Lack of release of bound anthocyanins and phenolic acids from carrot plant cell walls and model composites during simulated gastric and small intestinal digestion

Separately, polyphenols and plant cell walls (PCW) are important contributors to the health benefits associated with fruits and vegetables. However, interactions with PCW which occur either during food preparation or mastication may affect bioaccessibility and hence bioavailability of polyphenols. Binding interactions between anthocyanins, phenolic acids (PAs) and PCW components, were evaluated using both a bacterial cellulose-pectin model system and a black carrot puree system. The majority of available polyphenols bound to PCW material with 60-70% of available anthocyanins and PAs respectively binding to black carrot puree PCW matter. Once bound, release of polyphenols using acidified methanol is low with only similar to 20% of total anthocyanins to similar to 30% of PAs being released. Less than 2% of bound polyphenol was released after in vitro gastric and small intestinal (S.I.) digestion for both the model system and the black carrot puree PCW matter. Confocal laser scanning microscopy shows localised binding of anthocyanins to PCW. Very similar patterns of binding for anthocyanins and PAs suggest that PAs form complexes with anthocyanins and polysaccharides. Time dependent changes in extractability with acidified methanol but not the total bound fraction suggests that initial nonspecific deposition on cellulose surfaces is followed by rearrangement of the bound molecules. Minimal release of anthocyanins and PAs after simulated gastric and S.I. digestion indicates that polyphenols in fruits and vegetables which bind to the PCW will be transported to the colon where they would be expected to be released by the action of cell wall degrading bacteria.

Diet, cell signalling, and tumourigenesis in multiple intestinal neoplasia mice

The incidence of colon cancer is high in Western societies, and in Finland it is among the three most common cancer types in both females and males. Environmental factors, including diet, affect colon cancer development. During the last few years, a vast amount of new, functional foods have been introduced to the consumers. Several products are already available that are marketed as promoting intestinal health. To be able to reliably call a dietary compound a chemopreventive substance it is of fundamental importance to understand the mechanism by which it affects tumour formation and the integrity of the epithelial cells. In this thesis, three different dietary compounds were studied in an experimental model of colon cancer. Inulin is a non-digestible fibre found naturally in chicory roots, artichokes and onions, amongst others. Nowadays it is widely used as an added dietary fibre in several food products. Conjugated linoleic acid (CLA) is a conjugated form of the fatty acid linoleic acid. CLA is formed by bacterial fermentation of linoleic acid in the rumen of cows and other ruminants. Concomitantly, it can naturally be found in milk and meat of ruminants. White currant is a colourless berry low in phenolic compounds that are believed to prevent cancer formation. Contrary to what was expected, inulin and the conjugated linoleic acid isomer trans-10, cis-12, were tumour growth promoting dietary constituents when fed to Min mice. Both diets decreased the NF-kappaB levels in the mucosa, but physiological adenoma development did not affect NF-kappaB. Diet altered beta-catenin and p53 signalling in the adenomas, confirming their involvement in adenoma growth. White currant, on the other hand, was chemopreventive, despite its low contents of phenolic compounds. The chemopreventive effect was accompanied by increased p53 levels in the mucosa, and decreased beta-catenin and NF-kappaB levels in the adenoma. This could explain the reduced adenoma number and size. The results underline the importance of carefully testing new dietary compounds in different settings to reliably confirm their health benefits. In this study two compounds that are consumed and believed to add to our health proved to be cancer promotive. A berry with low phenolic contents, on the other hand, was chemopreventive.

The mechanism of intestinal absorption of phosphatidylcholine in rats

1. The mechanism of absorption of phosphatidylcholine was studied in rats by injecting into the intestine phosphatidylcholine specifically labelled either in the fatty acid or in the glycerol moiety or with 32P, when considerable amounts of 1-acyl-lysophosphatidylcholine were found in the intestinal lumen. 2-([14C]Acyl)phosphatidylcholine gave markedly more radioactive unesterified fatty acids in the lumen, compared with the 1-([14C]acyl) derivative. Some of the radioactivity from either the fatty acid or the glycerol moiety of the injected phosphatidylcholine appeared in the mucosal triacylglycerols. 2. Injection of 32P-labelled phosphatidylcholine or 32P-labelled lysophosphatidylcholine led to the appearance of radioactive glycerylphosphorylcholine, glycerophosphate and Pi in the mucosa. 3. Rat mucosa was found to contain a highly active glycerylphosphorylcholine diesterase. 4. It was concluded that the dietary phosphatidylcholine is hydrolysed in the intestinal lumen by the pancreatic phospholipase A to 1-acylglycerylphosphorylcholine, which on entering the mucosal cell is partly reacylated to phosphatidylcholine, and the rest is further hydrolysed to glycerylphosphorylcholine, glycerophosphate, glycerol and Pi. The fatty acids and glycerophosphate are then reassembled to give triacylglycerols via the Kennedy (1961) pathway.

Studies on the relative biopotencies and intestinal absorption of different apo-beta-carotenoids in rats and chickens.

1. The biopotencies relative to beta-carotene of several apocarotenoids, such as 8'-, 10'- and 12'-apo-beta-carotenal and methyl 8'-apo-beta-carotenoate, were investigated in rats, on a molar basis, by both curative-growth assay and liver-storage tests. 2. In the curative-growth assays, on a molar basis the biopotencies of 8'-, 10'- and 12'-apo-beta-carotenal and methyl 8'-apo-beta-carotenoate were 72, 78, 72 and 53% respectively, whereas on a weight basis the corresponding values were 93, 111, 111 and 63%, with respect to beta-carotene taken as 100%. In terms of yield of vitamin A, these values were much lower in the liver-storage tests. 3. When 8'-apo-beta-carotenal was fed, the unchanged aldehyde together with small amounts of the corresponding alcohol and larger proportions of the acid rapidly appeared in the tissues of both rats and chickens. The 8'-apocarotenol, 8'-apocarotenoic acid and its methyl ester were absorbed unchanged. The free acid disappeared most rapidly from the tissues, but its methyl ester persisted in the tissues longest. 4. On the basis of these observations it is suggested that most of an apocarotenal is oxidized to the corresponding acid, which, in turn, is mostly degraded to retinoic acid, with small proportions of it being attacked by the dioxygenase system giving retinal.

Novel matrix metalloproteinases in intestinal inflammation and in cancer of the gastrointestinal tract

Matrix metalloproteinases (MMPs) comprise a family of 23 zinc-dependent human endopeptidases that can degrade virtually all components of the extracellular matrix (ECM). They are classified into eight subgroups according to their structure and into six subgroups based on their substrate-specificity. MMPs have been implicated in inflammation, tissue destruction, cell migration, arthritis, vascular remodeling, angiogenesis, and tumor growth and invasion. MMPs are inhibited by their natural inhibitors, tissue inhibitors of metalloproteinases (TIMPs). Different MMPs function in the same tasks depending on the tissue or cancer subtype. I investigated the role of recently discovered MMPs, especially MMPs-19 and -26, in intestinal inflammation, in intestinal and cutaneous wound healing, and in intestinal cancer. Several MMPs and TIMPs were studied to determine their exact location at tissue level and to obtain information on possible functions of MMPs in such tissues and diseases as the healthy intestine, inflammatory bowel disease (IBD), neonatal necrotizing enterocolitis (NEC), pyoderma gangrenosum (PG), and colorectal as well as pancreatic cancers. In latent celiac disease (CD), I attempted to identify markers to predict later onset of CD in children and adolescents. The main methods used were immunohistochemistry, in situ hybridization, and Taqman RT-PCR. My results show that MMP-26 is important for re-epithelialization in intestinal and cutaneous wound healing. In colon and pancreatic cancers, MMP-26 seems to be a marker of invasive potential, although it is not itself expressed at the invasive front. MMP-21 is upregulated in pancreatic cancer and may be associated with tumor differentiation. MMPs-19 and -28 are associated with normal tissue turnover in the intestine, but they disappear in tumor progression as if they were protective markers . MMP-12 is an essential protease in intestinal inflammation and tissue destruction, as seen here in NEC and in previous CD studies. In patients with type 1 diabetes (T1D), MMPs-1, -3, and -12 were upregulated in the intestinal mucosa. Furthermore, MMP-7 was strongly elevated in NEC. In a model of aberrant wound repair, PG, MMPs-8, -9, and 10 and TNFα may promote ECM destruction, while absence of MMP-1 and MMP-26 from keratinocytes retards re-epithelialization. Based on my results, I suggest MMP-26 to be considered a putative marker for poor prognosis in pancreatic and colon cancer. However, since it functions differently in various tissues and tumor subtypes, this use cannot be generalized. Furthermore, MMP-26 is a beneficial marker for wound healing if expressed by migrating epithelial cells. MMP-12 expression in latent CD patients warrants research in a larger patient population to confirm its role as a specific marker for CD in pathologically indistinct cases. MMP-7 should be considered one of the most crucial proteases in NEC-associated tissue destruction; hence, specific inhibitors of this MMP are worth investigating. In PG, TNFα inhibitors are potential therapeutic agents, as shown already in clinical trials. In conclusion, studies of several MMPs in specific diseases and in healthy tissues are needed to elucidate their roles at the tissue level. MMPs and TIMPs are not exclusively destructive or reparative in tissues. They seem to function differently in different tissues. To identify selective MMP inhibitors, we must thoroughly understand the MMP profile (degradome) and their functions in various organs not to interfere with normal reparative functions during wound repair or beneficial host-response effects during cancer initiation and growth.

Acylation of lysolecithin in the intestinal mucosa of rats

1. The presence of an active acyl-CoA–lysolecithin (1-acylglycerophosphorylcholine) acyltransferase was demonstrated in rat intestinal mucosa. 2. ATP and CoA were necessary for the incorporation of free [1-14C]oleic acid into lecithin (phosphatidylcholine). 3. The reaction was about 20 times as fast with [1-14C]oleoyl-CoA as with free oleic acid, CoA and ATP. 4. With 1-acylglycerophosphorylcholine as the acceptor, both oleic acid and palmitic acid were incorporated into the β-position of lecithin; the incorporation of palmitic acid was 60% of that of oleic acid. 5. Of the various analogues of lysolecithin tested as acyl acceptors from [1-14C]oleoyl CoA, a lysolecithin with a long-chain fatty acid at the 1-position was most efficient. 6. The enzyme was mostly present in the brush-border-free particulate fraction of the intestinal mucosa. 7. Of the various tissues of rats tested for the activity, intestinal mucosa was found to be the most active, with testes, liver, kidneys and spleen following it in decreasing order.

Intestinal transport in the silkworm, Bombyx mori L., of amino acids from mixtures

The rate of absorption of amino acids from mixtures has been studied in the silkworm midgut by using an in vitro perfusion technique. The rates differ for individual amino acids. A characteristic absorption pattern is observed which is independent of the amino acid composition of the mixture used. The metabolic inhibitors dinitrophenol and cyanide have no effect on the amino acid transport from mixtures. Based on these results an energy-independent, carrier-mediated transport is postulated.

Evidence for the inability of the intestinal mitochondria of the silkworm, Bombyx mori L.to oxidize fatty acids

Conditions for the preparation of mitochondria from silkworm intestines have been standardized. The inability of mitochondria to oxidize fatty acids has been demonstrated. Evidence for the absence of an inhibitor in the mitochondria has been obtained.