Carotinoid-Bioaktivität (Subject) / Grundwissen (Lesson)

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• On what grounds is an interaction between carotenoids and TFs taking place? Carotenoids are lipophillic in nature, making them good candidates to transverse the cell membrane --> retinoic acid (RA) is an endogenous agonist of specific nuclear receptors, termed retinoid receptors all trans RA activates RAR 9 cis RA activates RXR these receptors modify transcription of specific genes or interfere with the activity of other TFs But other CCPs can interact with TFs as well!  -apo-10-lycopenoic acid has been shown  to inhibit lung cancer cell growth in vitro and suppress lung tumorogenesis in vivo -> it has also been shown to transactivate the RARß promotor (RA analogue) ß carotene metabolites such as ß-apo-14-carotenal are discussed as antagonists of NRs as well
• Which enzymatic possibilities for Carotenoid degradation do mammals possess? carotene-15,15'-oxygenase (BCO1) the key enzyme for retinoid production (tested in BCO1 deficient mouse model) Cytosolic protein, central cleavage of pro vitamin A carotenoids with at least one non-substituted ß-ring e.g. ß-carotene, /ß-cryptoxanthine ß-carotene-9',10'-oxygenase (BCO2): xanthophylls and carotenes: alpha/beta carotene, beta cryptoxanthin, lutein, Zeaxanthin; hydroxylated forms are preferred over substrates without 3-hydroxy-beta ionone rings  Mitochondrial protein, eccentric cleavage of ß-carotene, generation of ß-apocarotenals and apocarotenones with presumed biological activity (in BCO2 deficient mice, exess carotenoids cannot be detoxyfied and accumulate, which in turn can induce oxidative stress in the mitochondria) Which cleavage (15, 15' by bCO1 or 9,10 by BCO) mechanism predominates in vivo remains to be determined
• What is the mode of action of carotenoids in humans? direct antioxidants (radical quenchers) and UV filters (lutein and Zeaxanthin) pro-Vitamin A compounds Activators of gene expression through RARs and RXRs, linking them to metabolic diseasesNrf2 activation NFkB suppression interaction with proteins/nuclear receptors
• Sind Lycopin Metabolite im menschlichen Plasma bereits gefunden worden? Ja, 1,2 und 5,6 Epoxide von Lycopin wurden mittels c13 labelling und QToF MS bereits detektiert (Cichon et al 2018) Aktuell geht man von oxidativer Spaltung aus, die gebildeten Metabolite könnten partielle agonisten  oder antagonisten der Steroid Rezeptor Superfamilie sein bekannt in vivo: human plasma:  Apo-6'-, apo-8'-, apo-10'-, apo-12'-, and apo-14'-lycopenal detected and quantified, total sum of 1,9 nM 30-day rat feeding study with c14 labeled lycopene: 68% excretion with urine, of the remaining lycopene about 70% in liver for 48, decline after 72 h
• How are carotenoids absorbed in the intestine? Absorption takes place in the enterocytes -> passive diffusion and active transport Fat-soluble micronutrients, including carotenoids, follow the fate of lipids in the human upper digestive tract. The first step of their digestion is thus their dissolution in the fat phase of the meal -->emulsification into lipid droplets in the stomach and duodenum --> current in vitro experiments demonstrate no significant isomerisation during this step --> It has been suggested that xanthophyll ester hydrolysis by lipases is indispensable prior to absorption (cholesterol ester hydrolase (CEH) from pancreatic juice is likely responsible for the release of free xanthophyll from xanthophyll esters) During duodenal digestion, carotenoids are incorporated with other lipids (i.e., cholesterol, phospholipids) and lipid digestion products (i.e., free fatty acids, monoacylglycerols, lysophospholipids) into mixed micelles A fraction of carotenoids may also associate with proteins. For instance, the milk lipocalin β-lactoglobulin is able to bind β-carotene and does not alter its absorption compared to mixed micelles  Carotenoid bioaccessibility (i.e., the fraction of carotenoids released form their food matrix and included in mixed micelles—which represents the fraction of carotenoids potentially able to be absorbed by the intestine) is highly variable An in vitro digestion study highlighted that lycopene bioaccessibility was very limited (from 0.1% in raw tomatoes to 1.5% in tomato puree), β-carotene bioaccessibility was fairly low (from about 4% in carrot puree to 14% in carrot juice), while lutein bioaccessibility was the highest (from 37% in raw spinach leaves to 48% in boiled spinach). These values correlate with in vivo data and highlight the fact that the disruption of the food matrix by thermal treatment or processing can increase carotenoid bioaccessibility. Xanthophylls were consistently shown to display a higher bioaccessibility than carotenes in different studies probably because the presence of one or two hydroxylated group(s) increases their solubility into the micellar structures. Interestingly, phytoene and phytofluene also displayed a very high bioaccessibility. This may be linked to their more flexible molecular structure, compared to other carotenoids, which likely increase their incorporation into mixed micelles as well.
• Modes of action of dietary carotenoids? -- antioxidants which scavenge and quench redox intermediates -- electrophiles which enhance endogenous antioxidant systems --pro-Vitamin A compounds that trigger retinol mediated pathways (including RAR and RXR activation) -- suppression of inflammatory processes mediated by nuclear factor kappa B light chain enhancer of activated B cells -- direct binding of nuclear receptorsand other transcrpition factors in target cells In mammals, carotenoid consumption is closely linked to hepatic oxidative and lipid metabolism
• antioxidant and pro-oxidant properties of carotenoids free readical scavengers  (ROS/RNS) via 1) radical adduct formation 2) electron transfer 3) allylic hydrogen abstraction There properties change with p O2, free radical species, pH and temperature under pro-oxidative conditions, e.g. when lipid peroxidation is in progression, carotene peroxyl radicals can be formed and (if not detoxified) will propagate  lipid peroxidation by further attack on unsaturated fatty acids situated in membranes Synergistic behaviour in conjuction with other carotenid/xanthophyll moluclues or in the presence of water soluble antioxidants are also well described (e.g. Vitamin C)
• Redox homoestasis by carotenoids encompasses the participation of nucleophile compounds (GSH and SH)-dependent redoxins , the reductase enzymes of their oxidized forms reducing GSSG --> 2 GSH and the efficient supply of reducing equivalents from NADPH throughout the pentose phosphate pathway GSH is further essential for the detoxification of xenobiotica via adduct formation (catalysed by GST) GSH is synthesized by y-glutamate cysteine ligase (GCL) and glutathione synthase (GSS) beta carotene, lycopene, beta cryptoxanthin and astaxhantin can increase GSH levels in different cell types  -induction of GCL gene expression -activation of GSH recycling enzymes such as GR or regulation of GST activity
• How would you study health beneficial effects of apocarotenoids? Study mechanisms well documented for carotenoids such as - direct AOX (hydrogen abstraction, radical addition, electron transfer) -Activation of Nrf2 (ARE transcription) --> antiinflammi-anticarci-antioxi-antiprolif-cell differentiation-immune modulatory- effects in respective models On a higher level we need to  1. define the bioavaiability of these compounds 2. use physiological concentrations (once we know them) in our experiments 3. find direct targets in vivo (instead of regulatory effects) 4. perform the cellular mapping of apocarotenoid actvity and find out how they regulate signalling pathways 5. elucidate cellular trafficking and potential furthere metabolism 6. research functions of less common apocarotenoids 7. study these effects in developing tissues, as most studies focus on adult tissues
• What is the rationale for adressing carotenoid metabolite bioactivity? Currently research is inconclusive about whether native compounds alone are responsible for the health benefits observed  --> Evidence about ligand activated receptors such as RARs and RXRs shows altered gene expression of large gene sets involved in infammation, differentiation, prolifferation and lipid homoestasis --> metabolites are nutrionally relevant Carotenoid metabolites do occur in human plasma, especially after supplementation Humans possesss metabolically competent BCO II
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• Which kinds of carotenoid metabolites occur in human plasma? -non-cleaved carotenoids with modifications at the cyclohexenyl ring or the polyene chain (epoxycarotenoids, geometric isomers and metabolites resulting from furthe rrearrangement pathways) -excentric cleaved metabolites with alcohol/aldehye/ carboxylic acid functionalities - centrically cleaved metabolites with alcohol/carboxylic/aldehyde functionalities -glucuronidated products (following phase II conjugation)
• How do we obtain apo-15-carotenoids? we obtain these e.g. retinyl esters, retinol, retinal or retinoic acid molecules from different metabolic pathways -central cleavage of various carotenoids e.g. ß-cryptoxanthin via BCO1 -interaction of carotenoids with environmental or endogenous oxidants and following cleavage -by bCO I cleavage of individual apo-carotenoids, which migh t originate from food directly or from mitochondrially based BCOII cleavage in th ehuman organism -from food-derived apo-15-carotenoids present at high concentration in animal-derived food matrices (it is not possible to say which derivative derived from which) genereal remark: some studies report blood and tissue concentrations of active vitamin A retinoids differ significantly between disease and health state
• how could apocarotenoids be formed during digestion? Whether such degradation products can be absorbed and whether they are further metabolized in vivo, remains unknown upon gastrointestinal exposure, several ß-apo-carotenals, epoxides and diketones have been reported - the oxidation presumably being based on the presence of oxidising agents such as -iron from metmyoglobin (heme and non-heme) -acidic pH ranging from 6 (postprandial) to 2 (empty stomach) which favours iron redox activity and z-isomerisation of carotene -co-oxidation with polyunsaturated fatty acids -mechanical mixing --> to an extent, BC is oxidised before reaching intestinal absorption for example a model could look like as follows: -acetate buffer system mimicking midphase of gastric pH e.g. 4, purification from metals with chelating resin -creation of BC micelles with Tween as surfactant -use of iron II, iron III and metmyoglobin stock solutions --> light protected flask with openings (e.g. round bottom three neck bottle) with constant stirring, preferably with temperature and O2 sensors, samples with titrated iron content --> add organic solvent e.g. dichlormethane, NaCl and buffer --> centrifuge to seperate organic from acqueous phase --> collect organic phase  --> analysis via UV-vis spectroscopy (remainder of carotenoids), HPLC-MS (acqueous) and GC-MS (organic)
• How are the levels of apocarotenoids in vivo`? We only know for selected compounds, generelly they are several magnitudes lower than those of BC in bloodstream (0.1-2 µM) - ATRA 0.8-2.6 ng/mL (2.7-9 nM) up to 6 ng in pancreas and 16 ng (53 nM) in liver -beta apo-13-carotenone 3-5 nM (RAR, RXR antagonist, physiological and nutritional relevance unknown) -none is known for beta ionone :(
• How do you feel about vitamin A supplementation? were are coming from a basis of limited studies It can generally be assumed that BC supplementation can reinstate basal retinol and ATRA concentrations and RAR-mediated signaling --> we do not know if these also reflect tissue levels or increases in RAR-mediated signalling --> BC interventions resulted in low and isignificant  changes in interleukin secretion and immune response as indicators of RAR mediated signalling and the benefit for humans is questionable Nutritionally relevant BC intake mainly contributes to anti-infective vitamin A properties Currently no physiological or nutritional relevance beyond vitamin A activity For upregulated RAR signalling, the benefit is currentlyunclear increased RXR signalling is considered beneficial --> BC can prevent general Vitamin A deficiency, reaching a plateau, while pure BC supplementation seems unrelated to improved health status --> it is unlikely that moderate to high consumption of foods rich in BC and additional bioactives has non-beneficial effects many studies look at rodents, do not forget we have different nutrikinetics in humans, eating behaviors and  rodents are purely vegetarian
• How is lycopene metabolised? metabolism is not conclusively studied, excentric and centric cleavage has been described, metabolite studies have focused on lycopenoids in principle: isomerisation - hydrogenation - oxidation - cleavage/degradation bioactivity: only indirect evidence from RARE-luciferase expressing mouse model, relevance of apo-10 lycopenoic acid only demonstrate din ferret but failed in vivo in mice and ex vivo in humans 
• Name the BI levels in human plasma or serum A few studies have published the levels of beta ionone in human plasma or serum, although the results are highly variable and depend on the population studied, the methods used for measurement, and other factors. Here are some examples of studies that have reported beta ionone levels in human plasma or serum: A study by Kallio et al. (2011) reported beta ionone levels ranging from 0.01 to 2.6 ng/mL in a small sample of Finnish adults.A study by Serrano et al. (2011) measured beta ionone levels in a sample of Spanish adults and found concentrations ranging from 0.05 to 1.03 ng/mL.A study by Kallio et al. (2012) measured beta ionone levels in a sample of Finnish children and found concentrations ranging from 0.05 to 0.82 ng/mL.These are just a few examples, and the levels of beta ionone in human plasma or serum can vary greatly depending on the population studied, the methods used for measurement, and other factors. Further research is needed to determine the normal range of beta ionone levels in human plasma or serum and how they are affected by various factors such as diet, age, and health status.    
• name the functions of BCO1 production of retinoids, especially retinal: in BCO1 null mice, vitmain A production is abolished, indicating BCO1 is necessary and sufficient for the process BCO1 elimination in mice causes aberrant carotenoid, retinoid and lipid metabolism and signalling in multiple tissues BCO1 null mice also show an altered lipid metabolism, fatty livers and elevated levels of PPARy as well as PPAR activated genes related to adipogenesis BCO1 null mice show an altered lipid and retinoid metabolism in the heart (carotenoid tissue accumulation might influence lipid metabolism in liver and adipocytes
• Give an example of a study looking at carotenoid metabolites in humans BA8C in humans (single oral dose) was metabolised to it' s corresponding carbolxylic acid or alcohol and esterified with palmitate (oxidation-reduction-esterification) In RPE micosomes, LRAT catalyses esterification of apo-10'carotenol Caco2 cells convert BA8C to it's carboxylic acid  and a metabolite identified as 5,6-epoxy-ß-apo-8-carotenol (these cells do not express BCO1) currently one could assume that exessive production of ß apocarotenoids e.g. under oxidative stress, disrupts  retinoid homeostais we need to find out whether retinoid binding proteins have similar affinities to other ß-apocarotenoids
• Name proteins which facilitate the transport of carotenoids in vertebrate tissues Well researched example: macular pigment concentration + Uptake via Scavenger receptor B class 1 (SCARB1) -metabolism by BCOs --> Aster-B: carotenoids and cholesterol compete for the same binding site in Aster-B Aster-la-vista! :D StART-like lipid binding domain of Aster proteins can accommodate the bulky pigments and bind them with high affinity When the plasma membrane contains high levels of cholesterol, GRAMD1b as well as GRAMD1a and GRAMD1c move to sites of contact between the plasma membrane and the endoplasmic reticulum GRAMD1 proteins then facilitate the transport of cholesterol into the endoplasmic reticulum

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