Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology
Carotenol fatty acid esters: easy substrates for digestive enzymes?
Introduction
Carotenoids and carotenoid esters are lipid soluble and follow the same absorption mechanism as dietary triacylglycerides and other fatty minor components (Furr and Clark, 1997). It could be shown that β,β-carotene is readily ingested without metabolic conversion in the human duodenum (Bowen et al., 1993). In many yellow/orange fruits and vegetables β,β-carotene is accompanied by other carotenes (e.g. lycopene) and xanthophylls (e.g. lutein, zeaxanthin, and β-cryptoxanthin), which, depending on the respective food plant, occur in free form and as fatty acid derivatives. Surprisingly, little information is given in the literature about the metabolism of xanthophyll esters. It has been suggested that ester hydrolysis by lipases is indispensable prior to absorption (Bowen et al., 1993, Furr and Clark, 1997, Wingerath et al., 1995, Wingerath et al., 1998), and that the required enzymes are generated by the pancreas and secreted into the gut. The specific enzymes involved in the gastric hydrolysation process of carotenoid esters are yet unknown. Thus, it is hardly possible to estimate the (bio)availability while the rate of conversion is not known. Diminished amounts of unesterified carotenoids, present for direct incorporation into lymphatic lipoproteins, may reduce the health benefits attributed to xanthophylls: β-cryptoxanthin possesses vitamin A activity and has been shown to exhibit an antioxidant capacity in the TEAC assay comparable to that of β,β-carotene (Van den Berg et al., 1999) whereas lutein and zeaxanthin are expected to retard macular degeneration (Landrum and Bone, 2001). The antioxidant activity of capsanthin and its esters, isolated from red paprika, has been studied by Matsufuji et al. (1998). Their results suggest that both, free and esterified capsanthin, are good radical scavengers.
Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3), widely distributed in animals, plants, and prokaryotes, preferentially hydrolyze glycerol esters of long chain fatty acids. Thus, triacylglycerol and other lipophilic dietary fat components are their natural substrates. Pancreatic lipase is the best known and most investigated of all lipolytic enzymes. It has found extensive use as a research tool in lipid chemistry owing to the fact that it readily hydrolyzes primary ester groups of triacylglycerides (Brockerhoff and Jensen, 1974). Secondary alcohols, e.g. 2-monoglycerides, are hydrolyzed during the human digestion process after acyl migration of the acid from position 2 to position 1 or 3. In contrast to most other enzymes, lipases exert their biological activity at the lipid–water interface of micellar or emulsified substrates. In the course of digestion, micelles are generated by the presence of bile salts, enhancing the hydrolytic activity of lipases (interfacial activation). Additional stimulation of lipolysis is caused by colipase, which is synthesized in the pancreas and activates the classic pancreatic lipase in presence of bile salts (Maylié et al., 1971).
Pancreatic cholesterol esterase (EC 3.1.1.13) is responsible for the hydrolysis of dietary cholesterol esters, esters of fat soluble vitamins, phospholipids, and mono-, di- as well as triacylglycerides. One reason for the high activity of cholesterol esterase towards several substrates is attributed to its unusual structural features. Chen et al. (1998), who investigated the structure of bovine cholesterol esterase, found that this enzyme possesses no helical lid, covering the catalytic triade of most lipases. This implies a high affinity towards a wide range of lipophilic substrates, since a wide gap in the center of the molecule is accessible. Jacobs et al. (1982) used cholesterol esterase from Pseudomonas fluorescens for facile and specific hydrolysis of carotenoid esters occurring in algae and crayfish, e.g. astaxanthin diesters and fucoxanthin, yielding by-products under chemical hydrolysis procedures. They found cholesterol esterase to react rather with long chain astaxanthin diesters than with carotenoids possessing short chain residues as fucoxanthin, a carotenoid acetate. Another attempt to hydrolyze carotenoid acetates was made successfully by Aakermann et al. (1996) who used pig liver esterase in Tris–HCl buffer to hydrolyze peridinin, pyrrhoxanthin, and other acetates. The possible role of cholesterol esterase in carotenoid ester digestion has not been discussed yet.
Lipase from Candida rugosa (formerly C. cylindracea) is an almost universal lipolytic biocatalyst. Benjamin and Pandey (1998) discussed its numerous versatile catalytic reactions. Since C. rugosa lipase is known to cleave carotenoid esters with high yields (Liu et al., 1998, Breithaupt, 2000) we used this yeast lipase as a reference enzyme for assay control.
To study the hydrolyzing capacity of typical gastric lipases on natural carotenoid esters, an enzyme assay comprising bile salts as emulsifying agent and calcium ions as activator of the lipases was applied. As substrates we employed commercially available oleoresins from marigold (Tagetes erecta L.; lutein diesters) and red paprika (Capsicum annuum L.; mainly capsanthin diesters), as well as self made oleoresins from papaya (Carica papaya L.) and loquat (Eriobotrya japonica Lindl.), both comprising high amounts of β-cryptoxanthin esters. Since vitamin A is preferentially stored in human liver cells as palmitate ester, we used retinyl palmitate as carotenoid derived substrate, too. The best suited enzyme for digestion of triacylglycerides is pancreatic lipase. Therefore porcine pancreatic lipase and pancreatin from porcine pancreas were employed. Furthermore, porcine cholesterol esterase and human pancreatic lipase were investigated. Colipase was not used as an additive in our enzymatic assay; however, porcine pancreatic lipase, which is a crude product from porcine pancreas, and pancreatin may contain uncontrolled levels of native colipase since it is rather difficult to completely separate this protein cofactor from lipase.
Section snippets
Chemicals
The following lipases were used (activity given by the manufacturer): Lipase type VII from C. rugosa (12.1 U/mg solid using olive oil as substrate at pH 7.2, 37 °C, it contains lactose as an extender), lipase type II from porcine pancreas (crude product, it contains approx. 25% protein, 3.7 U/mg protein using olive oil as substrate at pH 7.7, 37 °C), lipase from human pancreas (0.07 mg in 0.07 ml 0.1 M Tris buffer, 765 U/mg protein using 1,2-diglyceride as substrate at pH 8.1, 37 °C, diluted
Carotenoid ester pattern of oleoresins used in this study
Since papaya and loquat are known to be good sources for β-cryptoxanthin, the carotenoid ester pattern of both fruits was investigated using an HPLC-MS (APCI detection; pos. mode) method previously described (Breithaupt and Schwack, 2000). As far as we know, the carotenoid ester pattern of papaya and loquat was never investigated by LC/MS before. Fig. 1, Fig. 2 show typical chromatograms of non-saponified extracts of loquat and papaya, respectively. All-trans-β,β-carotene dominated the
Conclusions
Many authors consider the free form of carotenoids as crucial for their biological functions. Hence, it is important to know about the ‘bioaccessibility’ of carotenoid esters, which, according to this statement, is given by the rate of hydrolysis of carotenoid esters during the intestinal passage. As expected, a test kit, based on 1,2-diglycerides as lipase substrate, proved human pancreatic lipase to be very effective towards its common target. However, this enzyme did not accept carotenoid
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