[16] Stability of β-carotene under different laboratory conditions

[ 16]

fl-CAROTENE STABILITY

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[ 16] Stability of fl-Carotene under Different Laboratory Conditions By

GIORGIO

SCITA

Carotenoids are a well-characterized class o f compounds present in microorganisms, algae, higher plants, animals, and humans.~,2 More than 600 carotenoids occur in nature. About 38 are precursors o f vitamin A. O f these, only a few occur in sufficient concentration to play a significant role in the h u m a n diet. The most plentiful carotenoid is/?-carotene (BC), which also has the highest vitamin A activity. Multiple biological functions beside the provitamin A activity have been ascribed to this compound, for instance, the activity as singlet oxygen or free-radical scavenger) as a stimulant o f the i m m u n o response, 4 and the action as anticarcinogenic agent. ~ In consequence of this variety o f functions and in correlation with the nontoxicity o f BC, its biological role and metabolic fate have been the subject of emphasis in recent research. Nevertheless, little has been done to test the stability of BC under conditions other than food storage or processing. Simpson and Chichester, 6 for instance, reviewed the metabolic degradation of BC in senescent tissues, showing also how a pure solution of this pigment was destroyed or altered by acid and, in some cases by alkalies. EI-Tinay and Chichester 7 studied the oxidation o f BC in toluene at various temperatures, finding that the rate o f BC loss indicated a zero-order reaction in the presence o f excess oxygen. Carnevale et al. s showed that fluorescent light catalyzed the autooxidation o f BC dissolved in fatty acid. A free-radical destruction of BC was shown to occur during the aerobic conversion o f sulfite, a chemical widely used in foods, to sulfate, a process known to generate radicals) The purpose o f this chapter is to define the conditions to be used in T. W. Goodwin, "The Comparative Biochemistry of Carotenoids." Chapman and Hall, London, 1952. 2 T. W. Goodwin, "The Biochemistryof the Carotenoids," 2nd Ed., Vol. 1. Chapman and Hall, London, 1980. G. W. Burton and K. U. Ingold, Science 224, 569 0984). 4A. Bendich, Clin. Nutr. 7, 113 (1988). 5N. I. Krinsky, Clin. Nutr. 7, 107 (1988). 6 K. L. Simpson and C. O. Chichester,Annu. Rev. Nutr. 1, 351 (1982). 7A, H. El-Tinay and C. O. Chichester, J. Org. Chem. 35, 2290 (1970). s j. Carnevale, E. R. Cole, and G. Crank, J. Agric. FoodChem, 27, 462 (1979). 9 G. D. Peiser and S. F. Yang, J. Agric. Food Chem. 27, 446 (1979). METHODS IN ENZYMOLOGY, VOL 213

Copyright © 1992 by Academic Press, Inc. All rights ofr~roduction in any form reserved.

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laboratory research on fl-carotene that could prevent its degradation. To do this, (1) BC stability was examined under conditions usually used in an experimental setting to investigate its metabolic or functional relevance both in vitro and in cell culture systems; (2) the effect of some antioxidants in preventing this degradation was tested; and (3) its storage stability under controlled conditions with regard to the type of solvent, light, temperature, and atmosphere was tested.

Reagents fl-[14C]carotene (BC) was a gift from Hoffmann-La Roche (Nutley, NJ). Unlabeled BC was purchased from Fluka Biochemica (Buchs, Switzerland), a-Tocopherol, butylated hydroxytoluene (BHT), and taurodeoxycholic acid (TDCA) were from Sigma (St. Louis, MO). All the organic solvents were Fischer-brand (Santa Clara, CA) high-performance liquid chromatography (HPLC) grade. The HPLC column was an Ultrasphere ODS, 5/tin, 4.6 × 150 mm, from Beckman (Fullerton, CA). Separation was performed on a Beckman/Altex HPLC system. Citoscynt, the scintillation liquid, was from ICN (Irvine, CA).

High-Performance Liquid Chromatography The extracts from each experiment (see below) were analyzed by HPLC in the following way: after evaporating to dryness under a stream of N2, the samples were reconstituted in a mixture of toluene-methanol (1:3) and injected into the HPLC column. The optimized HPLC analysis was performed at a flow rate of 1.5 ml/min, using a linear gradient elution system from 100% solvent A (methanol plus 0.5% ammonium acetate) to 80% solvent A plus 20% toluene, developed in 12 min. The retention time of BC was 12 rain. The effluent was monitored at 452 nm.

Stability of BC under Simulated Incubation Conditions Two different micellar solutions of BC were prepared. The first was a modification of the method of Bertram ~° obtained by rapidly mixing 20 #1 of a 2 m M solution of BC in tetrahydrofuran-dimethylsulfoxide ( T H F 10 R. V. Cooney and J. S. Bertram, this series, Vol. 214 [6].

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fl-CAROTENESTABILITY

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DMSO) (1 : 1, v/v) plus 5/tl of a 0.5 m M [I*C]BC solution in T H F - D M S O (1:1, v/v) into 5 ml of 90% Iscove's modified Dulbecco's medium (IMDM) with 10% bovine fetal serum and stirring it for 30 min. The second was prepared according to a modified procedure by Westergaard and Dietschy. ~1 Crystalline BC was dissolved in T H F to provide a final concentration of 8 mM. This solution (400/tl) was evaporated to dryness under ]'42 and the resulting precipitated crystals were resuspended in 50/tl of T H F and added to 55 ml of 40 m M taurodeoxycholic acid (TDCA) in 40 m M Tris, pH 7.4. The mixture was gently stirred overnight in the dark at room temperature, then diluted 1:1 with the same buffer without TCDA and sonicated for 15 min using a Branasonic (Branson Sonic Power, Danbury, CN) 220 sonicator bath. The BC recovered in micellar solution was 60%. Both types ofmicellar solutions were incubated at 37 ° in Coming (Coming, NY) dishes (60-mm diameter) in the dark, in presence of a 5% CO2 in air atmosphere. At each time point a volume of ethanol plus 0.025% BHT was added to aliquots of each solution and, after saturating with NaC1, extracted twice with 6 vol ofhexane. The samples were then evaporated to dryness and reconstituted either in toluene- methanol ( I : 3, v/v) when analyzed by HPLC, or in benzene when the BC concentration was determined by spectrophotometer at 452 nm. When [~4C]BC was used, each fraction from the HPLC elution was collected, mixed with scintillation liquid, and counted by a liquid scintillation counter.

Exposure of BC to Light Crystalline BC was dissolved in a solution of toluene with or without 0.025% BHT or a-tocopherol to provide a final concentration of 4 ltM. This solution (50 ml) was put into a 200-ml beaker to produce a layer 1.5 cm deep and a surface area of 45.3 cm 2. Exposure was to a fluorescent cold lamp (40 W) giving an intensity of 6500 Ix at the surface of the sample from a distance of 10 cm. A UV lamp at 325 nm was used for UV treatment, giving an intensity of 750 pW/cm 2 with the sample at a 30-cm distance. The BC concentration was tested at different times by monitoring the absorbance at 463 nm. The experiment with a smaller air exchange was carded out under the same conditions except that a test tube with an exchange surface of 0.785 cm 2 was used.

~1 H. Westergaard and J. M. Dietschy, J. Clin. Invest. 58, 97 (1976).

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Stability of BC u n d e r Simulated Incubation Conditions This study was carried out to test the stability of two different micellar BC solutions at different times, under dark conditions at 37 °. Detection limits of less than 1 pmol were obtained by the use of liquid chromatographic techniques together with the utilization of radioactive BC. Furthermore, as a double check, a spectrophotometric monitoring system was used. As shown in Fig. 1 the BC appeared to be fairly stable over 24 hr of incubation (less than 4%) with no significant differences between the two different methods used to prepare micellar solutions. Even after 48 hr of incubation, the loss of the pigment did not exceed 10 and 15%, respectively, for the TCDA and the THF miceUar solutions. No known putative metabolic products were detected in the breakdown products of p-carotene. To have shown the BC is quite stable under conditions of relatively high temperature and for a long time is relevant in relation to the widespread use of cell-free and cell culture systems, which require similar incubation conditions for the study of BC metabolism (uptake and cleavage) and for the investigation of a direct anticarcinogenic action of this pigment.

Light Degradation and Antioxidant Effect To explore the factors that could cause BC degradation, the effects of different kinds of light exposure were investigated. As shown in Figs. 2 and 3, the exposure of BC both to UV and fluorescent light, concomitant with experimental conditions that allowed a good atmospheric oxygen exchange, was highly damaging. The loss of the pigment was complete after 48 hr, resulting in total bleaching under both conditions: UV light was three times more effective (50% loss occurs after 8 hr) than fluorescent light (50% loss occurs after 24 hr). In this respect the present results are in accord with those previously obtained by Carnevale et al. s for BC autooxidation catalyzed by fluorescent light. Nevertheless, it is not easy to compare their rates of BC loss to the present data because the composition of lipids used as solvent system by these authors highly affected this rate (50% loss occurred after 15 hr in laurate solution, while no loss was observed in oleate solution), showing that a protective effect had been introduced with greater efficiency as unsaturation was increased. Having established the degradation rate induced by light-atmospheric air exposure on BC, the protective effect of two common antioxidants, butylated hydroxytoluene (BHT) and a-tocopherol, were further investigated. BHT, at the usual concentration used in biological assays (1 mM), accounted for a loss reduction that ranged from more than 210% (50% loss

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TIME(hr) FIG. 1. Rate of degradation of fl-carotene from a micellar solution under simulated incubation conditions. (A) a micellar solution in T H F - D M S O (1:1, v/v) consisting of unlabeled (2 raM) and labeled BC (0.5 mM), was mixed with cell culture medium and incubated at 37* in the dark under a 5% CO2:95% air atmosphere. An aliquot of each sample was extracted at different times and analyzed both for the unlabeled BC content (O), by measuring the absorbance at 452 nm in benzene, and for the labeled BC content (4,), by a monitoring system described in the text. (B) Micellar solutions of fl-carotene (17#M) in taurodeoxycholic acid were incubated and analyzed as described above. Each value is the mean of at least two determinations, which differed by no more than 5% of the mean.

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[ 16]

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FIG. 2. Rate of degradation of a toluene solution offl-earotene exposed to different light sources in the presence (0) or in the absence (O) of butylated hydroxytoluene. Solutions of BC (4 g M ) were put in a 200-ml beaker and exposed to ultraviolet light (A) or to fluorescent light (B) for different times. At each time point an aliquot was taken and the BC content was determined by spectrophotometer at 463 n m in toluene. Each data point is the mean of two determinations, which differed by no more than 5% of the mean.

[ 16]

fl-CAROTENE STABILITY

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FIG. 3. Effect of different concentrations of c~-tocopherol on BC degradation by air-light exposure. Solutions of BC (4 pM) in toluene were exposed to UV light (A) or to fluorescent light (B) in the presence of different concentrations of t~-tocopherol: control, no c~-tocopherol (O); 0.1 m M ct-tocopherol (0); 1 m M a-tocopheroi (A); 10 m M ot-tocopherol (A). The BC content was determined by measuring the absorbance at 463 nm in toluene. Each value is the mean of two determinations, which differed by no more than 4% of the mean.

182

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occurs after 17 hr compared to 8 hr in the absence of antioxidant) to more than 140% (50% loss after 33 hr compared to 23 hr in the absence of antioxidant), respectively, on UV light and fluorescent light exposure. The effect of the most potent natural antioxidant, a-tocopherol, is shown in Fig. 3. There was a concentration-dependent effect in both conditions assayed. At the same concentration of BHT (1 mM) the 50% BC loss occurs by 48 and 40 hr, respectively, on UV and fluorescent light exposure, showing that a-tocopherol had a much stronger relative potency to prevent BC degradation than BHT under our exposure conditions. The protective effect of t~-tocopherol and BHT was also shown by Peiser and Yang 9 in an experimental system in which BC destruction was induced by free radicals formed from the transformation of sulfite to sulfate. This might suggest that even in the present incubation, BC degradation could be a free radicalmediated process that could be promoted by light absorption either by BC or by the toluene used as a solvent. Nevertheless, because a cis-trans isomerization has been suggested to occur in the autooxidation process of carotenoids, 12,1a further investigation will be required to assess the relevance of this interconversion in reducing the maximum absorbance used as monitoring signal of BC degradation in the present assay. Interestingly, there is a marked difference in the disappearance curves for BC under UV and fluorescent illumination. The BC concentration, in the absence of antioxidant, decreased in a hyperbolic fashion when exposed to UV light and linearly under fluorescent illumination. Thus UV light appears to operate by a first-order kinetic model, while the action of fluorescent light is best described as zero order. Speculation about this difference in terms of mechanism requires further investigation, although the direct absorption of fluorescent light by BC ( 2 ~ = 450) may account for such a difference. The stability of BC exposed to fluorescent light in relation to different rates of air-solution exchange was also studied to investigate the effect of oxygen. The rate of gas-liquid exchange is affected by the size of the surface of the solution exposed to air. An experiment was carried out comparing the degradation rate of BC solution in a test tube (exchange surface of 0.785 cm 2) and in a 200-ml beaker (exchange surface of 45.3 cm2). As shown in Fig. 4 the 50% loss occurs after 72 hr for the solution in the test tube and after 23 hr for the beaker.

t2 R. T. Holman, Arch. Biochem. 21, 51 (1949). ,3 p. Budowski and A. Bondi, Arch. Biochem. Biophys. 89, 66 (1960).

[ 16]

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~-CAROTENE STABILITY 100~

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FIG. 4. Rate of degradation of toluene solution of ~-carotene exposed to fluorescent light under conditions of varying air exchange. Solutions of BC (4 pM) in toluene in the presence (filled symbols) or in the absence (open symbols) of 0.025% BHT were put into test tubes (exchange surface, 0.785 cm2: open and filled circles) or a 200-ml beaker (exchange surface, 45.3 cm2: open and filled triangles) and exposed to fluorescent light. At each time point the BC content was analyzed as described in Fig. 3. The values are the mean of two determinations, which differed by no more than 6% of the mean.

Stability of BC under Storage Conditions To test the stability under conditions o f prolonged storage, BC in T H F solution was kept at - 2 0 °, avoiding any illumination by using a double brown container (two brown bottles, one inside the other), in a nitrogen atmosphere. The concentration was analyzed at different times both by liquid chromatographic and spectrophotometric systems. The rate o f BC loss ranged f r o m 1. 1%/month in the presence o f B H T to 1.55%/month in the absence o f a n t i o x i d a n t during the 4 - m o n t h experiment as shown in Fig. 5. This clearly strengthens the i m p o r t a n c e o f controlling air, light exposure, and t e m p e r a t u r e to reduce any degradative processes. An antioxidant like B H T can effectively i m p r o v e the storage life o f BC even when the degradative factors are minimized.

184

SEPARATIONAND QUANTITATION

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Fio. 5. Stability of#-carotene under storage conditions. Solutions (I r a M ) of BC in T H F in the presence (open symbols) or in the absence (filled symbols) of 0.025% BHT were kept at - 20 ° in a double brown bottle in the dark under N 2. At each time point an aliquot was taken and analyzed for BC content both by a liquid chromatographic system (circles) and a spectrophotometric monitoring system at 452 n m in benzene (triangles). Percentage of maxim u m peak area on HPLC separation was calculated on the basis of the nanomoles (5 n m o l ) of BC detected by the liquid chromatographic system. Each data point is the mean of two determinations, which differed by no more than 4% of the mean.

Conclusion The present work is to my knowledge the first attempt at systematic investigation of the factors that could affect the stability of BC under the common laboratory conditions generally used to evaluate its biological role and to investigate its metabolic pathway. BC remained fairly stable during 48 hr of what is defined as "incubation conditions" (37 °, 5% CO2, in the dark), generally used for cell-free or cell culture model systems. Besides, as great emphasis has been put on the anticarcinogenic properties of BC, independently of a vitamin A-mediated action, it was important to show that, under the condition used, the integrity of the molecule is preserved. BC is rapidly degraded by ultraviolet and visible light in the presence of atmospheric oxygen, the degradation rate being lower with a lower air-oxygen exchange. This points to the following laboratory working conditions to preserve BC solutions from degradation: use of a subdued fight environment and the use of a dark container with a small air-surface exchange. A significant improvement in

[17]

CAROTENOID REVERSED-PHASEHPLC METHODS

185

BC stability is also given by the routine use of antioxidants such as the two well-known radical trapping agents, BHT and vitamin E. This, together with the absence of any sensitizer (to rule out the involvement of singlet oxygen), suggests that the light-induced degradation of this pigment could involve a chain-radical mechanism. Finally, the relative stability of BC was confirmed when the degradative factors were strictly controlled and antioxidants were present, suggesting conditions to be used to prolong its storage life. Acknowledgments I thank Dr. George Wolf for a careful review and stimulating discussion of the manuscript. I am grateful to Dr. H. N. Bhagavan (Hoffmann-La Roche, Inc.) for supplying the fl-[14C]carotene. Supported by USDA Grant No. 87-CRCR-I-2593.

[17] C a r o t e n o i d R e v e r s e d - P h a s e H i g h - P e r f o r m a n c e Liquid Chromatography Methods: Reference Compendium By NEAL E. CRAFT

Introduction High-performance liquid chromatographic (HPLC) methods for the separation of carotenoids were first reported in 1970) A positive pressure of nitrogen gas was applied to a traditional open column system to speed the separation and reduce oxygen exposure. By the standards of today this would constitute low-pressure liquid chromatography. The sophistication of carotenoid HPLC methods has evolved as HPLC columns and equipment have improved. The first report of carotenoids separated using highpressure pumping systems was in 1971, when Stewart and Wheaton2 employed gradient elution normal-phase HPLC to separate 23 different carotenoid peaks from citrus peel using a zinc carbonate column over a period of 5 hr. Their accomplishment constituted a phenomenal improvement over previous separations. By comparison, most current HPLC separations of carotenoids are performed using short (< 20 min) isocratic conditions with commercially prepared reversed-phase columns) -6 This is J. P. Sweeney and A. C. Marsh, J. Assoc. Off. Anal. Chem. 53, 937 (1970). 2 I. Stewart and T. Wheaton, J. Chromatogr. 55, 325 (1971). 3 W. Driskell, M. Bashor, and J. Neese, Clin. Chem. 29, 1042 (1983).

METHODS IN ENZYMOLOGY, VOL 213

Copyright© 1992by AcademicPress, Inc. All rightsof reproductionin any form t~served.