestinal (GI) tract and head and neck cancers (62 %). Half of the patients (49 %) experienced weight loss >5 %. The main treatment-induced side effects were fatigue, nausea, dry mouth, hypersensitivity to odors and GI tract transit disorders. These discomforts affected eating and drinking in 83 % of patients, inducing appetite loss and selected food aversion. Food preference appeared heterogeneous. Food taste, odor and finally appearance stimulated appetite. Finally, dietary behaviors and satisfaction were driven by the extent to which food was enjoyed. CONCLUSIONS: During oncologic treatments, eating and drinking were affected in more than three-quarters of patients. As recommended by practice guidelines, nutritional assessment and follow-up are required. Personalized nutritional counseling should include the role of the family, patient's meal traditions, and food habits.

A phase III study evaluating oral glutamine and transforming growth factor-beta 2 on chemotherapy-induced toxicity in patients with digestive neoplasm.

BACKGROUND: Patients with gastrointestinal (GI) cancer are exposed to cachexia, which is highly correlated with chemotherapy-induced side effects. Research suggests that specific immunonutrients could prevent such toxicities. AIMS: The primary objective of this phase III study was to evaluate the efficacy of glutamine and transforming growth factor-ß2 (TGF-ß2) in the prevention of grade 3-4 non-hematological toxicities induced by chemotherapy in patients with GI cancer. PATIENTS AND METHODS: We designed a double-blind, randomized, controlled and multicenter trial stratified according to center, type of chemotherapy, presence of cachexia, and age. Patients were randomized to receive either Clinutren Protect(®) (CP) or a control isocaloric diet (without TGF-ß2 or glutamine). RESULTS: Between November 2007 and October 2011, 210 patients were enrolled in the study, of which 201 were included in the intention-to-treat analysis. Grade 3-4 non-hematological toxicities were not significantly different between the CP and control groups when evaluated by univariate and multivariate analyses. Likewise, no difference was observed regarding grade 3-4 hematological toxicities or reasons for treatment interruption. CONCLUSION: This randomized study does not support the hypothesis that oral glutamine and TGF-ß2 supplementation is effective to reduce grade 3 or 4 non-hematological toxicities induced by chemotherapy in patients with GI neoplasm.

Reproductive Decision-Making in MMR Mutation Carriers After Results Disclosure: Impact of Psychological Status in Childbearing Options.

Reproductive techniques such as prenatal diagnosis (PND) or preimplantation genetic diagnosis (PGD), although debated, are legally forbidden in France in case of Lynch syndrome. The preference of mutation carriers about their reproductive options is not systematically considered in France. We aimed to prospectively assess the reproductive preferences of mismatch repair mutation carriers consulting in our institution (2003-2010, n = 100). We also considered the short- and long-term post-disclosure psychological impact using the Impact of Events Scale-Revised questionnaire to measure the prevalence of posttraumatic stress disorder (PTSD) in those patients. Complete data were obtained for 34 respondents (17 males, 17 females, median age of 33.5 years [22-59]). Seventeen respondents (57 %) preferred spontaneous natural conception versus 28 % and 35 % choosing PND and PGD, respectively. At results disclosure, respondents mainly explained their distress by fear of premature death (43 %) and transmitting mutated genes (42 %). One year later, this last fear remained predominant in 55 % of subjects. None of the main socio-demographical, psychological or medical variables (including fear of transmitting mutations) was significantly associated with the reproductive preferences. Results disclosure had a real and time-decreasing psychological impact on mutation carriers. Reproductive techniques, expected to decrease the hereditary risk, were not significantly preferred to natural conception.

Stability and bioaccessibility of different forms of carotenoids and vitamin A during in vitro digestion.

Vitamin A deficiency is a public health issue in developing countries and promoting dietary carotenoids as precursors is a promising strategy. However, carotenoids present in numerous fruits and vegetables are unstable and poorly bioaccessible. This study evaluated these two parameters during in vitro digestion of carotenoids and retinoids from carrot juice, raw and cooked spinach, micronutrient-fortified flour and standards without food matrix. Standards were unstable whereas vitamin A from fortified flour and native food carotenoids were generally better protected by the food matrix (30-100% remaining versus 7-30% for standards). Hydrothermal cooking did not influence spinach carotenoid digestive stability but decreased their contents, phenomenon compensated by a significantly better micellarisation from 15-fold for ß-carotene to 72-fold for lutein. Finally, carrot juice provided the greatest amount of bioaccessible provitamin A with 1850 µg/100g dry matter (DM) versus 790 and 80 µg/100g DM in cooked and raw spinach, respectively.