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Carotenol fatty acid esters: easy substrates for digestive enzymes?
Comparative Biochemistry and Physiology Part B 132 (2002) 721–728
Carotenol fatty acid esters: easy substrates for digestive enzymes? Dietmar E. Breithaupt*, Ameneh Bamedi, Ursula Wirt ¨ Hohenheim, Institut fur ¨ Lebensmittelchemie, Garbenstr. 28, 70593 Stuttgart, Germany Universitat Received 6 March 2002; received in revised form 30 April 2002; accepted 30 April 2002
Abstract To study the specificity of gastric lipases on carotenoid mono- and diesters, an enzymatic assay was applied. Digestions were carried out in phosphate buffer at pH 7.4 and 37 8C. As substrates we employed oleoresins from marigold (Tagetes erecta L.; lutein diesters), red paprika (Capsicum annuum L., mainly capsanthin diesters), papaya (Carica papaya L.; b-cryptoxanthin esters), and loquat (Eriobotrya japonica Lindl.; b-cryptoxanthin esters) as well as retinyl palmitate. These were reacted with porcine pancreatic lipase, porcine pancreatin, porcine cholesterol esterase, and human pancreatic lipase. As reference enzyme a yeast lipase from Candida rugosa was applied. A high turnover could be observed with porcine pancreatic lipase and porcine cholesterol esterase, indicating cholesterol esterase to be a plausible candidate for generation of free carotenoids in the gut. Human pancreatic lipase accepted only retinyl palmitate as substrate, carotenoid mono- and diesters were not hydrolyzed. The assay permits an approach for calculation of enzymatic activities towards carotenoid esters as substrates for the first time, which is based on the amount of enzyme formulation, present in the assay (Uymg solid). Furthermore, these studies provide deeper insight into carotenoid ester bioaccessibility. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Bioaccessibility; Carotenoid esters; Cholesterol esterase; Human pancreatic lipase
1. 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 b,b-carotene is readily ingested without metabolic conversion in the human duodenum (Bowen et al., 1993). In many yellowyorange fruits and vegetables b,b-carotene is accompanied by other carotenes (e.g. lycopene) and xanthophylls (e.g. lutein, zeaxanthin, and b-cryptoxanthin), which, depending on the respective food plant, occur in free form and as fatty acid deriva*Corresponding author. Tel: q49-711-459-4094; fax: q49711-459-4096. E-mail address: [email protected] (D.E. Breithaupt).
tives. 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, 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: bcryptoxanthin possesses vitamin A activity and has been shown to exhibit an antioxidant capacity in the TEAC assay comparable to that of b,b-carotene (Van den Berg et al., 1999) whereas lutein
1096-4959/02/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 6 - 4 9 5 9 Ž 0 2 . 0 0 0 9 6 - 9
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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 (Maylie´ 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 resi-
dues 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 b-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. 2. Materials and methods 2.1. Chemicals The following lipases were used (activity given by the manufacturer): Lipase type VII from C. rugosa (12.1 Uymg solid using olive oil as substrate at pH 7.2, 37 8C, it contains lactose as an extender), lipase type II from porcine pancreas (crude product, it contains approx. 25% protein,
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3.7 Uymg protein using olive oil as substrate at pH 7.7, 37 8C), lipase from human pancreas (0.07 mg in 0.07 ml 0.1 M Tris buffer, 765 Uymg protein using 1,2-diglyceride as substrate at pH 8.1, 37 8C, diluted with 1.1 ml ultrapure water), and the lipase test kit ‘Lipase-PS娃 were purchased from Sigma–Aldrich (Taufkirchen, Germany). Pancreatin from porcine pancreas (lipase: 53 Uy mg solid, determined according to F.I.P. (Stellmach et al., 1988) using olive oil as substrate at pH 9.0, 37 8C) was a gift from Extrakt-Chemie (Stadthagen, Germany). Cholesterol esterase (from porcine pancreas, 44.4 Uymg protein using cholesterol acetate as substrate at pH 7.0, 37 8C), retinyl palmitate, lycopene, and b,b-carotene were obtained from FlukaySigma (Taufkirchen, Germany). Lutein, b-cryptoxanthin, and capsanthin were provided as a gift by the Roche Vitamins AG (Basel, Switzerland). Oleoresins from marigold (Tagetes erecta L.) and paprika (Capsicum annuum L.) were kindly supplied by Euram Food GmbH (Stuttgart, Germany). The oils were manufactured in the country of origin from dried petals or fruits by solvent extraction. Fully ripe fruits of papaya (Carica papaya L.) and loquat (Eriobotrya japonica Lindl.) as well as olive oil (cold pressed) were purchased from a supermarket. Ultrapure water was obtained from a Milli-Q 185 apparatus (Millipore, Eschborn, Germany). 2.2. Preparation of samples To prepare papaya and loquat oleoresins, edible portions (500 g each) were homogenized for 1 min by an Ultra Turrax T 25 (Janke and Kunkel, Germany) and extracted in aliquots (50 g) with light petroleum etherydiethyl ether (1:1 vyv, 4=50 ml), until the extracts were colorless. The combined extracts (2 l) were dried with anhydrous sodium sulfate (30 g), passed through a folded filter, and evaporated to dryness in vacuum at 30 8C. The oily residues were dissolved in light petroleum ether (50 ml). For determination of the total b-cryptoxanthin content, an aliquot (1 ml) was transferred to a flat bottomed flask, dried in a stream of nitrogen, dissolved in diethyl ether (100 ml), and saponified at room temperature overnight with methanolic potassium hydroxide (30% wyv, 5 ml). For complete removal of alkali, the solution was washed with water (2=100 ml). The organic layer was dried over anhydrous sodium sulfate (20 g), passed through a folded filter, evaporated to
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dryness, the residue dissolved in tert-butylmethyl etherymethanolybutylated hydroxytoluene (1:1:0.01 vyvyw, 2 ml), and subjected to HPLC analysis. Typically, the oleoresin solutions showed b-cryptoxanthin concentrations of approximately 50 mgyml for papaya and 35 mgyml for loquat. To prepare marigold and paprika oleoresin solutions, commercially available marigold and paprika oleoresins (140 and 180 mg, respectively) were dissolved in light petroleum ether (50 ml). For determination of the total lutein (marigold) and capsanthin (red paprika) content, aliquots (1 ml) were chemically saponified and treated as described above. The concentrations of both stock solutions were in the range of 50 mgyml for lutein and capsanthin, respectively. To prepare a retinyl palmitate solution in the same concentration range, 9.2 mg retinyl palmitate and 500 mg olive oil were dissolved in light petroleum ether (100 ml). 2.3. Enzymatic assay Aliquots of the solutions, equal to 100 mg carotenoid (paprika and marigolds1 ml each; papaya and retinyl palmitates2 ml each; loquats 3 ml) were transferred in sealable glass tubes (50 ml volume). The solvent was evaporated in a gentle stream of nitrogen. The enzymatic assay followed a protocol, developed in our laboratory (Breithaupt, 2000). In brief: Phosphate buffer (0.1 M, pH 7.4, 10 ml), bile salts (30 mg), and a sodium chlorideycalcium chloride solution (250 ml) were added to the residue and the mixture preincubated at 37 8C for 30 min. For enzymatic hydrolysis, an aliquot of a suspension containing lipase in calcium chloride solution (5 mM, 50–60 Uyassay; in the case of lipase from human pancreas: 9 Uyassay) was added and the mixture incubated at 37 8C for different time intervals (see Table 1). All enzyme solutions were prepared freshly every day. To stop the reaction, methanoly ethyl acetateylight petroleum ether (1:1:1, vyvyv, 20 ml) was added and the carotenoids extracted as described earlier (Breithaupt and Schwack, 2000). The residue was dissolved in methanoly tert-butylmethyl etherybutylated hydroxytoluene (1:1:0.01 vyvyw, 2 ml) and subjected to HPLC analysis. For each data point (one tube), three replicates were performed. 2.4. HPLC and LCyMS analyses HPLC was performed on an HP1050 HPLC system which comprised an autosampler, a gradient
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pump, a diode array detector (DAD) module (Hewlett–Packard, Waldbronn, Germany), and a column thermoregulator (Mistral, Spark, The Netherlands). UV absorbance was monitored at 450 nm (carotenoids) or 325 nm (retinol). A YMC (Schermbeck, Germany) analytical column (RPC30, 250=4.6 mm, 5 mm) equipped with a precolumn (RP-C30, 10=4.0 mm, 5 mm) was used and kept at 35 8C; the injection volume was 20 ml. The mobile phases were described previously (Breithaupt, 2000). LCyMS was run on an HP1100 HPLC system, modules as given above, coupled to a Micromass (Manchester, UK) VG platform II quadrupole mass spectrometer equipped with an Atmospheric Pressure Chemical Ionization (APCI)-interface; the MS parameters were detailed by Breithaupt and Schwack (2000). 2.5. Determination of lipase activities in enzyme formulations For determination of enzymatic activities we used a commercially available test kit, which is based on a photometric method of Imamura et al. (1989), using 1,2-diglyceride as substrate. All enzyme formulations were dissolved in ultrapure water (50 mgy100 ml; in the case of cholesterol esterase: 10 mgy100 ml) and used without further dilution. Measurements were carried out at 550 nm in micro disposable cuvettes (1.0=0.4=4.5 cm; 100 mm pathlength) from Ratiolab (Dreieich, Germany). To express the results determined at 37 8C, all values obtained at 25 8C were multiplied by a factor of 1.82. 3. Results and discussion 3.1. Carotenoid ester pattern of oleoresins used in this study Since papaya and loquat are known to be good sources for b-cryptoxanthin, the carotenoid ester
Fig. 1. Typical HPLC chromatogram (DAD, 450 nm) of a nonsaponified extract of loquat (Eriobotrya japonica Lindl.). Peak assignment: (1)sall-trans-b-cryptoxanthin; (2)s13-cis-b,bcarotene; (3)sall-trans-b,b-carotene; (4)s9-cis-b,b-carotene; (5)sb-cryptoxanthin laurate; (6)sb-cryptoxanthin myristate; (7)sb-cryptoxanthin oleate; (8)sb-cryptoxanthin palmitate; and (9)sb-cryptoxanthin stearate.
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 LCyMS before. Figs. 1 and 2 show typical chromatograms of non-saponified extracts of loquat and papaya, respectively. All-trans-b,bcarotene dominated the carotenoid spectrum of loquat (Fig. 1), accompanied by several cis-isomers, whereas 9-cis- and 13-cis-b,b-carotene were identified due to the intensity of the corresponding ‘cis-peaks’ of their UV spectra and the typical retention times on the RP-C30 column. The bcryptoxanthin ester profile was dominated by bcryptoxanthin laurate, myristate, oleate, and palmitate in similar amounts whereas b-cryptoxanthin stearate was present as trace component. In addition, very low amounts of free b-cryptoxanthin were detectable. Extracts of papaya (Fig. 2) showed a different carotenoid spectrum: besides
Table 1 Concentrations of enzyme solutions and reaction times, used in the enzymatic assay
Cholesterol esterase C. rugosa lipase Porcine pancreatic lipase Pancreatin from porcine pancreas Human pancreatic lipase a b
Concentration of enzyme solution wmgyml (ml)x
Reaction time wminx
10 (110)a 48 (100) 328 (200) 340 (200) 0.06 (200)
15y30y45y60b 60y120y180 60y120y180 60y120y180 240y1440
In parentheses: volumes used per assay. In the case of marigold and paprika oleoresins additional assays with reaction times of 120 and 180 min were conducted.
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Fig. 2. Typical HPLC chromatogram (DAD, 450 nm) of a nonsaponified extract of papaya (Carica papaya L.). Peak assignment: (1)sall-trans-b-cryptoxanthin; (2)sall-transb,b-carotene; (3)sb-cryptoxanthin caprate; (4)sb-cryptoxanthin laurate; (5)sb-cryptoxanthin myristate; (6)s b-cryptoxanthin palmitate; and (7)sall-trans-lycopene.
b,b-carotene and lycopene, significant amounts of free b-cryptoxanthin were present. The b-cryptoxanthin ester pattern was dominated by b-cryptoxanthin caprate, laurate, myristate, and palmitate with b-cryptoxanthin laurate being the carotenoid with the highest concentration in the native fruit extract. These findings are in accordance with results reported by Philip and Chen (1988) who used gas chromatography for identification of the fatty acid composition of b-cryptoxanthin esters of papaya. Minor peaks present in the chromatogram (Fig. 2) may be due to esters of b-cryptoxanthin-5,6-epoxide and antheraxanthin (Philip and Chen, 1988; Cano et al., 1996). Due to their low concentration they were not investigated here. The carotenoid ester pattern of red paprika and marigold are described in the literature (e.g. Gregory et al., 1987; Rivas, 1989) and were verified by own HPLC-MS (APCI detection) analyses, too: oleoresins from red paprika consist mainly of capsanthin diesters (capsanthin laurate, myristate, and palmitate; Breithaupt and Schwack, 2000) whereas marigold oleoresins are rich sources of lutein diesters with lutein dipalmitate being the major compound (Breithaupt et al., 2002). To investigate the behavior of retinyl palmitate in the enzymatic assay we used a solution of synthetic retinyl palmitate and added olive oil, simulating the usual fat content present in ‘natural’ oleoresins. 3.2. Enzymatic reactions with carotenoid esters and retinyl palmitate After HPLC analysis, using an RP-C30 phase, the amounts of enzymatically released lutein (mar-
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Fig. 3. Turnover of retinyl palmitate, b-cryptoxanthin esters from papaya and loquat, lutein diesters from marigold, and capsanthin diesters from paprika using porcine pancreatic lipase in the enzymatic assay.
igold), capsanthin (red paprika), b-cryptoxanthin (papaya and loquat), or retinol (retinyl palmitate), respectively, were determined in relation to the total quantities of carotenoids or retinol produced by treatment of sample aliquots with methanolic KOH (areas100%). With the exception of human pancreatic lipase, all enzymes accepted all substrates, but significantly different yields were observed. Figs. 3 and 4 illustrate some of the results. Porcine pancreatic lipase (Fig. 3) showed a high turnover with retinyl palmitate as substrate whereas b-cryptoxanthin esters were hydrolyzed to a lower extent. A very small turnover was observed with lutein and capsanthin diesters, respectively. Although carotenoid esters are esters of secondary long chain alcohols, they were accepted as substrates by porcine pancreatic lipase. With regard to carotenoid diesters it must be realized that only
Fig. 4. Turnover of retinyl palmitate, b-cryptoxanthin esters from papaya and loquat, lutein diesters from marigold, and capsanthin diesters from paprika using porcine cholesterol esterase in the enzymatic assay.
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the non-esterified form was used to calculate the turnover presented here; monoesters which were formed as by-products and which are present in both cases were not taken into account. Porcine cholesterol esterase (Fig. 4) showed in many respects similar results as porcine pancreatic lipase: retinyl palmitate and b-cryptoxanthin esters were readily hydrolyzed whereas lutein and capsanthin diesters showed very low hydrolysis rates. However, compared to porcine pancreatic lipase, the reaction with b-cryptoxanthin esters was much faster (after 1 h: cholesterol esterases62–77%; pancreatic lipases17–20%) whereas the conversion of retinyl palmitate was comparable in both cases (after 1 h: approx. 70% each). Using pancreatin from porcine pancreas, only low conversions were observed with exception of retinyl palmitate, which reacted again easily (74% after 1 h). Lipase from C. rugosa reacted readily with retinyl palmitate (89% after 1 h). Interestingly, the conversion of all other carotenoid esters was found to be almost identical (32–48% after 3 h), indicating a low specificity of this enzyme. Grochulski et al. (1993) speculated that acyl chains of substrates unfold the binding region of C. rugosa lipase, explaining the large variation in substrate specificity of this enzyme. The lipase from human pancreas showed no effect on carotenoid esters. The only substrate which was hydrolyzed was retinyl palmitate (15% after 4 h; 47% after 24 h). Compared to retinyl palmitate (C36, six double bonds), lutein mono(C56, 11 double bonds)- and dipalmitate (C72, 11 double bonds) are substrates which require a hydrophobic region large enough to accommodate long acyl chains. Additionally, the stability of carotenoid esters in acidic medium was checked by incubating aliquots with artificial gastric fluid at pH 1.2 for 24 h at ¨ 37 8C (List and Horhammer, 1967). The results proved carotenoid esters to be stable under these conditions (data not shown). Thus, cleavage of carotenoid esters caused by acidic conditions of the stomach can be excluded. 3.3. Calculation of enzymatic activities The activity of enzymes is usually calculated according to the following definition: 1 Unit is the amount of enzyme which converts 1 mmol substrateymin. For the use of this equation, the know-
ledge of the absolute amount of enzyme present in the assay is a prerequisite. This is often problematic since the composition of most commercial enzyme preparations is prevalently dominated by non- protein material such as carbohydrates, sodium chloride, and maltodextrin being the main compounds. Moreover, the protein fraction of many lipases frequently contains non-active proteins. Bjurlin et al. (2001) showed that formulations from porcine pancreas lipase contained approximately 20 isophoretically distinguishable protein bands. Furthermore, activities given by the manufacturer are dependent on the choice of substrate (mostly olive oil) and the method used for determination. Consequently, it is hardly possible to calculate the enzymatic activity based on the absolute amount of enzyme present in an assay. Therefore, we decided to calculate the activity on the basis of the amount of enzyme formulation, used in one assay (Uymg solid). Reactions were carried out in an air oven at 37 8C, simulating quasi physiological conditions. For calculation of enzymatic activities using carotenoid diesters as substrates (marigold and red paprika), the amounts (mmol) of free lutein or capsanthin were duplicated because two enzymatic steps are required to produce free carotenoids. Table 2 shows the calculated results and demonstrates again, that retinyl palmitate is the substrate accepted best by all investigated enzymes (10y2 – 10y4 Uymg solid). The activities of porcine pancreatic lipase and porcine pancreatin are in the range of 10y5 –10y6 Uymg solid, pointing out a comparatively low reactivity towards these carotenoid esters. Human pancreatic lipase showed relatively high activity towards retinyl palmitate (2.0=10y2 Uymg solid), too, comparable to those of porcine cholesterol esterase (1.2=10y2 Uymg solid). Since no conversion of carotenoid esters was observed using human pancreatic lipase in our assay, the respective activities could not be calculated. To get an idea of the enzymes activity towards more usual lipase substrates, we used a commercially available test kit intended for the quantitative determination of pancreatic lipase activity in serum. In this assay, 1,2-diglycerides serve as substrate, porcine colipase and deoxycholate are used as activators, human pancreatic lipase is used as standard enzyme. Since this assay uses one single substrate and identical conditions for each preparation, results are comparable. The following
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Table 2 Activities (Uymg solid) of cholesterol esterase, C. rugosa lipase, porcine pancreatic lipase, pancreatin from porcine pancreas, and human pancreatic lipase using lutein diesters from marigold (Tagetes erecta L.), capsanthin diesters from red paprika (Capsicum annuum L.), b-cryptoxanthin esters from papaya (Carica papaya L.) and loquat (Eriobotrya japonica Lindl.), as well as retinyl palmitate as substrates Enzyme
Cholesterol esterase Candida rugosa lipase Porcine pancreatic lipase Pancreatin from porcine pancreas Human pancreatic lipase
Activity (Uymg solid) Marigold
Paprika
Papaya
Loquat
Retinyl palmitate
8.1=10y4 3.5=10y4 5.5=10y6 4.1=10y6 –a
4.8=10y3 3.8=10y4 3.8=10y6 1.1=10y5 –a
2.8=10y3 1.1=10y4 5.8=10y6 3.4=10y6 –a
3.3=10y3 1.6=10y4 5.8=10y6 1.6=10y6 –a
1.2=10y2 1.7=10y3 1.2=10y4 1.2=10y4 2.0=10y2
a
No reaction was observed within 4 and 24 h. Results are given as average values of three replicates based on the reaction times given in Table 1.
activities were obtained: porcine cholesterol esterases44.8 Uymg; porcine pancreatins19.8 Uymg; porcine pancreatic lipases10.4 Uymg; C. rugosa lipases2.4 Uymg; human pancreatic lipases765 Uymg (determined by the manufacturer according to the same method). This demonstrates that all lipases investigated here accepted 1,2-diglycerides as substrate, human pancreatic lipase being the most effective. Lipase from C. rugosa showed only weak activity (2.4 Uymg) whereas porcine cholesterol esterase reacted well (44.8 Uymg); all other enzymes showed values between these. 4. 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 1,2diglycerides as lipase substrate, proved human pancreatic lipase to be very effective towards its common target. However, this enzyme did not accept carotenoid esters at all whereas retinyl palmitate was hydrolyzed. Thus, conversion of carotenoid esters by pancreatic lipase, as suggested by many authors, seems not to proceed smoothly. Our investigations show that cholesterol esterase, which is known to cleave secondary alcohols (e.g. retinyl esters) with high yields, is a plausible candidate for the generation of free carotenoids in the gut, rather than pancreatic lipase. This enzyme cleaves retinyl palmitate and b-cryptoxanthin esters with high yields. Concerning the determination of the accurate rate of conversion of cho-
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Gregory, G.K., Chen, T.-S., Philip, T., 1987. Quantitative analysis of carotenoids and carotenoid esters in fruits by HPLC: red bell peppers. J. Food Sci. 52, 1071–1073. Grochulski, P., Li, Y., Schrag, J.D., et al., 1993. Insights into interfacial activation from an open structure of Candida rugosa lipase. J. Biol. Chem. 268, 12843–12847. Imamura, S., Hirayama, T., Arai, T., Takao, K., Misaki, H., 1989. An enzymatic method using 1,2-diglyceride for pancreatic lipase test in serum. Clin. Chem. 35, 1126. Jacobs, P.B., LeBoeuf, R.D., McCommas, S.A., Tauber, J.D., 1982. The cleavage of carotenoid esters by cholesterol esterase. Comp. Biochem. Physiol. 72B, 157–160. Landrum, J.T., Bone, R.A., 2001. Lutein, zeaxanthin, and the macular pigment. Arch. Biochem. Biophys. 385, 28–40. ¨ List, P.H., Horhammer, L., 1967. Hagers Handbuch der Pharmazeutischen Praxis. Springer Verlag, Berlin, Heidelberg, New York p. 733. Liu, Y., Xu, M.J., Canfield, L.M., 1998. Enzymatic hydrolysis, extraction, and quantitation of retinol and major carotenoids in mature human milk. J. Nutr. Biochem. 9, 178–183. Matsufuji, H., Nakamura, H., Chino, M., Takeda, M., 1998. Antioxidant activity of capsanthin and the fatty acid esters in paprika (Capsicum annuum). J. Agric. Food Chem. 46, 3468–3472.
´ M., Charles, M., Gache, C., Desnuelle, P., 1971. Maylie, Isolation and partial identification of a pancreatic colipase. Biochem. Biophys. Acta 229, 286–289. Philip, T., Chen, T.-S., 1988. Quantitative analyses of major carotenoid fatty acid esters in fruits by liquid chromatography: persimmon and papaya. J. Food Sci. 53, 1720–1722. Rivas, J.D.L., 1989. Reversed-phase high-performance liquid chromatographic separation of lutein and lutein fatty acid esters from marigold flower petal powder. J. Chromatogr. 464, 442–447. Stellmach, B., Gottschick, W., Battermann, F., Zabel, K., 1988. Bestimmungsmethoden Enzyme. Steinkopff Verlag, Darmstadt p. 169. Van den Berg, R., Haenen, G.R.M.M., van den Berg, H., Bast, A., 1999. Applicability of an improved trolox equivalent antioxidant capacity (TEAC) assay for evaluation of antioxidant capacity measurements of mixtures. Food Chem. 66, 511–517. Wingerath, T., Stahl, W., Sies, H., 1995. beta-Cryptoxanthin selectively increases in human chylomicrons upon ingestion of tangerine concentrate rich in beta-cryptoxanthin esters. Arch. Biochem. Biophys. 324, 385–390. Wingerath, T., Sies, H., Stahl, W., 1998. Xanthophyll esters in human skin. Arch. Biochem. Biophys. 355, 271–274.
Carotenol fatty acid esters: easy substrates for digestive enzymes? Dietmar E. Breithaupt*, Ameneh Bamedi, Ursula Wirt ¨ Hohenheim, Institut fur ¨ Lebensmittelchemie, Garbenstr. 28, 70593 Stuttgart, Germany Universitat Received 6 March 2002; received in revised form 30 April 2002; accepted 30 April 2002
Abstract To study the specificity of gastric lipases on carotenoid mono- and diesters, an enzymatic assay was applied. Digestions were carried out in phosphate buffer at pH 7.4 and 37 8C. As substrates we employed oleoresins from marigold (Tagetes erecta L.; lutein diesters), red paprika (Capsicum annuum L., mainly capsanthin diesters), papaya (Carica papaya L.; b-cryptoxanthin esters), and loquat (Eriobotrya japonica Lindl.; b-cryptoxanthin esters) as well as retinyl palmitate. These were reacted with porcine pancreatic lipase, porcine pancreatin, porcine cholesterol esterase, and human pancreatic lipase. As reference enzyme a yeast lipase from Candida rugosa was applied. A high turnover could be observed with porcine pancreatic lipase and porcine cholesterol esterase, indicating cholesterol esterase to be a plausible candidate for generation of free carotenoids in the gut. Human pancreatic lipase accepted only retinyl palmitate as substrate, carotenoid mono- and diesters were not hydrolyzed. The assay permits an approach for calculation of enzymatic activities towards carotenoid esters as substrates for the first time, which is based on the amount of enzyme formulation, present in the assay (Uymg solid). Furthermore, these studies provide deeper insight into carotenoid ester bioaccessibility. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Bioaccessibility; Carotenoid esters; Cholesterol esterase; Human pancreatic lipase
1. 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 b,b-carotene is readily ingested without metabolic conversion in the human duodenum (Bowen et al., 1993). In many yellowyorange fruits and vegetables b,b-carotene is accompanied by other carotenes (e.g. lycopene) and xanthophylls (e.g. lutein, zeaxanthin, and b-cryptoxanthin), which, depending on the respective food plant, occur in free form and as fatty acid deriva*Corresponding author. Tel: q49-711-459-4094; fax: q49711-459-4096. E-mail address: [email protected] (D.E. Breithaupt).
tives. 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, 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: bcryptoxanthin possesses vitamin A activity and has been shown to exhibit an antioxidant capacity in the TEAC assay comparable to that of b,b-carotene (Van den Berg et al., 1999) whereas lutein
1096-4959/02/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 6 - 4 9 5 9 Ž 0 2 . 0 0 0 9 6 - 9
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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 (Maylie´ 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 resi-
dues 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 b-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. 2. Materials and methods 2.1. Chemicals The following lipases were used (activity given by the manufacturer): Lipase type VII from C. rugosa (12.1 Uymg solid using olive oil as substrate at pH 7.2, 37 8C, it contains lactose as an extender), lipase type II from porcine pancreas (crude product, it contains approx. 25% protein,
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3.7 Uymg protein using olive oil as substrate at pH 7.7, 37 8C), lipase from human pancreas (0.07 mg in 0.07 ml 0.1 M Tris buffer, 765 Uymg protein using 1,2-diglyceride as substrate at pH 8.1, 37 8C, diluted with 1.1 ml ultrapure water), and the lipase test kit ‘Lipase-PS娃 were purchased from Sigma–Aldrich (Taufkirchen, Germany). Pancreatin from porcine pancreas (lipase: 53 Uy mg solid, determined according to F.I.P. (Stellmach et al., 1988) using olive oil as substrate at pH 9.0, 37 8C) was a gift from Extrakt-Chemie (Stadthagen, Germany). Cholesterol esterase (from porcine pancreas, 44.4 Uymg protein using cholesterol acetate as substrate at pH 7.0, 37 8C), retinyl palmitate, lycopene, and b,b-carotene were obtained from FlukaySigma (Taufkirchen, Germany). Lutein, b-cryptoxanthin, and capsanthin were provided as a gift by the Roche Vitamins AG (Basel, Switzerland). Oleoresins from marigold (Tagetes erecta L.) and paprika (Capsicum annuum L.) were kindly supplied by Euram Food GmbH (Stuttgart, Germany). The oils were manufactured in the country of origin from dried petals or fruits by solvent extraction. Fully ripe fruits of papaya (Carica papaya L.) and loquat (Eriobotrya japonica Lindl.) as well as olive oil (cold pressed) were purchased from a supermarket. Ultrapure water was obtained from a Milli-Q 185 apparatus (Millipore, Eschborn, Germany). 2.2. Preparation of samples To prepare papaya and loquat oleoresins, edible portions (500 g each) were homogenized for 1 min by an Ultra Turrax T 25 (Janke and Kunkel, Germany) and extracted in aliquots (50 g) with light petroleum etherydiethyl ether (1:1 vyv, 4=50 ml), until the extracts were colorless. The combined extracts (2 l) were dried with anhydrous sodium sulfate (30 g), passed through a folded filter, and evaporated to dryness in vacuum at 30 8C. The oily residues were dissolved in light petroleum ether (50 ml). For determination of the total b-cryptoxanthin content, an aliquot (1 ml) was transferred to a flat bottomed flask, dried in a stream of nitrogen, dissolved in diethyl ether (100 ml), and saponified at room temperature overnight with methanolic potassium hydroxide (30% wyv, 5 ml). For complete removal of alkali, the solution was washed with water (2=100 ml). The organic layer was dried over anhydrous sodium sulfate (20 g), passed through a folded filter, evaporated to
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dryness, the residue dissolved in tert-butylmethyl etherymethanolybutylated hydroxytoluene (1:1:0.01 vyvyw, 2 ml), and subjected to HPLC analysis. Typically, the oleoresin solutions showed b-cryptoxanthin concentrations of approximately 50 mgyml for papaya and 35 mgyml for loquat. To prepare marigold and paprika oleoresin solutions, commercially available marigold and paprika oleoresins (140 and 180 mg, respectively) were dissolved in light petroleum ether (50 ml). For determination of the total lutein (marigold) and capsanthin (red paprika) content, aliquots (1 ml) were chemically saponified and treated as described above. The concentrations of both stock solutions were in the range of 50 mgyml for lutein and capsanthin, respectively. To prepare a retinyl palmitate solution in the same concentration range, 9.2 mg retinyl palmitate and 500 mg olive oil were dissolved in light petroleum ether (100 ml). 2.3. Enzymatic assay Aliquots of the solutions, equal to 100 mg carotenoid (paprika and marigolds1 ml each; papaya and retinyl palmitates2 ml each; loquats 3 ml) were transferred in sealable glass tubes (50 ml volume). The solvent was evaporated in a gentle stream of nitrogen. The enzymatic assay followed a protocol, developed in our laboratory (Breithaupt, 2000). In brief: Phosphate buffer (0.1 M, pH 7.4, 10 ml), bile salts (30 mg), and a sodium chlorideycalcium chloride solution (250 ml) were added to the residue and the mixture preincubated at 37 8C for 30 min. For enzymatic hydrolysis, an aliquot of a suspension containing lipase in calcium chloride solution (5 mM, 50–60 Uyassay; in the case of lipase from human pancreas: 9 Uyassay) was added and the mixture incubated at 37 8C for different time intervals (see Table 1). All enzyme solutions were prepared freshly every day. To stop the reaction, methanoly ethyl acetateylight petroleum ether (1:1:1, vyvyv, 20 ml) was added and the carotenoids extracted as described earlier (Breithaupt and Schwack, 2000). The residue was dissolved in methanoly tert-butylmethyl etherybutylated hydroxytoluene (1:1:0.01 vyvyw, 2 ml) and subjected to HPLC analysis. For each data point (one tube), three replicates were performed. 2.4. HPLC and LCyMS analyses HPLC was performed on an HP1050 HPLC system which comprised an autosampler, a gradient
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pump, a diode array detector (DAD) module (Hewlett–Packard, Waldbronn, Germany), and a column thermoregulator (Mistral, Spark, The Netherlands). UV absorbance was monitored at 450 nm (carotenoids) or 325 nm (retinol). A YMC (Schermbeck, Germany) analytical column (RPC30, 250=4.6 mm, 5 mm) equipped with a precolumn (RP-C30, 10=4.0 mm, 5 mm) was used and kept at 35 8C; the injection volume was 20 ml. The mobile phases were described previously (Breithaupt, 2000). LCyMS was run on an HP1100 HPLC system, modules as given above, coupled to a Micromass (Manchester, UK) VG platform II quadrupole mass spectrometer equipped with an Atmospheric Pressure Chemical Ionization (APCI)-interface; the MS parameters were detailed by Breithaupt and Schwack (2000). 2.5. Determination of lipase activities in enzyme formulations For determination of enzymatic activities we used a commercially available test kit, which is based on a photometric method of Imamura et al. (1989), using 1,2-diglyceride as substrate. All enzyme formulations were dissolved in ultrapure water (50 mgy100 ml; in the case of cholesterol esterase: 10 mgy100 ml) and used without further dilution. Measurements were carried out at 550 nm in micro disposable cuvettes (1.0=0.4=4.5 cm; 100 mm pathlength) from Ratiolab (Dreieich, Germany). To express the results determined at 37 8C, all values obtained at 25 8C were multiplied by a factor of 1.82. 3. Results and discussion 3.1. Carotenoid ester pattern of oleoresins used in this study Since papaya and loquat are known to be good sources for b-cryptoxanthin, the carotenoid ester
Fig. 1. Typical HPLC chromatogram (DAD, 450 nm) of a nonsaponified extract of loquat (Eriobotrya japonica Lindl.). Peak assignment: (1)sall-trans-b-cryptoxanthin; (2)s13-cis-b,bcarotene; (3)sall-trans-b,b-carotene; (4)s9-cis-b,b-carotene; (5)sb-cryptoxanthin laurate; (6)sb-cryptoxanthin myristate; (7)sb-cryptoxanthin oleate; (8)sb-cryptoxanthin palmitate; and (9)sb-cryptoxanthin stearate.
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 LCyMS before. Figs. 1 and 2 show typical chromatograms of non-saponified extracts of loquat and papaya, respectively. All-trans-b,bcarotene dominated the carotenoid spectrum of loquat (Fig. 1), accompanied by several cis-isomers, whereas 9-cis- and 13-cis-b,b-carotene were identified due to the intensity of the corresponding ‘cis-peaks’ of their UV spectra and the typical retention times on the RP-C30 column. The bcryptoxanthin ester profile was dominated by bcryptoxanthin laurate, myristate, oleate, and palmitate in similar amounts whereas b-cryptoxanthin stearate was present as trace component. In addition, very low amounts of free b-cryptoxanthin were detectable. Extracts of papaya (Fig. 2) showed a different carotenoid spectrum: besides
Table 1 Concentrations of enzyme solutions and reaction times, used in the enzymatic assay
Cholesterol esterase C. rugosa lipase Porcine pancreatic lipase Pancreatin from porcine pancreas Human pancreatic lipase a b
Concentration of enzyme solution wmgyml (ml)x
Reaction time wminx
10 (110)a 48 (100) 328 (200) 340 (200) 0.06 (200)
15y30y45y60b 60y120y180 60y120y180 60y120y180 240y1440
In parentheses: volumes used per assay. In the case of marigold and paprika oleoresins additional assays with reaction times of 120 and 180 min were conducted.
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Fig. 2. Typical HPLC chromatogram (DAD, 450 nm) of a nonsaponified extract of papaya (Carica papaya L.). Peak assignment: (1)sall-trans-b-cryptoxanthin; (2)sall-transb,b-carotene; (3)sb-cryptoxanthin caprate; (4)sb-cryptoxanthin laurate; (5)sb-cryptoxanthin myristate; (6)s b-cryptoxanthin palmitate; and (7)sall-trans-lycopene.
b,b-carotene and lycopene, significant amounts of free b-cryptoxanthin were present. The b-cryptoxanthin ester pattern was dominated by b-cryptoxanthin caprate, laurate, myristate, and palmitate with b-cryptoxanthin laurate being the carotenoid with the highest concentration in the native fruit extract. These findings are in accordance with results reported by Philip and Chen (1988) who used gas chromatography for identification of the fatty acid composition of b-cryptoxanthin esters of papaya. Minor peaks present in the chromatogram (Fig. 2) may be due to esters of b-cryptoxanthin-5,6-epoxide and antheraxanthin (Philip and Chen, 1988; Cano et al., 1996). Due to their low concentration they were not investigated here. The carotenoid ester pattern of red paprika and marigold are described in the literature (e.g. Gregory et al., 1987; Rivas, 1989) and were verified by own HPLC-MS (APCI detection) analyses, too: oleoresins from red paprika consist mainly of capsanthin diesters (capsanthin laurate, myristate, and palmitate; Breithaupt and Schwack, 2000) whereas marigold oleoresins are rich sources of lutein diesters with lutein dipalmitate being the major compound (Breithaupt et al., 2002). To investigate the behavior of retinyl palmitate in the enzymatic assay we used a solution of synthetic retinyl palmitate and added olive oil, simulating the usual fat content present in ‘natural’ oleoresins. 3.2. Enzymatic reactions with carotenoid esters and retinyl palmitate After HPLC analysis, using an RP-C30 phase, the amounts of enzymatically released lutein (mar-
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Fig. 3. Turnover of retinyl palmitate, b-cryptoxanthin esters from papaya and loquat, lutein diesters from marigold, and capsanthin diesters from paprika using porcine pancreatic lipase in the enzymatic assay.
igold), capsanthin (red paprika), b-cryptoxanthin (papaya and loquat), or retinol (retinyl palmitate), respectively, were determined in relation to the total quantities of carotenoids or retinol produced by treatment of sample aliquots with methanolic KOH (areas100%). With the exception of human pancreatic lipase, all enzymes accepted all substrates, but significantly different yields were observed. Figs. 3 and 4 illustrate some of the results. Porcine pancreatic lipase (Fig. 3) showed a high turnover with retinyl palmitate as substrate whereas b-cryptoxanthin esters were hydrolyzed to a lower extent. A very small turnover was observed with lutein and capsanthin diesters, respectively. Although carotenoid esters are esters of secondary long chain alcohols, they were accepted as substrates by porcine pancreatic lipase. With regard to carotenoid diesters it must be realized that only
Fig. 4. Turnover of retinyl palmitate, b-cryptoxanthin esters from papaya and loquat, lutein diesters from marigold, and capsanthin diesters from paprika using porcine cholesterol esterase in the enzymatic assay.
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the non-esterified form was used to calculate the turnover presented here; monoesters which were formed as by-products and which are present in both cases were not taken into account. Porcine cholesterol esterase (Fig. 4) showed in many respects similar results as porcine pancreatic lipase: retinyl palmitate and b-cryptoxanthin esters were readily hydrolyzed whereas lutein and capsanthin diesters showed very low hydrolysis rates. However, compared to porcine pancreatic lipase, the reaction with b-cryptoxanthin esters was much faster (after 1 h: cholesterol esterases62–77%; pancreatic lipases17–20%) whereas the conversion of retinyl palmitate was comparable in both cases (after 1 h: approx. 70% each). Using pancreatin from porcine pancreas, only low conversions were observed with exception of retinyl palmitate, which reacted again easily (74% after 1 h). Lipase from C. rugosa reacted readily with retinyl palmitate (89% after 1 h). Interestingly, the conversion of all other carotenoid esters was found to be almost identical (32–48% after 3 h), indicating a low specificity of this enzyme. Grochulski et al. (1993) speculated that acyl chains of substrates unfold the binding region of C. rugosa lipase, explaining the large variation in substrate specificity of this enzyme. The lipase from human pancreas showed no effect on carotenoid esters. The only substrate which was hydrolyzed was retinyl palmitate (15% after 4 h; 47% after 24 h). Compared to retinyl palmitate (C36, six double bonds), lutein mono(C56, 11 double bonds)- and dipalmitate (C72, 11 double bonds) are substrates which require a hydrophobic region large enough to accommodate long acyl chains. Additionally, the stability of carotenoid esters in acidic medium was checked by incubating aliquots with artificial gastric fluid at pH 1.2 for 24 h at ¨ 37 8C (List and Horhammer, 1967). The results proved carotenoid esters to be stable under these conditions (data not shown). Thus, cleavage of carotenoid esters caused by acidic conditions of the stomach can be excluded. 3.3. Calculation of enzymatic activities The activity of enzymes is usually calculated according to the following definition: 1 Unit is the amount of enzyme which converts 1 mmol substrateymin. For the use of this equation, the know-
ledge of the absolute amount of enzyme present in the assay is a prerequisite. This is often problematic since the composition of most commercial enzyme preparations is prevalently dominated by non- protein material such as carbohydrates, sodium chloride, and maltodextrin being the main compounds. Moreover, the protein fraction of many lipases frequently contains non-active proteins. Bjurlin et al. (2001) showed that formulations from porcine pancreas lipase contained approximately 20 isophoretically distinguishable protein bands. Furthermore, activities given by the manufacturer are dependent on the choice of substrate (mostly olive oil) and the method used for determination. Consequently, it is hardly possible to calculate the enzymatic activity based on the absolute amount of enzyme present in an assay. Therefore, we decided to calculate the activity on the basis of the amount of enzyme formulation, used in one assay (Uymg solid). Reactions were carried out in an air oven at 37 8C, simulating quasi physiological conditions. For calculation of enzymatic activities using carotenoid diesters as substrates (marigold and red paprika), the amounts (mmol) of free lutein or capsanthin were duplicated because two enzymatic steps are required to produce free carotenoids. Table 2 shows the calculated results and demonstrates again, that retinyl palmitate is the substrate accepted best by all investigated enzymes (10y2 – 10y4 Uymg solid). The activities of porcine pancreatic lipase and porcine pancreatin are in the range of 10y5 –10y6 Uymg solid, pointing out a comparatively low reactivity towards these carotenoid esters. Human pancreatic lipase showed relatively high activity towards retinyl palmitate (2.0=10y2 Uymg solid), too, comparable to those of porcine cholesterol esterase (1.2=10y2 Uymg solid). Since no conversion of carotenoid esters was observed using human pancreatic lipase in our assay, the respective activities could not be calculated. To get an idea of the enzymes activity towards more usual lipase substrates, we used a commercially available test kit intended for the quantitative determination of pancreatic lipase activity in serum. In this assay, 1,2-diglycerides serve as substrate, porcine colipase and deoxycholate are used as activators, human pancreatic lipase is used as standard enzyme. Since this assay uses one single substrate and identical conditions for each preparation, results are comparable. The following
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Table 2 Activities (Uymg solid) of cholesterol esterase, C. rugosa lipase, porcine pancreatic lipase, pancreatin from porcine pancreas, and human pancreatic lipase using lutein diesters from marigold (Tagetes erecta L.), capsanthin diesters from red paprika (Capsicum annuum L.), b-cryptoxanthin esters from papaya (Carica papaya L.) and loquat (Eriobotrya japonica Lindl.), as well as retinyl palmitate as substrates Enzyme
Cholesterol esterase Candida rugosa lipase Porcine pancreatic lipase Pancreatin from porcine pancreas Human pancreatic lipase
Activity (Uymg solid) Marigold
Paprika
Papaya
Loquat
Retinyl palmitate
8.1=10y4 3.5=10y4 5.5=10y6 4.1=10y6 –a
4.8=10y3 3.8=10y4 3.8=10y6 1.1=10y5 –a
2.8=10y3 1.1=10y4 5.8=10y6 3.4=10y6 –a
3.3=10y3 1.6=10y4 5.8=10y6 1.6=10y6 –a
1.2=10y2 1.7=10y3 1.2=10y4 1.2=10y4 2.0=10y2
a
No reaction was observed within 4 and 24 h. Results are given as average values of three replicates based on the reaction times given in Table 1.
activities were obtained: porcine cholesterol esterases44.8 Uymg; porcine pancreatins19.8 Uymg; porcine pancreatic lipases10.4 Uymg; C. rugosa lipases2.4 Uymg; human pancreatic lipases765 Uymg (determined by the manufacturer according to the same method). This demonstrates that all lipases investigated here accepted 1,2-diglycerides as substrate, human pancreatic lipase being the most effective. Lipase from C. rugosa showed only weak activity (2.4 Uymg) whereas porcine cholesterol esterase reacted well (44.8 Uymg); all other enzymes showed values between these. 4. 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 1,2diglycerides as lipase substrate, proved human pancreatic lipase to be very effective towards its common target. However, this enzyme did not accept carotenoid esters at all whereas retinyl palmitate was hydrolyzed. Thus, conversion of carotenoid esters by pancreatic lipase, as suggested by many authors, seems not to proceed smoothly. Our investigations show that cholesterol esterase, which is known to cleave secondary alcohols (e.g. retinyl esters) with high yields, is a plausible candidate for the generation of free carotenoids in the gut, rather than pancreatic lipase. This enzyme cleaves retinyl palmitate and b-cryptoxanthin esters with high yields. Concerning the determination of the accurate rate of conversion of cho-
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